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ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

ICAR-IVRI, IZATNAGAR
DIVISION OF VETERINARY MEDICINE
ICAR-INDIAN VETERINARY RESEARCH INSTITUTE
IZATNAGAR, BAREILLY-243122

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OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
ICAR-IVRI, IZATNAGAR
DIVISION OF VETERINARY MEDICINE
ICAR-INDIAN VETERINARY RESEARCH INSTITUTE
IZATNAGAR, BAREILLY-243122, UP

ANNUAL REPORT (2016-2017)

1. Name of Centre : IVRI, IZATNAGAR


2. Name of Investigators: Dr S. Dey, Dr Mohini Saini (Biochemistry), Dr A. K. Sharma
(Pathology), Dr K. Mahendran (Medicine) and Dr Sumit Mahajan (Medicine)
3. Approve activity (2016-17):
Scientific validation of antiurolithiatic activity of selected medicinal plants used by
ethnomedicine.
Scientific validation of wound healing potential of 4 pure compounds isolated from
medicinal plant from IICT Hyderabad.
In vivo anti trypanosomal potential of 2 pure compounds isolated from ORP-EVM-
AD 005 and their interaction with standard antitrypanosomal drug.
4. Salient achievement
Hydro-ethanol extract of dry fruits of ORP-EVM 21 possess potent anti-
Urolithiasis activity in vivo
Ec 50 of the extract was found as 250 mg/kg orally
Besides anti-lithiatic property the extract posses Reno-protective potential as
indicated in renal function test
The extract bears potent antioxidant property and could be a possible mechanism
for its Reno-protective potential
ORP EVM-21 reduced the immunohitochemical expression of matrix glycoprotein,
Osteopontin
Out of 4 compound isolated from Ethanol Extract of IICT #001 compound B has
excellent wound healing property followed by Compound D and Compound C
Compound B has increased Hydrohy-proline and d-glucosamine content
significantly and shown excellent wound healing in histopathology.

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Compound B has up- regulated glucosamine (UDP-N-acetyl)-2-Epimerase/N-
acetylmannosamine kinase gene up to 30 fold compared to control in healing tissue
followed by compound D and compound C
In vitro assay to evaluate antitrypanosomal activity of Purified compound isolated
from AD-005 had IC50 values of 6.65g/ml.
The interaction between Purified compound isolated from AD-005 with diminazene
aceturate, isometamidium chloride and quinapyramine sulphate revealed reduction
in the concentration of standard drugs and test agents required to inhibit
trypanosomes to a specific percent.
The purified compound showed no trypanocidal activity but a 24 hr increase in the
life span of infected mice could be noted in purified compound treated group.

5. Concise progress Report (01.04.2016 to 31.03.2017)

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5.1 Experiment 1: Antiurolithiatic potential of Cucumis callosus (Rottl.) Cogn. fruit
extract in ethylene glycol induced urolithiatic rats
The Cucumis callosus (Rottl.) Cogn is aherb of family Cucurbitaceaeand
cultivated during rainy season throughout arid regions, especially north western part of
India. The herb is locally known as Kachri in the state of Rajasthan. Ethnic people of
Bikaner district of Rajasthan state used Cucumis callosus (Rottl.) Cogn as a folk remedies
for problems like pain or irritations in urethra, dribbling or stoppage of urine and other
urinary affliction.Such clinical symptoms are also recorded in urolithiasis. However, its
antiurolithiatic efficacy was not explored scientifically. Therefore, this study was
designed for scientific validation ofkachari fruits against urolithiasis to preserve the ethnic
medicinal valuesand to explore the possible mechanism of antiurolithiatic effect of Kachri
to rationalize its medicinal use.

5.1.1 Plant material collection and authentication


The Cucumis callosus (Rottl.) Cogn dry fruits were collected from Bana and Sadhasar
Villages of Bikaner, Rajasthan. The fruits were identified and authenticated from
Botanical Survey of India, Central National Herbarium, Howrah, India.
5.1.1.1 Extraction method
The dry fruits were uniformly powdered using an electric grinder. Cucumis callosus
hydro-ethanolic extract (CCHEE) was prepared using ethanol and distilled water (1:1) as
solvents for 6 hours on a magnetic stirrer at room temperature. The extract was filtered
using Whatman filter paper no. 40. The extract was dried in vacuoand stored in a well
closed container at 4 C until use (Williamson, 1996).The recovery of extract was found
to be 31.4% (w/w).
5.1.2 Animals
Healthy male albino rats of 12-13 weeks age andweighing 150-200g bred inthe
Laboratory Animal Research Division of the Institute were used for experiment. The
rats were housed in clean polypropylenecages at room temperature 25 2 0C, with 12
hours light -12 hours dark cycle throughout theexperiment and provided with standard
ration and ad libitum water.They were acclimatized well prior to starting of protocol.

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Whole study and animal care procedure were conducted according to standard
guidelines of Committee for the Purpose of Control and Supervision of Experiment on
Animals (CPSCSEA) after prior permission from Institute Animal Ethics Committee,
IVRI (Approval no-F.26-1/2015-16/JDR).
5.1.3 In vivo urolith induction and treatment
In vivo antiurolithiatic activity of the test plant material was investigated
usinghyperoxaluric rat model of calcium oxalate urolithiasisas described previously
(Panigrahi et al., 2016b; Khan et al., 2011; Shirfule et al., 2013). Thirty male rats were
dividedwith matched body weights into five groups of six rats each (n=6), and given
various treatments. The urolithiasis was induced by administering 0.75% (w/v) ethylene
glycol (EG) along with 1% (w/v) ammonium chloride in drinking water up to 14th day.
Only Group II rats were provided with addition 0.75% (w/v) EG alone treatment till 28th
day.
Group I: Healthy control, without receiving any treatment.
Group II: lithiatic control
Group III: Vehicle control, up to 14th day lithiatic treatment and thereafter ad libitum
distilledwater till 28th day
Group IV: Standard treatment, up to 14th day lithiatic treatment and thereafter Cystone at
100 mg/kg b.w. till 28th day through gastric gavages
Group V: Hydro-ethanolic plant extracttreated, up to 14th day lithiatic treatment and
thereafter Cucumis extract at EC50that is 250 mg/kg b.w.through gastric gavages (The
EC50 dose was calculated from earlier study, detail not presented in this manuscript).
5.1.4 Biochemical parameters
5.1.4.1 Analysis of urine
The rats were housed individually in metabolic cages for 24 hours period and urine samples
were collected in sterile vial on 0, 14th and 28th day of study. Immediately after collection,
urine pH and volume were measured. The half of urine was acidified using 1-2 drop of 3
N Hydrochloric acid and remaining was kept non-acidified. Then both sample were
centrifuged at 1500 rpm for 10 minutes to remove extraneous objects and only clear
supernatant were preserved at -200C until evaluation. Morning 3 hours non-acidified urine
sample was used for microscopic examination of crystal. The acidified urine was processed

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to assess urinary calcium, phosphorus, sodium and potassium using standard commercial
kit while urinary oxalate by usingquantitative ELISA kit (Quibiotech, China). Non
acidified urine were used for Urine urea nitrogen (UUN) and Creatinine estimation
(Panigrahi et al., 2016b).
5.1.4.2 Analysis of blood
The blood was collected in preheparinised sterile tube from retro orbital venous plexus of
rat using microhaematocrit capillaries (Sorg and Buckner, 1964; Sikarwar et al., 2017) on
day 0, 14th and 28th.Then blood was centrifuged at 3000 rpm for 20 minutes, plasma was
separated and stored at -200C until use. The blood urea nitrogen (BUN), creatinine,
calcium, phosphorous, total protein and albumin were measured using standard commercial
kit by spectrophotometry.
5.1.4.3 Oxidative stress indices
Lipid peroxidase (LPO) estimation was carried out in 10 % hemolysate using
spectrophotometric method of Placer et al. (1966).Lipidperoxidation activity is expressed
as nanomole of MDA per milliliter of hemolysate, using 1.56105 as extinction coefficient
(Utley et al., 1967). Supernatant of 10% RBC hemolysate was used for Superoxide
dismutase (SOD) evaluation in accordance of method described by Marklund et al. (1974)
including certain modification mentioned by Menami and Yoshikawa (1979). The each
unit of SOD activity is a measure of enzyme quantity required to inhibit auto oxidation of
pyrogallol by 50% under ideal condition. For estimation of catalase activity (CAT) of 10
% hemolysate at 240 nm wave length and appropriate dilution, Cohen et al.(1970)
spectrophotometric method was used. CAT values are expressed as units per milligram of
haemoglobin. DNTB (di-thiobis2-nitro benzoic acid) method of Prins and Loos (1969) was
used for evaluation Glutathione (GSH) level in packed RBC. GSH activity is expressed in
mmol of per milligram haemoglobin.
5.1.4.4 Histopathologyof kidney tissue
Left kidneyfrom eachrat at 28th day of study, was used for histopathogical evaluation and
fixed in 10% buffered formalin solution having pH of 7.4. Kidney was dehydrated in
graded alcohol and paraffin embodied. Tissue sections of 5 m size were prepared using
microtome machine and stained byHematoxylinEosin combination dye for microscopic
examination of kidney.

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5.1.4.5 Immunohistochemical protein expression
The osteopontin (OPN) protein expression were observed using laboratory standardized
procedure. The paraffinised kidney tissues were sectioned in 5m thickness on coated slide
and immersed in xylene to remove paraffin, then in graded alcohol for water removal.
Antigenic retrieval of sections were carried out by process of boiling for period of 10
minutes in 10 mM sodium citrate buffer of pH 6.0 and cooling at room temperature. The
sections were incubated in 0.3% hydrogen peroxide for 10 minutes to block endogenous
peroxidase activity and in 3% normal goat serum for 60 minutes to prevent non-specific
binding. The primary antibody of OPN (1: 50 dilutions,mouse monoclonal to osteopontin,
Santa Cruz Biotechnologies, USA) were used to stain kidneys section by overnight
incubation at 40C temperature. Then, sections were washed three times in PBS for 10
minutes period and incubated for 1 hour using horse radish peroxidaseconjugated
secondary antibody of goat-anti mouse immunoglobulin-G (1: 200 dilutions, Santa Cruz
Biotechnologies, USA). Then, again sectionswere washed three times with PBS and 3.30-
diamino-benzidine (Abcam Biotechnologies, India) dye were used to stain.Mayers
hematoxylin solution (Sigma Aldrich biochemical, USA) was used to counterstain and
CC/mount (Sigma Aldrich biochemical, USA) to mount the section. Under microscopic
examination cytoplasm of OPN positive cells showed brown coloration.
5.1.5 Statistical analysis
The results were analyzed statistically using software IBM SPSS Version 20. Values were
considered significant when P 0.05. All quantitative data were expressed as mean
Standard error of mean(SEM). The comparison between mean of different groups were
carried outusing one way analysis of variance (ANOVA) along withpost-hocDunnetts test.
5.1.6Results
5.1.6.1 Biochemical parameters
5.1.6.2 Urine
After stone inducing treatment on day 14, urine oxalate, volume, urea nitrogen and
creatinine values were significantly (p<0.01) increased in all four group rats as compared
with normal healthy control (Table 8). Further, on day 28 increase in these values were
observed in lithiatic and vehicle control groups due to continuous renal damage. But
treatment with test plant extract restored above parameters statistically similar to standard

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cystone drug when compared with healthy control on day 28. After urolith induction in all
four groups on day 14, urine calcium concentration were decreased significantly (p<0.01)
but reduction in pH values non-significant when compared with healthy control. While on
day 28, significant (p<0.05) lower values were observed in lithiatic and vehicle control
group as compared to healthy control. These values were increased significantly after
extract treatmenton day 28 similar to standard drug cystone when compared with lithiatic
control. Urine inorganic phosphorus, sodium, potassium values were not changes or
changes non-significantly throughout experiment.
Table 8 Effect of Cucumis callosus (Rottl.) Cogn. Hydro Ethanolic Extract (CCHEE) on
urinary biochemical parameters in ethylene glycol and ammonium chloride induced
urolithiasis
Parameters Day Group I Group II Group III Group IV Group V
Oxalate 14 0.730.04## 1.700.16** 1.880.03** 2.050.20** 1.970.15**
## ** **## ##
(mg/dl) 28 0.600.04 2.700.09 2.010.12 0.740.06 0.800.019##
Volume 14 6.531.09 11.331.45** 10.901.32** 10.700.81** 10.670.73**
(ml/24 hrs) 28 6.500.76 ##
14.870.93** 11.600.76**# 7.530.71## 8.000.50##
* * **
UUN 14 8.530.35 #
12.480.80 12.721.19 14.570.65 14.040.82**
** **
(g/dl) 28 7.330.74## 20.681.75 18.262.68 7.800.46## 10.920.25##
* ** **
Creatinine(g/d 14 0.340.05 #
0.680.09 0.800.03 0.810.09 0.820.07**
** **
l) 28 0.360.01 ##
1.390.03 1.270.06 0.550.04 ##
0.590.10##
** ** **
Calcium 14 6.930.45 ##
3.840.24 4.010.47 4.280.42 4.400.23**
(mg/dl) 28 7.240.70## 3.370.49** 3.670.33** 6.080.21## 6.550.37##
Phosphorus 14 4.960.42 6.100.63 5.680.36 5.260.75 6.020.47
(mg/dl) 28 5.010.19## 7.410.33** 6.500.29 5.080.31## 5.370.59##
Sodium 14 94.4413.27 102.0710.68 86.258.35 100.2621.67 98.1220.07
(mEq/L) 28 74.7812.50 91.9124.22 69.049.45 91.047.57 74.009.92
Potassium 14 10.220.15 9.480.22 9.840.25 10.400.39 10.061.01
(mEq/L) 28 13.060.12 12.270.33 11.480.38 12.560.38 12.250.80
pH 14 7.100.12 6.570.09 6.700.35 6.370.38 6.370.61
28 6.600.31# 5.470.44* 5.820.36* 6.600.31# 6.230.58#
Group I = Healthy control, Group II = Lithiatic control, Group III = Vehicle control, Group IV= 100
mg/kg standard drug cystone, Group V= CCHEE = 250 mg/kg Cucumis callosus Hydro Ethanolic
Extract. *p < 0.05 and **p < 0.01 vs Healthy control (group I), #p < 0.05 and ##p < 0.01 vs. Lithiatic
control (group II).

5.1.6.3 Blood
On day 0 or before start of study all biochemical values were statistically similar
between different groups, so not mentioned in the table. BUN, Creatinine and total
proteinlevels were increased significantly (p<0.01) in all the four group rats having lithiatic
treatmentas compared to healthy controlon day 14 (Table 9). At the end of study these

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values were significantly higher in lithiatic and vehicle control group. However, CCHEE
significantly reduced the above parameters and the values were similar to standard
treatment on day 28.The changes in serum calcium, phosphorus, sodium and potassium
values were remain non-significant throughout the study.
Table 9 Effect of Cucumis callosus (Rottl.) Cogn. Hydro Ethanolic Extract (CCHEE) on
plasma biochemical parameters in ethylene glycol and ammonium chloride induced
urolithiasis
Parameters Day Group I Group II Group III Group IV Group V
BUN(mg/dl) 14 26.190.53## 45.461.56** 51.301.96** 48.892.14** 44.263.48**
## ##
28 21.801.87 53.663.46** 36.581.81**## 23.781.60 27.191.90##
Creatinine(mg 14 0.320.03# 0.470.02* 0.540.05** 0.560.05** 0.590.03**
## ##
/dl) 28 0.280.05 1.45.08 **
0.800.04 **## 0.350.05 0.410.02##
## ** ** **
Total 14 4.240.21 5.950.24 5.130.19 # 5.380.11 5.810.14**
Protein(g/dl) 28 5.220.21## 6.740.18** 6.330.24** 5.630.17## 5.020.33##
Albumin(g/dl) 14 2.740.06 3.060.14 3.120.13 3.050.22 3.300.13*
#
28 3.150.17 3.520.17 3.360.10 3.130.07 2.940.05##
Calcium(mg/d 14 11.530.30 11.350.11 10.900.58 11.570.43 10.500.59
l) 28 10.370.44 11.040.37 10.520.66 9.070.95 9.570.72
Phosphorus(m 14 7.870.77 6.790.16 5.890.28* 5.450.25** 6.230.66
g/dl) 28 5.701.23 6.630.89 7.220.45 6.971.74 6.570.60
*
Sodium(mEq/ 14 113.498.67 103.879.75 127.094.98 148.119.44 ##
145.014.70*##
**
L) 28 134.944.46 146.753.29 106.645.58 ## 133.718.47 106.306.36**##
Potassium(mE 14 4.720.40 4.760.30 5.020.28 4.470.36 4.460.28
q/L) 28 7.201.03 7.960.99 5.950.38 7.181.02 6.820.54
Group I = Healthy control, Group II = Lithiatic control, Group III = Vehicle control, Group IV= 100
mg/kg standard drug cystone, Group V= CCHEE 250 mg/kg Cucumis callosus Hydro Ethanolic Extract.
*p < 0.05 and **p < 0.01 vs Healthy control (group I), #p < 0.05 and ##p < 0.01 vs. Lithiatic control
(group II).

5.1.6.4 Oxidative stress indices


The LPO activity wasincreasedsignificantly (p<0.01) on day 14 in all four groups
as compared to healthy control after stone inducing treatment. On day 28, it was
significantly (p<0.01) high in lithiatic control group and non-significantly elevated in
vehicle control group but LPO activities were decreased significantly in extract treated
CCHEE (group V) and standard cystone treated group and mean values were statistically
similar to healthy control group (Table 10). Antioxidants like GSH and CAT activities were
decreased significantly in all four stone induced groups on day 14 and further reduction
were noticed in Lithiatic and vehicle control group on day 28 compare to healthy control.
After treatment with plant extract antioxidant values were restored on day 28, statistically
similar to healthy control group and standard cystone treatment.

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Table 10. Effect of Cucumis callosus (Rottl.) Cogn. Hydro Ethanolic Extract (CCHEE) on
oxidative stress indices in ethylene glycol and ammonium chloride induced urolithiasis
Parameters Day Group I Group II Group III Group IV Group V
LPO 14 2.3680.275## 3.8870.193** 4.0630.190** 4.0950.171** 3.8260.279**
(nmol 28 5.7830.210** 4.3620.216#
##
MDA/mg Hb) 3.1550.175 3.1810.242## 3.2760.611##
* *
GSH 14 0.3040.034# 0.1620.032 0.1820.011 0.2060.048* 0.1420.016**
(mmol/mg 28
Hb) 0.3100.027## 0.1400.021** 0.1500.010** 0.2150.022* 0.2340.021##
CAT 14 1.2520.035## 0.7730.042** 0.8340.036** 0.7220.026** 0.5570.122**
(U/mg Hb) 28 1.2050.118## 0.5340.041** 0.6540.044* 1.1370.068# 0.9910.267
Group I = Healthy control, Group II = Lithiatic control, Group III = Vehicle control, Group IV= 100
mg/kg standard drug cystone, Group V= CCHEE = 250 mg/kg Cucumis callosus Hydro Ethanolic
Extract. *p < 0.05 and **p < 0.01 vs Healthy control (group I), #p < 0.05 and ##p < 0.01 vs. Lithiatic
control (group II).

5.1.6.5 Histopathology of kidney section


The healthy control group showed normal architect of kidney tissue, there was an
absence of crystals and renal damage during histopathological examination of H&E stained
renal tissues under light microscope. Administration of ethylene glycol and ammonium
chloride for urolith induction in lithiatic control and vehicle control group caused significant
calcium oxalate crystals deposition in all field of kidney section like tubules, glomeruli,
proximal and distal duct of renal cortex or medulla. There was severe infiltrations of
mononuclear cells along with necrosis, tubular degeneration, dilatation of tubules, casts or
fibrin deposits, severe hemorrhages and newer blood vessels growth in the kidney of lithiatic
and vehicle control rats. Crystal depositions caused significant damages to renal tubules
resulted in cellular infiltration in to the lumen.Group treated with Cucumis callosus plant
extract and standard cystone were showed significant reduction in number of crystals
deposition as well as mononuclear cells infiltration. The renal damage was restored markedly
after extract treatment similar to standard cystone treatment.
5.1.6.6 Immunohistochemical expression of protein OPN
The immunohistochemistry of renal tissue showed significantly increased expression
of OPN protein after stone induction in lithiatic control and vehicle control group . The
lithiatic rats showed increased expression in all field of kidney like tubules, glomeruli and
convoluted tubules. Cucumis callosus extract treated groups showed significant reduction
in OPN expression similar to standard cystone treated group when compared with lithiatic
control. Microscopic examination of kidney section of healthy control rats revealed

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absence of OPN expression While quantification of OPN expression there was significant
reduction in expression were observed in CCHEE group similar to standard cystone treated
group
5.1.7 Conclusion
Hydro-ethanol extract of dry fruits of ORP-EVM 21 possess potent anti-Urolithiasis
activity in vivo
Ec 50 of the extract was found as 250 mg/kg orally
Besides anti-lithiatic property the extract posses Reno-protective potential as indicated in
renal function test
The extract bears potent antioxidant property and could be a possible mechanism for its
Reno-protective potential

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5.2 Experiment 2: Evaluation of Wound Healing Property of four
compounds isolated from Plant Material (IICT)
Test material

Four pure compounds isolated from the plant material (IICT 001) and send to lead centre
for testing their wound healing property.

Animals
Healthy male albino rats of 12-13 weeks age and weighing 150-200g bred in the
Laboratory Animal Research Division of the Institute were used for experiment. The rats
were housed in clean polypropylene cages at room temperature 25 20C, with 12 hours
light -12 hours dark cycle throughout the experiment and provided with standard ration and
ad libitum water. They were acclimatized well prior to starting of protocol. Whole study
and animal care procedure were conducted according to standard guidelines of Committee
for the Purpose of Control and Supervision of Experiment on Animals (CPSCSEA) after
prior permission from Institute Animal Ethics Committee, IVRI (Approval no-F.26-
1/2015-16/JDR).

Experimental Design

Thirty adult rats were use for this study .They were divided into 5 equal groups of 6
animals each as per the following protocol

S.No Group No of Treatment


animals
I Control 6 No treatment
II Compound 6 Compound A @20 mg/kg orally
A daily for 7 days
III Compound 6 Compound B @20 mg/kg orally
B daily for 7 days
IV Compound 6 Compound C @20 mg/kg orally
C daily for 7 days
IV Compound 6 Compound D @20 mg/kg orally
D daily for 7 days
Surgical Procedure

The rats were anaesthetized by administrating Ketamine@5m/kg body weight


intramuscularly. One incision wound of thickness 15 x15 mm2 was created surgically on
the back of the rats. Treatment was carried out for 7 days for grs II & III.

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Clinical Observation

a) Measurement of wound area: wound area was measured at 0,7,14, 21 and 28 days post
operative period.
B) Wound contraction: percent contraction with respect to the original wound size was
calculated.
Percent contraction = 100 Total wound area on dayn / original wound area on day0 X 100
c) Exudation: the degree of exudation at the site of repair was graded on 1-4 scale .
1= None (Apparently dry wound)
2= Mild exudates (wound is moist, no oozing on pressing the wound).
3= Moderate exudates (wound is moist, slight oozing on pressing the wound)
4 = Extreme exudates (exudates is visible and pressing lead to extensive exudation)
Computerized Plainmetry

Colour photographs were taken ai days 0,3,7,14,21,28 with digital camera at a fixed
distance. Analysis of shape, irregularities and colour of the lesion was determined as
follows.The group having less gross observation score was considered the best.

A. Colour of wound: 1- pink, 2- pinkish pale,3- Pale, 4- Light brown, 5- dark brown, 6- black
B. Wound margin: 1- Pink 2- Brown, 3- Black.
C. Scar; 1- no scar, 2- Mild, 3- moderate,4- Severe

Estimation of hydroxyproline and glucosamine

The hydroxyproline content in the healing tissue on 28 post operative day was measured
as per method described by Reddy and Enwemeka (1996) using hydroxylproline stock
solution as a standard.

The glucosamine content in the healing tissue on 28 post operative day was measured as
per method described by C.J.M.Rondle andW.T.J.Morgan (1955) using glucosamine stock
solution as a standard.

Results

The effect of the Phytochemical on wound area is presented at table no 1. The compound
coded as B shown best results in reducing the wound areas from day 3,onward followed by
compound D ,C and A. It is worth mentioning that the wound area became invisible after
14 day post treatment in rats received Compound B

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Table 1 Mean SE of wound area (cm2) in different groups at different interval

Group Days post operation

0 3 7 14 21 28
I 1.38 1.00 0.88 0.10 0.02 0.0
(Control) 0.0 0.0 0.01 0.00 0.0 0.00
06 12 0 1 01

II 1.40 0.92 0.67 0.08 0.01 0.0


(Comp A) 0.0 0.0 0.00 0.00 0.0 0.00
04 08 6* 1* 02*

III 1.42 0.69 0.54 0.04 0.0 0.0


( Comp B) 0.0 0.0 0.00 0.00 0.0 0.00
04 10* 8** 04** 0
IV 1.38 0.90 0.70 0.07 0.02 0.0
( Comp C) 0.010 0.000 0.0 0.00
0.00 0.00 * 6** 02*
6 8 *
V 1.41 0.87 0.63 0.06 0.0 0.0
(Comp D) 0.006 0.001 0.00 0.00
0.00 0.00 ** 2**
4 6*
* & ** denoted the mean varies significantly from control group at same interval

Similarly the percent contraction of wound is presented in Table 2. The results denotes all the four
compound has effect on contraction of wound area .Highest contraction of wound area was recorded
in compound B followed by compound D, Compound -C and compound-A

Table 2 Percentage of wound contraction of wound area (cm2) in different groups in different
interval

Group Days post operation

3 7 14 21 28
I 0.08 0.22 0.70 0.87 Healed
(Control) 0.02 0.068**a 0.08* 0.04
*a 2** a

II 0.093 0.28 0.78 0. 84 Healed


(Comp A) 0.01 0.026**a 0.06* 0.00
*a 7** a

III 0.248 0.58 0.87 0.89 Healed


( Comp B) 0.04 0.048**b 0.024 0.014
** b ** a
IV 0.10 0.42 0.80 0.85 Healed
( Comp C) 0.01 0.039**b 0.06** 0.034
b ** a
V 0.15 0.46 0.81 0.87 Healed
(Comp D) 0.06 0.018**b 0.046* 0.014
*a ** a
Exudation, Colour of wound and wound margin were recorded daily till 28 days post
operation significant exudation was observed in rats of untreated control group up to day 5

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post operations. Mild to no exudation was observed in rats received test compound B,D
and after day 5 post operation similar was the response to pain scor

Fig 1. Wound area in rats receiving different treatment at different intervals

Histopathology

The Histopathological examination of the healing tissue revealed In untreated control


animal it was found a single wide crater with complete epithelisation. However skin
adenexa is absent and lining epithelium is not smooth. Lining epithelium is irregular with
variation in thickness.
In rats received Compound A, epithelisation wass complete with uniform thickening,
Keratinisation is complete and uniform,Craters- absent,Collagen compact but poorly
aligned ,skin adenexa- A pair of hair follicles is evident in healing tissue
In rats received Compound B; epithelisation is complete with uniform and smooth
epithelial lining ,granulation tissue adequate, keratinisation normal, collagen well
aligned but not compact and not present in bundles ,skin adenexa absent in granulation
and healing tissue and craters/fissures- absent

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In rats received Compound C ; epithelisation was complete over granulation tissue and
transition from normal compact epidermis to thicker newly form epithelial lining is evident
,fibrosis is marked and ongoing i.e. immature ,Collagen present but not compact and
bundles are absent ,Skin adenexa absent in healing tissue, Craters/fissures- absent and
Keratinisation-complete and uniform
In rats received Compound D; epithelisation is complete with uniform and smooth
epithelial lining surface. However, epidermis is thicker than normal and less compact,
keratinisation normal, collagen -well organized but less compact,skin adenexa absent
in healing tissue,Craters absent
Necrotic tissue and inflammatory changes could not be observed in any control or
treatment group

In vivo Assessment of wound healing activity


Test compounds : A, B, C, D
Histopathology

Control

A B C D

Compound B is better in healing of wounds than other 3 compounds

Effect on Expression of Glucosamine (UDP-N-Acetyl)-2-epimerase/ N-acetyl manno-


samine kinase gene

To explore the mechanism of healing in molecular level expression analysis of glucosamine


(UDP-N-acetyl)-2-Epimerase/N-acetylmannosamine kinase gene was performed in
healing tissue and the results are presented in Figure 3&4).It was foundthat compound B
has upregulated this gene up to 30 fold compared to control followed by compound D and
compound C

16
17
Conclusion

Out of the 4 compound isolated from Ethanol Extract of IICT #001 compound B has
excellent wound healing property Followed by Compound D and Compound C
Compound B has excellent wound healing was recorded in histopathology.
Compound B has up- regulated glucosamine (UDP-N-acetyl)-2-Epimerase/N-
acetylmannosamine kinase gene up to 30 fold compared to control in healing tissue
followed by compound D and compound C

18
5.3 Experiment 3: Evaluation of antitrypanosomal activity of selected medicinal
plants

1.1. Plant material

A compound isolated from the medicinal plant ORP-EVM AD005 and seeds of a
medicinal plant collected from Thiruvanthapuram district of Kerala were used for this
study and was coded as ORPEVM-22 for intellectual property right point of view. The
plant was identified and authenticated from Botanical Survey of India, Central National
Herbarium, Howrah, India.

1.2. Preparation of extracts of ORPEVM-22

The seeds were washed with water to remove dirt and superficial impurities and dried in
shade and powdered in electric grinder. Sequential extraction of the plant material in N-
hexane, methanol and water was carried out as described below.

1.2.1. Preparation of N-hexane extract: The extract was prepared from the powdered
plant material using N-hexane (Merck Specialities private limited, Mumbai) as solvent at
room temperature. The extract was filtered using filter paper (Whatmann No. 40). The
solvent was removed by using rotary evaporator. The extract was collected and dried in
vacuo and kept at 4C until use.

1.2.2. Preparation of Methanolic extract: The residue remaining in the filter paper after
preparation of N-hexane extract was used for preparation of methanolic extract using
methanol as solvent. (Merck Specialities private limited, Mumbai). The extract was
filtered using filter paper (Whatmann No. 40). The solvent was removed by using rotary
evaporator. The extract was collected and dried in vacuo and kept at 4C until use.

1.23. Preparation of Aqueous extract: The residue remaining in the filter paper after
extraction with n-hexane followed by methanol was used for preparation of aqueous
extract. Double distilled water was used as solvent and the extract was prepared as
described earlier.

1.3. Calculation of recovery percentage

The recovery percent for each extract was calculated using the formula,

19
Recovery percentage = X/Y x 100

Where X =Weight of the dried extract,

Y = Weight of the plant material taken for extraction

1.4. Evaluation of in vitro antitrypanosomal activity

1.4.1. Isolation of Trypanosoma evansi for in vitro studies


The cryostabilites were thawed to room temperature and viability of trypanosomes was
checked using light microscope under 400X magnification. One mouse was infected by
intraperitoneal injection of ~20000 organisms. Presence of parasites in peripheral blood
was from second day onwards in blood collected from tail vein and every day thereafter.
At peak parasitemia the animal was sacrificed with chloroform anaesthesia and 400L
blood was collected directly from heart into a sterile tube containing modified HMI-9
media (Hirumis Modified Iscoves media) (Hirumi and Hirumi, 1989). Centrifuged the
sample at 1500 rpm for 5 min and supernatant was collected and total number of live
trypanosomes were counted using haemocytometer. The supernatant was further diluted
with HMI-9 media to 105 trypanosomes per ml.
1.4.2. In vitro experimental design
In vitro studies were carried out in 96 well cell culture plates (BD Falcon) as per the
standard protocol (Oliveria et al., 2004). In all wells 50l of trypanosomes (5000
trypanosomes) were added. Extracts stock solutions were prepared by dissolving 50 mg
of each extract in 1ml DMSO and which was further diluted with 4 ml HMI-9 media so
that stock solutions contains 10 mg extract per ml. The stock solutions were diluted in
HMI-9 such that 1000L of media contained 0-1000g per ml of each extract. Fifty
microlitres of media containing varying concentrations of extract dilutions was dispensed
to the test wells in triplicate. A row of wells were maintained as vehicle control to
evaluate the effect of DMSO on Trypanosoma evansi viability. Diminazene aceturate
was used as standard drug at varying concentrations between 0.008 and 0.28 g per ml. A
row of wells were maintained as infected control without any drug or vehicle.
The plate was incubated at 37C in 5% CO2 incubator for 72 hrs. After , 1, 3, 6, 9, 24
and 48 hrs motility and longevity of trypanosomes was examined. The concentration of
each extract and the purified compound that kills 50% of the T. evansi within 24 hrs of

20
incubation (IC50) were calculated. The extract that showed lowest IC50 was selected for
further studies.
1.4.3. In vitro evaluation of interaction of extracts with standard trypanocidal agents
Interaction of selected medicinal plant extract with standard available antitrypanosomal
drugs namely, diminazene aceturate, isometamidium chloride and quinapyramine
sulphate was evaluated using the standard protocol (Williamson et al., 1982). In vitro
antitrypanosomal studies were carried out in 96 well plates containing 5000
trypanosomes/well. Serial dilutions of drugs and extract were prepared and were
dispensed alone and in combination into the wells containing 5000 trypanosomes. A row
of wells were maintained as infected control without any drug. The plates were incubated
at 37C in 5% CO2 incubator for 72 hrs. After incubation at 37C for 24 hrs the parasite
number and motility were checked under microscope and minimum inhibitory
concentration i.e., the lowest concentration of drugs kills and/or abolishing motility of
50% of the T. evansi was noted. Combination Index (CI) was calculated for drugs using
the equation (Chou, 2006)
CI = (D)1+ (D)2
(Dx)1 (Dx)2
where (Dx)1 is the dose of drug 1 alone that inhibit x%, (Dx)2 is the dose of drug 2 alone
that inhibit x%, (D)1 is the dose of drug 1 in the combination that inhibit x% and (D)2 is
the dose of drug 1 in the combination that inhibit x%; CI values <1 indicate synergism,
CI values >1 indicate antagonism CI=1 indicates additive effect.
1.5. In vivo evaluation of antitrypanosomal activity using mice model

The extracts were dissolved in dimethylsulfoxide (Friedburghas et al., 1996). Forty two
mice weighing about 20-25g (45-60 days old) procured from small animal breeding
facility of laboratory animal research division of IVRI, Izatnagar and maintained
throughout the study as per IAEC guidelines. Each mouse will be inoculated with 2104
trypomastigotes except those in non-infected group and divided into groups of 6 animals
and subjected to treatment as described in the Table 1. Appearance of parasites in
peripheral blood was evaluated by the method of Ercoli et al. 1980. Survival period and
survival rate of mice were recorded. Body weight of the animals were recorded to

21
evaluate cachexia and based on weight loss cachexia scores were given as mild (025),
moderate (2650), severe (5175), and terminal (76100)

Groups (n=6) Trypanosoma evansi Treatment

I Nil Placebo/water

II 2104 trypomastigotes IP Placebo/water

III 2104 trypomastigotes IP Berenil:7mg/kg1hr after Infection IP

IV 2104 trypomastigotes IP ORP-EVM 22 at the dose rate of 2IC IP


50

V 2104 trypomastigotes IP ORP-EVM22 at the dose rate of 10xIC


50

IP

VI 2104 trypomastigotes IP AD- 005 at the dose rate of 2IC , IP


50

VII 2104 trypomastigotes IP AD- 005 at the dose rate of 10x IC , IP


50

Table 1: Protocol for in vivo evaluation of anti-trypanosomal activity of the


methanolic extract of ORP EVM-22 and purified compound isolated from ORP
EVM-AD005

Haematology: Blood samples were collected in EDTA vacutainer from heart at the end
of the experiment and complete blood counting was carried out (Jain, 1986). Samples
were centrifuged at 3000 rpm 10 min to separate plasma and stored at -20C for further
biochemical analysis.

.1.5 Evaluation of prophylactic activity of extract if any


Twenty four Swiss albino mice weighing about 20-25 g were used for the study and
divided into 4 groups of 6 animals each. All mice except those in non-infected groups
were inoculated with 2104 trypomastigotes and treatment was carried out for 40 days as
described in Table 4. Prepatent period, survival rate, weight loss and longevity of mice

22
were evaluated along with motility and survivability of trypanosomes collected by tail

Group (n=6) Infection Treatment

I (Non infected Control) Nil Water

th
II (Infected Control) Water
20000 BT IP on 14 day

th
III (ORP-EVM 22) 2 wks prior to infection up to 28
20000 BT IP on 14 day
days post infection
2IC /day
50

th
IV (ORP EVM -AD 005 2 wks prior to infection upto 28
20000 BT IP on 14 day
Purified compound) days post infection

2IC /day
50

snip every day starting from second day after infection.

Table 2: Experimental design to evaluate prophylactic activity if any

1.6. Statistical analysis

The data of the study was expressed as mean SEM. Standard error of mean and p-
values were used to determine whether there is any significant difference among different
treatment groups using one-way analysis of variance (ANOVA) following standard
procedure (Snedecor and Cochran, 1994).

Result

In vitro evaluation of antitrypanosomal activity of selected medicinal plants

Antitrypanosomal activity of extracts of selected medicinal plant ORPEVM- 22 and


purified compound of ORP EVM AD-005 were evaluated in vitro in HMI 9 media. The
motility and viability of trypanosomes were examined under microscope 0.5, 1, 3, 6, 9,
24 and 48 hrs after incubation at 37C in CO2 (5%) incubator. The percentage reduction
in number of motile and viable trypanosomes at varying concentration for each extracts

23
(Table: 3) was calculated. The extracts had shown dose dependant antitrypanosomal
effect. The dose response curve (Fig.1, 2,3 and 4) was depicted as percent reduction in
number of parasites against various drug concentration (log dose) by fitting data points in
a sigmoidal (four parameter logistic) curve in GraphPad Prism (GraphPad Prism
Software, La Jolla, CA) and the effective concentration 50 (IC50) were calculated for each
extracts.
Table 3: Percent reduction of live trypanosomes by extracts of ORP EVM 22 and
purified compound AD 005 at varying concentrations

Drug Percent reduction in live trypanosomes


concentration N- hexane Methanolic Aqueous Purified compound
(g/ml) extract extract extract ORPEVM-AD-005
0.007 1.470.33 29.50.39 6.531.56 21.521.49
0.015 10.270.41 37.60.72 10.891.04 32.081.19
0.029 14.670.79 41.10.55 19.290.95 34.050.07
0.058 16.160.86 42.50.35 22.761.42 35.960.21
0.117 17.140.18 43.30.19 25.650.55 50.430.91
0.235 18.410.41 45.90.19 29.170.35 51.510.41
0.47 21.540.83 46.130.11 32.941.65 55.330.90
0.95 23.160.33 50.430.39 34.740.47 57.120.25
1.95 25.500.85 53.260.43 38.561.65 60.690.56
3.9 29.470.62 56.90.21 40.941.99 64.490.80
7.8 34.620.39 60.20.51 43.561.44 72.121.11
15.6 46.900.50 72.330.32 47.220.34 78.171.33
31.25 49.910.26 95.630.41 50.861.42 78.890.64
62.5 52.811.16 100 52.992.13 79.890.90
125 65.770.59 100 71.960.90 80.620.72
250 74.261.61 100 78.761.71 90.560.84
500 88.900.93 100 89.141.68 92.870.37
1000 94.371.31 100 95.001.02 95.690.80

The IC50 (Best-fit values) for each extracts were presented in Table 4. The methanolic
extract of ORPEVM-22 (12.711 g/ml) and purified compound isolated from AD005
(6.65 g/ml) were noted to have lowest IC50. Accordingly methanolic extract of
ORPEVM-22 and purified compound isolated from ORPEVM-AD005 were selected for
further studies.

24
Log dose response curve of N-hexane extract of ORPEVM 22 LOG DOSE RESPONSE CURVE OF METHANOLIC EXTRACT ORP EVM-22
110 120
% reduction in live trypanosomes

100 110

% reduction of live trypanosomes


90 100
80 90
N- hexane extract
70 80
60 70 Methanolic extract of
50 60 ORPEVM-22
40
50
30
40
20
30
10
20
0
0 1 2 3 4 5 6 7 10
log dose of N- hexane extract (ng/ml) 0
0 1 2 3 4 5 6
Log Dose (ng/ml)

Fig. 1. Log dose response curve N-hexane Fig.2. Log dose response curve methanolic
extract of ORPEVM-22 extract of ORPEVM-22

Log dose response curve aqueous extract of ORPEVM-22


Log dose response curve AD-005
% reduction in live trypanosomes

110
% Reduction in live trypanosomes

100 100
90
80
Aqueous extract of ORPEVM-22 75
70
60
50 50
40
30
20 25
10
0
0 1 2 3 4 5 6 7 0
Log dose (ng/ml) 0 1 2 3 4 5 6
log dose (ng/ml)

Fig. 3: Log dose response curve aqueous extract Fig. 4: Log dose response curve purified
of ORPEVM-22 compound from ORPEVM-AD005

25
Table4: The IC50Best-fit values (ug/ml) for each test extracts

S.NO. Name of extracts IC50 Best Fit Values (g/ml)

1 N-hexane extract ORPEVM22 37.28

2 Methanolic extract ORPEVM22 12.71

3 Aqueous extract ORPEVM22 79.81

4 Purified compound ORP EVM AD005 6.65

In vitro evaluation of interaction of extracts with standard trypanocidal agents

Interaction of methanolic extract of ORPEVM-22 was evaluated with standard


available antitrypanosomal drugs namely, diminazene aceturate, isometamidium chloride
and quinapyramine sulphate. In vitro trials carried out to study the effect of combination
therapy of diminazene aceturate with methanolic extract of ORPEVM-22 and purified
compound from AD-005 showed reduction in the concentration of drugs in combination
therapy to inhibit 100% motility as compared to each drug alone (Table 5). Synergy
experiment of methanolic extract and diminazene aceturate revealed lowest concentration
of methanolic extract in combination resulted in >75% reduction in motile trypanosomes
was 3.9 g/ml, whereas that in extract alone was 31.2 g/ml. Lowest concentration of
diminazene aceturate resulted in >75% reduction (IC75) in motility of trypanosomes in
combination trial with methanolic extract was 0.007 g/ml while 0.14 g/ml of
diminazene aceturate was noted to have >75% reduction when used alone. Combination
index (CI) was calculated for diminazene aceturate and methanolic extract of ORPEVM-
22 and was 0.175. Since CI value <1 indicates synergism between methanolic extract and
diminazene aceturate. The purified compound isolated from AD-005 showed >75%
reduction in trypanosomes in in vitro culture 24 hrs after incubation at 37C alone was
3.9 g/ml and in combination with diminazene aceturate was 15.6 g/ml. CI value for
purified compound from AD-005 and diminazene aceturate was 0.3 (CI value <1)
indicating synergism.

26
Table 5: Percent reduction in trypanosome count with in vitro combination therapy with
Diminazene aceturate and methanolic extract ORP EVM 22 and purified compound
from ORPEVM-AD005

Diminazene Percent reduction in Percent reduction in Diminazene


aceturate trypanosomes with methanolic trypanosomes with purified aceturate
extract ORP EVM 22 (g/ml) compound AD005(g/ml) control

Dose (g/ml) 125 62.5 31.2 15.6 3.9 125 62.5 31.2 15.6 3.9

0.56 100 100 100 100 100 100 100 100 100 100 100

0.28 100 100 100 100 100 100 100 100 100 100 100

0.14 100 100 100 100 100 100 100 100 100 100 100

0.07 100 100 100 100 100 100 100 100 84.6 73.68 46.62

0.03 100 100 100 100 60.54 87.38 84.32 81.69 78.45 65.32 27.72

0.015 100 100 100 87.5 54.6 86.58 80.54 80.04 78.16 63.32 18.86

0.007 100 100 95.8 72.15 52.16 82.18 80.54 79.75 77.98 62.84 11.77

Extract 100 100 95.2 69.8 51.6 81.69 79.86 78.64 77.94 62.16 0
/AD005

The in vitro trial with methanolic extract of ORPEVM 22 and purified compound
of AD-005 with quinapyramine sulphate revealed reduction in EC75 of methanolic extract
and purified compound of AD-005 when used in combination (Table 6). CI value for
methanolic extract and purified compound of AD-005 were 0.532 and 0.283 respectively
indicating synergism between drugs used for the study.

Table 6: Percent reduction in trypanosome count with in vitro combination therapy


with Quinapyramine sulphate and methanolic extract ORP EVM 22 and purified
compound from ORPEVM-AD005

27
Quinapyramine Percent reduction in Percent reduction in Quinapyramine
sulphate trypanosomes with trypanosomes with purified sulphate control
methanolic extract ORP compound AD005 (g/ml)
EVM 22 (g/ml)

Dose (g/ml) 100 25 12.5 100 25 6.25

0.4 100 96.14 79.86 100 88.56 76.61 75.15

0.2 100 95.4 70.58 100 87.74 75.5 70.58

0.1 100 91.64 67.87 100 86.57 69.80 64.32

0.05 100 89.58 65.46 91.90 82.14 70.0 60.00

0.025 100 89.24 64.71 85.65 80.46 67.87 55.88

0.013 100 88.63 61.29 83.46 78.90 63.14 51.61

Extract /AD005 100 87.5 58.29 80.61 78.6 63.23 0

The evaluation of synergy between isometamidium chloride along with methanolic extract
and the purified compound in HMI-9 media revealed CI value of 0.283 (Table 7).

Table 7: Percent reduction in trypanosome count with in vitro combination therapy


with and isometamidium chloride, methanolic extract ORP EVM 22 and purified
compound from ORPEVM-AD005

Isometamidium Percent reduction in Percent reduction in


trypanosomes with trypanosomes with purified

28
chloride methanolic extract ORP compound AD005 (g/ml)
EVM 22 (g/ml)

Dose (g/ml) 100 25 6.25 100 25 6.25

0.5 100 100 100 100 100 100

0.125 100 100 100 100 100 100

0.03 100 100 100 100 100 100

0.008 100 100 68.75 100 100 86.65

0.002 100 81.25 62.5 100 95.4 78.13

0.001 100 75 56.25 100 92.3 63.75

Extract /AD005 100 75 53.75 80.61 77.14 62.5

In vivo evaluation of antitrypanosomal activity using mice model

Antitrypanosomal activities of methanolic extract of ORPEVM-22 and purified


compound of AD-005 were studied using mice model. All groups except uninfected
untreated group were infected with 20000 trypomastigotes intraperitoneally. Group IV
animals were treated with methanolic extract intraperitoneally at a dose rate twice the IC50
value in vitro i.e., 25.42 mg/kg/day. Group V animals were treated with 105 (1012.71)
i.e., 127.1 mg/kg/day intraperitoneally. Animals in group VI and VII were treated with
AD-005 at a dose rate twice the IC50 (26.65) i.e., 13.3 mg/kg/day and ten times IC50
(106.65) i.e., 66.5 mg/kg/day intraperitoneally respectively. Group III animals were
treated with standard drug diminazene aceturate 7 mg/kg intraperitoneally. Prepatent
period in all infected groups were noted to be ranging between 4 and 5 days (mean=4.3
days). Mice in group IV, V, VI and VII showed parasitemia as similar to group II (infected
untreated) and their survival period were 5.5, 5.33, 6 and 6.66 days respectively.

Prophylactic activity of methanolic extract of ORPEVM-22 and purified compound


from AD-005

29
Prepatent period, survival rate of mice, alteration in body weight, longevity of mice were
evaluated to study prophylactic effect of the medicinal plants. The prepatent period in
animals of infected and untreated group (4.33 0.21 days) had no significant difference
(p>0.05) with that of group III i.e., treated with methanolic extract of ORPEVM 22 28
days prior to infection (5.00 0.26 days) and IV i.e., treated with purified of AD-005 28
days prior to infection (4.670.33 days). Survival period in group II animals were
5.330.42 days which was similar (p>0.05) to that of group III (6.16 0.17 days) and IV
(5.330.21 days). Since the test extract and the purified compound could not completely
remove Trypanosoma evansi from the mice, animals in group III and IV showed similar
survival rate as group II. Evaluation of the study compound and extract for the effect on
weight loss revealed animals treated with extract (group III) and infected untreated animals
(group II) showed mild cachexia with weight loss of 6.9% and 6.6% respectively. Animals
treated with purified compound of AD-005 had insignificant/no loss of weight (0.46%).

Conclusions
Based on the above findings the following conclusions were made.

1. In vitro assay to evaluate antitrypanosomal activity of n-hexane, methanolic and aqueous


extract of ORPEVM-22 revealed IC50 values of 37.28 g/ml, 12.71 g/ml and 79.81 g/ml
respectively. Purified compound isolated from AD-005 had IC50 values of 6.65g/ml.
2. The interaction between ORPEVM-22 ,Purified compound isolated from AD-005 with
diminazene aceturate, isometamidium chloride and quinapyramine sulphate revealed
reduction in the concentration of standard drugs and test agents required to inhibit
trypanosomes to a specific percent. The combination index values indicate synergism
between the standard drugs and test agents.
3. The two selected agent showed no trypanocidal activity but a 24 hr increase in the life span
of infected mice could be noted in purified compound treated group.
4. The safety study revealed no toxic effect in mice at ten times IC50 dose. Prophylactic
treatment to mice 14 days prior to infection improved the survival period by 24 hrs in both
methanolic extract and purified compound treated groups.
References:
Chou, T. C. 2006. Theoretical basis, experimental design, and computerized simulation of
synergism and antagonism in drug combination studies. Pharmacol. Rev. 58:621681.

30
Ercoli, N., Minell, E.B. and Olivo, N. 1980. Antitrypanosomal activity of trivalent
antimonial in vitro and its significance. Chemother.26: 254-262.
Oliveira, D.A., Pereira, D. G., Fernandes, A. M. A. P., De Castro, S. L. , Souza Brito,
A. R. M., De Souza, A. O. and Durn, N.2005.Trypanocidal activity of 2-propen-1-
amine derivatives on trypomastigotes culture and in animal model. Parasitol. Res.
95: 161-166.
Snedecor, G. W. and Cochran, W. G. 1994. Statistical methods. Iowa, USA, Iowa State
University Press, Ames. 593 p.
Williamson, J., March, J.C. and Scott-Finnigan, T.J. 1982. Drug synergy in experimental
African trypanosomiasis. Tropenmed. Parasitol. 33: 76- 82.

31
ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

AIZAWL COLLABORATING CENTRE, MIZORAM


DEPATMENT OF PHARMACOLOGY & TOXICOLOGY
CENTRAL AGRICULTURAL UNIVERSITY
SELESIH, AIZAWL-796014, MIZORAM

32
OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
AIZAWL COLLABORATING CENTRE, MIZORAM
DEPATMENT OF PHARMACOLOGY & TOXICOLOGY
CENTRAL AGRICULTURAL UNIVERSITY
SELESIH, AIZAWL-796014, MIZORAM

ANNUAL REPORT (2016-2017)

1. Name of Centre : Aizawl, Mizoram

2. Name of Investigators: Dr(s).C. Lalmuanthanga, T.K. Rajkhowa, Gunjan Das, M Ayub


Ali, T. K. Dutta and Kalyan Sharma.

3. Approved mandates of the Centre:


Wound Healing
Diabetes

4. Salient achievement:
Collected and authenticated 3 medicinal plants from Mizoram and and processed for
petroleum ether, chloroform and methanol extracts
All the 3 extract contain Flavinoids and terpenoids and negative for Anthraquinones
The HPTLC fingerprinting of plants, AZL/2016/OPEVM/01 and
AZL/2016/OPEVM/02 show presence of three distinct compounds.

5. Concise progress Report (01.04.2016 to 31.03.2017)

5.1 Plant Collection, Processing and Extraction of plants


Five different plants viz. Blumea lanceolaria (fig. 1), Securinega virosa (fig. 2),
Parkia timoriana (fig. 3), Scoparia dulcis (fig. 4) and Albesmoschus moschatus (fig. 5) were
collected from Champhai, Serchhip and Aizawl District and processed for petroleum ether,
chloroform and methanol extracts by using soxhlet apparatus to be studied for wound
healing in various different wound models in rats.
5.1.1 Blumea lanceolaria
Blumea lanceolaria (Fig. 1) is commonly known as Blumea camphor
(English), Kakaronda (Hindi), Kuk-sungh (Bengali), Mugongre (Assam), Tera paibi macha
(Manipuri), Buarze (Mizo).

33
5.1.2 Securinega virosa
Securinega virosa (Fig. 2) is commonly found in the hilly forests of all the seven
states of NE India (Handique, 2009). The common name of this plant is common bushweed
(English), Dalme (Hindi), Nilishila (Sanskrit), Bon-jetuka (Assamese), Saisiak (Mizo).
5.1.3 Parkia timoriana
Parkia timoriana (Fig. 3) is commonly known as Tree Bean (English),
Sapota/Khorial(Hindi), Khorial (Assamese), Yongchak (Manipuri), Zawngtah (Mizo).
5.1.4 Scoparia dulcis
Scoparia dulcis (Fig. 4) is commonly known as Sweet Broom Weed/Sweet Broom
Wort (English), Mithi patti/Ghoda tulsi (Hindi), Bon dhonya (Bengali), Garu-tulashi
(Assamese), Sumjit-mambi (Manipuri), Perhpawng-chaw/Hlothlum (Mizo).
5.1.5 Albesmoschus moschatus
Albesmoschus moschatus is commonly known as Okra/Annual hibiscus (English),
Gorokhia/koroi (Assamese), Muskdada (Bengali/Hindi), Uichhuhlo (Mizo).

Fig.1: Blumea lanceolaria Fig.2: Securinega virosa

Fig.3: Parkia timoriana Fig.4: Scoparia dulcis

34
Fig.5: Abelmoschus moschatus

5.2 Preparation of plant extracts


Extraction of Plant samples were done with methanol, chloroform and petroleum
ether as per standard method by using Soxhlet apparatus and concentrated in rotary
evaporator.
5.3 Phytochemical study
The qualitative phytochemical analysis of plant samples coded 01AZl2016,
02AZL2016 and 03AZL2016 were performed as per standard protocol (table 1 and figures
6, 7 and 8). The following are the result of phytochemical analysis (+ve= present, -ve=
Absent).

Table 1: result of Phytochemical Analysis of three different plants

Plants tannins Phlobatannins Saponins Flavanoids Terpenoids Anthraquinones

1 01AZl2016 -ve +ve -ve +ve +ve -ve

2 02AZL2016 +ve -ve +ve +ve +ve -ve

3 03AZL2016 +ve -ve -ve +ve +ve -ve

5.4 Isolation of Phytochemical Constituent of Plants:

The processing for the isolation of phytochemical constituent of plant coded


03AZL2016 was initiated. TLC of plant coded 03AZL2016 (figure 9) was performed by
using different ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 8:2 of Chloroform and Methanol
solvent for solvent selection for further processing in column chromatography for
separation of active phytochemical. It was found that the ratio of 6:4 of chloroform and

35
methanol has a better separation. Further processing for isolation of active component by
using column chromatography is ongoing. The separated component of the plants will be
further analyses with HPTLC, HPLC etc.

Fig. 6 Fig.7

Fig. 8 Fig. 9

5.5 HPTLC Fingerprinting:

The HPTLC Fingerprinting of plants, AZL/2016/OPEVM/01 and


AZL/2016/OPEVM/02 was carried-out by using CAMAG Linomat 5 applicator, CAMAG
TLC Scanner and winCATS. The solvent, Chloroform: Methanol (8:2) was used for
separation on HPTLC Plate silica gel 60 F 254. Both the plants show three distinct peak as
shown below (Figure 10, 11 and 12).

36
Fig.10: 3D of HPTC fingerprinting of plants, AZL/2016/OPEVM/01 and AZL/2016/OPEVM/02

Fig.11: Graph showing HPTC fingerprinting of plant AZL/2016/OPEVM/01 with RF Value of


0.56, 0.89 and 1.00

37
Fig. 12: Graph showing HPTLC fingerprinting of plant AZL/2016/OPEVM/02. RF Value:
0.78, 0.91 and 1.03

6. Conclusion

Collected and authenticated 3 medicinal plants from Mizoram and and processed for
petroleum ether, chloroform and methanol extracts
All the 3 extract contain Flavinoids and terpenoids and negative for Anthraquinones
The HPTLC fingerprinting of plants, AZL/2016/OPEVM/01 and
AZL/2016/OPEVM/02 show presence of three distinct compounds.

38
STATEMENT OF EXPENDITURE (2016-2017)

39
Outreach Programme on
Ethnoveterinary Medicine
Progress Report (April- 2016 to March-2017)
Cooperating Center
Department of Pharmacology and Toxicology
College of Veterinary Sciences & A.H.
Anand Agricultural University
Anand-388 001 (Gujarat)

Principal Investigator:
Dr. S.K. Raval
Professor
Dept. of Veterinary Medicine
Veterinary College, Anand.

Lead Center
Division of Medicine
Indian Veterinary Research Institute
Izatnagar-243 122 (UP)

1
40
OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
DEPARMENT OF VETERINARY MEDICIN,
VETERINARY COLLEGE, ANAND

ANNUAL REPORT (2016-2017)


1. Name of Centre: Anand (Gujarat)

2. Team: Dr(s) S.K. Raval, D.S. Nauriyal, P.V. Parikh, D.J. Ghodasara and K.A.
Sadariya
3. Approved mandate:
Validation of EVM and identification, isolation and standardization of active
components, especially of herbal remedies found effective against common
diseases/conditions of animals.
Evaluation of potential toxicity of EVM and assessment of interaction between
active components of EVM and allopathic drugs used for purposes.
Work assigned to this centre- evaluation of anti-urolithiatic and renoprotective
activity of plants.
4. Approved activity for this year: Evaluation of Therapeutic Efficacy of Plants
Bryophyllum calycinum and Solanum xanthocarpum on Ethylene Glycol and 2%
Ammonium chloride induced Urolithiasis in Wistar Rats.
5. Salient achievements for last year (1st April 2016 to 31st March-2017)
Aqueous and alcoholic extract of individual plant and biherbal extract were
evaluated in rats for antiurolithiatic activity in ethylene glycol and ammonium
chloride induced urolithiasis model.
The biherbal alcoholic extract of Bryophyllum calycinum and Solanum
xanthocarpum 1:1 at the dose rate of 300mg/kg body weight orally once in a day
for four weeks has antiurolithiatic effect on ethylene glycol and ammonium
chloride induced urolithiasis in wistar rats
6. Progress report of last year (1st April 2016 to 31st March-2017)
6.1 Antiurolithiatic activity of Biherbal extracts Solanum xanthocarpum and
Achyranthes aspera
During this period plant Bryophyllum calycinum (Crassulaceae) and Solanum
xanthocarpum were selected for scientific validation of their antilithiatic properties.

41
Aqueous and alcoholic extracts were prepared from the leaves of Bryophyllum
calycinum and ripen fruits of Solanum xantocarpum. After procurement of plant from its
natural habitat identification and authentication was carried out.
6.2 Preparation of extract of Solanum xanthocarpum and Bryophyllum calycinum
and its biherbal preparation:
Aqueous and alcoholic extracts were prepared from the leaves of Bryophyllum
calycinum and ripen fruits of Solanum xantocarpum. Leaves of Bryophyllum calycinum
were cut in small piecesed and fruits of Solanum xanthocarpum were also cut in small
piecesed and dried under shade, then powdered by mechanical grinder and stored in air
tight containers. Exactly 100g of coarse powdered material of both the plants were
successfully extracted in soxhlet extractor with water and also with alcohol. Extracts so
obtained were decanted in beaker and then concentrated to fullest extent in water bath.
The aqueous and alcoholic extracts were preserved in refrigerator at 40 C for further use.
Dose was calculated according to body weight of animal and administrated as per
concentration strength of formulation. Same procedure was followed for preparation of
biherbal alcoholic extract.
6.3 Safety assessment:

Safety assessment study involves administration of usually single dose of the


test drugs to a group of animals at different dose levels and to observe the effect of
test drug on functions of vital organs and general behavior. The approximate lethal
dose (ALD50) of a test drug i.e. the dose level at which 50% of the animals in group
attains death can be calculated.

6.4 Induction of urolithiasis in Wistar rats:

Rats were selected randomly and divided in to 15 groups (group I, II, III, IV, V,
VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV). All group consist of six rats. All the rats
were numbered group wise and individually group I served as normal control.
Urolithiasis was induced in group III, IV, V, VI, VII, VIII, IX rats using 0.75% (v/v)
ethylene glycol and 2% (w/v) ammonium chloride along with drinking water as stone
inducing agent for 28 days. Group I, II, X, XI, XII, XIII, XIV and XV animals were
given normal water.

42
6.5 Studies of therapeutic effect:

After 28 days of induction of urolithiasis group IV was given aqueous extract of


Bryophyllum calycinum @ 300 mg/kg, group V was given alcoholic extract of
Bryophyllum calycinum @ 300 mg/kg, and group VI was given aqueous extract of
Solanum xanthocarpum at 300 mg/kg and group VII was given alcoholic extract of
Solanum xanthocarpum at 300 mg/kg and group VIII was given aqueous biherbal extract
of Bryophyllum calycinum + Solanum xanthocarpum (1:1) 300 mg/kg and group IX was
given alcoholic biherbal extract Bryophyllum calycinum + Solanum xanthocarpum at
300 mg/kg. Group X was given aqueous extract of Bryophyllum calycinum XI was given
alcoholic extract of Bryophyllum calycinum, XII was given aqueous extract of Solanum
xanthocarpum, XIII was given alcoholic extract of Solanum xanthocarpum, XIV was
given aqueous biherbal extract of Bryophyllum calycinum + Solanum xanthocarpum,
while XV was given alcoholic biherbal extract of Bryophyllum calycinum + Solanum
xanthocarpum and were kept as extract control group and they were given aqueous,
alcoholic and biherbal extract of Bryophyllum calycinum, Solanum xanthocarpum for 28
days.
6.6 Research Protocol
Group No. of
Substance Dose (mg/kg)
No. Animals
1 Normal control --- 6
0.5% sodium bicarbonate in
2 Vehicle control 6
water
Ethylene glycol 0.75%v/v
Lithiatic Control (Ethylene and 2% W/V ammonium
3 6
glycol+ ammonium chloride) chloride p.o. along with
drinking water
4 Aqueous extract (plant A) 300mg/kg 6
5 Alcoholic Extract (Plant A) 300mg/kg 6
6 Aqueous extract (plant B) 300mg/kg 6
7 Alcoholic extract (plant B) 300mg/kg 6

8 Biherbal aqueous extract(A+B) 300mg/kg 6

Biherbal alcoholic extract(plant


9 300mg/kg 6
A+B)

43
Aqueous extract control (plant
10 300mg/kg 6
A)
Alcoholic extract control (plant
11 300mg/kg 6
A)
Aqueous extract control
12 300mg/kg 6
(plantB)
Alcoholic extract control
13 300mg/kg 6
(plant B)
Biherbal aqueous extract
14 300mg/kg 6
control (plant A+B)
Biherbal alcoholic extract
15 300mg/kg 6
control (plantA+B)
Total 90

Plant A= Bryophyllum calycinum; Plant B= Solanum xanthocarpum;

6.7 Collection of samples from rats:

Blood samples were collected twice: first after 28 days of induction of urolithiasis
and then on 56th day (after treatment) of experimental period. Blood samples were
collected from all the animals by retro-orbital plexuses puncture under light ether
anesthesia with the help of capillary tube. Blood samples (2 ml) collected in K3EDTA
test tubes were utilized for hematological evaluation. Blood samples (2 ml) collected in
centrifuge tubes without anticoagulant were allowed to clot at room temperature
(262 C). Serum was harvested by centrifugation at 3000 rpm for 15 minutes at
10 C (Eppendorf 5804 R, Germany) and stored - 40C for biochemical analysis.

6.7.1 Collection of urine:


All the rats were kept in metabolic cages and after induction of urolithiasis on
28th day urine samples were collected and analyzed. After the treatment on 56th day urine
samples were collected and analysed. Urine was acidified with a drop of concentrated
HCl and stored -20 C.

6.7.2 Collection of tissues:


After collection of blood samples on 56th day, all the rats were sacrificed by
cervical dislocation. All sacrificed animals were subjected to post mortem

44
examination to determine the presence/absence of gross and histopathological lesions.
Detailed post mortem lesions from all the animals were recorded. Tissue samples viz.,
kidney was collected and preserved in 10 % formalin solution for histopathological
examination.
6.8 Feed Consumption:
The measured quantity of feed was offered to all group of animals housed in
each cage based on the requirement and the same was recorded. Left over feed was
recorded daily to calculate feed consumption by each group.
6.9 Body Weight:
Body weight of all animals of Group I to XV was taken initially on day
before initiation of experiment and dosing (Day 0) and then on 7th, 14th, 21st, 28th ,
35th, 42nd , 49th and 56th day of experimental period.
6.10 Biochemical Study:
Serum biochemical parameters were estimated using standard assay kits
(Coral Clinical System, Goa, India) with the help of clinical serum biochemistry auto-
analyzer (BS-120) in the Department of Physiology and Biochemistry.

6.11 Urine Analysis:

A. Urine Calcium
Calcium was determined using following method
Reagents: 1% Methyl orange indicator (1 g methyl orange in 100 ml alcohol),
Concentrated HCL, saturated ammonium oxalate solution, 20 % sodium acetate, 2%
dilute ammonia, 1 N H2SO4, 0.01 N KMno4
Procedure:
In a centrifuge tube take 2 ml urine then add one drop of methyl orange, one drop of
conc. HCL, 1 ml saturated ammonium oxalate and 20% sodium acetate drop by drop
until indicator turns to yellow. Mix and keep for overnight then centrifuge again
discards the supernatant. Repeat above step once again. Add 4 ml 1 N H2SO4
dissolve precipitates by placing tube in waterbath at 70 C to 80 C titrate the solution
with 0.01 N KMno4 from a microburette till pink colour appears.

45
For Blank: Take 4 ml 0.01 N KMno4 used in titration is equal to 0.2 mg of calcium.
Calcium in urine = (X-B) 100
Where, Test reading = X, Blank reading = B

B. Phosphorus and Creatinine,


Phosphorus and creatinine were determined using commercially available kits
(Coral Clinical System, Goa, India).
Sr.No Name of parameter Method

1. . Phosphorus Mod. Gomrris

2. Creatinine Kinetic (Mod.Jaffes)

6.12 Kidney homogenate analysis


On 56th day the abdomen of the rats were cut open to remove both kidneys from
each animal. Isolated kidneys were cleaned off extraneous tissue and left side kidney was
preserved in 10 % formalin. The right side kidney was dried at 80c in a hot air oven. A
sample of 100mg dried kidney were boiled in 10 ml of 1N hydrochloric acid for 30 min
and homogenized. The homogenate was centrifuged at 2000 RPM for 10 min and the
supernatant was separated. From it calcium, phosphate and oxalate were determined
A. Oxalate Estimation in Kidney Homogenate
Two gm of kidney was weighed in a 250 ml volumetric flask. 190 ml of water and
10ml of 6 N HCl was added and boiled for 1 hr on boiling water inorder to digest it. It
was then allowed to cool and the supernatant was filtered. Fifty ml of filtrate was taken in
a beaker and 20 ml of 6N HCl was added. The mixture was then evaporated to about half
of its volume and filtered. Precipitate was washed several times to make the volume
about 125 ml. To the filtrate 3-4 drops of methyl red was added followed by concentrated
ammonia till the solution turns faint yellow. Mixture was heated to 90-100c and allowed
to cool then filtered to remove the precipitated ferrous impurities. Filtrate was boiled and
10 ml of 5% CaCl2 was added with constant stirring and allowed to stand overnight.
Mixture was filtered through filter paper (No. 41). Precipitates were washed several times
with hot water to make it free of Ca ions. Precipitates were transferred to original beaker
by washing with distilled water and sulphuric acid solution (1:4) was added till the

46
precipitate dissolved. The content was warmed and titrated with N/20 KMnO4 to the near
end point i.e. till the colour disappears by continuous stirring.
Formula:
Oxalate (mg/100g) = N/20 KMnO4 used (ml) 0.00225250/50 100

B. Calcium Estimation in Kidney Homogenate


Calcium estimation from kidney homogenate was done using method described by
(Medeiros and Mustafa, 1985).
C. Phosphate Estimation in Kidney Homogenate
Phosphate estimation from kidney homogenate was done using method described
by (Fiske and Subbarow, 1925).
D. Rat Kim 1 Elisa Assay
Kim 1 is type 1 transmembrane structural glycoprotein located in the renal proximal
tubule epithelial cells. These cells undergo regeneration after various forms of injury and
shed Kim-1 antigen into the serum. Thus Kim-1 is an early and specific biomarker for
tubular kidney injury. It was estimated by using Rat Kim ELISA kit Aviscera
Bioscience Inc., USA.

6.13 Ultrasonography:
Ultrasonography was performed using esaote MyLab40 VET (Esaote Europe
B.V., Philipsweg 1, 6227 AJ Maastricht, The Netherlands) ultrasonography machine at
Department of Veterinary Surgery and Radiology in real time B-mode with linear
transducers using a frequency ranging from 3.5 to 12 MHz. Sterile ultrasound coupling
gel (Carbogel, Brazil) was used in ample amount to ensure proper skin to transducer
contact.

6.14 Gross and histopathology:


After observing gross lesions, the tissues were fixed in the formalin. The
formalin fixed tissues were processed by paraffin wax embedding method of tissue
sectioning. Sections from all the tissues were cut at 5-6 microns thickness with
automatic section cutting machine (Leica, Automatic Microtome Machine, Germany) in
the Department of Pathology and were stained with Haematoxylin and Eosin (H &E)
8

47
stains (Luna, 1968). The H & E stained slides were observed under microscope and
lesions were recorded.

Results

I. Physical characteristics and Recovery percent

The aqueous extract of Bryophyllum calycinum was dark brown while alcoholic
extract was greenish brown. Aqueous extract of Solanum xanthocarpum was yellowish
brown while alcoholic extract was dark brown. The percent extractability of plant was
calculated by the following formula:
Weight of extract
Percent extractability = --------------------------------------------------------- 100
Weight of powder taken for extraction
Table 1: The percent extractability of plant extracts:

Name of plant Aqueous extract Alcoholic extract


Bryophyllum calycinum 37.5% 23.33%
Solanum xanthocarpum 20.83% 16.66%

The percent extractability of aqueous and alcoholic extracts of plant Bryophyllum


calycinum was 37.5 and 23.33%, respectively. The percent extractability of aqueous and
alcoholic extracts of plant Solanum xanthocarpum was 20.83% and 16.66%, respectively.
II. Feed Consumption:
The feed used in the present study was purchased from the Keval sales
corporation, Laboratory Animal Feed Suppliers, Vadodara. The feed was weighted every
day and offered at scheduled time. Feed consumption in different groups is presented in
Table 2.
Group III showed significant (P < 0.05) lower feed consumption on fourth week
as compare to group I. It may be due to the induction of urolithiatic condition and
being kept untreated. Group IV, V, VI, VII, VIII and IX showed significant (P < 0.05)
lower feed consumption during fourth week as compared to group I.

48
It may be due to treatment given by aqueous and alcoholic biherbal extract of
Bryophyllum calycinum and Solanum xanthocarpum showed significant (P < 0.05)
higher in feed consumption as compare to lithiatic group.

10

49
Table 2: Comparison of feed consumption (g/day) in different groups

Group 7th Day 14th Day 21st Day 28th Day 35th Day 42nd Day 49th Day 56th Day

I 13.21a 0.48 13.92ab 0.26 14.04b 0.32 16.66c 0.01 17.14cd 0.21 17.85de 0.10 18.09e 0.21 19.28f 0.10

II 10.95a 2.60 13.33b 1.04 14.28bc 0.26 15.00c 0.31 17.14d 0.21 17.38d 0.10 17.85d 0.31 18.10d 0.21

III 11.67ab 0.95 11.19ab 0.74 11.54ab 0.53 10.48ab 0.22 10.47ab 0.21 10.00a 0.21 09.68a 2.20 09.61ab 0.23

IV 10.71a 0.53 10.83a 0.05 10.47a 1.06 9.92a 0.45 10.95a 0.21 13.33b 0.21 13.33b 0.85 13.80b 0.21

V 10.95a 0.85 10.23a 0.53 10.22a 1.06 9.99a 0.42 12.85b 0.42 12.86b 0.21 13.57b 0.10 13.80b 0.21

VI 10.35a 0.63 10.23a 0.10 10.17a 0.21 10.13a 0.10 11.90ab 0.21 12.38bc 1.06 12.38bc 0.42 13.81c 0.22

VII 11.19ab 1.17 11.15ab 0.30 10.47a 0.64 10.43a 0.62 12.38abc 0.42 11.67ab 0.74 13.09bc 0.31 14.04c 0.31

VIII 10.00a 0.85 11.42abc 0.21 11.19ab 0.32 10.47a 0.42 13.33d 0.64 12.62bcd 0.32 12.85cd 0.21 12.86cd 0.64

IX 10.28a 0.32 10.47c 0.21 10.00c 0.21 9.76ab 0.16 10.00bc 0.21 11.42d 0.21 12.38e 0.21 12.85e 0.21

X 12.85b 0.63 11.66a 0.32 12.85b 0.63 13.09b 0.10 15.47c 0.52 15.83cd 0.37 16.66de 0.01 16.90e 0.10

XI 12.14a 0.31 12.73a 0.05 12.38a 0.856 13.33a 0.42 15.23b 0.00 15.71b 0.21 16.07b 0.47 16.08b 0.16

XII 12.38a 0.21 12.85a 0.64 12.86a 0.21 13.21a 0.05 15.71b 0.64 16.90c 0.10 16.55bc 0.26 17.14c 0.10

XIII 13.57a 0.53 13.57a 0.32 13.80ab 0.21 13.45a 0.48 14.28abc 0.64 14.99bc 0.10 15.47c 0.53 15.35c 0.15

XIV 12.73a 1.01 12.62a 0.74 13.57ab 0.74 14.64bc 0.16 15.47cd 0.53 15.71cd 0.00 15.12bcd 0.37 16.54d 0.27

XV 13.15a 0.05 13.92ab 0.15 13.92ab 0.32 14.40b 0.47 16.19c 0.21 15.95c 0.10 16.66c 0.21 17.61d 0.42

Mean with single superscript differs significantly P<0.05

11
50
III. Body weight:

Changes in the body weight have been s presented in table 3. Body weight of
group III on 8th week was significant (P < 0.05) lower as compare to group I. Group IV,
V, VI, VII, VIII, and IX showed significant increase in body weight as compare to group
III. Due to treatment given with aqueous, alcoholic and biherbal extract.

IV. Serum Biochemistry:

The changes in the serum protein, serum calcium, phosphorus is presented in the
table 4. In group-III, group-IV, group-V, group-VI, group-VII, group-VIII and group-IX
on 28th day serum protein level decreased in comparison to normal control group. After
the treatment on 56th day significant increase in serum protein level was found in group-
VI, group-VII, group-VIII and group-IX in comparision to 28th day.
In group-III, group-IV, group-V, group-VI, group-VII, group-VIII and group-IX
on 28th day serum calcium level increased in comparison to normal control group. After
the treatment on 56th day in group-IV, group-VI, group-VIII and group-IX serum calcium
level significantly decreased in comparision to 28th day.
Serum phosphorus level in group-III, group-IV, group-V, group-VI, group-VII,
group-VIII and group-IX on 28th day serum phosphorus level increased in comparison to
normal control group. After the treatment on 56th day in group-IV, group-V, group-VI,
group-VII, group-VIII and group-IX serum phosphorus level significantly decreased in
comparision to 28th day.
The changes in the BUN, uric acid and serum creatinine presented in the table 5.
BUN, uric acid and serum creatinine significantly decreased in comparision to 28th day
showing antiurolithiatic activity of extract of Bryophyllum calycinum and Solanum
xanthocapum.

V. Urine analysis
In group-III, group-IV, group-V, group-VI, group-VII, group-VIII and group-IX
on 28th day urine calcium and phosphorus increased in comparison to normal control
group. After the treatment on 56th day in group-IV, group-V, group-VI, group-VII, group-

12
51
VIII and group-IX urine calcium and phosphorus significantly decreased in comparision
to 28th day. Similarly after treatment on 56th day in group-IV, group-V, group-VI, group-
VII, group-VIII and group-IX urine creatinine significantly decreased in comparison to
28th day (Table 6).

VI. Kidney Homogenate Analysis:


The deposition of urolithiasis promoters in kidney such as calcium, phosphate
and oxalate was recovered. However, these promoters were found to be significantly
higher (p<0.05) in kidney homogenate in group-III in comparision to normal control
group. Treatment with different extract significantly reversed the deposition of
urolithiasis promoters compared with lithiatic control group (Table 7).
After the treatment on 56th day mean values of calcium in kidney homogenate in
group IV, V, VI, VII, VIII, IX significantly decreased in comparision to group-III
(lithiatic control). No significant changes were observed in aqueous and alcoholic
extracts control group.

13

52
Table 3: Comparison of body weight (g) in different groups

Group 0 Day 1st Week 2nd Week 3rd Week 4th Week 5th Week 6th Week 7th Week 8th Week

I 258.33ab 8.72 261.67 ab 9.28 265.00 a 9.12 270.83ab 9.86 278.33bc 10.30 278.33cde 10.62 293.33 cd 10.38 299.17 cd 9.25 306.67fg 9.89

II 291.67 b 13.51 295.83 b 13.31 297.50b 13.02 300.83 c 1 2.54 305.83b 12.54 305.83 e 11.79 312.50 d 11.74 317.50 d 11.74 321.67a 11.15

III 281.67 ab 7.03 279.17 ab 5.83 273.33 ab 6.28 269.17 ab 5.83 265.83 abc 5.97 265.83 abc 6.41 257.50 a 6.15 252.50 a 6.15 248.33 a 6.28

IV 268.33 ab 9.37 263.33 ab 9.37 259.17 a 9.95 254.17 ab 8.89 250.0 ab 8.46 250.00 a 8.02 251.67 a 7.03 254.17 a 7.12 257.50abc 6.29

V 266.67ab 6.14 263.33ab 5.27 259.17 a 5.38 255.00 ab 5.00 250.83 ab 4.55 250.83 a 4.61 256.67 a 4.94 260.83 a 4.36 267.50abcd 3.81

VI 256.67 ab 2.11 252.50 a 1.70 247.50 a 1.70 245.00 a 1.82 240.83 a 2.00 240.83 a 2.79 243.33 a 2.47 246.67 a 3.33 250.83 ab 2.71

VII 261.67 ab 4.77 258.33 a 4.41 254.17 a 3.96 249.17 ab 3.96 245.00 a 3.87 245.00 a 5.11 252.50 a 4.61 259.17 a 4.36 267.50abcd 3.35

VIII 285.00 ab 9.22 280.83 ab 7.35 274.17 ab 7.23 267.50 ab 7.04 261.67 abc 6.91 261.67 abc 6.54 262.50 ab 5.88 265.83 ab 5.97 269.17abcd 5.97

IX 268.33 ab 7.49 264.17 ab 7.12 259.17a 7.12 255.00 ab 5.91 248.33 ab 5.42 249.17 a 5.07 254.17 a 5.07 260.00 a 4.80 266.67abcd 4.59

X 278.33ab 6.66 272.50 ab 6.80 266.67ab 7.15 260.00 ab 6.58 254.17 ab 6.88 255.83 ab 6.51 258.33 ab 5.42 262.50 ab 5.88 267.50abcd 5.88

XI 272.50 ab 8.24 267.50 ab 8.24 261.67 a 7.81 255.83 ab 6.76 249.17 ab 6.51 250.83 a 6.25 258.33 ab 6.01 265.00 ab 5.77 274.17abcd 5.68

XII 268.33 ab 6.01 263.33 ab 6.01 255.83 a 6.76 251.67 ab 7.03 247.50 ab 6.55 250.0 a 6.58 254.17 a 7.00 259.17 a 7.90 263.33 abc 7.60

XIII 270.0 ab 10.32 266.67 ab 9.71 261.67 a 9.71 255.83 ab 9.87 250.83 ab 8.79 255.83 ab 7.57 262.50 ab 6.29 270.00 ab 5.77 277.50bcde 5.43

XIV 274.17 ab 5.07 269.17 ab 5.07 265.00 a 3.87 257.50 ab 3.35 250.00 ab 3.16 253.33 a 3.07 260.83 ab 3.51 269.17 ab 4.16 276.67abcde 4.77

XV 267.50 ab 5.73 263.33 ab 5.57 255.83 a 4.90 248.33 a 4.41 240.83 a 4.16 244.17 a 4.16 258.33 abc 3.33 270.83 abc 2.38 281.67 cdef 3.07

XVI 250.00 ab 6.83 253.33 a 6.41 258.33 a 6.41 262.50 ab 6.67 267.50 abc 6.68 265.83 abcd 5.69 268.33 abc 4.77 274.17 abc 4.73 280.00 cdef 4.83

XVII 261.67 ab 9.46 264.17 ab 8.00 268.33 ab 7.14 274.17 abc 7.35 278.33 abc 6.91 283.33 bcde 7.03 286.67 bcd 7.37 290.00 bc 8.66 293.33 def 9.36

XVIII 268.33ab 275.00ab 11.83 278.33ab 11.67 281.67bc 286.67 abc 10.77 291.67 de 11.78 295.00 cd 10.49 298.33 cd 9.97 303.33 ef 9.97

12.22 10.77

Mean with single superscript differs significantly P<0.05


14
53
Table 4. Changes in Total Protein and Serum Calcium and Phosphrus concentration before and after treatment (MeanSE)

Total Protein (g/dl) Serum Calcium (mg/dl) Serum Phosphorus (mg/dl)


Group
Group Name Changes Changes Changes on Changes on Changes Changes on
No.
on 28th day on 56th day 28th day 56th day on 28th day 56th day
I Normal Control 8.14 0.38 8.33 0.36 8.10 0.59 8.33 0.36 6.66 0.35 6.57 0.35
II Vehicle Control 7.39 0.36 7.70 0.37 7.93 0.80 7.79 0.80 6.87 0.17 6.77 0.22
III Lithiatic Control 6.74 0.28 6.66 0.18 10.300.70 10.36 0.81 9.47 0.20 9.77 0.19
IV AQ. EX. B.C.300mg/kg 6.64 0.60 7.20 0.42 9.83 0.36 8.90 0.29* 8.72 0.34 7.850.50**
V AlCH .EX.B.C.300mg/kg 6.39 0.57 7.77 0.48 9.26 0.44 9.08 0.38 7.59 0.31 6.80 0.33**
VI AQ.EX.S.X.300mg/kg 6.98 0.58 7.880.67* 9.73 0.80 8.80 0.38* 7.63 0.22 7.18 0.27*
VII AlCH.EX.S.X.300mg/kg 6.47 0.50 7.84 0.55 9.28 0.57 9.21 0.69 8.44 0.21 8.01 0.24*
VIII BIH.AQ.EX. (B.C.+S.X.) 300mg/kg 6.71 0.44 7.570.45* 10.540.33 8.930.50* 8.95 0.34 8.140.39*
IX BIH.AL.EX. (B.C.+S.X.) 300mg/kg 6.70 0.42 7.900.44* 10.50 1.72 8.360.34** 8.60 0.23 7.400.22**
X AQ.EX. Control B.C. 7.98 0.30 7.95 0.27 8.00 0.40 8.09 0.28 6.80 0.34 6.94 0.28
XI AlCH. EX. Control B.C. 7.73 0.63 7.67 0.41 7.49 0.50 7.80 0.49 6.98 0.24 6.74 0.13
XII AQ.EX. Control S.X. 8.17 0.47 8.70 0.64 7.72 0.67 7.78 0.64 7.02 0.16 6.93 0.15
XIII ALCH. EX. Control S.X. 8.18 0.71 8.08 0.54 8.42 0.45 8.46 0.44 6.72 0.19 6.55 0.16
XIV BIH.AQ.EX. Control (plant B.C.+S.X) 8.20 0.56 8.27 0.44 8.25 0.80 8.14 0.71 6.96 0.21 6.62 0.18
XV BIH.ALCH.EX. Control (plant B.C.+S.X) 8.11 0.49 8.26 0.65 8.05 0.79 7.90 0.57 6.83 0.25 6.70 0.15

54 15
Table 5. Changes in BUN, Uric Acid concentration and serum creatinine before and after treatment (MeanSE)

Blood Urea Nitrogen (BUN) Uric Acid Serum Creatinine


Group (mg/dl) (mg/dl) (mg/dl)
Group Name
No. Changes on Changes on Changes on Changes on Changes Changes on
th
28 day 56th day 28th day 56th day th
on 28 day 56th day
I Normal Control 12.800.38 12.87 0.26 2.00 0.17 2.30 0.20 0.49 0.04 0.57 0.01
II Vehicle Control 12.200.58 12.32 0.38 2.42 0.22 2.39 0.19 0.41 0.03 0.47 0.04
III Lithiatic Control 36.820.53 37.01 0.40 4.01 0.48 4.33 0.55 1.95 0.18 1.85 0.18
IV AQ. EX. B.C.300mg/kg 34.700.41 23.930.31* 4.57 0.44 4.19 0.40* 1.84 0.30 1.21 0.23*
V AlCH .EX.B.C.300mg/kg 33.470.80 21.890.50** 3.79 0.19 3.40 0.14* 1.91 0.02 1.11 0.20*
VI AQ.EX.S.X.300mg/kg 31.990.42 17.990.85** 4.70 0.28 4.28 0.30* 1.73 0.17 1.18 0.31*
VII AlCH.EX.S.X.300mg/kg 32.370.39 20.630.28** 3.99 0.41 3.03 0.34* 1.62 0.16 1.10 0.18*
BIH.AQ.EX. (B.C.+S.X.)
VIII 32.620.32 19.580.54** 4.29 0.35 3.590.29** 1.99 0.23 0.98 0.20**
300mg/kg
BIH.AL.EX. (B.C.+S.X.)
IX 35.790.40 20.290.83** 3.65 0.20 2.60 0.31* 1.98 0.09 0.86 0.12**
300mg/kg
X AQ.EX. Control B.C. 12.600.53 12.47 0.47 2.20 0.20 2.39 0.23 0.52 0.07 0.54 0.07
XI AlCH. EX. Control B.C. 12.270.38 12.88 0.55 2.23 0.26 2.01 0.30 0.50 0.07 0.51 0.07
XII AQ.EX. Control S.X. 11.660.38 11.81 0.37 2.00 0.24 1.89 0.21 0.56 0.06 0.58 0.06
XIII ALCH. EX. Control S.X. 10.902.20 14.02 0.46 2.22 0.21 2.34 0.17 0.48 0.04 0.52 0.10
BIH.AQ.EX. Control (plant
XIV 12.070.58 14.14 0.49 2.31 0.26 2.38 0.18 0.58 0.07 0.60 0.07
B.C.+S.X)
BIH.ALCH.EX. Control
XV 11.140.25 11.37 0.36 2.11 0.26 2.19 0.18 0.48 0.05 0.50 0.03
(plant B.C.+S.X)

*(P<0.05), **(P<0.01) B.C.= Bryophyllum calycinum S.X. = Solanum xanthocarpum

16
55
Table 6: Comparison between biochemical parameters of urine in rats induced with Urolithiasis and treated with Biherbal
extract for 28 days

Urine Calcium Urine Phosphorous Urine Creatinine


Mean SE(mg/dl) Mean SE(mg/dl) Mean SE(mg/dl)
Group Name After After After After After
GROUP After
induction biherbal induction biherbal biherbal
induction
on 28th treatment on 28th treatment on treatment on
on 28th day
day on 56th day day 56th day 56th day
I Normal Control 1.75 0.26 1.82 0.30 2.85 0.24 3.05 0.34 7.84 0.21 8.02 0.05
II Vehicle Control 1.72 0.27 1.87 0.20 2.87 0.45 2.98 0.48 8.04 0.05 7.93 0.06
III Lithiatic Control 3.40 0.47 4.10 0.47 4.55 0.80 4.50 0.60 11.90 0.14 12.20 0.08
IV AQ. EX. B.C.300mg/kg 3.62 0.65 2.94 0.46* 4.96 0.57 3.76 0.41* 11.06 0.11 9.81 0.17*
V AlCH .EX.B.C.300mg/kg 3.10 0.49 2.31 0.41* 4.62 0.76 3.51 0.71* 11.29 0.20 9.62 0.10*
VI AQ.EX.S.X.300mg/kg 3.16 0.31 2.75 0.51* 4.29 0.65 3.56 0.73* 11.80 0.20 9.760.19**
VII AlCH.EX.S.X.300mg/kg 3.22 0.37 2.39 0.37* 4.68 0.57 3.24 0.37* 11.36 0.24 9.280.24*
BIH.AQ.EX. (B.C.+S.X.)
VIII 3.38 0.36 1.98 0.35* 4.98 0.68 3.020.43** 11.80 0.27 8.600.09*
300mg/kg
BIH.AL.EX. (B.C.+S.X.)
IX 3.52 0.30 1.900.33** 4.76 0.25 2.98 0.24** 11.50 0.27 8.40 0.26**
300mg/kg
X AQ.EX. Control B.C. 1.80 0.20 1.78 0.27 2.89 0.37 3.02 0.49 7.98 0.11 8.09 0.07
XI AlCH. EX. Control B.C. 1.76 0.25 1.82 0.50 2.96 0.32 3.08 0.49 8.04 0.08 7.74 0.17
XII AQ.EX. Control S.X. 1.70 0.14 1.80 0.18 2.92 0.43 2.99 0.50 8.01 0.10 8.10 0.06
XIII ALCH. EX. Control S.X. 1.72 0.60 1.77 0.35 2.80 0.50 2.86 0.50 7.75 0.20 7.73 0.15
BIH.AQ.EX. Control
XIV 1.91 0.20 1.94 0.27 2.96 0.30 3.01 0.34 8.05 0.24 7.91 0.08
(plant B.C.+S.X)
BIH.ALCH.EX. Control
XV 1.79 0.15 1.85 0.16 3.01 0.50 3.04 0.62 7.69 0.21 7.74 0.21
(plant B.C.+S.X)
*(P<0.05),**

17
56
Table 7: Changes in biochemical parameter concentration in Kidney Homogenate after treatment

Calcium (mg/dl) Phosphate (mg/dl) Oxalate (mg/dl)


GROUP After treatment on After treatment on After treatment on
56th day 56th day 56th day
I Normal Control 3.63a 0.15 2.42a 0.21 2.30a 0.34
II Vehicle Control 3.66a 0.10 2.36a 0.23 2.64a 0.42
III Lithiatic Control 6.91e 0.21 4.64c 0.10 6.64d 0.49
IV AQ. EX. B.C.300mg/kg 6.00d 0.17 3.84b 0.16 3.77bc 0.40
V AlCH .EX.B.C.300mg/kg 4.74c 0.21 3.64b 0.12 3.45bc 0.60
VI AQ.EX.S.X.300mg/kg 4.60d 0.20 3.93b 0.18 3.83c 0.81
VII AlCH.EX.S.X.300mg/kg 4.76bc 0.14 3.70b 0.16 3.20c 0.76
BIH.AQ.EX. (B.C.+S.X.)
VIII 4.32c 0.20 3.03b 0.10 2.72bc 0.54
300mg/kg
BIH.AL.EX. (B.C.+S.X.)
IX 3.78c 0.21 2.83b 0.10 2.62bc 0.49
300mg/kg
X AQ.EX. Control B.C. 3.62a 0.13 2.41a 0.12 2.18a 0.26
XI AlCH. EX. Control B.C. 3.74a 0.24 2.56a 0.16 3.02ab 0.72
XII AQ.EX. Control S.X. 3.60a 0.16 2.50a 0.16 2.29a 0.55
XIII ALCH. EX. Control S.X. 3.51a 0.22 2.65a 0.21 2.25a 0.44
BIH.AQ.EX. Control
XIV 3.65ab 0.16 2.53a 0.21 2.56a 0.63
(plant B.C.+S.X)
BIH.ALCH.EX. Control
XV 3.64a 0.18 2.24a 0.11 2.14a 0.40
(plant B.C.+S.X)
(Mean with different superscript differs significantly P<0.05

18
57
VII. RAT KIM- 1 ELISA:
Kim-1 is a type I trans-membrane structural glycoprotein located in the renal proximal
tubule epithelial cells. These cells undergo regeneration after various forms of injury and
shed Kim-1 antigen into the serum. Thus Kim-1 is an early and specific biomarker for
tubular kidney injury (Table 8).

Table 8: Changes in KIM1 ELISA concentration before and after treatment (pg/ml)

Group Mean SE on Mean SE on 56th


Group Name
no 28th day day
I Normal Control 96.00 1.39 95.00 2.47
II Vehicle Control 95.50 1.72 97.28 1.03
III Lithiatic Control 962.33 4.47 1213.83 5.47
IV AQ. EX. B.C.300mg/kg 1021.33 7.65 597.22 5.66**
V Al CH .EX.B.C.300mg/kg 1014.66 7.49 744.83 4.41**
VI AQ.EX.S.X.300mg/kg 1047.50 4.31 740.83 5.51**
VII AlCH.EX.S.X.300mg/kg 1100.33 6.37 856.16 5.77**
VIII BIH.AQ.EX. (B.C.+S.X.) 300mg/kg 1078.83 3.36 694.50 7.35**
IX BIH.AL.EX. (B.C.+S.X.) 300mg/kg 1065.33 5.30 762.66 2.33**
X AQ.EX. Control B.C. 99.66 4.20 99.33 3.27
XI AlCH. EX. Control B.C. 87.00 1.25 89.50 3.50
XII AQ.EX. Control S.X. 96.33 2.14 95.16 1.18
XIII ALCH. EX. Control S.X. 97.50 3.60 98.50 2.35
XIV BIH.AQ.EX. Control (plant B.C.+S.X) 98.50 0.20 98.00 0.27
BIH.ALCH.EX. Control (plant
XV 96.83 4.15 95.16 3.16
B.C.+S.X)
*(P<0.05), **(P<0.01) B.C.= Bryophyllum calycinum S.X. = Solanum xanthocarpum

Non-significant change was observed in the mean values of KIM-1 ELISA in group I
(Normal control) on 28th day (before treatment) and on 56th day (after treatment). Similarly in
group II (Vehicle control) a non-significant change was observed on 28th day (before
treatment) and on 56th day (after treatment). It indicates that there is no adverse effect of 0.5%
sodium bicarbonate on the mean value of KIM-1 ELISA.
Mean values of KIM-1 ELISA in group III (lithiatic control) showed significant
increase in comparision to normal control group on 56th day.
Group X, XI, XII, XIII, XIV and XV showed non-significant change on 56th day (after
treatment) in comparision to 28th day. It indicates that there is no adverse effect of alcoholic

58
and aqueous extracts of Bryophyllum calycinum and Solanum xanthocarpum on the mean value
of urine KIM-1 ELISA.
Mean values of KIM-1 ELISA in group IV, V, VI, VII, VIII and IX showed showed
highly significant decrease (P<0.01) on 56th day (after treatment) in comparision to 28th day. It
may be due to the treatment given in these groups with aqueous, alcoholic and biherbal extract.
It indicates that both the extract of Bryophyllum calycinum and Solanum xanthocarpum have a
therapeutic potential against urolithiasis.

VIII. Ultrasonography
Ultrasonography is a good diagnostic tool for confirmation of urolithiasis.
Ultrasonography was done in kidney part in which kidney shows distinct cotico-medullary
junction in the normal control. While lithiatic control group showed presence of numerous of
hyperechoic foci at the cortico-medullary junction and indistinct cortico-medullary junction and
ecostic shadow like sturucture was seen at corticomedullary junction which indicated presence
of crystals. While after treatment by aqueous, alcoholic and biherbal extract the of Bryophyllum
calycinum and Solanum xanthocarpum hyperechoic foci were decreased and the
corticomedullary junction become distinct after treatment in group IV, V, VI, VII, VIII and IX
which shows a therapeutic potential of both the plants.
Distinct cortico-medulary junction can be seen in kidney of rats under normal control
while ultrasonography performed in group VIII and IX on 56th day which showed Cortico-
medullary junction becoming distinct as in normal kidney of rats (Plate 1, 2, 3 and 4).

59
Plate 1 - Kidney from Group I shows normal architecture with distinct cortico
medullary junction

Plate 2 - Kidney from lithiatic control group shows numerous hyperechoic foci of
uroliths

Plate 3 - Group VIII (Biherbal aqueous extract (Plant -A+B) treatment group)
shows nephrocalsinosis in the kidney before treatment and decreased
nephrocalsinosis in the kidney after treatment

60
Plate 4 Group IX (Biherbal alcoholic extract (Plant - A+B) treatment group) shows
nephrocalsinosis in the kidney before treatment and decreased nephrocalsinosis
in the kidney after treatment

IX. Histopathology:

Gross examination:
Kidney from lithiatic control group showed pale color in comparison to normal rat
kidney due to effect of urolithiasis which causes damage of kidney tissue while kidney from
normal control group showed pinkish-red color.
No gross and microscopic lesions were noted in liver and spleen of rats from different
groups indicating no action of any chemicals and drugs used in this experiment on those organs
during experiment.

Microscopic examination:
Histopathological examinations of kidney revealed many pathological alterations.
0.75% (v/v) EG and 2% (w/v) ammonium chloride treated group-III (lithiatic control)
showed pathological alterations like presence of crystals in the cystic spaces, necrotic
degeneration, inter tubular hemorrhage, cystic dilatation of tubular epithelium, tubular
epithelial hyperplasia and presence of cast in the lumen of tubules on H&E stain. Group VIII
and IX showed comparatively less patho logical alteration on histopathological examination in
comparision to group-III.

61
Plate A - Section of kidney from Group I showing normal architecture of the
kidney. H&E (120)

Plate B: Section of kidney from Group III showing severe haemorrhage, dilatation
of tubules and presence of crystals in cystic space. H&E (240)

Plate C - Section of kidney from Group VIII showing mild necrosis and
haemorrhage at corticomedullary junction. H&E (120)

62
Plate D- Section of kidney from Group IX showing mild haemorrhage at
corticomedullary junction. H&E (120)

7. Deliverables during this periods:

The facilities created in terms of laboratory, equipments, medicinal plants herbarium are
being used for teaching / training of UG and PG students about EVM.
The results of study will be of immense benefit to the livestock owners.

8. Research Paper published:

1. Akshatha, G. M., Raval, S. K., Ghodasara. D. J, Sadariya. K. A. and Nirav Amin.


(2016). Induction of Hepatocelluar Carcinoma using N-Nitrosodiethylamine in
Wistar Rats. Indian Journal of Natural Sciences. 7(19): 11746-11752.
2. Rathva, A. N., Raval, S. K., Karetha, H.B. and Prajapati, K. K. (2017). Effect of Extracts
of Solanum xanthocarpum and Achyranthes aspera on Feed Consumption and
Bodyweight Gain on Ethylene Glycol Induced Urolithiasis in Wistar Rats. Trends
in Biosciences. 10(19): 3397-3402.

9. Financial status for the Year 2016-2017 (April-16 to March-17)

Grant Received during


Grant Utilized upto Balance on
1st April 2015 to 31st
1st April 2016 st
31 March 2016
march-2016
Non-recurring - - -

Recurring 248000 246634 1366

TA 47000 31086 15914

Other - - -

Total 295000 277720 17280

63
10. Work to be undertaken:
As per the mandate work will be carried out

64
ANNUAL REVIEW REPORT
GOI-ICAR RESEARCH PROJECT
Outreach Programme on Ethnoveterinary
Medicine

2016-2017

PRINCIPAL INVESTIGATOR
Dr. (Ms) Chandana Choudhury Barua
Department of Pharmacology & Toxicology
Faculty of Veterinary Science, AAU
Khanapara, Guwahati -781022, Assam

65
Annual Report, ORP on EVM
Faculty of Veterinary Science, Assam Agricultural University,
Khanapara, Guwahati -781022.

1. Name of Centre: Guwahati, (Assam)


2. Team: Dr(s) Chandana C. Barua, D. C. Pathak, A. Phukan, P. Hussain.
3. Approved activity:
Memory enhancing property of AAU-EVM-NW-3 in mouse/rat model.
Large scale study for anthelmintic activity of AAU-EVM-NW-3
4. Salient achievement
i. AAU-EVM-NW-3 @200mg/kg showed positive effect in reverting the escapes
latency towards normal, along with reduction in transfer latency.
ii. The in vivo antioxidant property of the extract and Tacrine was able to destroy the
harmful free radical scavenging activity due to scopolamine.
iii. The extract efficiently restored the increased scopolamine induced AChE ,
amyloid, level
iv. Various pro apoptotic factors/genes like caspase 3, neurotrophic factors like BDNF,
TrKb were increased/ decreased in positive control group, were reverted towards
normal level
v. Expression of genes like Nrf2, AChE, HO1 genes were upregulated /downregulated
during scopolamine induced condition, but after treatment , they were significantly
suppressed/ expressed accordingly.
vi. Level of catecholamines like NE, Dopamine which usually diminish in impaired
memory, were altered /increased in treated group, indicating restored level.
5. Concise report (01.04.2016 to 31.03.2017)
Universally accepted memory models were studied to ascertain the memory
enhancing activity of methanol extract of AAU-EVM-NW-3. The standard drug used
for the purpose was Tacrine @ 3mg/kg b.wt and drug used for memory impairment for
the present study was Scopolamine @ 0.4mg/kg b. wt.

66
5.1 Study on effect of AAU-EVM-NW-3 on Cognitive Dysfunction in Radial Arm
Maze model in Rats

Memory is a faculty by which sensations, impressions, and ideas are stored and
recalled, where learning is a process by which brain acquires new information about the
events occurring in the given surroundings. Poor memory, lower retention and slow recall
are common problems in todays stressful and competitive world. Ages, stress, emotions,
are conditions that may lead to memory loss, amnesia, anxiety, high blood pressure,
dementia or to more threats like schizophrenia and Alzheimer disease. Learning and
memory impairment is the deficits in the memory conditions. The ability to find objects,
recall previous locations and navigate throughout the world is dependent upon spatial
learning and memory. Cognitive deficits have long been recognized as severe and
consistent neurological disorders associated with numerous psychiatric and
neurodegenerative states. Dementia is a syndrome of progressive nature, characterized by
impairment of memory and loss of intellectual ability. The dementing condition that has
received the utmost attention in the past decade is Alzheimers disease (AD), which is the
most common cause of dementia in the elderly, accounting for 6070% of all cases.
According to the World Health Organization (WHO), 5% of men and 6% of women aged
above 60 years suffer from dementia of AD worldwide.

Medicinal Plants are the valuable source of phytochemical agents and give
templates to develop synthetic or semi-synthetic drugs. The uses of medicinal plants or
herbal medicines are still popular in the field of drug development to treat AD. But more
research is needed to produce scientific support for efficacy and safety of medicinal
plants in the treatment of AD. Thus in our present study we evaluated the memory
enhancing property of AAU-EVM-NW-3 in rats for treating Alzheimer disease by
exploring its mechanism through behavioral, immunologica, biochemical and molecular
(RT-PCR and Immunoblotting) approach.

67
5.2 Experimental:

5.2.1 Plant Collection and Extract preparation

The seeds of AAU-EVM-NW-3 was collected and dried under shade. These dried
materials were mechanically powdered, sheaved using 80 meshes and stored in an
airtight container. These powdered materials were used for further extraction with
different solvents. Briefly, 100 gm of shade dried powdered plant material was
extracted using methanol in a soxhlet extractor at 60-80 C.
5.2.2 Animals
All experiments will be performed in accordance with the guidelines of
institutional animal ethical committee and care of the animals will be taken as per
guidelines of the Committee for the Purpose of Control and Supervision of
Experiments on Animals (770/ac/CPCSEA/FVSc,AAU/IAEC/11-12/118). Wistar rats
(male, age 4-5 months, weighing 150200 g) will be housed four animals per cage
with access to food and water ad libitum under controlled laboratory conditions.
Healthy mice and rats will be screened on the basis of the swimming ability and
normal behavior. Experiments will be conducted between 9:00 and 18:00 h in a semi
soundproof laboratory.
5.2.3 Drugs and chemicals

Scopolamine hydrobromide was purchased from SigmaAldrich (MO, USA).


Tacrine hydrochloride (9-Amino-1, 2, 3, 4-tetrahydro-acridine hydrochloride hydrate)
was purchased from SigmaAldrich (MO, USA). Acetylthiocholine iodide and 5, 5-
dithio- bis-nitro-benzoic acid (DTNB), TMB were obtained from Sigma (St. Louis,
USA). Reagents such as 2x Green Taq master mix ,cDNA synthesis kit, ladder, TRIzol
(Invitrogen) were purchased from Thermo Scientific (USA). Chemicals for western
blotting was procured from Merck.
5.2.4 Acute Toxicity Study

The study was directed as per the protocol of Organization for Economic
Cooperation and Development guidelines for testing of chemicals (OECD 423). The
animals (male Wistar rats, 8-12 weeks old/ albino mice weighing between 20-40 gm),
4

68
were fasted overnight and the extracts of were administered orally at the dose levels 5,
50, 300 and 2000 mg/kg body weight p.o. respectively in five different groups (n = 5)
along with a vehicle control group. Animals were observed individually after dosing
for a total of 14 days for any clinical signs of toxicity and mortality. Any change in
body weight, food and water intake was recorded. The body weight changes were
recorded weekly while food and water intake were observed daily. The extracts were
found to be safe upto 2 gm/kg body weight, p.o. Hence, 3 doses viz, 50,100 and 200
mg/kg were selected for various in vivo experiments.
Plant extracts were dissolved in distilled water and Tween 80 (0.1%) solutions.
Drugs were prepared fresh daily before administration. Preliminary behavioral study
was performed taking 3 doses viz. 50 100mg and 200mg/kg, p.o. methanolic AAU-
EVM-NW-3 based on acute toxicity studies as per OECD guidelines no. 423. Among
them, 100mg and 200mg/kg dose were found to be more potent.
5.3 Experimental design
Animals were divided into the following groups with 6 (six) numbers each.
Group I : Vehicle control (negative control).
Group II : Scopolamine treated (0.4 mg/kg i.p.)
Group III : Tacrine (3mg/kg i.p.) + Scopolamine
Group IV : AAU-EVM-NW-3 (100 mg/kg p.o.) + Scopolamine.
Group V : AAU-EVM-NW-3 (200 mg/kg p.o.) + Scopolamine.

5.4 Tissue Preparation


Hippocampus tissue from the rat brain was skillfully isolated and homogenized in
lysis buffer (RIPA buffer) / PBS (7.4 pH) using hand homogenizer. The sample was then
centrifuged in 15,000 rpm for 20min followed by collection of supernatant. The protein
content of the supernatent was calculated using Bradford reagent (Himedia).

5.5 Behavioral Study by Radial Arm Maze (Parle and Singh; 2007)

In the present study, baited and unbaited arms will be fixed throughout the tests.
The 1st, 3rd, 6th, and 8th arms will be baited while the 2nd, 4th, 5th, and 7th arms will
5

69
be unbaited. At the very beginning of each test session, each rat will placed in the
central starting platform of the RAM in the position facing towards the 1st arm. Food-
deprived rats will be expected to seek specific arms with rewards and subsequently
register and retain the memory of each entered arm where food is present. Each rat will
allowed to freely explore and consume food rewards for 3minutes or until all food
rewards of the four baited arms will be eaten, whichever occurred first. An entry will
recorded every time the rat placed all four paws into the initial part of the arm. The
maze will then thoroughly cleaned with 70% alcohol prior to the next test session in
order to minimize the effect of residual odours from the previous test.
Behavioral tests Radial Arm Maze was assessed. Data obtained from behavioral
tests were assessed through a video camera attached to a computerized tracking system
(ANY-maze TM software Stoelting Co. Video tracking).

5.6 Immunological, Biochemical and Molecular studies:

5.6.1 Immunological Assay


Cytokine estimation by ELISA was conducted for detection of IL-1 and TNF-
protein levels in serum using standard kit protocol (Ray Biotechnology). Beta- Amyloid
a hallmark for Alzheimer's disease was estimated in hippocampus by ELISA.
5.6.2 In vivo antioxidant Study
The levels of antioxidants related to depression such as Non- enzymatic MDA (The
production of this aldehyde is used as a biomarker to measure the level of
oxidative stress in an organism), Antioxidant enzyme (SOD, GSH, LPO, NO and
Catalase) were estimated in the hippocampus of treated and untreated rats.
5.6.3 Acetylcholinesterase enzyme estimation
The Acetylcholinesterase (AChE) was estimated using standard protocol (Ellman,
1991) in the hippocampus of treated and untreated rats.
5.7 HPLC

5.7.1 Measurement of NE, DA and 5-HT Levels in the Hippocampus


Twenty-four hours after completion of the final food consumption test, six of the
mice in each group was used for tissue assays of NE, DA and 5-HT. The animals were

70
decapitated and their brains rapidly were removed and dissected on an ice-chilled glass
plate to obtain the hippocampus. Each tissue sample was weighed and homogenized by
sonication in 1000L 0.4M perchloric acid. The homogenate was kept on ice for 1h
and then centrifuged at 12,000rpm (4C) for 20min. The supernatant was preserved,
and the concentrations of NE, DA and 5-HT were measured using high performance
liquid chromatography with electrochemical detection (HPLC-ECD) as with minor
modifications. The mobile phase consisted of 75mM sodium phosphate monobasic
monohydrate, 1.7mM 1-Octanesulfonic Acid sodium salt, 25M EDTA, 100l
R
Triethylamine, and 10% methanol, pH 3.0. An Acclaim C30 5 m (4.6250 mm)
(Dionex) was operated at 0.7mL/min. The Coulochem III detector 5011A cell was set
at: E1@-150mV: E2@+225mV,Dionex,Sunyvale, CA).External standard curves were
used to quantify the amounts of NE, DA and 5-HT in each sample, calculated using the
area under the curve. The injection volume was 20L.
5.7.2 Estimation of Caspase 3 using fluorogenic substrate
Caspase 3 activity was studied using specific substrate Ac-DEVD-AMC (Sigma) in
Fluoroskan Ascent (Thermo Scientific). The main chemistry of the fluorogenic study
was based on the capacity of the Caspase 3 protein to break the Ac-DEVD-AMC
substrate to Ac-DEVE and AMC. The AMC is the fluorogenic agent. Thus more the
caspase 3 protein more the AMC produced and thus calculated based on the production
of nMol of AMC/min/mL.
5.8 Immunoblotting

To study the possible molecular mechanism involved in the antidepressant


activity of AAU-EVM-NW-03 by detection of few molecular markers (genes involved in
depression mechanism) for depression. Western blotting was done for TrkB and BDNF
for mice hippocampus (30-50mg).
Samples (30 mg approx.) were homogenized using micro pestle (Tarsons) in 5 mL
of chilled lysis Buffer (RIPA Buffer, Amresco, USA) and were centrifuged at 23,000g
for 20 min at 40C. The protein concentration of the supernatants was quantified by
Bradford reagent (Himedia) with BSA as the standard. Fifty micrograms of total protein
was loaded and separated in 10% polyacrylamide gels containing SDS using the Hoefer

71
Midi Gel apparatus (Harvard Apparatus, Holliston, MA). Gels were electrophoresed at
150 V until the tracking dye reached the base of the gel. The fractionated proteins were
visualized by Coomassie blue staining or transferred to nitrocellulose membrane using
semi dry blotting apparatus (Hoefer). The membrane were then blocked using 10ml of
cold blocking buffer containing 3% BSA in TBST (Tris Buffer Saline with Tween 20) for
1hr. The membranes were incubated overnight (40C) with 5 mL of 1% BSA in TBST
containing antiserum (Sigma Chemical Co.) against BDNF and TrkB in a 1: 500
dilution. After overnight incubation, the blots were washed 4 times (5 min each) with 10
mL of TBST. The blots were then reacted with secondary antibody conjugated to HRP
(Santa Cruz Biotechnology) for 1 h. After rinsing with cold TBST, the color reaction on
the nitrocellulose membrane were obtained using commercially available Ultra TMB
Blotting Buffer (Pierce Biotech, USA).
5.8.1 Nf-kB

Nf-kB is a protein complex that controls transcription of DNA, cytokine production and
cell survival. Its expression is most prominent and any kind of inflammatory condition.
As depression also caused inflammatory response in brain tissue, the expression of NF-
kB can be observed in LPS induced depressed mice.
5.8.2 BAX
BAX is reported to interact with, and increase the opening of, the mitochondrial
voltage-dependent anion channel (VDAC), which leads to the loss in membrane
potential and the release of Cytochrome C. The expression of this gene is regulated by
the tumor suppressor P53 and has been shown to be involved in P53-mediated
apoptosis. Thus Bax, a pro-apoptotic member of the Bax/Bcl-2 family, may be a key
factor promoting cytochrome c release.
5.8.3 BDNF

Neurotrophic factors are critical regulators of the formation and plasticity of neuronal
networks. Brain-derived neurotrophic factor (BDNF) is abundant in the brain and
periphery, and is found in both serum and plasma. Animal studies have demonstrated
that stress reduces BDNF expression or activity in the hippocampus and that this
reduction can be prevented by treatment with antidepressant drugs. BDNF levels can
8

72
therefore be useful markers for clinical response or improvement of depressive
symptoms.
5.8.4 TrkB
TrkB is a receptor for brain-derived neurotrophic factor. It is a small protein growth
factors that induce the survival and differentiation of distinct cell populations.Brain-
derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B
(TrkB), signaling represent potential therapeutic targets for major depressive
disorder. Antidepressant drugs and electroconvulsive shock treatment increase the
expression of several molecules, which are associated with neuronal plasticity, in
particular the neurotrophin BDNF and its receptor TrkB.
5.9 Reverse Transcriptase -PCR

The gene expression was performed for few molecular markers (genes involved in
depression mechanism) for depression to study the possible molecular mechanism involved in the
antidepressant activity of. RT-PCR were done for NfKB, Nrf2, Caspase 3 and Bax genes in the
mice hippocampus (30-50mg) .
Hippocampus Tissues was homogenized in TRIzol using micro pestle (Tarsons).
The total RNA was isolated using TRIzo (Ambion). The RNA was then quantified using
drop plate in spectrophotometer (Thermo Scientific). The mRNA was reverse
transcribed by RevertAid First Strand cDNA synthesis kit (Thermo Scientific) with
random hexamer primers. Initial step included 6.5l reaction mixture containing 3l
RNA template, 0.5l Random hexamer and 3l nuclease free water with PCR condition
of 650C for 5 minutes. Subsequent step includes 2 l 5X buffer, 0.5 l dNTPs, 0.5 l
RNase Inhibitor (Ribolock) and 0.5 l Reverse Transcriptase making the total volume of
reaction to 10 l. The PCR condition in final step comprised of three phases, initiation at
250C for 5 min, elongation 420C for 60 min and termination at 700C for 15 min and 40C
hold for infinite time. Subsequently PCR was performed for above mentioned genes.
5.9.1 AChE
Acetylcholine is a neurotransmitter inhibited primarily by acetylcholinesterase
(AChE). The enzyme acetylcholinesterase (AChE) catalyses the hydrolysis of the ester
bound of acetylcholine (ACh) to terminate the impulse transmitted action of ACh through

73
cholinergic synapses. A number of AChE inhibitors have been considered as candidates
for the symptomatic treatment of diseases such as Alzheimer's disease(AD) as the most
useful relieving strategy (Howes et al., 2003). Thus it has become a prime enzyme to
profile the level of memory impairment and its mRNA expression levels was studied in
our present study.
5.9.2 7 nAChR
The alpha-7 nicotinic receptor, also known as the 7 receptor, is a type of nicotinic
acetylcholine receptor implicated in long term memory. Recent studies have correlated
nicotinic acetylcholine receptor (nAChR) dysfunction with the neurodegeneration and
cognitive deficits of Alzheimer's disease, epilepsy, schizophrenia and Parkinson's disease.
Thus in our study we have tried to correlate its mRNA expression levels in control and
drug treated groups.

5.9.3 Caspase 3
Caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of
many key cellular proteins. Caspase-3 is essential for normal brain development and is
important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or
death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of
apoptosis.
5.9.4 Nrf 2
It is already known that oxidative stress occurs when there is an imbalance between
ROS production and antioxidant defense system. Thus, in order to normalize the
condition nuclear factor erythroid 2-related factor (Nrf2), a transcription factor which
functions as the key controller of the redox homeostatic gene regulatory network come
into play under oxidative and electrophilic stresses, the Nrf2 signalling pathway is
activated to augment the expression of a multitude of antioxidant and phase II enzymes
that restore redox homeostasis.
5.9.5 HO-1
Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress
stimuli. The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant
protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response.
10

74
These two systems work together to combat oxidative stress and inflammation and are
attractive drug targets for counteracting different pathologies, including
neuroinflammation.
6 Result
Radial Arm Maze
6.1. Behavioral Study

6.1.1.1. Escape Latency (Sec)


As compared to the Scopolamine treated group the escape latency in sec. could
be observed to decline at significant (P< 0.001) levels in AAU-EVM-NW-3 (200mg/kg)
(36.03 2.474) followed by Tacrine (60.28 0.5200) and AAU-EVM-NW-3
(100mg/kg) (66.31 0.6386) treated groups .
6.1.1.2. Reference Memory Error
Number of error committed showed a decreasing trend at significant (P< 0.001) level in
AAU-EVM-NW-3 (200mg/kg) followed by Tacrine (0.6500 0.08145) and AAU-EVM-
NW-3 (100mg/kg) (1.317 0.03383) when compared to Scopolamine treated group
(Figure 1 and Table 1)

6.2. Immunological, Biochemical and Molecular studies:


6.2.1. Immunological Assay
Immunological assay by ELISA in rat hippocampus (memory loss induced by
scopolamine) revealed a significant (P< 0.001) elevation in levels of cytokines viz IL-1
and TNF- in Scopolamine treated group when compared to control while a significant
(P< 0.001) decline in the protein levels could be observed in Tacrine and AAU-EVM-
NW-3 (100mg/kg and 200mg/kg) when compared to Scopolamine treated group (Figure
2a, 2b). On the other hand -amyloid a hallmark for Alzheimer's disease showed also
showed a significant (P<0.001) elevation in the protein level in the rat hippocampus of
Scopolamine treated group when compared to control. While comparing the treated group
with that of the Scopolamine group a complete decline in the protein levels could be
observed in AAU-EVM-NW-3 (200mg/kg) (P<0.001) followed by AAU-EVM-NW-3
(100mg/kg) (P< 0.01) and Tacrine (P<0.01) (Figure 2c) .

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75
6.2.2. In-vivo Antioxidant Study
Antioxidants GSH, Catalase and SOD showed significant decline (P<0.001) in
Scopolamine treated group when compared to control. Whereas, the levels were found to
elevate significantly (P<0.001) in Tacrine, AAU-EVM-NW-3 (100mg/kg) and AAU-
EVM-NW-3 (200mg/kg) treated group when compared to Scopolamine treated group
(Figure 3a, Figure 3d, Figure 3e). On the other hand LPO and NO showed increased
(P<0.001) levels in Scopolamine treated group on comparison to control. A complete
reversal in LPO and NO levels could be observed in Tacrine, AAU-EVM-NW-3
(100mg/kg) and AAU-EVM-NW-3 (200mg/kg) treated group at significantly decreasing
(P<0.001) levels when compared to scopolamine treated group (Figure 3b, Figure 3c).
These levels thus ascertain the antioxidant property of our plant.
6.2.3. Acetylcholinesterase enzyme estimation
Acetylcholinesterase levels in Scopolamine treated rat hippocampus showed significant
increase (P< 0.001) when compared to control. On the other hand a significant decline
(P< 0.001) in the level of the enzyme could be observed in Tacrine, AAU-EVM-NW-3
(100mg/kg) and AAU-EVM-NW-3 (200mg/kg) treated groups (Figure 4). Thus the
results suggests AAU-EVM-NW-3 have inhibitory effect on the AChE thus maintaining
the level of Acetylcholine (neurotransmitter) in hippocampus.
6.3 HPLC

6.3.1 Effects of AAU EVM NW-3 on NE, DA and 5-HT levels in the hippocampus in
Scopolamine induced rat
The effect of AAU EVM NW-3 on NE, DA and 5-HT levels in the hippocampus in
Scopolamine induced mice (Figure 5). Scopolamine significantly reduced NE, DA and 5-
HT concentrations in the hippocampus compared with that in control animals.
Administration of AAU EVM NW-3(100 and 200mg/kg, p.o.) was able to reverse the
effects of scopolamine on NE, DA and 5-HT concentrations. The standard drug Tacrine
was able to reverse the effects of scopolamine on NE, DA and 5-HT.
6.3.2 Estimation of Caspase 3 using fluorogenic substrate
Fluorogenic assay of Caspase-3 revealed a significant decline in the levels in
Tacrine (P<0.001) and AAU-EVM-NW-3 (200mg/kg) (P<0.05) when compared to the

12

76
Scopolamine treated group. Thus confirming the antiapoptotic activity similar to that of
the standard drug (Figure 6).
6.4 Immunoblotting

6.4.1 NfkB
The NfkB expression was found to be significantly (P< 0.01) upregulated in
Scopolamine treated group when compared to control in rat hippocampus. Whereas, a
significant level of downregulation of the gene could be observed in standard drug
(Tacrine) treated group (P<0.001) followed by AAU-EVM-NW-3 (200mg/kg) (P< 0.001)
and AAU-EVM-NW-3 (100mg/kg) (P< 0.05) treated groups when compared to
Scopolamine treated group. Thus implicating its anti-inflammatory effect which might be
a contributing factor in combating neuronal damage resulting in memory enhancement
(Figure 7a and Table 2).
6.4.2 Bax
The level of expression of Bax was observed to be upregulated significantly
(P<0.001) in Scopolamine treated group when compared to control. While a gradual level
of downregulation of the gene could be observed in standard drug (Tacrine) treated group
(P<0.001) followed by AAU- AAU-EVM-NW-3 (100mg/kg) (P< 0.001) and EVM-NW-
3 (200mg/kg) (P< 0.01) treated groups when compared to Scopolamine treated group.
Thus showing anti-apoptotic property of AAU-EVM-NW-3 similar to standard drug
which cumulatively contributes in memory enhancing activity (Figure 7b and Table 2).
6.4.3 BDNF
The expression of BDNF showed a significant decline in Scopolamine treated group
when compared to control. But an upregulation in level of the gene could be observed in
standard drug (Tacrine) treated group (P<0.05) and AAU-EVM-NW-3 (200mg/kg) (P<
0.05) treated groups when compared to Scopolamine treated group. Thus the results
concluded that AAU-EVM-NW-3 has contributory effect in maintaining the plasticity of
the neuron (Figure 7c and Table 2).
6.4.4 TrkB
The expression of TrkB was found to significantly downregulate in Scopolamine
treated group when compared to control. While a gradual level of upregulation of the

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77
gene could be observed in standard drug (Tacrine) treated group (P<0.001) followed by
AAU-EVM-NW-3 (100mg/kg) (P< 0.001) and EVM-NW-3 (200mg/kg) (P< 0.01)
treated groups when compared to Scopolamine treated group. As TrkB is the receptor
for BDNF so similar trend to that of BDNF could be observed (Figure 7d and Table
2).
6.5 Reverse Transcriptase -PCR
6.5.1 AChE
AChE gene expression in Scopolamine treated rat hippocampus showed significant
increase (P< 0.01) when compared to control. On the other hand a significant
downregulation (P< 0.001) in the expression of the enzyme could be observed in
Tacrine, AAU- and AAU-EVM-NW-3 (200mg/kg) treated groups followed by EVM-
NW-3 (100mg/kg) treated group (Figure 8a and Table 3). Thus the results suggests
AAU-EVM-NW-3 have inhibitory effect on the AChE thus maintaining the level of
Acetylcholine (neurotransmitter) in hippocampus.

6.5.2 7 nAChR
7 nAChR gene expression in Scopolamine treated rat hippocampus showed significant
downregulate (P< 0.01) when compared to control. On the other hand a significant
upregulation (P< 0.001) in the expression of the was observed in Tacrine, AAU-EVM-
NW-3 (100mg/kg) and AAU-EVM-NW-3 (200mg/kg) treated group (Figure 8b and
Table 3).
6.5.3 Caspase3
The level of expression of Caspase3 was observed to be upregulated significantly
(P<0.001) in Scopolamine treated group when compared to control. While a
downregulation of the gene could be observed in standard drug (Tacrine) treated group
(P<0.001) followed by AAU-EVM-NW-3 (100mg/kg) (P< 0.001) and AAU- EVM-
NW-3 (200mg/kg) (P< 0.001) treated groups when compared to Scopolamine treated
group. Thus showing anti-apoptotic property of AAU-EVM-NW-3 similar to standard
drug which cumulatively contributes in memory enhancing activity (Figure 8c and
Table 3).

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78
6.5.4 Nrf2
A significant upregulation (P<0.001) in the expression of the antioxidant gene
was observed in Scopolamine treated group when compared to control. While on
contrary the standard and extract treated group viz. Tacrine, AAU-EVM-NW-3
(100mg/kg) and AAU- EVM-NW-3 (200mg/kg) showed significant downregulation
(P<0.001) in its levels when compared to Scopolamine treated group. Thus AAU-EVM-
NW-3 has antioxidant property which helps to neutralize the oxidizing agent and
ultimately reducing the levels of Nrf2 whose expression augments in any kind of
oxidative stress (Figure 8d and Table 3).
6.5.5 HO-1
A significant level of upregulation (P<0.05) in the expression of the antioxidant
gene was observed in Scopolamine treated group when compared to control. Whereas,
the standard and extract treated group viz. Tacrine, AAU-EVM-NW-3 (100mg/kg) and
AAU- EVM-NW-3 (200mg/kg) showed significant downregulation (P<0.001) in its
levels when compared to Scopolamine treated group. Thus AAU-EVM-NW-3 has
antioxidant property which helps to neutralize the oxidizing agent and ultimately
reducing the levels of HO-1 whose expression augments in any kind of oxidative stress
similar to that of Nrf2 (Figure 8c and Table 3).
6.6 Morris Water Maze
6.6.1 Behavioral Study by Morris Water Maze (Richard G. Morris, 1981)
The water maze contained a circular water pool with 150 cm diameter into
Northeast (NE), Southeast (SE), Southwest (SW), and Northwest (NW) equally spaced
quadrants along the circumference of the pool. In the NW quadrant, an escape platform
(10cm diameter) was kept, 2cm below the water surface. Throughout the acquisition trials
the platform was maintained in a constant location in NW quadrant. The mice were
trained to locate this hidden platform within 60 s, it was gently guided to the platform and
was allowed to stay there 15 s. Animals had four acquisition trials per day for four
consecutive days. To eliminate the quadrant effects, animals was positioned in each
quadrant during each trial. Animals that failed to reach the platform in 20 s on the 4th trial
day were excluded from the study. On the probe day (day 5), 24 hrs after the last

15

79
acquisition trial, escape platform was removed and retention trial was conducted. The
animals were allowed to swim for 60 s before the end of session. Data obtained from
behavioral tests were assessed through a video camera attached to a computerized tracking
TM
system (ANY-maze software Stoelting Co. Video tracking). Time to reach hidden platform
(escape latency), distance travelled to reach the platform were measured during retention trials.
6.6.2 Escape Latency (Sec)
As compared to the Scopolamine treated group the escape latency in sec declines
significantly (P< 0.001) in AAU-EVM-NW-3 (200mg/kg) (6.373 0.029) followed by
AAU-EVM-NW-3 (100mg/kg) (6.433 0.05) treated groups. (Figure 8a).
6.6.3 Time spent in target quadrant (Sec).

It is observed that during the probe trial, results clearly indicated that vehicle
treated animals spent an average time of (45.33 0.06) in the target quadrant. The
Scopolamine-treated animals spent lesser time (22.41 0.03) in the target quadrant in
comparison to control group. AAU-EVM-NW-3 (100mg/kg) (38.52 0.07) and AAU-
EVM-NW-3 (200mg/kg) (41.41 0.06) shows significant (P<0.001) reversal of
Scopolamine induced amnesia as the treated animals in the target quadrant. (Figure 8b)

Vehicle Control
Scopolamine (0.4mg/kg) Vehicle Control
Tacrine (3mg/kg) Scopolamine (0.4mg/kg)
Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg) AAU-EVM-NW-3 (100mg/kg)
AAU-EVM-NW-3 (200mg/kg) AAU-EVM-NW-3 (200mg/kg)
15
time spent in target quadrant(s)

50 ###
Escape Latency(Sec)

*** *** ###


###
***
40 ***
10
### ### ### 30
** *** ***
***
5 20

10
0
0
a Groups Groups b

Figure 8: Behavioral study by Morris Water Maze showing a) Escape Latency (Sec) b)
Time spent in target quadrant (Sec). All values represent mean standard error of mean
(n=4). Statistical significance was determined by one way ANOVA followed by Tukeys
post hoc test, ***P < 0.001, **P< 0.01, *P < 0.05 when compared to Control group, ###P <
0.001, ##P< 0.01, #P < 0.05 when compared to Scopolamine group.

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80
6.6.4 mmunological Assay

6.6.4.1 -amyloid: It is the hallmark for Alzheimer's disease showed also showed a
significant (P<0.001) elevation in the protein level in the mice hippocampus of
Scopolamine treated group when compared to control. While comparing the treated group
with that of the Scopolamine group a complete decline in the protein levels could be
observed in AAU-EVM-NW-3 (200mg/kg) (P<0.001) followed by AAU-EVM-NW-3
(100mg/kg) (P< 0.001) and Tacrine (P<0.001)(Figure 9).

Vehicle Control
Scopolamine (0.4mg/kg)
Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg)
1500 AAU-EVM-NW-3 (200mg/kg)

*** ###
### *** ###
1000 ***
**
ng/L

500

Be ta amyloid

Figure 9: Estimation of levels of proinflammatory cytokines a) Beta- Amyloid in mice


hippocampus by ELISA in Morris Water Maze model.Values are expressed as MeanSEM (n=3).
Statistical significance was determined by one-way ANOVA followed by Tukeys post
hoc test; P< ***, P<### when compared to control and Scopolamine treated group
respectively.

6.6.5 In-vivo Antioxidant Study


Antioxidants GSH, Catalase and SOD showed significant decline (P<0.001) in
Scopolamine treated group when compared to control. Whereas, the levels were found to
elevate significantly (P<0.001) in Tacrine, AAU-EVM-NW-3 (100mg/kg) and AAU-
EVM-NW-3 (200mg/kg) treated group when compared to Scopolamine treated group
(Figure 10a, Figure 10d, Figure 10e). On the other hand LPO and NO showed increased
(P<0.001) levels in Scopolamine treated group on comparison to control. A complete

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81
reversal in LPO and NO levels could be observed in Tacrine, AAU-EVM-NW-3
(100mg/kg) and AAU-EVM-NW-3 (200mg/kg) treated group at significantly decreasing
(P<0.001) levels when compared to scopolamine treated group (Figure 10b, Figure
10c).These levels thus ascertain the antioxidant property of our plant.
a b
Vehicle Control Vehicle Control
Scopolamine (0.4mg/kg) Scopolamine (0.4mg/kg)
Tacrine (3mg/kg)
Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg)
AAU-EVM-NW-3 100mg/kg
AAU-EVM-NW-3 200mg/kg
AAU-EVM-NW-3 (200mg/kg)
2500 250 ***
###
###
Microgm/mg Protein

2000 *** ### *** 200


*** ### ###
###

nMol/mg
1500 *** 150

1000 100

500 50

0 0

GSH LPO

c d
Vehicle Control
Scopolamine (0.4mg/kg) Vehicle Control
Tacrine (3mg/kg) Scopolamine (0.4mg/kg)
AAU-EVM-NW-3 (100mg/kg) Tacrine (3mg/kg)
AAU-EVM-NW-3 (200mg/kg) AAU-EVM-NW-3 (100mg/kg)
80 AAU-EVM-NW-3 (200mg/kg)
4
m Mol/min/mg of protein
Microgm/mg Protein

***
60 ### ### ###
3
*** ***
###
***
2 ### *** 40
***
1 20

0 0
Nitrite Level Catalase

e
Vehicle Control
Scopolamine (0.4mg/kg)
Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg)
8 AAU-EVM-NW-3 (200mg/kg)

### ###
###
**
U/mg of protein

6 *** ***

***
4

0
SOD

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82
Figure 10: Estimation of in-vivo Antioxidants a) GSH b) LPO c) NO d) Catalase and e) SOD in
mice hippocampus with Scopolamine induced memory loss in Morris Water Maze model.
Values are expressed as MeanSEM (n=3). Statistical significance was determined by one-
way ANOVA followed by Tukeys post hoc test; P< ***, P<### when compared to
control and Scopolamine treated group respectively.

6.6.6 Acetylcholinesterase enzyme estimation


Acetylcholinesterase levels in Scopolamine treated mice hippocampus showed
significant increase (P< 0.001) when compared to control. On the other hand, a
significant decline (P< 0.001) in the level of the enzyme could be observed in Tacrine,
AAU-EVM-NW-3 (100mg/kg) and AAU-EVM-NW-3 (200mg/kg) treated groups
(Figure 11). Thus the results suggests AAU-EVM-NW-3 have inhibitory effect on the
AChE thus maintaining the level of Acetylcholine (neurotransmitter) in hippocampus.

250
Vehicle Control
nM/min/mg of protein

*** Scopolamine (0.4mg/kg)


200
Tacrine (3mg/kg)
###
150 ### ### AAU-EVM-NW-3 (100mg/kg)
*
AAU-EVM-NW-3 (200mg/kg)
100

50

0
Acetylcholinesterase

Figure 11. Estimation of Acetylcholinesterase level in mice hippocampus with Scopolamine


induced memory loss. Values are expressed as MeanSEM (n=3). Statistical significance was
determined by one-way ANOVA followed by Tukeys post hoc test; P< ***, P<###
when compared to control and Scopolamine treated group respectively.

6.6.7 Immunoblotting

6.6.7.1 NfkB
The NfkB expression was found to be significantly (P< 0.001) upregulated in Scopolamine
treated group when compared to control in rat hippocampus. Whereas, a downregulation of
the gene was observed in standard drug (Tacrine) treated group followed by AAU-EVM-
NW-3 (200mg/kg) (P< 0.001) and AAU-EVM-NW-3 (100mg/kg) treated groups when
compared to Scopolamine treated group. Thus implicating its anti-inflammatory effect
19

83
which might be a contributing factor in combating neuronal damage resulting in memory
enhancement (Fig.12a).
6.6.7.2 BDNF
The expression of BDNF showed a significant decline in Scopolamine treated
group when compared to control. But an upregulation in level of the gene could be
observed in standard drug (Tacrine) treated group (P<0.05) and AAU-EVM-NW-3
(200mg/kg) treated groups when compared to Scopolamine treated group. Thus the results
concluded that AAU-EVM-NW-3 has contributory effect in maintaining the plasticity of
the neuron. (Fig.12.b)
6.6.7.3 TrkB
It was significantly downregulated in Scopolamine treated group when compared
to control. While a gradual level of upregulation of the gene could be observed in
standard drug (Tacrine) treated group (P<0.001) followed by AAU-EVM-NW-3
(100mg/kg) (P< 0.05) and AAU-EVM-NW-3 (200mg/kg) treated groups when compared
to Scopolamine treated group. As TrkB is the receptor for BDNF so similar trend to that
of BDNF could be observed. (Fig.12c)
6.6.7.4 Caspase3
The expression of Caspase3, an apoptotic factor, was upregulated significantly
(P<0.01) in Scopolamine treated group when compared to control. At the same time
downregulation of the gene observed in standard drug (Tacrine) treated group (P<0.05)
followed by AAU-EVM-NW-3 (100mg/kg) and AAU- EVM-NW-3 (200mg/kg)(P<
0.001) treated groups when compared to Scopolamine treated group. Thus showing anti-
apoptotic property of AAU-EVM-NW-3 similar to standard drug which cumulatively
contributes in memory enhancing activity. (Fig.12d).

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a b
Beta-Actin (42 kDa)

TrkB (95 kDa) BDNF(32kDa)

Vehicle Control 1.5 Vehicle Control


Scopolamine (0.4 mg/kg ) Scopolamine (0.4 mg/kg )
Tacrine (3mg/kg) Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg)
1.5 AAU-EVM-NW-3 (100mg/kg)

%Fold Change
AAU-EVM-NW-3 (200mg/kg)
AAU-EVM-NW-3 (200mg/kg)
### 1.0
#
%Fold Change

#
1.0
ns
ns ns
*** 0.5 ***
0.5

0.0 0.0
TrK BDNF

c d
Beta-Actin (42 kDa)

Caspase-3(20 kDa)
NFBp65 (70kDa)

Vehicle Control
Vehicle Control
Scopolamine (0.4mg/kg)
Scopolamine (0.4 mg/kg )
Tacrine (3mg/kg)
Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg AAU-EVM-NW-3 (100mg/kg)
2.0 AAU-EVM-NW-3 (200mg/kg)
AAU-EVM-NW-3 (200mg/kg) 1.5
ns
**
*** *
%Fold Change

1.5 ns #
ns
%Fold Change

1.0 ###
1.0
###

0.5 0.5

0.0 0.0
NfkB Caspase-3

Figure 12 a-d: Quantitative expression of a) TrkB b) BDNF c) NfkB d) Caspase-3 gene by


western blotting in different treatment groups viz. control, Scopolamine, Tacrine, AAU-EVM- 3
(100mg), AAU-EVM- 3 (200mg) in mice hippocampus with Scopolamine induced memory
loss.Values are expressed as percent fold change represented as meanSE (n=3). Statistical
significance was determined by one-way ANOVA followed by Tukey post hoc test; ***P<0.001
when compared to control, ### P <0.001 when compared to Scopolamine group.

21

85
6.6.8 Reverse Transcriptase PCR

6.6.8.1 AChE
AChE gene expression in Scopolamine treated rat hippocampus showed
significant increase (P< 0.05) when compared to control. On the other hand, a
significant downregulation (P< 0.001) in the expression of the enzyme could be
observed in Tacrine, AAU- and AAU-EVM-NW-3 (200mg/kg) treated groups
followed by AAU-EVM-NW-3 (100mg/kg) treated group.Thus the results suggests
AAU-EVM-NW-3 have inhibitory effect on the AChE thus maintaining the level of
Acetylcholine (neurotransmitter) in hippocampus.(Fig.13a.)
6.6.8.2 Nrf2
A significant upregulation (P<0.001) in the expression of the antioxidant
gene was observed in Scopolamine treated group when compared to control. While
on contrary, the standard and extract treated group viz.Tacrine, AAU-EVM-NW-3
(100mg/kg) and AAU- EVM-NW-3 (200mg/kg) showed significant downregulation
(P<0.001) in its levels when compared to Scopolamine treated group. Thus AAU-
EVM-NW-3 has antioxidant property which helps to neutralize the oxidizing agent
and ultimately reducing the levels of Nrf2 whose expression augments in any kind of
oxidative stress (Fig.13b).
6.6.8.3 HO-1
An upregulation in the expression of the antioxidant gene was observed in
Scopolamine treated group when compared to control. Whereas, the standard and
extract treated group viz.Tacrine, AAU-EVM-NW-3 (100mg/kg) and AAU- EVM-
NW-3 (200mg/kg) showed significant downregulation (P<0.001) in its levels when
compared to Scopolamine treated group. Thus AAU-EVM-NW-3 has antioxidant
property which helps to neutralize the oxidizing agent and ultimately reducing the
levels of HO-1 whose expression augments in any kind of oxidative stress similar to
that of Nrf2 (Fig.13c).

22

86
a b
GAPDH (130bp)

AChE( 331bp) Nrf2 (196bp)

Vehicle Control
Vehicle Control
Scopolamine (0.4 mg/kg ) Scopolamine (0.4 mg/kg )
Tacrine (3mg/kg) Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg) AAU-EVM-NW-3 (100mg/kg)
AAU-EVM-NW-3 (200mg/kg) AAU-EVM-NW-3 (200mg/kg)
1.5
* 2.0 ***
%Fold Change

### ###
1.0

%Fold Change
### 1.5 *** ***
* ###
### ***
### 1.0
0.5 *** ***

0.5

0.0
0.0
Ache Nrf2

c
GAPDH (130bp)

HO-1 (262bp)

Vehicle Control
Scopolamine (0.4mg/kg)
Tacrine (3mg/kg)
AAU-EVM-NW-3 (100mg/kg)
1.5 AAU-EVM-NW-3 (200mg/kg)

ns
%Fold Change

1.0

0.5 ###
*** ### ###
*** ***

0.0

HO-1

Figure 13 a-c: Quantitative expression of a) AChE, , b) Nrf2, c) HO-1genes by RT-PCR in


different treatment groups viz. control, Scopolamine, Tacrine, AAU-EVM- 3 (100mg), AAU-
EVM- 3 (200mg) in mice hippocampus with Scopolamine induced memory loss.Values are
expressed as percent fold change represented as meanSE (n=3).Statistical significance was
determined by one-way ANOVA followed by Tukey post hoc test ;***P<0.001 when compared
to control, ###P<0.001 when compared to Scopolamine group.

23

87
6.6.9 Catecholamine levels
During neurodegenerative disorders, dopamine and noradrenaline levels
are declined. We have estimated their levels in hippocampus of mice and the level of
dopamine is increased in the treated groups in comparison to the scopolamine treated
groups. It is known as mood hormone which is closely linked with memory.
Similarly, noradrenaline levels which were reduced in cognitive dysfunction, was
elevated in the groups than the scopolamine treated group. Hence the compounds are
presumed to act through modulation of HPA axis in the animals (Fig.14a-b).

DOPAMINE 5-HT
100 100
Vehicle Control *** vehicle Control
***
*** Scopolamine (0.4mg/kg) 80 Scopolamine (0.4mg/kg)

ng/gm of tissue
80
ng/gm of tissue

** Tacrine (3mg/kg)
*** *** Tacrine (3mg/kg)
60 ## AAU-EVM-NW-3 (100 mg/kg)
60 AAU-EVM-NW-3 (100mg/kg)
AAU-EVEM-NW-3 (200 mg/kg)
AAU-EVM-NW-3 (200mg/kg) 40
40
20
20
### 0
0
Fig.14a-b: Catecholamine (DA and 5HT)levels in the hippocampus of mice in different
treatment groups.

7. Conclusion
On the basis of consistent findings of AAU-EVM-NW-3 for its effect on
Scopolamine induced Cognitive Dysfunction in Radial Arm Maze in rats and Morris
water maze model in mice, AAU-EVM-NW-3 was found to be a good candidate plant
which showed satisfactory result in almost all the parameters under study thus confirming
its memory enhancing activity in cognitive dysfunction. AAU-EVM-NW-3 @200mg/kg
showed good potency as memory enhancer as based on immunological, antioxidant,
HPLC and gene expression profile.
In escape latency, the test compound and standard drug showed positive effect in
reverting the escapes latency towards normal, along with reduction in transfer
latency.
The in vivo antioxidant property of the extract and Tacrine was able to destroy the
harmful free radical scavenging activity due to scopolamine.

24

88
Level of AChE which was elevated following scopolamine administration, was
declined, which indirectly showed that level of Ach is restored.
amyloid, a hall mark of memory impairment which was reduced in treated group.
Various pro apoptotic factors/genes like caspase 3, neurotrophic factors like BDNF,
TrKb were increased/ decreased in positive control group, were reverted towards
normal level following treatment.
Expression of genes like Nrf2, AChE, HO1 genes were upregulated /downregulated
during scopolamine induced condition, but after treatment, they were significantly
suppressed/ expressed accordingly.
Level of catecholamines like NE, Dopamine which usually diminish in impaired
memory, were altered /increased in treated group, indicating restored level.
8. Deliverables
Provide a platform of neurobiology research for one MVSc student and one
PhD student

25

89
(Year 2016-17)

Dr. Yash Pal Sahni


Principal Investigator
College of Veterinary Science and Animal Husbandry
Nanaji Deshmukh Veterinary Science University
Jabalpur (M.P.)

90
OUTREACH PROGRAMME ON ETHNO-VETERINARY MEDICINE
INDIAN COUNCIL OF AGRICULTURAL RESEARCH, NEW DELHI
ANNUAL PROGRESS REPORT: 2016-17
JABALPUR CENTRE

1. Centre : Jabalpur Centre


College of Veterinary Science and A.H.
Nanaji Deshmukh Veterinary Science University
Jabalpur (M.P.)
2. Team : Dr(s). Yash Pal Sahni, P.C. Shukla and Vidhi Gautam

3. Approved activity for the Year 2016-17


To evaluate the dietary supplementation of Nigella sativa and Withania
somnifera, Selenium and Vitamin E on cholesterol, triglycerides and production
performance of birds (Cross validation).
To study the gene expression profile of SREBP-2 and LDLr on
hypocholesterolemic action of Nigella sativa and Withania somnifera
supplementation.
To analyse the cost economics of egg production with dietary enrichment of
Nigella sativa, Withania somnifera, Selenium and Vitamin E.
4. Salient Achievement
Dietary supplementation of Nigella sativa, Withania somnifera, selenium and
Vitamin E modified cholesterol level by causing reduction in egg and serum
cholesterol concentration to 18.48 and 8.97 per cent respectively.
Dietary supplementation of Nigella sativa, Withania somnifera, selenium and
Vitamin E produced enrichment of egg with increased concentration of Selenium
to 30.96 per cent and Vitamin E to 31.11 per cent.
The efficacy of dietary supplementation of herbs regarding Cholesterol,
Triglycerides, Selenium and Vitamin E concentrations in egg has been found
almost similar to the experiment conducted earlier in the year 2015-16.

91
Dietary supplementation with N. sativa, W. somnifera, Selenium and Vitamin E
did not alter egg production in control and treatment groups of birds.
Expression of SREBP-2 and LDLr indicated the involvement of these two genes
in hypocholestremic activity of Nigella sativa and Withania somnifera. The
mRNA expression of SREBP-2 and LDLr was up-regulated more in ovarian
follicles than in liver of layers.
The cost economics of designer/ enriched egg was calculated as Rs. 8.33 per egg.
5. Progress Report (2016-17)
5.1 Experimental Birds
The study was conducted on a total of 126 healthy Jabalpur colour birds of
thirty two weeks age. Birds were maintained and kept in individual cages under
standard management conditions at All India Coordinated Research Project
(A.I.C.R.P.) on Poultry Breeding, Adhartal Farm, Jabalpur. All the experimental birds
were kept under constant observations during the entire period (84 days) of study.
Birds were randomly divided into seven groups (T1 - T7) with 18 birds in each
group. The birds of different groups were kept separately in individual cages and
maintained under similar hygienic conditions. Group T1 was kept as control.
5.2 Dietary Supplementation
Nigella sativa : 20 gm/kg feed
Withania somnifera : 20 gm/kg feed
Tocopherol : 100 mg/kg feed
Seleno-l-methionine : 0.4 mg/kg feed
5.3 Design of Experiment
Groups No. of Treatment Sample collection
birds per
treatment
T1 18 Standard Diet Blood and eggs were collected on
day 0, 28, 56 and 84 of the study
Liver and ovarian follicles were
collected on day 84 for the gene
expression analysis
2

92
T2 18 Standard Diet+ Nigella sativa (20 Same as T1
gm/kg feed)
T3 18 Standard Diet+ Withania Same as T1
somnifera (20 gm/kg feed)
T4 18 Standard Diet+ Nigella sativa (10 Same as T1
gm/kg feed) + Withania
somnifera (10 gm/kg feed)
T5 18 Standard Diet+ Nigella sativa (20 Blood and eggs were collected on
gm/kg feed) + Selenium (0.4 day 0, 28, 56 and 84 of the study
mg/kg feed) + Vitamin E (100
mg/kg feed)
T6 18 Standard Diet+ Withania Same as T5
somnifera (20 gm/kg feed) +
Selenium (0.4 mg/kg feed) +
Vitamin E (100 mg/kg feed)
T7 18 Standard Diet+ Nigella sativa (10 Same as T5
gm/kg feed) + Withania
somnifera (10 gm/kg feed) +
Selenium (0.4 mg/kg feed) +
Vitamin E (100 mg/kg feed)

93
Experiment - 1

Evaluation of dietary supplementation of Nigella sativa and Withania


somnifera, Selenium and Vitamin E on cholesterol, triglycerides
and production performance

(Cross Validation)

94
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on serum cholesterol of Jabalpur colour bird
Serum cholesterol (mg/dl) MeanSE
Pre Per cent reduction
Group Treatment Post treatment
treatment
Day 0 Day 28 Day 56 Day 84 Day 28 Day 56 Day 84
T1 Untreated control (Standard Diet) 160.5A1.25 160.4A1.25 161.1A1.37 160.4A1.48 - - -
T2 Standard Diet + N. sativa (20 gm/kg feed) 161.4A1.10 156.6AB1.50 153.2B1.29 151.7B1.60 2.97 5.08 6.01
T3 Standard Diet +W. somnifera (20 gm/kg feed) 160.2A1.48 156.8AB1.56 154.5AB1.78 152.0B1.35 2.12 3.87 5.12
Standard Diet + N. sativa (10 gm/kg feed) + W.
T4 159.9A1.14 155.7AB1.38 154.4BC1.10 150.7C1.85 2.63 4.07 5.75
somnifera (10 gm/kg feed)
Standard Diet + N. sativa (20 gm/kg feed)+
T5 Selenium (0.4 mg/kg feed) +Vitamin E (100 mg/kg 160.8A1.85 155.2B1.50 151.8BC1.54 148.8C2.04 3.48 5.60 7.46
feed)
Standard Diet + W. somnifera (20 gm/kg feed) +
T6 Selenium (0.4 mg/kg feed) +Vitamin E (100 mg/kg 160.9A1.40 156.3AB1.48 153.0BC1.80 151.0C1.40 2.86 4.91 6.15
feed)
Standard Diet + N. sativa (10 gm/kg feed)+
T7 W. somnifera (10 gm/kg feed)+ Selenium (0.4 161.6A1.35 154.6B1.08 150.0C1.85 147.1C1.78 4.33 7.18 8.97
mg/kg feed) +Vitamin E (100 mg/kg feed)
Values are mean SE (n=18).
Values in column (uppercase) with different superscripts differ significantly (p<0.05)

95 9
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on egg cholesterol of Jabalpur colour bird
Egg cholesterol (mg/gm yolk) MeanSE
Pre Per cent reduction
Group Treatment Post treatment
treatment
Day 0 Day 28 Day 56 Day 84 Day 28 Day 56 Day 84
T1 Untreated control (Standard Diet) 18.6A0.40 18.6A0.45 18.8A0.28 18.2A0.29 - - -
T2 Standard Diet + N. sativa (20 gm/kg feed) 18.1A0.45 17.4AB0.52 16.8B0.29 16.1B0.25 3.87 7.18 11.05
T3 Standard Diet +W. somnifera (20 gm/kg feed) 18.0A0.42 17.6AB0.50 17.0AB0.25 16.5B0.22 2.22 5.56 8.33
T4 Standard Diet + N. sativa (10 gm/kg feed) + W.
18.4A0.40 17.0AB0.47 16.3BC0.20 16.1C0.25 7.61 11.41 12.50
somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) + Vitamin E (100 mg/kg 18.0A0.48 16.8B0.46 15.8BC0.22 15.0C0.25 6.67 12.22 16.67
feed)
T6 Standard Diet + W. somnifera (20 gm/kg feed)
+ Selenium (0.4 mg/kg feed) + Vitamin E (100 18.2A0.57 17.3A0.40 16.04B0.25 15.8B0.29 4.95 12.09 13.19
mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg feed)+
W. somnifera (10 gm/kg feed)+ Selenium (0.4 18.4A0.55 16.8B0.44 15.6C0.25 15.0C0.27 8.70 15.22 18.48
mg/kg feed) + Vitamin E (100 mg/kg feed)
Values are mean SE (n=18).
Values in columns (uppercase) with different superscripts differ significantly (p<0.05)

96 10
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on serum triglycerides of Jabalpur colour bird
Serum triglycerides (mg/dl) MeanSE
Pre Per cent reduction
Group Treatment Post treatment
treatment
Day 0 Day 28 Day 56 Day 84 Day 28 Day 56 Day 84
T1 Untreated control (Standard Diet) 471.8A1.60 470.5A1.70 469.9A1.66 470.6A1.85 - - -
T2 Standard Diet + N. sativa (20 gm/kg feed) 473.5A1.55 469.8AB1.80 466.5BC1.68 462.2C2.00 1.00 1.48 2.39
T3 Standard Diet + W. somnifera (20 gm/kg feed) 474.0A2.01 470.8A2.01 466.1AB1.80 464.6B1.88 1.00 1.67 1.98
T4 Standard Diet + N. sativa (10 gm/kg feed) + W.
473.2A1.86 467.1AB2.05 464.9BC1.85 460.6C1.72 1.29 1.75 2.66
somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) +Vitamin E (100 mg/kg 472.4A1.82 468.0B1.74 463.7BC1.84 459.9C1.88 1.00 1.84 2.65
feed)
T6 Standard Diet + W. somnifera (20 gm/kg feed)
+ Selenium (0.4 mg/kg feed) +Vitamin E (100 474.5A1.84 466.3B1.88 462.1BC1.75 460.5C1.80 1.52 2.41 2.75
mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg feed)+
W. somnifera (10 gm/kg feed)+ Selenium (0.4 475.7A1.78 462.0B2.87 455.5C1.54 450.9C1.78 2.88 4.25 5.21
mg/kg feed) + Vitamin E (100 mg/kg feed)
Values are mean SE (n=18).
Values in column (uppercase) with different superscripts differ significantly (p<0.05)

97 11
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on egg triglycerides of Jabalpur colour bird
Egg triglycerides (mg/gm) MeanSE
Pre Per cent reduction
Group Treatment Post treatment
treatment
Day 0 Day 28 Day 56 Day 84 Day 28 Day 56 Day 84
T1 Untreated control (Standard Diet) 188.8A0.72 189.1A0.88 189.5A0.94 188.2A1.02 - - -
T2 Standard Diet + N. sativa (20 gm/kg feed) 188.5A0.88 183.8AB1.02 180.04BC1.10 177.7C1.05 2.49 4.51 5.73
T3 Standard Diet + W. somnifera (20 gm/kg feed) 188.2A0.82 186.7AB0.87 183.8BC1.15 181.8C1.04 1.00 2.34 3.40
T4 Standard Diet + N. sativa (10 gm/kg feed) +
188.0A0.95 183.5B0.88 179.0C1.04 177.2C122 2.39 4.79 5.74
W. somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) + Vitamin E (100 mg/kg 189.6A0.92 183.4B1.05 176.9BC0.88 175.4C1.24 3.27 6.70 7.49
feed)
T6 Standard Diet + W. somnifera (20 gm/kg feed)
+ Selenium (0.4 mg/kg feed) + Vitamin E (100 189.2A0.12 184.4B0.82 179.7Co.72 175.0C0.82 2.54 5.02 7.51
mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg feed)+
W. somnifera (10 gm/kg feed)+Selenium (0.4 189.5A1.02 180.4B0.75 175.5C0.83 170.3D0.84 4.80 7.39 10.13
mg/kg feed) + Vitamin E (100 mg/kg feed)
Values are mean SE (n=18).
Values in columns (uppercase) with different superscripts differ significantly (p<0.05)

98 12
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on concentration of Selenium in egg yolk of
Jabalpur colour bird
Concentration of Selenium in egg yolk (ng/gm) MeanSE
Per cent increase
Group Treatment Pre treatment Post treatment

Day 0 Day 28 Day 56 Day 84 Day 28 Day 56 Day 84


T1 Untreated control (Standard Diet) 240.7Ab1.44 242.5Ae1.15 243.6Ad1.45 243.8Ad1.40 - - -
T2 Standard Diet + N. sativa (20 gm/kg feed) 244.4Aab1.20 244.5Ade1.25 244.8Acd1.44 243.6Ad1.25 - - -
T3 Standard Diet +W. somnifera (20 gm/kg feed) 244.8Aab1.55 245.6Ade1.44 245.2Acd1.65 245.8Ad1.34 - - -
T4 Standard Diet + N. sativa (10 gm/kg feed) +
247.9Aa1.75 249.3Ad1.82 249.8Ac1.80 247.8Ac1.65 - - -
W. somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) + Vitamin E (100 243.1Da1.62 295.6Ca1.42 308.3Ba1.82 315.7Aa1.20 21.60 26.82 29.86
mg/kg feed)
T6 Standard Diet + W. somnifera (20 gm/kg feed)
+ Selenium (0.4 mg/kg feed) + Vitamin E (100 244.9Dab1.42 281.7Cc101 296.2Bb1.12 305.7Ab1.34 15.03 20.95 24.83
mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg feed)+
W. somnifera (10 gm/kg feed)+ Selenium (0.4 240.3Dab1.60 301.3Cb1.05 306.2Ba1.26 314.7Aa1.25 25.38 27.34 30.96
mg/kg feed) + Vitamin E (100 mg/kg feed)
Values are mean SE (n=18).
Values in rows (lowercase) with different superscripts differ significantly (p<0.05)
Values in columns (uppercase) with different superscripts differ significantly (p<0.01)

99 13
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on concentration of Vitamin E in egg yolk of
Jabalpur colour bird
Concentration of Vitamin E in egg yolk (g/gm) MeanSE
Pre Per cent increase
Group Treatment Post treatment
treatment
Day 0 Day 28 Day 56 Day 84 Day 28 Day 56 Day 84
T1 Untreated control (Standard Diet) 54.3Aa1.27 55.5Ab1.01 54.8Ab1.25 54.5Ab1.02 - - -
T2 Standard Diet + N. sativa (20 gm/kg feed) 53.4Aa1.22 53.7Ab1.08 54.0Ab1.02 54.1Ab0.85 - - -
T3 Standard Diet +W. somnifera (20 gm/kg feed) 54.0Aa1.20 52.9Ab1.22 54.2Ab1.33 54.7Ab0.76 - - -
T4 Standard Diet + N. sativa (10 gm/kg feed) +
53.8Aa1.03 54.5Ab1.10 54.2Ab1.01 54.5Ab0.89 - - -
W. somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) +Vitamin E (100 mg/kg 53.4Ca0.85 62.8Ba0.82 69.1Aa0.76 69.8Aa0.59 17.60 29.40 30.71
feed)
T6 Standard Diet + W. somnifera (20 gm/kg feed)
+ Selenium (0.4 mg/kg feed) +Vitamin E (100 53.9Ca0.82 63.8Ba0.98 68.3Aa0.75 69.7Aa0.76 18.37 26.72 29.31
mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg feed)+
W. somnifera (10 gm/kg feed)+ Selenium (0.4 54.0Ca0.76 65.2Ba0.88 69.2Aa0.68 70.8Aa0.82 20.74 28.15 31.11
mg/kg feed) + Vitamin E (100 mg/kg feed)
Values are mean SE (n=18).
Values in rows (lowercase) with different superscripts differ significantly (p<0.05)
Values in columns (uppercase) with different superscripts differ significantly (p<0.01)

100 14
Efficacy of N. sativa, W. somnifera, Selenium and Vitamin E on egg production of Jabalpur colour bird

Egg Production (No. of eggs/bird) MeanSE


Group Treatment
Period I Period II Period III
Day 0-Day 28 Day 28-Day 56 Day 56-Day 84
T1 Untreated control (Standard Diet) 14.2B0.35 17.6A0.37 18.8A0.44
T2 Standard Diet + N. sativa (20 gm/kg feed) 14.5B0.40 17.8A0.28 18.9A0.35
T3 Standard Diet +
14.8B0.72 17.4A0.55 18.7A0.60
W. somnifera (20 gm/kg feed)
T4 Standard Diet + N. sativa (10 gm/kg feed) +
13.9B0.40 17.5A0.36 18.4A0.55
W. somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) + 13.9B0.34 16.8A0.48 18.6A0.52
Vitamin E (100 mg/kg feed)
T6 Standard Diet + W. somnifera (20 gm/kg feed)+
Selenium (0.4 mg/kg feed) + 14.1B0.22 16.9A0.50 18.8A0.55
Vitamin E (100 mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg feed)+
W. somnifera (10 gm/kg feed)+
Selenium (0.4 mg/kg feed) + 14.2B0.35 17.2A0.50 18.5A0.48
Vitamin E (100 mg/kg feed)

Values are mean SE (n=18).


Values in columns (uppercase) with different superscripts differ significantly (p<0.01)

101 15
Efficacy of Dietary Supplementation on Enrichment of Egg
(Cross Validation)

Experiment
S. No. Parmeters
Year 2016-17 Year 2015-16
1. Egg Cholesterol ( ) 18.48 per cent 17.30 per cent
2. Serum Cholesterol ( ) 8.97 per cent 8.92 per cent
3. Egg Triglycerides ( ) 10.13 per cent 9.60 per cent
4. Serum Triglycerides( ) 5.21 per cent 4.40 per cent
5. Selenium ( ) 30.96 per cent 28.65 per cent
6. Vit. E. ( ) 31.11 per cent 30.35 per cent

12

102
Experiment - 2

To study the gene expression profile of SREBP-2 and LDLr on hypocholesterolemic


action of Nigella sativa and Withania somnifera supplementation.

The cDNA samples prepared by Reverse Transcriptase PCR (RT-PCR) were tested by in
vitro amplification of 543 bp fragment of GAPDH gene (house keeping/ endogenous
control/ reference gene) for purity and integrity.

The gene specific primer set was used to amplify 339 bp fragment of SREBP-2 gene and
108 bp fragment of LDLr gene using representative cDNA samples.

The mRNA expression level of SREBP-2 gene in liver and ovarian follicle samples of
Jabalpur colour bird was significantly (p<0.05) up-regulated in all treatment groups as
compared to control group. In liver samples maximum up- regulation was found in group
T4 (4.2 fold) supplemented with N. sativa and W. somnifera (10 gm/kg feed each),
followed by group T2 (4.1 fold), group T3 (2.7 fold) was observed relative to control
group. In ovarian follicle samples maximum up- regulation was found in group T4 (8.5
fold) supplemented with N. sativa and W. somnifera followed by group T2 (7.1 fold) and
group T3 (5.3 fold).

The mRNA expression level of LDLr gene in liver and ovarian follicle samples of
Jabalpur colour bird was significantly (p<0.05) up-regulated in all treatment groups as
compared to control group. In liver samples maximum up- regulation (5.2 fold) was
found in group T4 supplemented with N. sativa and W. somnifera (10 gm/kg feed each),
followed by group T2 (4.9 fold), group T3 (3.0 fold) was observed relative to control
group. In ovarian follicle samples maximum up- regulation was found in group T4 (8.7
fold) supplemented with N. sativa and W. somnifera followed by group T2 (7.9 fold) and
group T3 (5.9 fold).

13

103
mRNA expression of SREBP-2 gene in liver and ovary of Jabalpur colour bird

T1 T2 T3 T4
Tissues
Ct 2-Ct Ct 2-Ct Ct 2-Ct Ct 2-Ct

Liver 3.9 A 0.04 1 1.9C 0.04 4.1 2.5 B 0.04 2.7 1.8C 0.04 4.2

Ovary 5.1A 0.03 1 2.2C 0.03 7.1 2.7 B 0.03 5.3 2.0D 0.03 8.5

Means bearing different superscripts within same row differ significantly (p<0.05)

T1- Control T2- N.sativa (20 gm/kg feed)


T3- W. somnifera (20 gm/kg feed) T4- N.sativa (10 gm/kg feed)+ W. somnifera (10 gm/kg feed)

mRNA expression of LDLr gene in liver and ovary of Jabalpur colour bird

T1 T2 T3 T4
Tissues
Ct 2-Ct Ct 2-Ct Ct 2-Ct Ct 2-Ct

Liver 3.5A 0.04 1 1.2C 0.04 4.9 1.9B 0.04 3.0 1.1C0.04 5.2

Ovary 4.9A 0.04 1 1.9C0.04 7.9 2.3B 0.04 5.9 1.7D 0.04 8.7

Means bearing different superscripts within same row differ significantly (p<0.05)

T1- Control T2- N.sativa (20 gm/kg feed)


T3- W. somnifera (20 gm/kg feed) T4- N.sativa (10 gm/kg feed)+ W. somnifera (10 gm/kg feed)

14

104
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 N M

1000 bp

339 bp 400 bp
300 bp
200 bp
100 bp

Agarose gel electrophoresis confirming amplification of SREBP-2 gene (339 bp) of


Jabalpur colour bird

Lane M : 100 bp ladder


Lane 1-16 : Gene specific amplified product
Lane N : No template (negative) control

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 N M

1000 bp
200 bp
108 bp 100 bp

Agarose gel electrophoresis confirming amplification of LDLr gene


(108 bp) of Jabalpur colour bird

Lane M : 100 bp ladder


Lane 1-16 : Gene specific amplified product
Lane N : No template (negative) control

15

105
Activity- 3
Analyses of cost economics of egg production with dietary enrichment of Nigella sativa,
Withania somnifera, Selenium and Vitamin E
Economics of egg production of Jabalpur colour bird with standard and enriched diet
Cost of Cost of dietary supplements (Rs.)
feed (Rs.) (Treatment-wise) No. of eggs Cost per
Group Treatment Total cost (Rs.)
per N. W. produced egg (Rs.)
treatment Selenium Vitamin E
sativa somnifera
T1 Untreated control (Standard Diet) 2740 - - - - 2740 612 4.48
T2 Standard Diet + N. sativa (20 gm/kg feed) 2740 380 - - - 3120 610 5.11
T3 Standard Diet + W. somnifera (20 gm/kg
2740 - 920 - - 3660 615 5.95
feed)
T4 Standard Diet + N. sativa (10 gm/kg feed)
2740 190 460 - - 3390 609 5.52
+ W. somnifera (10 gm/kg feed)
T5 Standard Diet + N. sativa (20 gm/kg
feed)+ Selenium (0.4 mg/kg feed) + 2740 380 - 520 1225 4865 607 8.01
Vitamin E (100 mg/kg feed)
T6 Standard Diet + W. somnifera (20 gm/kg
feed)+ Selenium (0.4 mg/kg feed) + 2740 - 920 520 1225 5405 618 8.74
Vitamin E (100 mg/kg feed)
T7 Standard Diet + N. sativa (10 gm/kg
feed)+W. somnifera (10 gm/kg feed)+
2740 190 460 520 1225 5135 616 8.33
Selenium (0.4 mg/kg feed) + Vitamin E
(100 mg/kg feed)

16

106
7. Conclusion
Dietary supplementation of Nigella sativa, Withania somnifera, selenium and Vitamin E
modified cholesterol level by causing reduction in egg and serum cholesterol
concentration to 18.48 and 8.97 per cent respectively.
Dietary supplementation of Nigella sativa, Withania somnifera, selenium and Vitamin E
produced enrichment of egg with increased concentration of Selenium to 30.96 per cent
and Vitamin E to 31.11 per cent.
The efficacy of dietary supplementation of herbs regarding Cholesterol, Triglycerides,
Selenium and Vitamin E concentrations in egg has been found almost similar to the
experiment conducted earlier in the year 2015-16.
Dietary supplementation with N. sativa, W. somnifera, Selenium and Vitamin E did not
alter egg production in control and treatment groups of birds.
Expression of SREBP-2 and LDLr indicated the involvement of these two genes in
hypocholestremic activity of Nigella sativa and Withania somnifera. The mRNA
expression of SREBP-2 and LDLr was up-regulated more in ovarian follicles than in liver
of layers.
The cost economics of designer/ enriched egg was calculated as Rs. 8.33 per egg.

8. Deliverable; Development of designer egg is in progress

9. Budget : Statement of Expenditure (Year 2016-17)

1 2 3 4 5 6
Head wise Sanctioned Remittance Actual Closing Funds for
budget during the Expenditure balance revalidation
year from as on as on
council 31-03-2017 31-03-
2017
(a) Contingency 2,48,000=00 2,48,000=00 2,44,107=00 3,893=00
(b) T.A. 37,000=00 37,000 =00 3,930=00 33,070=00
Total (Rs.) 2,85,000=00 2,85,000=00 2,48,037=00 36,963=00 36,963=00

Y.P. Sahni
P.I.-EVM Project, Jabalpur Centre

17

107
ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

Department of Clinical Veterinary Medicine


Kerala Veterinary and Animal Sciences University
College of Veterinary and Animal Sciences
Mannuthy, Thrissur, Kerala

108
Department of Clinical Veterinary Medicine
Kerala Veterinary and Animal Sciences University
College of Veterinary and Animal Sciences
Mannuthy, Thrissur, Kerala

1. Name of Centre : Kerala Veterinary and Animal Sciences University, Kerala


2. Name of Investigators: Dr(s). Usha Narayana Pillai, N. Madhavan Unny, Deepa A.
K., Mr. Vijeesh V
3. Approved activity (2015-2016)
i. Isolation and purification of active compounds
ii. In vitro analysis of isolated compounds
iii. Clinical trail
4. Salient Achievements
Antifungal activity of various fractions were completed and active molecule
based studies are going on
Anti-bacterial activity of three crude extracts of hexane, chloroform and ethyl
acetate were completed
5. Concise Report
5.1 Conduct Annual review meet 2015-16
Our centre hosted the annual review meeting of 2015-16 on September 17th
2016 at Hotel Highway Palace, Pattikadu, Thrissur, Kerala.
5.2 Plant selection
The plant Artemisia japonica (KVASU/CM/003) (Fig.1) was selected for the study
based on the previous year results. Aerial part of the plant was collected, washed
thrice in pure water to remove dust and unwanted materials then kept it for drying.
After drying the plant part crushed in to powder form and stored in an airtight
container.

Fig. 1. Artemisia japonica (KVASU/CM/003)

109
5.3 Garden maintenance
The herbal garden was setup in previous year and maintaining very well. From this
garden we got yield of 6.5 Kg of dried plant research material Artemisia japonica
(KVASU/CM/003) were send to the Project Coordinating Centre, IVRI unit for
further research works.
5.4 Experimental Procedures
5.4.1 Active compound based studies
5.4.1.1 Preparation of extract
Acetonic extract was prepared from 25 gm of dried plant materials for the study.
After the soxhlation (Fig. 2.) the extract kept it for drying and the dried material
used for the further studies.

Fig. 2. Soxhlation apparatus

5.4.1.2 Column Chromatographic studies

Twenty five grams of 60 mesh Silica gel was used for column chromatography.
The silica gel slurry was prepared using hexane. The lower part of the glass
column stocked with glass wool and the column packed carefully without
formation of air bubbles. The tap of the glass column was left open to allow free
flow of solvent into a conical flask below the setup was seen to be in order when
the solvent drained freely without carrying glass wool or silica gel in to the tap.
At the end of the packing process the tap was locked the column was allowed 24
hour to stabilize after which the clear solvent on the top of the silica gel was
allowed to drain down to the silica gel meniscus. 1 gram of dried acetonic extract
gently layered on the top of the column for fractionation. The column tap was
opened to allow elute to flow at the rate of 40 drops per minutes. The elution of
the extract was done with solvent systems of gradually increasing polarity and the

110
combinations of solvents such as Hexane, Hexane:Chloroform (50:50),
Chloroform, Chloroform:Ethylacetate (50:50), Ethylacetate, Ethylacetate:Acetone
(50:50), Acetone, Acetone:Methanol (50:50) , Methanol, Methanol:Water
(50:50), Water. Measured volume of (100ml) of each solvent and solvent
combination was collected gradually with 10ml syringe and sprayed uniformly by
the sides of the glass into the column each time. The elute fractions were
collected in aliquots of 10 ml tubes.

5.4.2 In vitro analysis of antifungal effect


Lyophilized crushable pellets of fungal cultures; Trichophyton mentagrophytes and
Microsporum canis were purchased from HiMedia chemicals and kept at 2-8 C for
In vitro analysis.
5.4.2.1 Revival of cultures
Taken one pellet of fungal culture put it into 0.5ml of sterile water and crushed the
pellet using sterile swab, kept for 20 minutes for revival.
5.4.2.2 Antifungal effect on Trichophyton mentagrophytes (TM) and
Microsporum canis (MC)

The disc diffusion method was performed for studying the antifungal effect of
various fractions obtained from column chromatography. Sabourauds dextrose agar
and sabourauds dextrose agar (SDA) with CC supplement were used for culturing
MC and TM respectively by swab plate technique. After plating, sterile discs were
placed and 30l of each fraction was poured over the sterile disc. The plates were
labelled and incubated at 37oC for 48 hours. Zone of inhibition was calculated after
the incubation period.

111
Figure 3. Anti Trichophyton mentagrophytes activity of various fractions

Figure 4. Anti Microsporum canis activity of various fractions

112
Name of the Solvent extract Zone of inhibition of fractions (mm)
organism
1 2 3 4 5 6 7 8 9 10

Microsporum Hexane - - - - - - - - - -
canis
Hexane : Chloroform 7 7 8 9 9 10 8 8 8 9

Chloroform 7 9 8 7 8 10 11 9 11 7

Chloroform : Ethyl - 9 8 8 10 9 8 9 9 11
acetate

Ethyl acetate 7 8 9 9 9 9 8 10 11 12

Ethyl acetate: Acetone 10 9 8 9 9 11 13 10 10 9

Acetone 6 7 11 10 8 9 8 7 7 6

Acetone : Methanol 10 9 8 8 10 9 8 7 - 9

Methanol - - - - - - - - - -

Methanol : Water - - - - - - - - - -

Water - - - - - - - - - -

Trichophyton Hexane - - - - - - - - - -
mentagarophy
te Hexane : Chloroform - - 8 9 9 16 12 11 13 13

Chloroform 13 13 11 13 15 11 13 12 13 17

Chloroform : Ethyl 9 10 11 11 11 11 10 12 14 13
acetate

Ethyl acetate - 14 13 12 13 - - - - -

Ethyl acetate: Acetone 7 10 11 12 12 10 11 11 11 11

Acetone 11 12 11 10 11 11 11 12 12 12

Acetone : Methanol 10 13 15 10 14 18 14 15 16 17

Methanol 8 11 10 11 10 - - - - -

Methanol : Water - - - - - - - - - -

Water - - - - - - - - - -

Table 1. Zone of inhibition of fractions

113
5.5 Result

After the incubation, inhibitory activity of various fractions against Trichophyton


mentagarophyte and Microsporum canis were measured and documented (Table
No.1, Fig 3 and Fig 4). The solvent fractions of Hexane: Chloroform, Chloroform,
Chloroform- Ethyl acetate, Ethyl acetate, Ethyl acetate: Acetone, Acetone, Acetone:
Methanol and Methanol were showed inhibitory activity. Within the group of active
fractions the Hexane: Chloroform, Chloroform, Chloroform: Ethyl acetate, Ethyl
acetate and Acetone: Methanol are more active against Trichophyton mentagarophyte.
The fractions of Hexane, Water: Methanol and water have no inhibitory activity.
Sixth fraction of Acetone:Methanol showed maximum inhibitory activity of 18
mm followed by tenth fraction of chloroform (17mm) when compared to
standard ketconazole of 24mm.
The activity against Microsporum canis was observed that the fractions of
Hexane: Chloroform, Chloroform, and Chloroform: Ethyl acetate, Ethyl acetate, Ethyl
acetate: Acetone, Acetone and Acetone: Methanol showed inhibitory activity. Within
the group of active fractions; Ethyl acetate and Ethyl acetate: Acetone posses
promising inhibitory activity against Microsporum canis. The fractions of Hexane,
Methanol: methanol: Water and Water have no inhibitory activity against
Microsporum canis. The Seventh fraction of Ethyl acetate:Acetone showed
maximum inhibitory activity of 13 mm when compared to standard ketconazole
of 24mm.
5.6 Thin Layer Chromatography of active fractions
Thin-layer chromatography was carried out using TLC pre-coated plates
(silica gel 60F254). The plates were cut with scissors and marked with pencil about
1cm from the bottom of the plate. Combination of ten fractions of each sample was
loaded using capillary tubes on the plates and allowed to dry. The plates were
developed in a chromatographic tank using the different solvent systems including;
(1) hexane (100%), (2) hexane: ethyl acetate (9:1; 8:2, 7:3), (3) chloroform:
methanol (30:1, 15:1), (4) chloroform: ethyl acetate: methanol: water (15: 8: 4: 1).
The plates were dried and visualized under normal day light, ultraviolet light
(254nm & 366nm). The retention factor (Rf) was calculated for each fraction using
the following formula (Table 2.);
TLC was carried out using selected active fractions and results of various
solvent systems were represented in the Fig No.5 and Rf value was calculated

114
(Table 2). Greenish yellow and dark green spots appeared on TLC plate. Screen out
the samples with spots for further examination. Phytochemical constituents give
different Rf values in different solvent system. This variation in Rf values provides a
very important clue in understanding of their polarity and also helps in selection of
appropriate solvent system for further purification of active compounds.
Rf = Distance moved by the solute/compound

Distanced moved by the solvent (solvent front)

Fig 5. Results of TLC

366nm 254 nm

Result of Solvent system I

Result of Solvent system II

115
Result of Solvent system III -

Result of Solvent system IV

Result of Solvent system V

116
Result of Solvent system VI

Result of Solvent system VII

117
5.7 Preliminary antimicrobial activity of Artemisia japonica
5.7.1 Preparation of extracts
Soxhlet apparatus was used for the polarity based solvent extraction of the plant
from least polar to highly polar. 25g of dried plant material was used for 250 ml of
each solvent, such as n-Hexane, Chloroform, Ethyl acetate, Acetone, Methanol,
water and heating the solvents to the boiling point of the respective solvent. The
extracts obtained was concentrated using rotary vacuum evaporator and yield of
respective extracts were calculated and stored it in refrigeration temperature.

Plant No Solvent used Yield (%)

Hexane 0.94

Chloroform 4.92

Ethyl acetate 4.57


KVASU/CM/001 Acetone 4.14

Methanol 10.00

Aqueous 17.66

5.7.2 In vitro Antibacterial studies


The common bacteria staphylococcus aureus was selected for the study. Bacterial
pure culture was procured from the Department of Veterinary Microbiology, College
of Veterinary and Animal Sciences, Mannuthy. The culture stored in refrigeration
temperature and sub cultured into nutrient broth. In vitro antibacterial activity of the
various extracts was assessed by disc diffusion method.
5.7.2.1 Results
The crude extracts of hexane, chloroform and ethyl acetate possess antibacterial
activity and zone of inhibition were 11mm, 12mm and 13mm respectively (Fig 6.).

118
Fig 6. Zone of inhibition of crude extracts

6. Publication
Pillai Usha N, Vijeesh V , Mathew Manju K. (2016). In vitro antifungal activity of
selected plant extract against Candida albicans. Proceedings of 2nd International
seminar on Veterinary Ayurveda in 7th World Ayurveda Congress and Arogya Expo
2016, 1-4 December 2016. Science City, Kolkata.
Pillai Usha N, Vijeesh V , Mathew Manju K. (2017) Phytochemical Analysis and In
vitro Antifungal Studies of Medicinal Plants Elephantopus scaber, Cyclea peltata and
Artemisia japonica International Journal of Pharmacognosy and Phytochemical
Research. 9(3); 319-322.

119
7. Details of budget utilization 2016-2017

Amount sanctioned for the centre: Rs. 2, 85,000/-

Additional fund sanctioned for the centre: Rs. 1, 94,195

Allotted
Sl Expenditure Balance
Head (Rs in
No. (Rs) (Rs)
Lakh)

a. Recurring

1. TA 0.37 0 0.37

Recurring and research contingency


2.48
for chemicals/drugs/expl. Animals and
2. + 4.42195 0
contractual works including research
1.94195
associate

Total (A) 4.79195 4.42195 0.37

b. Non-recurring

3. Equipment 0 0 0

4. Work/renovation 0 0 0

Total (B) 0 0 0

Grand Total (A+B) 4.79195 4.42195 0.37

120
ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

Mathura, DUVASU

121
OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
Mathura, DUVASU

ANNUAL REPORT (2016-2017)

1. Name of Centre: Mathura, DUVASU


2. Name of Investigators: Dr(s) Satish K. Garg, Soumen Choudhury, Shanker Singh,
Naveen Kumar,
3. Approved mandates of the Centre:
To develop a mono or polyherbal formulation for uterine disorders in animals
possessing primarily promising antibacterial, anti-inflammatory and oxytocic
activities;

To study the safety of above formulation; and

Drug delivery system for the evolved formulation, if possible.

4. Salient Research Achievements in ICAR-EVM Outreach programme:


Phytochemical analysis of the methanolic extract of Eucalyptus robusta leaves using
UP-LC and HPLC assay techniques revealed the presence of 18 different marker
compounds; out of which betulinic acid was found to be present in highest
concentration, followed by ellagic acid, gallic acid, shikimic acid and quinic acid. But
2,5-Dihydroxybenzoic acid and polydatin were found to be present but below the
detection limits.
Phytochemical analysis of the methanolic extract of Polyalthia longifolia leaves using
UP-LC and HPLC assay techniques revealed the presence of 17 different marker
compounds; out of which quinic acid was found to be present in highest concentration,
followed by shikimic acid, ellagic acid and betulinic acid. But 2,5-Dihydroxybenzoic
acid, gallocatechin and polydatin were found to be present but below the detection
limits.
Polyherbal formulation Pyodermacare-G was found to be very effective against
generalized demodicosis as well as bacterial and fungal pyoderma and canines.
Polyherbal formulation Pyodermacare-G has promising antibacterial, antifungal,
antiviral and immune-modulatory potential.
Possible target(s)/mechanism of action of methanolic extract of Polyalthia longifolia
leaves against paramyxoviruses, namely peste des petits ruminants virus (PPRV) and
Newcastle disease virus (NDV) was studied. At noncytotoxic concentration, the extract

122
was found to inhibit the replication of PPRV and NDV at the level of viral entry and
budding, whereas other steps of viral life cycle such as attachment and RNA synthesis
remained unaffected.

5. Progress Report (01.04.2016-31.03.2017)


Summary of the research work undertaken during 2016-17
Phytochemical analysis of the selected plant extracts using hitech analytical equipments
Clinical efficacy study against generalized demodicosis in dogs
Clinical efficacy study against bacterial and fungal pyoderma in dogs
Possible mechanism of action of polyherbal formulation
Studies on the mechanism of antiviral activity of selected plant extract.

5.1 Phytochemistry of Selected Plant Extracts


Methanolic Extracts of Leaves of Eucalyptus robusta (Sample Code A) and Polyalthia
longifolia (Sample Code B)
Chemicals used:
LCMS grade methanol, acetonitrile and formic acid, purchased from SigmaAldrich (St.
Louis, MO, USA), were used in the mobile phase and sample preparation throughout the
LCMS studies. AR grade ethanol, purchased from Merck Millipore (Darmstadt,
Germany), was used in the preparation of ethanolic extract. Ultra-pure water, obtained
from Direct-Q water purification system (Millipore, Billerica, MA, USA), was used
throughout the analysis.

The reference standards were purchased from Sigma Aldrich Ltd. (St. Louis, MO, USA).
The reference standard of chebulagic acid was purchased from Natural Remedies Pvt. Ltd.
The purity of all the reference standards was 95%.

Sample preparation

Stock solutions (1 mg/mL) of each standard compound/sample were prepared in methanol


and filtered through a 0.22-m PVDF membrane (Merck Millipore, Darmstadt, Germany).

The calibration curves were constructed by plotting the value of peak areas versus
concentrations of each analyte. All stock solutions were stored in the refrigerator at -20C
until use.

123
UPLC-ESI-QqQLIT-MS conditions

Quantitative analysis was performed on a 4000 QTRAP MS/MS system, hybrid triple
quadrupole-linear ion trap mass spectrometer (Applied Biosystem; Concord, ON, Canada),
hyphenated with a Waters ACQUITY UPLC system (Waters; Milford, MA, USA) via an
electrospray (Turbo V) interface. Waters ACQUITY UPLCTM system was equipped with
binary solvent manager, sample manager, column compartment and photodiode array
detector (PAD) (Waters, Milford, MA).

Chromatographic separation of compounds was obtained with an ACQUITY UPLC


BEH C18 column (1.7 m, 2.1 100 mm) operated at 35C. The mobile phase
consisted of a 0.1% formic acid aqueous solution (A) and methanol (B), was delivered at a
flow rate of 0.350 mL/min under a gradient program: 0-5% (B) initial to 0.5 min, 5-15%
(B) from 0.5 min to 2.0 min, 15-30% (B) from 2.0 min to 5.0 min, 30-45% (B) from 5.0
min to 7.0 min, 45-90% (B) from 7.0 min to 8.0 min, 90-90% (B) from 8.0 min to 10.0 min
and back to initial condition from 10.0 min to 10.5 min. The sample injection volume was
1 L.

The compound-dependent parameters such as declustering potential (DP), entrance


potential (EP), collision energy (CE) and cell exit potential (CXP) were optimized for each
compound by direct infusion of 20 ng/mL solutions of the each analyte using a Harvard
22 syringe pump (Harvard Apparatus, South Natick, MA, USA) in negative ionization
mode.

The results obtained for various parameters have been shown in Table 1. Quadrupole 1 and
quadrupole 2 were maintained at unit resolution. Quantitative analysis was performed using
multiple-reaction monitoring (MRM) mode.

The optimized source dependent parameters were as follows: the Ion Spray voltage (IS)
was set to -4200 V; the turbo spray temperature (TEM) was 550C; nebulizer gas (GS 1) -
50 psi; heater gas (GS 2) - 50 psi; the curtain gas (CUR) - 20 psi and the collision-activated
dissociation gas (CAD) was set as medium and the interface heater was on. High-purity
nitrogen was used for all the processes.

HPLC-ESI-QTOF-MS conditions

An Agilent 1200HPLC system hyphenated with an Agilent 6520 QTOF-MS/MS system


(Agilent Technologies, USA) was used for high resolution mass detection of

124
compounds. The 1200HPLC system consisted of a quaternary pump (G1311A), online
vacuum degasser (G1322A), autosampler (G1329A), and a diode-array detector
(G1315D).

The output of the diode-array detector was introduced into the ESI source of the Agilent
6520 accurate mass QTOF-MS/MS system, operated at a resolving power above 10,000
(FWHM) in negative-ion mode from m/z 1001000 in MS analysis. The HPLC
separation was carried out on a Thermo Betasil C8 column (250 mm 4.5 mm, 5)
operated at 25C.

The mobile phase, consisted of 0.1% formic acid aqueous solution (A) and acetonitrile
(B), was delivered at a flow rate of 0.5 mL min1 under a gradient program: 1090%
(B) from 0.1 min to 20 min, 9090% (B) from 20 min to 25 min, 9010% (B) from 25
min to 30 min, and 1010% (B) from 30 min to 35 min. The sample injection volume
was 1L. In the ESI source, nitrogen was used as nebulizing, drying and collision gas.
The heated capillary temperature was set to 300C and nebulizer pressure to 30 psi. The
drying gas flow rate was 10 L min1. Ion source parameters, VCap, fragmentor,
skimmer and octapole RF peak voltages were set to 3500 V, 175 V, 65 V and 750 V,
respectively.

The chromatographic and mass spectrometric analyses, including the prediction of


chemical formula and exact mass calculation, were performed by using Mass Hunter
software version B.04.00 build 4.0.479.0 (Agilent Technologies).

125
Table 1: The optimized compound dependent MRM parameters and transitions for each analyte in the UPLC-ESI-MS/MS analysis

Peak RT Analyte Precursor DP EP CE Quantifiera Qualifiera


No. (min) ion (V) (V) (eV)
[M-H]-
1 0.74 Shikimic acid 173.1 -50 -2.7 -28.9 173.193.0 (-16) 173.1137.1 (-12)
2 0.99 (-)-Quinic acid 191.0 -67 -12 -33 191.084.9 (-6) 191.092.9 (-15)
3 1.85 Gallic acid 168.7 -59 -10 -22 168.7124.8 (-11) 168.796.7 (-17)
305.1 -93 -7 -28 305.1124.9 (- 305.1167 (-11)
4 2.20 (-)-Gallocatechin 11.9)
5 2.54 2-Furoic acid 111.1 -37 -8 -16 111.166.9 (-11) 111.180.0 (-8)
6 2.66 (-)-Epigallocatechin 304.9 -89 -10 -27 304.9125.0 (-11) 304.9166.5 (-29)
7 3.02 (+)-Catechin 288.9 -110 -10 -29 288.9203.1 (-8) 288.9244.8 (-12)
8 3.08 2,5-Dihydroxybenzoic acid 153.2 -50 -3 -29 153.2107.9 (-11) 153.281.1 (-13)
9 3.21 Methyl gallate 183.1 -64 -7 -30 183.1123.9 (-12) 183.1105.8 (-9)
10 3.52 (-)-Epicatechin 288.6 -120 -9 -29 288.6203.0 (-6) 288.6244.7 (-12)
11 4.19 Ellagic Acid 300.9 -62 -4 -56 300.9145.0 (-12) 300.9199.5 (-11)
12 4.25 Polydatin 389.1 -94 -9 -19 389.1227.0 (-6) 389.1184.9 (-14)
13 5.40 3-O-methyl ellagic acid 315.1 -72 -10 -33 315.1299.6 (-13) 315.1216.2 (-9)
14 5.72 Salicylic acid 136.9 -85 -6 -23 136.993.1 (-16) 136.964.9 (-11)
15 6.46 Luteolin 285.1 -139 -10 -41 285.1132.9 (-21) 285.1150.8 (-13)
16 6.67 Quercetin 301.0 -107 -9 -34 301.0150.9 (-12) 301.0178.9 (-15)
17 7.39 Kaempferol 285.2 -95 -5 -49 285.0158.9 (-10) 285.0143.0 (-8)
18 9.48 Betulinic Acid 455.2 -140 -9 -7 455.2455.2 (-15) 455.2189.0 (-11)
RT: Retention Time; DP: Declustering Potential; EP: Entrance Potential; CE: Collision Energy;
a
Cell Exit Potential (CXP in V) is given in brackets

126
Table 2: Linearity, LOD, LOQ, precisions, stability and recovery results of investigated components
Analytes Regression R2 Linear LOD LOQ Precision RSD (%) Stability Recovery
equation Range (ng/ml) (ng/ml) RSD
(ng/ml) Intraday Interday (%) Mean RSD
(n=6) (n=9) (n=5) (n=6) (%)
y = 297*x +
0.9999 0.5-25 0.15 0.45 0.12 0.21 1.58 104.15 1.21
Shikimic acid 84.8
y = 191*x -
0.9999 0.25-20 0.04 0.12 1.32 0.95 0.78 101.66 0.96
(-)-Quinic acid 145
y = 314*x
1.0000 2-1000 0.36 1.10 1.74 1.07 2.21 102.32 1.31
Gallic acid 95.8
y = 81.4*x +
1.0000 0.25-200 0.07 0.21 1.39 1.84 2.14 98.01 2.14
(-)-Gallocatechin 32.6
y = 756*x +
0.9972 5-100 0.46 1.39 0.98 1.65 2.06 95.09 0.79
2-Furoic acid 112
y = 300*x +
1.0000 1-125 0.30 0.91 0.61 1.20 2.80 96.95 1.52
(-)-Epigallocatechin 48.6
y = 197*x -
0.9997 10-125 0.41 1.25 0.51 0.98 1.51 99.42 1.76
(+)-Catechin 44
2,5-Dihydroxybenzoic y = 412*x -
0.9995 5-100 0.70 2.12 1.23 0.98 2.40 101.58 1.73
acid 115
y = 855*x +
0.9996 0.5-25 0.12 0.36 0.11 1.01 1.56 99.81 2.10
Methyl gallate 413
y = 223*x +
1.0000 10-100 0.69 2.10 2.17 1.99 1.70 100.52 2.46
(-)-Epicatechin 91.9
y = 7.88*x +
0.9999 5-500 0.53 1.62 1.39 1.50 1.66 98.41 1.08
Ellagic Acid 3.98
y = 988*x -
1.0000 0.5-100 0.10 0.29 0.75 1.21 1.89 100.35 2.89
Polydatin 150

127
y = 433*x -
1.0000 0.25-50 0.06 0.18 1.88 0.65 2.50 102.65 1.85
3-O-methyl ellagic acid 18
y = 13000*x
0.9997 5-100 0.37 1.12 2.70 2.65 2.01 98.84 0.92
Salicylic acid - 1300
y = 138*x
0.9995 0.5-200 0.04 0.11 0.89 1.50 1.15 105.84 1.88
Luteolin 7.64
y = 379*x
1.0000 1-50 0.19 0.58 1.69 2.70 1.31 101.63 1.95
Quercetin 75.9
y = 51.5*x +
0.9989 2.25-250 0.39 1.19 1.46 1.16 1.33 97.12 1.65
Kaempferol 33.3
y = 10300*x
0.9999 0.25-25 0.05 0.14 0.53 3.01 1.32 100.98 1.45
Betulinic Acid - 1620
y: peak area; x: concentration of compound (ng/ml);
LOD: limit of detection: S/N = 3.3;
LOQ: limit of quantification: S/N = 10

128
Table 3: Contents (in g/g) of 18 bioactive compounds in methanolic extracts of leaves
of Eucalyptus robusta (Sample Code A) and Polyalthia longifolia(Sample Code B)

Marker compounds Eucalyptus robusta Polyalthia longifolia

Shikimic acid 1833.33 3220.00

(-)-Quinic acid 1660.00 21200.00

Gallic acid 2550.00 53.10

(-)-Gallocatechin 5.06 Bdl

2-Furoic acid 4.15 24.80

(-)-Epigallocatechin bdl 3.61

(+)-Catechin 207.00 89.33

2,5-Dihydroxybenzoic acid bdl Bdl

Methyl gallate 796.33 Nd

(-)-Epicatechin 258.00 52.60

Ellagic Acid 4573.33 190.00

Polydatin bdl Bdl

3-O-methyl ellagic acid 351.67 14.80

Salicylic acid 15.77 18.80

Luteolin 51.57 5.41

Quercetin 182.00 41.60

Kaempferol 82.37 53.70

Betulinic Acid 11433.33 151.67

Total 24010.39 25126.92

129
Table 4: Compounds identified by HPLC-ESI-QTOF-MS and found to be present in the
methanolic extracts of leaves of Eucalyptus robusta (A) and Polyalthia longifolia (B)

S.N RT Analytes Molecular Exact Exact Erro A B


o. (mi Formula mass (cal) mass r
n) [M-H]- (obs) [M- (pp
H]- m)
1 5.5 (-)-Quinic acid C7H12O6 191.0561 191.0557 2.23 + +
2 6.2 Shikimic acid C7H10O5 173.0455 173.0452 1.89 + +
3 8.7 Gallic-Acid C7H6O5 169.0142 169.0141 0.99 + +
4 10.4 (-)-Gallocatechin C15H14O7 305.0667 305.0667 -0.16 - -
5 11.3 (-)-Epigallocatechin C15H14O7 305.0667 305.0663 1.22 - -
6 12.3 (+)-Catechin C15H14O6 289.0718 289.0712 1.99 + +
7 12.8 (-)-Epicatechin C15H14O6 289.0718 289.072 -0.79 + +
8 13.3 2-Furoic acid C5H4O3 111.0088 111.0089 -1.56 + +
9 13.4 Methyl gallate C8H8O5 183.0299 183.0298 0.51 + -
10 13.7 Ellagic acid C14H6O8 300.999 300.9991 -0.21 + +
11 13.9 Polydatin C20H22O8 389.1242 389.1244 -0.54 - -
12 2,5-Dihydroxybenzoic C7H6O4
14 acid 153.0193 153.0194 -0.14 - -
13 15.7 3-O-Methyl ellagic acid C15H8O8 315.0146 315.0145 0.6 + -
14 17 Luteolin C15H10O5 285.0405 285.0404 -0.23 + +
15 17.2 Quercetin C15H10O7 301.0354 301.0344 3.21 + +
16 18.6 Kaempferol C15H10O5 285.0405 285.0406 -0.56 + +
17 18.6 Salicylic acid C7H6O3 137.0244 137.0243 0.52 + +
18 30.9 Betulinic acid C30H48O3 455.3531 455.3533 -0.37 + +
+ present; - absent

130
Figure 1a- Base peak chromatogram (BPC) of standard mixture (18 analytes) in
negative ion mode using HPLC-ESI-QTOF-MS

Figure 1b- Base peak chromatogram (BPC) of sample code A in negative ion mode
using HPLC-ESI-QTOF-MS

Figure 1c- Base peak chromatogram (BPC) of sample code B in negation ion mode
using HPLC-ESI-QTOF-MS

131
Figure 2a- Extended BPCs of (a) standard mixture; (b) sample code A; (c) sample
code B

132
5.2. Clinical Efficacy Study of Poly-herbal Formulation (PyodermaCare-G)
i) Efficacy against generalised demodicosis in Dogs
1. Selection of animals and design of the study

Client-owned pet dogs presented with dermatological ailments at the Teaching Veterinary
Clinical Complex (Kothari Hospital) of the Veterinary College, Mathura for treatment
were examined and diagnosis of demodicosis was made based on the detection of mature
and immature Demodex canis mites in skin-scrapings and/or hair pluck samples from
lesional skin. The dogs were diagnosed with generalized demodicosis (GD) when they
either had minimum of five affected areas (>10 cm2 each) or had a single-affected body
region (>100 cm 2) or had at least one affected paw (pododemodicosis) (Paterson et al.
2009).

2. Animal inclusion criteria

The dogs suffering with generalized demodicosis, but not having been treated with
ectoparasiticides or steroidal anti-inammatory drugs during the last 30 days prior to
presentation in our clinics, were included in this study following thorough clinical
examination of all the animals included in this study. Diseased dogs had the history of
regular and routine de-worming and were free from any other concurrent disease(s). The
participated demodicosed dogs were also free of other ecto-parasites infestations, except
for D. canis mites. Dogs were also found negative for haemoprotozoa on thin blood
smears examination and had the physiological parameters like body temperature,
respiratory-rate and heart-rate within the normal reference range. When skin cytology of
the impression smears revealed presence of neutrophils and intracellular cocci, a
diagnosis of concurrent secondary pyoderma was made.

3. Treatment plan

The dogs diagnosed with generalized demodicosis were allocated into two groups,
namely- Groups 1 and 2, all were demodicosed dogs. Group I included 09 demodicosed
dogs while group Group 2 included 10 demodicosed dogs. The demodicosed dogs of
Group 1 were treated with 0.0375% solution v/v of amitraz rinse at weekly intervals for 8
weeks. Whereas, demodicosed dogs of Group 2 were treated with 0.0375% solution v/v
of amitraz rinse at weekly intervals along with polyherbal formulation (Pyodermacare-G

133
capsules), one capsule (250 mg) orally twice a day for eight weeks. Pyodermacare-G is a
polyherbal preparation developed under ICAR outreach programme on Ethnoveterinary
Medicine (Grant No.1-72/(EVM-Outreach Programme)/2009/Med dated 05.02.2010) by
Department of Pharmacology and Toxicology, College of Veterinary Science and Animal
Husbandry, DUVASU, Mathura. A patent has been filed for the polyherbal
(Pyodermacare-G) preparation.

Demodicosed dogs of both the groups were bathed thoroughly with 2.5% benzoyl
peroxide shampoo and towel dried before application of amitraz rinse. Demodicosed dogs
diagnosed with concurrent pyoderma were also treated with injection lincomycin at a
dose rate of 20 mg per kg of body weight once a day, intramuscularly for a period of
minimum five days and first amitraz rinse was applied on day fifth in these dogs. The
diseased dogs were clinically examined at the intervals of 30 03 days post-therapy for
clinical and parasitological recovery assay.

4. Dermatological investigation

4.1. Mites count

Mites count was performed in skin scrapings material obtained from the two different
skin-scraping sites of one squire centimetre. The same sites were sampled on each
subsequent examination (at 4 wks intervals). For taking skin scrap, the selected 1 cm2
area was squeezed by holding the area of skin between thumb and fore-finger. Further,
with the help of blunted knife a deep skin scrap was taken until the blood ooze out. The
scraped sample was transferred into a glass test tube. A 10% potassium hydroxide
solution (2-3 ml) was added into the scraped samples and subjected for mild heating over
a spirit lamp until 1or 2 bumps arised in the KOH solution. The tube was allowed to stand
for few min at room temperature to make it cool down and further subjected for
centrifugation at 2500 rpm for 5 min. The supernatant was discarded and the aliquot was
used for counting the mites under microscope (10X). Absolute counts for each life stage
(adult, larvae and egg) were recorded as per method suggested by Paterson et al. (2009)

134
4.2. Skin impression smears cytology

Pressure impression smear was prepared from the skin lesions by using the clean grease-
free glass slides. The fixed smear was stained with Field Stain B (Red Stain)
{approximately 1ml} for 30 seconds. After washing under tap water and the slide was
flooded over with Field Stain A (Blue Stain) {approximately 1ml} for 10 to 20 seconds.
Following washing under running tap water and air drying, the slides were examined
under microscope.

4.2.1 Effects on parasitological recovery

The parasitological recovery e.g. reduction in number of total mites counts (developing
and adult stages of Demodex mites) and percentage reduction in total mite counts of both
the groups of demodicosed dogs is depicted in Table 5. On day 30 post-therapy, the per
cent reduction in total mites counts in Pyodermacare-G supplemented dogs (group 2) was
significantly (P<0.0001) higher as compared with the values in non-supplemented group
(Group 1). Similarly following 60 days therapy also, the per cent reduction in total mites
count in Pyodermacare-G supplemented dogs of group 2 was also significantly
(P<0.0001) higher as compared with that of the values observed in dogs of group I i.e.
non-supplemented group. The per cent reduction in mites count was 52.423.83 % and
92.872.3 % on days 30 and day 60s post-therapy, respectively in dogs of control group.
Whereas, per cent reduction in mites counts in demodicosed dogs treated with
Pyodermacare-G capsules was 82.271.27 and 99.50.28 at day 30 and 60 post-therapy,
respectively. None of the dogs of control group were found negative for the presence of
mites and their developmental stages after 60 days of therapy. Whereas, out of 10
demodicosed dogs supplemented with Pyodermacare-G, nine dogs were found negative
for the presence of mites and their developmental stages 60 days post-therapy.

135
Table 5: Effects of Pyodermacare-G supplementation on parasitological recovery of
dogs with generalized demodicosis.

Days Control Pyodermacare-G treated P values


post-therapy (Group 1; n=09) (Group 2; n=10 )
(% reduction in (% reduction in
mites count) mites count)
Day 30 52.423.83 92.872.3 0.0001

Day 60 82.271.27 99.50.28 0.0001

Significant (P<0.0001) difference when compared with the same day post-treatment values
of Group 1.
4.3. Clinical recovery score analysis

The presence and severity of Demodex-induced skin lesions were recorded. Clinical
manifestations like extent of erythema, scalescrusts, comedonespapules/pustules and
alopecia were assessed and rated on a scale from 0 (absent) to 6 (extremely severe) with a
maximum total score of 24 at one sits. All the four scores were summed up for each
affected area of the body and expressed as Demodex-induced skin lesions score (DSLS) for
each affected site. The mean of DSLS at different sites of affected areas was calculated and
used for assessment of clinical recovery for each of the diseased dog. Per cent clinical
recovery was calculated by using formula e.g., % clinical recovery = [(Day 0 DSLS
DSLS at day post-therapy) / Day 0 DSLS] X 100.

The clinical recovery in skin lesions at various days of therapy of control has been
shown in Fig. 2.1, 2.2, 2.3 and 3.1, 3.2 and 3.3 while the clinical recovery in skin lesions at
various days in Pyodermacare-G treated dogs has been shown in Fig. 4.1, 4.2, 4.3, 5.1, 5.2,
5.3, 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.2, 8.3, and 9.1, 9.2, 9.3. The clinical recovery e.g.
improvement in Demodex-induced skin lesions score (DSLS) of diseased of both the
studied groups of demodicosed is summarized in Table 6. On day 30 post-therapy, the per
cent improvement DSLS in Pyodermacare-G supplemented dogs was significantly
(P<0.0001) higher as compared with the same days values in non-supplemented dogs of
Group 1. Similarly on day 60 post-therapy, the per cent improvement DSLS in
Pyodermacare-G supplemented dogs was also significantly (P<0.0001) higher as compared

136
with the same days values in non-supplemented group (Group 1). Albeit, 44.313.23 %
and 86.292.88 % improvement in DSLS were revealed by the dogs of control group on
day 30 and day 60 post-therapy, respectively. But compared to this, the per cent
improvements in DSLS of Pyodermacare-G supplemented group dogs were 73.193.44
and 98.010.85 on day 30 and 60 post-therapy, respectively. Apart from the better
improvement in DSLS, an appreciable improvement in food intake and body coat lustre
was exhibited by the dogs of Pyodermaccare-G supplemented group compared to that in
dogs of control group.

Further, important aspect is that none of the diseased dogs of Pyodermacare-G


supplemented group revealed any recurrence of the clinical disease during the 3-6 months
of follow-up period. Whereas out of nine enrolled dogs of control group, seven dogs
revealed recurrence of the clinical diseases within 3 months of the withdrawal of miticidal
therapy. Additionally, the demodicosed dogs of control group required 120-150 days to
become negative for the presence of the mites on skin scraping examination during the
recommended regimen of miticidal application. The demodicosed dogs of control group
with concurrent pyoderma required antibiotic therapy for prolonged period (10-20 days),
whereas Pyodermacare-G supplemented demodicosed dogs with concurrent pyoderma
required antibiotic therapy only for five days. Marked improvement in appetite and body
coat lustre was also noticed in demodicosed dogs supplemented with Pyodermacare-G.

Table 6: Effects of Pyodermacare-G supplementation on clinical recovery of dogs


with generalized demodicosis.

Days Controls Pyodermacare-G P values


supplemented
post-therapy (Group 1; n=09)
(% improvement in DSLS) (Group 2; n=10 )
(% improvement in DSLS)

Day 30 44.313.23 86.292.88 0.0001

Day 60 73.193.44 98.010.85 0.0001



Significant (P<0.0001) difference when compared with same day post-treatment values of
Group 1

137
4.4 Blood sample collection

With the informed verbal consent of the pet owners, approximately 3 mL of blood
samples were obtained from each dog in clot activators containing tubes on every
examination during the period of the therapy and used for harvesting serum for
biochemical estimation using automated biochemistry analyzer (BS-120 Chemistry
Analyzer; 2007- 2010 Shenzhen Mindray Biochemical Electronics Co. Ltd.) with
commercially available kits (Span Diagnodtics). Additionally, 2 mL blood samples were
also obtained into vials containing EDTA from each of the diseased dog on every
examination during the period of the therapy and were subjected for routine haematology
by using fully automated haematology analyzer (BS-2800 Vet Haematology analyzer,
Mindray Electronic Co. Ltd). Blood samples were collected on day 0 i.e. before start of the
therapy, Day 30 post-therapy and day 60 post-therapy. In similar manner blood samples
were also obtained from the healthy dogs and were used as reference value (Group 3).

4.4.1. Effect on Haematology

The haematological data of diseased dogs on days 0, 30 And 60 is depicted in Table


7. The ameliorative potential of Pyodermacare-G on haematology of dogs with generalised
demodicosis was evaluated in terms of alterations in the both leukograms and haemograms.
Remarkable alterations in the both leukograms and haemograms were not recorded on day
0 (before start of the therapy). Whereas, remarkable alterations in the leukograms were
recorded in both of the groups on day 30 and day 60 post-therapy when compared with
their own day 0 values (Table 7). Moreover, remarkable alterations in the both leukograms
and haemograms were also recorded in both of the groups on day 60 when compared with
their own day 30 values (Table 7 and 8).

The demodicosed dogs of control group (Group 1), that were not supplemented
with Pyodermacare-G, found have no significant reduction in total leukocyte counts (TLC)
at day 30 and day 60 post-therapy as compared with their own day 0 values. Moreover,
significant amelioration in other panels of the leukograms for instance lymphocytes,
granulocytes and monocytes counts as well as lymphocytes, neutrophils, monocytes and
eosinophils percentage was also not recorded in this groups at day 30 and day 60 post-
therapy as compared with their own day 0 values. Likewise, significant amelioration in all

138
the panels of leukograms were not revealed by the dogs of control group at day 60 post-
therapy as compared with their own day 30 values. Whereas, the demodicosed dogs
supplemented with Pyodermacare-G (Group 2) found have significant (P<0.001) reduction
in TLC at day 30 and day 60 post-therapy as compared with their own day 0 values.
Moreover, significant (P<0.001) reduction in granulocytes counts was recorded in this
groups at day 30 and day 60 post-therapy as compared with their own day 0 values. At
day 30 post-therapy, significant (P<0.001) reduction lymphocytes counts was revealed by
Pyodermacare-G supplemented dogs as compared with their own day 0 values. Moreover
at day 60 post-therapy these dogs also revealed significant (P<0.001) reduction in the both
lymphocytes and monocytes counts as compared with their own day 0 values. The dogs of
this group also found to have significantly (P<0.05) lower percentage of neutrophils at day
60 post-therapy as compared with their own day 0 and day 30 values. Whereas,
significantly higher percentage of lymphocytes (P<0.01) and monocytes (P<0.05) at was
recorded in this group at day 60 post-therapy as compared with own day 0 values.
Remarkable reduction in eosinophis percentage was not recorded in dogs of both of the
groups at day 30 and day 60 post-therapy as compared with their own day 0 values.

At day 30 post-therapy, the dogs supplemented with Pyodemacare-G were found to


have significantly (P<0.01) lower values of TLC, granulocytes and lymphocytes as
compared with day 30 post-therapy values of non-supplemented dogs (Group 1).
Additionally at day 30 post-therapy, percentage of neutrophils was significantly (P<0.05)
lower in the dogs supplemented with Pyodemacare-G as compared with the same day
values of Group 1. Whereas, Pyodemacare-G supplemented dogs found to have
significantly (P<0.001) elevated percentage of monocytes at day 30 post-therapy as
compared with same days values of non-supplemented group. Moreover at day 60 post-
therapy, the dogs supplemented with Pyodemacare-G found to have significantly lower
values of TLC (P<0.001), granulocytes (P<0.001), lymphocytes (P<0.001) and monocytes
(P<0.05) as compared with day 60 post-therapy values of non-supplemented dogs (Group
1). Additionally at day 60 post-therapy, significantly lower percentage of neutrophils
(P<0.005) and eosinophils (P<0.01) was recorded in the dogs supplemented with
Pyodemacare-G as compared with the same day values of Group 1. Whereas,
Pyodemacare-G supplemented dogs found to have significantly elevated percentage of

139
lymphocytes (P<0.02) and monocytes (P<0.001) at day 60 post-therapy as compared with
same days values of non-supplemented group.

Remarkable alterations in haemograms were recorded in the both within and


between the studied groups (Table 8).The demodicosed dogs of control group (Group 1),
that were not supplemented with Pyodermacare-G, found have no significant amelioration
haemograms panels for instance TEC, Hb, HCT, MCV, MCH and MCHC at day 30 and
day 60 post-therapy as compared with their own day 0 values. Whereas, significant
improvement in haemograms panels for instance TEC (P<0.001), Hb (P<0.001), HCT
(P<0.001), MCH (P<0.01) and MCHC (P<0.04) at day 30 post-therapy was revealed by the
dogs of Pyodermacare-G supplemented group as compared with their own day 0 values.
Additionally, the dogs of this group also found to have significant improvements in TEC
(P<0.001), Hb (P<0.001), HCT (P<0.001), MCH (P<0.04) and MCHC (P<0.004) at day 60
post-therapy as compared with their own day 0 values. Likewise at day 60 post-therapy the
dogs of this group found to have further significant improvements in TEC (P<0.03) and Hb
(P<0.01) as compared with their own day 30 values.

At day 30 post-therapy, the dogs supplemented with Pyodemacare-G found to have


significantly higher Hb (P<0.01), HCT (P<0.004) and MCH (P<0.01) as compared with
day 30 post-therapy values of non-supplemented dogs (Group 1). In tandem, at day 60
post-therapy the dogs supplemented with Pyodemacare-G found to have significantly
higher TEC (P<0.003), Hb (P<0.001), and MCH (P<0.004) as compared with day 30 post-
therapy values of non-supplemented dogs (Group 1).

140
Table 7:- Effect of Pyodermacare-G supplementation on leukogram of dogs with generalised demodicosis.

Panels Controls Pyodermacare-G supplemented


(Group 1; n=09) (Group 2; n=10 )

Day 0 Day 30 Day 60 Day 0 Day 30 Day 60

TLC (103/L) 27.262.64 24.102.9A 20.211.85A,B 24.401.9 11.570.64a, 7.670.37a,B,

Lymphocytes (103/L) 5.260.41 4.450.32A 4.540.40A,B 5.010.52 2.480.21a, 2.030.10a,B,

Monocytes (103/L) 0.970.14 0.680.07A 0.630.08A,B 1.060.16 0.680.14A 0.450.03a,B,

Granulocytes (103/L) 20.772.40 18.333.01A 14.661.52A,B 18.341.70 7.960.47a, 5.040.26a,B,

Lymphocytes (%) 20.072.02 20.361.60A 22.831.42A,B 20.711.71 23.081.41A 26.640.65a,B,

Monocytes (%) 3.710.45 2.980.26A 3.36021A,B 4.370.61 5.090.32A, 5.790.19b,B,

Neutrophils (%) 72.602.08 73.681.82A 70.811.57A,B 71.772.53 68.861.17A, 65.730.51b,c,

Eosinophils (%) 3.360.57 3.180.52A 3.870.71A,B 3.050.68 2.050.46A 1.890.24A,B,


A
Non-significant difference, when compared with day 0 values of the same group; BNon-significant difference, when compared with
day 30 values of the same group; aSignificant (P<0.01) difference, when compared with day 0 values of the same group; bSignificant
(P<0.05) difference, when compared with day 0 values of the same group; cSignificant (P<0.05) difference, when compared with day
30 values of the same group; Significant (P<0.01) difference, when compared with same day post-treatment values of Group 1;

Significant (P<0.05) difference, when compared with same day post-treatment values of Group 1; Non-significant difference, when
compared with day 0 values between the groups.

141
Table 8: Effect of Pyodermacare-G supplementation on haemogram of dogs with generalised demodicosis.

Panels Controls Pyodermacare-G supplemented


(Group 1; n=09) (Group 2; n=10 )

Day 0 Day 30 Day 60 Day 0 Day 30 Day 60

RBC (106/L) 5.120.5 5.650.36A, 6.200.29A,B 4.880.37 6.370.13a 7.330.16a,c,

HB (gm/dL) 9.250.96 9.940.82A 11.020.48A,B 8.70.60 12.140.15a, 13.790.24a,d,

HCT (%) 38.583.4 39.302.39A 42.902.20A,B 35.992.5 47.340.84a, 52.781.93a

MCV (fL) 71.931.5 70.261.3A 69.151.4A,B 75.581.92 75.512.34A 71.991.32A,B

MCH (pg) 19.61.18 16.740.75A 17.430.34A,B 17.370.5 19.080.41a, 18.790.23b,

MCHC (g/dL) 24.850.86 24.351.0A 25.760.46A,B 24.010.29 25.620.27b 26.260.66a

Platelets (103/L) 44291 27622A 31627A,B 31740 26917A 28821A,B

A
Non-significant difference, when compared with day 0 values of the same group; BNon-significant difference, when compared with
day 30 values of the same group; aSignificant (P<0.01) difference, when compared with day 0 values of the same group; bSignificant
(P<0.05) difference, when compared with day 0 values of the same group; cSignificant (P<0.05) difference, when compared with day
30 values of the same group; dSignificant (P<0.01) difference, when compared with day 30 values of the same group; Significant
(P<0.01) difference, when compared with same day post-treatment values of Group 1; Non-significant difference, when compared
with day 0 values between the groups.

142
4.4. 2. Effects on Serum biochemistry

The ameliorative potentials of Pyodermacare-G capsules on serum biochemical panels of


dogs with generalised demodicosis are depicted in Table 9. Demodicosed dogs of the
control group (Group 1) have not revealed remarkable alteration in most of the altered
serum biochemical panels including cholesterol, triglycerides, and albumin contents at day
30 and day 60 post-therapy as compared with their own day 0 values. However, significant
(P<0.05) amelioration in ALKP activity was recorded in this group at day 60 post-therapy
as compared with their own day 0 values. But remarkable reduction in serum ALKP
activity was not estimated in this group at day 30 post-therapy as compared with their own
day 0 values.

Whereas, the demodicosed dogs supplemented with Pyodermacare-G (Group 2) were


found to have significant reduction in total cholesterol level (P<0.05) on day 60 post-
therapy as compared with their own day 0 values. But at day 30 post-therapy, significant
amelioration in serum total cholesterol level was not revealed by the dogs of this group at
day therapy as compared with their own day 0 values. Moreover, significant increment in
total protein (P<0.001), albumin (P<0.001) and globulins (P<0.05) was revealed by the
dogs of this group at day 60 pot-therapy as compared with their own day 0 values.
Significant increment in total protein (P<0.05) and albumin (P<0.01) was also recorded at
day 30 post-therapy as compared with their own day 0 values. Additionally at day 60 post-
therapy, significant (P<0.05) increment in albumin content was also revealed by the dogs
of this group as compared with their own day 30 post-therapy values.

On day 30 post-therapy, the dogs supplemented with Pyodermacare-G were found to have
significantly (P<0.02) lower levels of total cholesterol and triglycerides as compared with
day 30 post-therapy values of the non-supplemented dogs (Group 1). Moreover at day 60
post-therapy, the dogs supplemented with Pyodemacare-G found to have significantly
lower levels of total cholesterol (P<0.02) and triglycerides (P<0.001) as compared with the
same day post-therapy values of non supplemented group (Group 1). Contrarily, the dogs
supplemented with Pyodemacare-G found to have significantly (P<0.03) higher albumin
level as compared with the day 60 post-therapy values of non-supplemented dogs (Group
1).

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Table 9: Effect of Pyodermacare-G supplementation on serum biochemistry of dogs with generalised demodicosis.

Panels Controls Pyodermacare-G supplemented


(Group 1; n=09) (Group 2; n=10 )
Day 0 Day 30 Day 60 Day 0 Day 30 Day 60
Glucose (mg/dL) 88.6712.54 76.886.76A 72.226.63A,B 65.903.42a 62.802.97a,A 73.103.54a,A,B
Cholesterol (mg/dL) 188.421.3 179.7716.6 A 172.7720.7A, 151.47.2a 136.28.1b,A 119.69.2b,C,B
B

Triglycerides (mg/dL) 170.519.4 157.3318.8A 148.015.6A,B 129.419.1a 10212.9b,A 77.909.6c,A,B


Total Proteins (g/dL) 6.820.32 7.250.33A 7.850.37A,B 6.930.34a 7.760.22a,C 8.560.14a,D,B
Albumin (g/dL) 2.110.07 2.220.10A 2.430.11A,B 2.040.05a 2.440.10a,D 2.800.11b,D,C
Globulins (g/dL) 4.70.31 5.020.32A 5.42037A,B 4.90.29a 5.310.19a,A 5.760.21a,C,B
Creatinine (mg/dL) 0.520.08 0.540.05A 0.590.06A,B 0.550.04a 0.490.05a,A 0.550.04a,A,B
Urea (mg/dL) 12.470.80 13.020.62A 12.931.02A,B 11.980.4a 13.100.86a,A 13.911.35a,A,B
Aspartate Aminotransferase 31.34.7 28.024.6A 29.413.9 A,B 26.343.18a 28.823.2a,A 24.832.2a,A,B
(AST) (u/L)
Alanine Aminotransferase (ALT) 31.25.1 24.413.2A 23.502.42A,B 25.113.3a 20.191.3a,A 18.751.5a,A,B
(u/L)
Alkaline Phosphatase (ALKP) 64.74.6 57.334.35A 48.04.4C, B 58.98.5a 47.14.4a,A,B 44.84.1a,A,B
(u/L)
A
Non-significant difference, when compared with day 0 values of the same group; BNon-significant difference, when compared with day 30 alues
of the same group; CSignificant (P<0.05) difference, when compared with day 0 values of the same group; DSignificant (P<0.01) difference,
when compared with day 0 values of the same group; a Non-significant difference, when compared with values of same days of treatment of
Group 1; bSignificant (P<0.05) difference, when compared with values of same days of treatment of Group 1; cSignificant (P<0.01) difference,
when compared with values of same days of treatment of Group 1.

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4.4.3. Effects on circulatory cytokines

Circulatory levels of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-) and


interferon-gamma (IFN-) were estimated at day 0 and day 60 post-therapy by using canine
specific ELISA kits (RAB0524-IL-10; RAB0526-TNF-; RAB0523-IFN-, Sigma-Aldrich,
USA) following the procedure as described by the manufacturer.

The effects of supplementation with Pyodermacare-G on circulatory cytokines levels


of dogs with generalised demodicosis are depicted in Table 10. The demodicosed dogs of
control group, that were not supplemented with Pyodermacare-G, found have significant
(P<0.02) reduction in IL-10 level at day 60 post-therapy as compared with their own day 0
values. Whereas remarkable amelioration in circulatory levels of TNF- and IFN- was not
revealed by the dogs of this group at day 60 post-therapy as compared with their own day 0
values.

The demodicosed dogs supplemented with Pyodermacare-G (Group 2) found have


significant (P<0.001) reduction in circulatory IL-10 and significant (P<0.001) increment in
IFN- levels at day 60 post-therapy as compared with their own day 0 values. In tandem,
remarkable reduction in circulatory level of TNF- was not revealed by this group at day 60
post-therapy as compared with their own day 0 values.

Noticeably at day 60 post-therapy, the demodicosed dogs supplemented with


Pyodermacare-G found to have significantly (P<0.001) lower circulatory IL-10 level as
compared with day 60 post-therapy values of control group (Group 1). Agreeably,
Pyodermacare-G supplemented dogs also revealed significantly higher circulatory TNF-
(P<0.02) and IFN- (P<0.001) levels as compared with the corresponding day values of
control dogs (Group 1).

145
Table 10: Effects of Pyodermacare-G supplementation on circulatory cytokines of dogs
with generalised demodicosis.

Estimated Cytokines Controls Pyodermacare-G


supplemented
(Group 1; n=09)
(Group 2; n=10 )

Day 0 Day 60 Day 0 Day 60

Interleukin-10 28.878.7 7.330.75A 25.974.3 3.130.02C,


(ng/mL)

Tumour necrosis factor- 153.0527.5 107.559.8B 154.4827.9 145.1511.3B,


(pg/mL)

Interferon- 3.830.08 3.840.09B 3.810.09 6.430.65C,


(ng/mL)
A
Significant (P<0.05) difference, when compared with day 0 values of the same group; BNon-
significant difference, when compared with day 0 values of the same group; C Significant
(P<0.01) difference, when compared with day 0 values of the same group; Non-significant
difference, when compared with day 0 values between the groups. Significant (P<0.01)
difference, when compared with day 60 values between the groups; Significant (P<0.05)
difference, when compared with day 60 values between the groups.

ii) Efficacy of Pyodermacare-G against bacterial and fungal pyoderma in dogs

Materials and Methods

Twelve dogs of different breed presented to TVCC with dermatological ailments were
included in this study. All the dogs had the history of having been treated previously with
antibiotics and/or antifungal drugs with mild clinical improvement and/or recurrence of the
clinical condition after cessation of antimicrobial therapy. Skin swabs (from clinical lesions)
and skin-scrapings were collected from each dog pre- and post-therapy. Swabs and scrapings
were subjected for bacterial and fungal culture. All diseased dogs were free of any ecto-
parasitic infestation. Seven dogs were found to have bacterial dermatitis and five dogs were
found to have the mycotic dermatitis. With the consent of the pet owners, approximately 5 ml

146
blood samples were collected by usual technique from each dog at pre- and post-therapy and
used for evaluating the immune status of the dogs.

All the dogs were treated with herbal capsule (Pyodermacare-G) at a dose regimen of
two capsules PO BID for 21-28 days. To evaluate the circulatory cytokines markers of
immune status e.g., interleukin-10 (IL-10), interferon-gamma (IFN-) and tumor necrosis
factor-alpha (TNF-) was estimated pre- and post-therapy following the manufacturers
protocol (Sigma-Aldrich, USA). Data generated has been summarized in Table 11

Table 11: Serum levels of different cytokines in dogs suffering with bacterial and fungal
pyoderma before and 28 days after treatment with Pyodermacare-G.

Cytokines Pre-therapy Post-therapy P value

IL-10 (ng/mL) 2.1320.27 0.5490.06* 0.0001


TNF- (pg/mL) 214.7917.08 115.882.36* 0.0001

IFN- (ng/mL) 3.830.042 4.2760.082* 0.0001


*significantly differ with pre-therapy values

Perusal of the data presented in Table 11 revealed:

Significant (P<0.0001) reduction in circulatory levels of immune-suppressive cytokine


(IL-10) and pro-inflammatory cytokine (TNF-) in dogs 28 days post-therapy as compared
with their own day 0 values.

Significant (P<0.0001) increase in circulatory level of IFN- level was recorded in these
dogs 28 days 28 post-therapy as compared to their own day 0 values.

Based on the above results, it is apparent that the Pyodermacare G herbal formulation has
excellent immune-modulatory potential. Immuno-modulatory response along with its
antibacterial and antimycotic activity helps the dogs in recovering from bacterial and mycotic
dermatitis. Further, all the dogs completely recovered by 28th day of therapy and none of the
pet-owners had reported for any recurrence of the clinical conditions during the follow-up
period of six month post-therapy. In addition, remarkable improvement in the appetite and
general activities of dogs was also reported by the pet owners along with Faster hair re-growth,
improved coat luster, remarkable reduction in dandruff and hair fall in all the treated dogs.

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Figures 3.1, 3.2 A and 3.2B illustrate the micrographs of skin scrapping showing various
stages of Demodex canis mites, adult Demodex mite and impression smear showing engulfed
bacterial cocci by WBCs respectively. Figures----- illustrate the photographs of dogs before their
treatment with Pyodermacare-G i.e. day 0, and 30 and 60 days post-treatments.

Immature

Adult
Egg

Fig. 3.1: Micrograph of skin scrapping showing various stages of Demodex canis mites

(A) (B)
Fig.3.2:- Micrograph of an adult Demodex mite (A) and impression smear showing engulfed
bacterial cocci by WBCs (B)

148
(A) (B)

(C)
Fig. 4: A 18-month-old male demodicosed German Shepherd dog of control group before therapy (A),
30 days post-therapy (B) and 60 days post-therapy (C) with Pyodermacare-G.

(A) (B)

(C)
Fig. 5: A 12-month-old female demodicosed Mongrel dog of control group before therapy (A), 30 days
post-therapy (B) and 60 days post-therapy (C) with Pyodermacare-G.

149
(A) (B)

(C)
Fig. 6: A 24-month-old female demodicosed German Shepherd dog before treatment (A),
30 days post-treatment (B) and 60 days post-treament (C) with Pyodermacare-G.

(A) (B)

(C)
Fig. 7: A 30-month-old female demodicosed Labrador Retriever dog before treatment (A), 30 days
posty-treatment (B) and 60 days post-treatment (C) with Pyodermacare-G.

150
(A) (B)

(C)

Fig. 8: Another view of a 30-month-old male demodicosed Labrador Retriever dog of before therapy
(A), 30 days post-treatment (B) and 60 days post-treatment with Pyodermacare-G.

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ADDITIONAL STUDIES

A. Studies on antiviral activity

1. Plant Material

Leaves of Polyalthia longifolia were collected from Veterinary College Campus, Mathura,
India. The identity of the plant material was confirmed by CSIR-National Botanical
Research Institute, Lucknow based on the taxonomic features of the whole plant including
leaves (Specimen accession No. LWG-71).

2. Extraction of Plant Material

Hot-methanolic extract of shade-dried and coarsely powdered leaves of Sarca indica was
prepared in soxhlet apparatus by hot percolation method. The extract was concentrated to
dryness using rotatory evaporator under reduced pressure and low temperature (<40C). The
extract was kept in air tight containers and stored at 4C for further studies.

3. Cells and viruses

Vero cells were grown in Dulbeccos Modifeid Eagles Medium (DMEM) supplemented
with 10% fetal bovine serum and antibiotics (penicillin and streptomycin). PPRV and NDV
available in our laboratory and described in our earlier publication (Kumar et al., 2016)
were used for evaluating the antiviral activity of test extract.

4. Determination of cytotoxicity (MTT assay)

Cytotoxicity of the test extract was assessed by employing the standard assay procedure
(Kumar et al., 2011). Vero cells in 96-well plates were treated with 3-fold serial dilutions of
the plant extract (starting from 4000 g/ml) or equal volume of methanol (vehicle control),
in triplicates, in a total of 100 l growth medium for 96 h. An amount of 20 l of the freshly
prepared 5 mg/ml MTT [3-(4, 5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium
bromide] solution was added to each well, and cells were incubated at 37C for 5 h. The
medium was removed and 200 l of DMSO was added to each well to dissolve the purple
formazan product and the plates were incubated at 37C for another 5 min to remove any air
bubbles. MTT signals were measured photometrically at an absorbance of 550 nm.

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As shown in Fig. 9a, no significant cytotoxicity was observed at concentrations 12.8
g/ml. Significant cyto-toxicity was observed at a concentration of 64 g/ml. Therefore,
in all the subsequent experiments in various in vitro assays, a sub-cytotoxic concentration of
10 g/ml was used

5. Determination of the antiviral activity

Vero cells were incubated with 10 g/ml of the plant extract or equal volume of methanol
(vehicle control) for 1 h and subsequently infected with PPRV/NDV at moi of 0.1 for 1 h at
37oC after which the cells were washed 3 times with phosphate buffer saline (PBS) and
replaced with fresh DMEM containing either vehicle-control or indicated concentration of
the plant extracts. Viral titers in the supernatant were determined by plaque assay on Vero
cells.
As shown in Fig. 9b, viral titers were comparable in the extract and vehicle control-treated
groups suggesting that extract has no virucidal effect on cell free virions (PPRV and NDV).
To determine the in vitro antiviral efficacy of the test extract, yields of infectious virions
were measured in the presence of the extract or vehicle-control. At non-cytotoxic
concentration (10 g/ml), test extract was found to significantly inhibit PPRV (Fig. 9c) and
NDV (Fig. 9d) replication suggesting its broad-spectrum antiviral efficacy against these
paramyxoviruses. Since the extract did not exhibit any virucidal activity even up to 100
g/ml (about 10-times the cytotoxic dose), possibility of the antiviral effect of the test
extract due to inhibition of the virus replication inside the host cell cannot be ruled out.

6. Determination of the virucidal activity

Virucidal activity of the plant extract was evaluated employing the standard experimental
procedure (Kumar et al., 2011). Briefly, virus suspensions containing approximately 106 pfu
of PPRV/NDV were incubated in serum-free medium containing either methanol of plant
extract (100 g/ml or 10 g/ml) for 1.5 h at 37C. The mixed samples were chilled at 4C
and diluted to 10-4, 10-5, and 10-6 with serum-free medium. These were applied onto Vero
cells in 6-well plates for 1h at 37oC followed by addition of agar overlay medium. Viral
titers were expressed in pfu/ml.

To evaluate the effect of test extract on specific step(s) of viral life cycle, a time-course
assay was performed, where extract was added at different times post-infection and the

153
resultant virus particles released into the supernatant were quantified by plaque assay. For
both PPRV (Fig. 10a) and NDV (Fig. 10b), highest inhibition was observed when the test
extract was applied before virus infection. Thereafter, the magnitude of inhibition gradually
declined, suggesting that multiple steps of viral life cycle may be affected by the test extract.
In virus attachment-specific assay, we did not observe any difference in the viral titers
between the extract and vehicle-control-treated cells in both NDV and PPRV , which
confirmed that the extract did not inhibit the attachment of virus to the host cells.

To determine if pre-attached virus was able to enter the cells in the presence of test extract,
standard entry assay was performed. As compared to the vehicle control-treated cells,
statistically significant reduction in the PPRV (Fig. 10c), and NDV (Fig. 10d) yield was
observed in the extract-treated cells which suggests that the extract inhibits the entry of
virus into the host cells.

In order to determine the effect of extract on synthesis of viral nucleic acid, it was applied to
the virus infected cells when early steps of virus life cycle (attachment/entry) were expected
to occur (>2h). No significant reduction in relative viral RNA/DNA copy number (extract-
treated versus vehicle-control) was observed in both PPRV and NDV infected cells
suggesting that extract did not inhibit the synthesis of viral RNA.

We also analyzed the effect of test extract on release of the progeny virus particles from
infected cells where extract was applied towards the end of the viral life cycle viz. 10 hpi
and 36 hpi respectively for NDV and PPRV (when virus is believed to be released from the
virus infected cells). As compared to the vehicle-control, treatment of cells with the extract
lead to reduction in both PPRV (Fig. 10e) and NDV (Fig. 10f) titers suggesting that the
extract inhibited viral budding (release) from the infected cells.

Summarily, our study evidently suggests that Polyalthia longifolia leaves methanolic extract
blocks virus replication at the level of viral entry and budding. As both these events of viral
replication cycle occur at the plasma membrane of the host, therefore, it is tempting to
speculate that Asoka leaves extract probably targets the host machinery involved in lipid
biogenesis, the integral components of plasma membrane (Abrescia et al., 2008). Asoka
leaves are endowed with several phytochemo molecules, namely- alkaloids, glycosides,
terpenoids, steroids, flavonoids, tannins, saponins, polyphenolics and carbohydrates (Singh

154
et al., 2015). But characterization of the active phytobiomolecule(s) responsible for antiviral
activity of the methanolic extract of Ashoka leaves in the present study remains elusive,
therefore, further studies on activity-guided purification of the active chemical moiety along
with the precise target(s) in the host are
warranted.

Fig. 9: (a) Cytotoxicity: Vero cells were treated with 5-fold serial dilutions of the plant
extracts or vehicle control for 96 h and the percentage cell viability was determined by MTT
assay.
(b) Virucidal activity: Virus suspensions containing approximately 106 pfu of virus were
incubated in serum-free medium containing either 10 or 100 g/ml of plant extract of equal
volume of vehicle control for 90 min at 37C. The mixed samples were chilled at 4C and
titrated by plaque assay on Vero cells.
(c) Antiviral efficacy: Vero cells were incubated with indicated concentrations of the extracts
or vehicle-control for 1 h. Subsequent infection with PPRV/NDV was carried out at a moi of
0.1 for 1 h followed by washing with PBS and addition of fresh media having plant extracts
or vehicle-control. The viral titers in the supernatant were determined by plaque assay on
Vero cells.

155
Fig. 10.
Time-of-addition assay: Vero cells were infected, in triplicates with indicated virus at moi of
5, washed with PBS followed by addition of fresh media containing either 10 g/ml of extract
or vehicle control. The infectious virus released in the infected cell culture supernatant at
indicated time points were quantified by plaque assay. Time-of-addition assays of PPRV (a)
and NDV (b) are shown.

Entry: Confluent monolayers of Vero cells were infected with indicated virus at moi of 5 in
extract-free medium for 1h at 4oC to permit attachment. Thereafter, the cells were washed
with chilled PBS to remove unattached virus and the fresh medium containing either extract
or vehicle-control were added. The entry was allowed to proceed at 37oC for 1 h after which
cells were washed again with PBS to remove any extracellular virus and incubated with the
cell culture media without extract. The cell culture supernatants were titrated for presence of
infections virus particles in extracted-treated and untreated cells. Viral entry assay for PPRV
(c) and NDV (d) is shown.

Virus release: Confluent monolayers of Vero cells were infected with indicated virus, in
triplicates, for 2h at moi of 5 followed by washing and addition of fresh media. At 36 hpi and
10 hpi respectively for PPRV and NDV, cells were washed 6 times with chilled PBS followed
by addition of fresh media containing extract or vehicle-control. PPRV (e) and NDV (f)
released in the supernatant at indicated time points were quantified by plaque assay. Error
bars indicate SD. Statistical analysis was conducted with Students t test (** = P<0.01).

156
Deliverables:
Publications: 1

Ruchi Tiwari, Shanker Kumar Singh, Soumen Choudhury and Satish Kumar Garg (2017).
Antifungal activity of methanolic extracts of leaves of Eucalyptus citriodora and Saraca
indica against fungal isolates from dermatological disorders in canines. International Journal
of Pharmacology, DOI: 10.3923/ijp.2017 (1-6) (NAAS Rating 6.753)

Budget Utilization:
Balance amount of Rs. 20,961/ has already been refunded to the Principal Coordinator
through demand draft No. 874730872 dated 25.05.2017 in favour of ICAR Unit, IVRI,
Izatnagar and acknowledgement received from the office of Principal Coordinator of the
project..

157
ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

PALAMPUR CENTRE

158
OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
PALAMPUR

ANNUAL REPORT (2016-2017)

1. Name of Centre: Palampur Centre

2. Name of Investigators: Dr(s). R.K. Asrani (Pathology), Subhash Verma, Dinesh


Sharma, Parveen Kumar Sharma and R.D. Patil

3. Approved mandates of the Centre:


To document the plants/herbs based on ethno-veterinary medicinal
practices of the state
To study the antibacterial/hepatoprotective activities of plant extracts

4. Salient achievement

S. Typhimurium found to be highly pathogenic and produced significant hepatic lesion


in the mice model during in vivo experimentation.
Saussurea lappa has shown good in vitro antimicrobial activity against S.
Typhimurium.
Addition of lyophilized methanolic extract of Saussurea lappa @ 2000mg per kg body
weight of drinking water was found safe as it did not produce any adverse effect on
growth, mortality pattern and various organs of the mice.
Saussurea lappa (@1000mg) showed promising hepatoprotective and
immunomodulatory effect of in mice experimentally infected with S. Typhimurium.

5. Concise progress report (01.04.2017 to 31.03.2017)

5.1 Survey pertaining to ethno-veterinary medicinal practices


Field surveys were conducted in different regions of Lahaul district and
information pertaining to ethnomedicinal plants was collected from local people and
traditional healers. Sixty four plants were documented and their specimens were submitted
to CSIR, IHBT, Palampur for identification. Arnebia euchroma, Artemisia maritima,
Taraxacum officinale, Picrorhiza kurrooa, Asparagus filicinus, Thymus linearis,
Podophyllum hexandrum, Angelica glauca, Rheum webbianum, Berginia stracheyi and
Origanum vulgare were the most important medicinal plants used for treating various
ailments.

159
5.2 Isolation of Salmonella enterica serovar Typhimurium
The strain of Salmonella enterica serovar was isolated by the Department
of Veterinary Microbiology COVAS, Palampur from an outbreak among colony of mice.
The isolate was confirmed as Salmonella enterica serovar Typhimurium 4,12:i:1,2 at
Central Research Institute, Kasauli. This serotype was subjected to antibiotic susceptibility
test (Table 1).
Table 1. Antibiotic susceptibility test for Salmonella Typhimurium

Antibiotic Zone of inhibition* (mm)


Ceftriaxone (CFX30) 13.50.28
Ciprofloxacin (CIP10) 25.80.44
Cefalexin (CN30) 22.00.71
Amikacin (AK30) 23.50.16
Chloramphenicol (C30) 23.00.28
Gentamicin (GEN10) 22.50.28
Amoxicillin (AMX30) 24.00.17
Ampicillin (AMP30) 11.00.00
Erythromycin (E10) 0.000.00
*Data represented as mean S.D.
Salmonella Typhimurium showed maximum susceptibility for Ciprofloxacin
followed by Amoxicillin, Amikacin and Chloramphenicol, whereas exhibited resistance
against Erythromycin.

5.3 Antibacterial activities of medicinal plant against Salmonella typhimurium


Plants screened for the assessment of in vitro antimicrobial activity against
Salmonella typhimurium is presented in Table 1. Extracts of the plant samples were
prepared in hexane, chloroform, ethanol, methanol and water (concentration 100 mg/ml
DMSO). The antibacterial activity of these extracts was determined against Salmonella
Typhimurium by Kirby-Bauer disc diffusion method.
The plant extracts were dissolved in DMSO to obtain a concentration of 100 mg/ml.
The antimicrobial activities of different extracts were estimated by measuring the diameter
of clear inhibition zone in millimeters. Discs impregnated with solvent were used as
negative control. Antibiotic discs (Enrofloxacin @ 10g/disc) served as positive control.
Table 2 represents the in vitro antibacterial activities of plant extracts against Salmonella
typhimurium. The methanolic extract of Saussurea lappa showed promising in vitro
antibacterial activity against Salmonella typhimurium with 14.0 mm zone of inhibition and
used further for the in vivo assessment of protective effects against Salmonella
Typhimurium infection in mice.

160
Table 2. In vitro antibacterial activity of various plant extracts against Salmonella
Typhimurium

Plant samples (s) Zone of inhibition (mm)


Hexane Chloroform Methanol Ethanol Aqueous
extract extract extract extract extract
Saussurea lappa 6.0 0 14.0 8.0 0
Ajuga parviflora 0 0 0 0 0
Rubus sp. 0 0 0 0 10.0
Plantago lanceolata 0 7.0 0 0 0
Calotropis procera 0 7.0 0 0 0
Zingiber chrysanthum 0 0 0 0 0
(rhizome)
Zingiber chrysanthum 8.0 8.0 0 0 0
(flowers)
Prunus cerasoides 0 0 0 0 0
Viola canascens 0 0 0 0 0
Pistacia integerrima 0 0 0 0 0
Tinospora cordifolia 0 0 0 0 0
Persicaria nepalensis 0 0 0 0 0
Grewiadi sperma 0 8.0 0 0 0
Valerian jatamansi 0 0 0 0 0
Solanum viarum 0 0 0 0 0

5.4 Preliminary phytochemical screening of methanolic extract of S. lappa

The methanolic extract of S. lappa was subjected to preliminary phytochemical


screening based on following tests:

Test for glycosides: To 1ml extract, 1ml concentrated sulphuric acid was added. The
mixture was allowed to stand for 2 min. Appearance of red color precipitates
confirmed the presence of glycosides in the extract.
Test for alkaloids (Wagners test): To 1ml extract, 1ml Wagners reagent (2g
iodine and 6g potassium iodide dissolved in 100 ml distilled water) was added. No
reddish brown precipitates were observed.
Test for flavonoids (Sodium hydroxide test): To 1ml extract 10% sodium
hydroxide was added. Appearance of yellow color confirmed the presence of
flavonoids in the extract.

161
Test for phenols (Ferric chloride test): To 2ml extract, few drops of 10% ferric
chloride were added. Emergence of blue green color confirmed the presence of
phenols in the extract.
Test for phlobatannins: To 1 ml extract, few drops of 2% HClwere added. The
mixture was boiled for few minutes. No red precipitates were observed.
Test for saponins (Froth test): About 2ml extract was shaked vigorously resulting
in the formation of froth confirming the presence of saponins.
Test for anthocyanins: To 2ml extract, 2ml HCl (2N) was added followed by 2ml
ammonia. No pinkish coloration was observed.
Test for anthraquinones: To 3ml extract, 3ml benzene and 5ml ammonia (10%)
was added. Appearance of pinkish violet or red coloration in ammonical layer
confirmed the presence of anthraquinones.
Test for sterols: To 2ml extract, 2ml chloroform was added followed by 2ml
concentrated sulphuric acid. Appearance of reddish brown ring at the junction
confirmed the presence of sterols.
The methanolic extract of S. lappawas found to be strongly positive for alkaloids,
flavonoids, phenols, phlobatannins and sterols; moderately positive for glycosides,
saponins and anthocyanins; negative for anthraquinones (Table 3).
Table 3. Phytochemical constituents of methanolic extract of S. lappa

Phytochemicals Methanolic extract


(100 mg/ml)
Glycosides +
Alkaloids ++
Flavonoids ++
Phenols ++
Phlobatannins ++
Saponins +
Anthocyanins +
Anthraquinones -
Sterols ++
*(++) Strongly positive; (+) Moderately positive; (-) Negative

5.5 Assessment of in vivo antibacterial activity of methanolic extract of S. lappa


against Salmonella typhimurium in mice
5.5.1 Pilot experiment to determine LD50 of Salmonella typhimurium
Adult Albino mice weighed around 25-35g were procured from Institute of Himalayan
Bioresource Technology, Palampur (H.P.) and acclimatized u pto 7 days under strict
hygienic conditions. The mice were randomly divided into 7 groups, comprising of 4 mice

162
in each group. The animals were orally gavaged by gastric intubation using specialized
gavage needles with appropriate length on 7th day. The mice in first six groups were
infected with 0.5 ml of the normal saline solution containing different concentrations of
the organism (5x107, 5x106, 5x105, 5x104, 5x103 and 5x102 cfu ml-1 respectively). The
seventh group served as control with no infection. Duration of the pilot experiment was 14
days. Peak mortality was achieved at 7th day post infection. LD50 dose was then calculated
by the method of Reed and Muench (1938). The dose was found to be 1.35x103 cfuml-1,
which was used in the final experiment.
5.5.2 Final experiment

For final experiment, adult albino mice (200 No.) were procured from Central
Research Institute Kasauli, (H.P.) and Institute of Himalayan Bioresource technology,
Palampur (H.P.) and reared under strict hygienic conditions. The mice were randomly
divided into different groups given below.

Various treatments in different groups of mice

Group(s) Treatment(s) Dosing level of infection + No. of


methanolic extract mice
Group 1 Control group 0+0 20
Group 2 Methanolic extract of S. lappa 2000 mg extract /kg body 30
weight
Group 3 S. Typhimurium infection 1.35 x 103cfu /ml 30
Group 4 S. Typhimurium infection + 1.35 103cfu/ml +100mg/kg 30
Ciprofloxacin body weight
Group 5 S. Typhimurium infection 1.35 103cfu/ml + 30
+Methanolic extract of S. 200mg/kg body weight
lappa
Group 6 S. Typhimurium infection 1.35 103cfu/ml + 1000 30
+Methanolic extract of S. mg/kg body weight
lappa
Group 7 S. Typhimurium infection 1.35 103cfu/ml + 2000 30
+Methanolic extract of S. mg/kg body weight
lappa
The infection was given to mice after 7th day of acclimatization period and was
considered as zero-day post infection. The mice were closely observed up to14 DPI.

Statistical analysis: Data pertaining to body weight, biochemical analysis, gross lesion
scoring and microscopic lesion scoring was subjected to ANOVA by the SPSS statistical
program and the means were compared by Kruskal-Walis Test (P 0.05).

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5.5.2.1 Clinical signs and mortality pattern

The animals in groups 1, 2 and 4 (plain control, plant extract and standard drug
control, respectively) were completely healthy and normal throughout the experiment.
Clinical signs were mainly apparent in mice of infected groups 3, 5, 6 and 7. At day 1 post-
infection, less feed and water intake were observed in the mice of group 3 and the severity
of clinical signs increased with progression of the disease. Around 3rd day, most of the mice
were dull and depressed in group 3 along with other clinical signs such as ruffled hair coat,
laboured breathing, staggering gait, hunched position, soiled anal area, soft faeces and were
reluctant to move and often exhibited discharge from the eyes.
The clinical signs were less severe in the mice of infected groups given methanolic
extract of S. lappa, i.e., in group(s) 5, 6 and 7. Majority of mice showed clinical signs like
ruffled fur, shallow abdominal breathing, reduced feed and water intake and soft faeces as
early as 3 DPI. After 7 DPI, these signs dramatically declined, though were observed
occasionally up to 10 DPI. From 10 DPI onwards, mice were appeared normal in all the
three S. lappa given infected groups.

No mortality was observed in mice of groups 1 and 2 (plain control and plant extract
alone, respectively) throughout the experiment. Mortality was recorded in the infected
group(s), i.e. group(s) 3 to 7 during first week post infection. The overall mortality in the
infected group(s) 3, 4, 5, 6 and 7 was 33.33%, 3.33%, 3.33%, 6.66% and 16.66%,
respectively (Table 4). The mortality in the infected groups was observed as early as 2 DPI
reaching its peak at 7 DPI. Afterwards, no mortality was observed in any of the infected
groups.

Table 4. Mortality pattern in different groups of mice

DPI Number of mice died at different intervals


Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7
1 0 0 0 0 0 0 0
2 0 0 1 0 0 0 0
3 0 0 2 0 1 0 0
4 0 0 1 0 0 1 2
5 0 0 1 1 0 0 0
6 0 0 1 0 0 1 2
7 0 0 4 0 0 0 1
Total 0 0 10 1 1 2 5
*Group 1: Control; Group 2: Plant extract @ 2000mg; Group 3: S. Typhimurium infection; Group 4: S. Typhimurium
infection + ciprofloxacin @ 200 mg; Group 5: S. Typhimurium infection + plant extract @ 200 mg; Group 6: S.
Typhimurium infection + plant extract @ 1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

164
5.5.2.2 Growth response

The mean body weight in different groups of mice at different DPI is presented in
Table 5. The difference in mean body weight between different infected groups was not
found at any stage of the experiment

Table 5. Effect of methanolic extract of S. lappa on body weight (g) of mice infected
with S. Typhimurium
DPI Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

0 30.800.49ab 31.800.90a 30.000.02b 30.400.40b 30.000.04b 30.000.01b 30.000.05b

3 31.001.18a 30.000.63a 29.200.49bc 30.000.05ab 30.000.05ab 29.200.49bc 28.000.03c

7 35.600.60a 31.001.18bc 31.001.18bc 32.401.12b 31.001.00bc 30.000.03bc 28.800.49c

14 34.001.00a 31.001.18b 30.000.63b 31.601.56ab 31.001.00b 30.000.05b 29.200.49b

*Group 1: Control; Group 2: Plant extract @ 2000mg; Group 3: S. Typhimurium infection; Group 4: S. Typhimurium
infection + ciprofloxacin @ 200 mg; Group 5: S. Typhimurium infection + plant extract @ 200 mg; Group 6: S.
Typhimurium infection + plant extract @ 1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

5.5.2.3 Serum biochemicals

Three mice were selected randomly from each group, weighed and blood was
collected from posterior vena cava after giving anesthesia with diethyl ether in a closed
chamber before sacrifice at 3, 7, 10 and 14 DPI for estimation of serum biochemicals, viz.
alanine aminotransferase (ALT), creatinine, total protein and albumin. Estimation was
done using diagnostic kits (Agappe diagnostics Ltd.) in a Semi-Automatic Biochemistry
Analyser (Model AGAPPE MISPA neo) according to the manufacturers instructions.

5.5.2.3.1 Alanine aminotransferase (ALT)

Mean serum ALT activity was observed to be highest in group 3 (S. typhimurium
infection alone) as compared to other infected group(s) 4, 5, 6 and 7 at all the intervals of
the experiment.

At 3 DPI and 7DPI a significant increase in the values of ALT was observed in
groups 3, 4, 5, 6 and 7 (Table 6). At 14 DPI, the ALT values returned to normal range in
the infected groups 6 and 7 with no significant difference in the values in comparison to
control groups 1 and 2. ALT activity of group 2 was at par with group 1 showing no adverse
effect of S. lappa extract on the liver.

165
Table 6. Effect of methanolic extract of Saussurea lappa on serum ALT activity (U/l)
in mice infected with S. typhimurium
DPI Group1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

d d a b b bc cd
3 76.182.49 82.217.05 269.1931.72 167.5418.50 184.8329.0 142.3319.31 100.7711.54
d d a b b bc cd
7 76.421.88 92.907.27 269.5242.62 149.2217.19 183.4384.3 118.4416.40 101.3310.07
d d a b b d d
14 79.454.60 73.713.21 193.2950.15 130.2511.22 144.31.7 95.6 0.96 85.336.45
*Group 1: Control; Group 2: Plant extract @ 2000mg; Group 3: S. Typhimurium infection; Group 4: S. Typhimurium
infection + ciprofloxacin @ 200 mg; Group 5: S. Typhimurium infection + plant extract @ 200 mg; Group 6: S.
Typhimurium infection + plant extract @ 1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

5.5.2.3.2 Creatinine, total protein and albumin


Creatinine content was observed to be significantly higher in all the infected group
(s) 3, 4, 5, 6 and 7 as compared to control group(s) 1 and 2 (plain control and plant extract
only, respectively) at all the intervals of the experiment (Table 7).
Total serum protein content was found to be statistically non-significant among all
the group(s) at almost all the intervals of the experiment (Table 8).
There was a significant decrease in the albumin content in all infected group (s) 3, 4,
5, 6 and 7 as compared to control group(s) 1 and 2 (plain control and plant extract only,
respectively) at 3 DPI (Table 9).
Table 7. Effect of methanolic extract of S. lappa on serum creatinine level (mg/dl) in
mice infected with S. typhimurium

DPI Group1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7


3 c c a ab a a a
0.260.02 0.260.03 0.460.01 0.390.15 0.450.03 0.410.03 0.460.04
7 c c a b ab b a
0.270.01 0.270.03 0.460.05 0.350.02 0.370.05 0.350.02 0.400.01
c c a ab c ab
14 0.300.01 0.260.02 0.470.00 0.430.00 0.370.04 0.310.01 0.390.01
a

*Group 1: Control; Group 2: Plant extract @ 2000mg; Group 3: S. Typhimurium infection; Group 4: S. Typhimurium
infection + ciprofloxacin @ 200 mg; Group 5: S. Typhimurium infection + plant extract @ 200 mg; Group 6: S.
Typhimurium infection + plant extract @ 1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

Table 8. Effect of methanolic extract of S. lappa on total serum protein concentration


(g/dl) in mice infected with S. typhimurium

DPI Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7


a a a a a a a
3 3.901.82 4.000.63 3.150.12 3.230.50 3.51+0.16 3.300.05 3.260.15
a a a a a a a
7 3.790.79 3.690.36 3.701.25 6.561.16 4.620.72 4.620.72 5.230.53
a a a a a a a
14 3.940.45 3.990.37 4.580.02 4.110.44 4.020.26 4.020.26 4.340.14
*Group 1: Control; Group 2: Plant extract @ 2000mg; Group 3: S. Typhimurium infection; Group 4: S. Typhimurium
infection + ciprofloxacin @ 200 mg; Group 5: S. Typhimurium infection + plant extract @ 200 mg; Group 6: S.
Typhimurium infection + plant extract @ 1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

166
Table 9. Effect of methanolic extract of S. lappa on serum albumin level (g/dl) in
mice infected with S. typhimurium

DPI Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7


3 a ab
1.750.11c c c c c
2.590.47 2.270.03 1.730.07 1.611.19 1.760.07 1.680.06
7 ab bc bc bc bc c c
2.280.10 2.170.13 2.010.09 2.130.08 2.040.07 1.770.07 1.720.04
14 bc bc ab a a ab a
2.190.03 2.170.07 2.200.07 2.380.03 2.310.03 2.250.05 2.320.02
*Group 1: Control; Group 2: Plant extract @ 2000mg; Group 3: S. Typhimurium infection; Group 4: S. Typhimurium
infection + ciprofloxacin @ 200 mg; Group 5: S. Typhimurium infection + plant extract @ 200 mg; Group 6: S.
Typhimurium infection + plant extract @ 1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.
5.5.2.3 Pathological changes
5.5.2.3.1 Gross pathological changes
Group 1 and 2 (Control groups):
No significant gross lesions were observed in the liver, spleen and other organs of the mice,
when sacrificed at different intervals. It may be concluded that addition of S. lappa
methanolic extract @ 2000mg/kg did not induce any significant gross lesion in any of the
organs.
Group 3
Liver:
Hepatomegaly was a consistent gross observation in group 3 given S. typhimurium
infection alone. The mild hepatomegaly was grossly visible as early as 3 DPI (Plate-4A).
It was more pronounced with the progression of disease up to 7 DPI and thereafter a
declining trend was recorded in the intensity of the lesion and number of mice showing the
lesion. At 7 DPI, hepatomegaly was characterized by marked enlargement of the liver with
rounding of its edges (Plate-5A). Total gross lesion score and mean lesion score for
hepatomegaly in group 3 given S. typhimurium infection alone indicated in Table 12 and
13. Hepatomegaly was significantly (P 0.05) comparable to other infected groups 4, 5, 6
and 7 at 3 and 7 DPI (Table 11). These findings were clearly supported by histopathological
study, which showed congested blood vessels, dilated sinusoids and inflammatory cell
infiltration in the liver parenchyma. Necrotic foci over the liver parenchyma were a
consistent gross observation in this group. The lesions were visible as early as 3 DPI, which
included severe necrotic foci (Plate-4A). Necrotic foci were more pronounced with the
progression of disease up to 7 DPI and thereafter, a declining trend was recorded in the
intensity of the lesion and number of mice showing the lesion. At 3DPI and 7 DPI, hepatic
necrosis was characterized by multifocal to coalescing, creamish to off white coloured foci
of variable size and almost covered the caudo-dorsal side of liver (Plate-4A, 5A).

167
Total gross lesion score and mean lesion score indicated in Table 12. Necrotic foci
were significantly higher in group 3 (P 0.05%) as compared to other infected groups 4,
5, 6 and 7 at 3 and 7 DPI (Table 13). These findings were clearly supported by
histopathological study, which showed multifocal to coalescing areas of necrosis,
infiltrated with inflammatory cells in the liver parenchyma. Liver paleness was a consistent
gross observation in this group at 3 and 7 DPI. It was visible as early as 3DPI, which
included moderate liver paleness (Plate-5A). It was less pronounced with the progression
of disease up to 7 DPI, thereafter appeared to decline, and almost returned to normal colour
at 14 DPI. At 3 and 7 DPI majority of mice showed mild to moderate pale discolouration
(Plate- 4A, 5A). Total gross lesion intensity and mean lesion score of liver paleness
indicated in Table 14 and mean lesion score tabulated in Table 15). Liver paleness may be
probably due to the hepatocellular damage produced by the invading organism. Although
liver paleness was, non-significantly comparable in this group (P 0.05) as compared to
other infected groups 4, 5, 6 and 7 at different intervals (Table 15).
Spleen
Splenomegaly was a consistent gross observation in this group given S. typhimurium
infection alone. Spleen was moderately congested and swollen at 3 DPI (Plate- 4A). But
in subsequent DPIs mild to moderate splenomegaly was observed. There was no significant
difference observed in splenomegaly except at 7 DPI, where higher values were obtained
in group 3 as compared to other groups ((Plate- 5A). Total gross lesion score was highest
in this group at 7 DPI (Table 10).
Table 10. Hepatomegaly indicated by total gross lesion score in different groups of mice infected with
S. typhimurium at different intervals

Hepatomegaly
Time interval Group Group Group Group Group Group Group
1 2 3 4 5 6 7
3 DPI 0 0 3 12 3 0 3
7 DPI 0 0 6 0 4 4 1
10 DPI 0 0 0 3 2 2 0
14 DPI 0 0 0 0 0 0 0
Grand total score 0 0 9 15 9 6 4
*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract@2000 mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg; Group 5: S. Typhimurium infection +
plant extract@200 mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant
extract @ 2000 mg.

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Table 11. Hepatomegaly indicated by mean lesion score in different groups of mice infected with S.
typhimurium at different intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
3 DPI 0.00 0.00d 0.00 0.00d 0.75 0.00cd 3.00 0.00a 0.75 0.70cd 0.00 0.00 0.750.25cd

7 DPI 0.00 0.00d 0.00 0.00d 1.50 0.86b 0.00 0.00d 1.00 0.70bc 1.00 0.57bc 0.250.15d

10 DPI 0.00 0.00d 0.00 0.00d 0.00 0.00d 0.750.75cd 0.50 0.50cd 0.50 0.50cd 0.500.25cd

14 DPI 0.00 0.00 d 0.00 0.00 d 0.00 0.00 d 0.00 0.00d 0.00 0.00d 0.00 0.00d 0.000.00d

*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium
infection alone; G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6:
S. Typhimurium infection+ plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Table 12. Necrotic foci indicated by total gross lesion score in different groups of mice infected with S.
typhimurium at different intervals
Necrotic foci

Time interval Group Group Group Group Group Group Group


1 2 3 4 5 6 7
3 DPI 0 0 8 0 0 0 0
7 DPI 0 0 12 0 1 1 1
10 DPI 0 0 0 2 1 0 0
14 DPI 0 0 0 0 0 0 0

Grand total score 0 0 20 2 2 1 1


*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group 2: plant extract@2000 mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg; Group 5: S. Typhimurium infection +
plant extract@200 mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant
extract @ 2000 mg.
Table 13. Necrotic foci indicated by mean lesion score in different groups of mice infected with S.
typhimurium at different intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d ab d d d
3 DPI 0.00 0.00 0.00 0.00 2.00 0.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00d

7 DPI 0.00 0.00d 0.00 0.00d 3.00 0.90a 0.00 0.00d 0.25 0.25d 0.50 0.50c 0.250.25d

10 DPI 0.00 0.00d 0.000.00d 0.000.00d 0.500.50c 0.25 0.25d 0.50 0.50c 0.000.00d

14 DPI 0.00 0.00d 0.00 0.00d 0.00 0.00d 0.00 0.00d 0.00 0.00d 0.00 0.00d 0.00 0.00d
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium
infection alone; G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200mg; G6:
S. Typhimurium infection+ plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Table 14. Liver paleness indicated by total gross lesion score in different groups of mice infected with
S. typhimurium at different intervals

169
Liver paleness
Time interval G1 G2 G3 G4 G5 G6 G7
3 DPI 0 0 4 8 8 0 8
7 DPI 0 2 3 0 1 1 2
10 DPI 0 0 0 0 0 0 2
14 DPI 0 0 0 0 0 0 0

Grand total score 0 2 7 8 9 1 12


*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract @2000 mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg; Group 5: S. Typhimurium infection +
plant extract @200 mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant
extract @ 2000 mg.

Table 15. Liver paleness indicated by mean lesion score in different groups of mice infected with S.
typhimurium at different intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
c c ab a a c
3 DPI 0.00 0.00 0.00 0.00 1.00 1.00 2.00 2.00 2.00 2.00 0.00 0.00 2.002.00a

7 DPI 0.000.00c 0.000.00c 0.75 0.75ab 0.000.00c 0.25 0.25c 0.250.25a 0.50 0.50bc

10 DPI 0.00 0.00c 0.00 0.00c 0.00 0.00c 0.00 0.00c 0.00 0.00a 0.000.00c 0.50 0.50bc

14 DPI 0.00 0.00c 0.00 0.00c 0.00 0.00c 0.00 0.00c 0.00 0.00c 0.00 0.00c 0.00 0.00c
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium
infection alone; G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6:
S. Typhimurium infection+ plant extract@1000 mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Table 16. Splenomegaly indicated by total gross lesion in different groups of mice infected with S.
typhimurium at different intervals

Splenomegaly

Time interval Group Group Group Group Group Group Group


1 2 3 4 5 6 7
3 DPI 0 0 4 3 3 0 8

7DPI 0 0 4 0 3 2 2

10 DPI 0 0 0 1 3 0 4

14 DPI 0 0 0 0 0 0 0

Grand total score 0 0 8 4 9 2 14

*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract@2000mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S.Typhimurium infection +
plant extract@200mg; Group 6: S. Typhimurium infection + plant extract @1000mg; Group 7: S. Typhimurium infection + plant
extract @ 2000mg.

Table 17. Splenomegaly indicated by mean lesion score in different groups of mice infected with S.
typhimurium at different intervals

DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7

170
3 DPI 0.00 0.00a 0.00 0.00a 1.00 1.00bc 0.75 0.75a 0.75 0.75a 0.00 0.00a 2.000.10b
7 DPI 0.00 0.00a 0.00 0.00a 1.00 0.25bc 0.00 0.00a 0.75 0.25a 0.50 0.15a 0.50 0.12a
a a a a a a
10 DPI 0.00 0.00 0.00 0.00 0.00 0.00 0.75 0.25 0.25 0.25 0.00 0.00 1.000.40bc
14 DPI 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.000.00a
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium
infection alone; G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6:
S. Typhimurium infection+ plant extract @1000 mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Group 4
Liver:
Hepatomegaly was a consistent gross observation in this group given S. typhimurium
infection along with Ciprofloxacin @ 200mg. The lesion was visible as early as 3 DPI,
which included the severe hepatomegaly. It was less pronounced with the progression of
disease up to 10 DPI and thereafter a declining trend was recorded in the intensity of the
lesions and numbers of mice showing the lesions. At 3 and 7 DPI hepatomegaly was
characterized by marked enlargement with rounding of its edges and occupied almost
whole of the abdominal cavity (Plate-4B, 6C). Total gross lesion score and mean lesion
score for hepatomegaly indicated in Table 10 and 11). Hepatomegaly was significantly (P
0.05) higher in this infected group (Ciprofloxacin @ 200 mg/kg) to other infected groups
3, 5, 6 and 7 at 3 DPI (Table 11). These findings were clearly supported by
histopathological study, which showed severely congested blood vessels, dilated sinusoids
and inflammatory cell infiltration in the liver parenchyma. Necrotic foci were not a
consistent gross finding in this group 4. The lesion was visible at later stage at 10 DPI,
which included occasional focal pinpoint necrotic foci in two animals. The necrotic foci
were not grossly present at the early stages, this may be due to the bactericidal action of
the Ciprofloxacin but the prolonged use of the Ciprofloxacin also showed multifocal areas
of pinpoint necrosis and inflammatory cells infiltration in the liver parenchyma on
histopathology. Total gross lesions intensity and mean lesion score was comparable to the
control groups 1 and 2 (plain control and plant control @ 2000 mg/kg) indicated in Table
12 and 13. Liver paleness was not a consistent gross lesion in this group except at 3 DPI.
The lesion was visible as early as 3 DPI, which included moderate liver paleness. This may
be probably due to the endotoxin and hepatocellular damage produced by the organism
and increased burden of Ciprofloxacin metabolism over liver at 3 DPI. Total gross lesions
intensity and mean lesion score for liver paleness indicated in Table 14 and 15. Although
the liver paleness was non-significant in this group when compared to the other infected
groups 3, 5, 6 and 7 (P 0.05) at 3 DPI (Table 15).
Spleen

171
Splenomegaly was a non-consistent gross lesion in this infected group given Ciprofloxacin
@ 200 mg/kg. Spleen was severely congested and swollen at 3 DPI (Plate- 4B).But at 10
DPI severe splenomegaly was also observed in one animal (Plate- 6C). There was no
significant difference observed in splenomegaly except at 10 DPI. Total gross lesion score
and mean lesion score was more or less comparable to the control groups 1 and 2 (plain
control and plant control @ 2000 mg/kg) indicated in Table 16 and 17.
Group 5
Liver:
Hepatomegaly was a consistent gross lesion in this infected group given plant extract
@200mg/kg (Table 10 and 11). The lesion was visible as early as 3 DPI, which included
mild hepatomegaly. Hepatomegaly was more pronounced with the progression of disease
up to 7 DPI and thereafter a declining trend was recorded in the intensity of the lesions and
numbers of mice showing the lesions. At 7 DPI, severe hepatomegaly was observed in one
mice, characterized by severe enlargement along with rounding of its edges, which
occupied the almost whole of the abdominal cavity . After 7 DPI, lesion appeared to be
decrease in intensity and return to normal size at 14 DPI. These findings were clearly
supported by histopathological study. Total gross lesions intensity and mean lesion score
was more or less similar to the plain infection group (Table 10 and 11). Hepatic necrosis
was a consistent gross lesion in this infected group given plant extract @ 200 mg/kg. The
lesions were grossly visible at 7 DPI, which included mild necrotic foci in one animal and
characterized by focal pinpoint, creamish to off white coloured foci of variable size and
present on the dorsal lobe of liver parenchyma. Total gross lesions intensity and mean
lesion score was comparable to the other infected groups 3, 4, 6 and 7 indicated in Table
12 and 13. Intensity and number of mice showed necrotic foci was less as compared to
infection group 3 (S. typhimurium infection alone). Liver paleness was a consistent gross
lesion in this infected group given plant extract @ 200 mg/kg. The lesions were grossly
visible as early as 3 DPI, which included mild liver paleness (Plate-4C). Liver paleness
was less pronounced with the progression of disease up to 7 DPI and thereafter returned to
normal colour at 10 DPI. Majority of mice showed mild level of pale discolouration at 3
DPI to 7 DPI. Total gross lesions intensity and mean lesion score indicated in Table 14 and
mean lesion score tabulated in Table 15. Although liver paleness showed non-significant
difference statistically when compared to other infected groups 3, 4, 6 and 7 (P 0.05) at
different intervals (Table 15).
Spleen

172
Splenomegaly was a consistent gross lesion in this infected group given plant extract
@ 200mg/kg. Spleen was mildly congested and swollen at 3 DPI. But in 7 DPI severe
splenomegaly was observed in one animal (Plate-5C). Total gross lesion score intensity
and mean lesion score indicated in Table 16 and mean lesion score tabulated in Table 17).
There was no significant difference (P 0.05) observed in splenomegaly except at 7 DPI
(Table 17).
Group 6
Liver:
Hepatomegaly was a consistent gross lesion in this infected group given plant extract
@1000 mg /kg. The lesion was visible at 7 DPI, which included moderate hepatomegaly,
characterized by marked enlargement along with rounding of its edges . After 10 DPI,
lesion appeared to decrease and return to normal size at 14 DPI. Total gross lesions
intensity and mean lesion score indicated in Table 10 and 11. The intensity of
hepatomegaly was less as compared to the plain infection group and other infected groups
4, 5 and 7. These findings were clearly supported by histopathological study, which
showed organized hepatic parenchyma in this group at almost all the stages. Necrotic foci
was not a consistent gross lesion in this group given plant extract @ 1000 mg/kg. The
lesions were visible at 7 DPI, which included severe necrotic foci in one animal (Plate-
5D). At 7 DPI, where liver showed focal pinpoint, creamish to off white coloured foci of
variable size and on the dorsal side of liver parenchyma. Total gross lesions intensity and
mean lesion score indicated in Table 12 and 13. These findings were clearly supported by
histopathological study, which showed occasional focal areas of pin-point necrosis.

173
Infiltrated with inflammatory cells in the liver parenchyma. Liver paleness was not a
consistent gross lesion in this infected group (plant extract @ 1000 mg/kg) as it was present
only at 7 DPI (Table 10 and 11). The lesion was visible at 7 DPI, which included mild to
moderate liver paleness. Liver paleness was less pronounced with the progression of disease
up to 7 DPI and thereafter returned to the normal at 10 DPI. Total gross lesions intensity and
mean lesion score indicated in Table 16 and 17. However, liver paleness showed non-
significant difference when compared to the other infected groups 3, 4, 5 and 7.
Spleen
Splenomegaly was a non-consistent gross lesion in this infected group given plant
extract @ 1000mg/kg (Table 10 and 11). Spleen was mildly congested and swollen at 7 DPI
and no lesion was observed at any DPI. Total gross lesion score intensity and mean lesion
score was more or less comparable to the control groups 1 and 2 (plain control and plant
control) indicated in Table 18 and mean lesion score tabulated in Table 19. There was no
significant difference observed in splenomegaly except at 7 DPI (Table 19).
Group 7
Liver:
Hepatomegaly was a consistent gross lesion in this infected group given plant extract
@ 2000 mg/kg (Table 10 and 11). The lesions were grossly visible at 3 DPI, which included
mild hepatomegaly (Plate-4E). At 7 DPI, hepatomegaly was characterized by marked
enlargement in size along with its round edges. After 7 DPI, lesions appeared to decrease and
return to normal size at 10 DPI. Total gross lesions intensity and mean lesion score indicated
in Table 12 and 13. The intensity of hepatomegaly was less as compared to the control groups
1 and 2 (plain control and plant control) and other infected groups 3, 4, 5, 6 and 7 (Table 13),
this may be probably due to hepatoprotective effect of the plant. These findings were clearly
supported by histopathological study, which showed intact hepatic cord structure and hepatic
parenchyma in this group at almost all the stages. Hepatic necrosis was not a consistent gross
lesion in this infected group given plant extract @ 2000 mg/kg (Table 10 and 11). The lesion
was visible at 7 DPI, which included moderate necrotic foci in one animal. Total gross lesions
intensity and mean lesion score indicated in Table 14 and 15. Intensity and number of mice
showed necrotic foci was very less as compared to infection group given S. Typhimurium
alone (Table 10 and 11). These findings were clearly supported by histopathological study,
which showed occasional focal areas of pinpoint necrosis, occasionally infiltrated with
inflammatory cells in the liver parenchyma with intact hepatic cord structure. Liver paleness
was a consistent gross lesion in this infected group given plant extract @ 2000mg/kg (Table

174
10 and 11). The lesion was grossly visible as early as at 3 DPI, which included moderate liver
paleness. Total gross lesions intensity and mean lesion score was highest in this group as
compared to other infected groups 3, 4, 5, 6 and 7 (Table 16 and 17). Liver paleness was less
pronounced with the progression of disease up to 10 DPI and thereafter a declining trend was
recorded in the intensity of the lesions and numbers of mice showing the lesions (Table 17).
Spleen
Splenomegaly was a consistent gross lesion in this group given plant extract @ 2000
mg/kg Table 9 and 10. Spleen was moderately congested and swollen at 3 DPI. Total gross
lesion score intensity and mean lesion score was highest among all groups (Table18 and 19).
There was a significant difference observed in splenomegaly up to at 10 DPI (Table 19).

5.6 Histopathological changes


Liver
Group 1 and 2: No major change was observed in the liver of control mice except for the
mild vacuolar changes at 14 DPI. Liver was normal with well-maintained hepatic cord
structure and hepatocytes. There was no evidence of necrotic foci or neutrophilic infiltration
at any time interval during the experiment. At 14 DPI, mild mononuclear infiltration around
bile duct was found in group 2.
Group 3: The detailed microscopic lesions in the liver tissues of infected group along with
their lesion score and intensity are presented in Table 16 and 17. In the liver, focal areas of
necrosis were, distributed throughout the hepatic parenchyma, they were variable in size small
as pin head, evident as early as 3 DPI. Multifocal areas of necrosis were infiltrated with
neutrophils and lymphocytes. In addition to this karyorhexis and karyomegaly was evident at
places of necrosis along with pyknotic hepatic nuclei. Severe sinusoidal dilatation and
congestion was evident in three animals. Hepatocytes were markedly swollen with increased
cytoplasmic granularity. Portal, periportal area and bile ducts were infiltrated with neutrophils
and lymphocytes in one mice as early as 3 DPI.

Table 18. Necrotic foci indicated by total microscopic lesion score of in mice liver at different intervals

175
Time interval Group Group Group Group Group Group Group
1 2 3 4 5 6 7
3 DPI 0 0 8 7 7 0 0
7 DPI 0 0 8 6 4 2 3
10 DPI 0 0 5 9 2 5 2
14DPI 0 0 6 1 1 0 0
Grand total score 0 0 27 23 14 7 5

*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract@2000 mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg; Group 5: S. Typhimurium infection + plant
extract@200 mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000
mg.

Table 19. Necrotic foci in liver indicated by mean lesion score in different groups of mice
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
a a bc bc bc a a
3DPI 0.00 0.00 0.00 0.00 2.00 1.41 1.751.43 1.75 0.62 0.00 0.00 0.00 0.00
a a bc a cd a a
7DPI 0.000.00 0.000.00 2.00 1.41 1.25 0.95 1.00 0.70 0.00 0.00 0.750.47
a a cd b a a cd
10DPI 0.00 0.00 0.00 0.00 1.25 0.75 2.25 1.31 0.50 0.50 0.750.25 1.250.50
a a cd a a a a
14DPI 0.00 0.00 0.00 0.00 1.50 1.43 0.25 0.25 0.00 0.00 0.00 0.00 0.000.00

*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection
alone; G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6: S.
Typhimurium infection+ plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000 mg (P0.05%).

Table 20. Inflammatory cell infiltration indicated by total microscopic lesion score in different groups of
mice at different intervals
Time interval Group Group Group Group Group Group Group
1 2 3 4 5 6 7
3 DPI 0 0 9 4 7 0 0
7DPI 0 0 8 6 4 2 4
10 DPI 0 0 6 9 2 2 3
14 DPI 0 1 10 1 4 0 0
Grand total score 0 1 33 20 17 4 7
*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract@2000mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S. Typhimurium infection + plant
extract@200mg; Group 6: S. Typhimurium infection + plant extract @1000mg; Group 7: S. Typhimurium infection + plant extract @
2000mg.

Plate 7: Histological changes in liver of mice at 3DPI

A. G2: Photograph of liver showing normal hepatocytes and hepatic cord structure. H&
E 66X.
B. G3: Photograph of liver showing multifocal necrotic foci along with inflammatory cell
infiltration (arrows). H& E 33X.
C. G3: Photograph of liver showing multifocal, variable size necrotic foci with infiltration
of neutrophils and lymphocytes (encircled area). H& E 132X.

176
D. G4: Photograph of liver showing multifocal small sized necrotic foci (encircled areas).
H& E 66X.
E. G4: Photograph of liver showing focal necrotic area with infiltration of inflammatory
cells (encircled area). H& E 66X.
F. G5: Photograph of liver showing focal necrotic area with infiltration of neutrophils and
lymphocytes (encircled area). H& E 66X.

177
Histopathological changes in the liver of mice at 3DPI

A B

C D

F
E

Histological changes in liver of at 14 DPI


A. G3: Photograph of liver showing diffuse necrosis along with infiltration of neutrophils
and lymphocytes (encircled). H& E 66X.

178
B. G3: Photograph of liver showing diffuse necrosis along with infiltration of neutrophils
lymphocytes with destruction of hepatic parenchyma (Higher magnification of above
picture (arrow). H& E 330X.
C. G4: Photograph of liver showing severe dilatation of sinusoids along with necrotic foci
(encircled). H& E 66X.
D. G4: Photograph of liver showing severe dilatation of sinusoids along with necrotic foci
(Higher magnification of above picture) (encircled, arrow) .H& E 132X.
E. G6: Photograph of liver showing normal hepatic parenchyma with mild cytoplasmic
granularity. H& E 66 X.
Histopathological changes in the liver of mice at 14 DPI

A B

C D

179
180
Histological changes in liver of at 14 DPI
A. G6: Photograph of liver showing normal hepatic parenchyma with mild cytoplasmic
granularity. H& E 66X.
B. G7: Photograph of liver showing normal hepatic parenchyma with mild degenerative
changes. H& E 33X.
Histopathological changes in the liver of mice at 14DPI

A B

Table 21. Inflammatory cell infiltration indicated by mean lesion score in different groups of mice
infected with S. Typhimurium

DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7


d d a ab ab d d
3DPI 0.00 0.00 0.00 0.00 2.25 1.31 1.751.43 1.75 0.62 0.00 0.00 0.00 0.00
d d ab ab bc d d
7DPI 0.000.00 0.000.00 2.00 1.41 1.50 0.95 1.00 0.70 0.50 0.50 1.001.00
d d ab a bc d d
10DPI 0.00 0.00 0.00 0.00 1.50 0.86 2.25 1.13 1.00 0.50 0.500.50 0.750.47
d d a d bc d
14DPI 0.00 0.00 0.25 0.25 2.50 2.17 0.25 0.25 1.00 0.57 0.00 0.00 0.000.00

*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000mg alone; G3: S Typhimurium infection
alone; G4: S. Typhimurium infection + ciprofloxacin @200mg; G5: S. Typhimurium infection + plant extract @200mg; G6: S.
Typhimurium infection+ plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).
Table 22. Degenerative changes indicated by total microscopic lesion score in different groups of mice at
different intervals
Time interval Group Group Group Group Group Group Group
1 2 3 4 5 6 7
3 DPI 0 2 8 8 6 0 2
7DPI 0 2 8 6 5 1 3
10 DPI 0 0 10 10 4 4 6
14 DPI 0 2 10 5 5 2 2
Grand total score 0 6 36 29 20 7 13
*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract@2000mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S. Typhimurium infection + plant
extract@200mg; Group 6: S. Typhimurium infection + plant extract @1000mg; Group 7: S. Typhimurium infection + plant extract @
2000mg.

181
Table 23. Degenerative changes indicated by mean lesion score in different groups of mice infected with
S. Typhimurium
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d ab ab d a d
3 DPI 0.00 0.00 0.50 0.50 2.00 1.41 2.000.91 1.50 0.80 0.00 0.00 0.50 0.00
d d ab bc d d d
7 DPI 0.000.00 0.500.50 2.00 2.00 1.50 0.95 1.25 0.94 0.25 0.25 0.750.47
d d a cd cd cd bc
10 DPI 0.00 0.00 0.00 0.00 2.50 1.50 1.00 0.95 1.00 0.97 1.000.57 1.501.50
d d a bc bc d d
14 DPI 0.00 0.00 0.50 0.50 2.50 1.50 1.50 1.50 1.50 0.75 0.50 0.50 0.500.50
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection
alone; G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6: S.
Typhimurium infection+ plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Table 24. Congestion indicated by total microscopic lesion score in different groups of mice at different
intervals
Time interval Group Group Group Group Group Group Group
1 2 3 4 5 6 7
3 DPI 0 0 8 9 6 0 0
7DPI 0 0 9 8 5 4 3

10 DPI 0 0 9 9 4 4 2
14 DPI 0 4 10 9 5 5 1
Grand total score 0 4 36 35 20 13 6

*No. of mice in each group (n=4). Group1: No infection and no plant extract; Group2: plant extract@2000 mg alone; Group 3: S
Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S. Typhimurium infection + plant
extract@200 mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000
mg.

Table 25. Congestion indicated by mean lesion score in different groups of mice infected with S.
Typhimurium at different time intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d a ab bc d d
3DPI 0.00 0.00 0.00 0.00 2.75 1.41 2.251.31 1.50 0.86 0.00 0.00 0.00 0.00
d d ab ab bc d d
7DPI 0.000.00 0.000.00 2.25 1.43 2.00 2.00 1.25 0.94 1.00 1.00 0.750.47
d d ab ab d d d
10DPI 0.00 0.00 0.00 0.00 2.25 1.43 2.25 2.25 1.00 1.00 1.001.00 0.500.50
d d a ab bc bc d
14DPI 0.00 0.00 1.00 1.00 2.50 2.17 2.25 1.31 1.25 0.75 1.25 0.75 0.250.25
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection
alone; G4:S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200mg; G6: S.
Typhimurium infection+ plant extract@1000 mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

At 7 DPI, necrotic foci areas were comparatively enlarged those seen at 3 DPI. At some
places, necrotic areas revealed the coalescing pattern and distributed throughout the hepatic
parenchyma . Karyorrhexis and karyomegaly was evident at many places of necrosis with
presence of numerous pyknotic hepatic nuclei. In addition to this, colonies of bacilli were
evident at necrotic areas, which was surrounded by the thin zone of degenerated neutrophils
and lymphocytes . Hepatic parenchyma was distorted because of massive infiltration of
horseshoe shaped band cells in the hepatic parenchyma. Hepatocytes were markedly swollen

182
with increase in the cytoplasmic granularity. Moderately to severely dilatated, sinusoids were
evident where sinusoids were trapped with the multilobed neutrophils. Periportal areas were
infiltrated with majority of neutrophils and occasional lymphocytes in one animal at 7 DPI
(Plate-9D). At 10 DPI, severe vacuolar changes, increased cytoplasmic granularity of
hepatocytes, sinusoidal congestion and focal infiltration of lymphocytes with occasional
neutrophils were evident in three mice. Signet ring appearance of hepatocytes was evident in
one animal due to the severe vacuolar changes. Pinpoint areas of infiltration have lymphocytes
and occasional macrophages. Portal areas and particularly around the bile duct showed
infiltration of lymphocytes, macrophages and occasional neutrophils were evident in one
mice. In addition, thin areas of necrosis were present in hepatic parenchyma of two mice. At
14 DPI hepatic cords, structure was well intact. Multifocal to coalescing areas of coagulative
necrosis of severe intensity were present in two mice. In addition to this focal collection of
neutrophils, lymphocytes and macrophages were evident in one animal. Vacuolar changes of
mild intensity were present. Severe sinusoidal dilatation with blood in the sinuses was evident
at 14 DPI. Haemorrhages were present at some places.

Group 4: The detailed microscopic lesions in the liver tissues of infected group along with
their lesion score and intensity are presented in Table 18 and 19. At 3 DPI, liver parenchyma
has focal areas of necrosis, distributed throughout the hepatic parenchyma, but often smaller
as compared to the group 3 . Focal area of necrosis was moderately infiltrated with neutrophils
and lymphocytes. Ground glass appearance of the hepatic parenchyma was evident due to
marked hepatocyte swelling. Karyorhexis and karyomegaly was present at many places in
area of necrosis with pyknotic hepatic nuclei. In addition, mild infiltration in periportal area
with neutrophils and lymphocytes in one animal were present as early as 3 DPI. At 7 DPI,
necrotic foci areas were comparatively smaller than as seen at 3 DPI but they were multiple
(9-10) in number on lower field or even more, infiltrated with mononuclear cell infiltration .
The areas of coagulative necrosis were appeared as homogenous pink infiltrated with
neutrophils and lymphocytes only, characterized as indistinct granuloma. In addition to this
mild periductal infiltration of lymphocytes and occasional neutrophils were present .
Dilatation of sinusoids, sinusoidal congestion and haemorrhages were of severe intensity
present in three mice while one showed moderate dilatation. But necrotic foci, inflammatory
changes were of less severity when compared with plain infection group. At 10 DPI, mild to
severe multifocal to coalescing necrotic areas were evident in 2 mice. Severe, sinusoidal
congestion and dilation were evident in all mice. In addition to these features moderate

183
Kupffer cell hyperplasia, karyorhexis and karyomegaly were evident at some places in two
mice . There was occasional infiltration of inflammatory cells under the capsule and
connective tissue, evident in one animal . Sharply defined multiple vacuoles along with
degenerative changes were present in one animal. At 14 DPI, necrotic foci were much thinner
as compared to the plain infection group . Multiple small, sharply defined vacuoles were
present in the hepatic parenchyma. Mild infiltrations of inflammatory cells were present in
one animal.
Group 5: The detailed microscopic lesions in the liver of S. Typhimurium infected mice kept
on methanolic Saussurea lappa extract (@ 200 mg/kg) along with their lesion score and
intensity are presented in Table 18 and 19. At 3 DPI, liver parenchyma has multifocal to
coalescing areas of necrosis, distributed throughout the hepatic parenchyma, largely
infiltrated with lymphocytes, macrophages and neutrophils apart from this fibroblast were
also present in one animal only. Although hepatocytes were markedly swollen with, increased
cytoplasm granularity and mild, vacuolar changes were evident in one animal. At 7DPI,
occasional pyknotic nuclei were evident in mice of this group. Apoptosis was evident at this
stage. No necrotic foci and infiltration was present . At 10 DPI, thin foci of necrosis and mild
infiltration of lymphocytes and neutrophils were present which were comparatively very mild
as seen at 3DPI . In addition to this, binucleated cells were predominant throughout the hepatic
parenchyma. No vacuolar changes were evident. Hepatic cord structure was well maintained.
At 14 DPI, mild infiltration of mononuclear cells was present around the bile duct in two
mice. Thin foci of lymphocytes, macrophages and occasional neutrophils were present around
the congested blood vessels. While one mice showed severe necrosis along with severe
infiltration of inflammatory cells in the liver parenchyma. Normal hepatic cord structure was
maintained in other animals.
Group 6: The detailed microscopic lesions in the liver of S. typhimurium infected mice kept
on methanolic Saussurea lappa extract (@ 1000 mg/kg) along with their lesion score and
intensity are presented in Table 18 and 19. At 3 DPI, liver have pallor stained parenchyma
around the centrilobular area, which showed the multiple vacuoles as compared to the portal
area. Binucleated hepatocytes were evident throughout the hepatic parenchyma, indicated
towards the regeneration. Hepatic parenchyma was more or less similar to the control group
among all mice. At 7 DPI, area of inflammatory cell reaction were present largely comprised
of neutrophils besides lymphocyte, macrophages distributed throughout the hepatic
parenchyma in one animal. Mild increase in the granularity with eosinophilic cytoplasm,
cellular swelling and moderate congestion was present in other mice of the group. Intensity

184
of the lesions was less severe when compared with the plain infection group. At 10 DPI,
occasional focal areas of coagulative necrosis, infiltrated largely with lymphocytes however
focal collection of inflammatory cell have largely of neutrophils were present without
appreciable necrosis present in one mice . Other mice have mild granularity in the cytoplasm.
No other changes were present. At 14 DPI, occasional focal areas of inflammatory cells were
present revealed largely of lymphocytes and macrophages in one animal. No other changes
were present. It was more or less similar to the control group . Normal hepatic cord structure
was maintained.
Group 7: The detailed microscopic lesions in the liver of S. Typhimurium infected mice kept
on methanolic Saussurea lappa extract (@ 2000mg/kg) along with their lesion score and
intensity are presented in Table 18 and 19. At 3 DPI, liver have mild to moderate vacuolar
changes were evident in all mice. Hepatic parenchyma was more or less similar to the control
group among all mice . At 7 DPI, thin foci of necrosis and inflammatory cell reaction were
present, largely comprised of neutrophils . Mild increase in the granularity with eosinophilic
cytoplasm, cellular swelling, karyomegaly and moderate congestion was present in other mice
of the group. Intensity of the lesions was very less severe when compared with the plain
infection group. While other animals showed normal parenchyma along with normal hepatic
cord structure. At 10 DPI, multifocal to coalescing necrotic foci and inflammatory cell
infiltration were present in one animal only. Other mice have the normal hepatic structure. At
14 DPI, moderate vacuolar changes were present. It was more or less similar to the control
group. Normal hepatic cord structure was maintained.
The total lesion score and mean lesion score for necrotic foci for all groups were
indicated in Table 20 and 21. The total lesion score was significantly higher for the plain
infected group 3 (S. typhimurium infection alone) as compared to the other infected groups 4,
5, 6 and 7 at all stages. However, plant extract given groups have mean lesion score
significantly lower (P 0.05) when compared to the plain infection group. Similarly, for the
inflammatory cell infiltration the total lesion score and mean lesion score was significantly
higher for the plain infection group 3 and lower for the plant treated groups 5, 6 and 7 (Table
22 and 23). Although infected group 6 given extract @ 1000 mg/ kg showed the significantly
lower inflammatory cell infiltration (P 0.05) when compared to the other infected groups 5
and 7 given plant extracts @ 200 and 2000 mg/kg at all the time intervals. The total lesion
score and mean lesion score for the degenerative changes were significantly higher (P 0.05)
for the group 3 (S. typhimurium infection alone) at 10 and 14 DPI (Table 24 and 25). While
in the plant treated groups 5, 6 and 7 showed significantly lower (P 0.05) degenerative

185
changes as compared to the plain infection group3 and infected group 4 given Ciprofloxacin
@ 200mg/kg. The total lesion score and mean lesion score for congestion in liver was
significantly higher (P 0.05) for the infected group 3 and 4 (S. typhimurium infection alone
and Ciprofloxacin @ 200 mg/kg respectively) indicated in Table 26 and 27. While the
congestion in the plant given groups 5, 6 and 7 showed significantly less congestion when
compared to the plain infection group 3.
Spleen
Group 1 and 2: No change was observed in the spleen of control mice. Spleen was normal
with red and white pulp intact. Splenic cord structure and architecture was completely intact.
Megakaryocytic and erythropoietic cell aggregates were in abundant. As compared to the
group 1, in group two lymphoid element was more compact, cellular and discrete at all the
stages. Haemosiderin was present in abundance in the red pulp.
Histological changes in spleen of mice at 3 DPI
A. G2: Photograph of spleen showing normal splenic parenchyma with compact white pulp
(arrow).H& E 33X.
B. G3: Photograph of spleen severe proliferation of trabecular vessels in the red pulp with
destruction of spleen parenchyma (arrow). H& E 66X.
C. G3: Photograph of spleen showing mild reticular cell hyperplasia and erythropoietic
cell depletion (arrow). H& E 33X.
D. G3: Photograph of spleen showing mild infiltration of neutrophils along with depleted
megakaryocytes (encircled). H& E 66X.
E. G3: Photograph of spleen showing mild infiltration of neutrophils along with depleted
megakaryocytes (Higher magnification of above picture) (arrow). H& E 330X.
F. G3: Photograph of spleen showing moderate necrosis along with degenerated
neutrophils (encircled). H& E 330X.

186
Histopathological changes in the spleen of mice at 3DPI

A B

C D

E F

187
Histological changes in spleen of mice at 3 DPI
B. G4: Photograph of spleen showing apoptosis in the white pulp (arrow). H& E 66X.
C. G5: Photograph of spleen showing severe neutrophil infiltration along with depletion
(encircle). H& E 132X.
D. G6: Photograph of spleen showing severe white pulp hyperplasia (arrow). H& E 66X.
F. G7: Photograph of spleen showing intact follicle with apoptosis in the white pulp
hyperplasia (arrow). H& E 66X.
Histopathological changes in the spleen of mice at 3DPI

B C

D F

188
Histological changes in spleen of mice at 7 DPI
A. G2: Photograph of spleen showing compact white pulp follicles (arrow).H& E 33X.
B. G3: Photograph of spleen mild neutrophil infiltration in the spleen parenchyma
(encircled). H& E 66X.
E G3: Photograph of spleen showing severe depletion of white pulp along with neutrophil
infiltration in spleen parenchyma (encircle). H& E 66X.
F G4: Photograph of spleen showing severe depletion of white pulp along with multifocal area
of necrosis in spleen parenchyma (encircle). H& E 66X.
Histopathological changes in the spleen of mice at 7DPI

A B

E F

Histological changes in spleen of mice at 7 DPI


A. G4: Photograph of spleen showing less distinct follicle in spleen parenchyma (arrow).
H& E 132X.
B. G5: Photograph of spleen showing compact and intact follicle (arrow). H& E 132X.
C. G5: Photograph of spleen showing normal spleen parenchyma along with abundant
megakaryocytes and erythropoietic cells (arrow, encircle). H& E 132X.
D. G6: Photograph of spleen showing normal red and white pulp along with haemosiderin
(arrow). H& E 132X.
E. G7: Photograph of spleen showing severe white pulp hyperplasia where white pulp is
extending from main follicle (arrow). H& E 13.25X.

189
Histopathological changes in the spleen of mice at 7DPI

A B

C D

190
Histological changes in spleen of mice at 14DPI
A. G2: Photograph of spleen showing intact follicles and moderate white pulp hyperplasia
(arrow). H& E 66X.
B. G3: Photograph of spleen showing severe trabecular sinus proliferation around the
white pulp and severe white pulp depletion (arrow). H& E 66X.
C. G4: Photograph of spleen showing severe depletion of white pulp, only red pulp is
evident in the parenchyma (encircle). H& E 13.25X.
D. G5: Photograph of spleen showing intact white pulp follicles and normal spleen
parenchyma (arrow). H& E 66 X.
E. G6: Photograph of spleen showing intact white pulp follicles and white pulp hyperplasia
(arrow). H& E 66X.
F. G7: Photograph of spleen showing severe white pulp hyperplasia (arrow) H& E 66X

191
Histopathological changes in the spleen of mice at 14DPI

A B

C D

E F

192
Group 3: The detailed microscopic lesions in the spleen tissues of infected group along with
their lesion score and intensity are presented in Table 26 and 27. In the spleen, profuse
reticular cell hyperplasia was evident, red pulp showed sheet like areas of reticular cell
hyperplasia as early at 3 DPI . White pulp was massively depleted of lymphocytes. There was
reduction in the erythropoietic cell aggregates throughout the parenchyma. Severe
granulocyte infiltration was present in the splenic parenchyma. Golden coloured granular
haemosiderin was greatly increased in one animal. Follicles were lack of germinal centres and
red pulp was expanded showed hyperplastic activity of endothelial cells. There were severe
proliferation of trabecular vessels and blood sinuses present as early at 3 DPI. At 7 DPI,
lymphoid element and marginal zone were not that much compact. Neutrophilic infiltration
was present in the white pulp. In red pulp, trabecular vessel proliferation was severe and
arteritis was present with infiltration of neutrophils and lymphocytes in two mice.
Erythropoietic cells were depleted, present in small numbers along with megakaryocytes.
Germinal centers have neutrophilic infiltration and depleted very much as compared to 3 DPI.
Severe red pulp hyperplasia and thin area of necrosis was also present in one animal. At 10
DPI, mild depletion of lymphoid element and severe red pulp hyperplasia were present.
Haemosiderin pigment was present in plenty. Germinal centres were depleted from
lymphocytes. Mild areas of necrotic foci were present in the red pulp in one animal. Intensity
of lesion was less severe as compared to the earlier stages. At 14 DPI, marked trabecular
vessel proliferation was present in two mice. Severe red pulp hyperplasia was present in one
animal. No other changes were evident at this stage
Group 4: The detailed microscopic lesions in the spleen tissues of infected with S.
Typhimurium group kept on Ciprofloxacin (@ 200 mg/kg body weight along with their
lesion, score and intensity are presented in Table 30 and 31. Severe granulocyte infiltration
was evident in spleen parenchyma along with it cystic spaces were present in the white pulp
as early at 3DPI. Megakaryocytic and erythropoietic cell activity was markedly increased.
Venous sinuses were severely dilated. In other mice, although lymphoid element was discrete
but appeared to be reduced in size and depleted of lymphocyte. There was massive
proliferation of trabecular network, which replaced the splenic details of parenchyma .
Hyperplastic sheets of reticular endothelial tissue were evident along with infiltration of
macrophages, granulocytes and lymphocytes. Erythropoietic cells were uniformly distributed
but appeared thin as compared to the other mice of the same group. Megakaryocytes were
hardly visible in this section of spleen.

193
Table 26. Number of mice showing microscopic lesions of variable intensity in the spleen from each group at 3 and 7 DPI
3DPI 7DPI
Type of lesion Intensity G1 G2 G3 G4 G5 G6 G7 G1 G2 G3 G4 G5 G6 G7
Mild 0 0 0 0 0 1 1 1 1 0 2 0 0 2
Congestion Moderate 0 0 0 0 2 0 0 0 0 1 0 3 1 0
Severe 0 0 0 1 1 0 1 0 0 3 2 0 1 1
Mild 0 0 0 1 2 2 2 1 3 1 1 2 1 1
Granulocyte infiltration Moderate 0 0 2 0 0 0 0 0 0 0 2 1 0 2
Severe 0 0 2 3 1 0 0 0 0 3 1 0 0 0
Mild 0 0 0 1 0 0 0 0 0 2 1 0 0 0
Necrotic foci Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Mild 0 0 2 1 1 1 2 0 1 2 2 0 0 0
Lymphoid depletion Moderate 0 0 2 3 2 0 0 0 0 2 0 0 0 1
Severe 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Mild 0 0 0 1 1 1 0 0 2 1 2 1 3 1
RE cell Moderate 0 0 0 0 1 0 0 0 0 0 1 1 0 1
hyperplasia Severe 0 0 2 3 2 1 1 0 0 3 1 1 0 1
*Data represent (n=4): Group1: No infection and No plant infection; Group2: plant extract @2000 mg alone; Group 3: S Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200
mg; Group 5: S. Typhimurium infection + plant extract @200 mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

194
Table 27. Number of mice showing microscopic lesions of variable intensity in the spleen from each group at 10 and 14DPI
10DPI 14DPI
Type of lesion Intensity G1 G2 G3 G4 G5 G6 G7 G1 G2 G3 G4 G5 G6 G7
Mild 1 1 0 0 2 0 0 0 1 0 2 0 0 1
Congestion Moderate 0 0 2 1 1 0 1 0 1 2 0 2 2 1
Severe 0 0 2 3 0 0 0 0 0 2 1 1 0 0
Mild 1 1 2 1 2 3 0 0 1 0 0 1 0 2
Granulocyte infiltration Moderate 0 1 2 1 0 0 2 0 2 1 3 3 1 0
Severe 0 0 0 2 0 0 1 0 0 3 0 0 1 0
Mild 0 0 2 2 0 1 1 0 0 0 1 0 1 0
Necrotic foci Moderate 0 0 0 1 0 0 0 0 0 3 0 0 0 0
Severe 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Mild 0 0 4 2 0 0 1 0 0 1 0 1 0 0
Lymphoid depletion Moderate 0 0 0 0 0 0 0 0 0 3 2 1 0 0
Severe 0 0 0 2 0 0 0 0 0 0 0 0 0 0
Mild 0 0 2 0 3 3 3 0 0 0 1 2 2 1
RE cell Moderate 0 0 2 1 0 0 0 0 0 3 1 0 0 1
hyperplasia Severe 0 0 0 3 0 0 0 0 0 1 0 0 0 0
*Data represent (n=4): Group1: No infection and No plant infection; Group2: plant extract @2000 mg alone; Group 3: S Typhimurium infection alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg;
Group 5: S. Typhimurium infection + plant extract @200mg; Group 6: S. Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg

195
Splenic cords were distorted due to the massive proliferation of trabecular network. In another
animal, lymphoid element was moderately depleted and lymphocytes showed karyorhexis,
pyknosis and apoptosis. In addition to this, immediately around the lymphoid follicles blood
sinuses were severely dilated and were filled with the blood. Mild small areas of homogenous
eosinophilic necrosis were evident in one animal at 3DPI. At 7 DPI, white pulp was severely
depleted along with it red pulp proliferation was massive. Moderate RE cell hyperplasia was
evident in one animal. In another animal, severe granulocyte infiltration was present in splenic
parenchyma along with the mild depletion of lymphocytes in the white pulp. While
megakaryocytic and erythropoietic cells were abundant in spleen parenchyma. In other mice,
severe red pulp hyperplasia was present along with severe blood sinuses dilatation around the
follicles, which showed infiltration of neutrophils, macrophages and lymphocytes.
Neutrophils were infiltrated in the white pulp along with the occasional homogenous pink
areas of necrosis evident in the red pulp in one mice. At 10 DPI, Severe multifocal areas of
necrosis along with degenerated neutrophils and lymphocytes were evident in one animal. In
another mice necrosis was present but of mild intensity as compared to the first mice of the
same group. Severe infiltration of neutrophils and lymphocytes were evident in splenic
parenchyma.

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Table 28. Congestion indicated by total microscopic lesion score in different groups of mice at different time
intervals
Time interval Group1 Group2 Group3 Group4 Group5 Group6 Group7
3 DPI 0 0 0 3 7 1 4
7DPI 1 1 8 8 6 5 5
10 DPI 1 1 10 8 4 0 2
14DPI 0 3 10 5 4 4 3
Grand total score 2 5 28 24 21 10 14
*No. of mice in each group (n=4). Group1: No infection and No plant infection; Group2: plant extract @2000 mg alone; Group 3: S Typhimurium infection
alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg; Group 5: S. Typhimurium infection + plant extract @200 mg; Group 6: S. Typhimurium
infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

Table 29.Congestion indicated by mean lesion score in different groups of mice infected with S. Typhimurium
at different time intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d a ab bc d d
3DPI 0.00 0.00 0.00 0.00 2.75 1.41 2.251.31 1.50 0.86 0.00 0.00 0.00 0.00
d d ab ab bc d d
7DPI 0.000.00 0.000.00 2.25 1.43 2.00 2.00 1.25 0.94 1.00 1.00 0.750.47
d d ab ab d d d
10DPI 0.00 0.00 0.00 0.00 2.25 1.43 2.25 2.25 1.00 1.00 1.001.00 0.500.50
d d a ab bc bc d
14DPI 0.00 0.00 1.00 1.00 2.50 2.17 2.25 1.31 1.25 0.75 1.25 0.75 0.250.25
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000mg alone; G3: S Typhimurium infection alone;
G4: S. Typhimurium infection + ciprofloxacin @200mg; G5: S. Typhimurium infection + plant extract @200mg; G6: S. Typhimurium infection+
plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Table 30. Granulocyte infiltration indicated by total microscopic lesion score in different groups of mice at
different intervals
Time interval Group1 Group2 Group3 Group4 Group5 Group6 Group7
3 DPI 1 1 10 11 11 2 2
7DPI 1 1 10 8 4 1 5
10 DPI 1 3 6 9 2 3 4
14 DPI 0 5 12 6 7 5 2
Grand total score 3 10 38 34 24 11 13
*No. of mice in each group (n=4). Group1: No infection and No plant infection; Group2: plant extract @2000mg alone; Group 3: S Typhimurium infection
alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S. Typhimurium infection + plant extract @200mg; Group 6: S. Typhimurium
infection + plant extract @1000mg; Group 7: S. Typhimurium infection + plant extract @ 2000mg.

40

197
Table 31. Granulocyte infiltration indicated by mean lesion score in different groups of mice infected with S.
Typhimurium at different time intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d a a d d d
3DPI 0.00 0.00 0.00 0.00 2.50 1.50 2.502.14 1.25 1.56 0.500.50 0.50 0.50
d d a ab d d d
7DPI 0.250.25 0.750.75 2.50 2.17 2.00 0.91 1.00 0.50 0.25 0.25 1.250.95
d d d ab d d d
10DPI 0.25 0.25 0.75 0.48 1.50 0.95 2.25 1.31 0.50 0.50 0.750.75 1.001.00
d d a bc bc d d
14DPI 0.00 0.00 1.25 0.94 2.75 2.04 1.50 1.50 1.75 1.43 1.25 0.75 0.500.50

*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection alone;
G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6: S. Typhimurium infection+
plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000 mg (P0.05%).

Table 32. Necrotic foci indicated by total microscopic lesion score in different groups of mice at different time
intervals
Time interval Group1 Group2 Group3 Group4 Group5 Group6 Group7
3 DPI 0 0 0 1 0 0 0
7DPI 0 0 2 1 0 0 0
10 DPI 0 0 2 7 0 1 0
14 DPI 0 0 6 1 0 1 0
Grand total score 0 0 10 10 0 2 0
*No. of mice in each group (n=4). Group1: No infection and No plant infection; Group2: plant extract @2000 mg alone; Group 3: S Typhimurium infection
alone; Group 4: S. Typhimurium infection + ciprofloxacin @200 mg; Group 5: S. Typhimurium infection + plant extract @200 mg; Group 6: S.
Typhimurium infection + plant extract @1000 mg; Group 7: S. Typhimurium infection + plant extract @ 2000 mg.

Table 33. Necrotic foci indicated by mean lesion score in different groups of mice infected with S.
Typhimurium at different time intervals
DPI Group1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7
a a a a a a a
3DPI 0.00 0.00 0.00 0.00 0.00 0.00 0.250.25 0.00 0.00 0.000.00 0.00 0.00
a a a a a a a
7DPI 0.000.00 0.000.00 0.50 0.50 0.25 0.25 0.00 0.00 0.00 0.00 0.000.00
a a
10DPI 0.00 0.00 0.00 0.00 0.50 0.50a 1.75 0.62
b
0.00 0.00
a
0.250.25
a
0.250.25
a

a a b a a a a
14DPI 0.00 0.00 0.00 0.00 1.50 1.50 0.25 0.25 0.00 0.00 0.25 0.25 0.000.00

*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection alone;
G4: S. Typhimurium infection + ciprofloxacin @200mg; G5: S. Typhimurium infection + plant extract @200 mg; G6: S. Typhimurium infection+
plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%)

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198
Table 34. Lymphoid depletion indicated by total microscopic lesion score in different groups of mice at different
intervals
Time interval Group1 Group2 Group3 Group4 Group5 Group6 Group7
3 DPI 0 0 6 7 5 1 2
7DPI 0 0 6 5 0 0 1
10 DPI 0 1 4 8 0 0 0
14 DPI 0 0 7 4 2 0 0
Grand total score 0 1 23 24 7 1 3
*No. of mice in each group (n=4). Group1: No infection and No plant infection; Group2: plant extract @2000mg alone; Group 3: S Typhimurium infection
alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S. Typhimurium infection + plant extract @200mg; Group 6: S. Typhimurium
infection + plant extract @1000mg; Group 7: S. Typhimurium infection + plant extract @ 2000mg.

Table 35. Lymphoid depletion indicated by mean lesion score in different groups of mice infected with S.
Typhimurium at different time intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d ab ab bc d d
3 DPI 0.00 0.00 0.00 0.00 1.50 0.95 1.751.44 1.25 0.95 0.250.25 0.50 0.50

d d ab bc bc d d
7 DPI 0.000.00 0.250.25 1.50 0.95 1.25 0.75 1.25 0.75 0.00 0.00 0.500.50

d d bc a d d d
10 DPI 0.00 0.00 0.00 0.00 1.00 1.00 2.75 1.41 0.00 0.00 0.000.00 0.250.25

d d ab bc d d d
14 DPI 0.00 0.00 0.00 0.00 1.75 1.43 1.00 1.00 0.75 0.47 0.00 0.00 0.000.00
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection alone;
G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6: S. Typhimurium infection+
plant extract@1000mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Table 36. RE cell hyperplasia indicated by mean lesion score in different groups of mice at different time
intervals
Time interval Group1 Group2 Group3 Group4 Group5 Group6 Group7
3 DPI 0 0 6 7 5 1 2
7DPI 0 0 6 5 0 0 1
10 DPI 0 1 4 8 0 0 0
14 DPI 0 0 7 4 2 0 0
Grand total score 0 1 23 24 7 1 3
*No. of mice in each group (n=4). Group1: No infection and No plant infection; Group2: plant extract @2000mg alone; Group 3: S Typhimurium infection
alone; Group 4: S. Typhimurium infection + ciprofloxacin @200mg; Group 5: S. Typhimurium infection + plant extract @200mg; Group 6: S. Typhimurium
infection + plant extract @1000mg; Group 7: S. Typhimurium infection + plant extract @ 2000mg.

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199
Table 37. RE cell hyperplasia indicated by mean lesion score in different groups of mice infected with S.
Typhimurium at different time intervals
DPI Group1 Group2 Group3 Group4 Group5 Group6 Group7
d d a a a bc ab
3DPI 0.00 0.00 0.00 0.00 2.50 1.50 2.501.44 2.25 1.31 1.001.44 1.25 0.75
d d a ab ab bc ab
7DPI 0.000.00 0.500.50 2.50 1.43 1.75 0.63 1.50 0.64 0.75 0.75 1.500.64
d d ab ab d bc bc
10DPI 0.00 0.00 0.00 0.00 1.50 0.95 2.00 1.41 0.75 0.75 0.750.75 0.750.75
d d a d d d bc
14DPI 0.00 0.00 0.00 0.00 2.25 1.43 0.75 0.47 0.50 0.50 0.50 0.50 0.750.47
*Data represent Mean S.E. (n=4). G1: No infection and no plant extract; G2: Plant extract@2000 mg alone; G3: S Typhimurium infection alone;
G4: S. Typhimurium infection + ciprofloxacin @200 mg; G5: S. Typhimurium infection + plant extract @200 mg; G6: S. Typhimurium infection+
plant extract@1000 mg; G7: S. Typhimurium infection + plant extract @ 2000mg (P0.05%).

Lymphoid hyperplasia of moderate to severe intensity was present in two mice. In


addition to this haemopoietic cell, aggregates were in abundant. At 14 DPI, moderate to
severe red pulp hyperplasia and white pulp was severely depleted in all mice.
Erythropoietic islands were in abundant.
Group 5: The detailed microscopic lesions in the spleen tissues of infected mice with S.
Typhimurium group kept on methanolic extract of Saussurea lappa (@ 200 mg/kg body
weight) along with their lesion score and intensity are presented in Table 26 and 27. At 3
DPI, lymphoid follicles were abundant but apoptosis was evident in white pulp and
periarteriolar lymphoid sheath region. Around the apoptotic, areas there were infiltration
of neutrophils. Moderate red pulp hyperplasia was evident along with it megakaryocytic
and erythropoietic cells were in abundant in whole parenchyma of one animal. In other
mice, lymphoid element was distinct but depleted at places along with severe infiltration
of neutrophils in white pulp of one animal. In addition to this, megakaryocytic and
erythropoietic cells were in plenty along with the haemosiderin pigment in splenic
parenchyma. In other mice lymphoid element was compact, normal structure was
maintained, and no apoptosis was present. At 7 DPI, moderate to severe lymphoid
hyperplasia was evident in all mice. Follicles were expanded from the white pulp,
appeared more compact and discrete. Along with it moderate to severe intensity red pulp
hyperplasia was also evident in all mice. Haemopoietic cell islands were abundant in
whole parenchyma of spleen in all mice. No other significant change was evident at 7
DPI. At 10 DPI, normal structure of spleen was maintained. White pulp appeared to be
compact, more or less extended in to the parenchyma along with it germinal center were
very prominent in all the mice . No other changes were evident. In addition to this,

43

200
haemopoietic cell aggregates were in abundant. At 14 DPI, it was more or less similar to
control group. Erythropoietic islands were in abundant. No other changes were evident at
this stage.
Group 6: The detailed microscopic lesions in the spleen tissues of infected mice with S.
Typhimurium group kept on methanolic extract of Saussurea lappa (@1000 mg/kg) along
with their lesion score and intensity are presented in Table 26 and 27. At 3 DPI, lymphoid
follicles were abundant, discrete and hyperplastic . Blood sinuses were severely dilated
around the follicles. Marginal zone was increased in one animal along with occasional
infiltration of neutrophils in one animal. In other mice normal splenic parenchyma was
maintained, no significant change was evident. Megakaryocytic and erythropoietic cells
were in plenty along with the haemosiderin pigment in splenic parenchyma. At 7 DPI,
follicles were expanded from the white pulp, appeared more compact and discrete .
Erythropoietic cell activity was abundant but less than the previous group. Mild RE cell
hyperplasia was evident in one animal. No other significant change was evident at 7DPI.
At 10 DPI, normal structure of spleen was maintained. White pulp appeared to be
compact, more or less extended in to the parenchyma.In addition to this, haemopoietic
cell aggregates were in abundant. At 14 DPI, it was more or less similar to control group
. Mild red pulp hyperplasia was present. Erythropoietic islands were in abundant.
Group 7: The detailed microscopic lesions in the spleen tissues of infected mice with S.
typhimurium group kept on methanolic extract of Saussurea lappa (@ 2000 mg/kg ) along
with their lesion score and intensity are presented in Table 26 and 27. At 3 DPI, lymphoid
follicles were intact but showed apoptosis . Blood sinuses adjoining to the white pulp
were engorged with the RBCs and lymphocytes along with mild RE cell hyperplasia.
Megakaryocytic and erythropoietic cells were in plenty along with the haemosiderin
pigment in splenic parenchyma. In other mice along with the depletion in lymphoid
follicles, apoptotic bodies were evident. In addition to this, lymphocytes were
degenerated and fragmented in the spleen parenchyma. At 7 DPI, lymphoid cell
hyperplasia of moderate intensity was evident in all mice. It was similar to the control
group . No other significant change was evident at 7DPI. At 10 DPI, normal structure of
spleen was maintained. Severe lymphoid hyperplasia and moderate white pulp
hyperplasia of moderate intensity was present in all mice. While severe necrosis along

44

201
with infiltration of neutrophil was evident in one animal only. No other changes were
evident. In addition to this haemopoietic cell, aggregates were in abundant. At 14 DPI, it
was more or less similar to control group. White pulp hyperplasia was evident. Mild red
pulp and lymphoid hyperplasia was present. Erythropoietic islands were in abundant. No
other changes were evident at this stage.
The total lesion score and mean lesion score for congestion in spleen was significantly
higher in group 3 given S. typhimurium infection alone as compared to the other infected
groups 4, 5, 6 and 7 (Table 28 and 29). While in plant treated groups 5, 6 and 7 mean
lesion score was significantly lower (P0.05) than the other infected groups 3 and 4 (plain
infection group and Ciprofloxacin @ 200 mg/kg). Similarly for the granulocyte
infiltration the total lesion score and mean lesion score was significantly lower (P0.05)
for the plant treated infected groups 5, 6 and 7 when compared to the other infected groups
3 and 4 at all stages (Table 30 and 31). However, the total lesion score and mean lesion
score was non-significant (P0.05) among all the infected groups 3, 4, 5, 6 and 7 at all
the time intervals except for the infected group 4 given Ciprofloxacin @ 200 mg /kg at
10 DPI (Table 30 and 31). Similarly the total lesion score and mean lesion score for
lymphoid depletion of plant treated groups 5, 6 and 7 was significantly lower (P0.05)
when compared to the infected group 3 and 4 (S. typhimurium infection alone and
Ciprofloxacin @ 200 mg/kg respectively) at all stages (Table 34 and 35). The total lesion
score and mean lesion score for RE cell hyperplasia was significantly lower (P0.05) in
infected groups 5, 6 and 7 given plant extract @ 200, 1000 and 2000mg/kg respectively
when compared to infected groups 3 and 4 at all intervals of sacrifice (Table 36 and 37).
5.7 Conclusions

S. typhimurium found to be highly pathogenic and produced significant hepatic


lesion in the mice model during in vivo experimentation.
Saussurea lappa has shown good in vitro antimicrobial activity against S.
Typhimurium.
Addition of lyophilized methanolic extract of Saussurea lappa @ 2000mg per kg
body weight of drinking water did not produce any adverse effect on growth,
mortality pattern and various organs of the mice.

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202
Based on the clinical signs, mortality, serum biochemistry, gross and
histopathological lesions strongly suggested promising hepatoprotective and
immunomodulatory effect of Saussurea lappa (@1000mg) in mice experimentally
infected with S. Typhimurium.
Saussurea lappa can be used as a medication for the therapeutic purpose and needs
to be explored its possible mechanisms in future.
6. Publications

Research Paper

1. Thakur M., Asrani R.K., Thakur S., Sharma P.K., Patil R.D., Lal B., Om Parkash
(2016) Observations on traditional usage of ethnomedicinal plants in humans and
animals of Kangra and Chamba districts of Himachal Pradesh in North-Western
Himalaya, India. Journal of Ethnopharmacology 191: 280-300.

Poster Presentation

1. Asrani R.K., Thakur M., Thakur S., Gautam H., Patil R.D., Sharma P.K. (2017)
Protective effects of Artemisia nilagirica leaves extract in broiler chickens against
Escherichia coli. 20th World Veterinary Poultry Association Congress at
Edinburgh U.K. from 3rd to 9th September, 2017.

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203
ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

PANTNAGAR

204
OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
PANTNAGAR

ANNUAL REPORT (2016-2017)

1. Name of Centre : Pantnagar


2. Name of Investigators: Dr(s). Mahesh Kumar. A.H. Ahmad and A.K. Thathoo.
3. Approved activity:
Nutraceutical and antidiabetic activity of medicinal plant
4. Salient Achievement

Root of EVM-GBPU-NUT 3 contains alkaloides, anthraquinone, flavanoides,


glycosides, proteins, triterpines, tannins, glycosides, proteins, sterols, triterpines
Resins and saponines.

Dose up to 2000 mg/kg body weight orally donot have any toxicity symptoms,
therefore, both the extracts were considered safe for long term administration.

Aqueous extracts of EVM-GBPU-NUT 3 bears the potential of inhibition of -


amylase & -glucosidase activity in a dose dependent manner and the inhibition
was almost parallel (31.03 0.97) to the standard drug metformin at 50 mg/ml (33.02
0.92)
The methanol and ethanol extracts did not reveal good results as compared to the
aqueous extract.

Aqueous extracts of EVM-GBPU-NUT 3 were further fractioned in column


chromatography

5. Progress Report 01.04.2016 to 31.03.2017

During the period, studies were continued on the roots of plant EVM-GBPU-NUT
3. Of aqueous, ethanol and methanol extracts, the yield was high in aqueous extract. The
phenolic and flavonoid contents were also high in the aqueous extract. The toxicity studies
had revealed that the plant was safe to the experimental mice and was well tolerated up to
1 gm/kg body weight which is 10 times more of the normal dose of plant used in earlier
studies.

205
Anti-diabetic activity of aqueous, ethanol and methanol extracts was studied
through in vitro trial using the glucose uptake and glucose utilization. These studied
suggested that the aqueous and methanolic extracts of the roots of plant EVM-GBPU-
NUT 3 possess antidiabetic properties. The antidiabetic activity was further confirmed in
vitro through inhibition assay for -amylase and -Glucosidase activity using metformin
as the standard antidiabetic drug.

5.1 Inhibition assay for -amylase activity (DNSA):

The test was conducted using five concentrations of plant extracts (10, 50, 100,
200 and 300 mg/ml). Total 250 l of plant extract and 250 l of 0.02M sodium phosphate
buffer containing -amylase solution (0.5 mg/ml) were incubated for 10 min at 25C.
After pre-incubation, 250 l of 1% starch solution in 0.02M sodium phosphate buffer was
added to each tube at five seconds intervals. This reaction mixture was then incubated for
10 min at 25C and then 1 ml of DNSA colour reagent was added to stop the reaction.
These test tubes were then incubated in a boiling water bath for 5 min and cooled to room
temperature. Finally this reaction mixture was diluted by adding 10 ml distilled water
following which absorbance was measured at 540 nm. The inhibition of -amylase
activity was measured by the following formula:

% Inhibition = {(Ac Ae)/Ac}x 100


where Ac and Ae are the absorbance of the control and extract, respectively.
The inhibition of -amylase activity was dose dependent and the aqueous extract
revealed the inhibition almost parallel (31.03 0.97) to the standard drug metformin at 50
mg/ml (33.02 0.92) concentration (Table 1). At 100, 200 and 300 mg/ml concentration,
the aqueous extract revealed better activity then the standard drug (Table 1). The methanol
and ethanol extracts did not reveal good results as compared to the aqueous extract.

5.2 Inhibitory assay -Glucosidase activity:


The substrate solution p-nitrophenyl glucopyranoside (pNPG) (3.0 mM) was prepared
in 20 mM phosphate buffer. Then 100 l of -glucosidase (1.0 U/ml) was pre-incubated
with 50 l of the different concentrations (10, 50, 100, 200 and 300 mg/ml) of the
aqueous, methanol and ethanol extracts for 10 min. Subsequently, 50 l of 3.0 mM
pNPG as a substrate dissolved in 20 mM phosphate buffer (pH 6.9) was added to start

206
the reaction. The reaction mixture was incubated at 37oC for 20 min and stopped by
adding 2 ml of 0.1 M Na2CO3. The -glucosidase activity was determined by measuring
the yellow coloured para-nitrophenol released from pNPG at 405 nm. The inhibition of
-glucosidase activity was measured by the following formula:

% Inhibition = {(Ac Ae)/Ac}X 100


Where Ac and Ae are the absorbance of the control and extract, respectively.
The inhibition of -glucosidase activity was also dose dependent and the aqueous extract
revealed the inhibition almost parallel (38.83 1.04) to the standard drug metformin at
100 mg/ml (43.52 0.93) concentration (Table 2). At 200 and 300 mg/ml concentration,
the aqueous extract revealed better activity then the standard drug (Table 2). The
methanol and ethanol extracts did not reveal good results as compared to the aqueous
extract.
5.3 In vivo anti-diabetic activity

Anti-diabetic activity of aqueous and methanol extracts was studied in alloxan-


induced diabetic mice. Diabetes was induced in adult fasted mice of either sex by a
single intraperitoneal injection of 120 mg/kg of alloxan monohydrate in sterile saline.
The diabetic mice (glucose level >300 mg/dl) were taken and divided into 7 groups of
six mice each. Group C1 served as healthy control and was given normal saline; group
C2 served as untreated diabetic control and was also given normal saline whereas groups
C3 and C4 were administered aqueous extract residues at @ 100 and 200 mg/kg body
weight. Groups C5 and C6 received methanol extract residues at @ 100 and 200 mg/kg
body weight. The standard anti-diabetic drug, metformin was administered orally at a
dose of 50 mg/kg to group C7 animals. The mice of all groups were given glucose (2
g/kg body weight, orally) 30 min after administration of the drug. Blood glucose levels
were measured at 0, 30, 90 and 150 min of glucose loading.

The results of the trial indicated that aqueous and methanol extracts of EVM-
GBPU-NUT 3 revealed good antidiabetic properties (Table 3).

5.4 Fractionation through column chromatography

207
The aqueous extract was subjected to column chromatography using silica gel
(60-120 mesh). Total 60 fractions of 60 ml each were collected. Among these 60
fractions, 26 fractions (10-26 and 30-38) were thought to be pure, showing single spot
on TLC. Fractions 10-26 were pooled and re-chromatographed and eluted. Fractions 30-
38 were also pooled separately and eluted.

Table 1. Inhibition of -amylase activity by extracts of EVM-GBPU-NUT 3


Conc. % Inhibition of -amylase
mg/ml Standard Aqueous Methanol Ethanol
10 25.33 1.82 20.78 0.85 9.52 0.80 11.90 1.38
50 33.02 0.92 31.03 0.97 13.05 0.30 15.68 0.57
100 39.63 0.89 42.34 1.05 17.71 0.53 19.43 0.20
200 52.48 1.24 61.27 1.07 21.54 0.50 23.72 0.33
300 63.56 1.92 75.81 1.37 29.37 0.70 27.50 0.61

Table 2. Inhibition of -glucosidase activity by extracts of EVM-GBPU-NUT 3


Conc. % Inhibition of - glucosidase
mg/ml Standard Aqueous Methanol Ethanol
10 23.96 1.03 19.01 1.63 10.29 1.81 11.07 1.04
50 31.78 1.42 25.33 0.68 19.48 1.03 19.35 0.21
100 43.52 0.93 38.83 1.04 23.01 1.72 22.00 0.37
200 61.39 1.01 66.01 1.06 31.44 0.47 27.12 0.51
300 73.04 1.12 81.01 1.45 37.09 0.64 33.43 0.81

208
Table 3. Blood glucose level (mg/dl) in different groups of mice before and after
treatment

Groups Days of observation

0 7 days after 15 days after 30 days after 60 days after


giving alloxan treatment treatment treatment

C1 88.46 3.72 87.35 4.91 92.78 6.25 96.21 5.73 92.05 4.36

C2 91.54 2.78 347.54 9.82 398.12 16.76 457.44 17.82 473.50 13.46

C3 92.24 3.12 387.42 18.33 278.25 16.73 226.57 14.40 162.55 9.15

C4 90.85 2.75 385.06 15.82 187.87 14.65 147.18 11.42 112.45 10.40

C5 85.91 4.08 362.92 18.22 297.05 12.24 265.25 16.71 238.66 11.70

C6 94.60 2.84 380.84 17.50 258.92 14.31 219.72 10.44 185.74 10.50

C7 93.22 4.72 379.35 13.90 176.12 11.62 142.80 9.69 120.05 12.50

5.5 Conclusion:

The phenolic and flavonoid contents were high in the aqueous extract.
The toxicity studies had revealed that the plant was safe to the experimental mice
and was well tolerated up to 1 gm/kg body weight which is 10 times more of the
normal dose of plant used in earlier studies.
Anti-diabetic activity of aqueous, ethanol and methanol extracts suggested that the
aqueous and methanolic extracts of the roots of plant EVM-GBPU-NUT 3 possess
antidiabetic properties.
The antidiabetic activity was further confirmed in vitro through inhibition assay for
-amylase and -Glucosidase activity using metformin as the standard antidiabetic
drug.

209
The results of the trial indicated that aqueous and methanol extracts of EVM-
GBPU-NUT 3 revealed good antidiabetic properties
The aqueous extract was subjected to column chromatography and fractioned into
38 sub
6. Deliverable: NIL
7. Financial statement

Expenditure Statement for the year 2016-2017 (up to 31.3.2017)

A. Opening Balance as 01.4.2016 Nil

B. Grant received during 2016-17 Rs. 2,95,000.00

C. Total Rs. 2,95,000.00

D. Expenditure during the year 2016-167

i. TA Rs. 24,570.00

ii. Recurring Contingency Rs. 2,48,005.00

iii. Non-recurring Contingency Nil

E. Total Expenditure during the year 2016-17 Rs. 2,72,575.00

F. Closing Balance as on 01.04.2017 Rs. 22,425.00

210
ANNUAL REPORT (2016-2017)

ON

OUTREACH PROGRAMME
ON
ETHNOVETERINARY MEDICINE

BY

TAMIL NADU VETERINARY AND


ANIMAL SCIENCES UNIVERSITY
THANJAVUR, CENTRE
1
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211
OUTREACH PROGRAMME ON ETHNOVETERINARY MEDICINE
TAMIL NADU VETERINARY ANDANIMAL SCIENCES UNIVERSITY
THANJAVUR CENTRE

ANNUAL REPORT (2016-2017)

1. Name of Centre : Thanjavur Centre


2. Name of Investigators: N. Punniamurthy and N. Ramakrishnan
3. Approved activity:
i. Documentation of Ethno-Veterinary Medicinal recipes employed by
traditional healers
ii. Assessment of selected Ethno-Veterinary medicinal practices using animals
in field conditions

4. Salient Findings 2012-2017

In treatment of mastitis, Aloe vera, Curcuma longa and calcium hydroxide is found
to be clinically effective. It restores Somatic Cell Count, pH, Electric Conductivity of
and ABST of pathogen near to normal level.The combination possesses to have anti-
inflammatory, anti-oxidant and anti- microbial activities.
Supplementation with fresh Murraya koenigii leaves and Pedalium murex, can induce
multiple follicular development
Staphylococcus aureus is a major pathogen in the pathogenesis of bovine mastitis.
Biotin protein ligase, Penicillin binding protein, opuCB, sirA, sortase A and DNA
gyrase of S. aureus were found to be potential drug targets as they are necessary for
the survival and pathogenesis of the organism.
The molecular docking study revealed that the active ingredients from aloe vera and
turmeric interact with the proteins those play crucial role in Staphylococcus aureus.
Hence the mixture of aloevera, turmeric and lime can be used to treat bovine mastitis.

5. Concise report (01.04.2016 to 31.03.2017).

Plan of work for the period 2016-2017


1. Investigating the stability and physiochemical properties of fresh and dry anti-
mastitis herbal preparations.
2. Standardizing the quality of fresh and dry anti-mastitis herbal preparations.
3. Evaluating the impact of fresh and dry anti-mastitis herbal preparations on biofilm
2
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formation of clinical isolates from mastitis, subject to funds

212
Anti mastitis ethno veterinary herbal formulation was hard in texture, smooth
in touch, brick red in color, characteristic basic odour and pungent taste. Diagnostic
characters of microscopic analysis of test drug shows the presence of idioblast
containing raphides, Aloe exudates, chloronchymatous and aquiferous layers, Cork
and Fiber indicated the presence of Aloe vera. Elongated xylem fiber, reticulate xylem
vessel, parenchymatous cells filled with starch grains, brick layer like cork cells
indicated the presence of Curcuma longa.

Physico chemical parameters of herbal preparation were tabulated in Low


moisture content is always desirable for higher stability of drugs. The result of loss on
drying at 110 C of the formulation was 6.39%. A high ash value was obtained and
indicated the presence of in organic material such as calcium which is involved in the
formulation.

The extractive value of aqueous and ethanol was found to be 15.9 % and 9.5%
respectively. Phytochemical screening and quantitative analysis were performed. The
flavanoid content was found to be higher than phenolic and other phytochemicals. In
TLC analysis the Methanolic extract was used to spot and allow to run and 6 spots
observed in 366 nm and 3 spots observed in 254 nm using CAMAG TLC UV cabinet.
The Rf value is 0.43, 0.79 and 0.26 recognized the Aloin, Aloe emodin and Curcumin
respectively.

Stability study for fresh preparation


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213
DURATION

PARAME
Initi 24 hours 48 hours 72 hours 1 week Rema
TERS
al rks
4C 37C 4C 37C 4C 37C 4C 37C
Colour brow brow brow brow brow brow pale brow pale
n n n n n n n
Odour Pleas Pleas Pleas Pleas Pleas Pleas Pleasa Pleas Pleasa
ant ant ant ant ant ant nt ant nt
Taste Basic Basic Basic Basic Basic Basic Basic Basic Basic

pH 10.1 10 10 9.8 9.8 9.8 9.6 9.0 9.0


A.S.E 2.72 4.50 2.71 4.50 2.70 4.50 2.70% 4.48 2.70%
% % % % % % %
W.S.E 6.01 8.38 5.89 8.38 5.89 8.38 5.89% 8.36 5.86%
% % % % % % %

Curcumin 9.90 6.80 3.90 5.35 2.38 3.575 1.905 1.78 0.95%
% % % % % %
Anthraqu 0.54 0.47 0.39 0.31 0.31 0.23 0.15% 0.15 0.13%
inone % % % % % % %
Vitamin - 2mg/ 1.9mg 1.8mg 1.9mg 1.7mg 1.8mg 1.7mg/ 1.6mg 1.2mg/
C gm /gm /gm /gm /gm /gm gm /gm gm
Tot .Anti 0.130 0.066 0.106 0.063 0.102 0.063 0.097 0.033 0.045
Oxidant
vitamin
C equ.
Tot 0.050 0.040 0.036 0.030 0.024 0.02 0.016 0.010 0.008
Flavanoid
Mg
Quercitin
equ/gm
Tot 0.214 0.214 0.206 0.214 0.206 0.210 0.200 0.163 0.184
Phenol
Mg gallic
acid
equ/gm

Bacteria - - - - 9cfu/ - 17cfu/ - 400cfu


Count gm gm /gm
Fungi - - - - 3cfu/ - 12cfu/ - 200cfu
Count gm gm /gm
4
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Stability study of mastitis formulation

214
(Dry powder formulation)

Parameters Initial 12th month 24th month

Colour Brown Brown Pale Brown


Odour Pleasant Pleasant Woody
Taste Basic Basic basic
Ph 8.7 8.7 7.5
Water soluble 20.07% 18.50% 14.07%
extractive value
Alcohol soluble 7.52% 7.36% 6.89%
extractive value
Curcumin content 10.05% 9.52% 8.86%
Barbaloin content 0.94% 0.19 0.02%
Total antioxidant 0.17 0.14 0.10
activity
Total count
Bacteria absent absent 50cfu/gm
Fungi absent absent 30cfu/gm

Pharmacognostical and physiochemical analysis of antimastitis herbal formulation

Table 1: Ingredients of anti-mastitis herbal formulation

Name of ingredients Parts used Ratio


Aloe vera Whole plant 25 Parts
Curcuma longa Powder 5 Parts
Calcium oxide Powder 1 Part

Table 2: Organoleptic Character of antimastitis herbal formulation

Parameters Results
Color Brick red color
Odour Characteristic odour - Basic
Taste Pungent
Touch Soft
Texture Hard
5
Page

Table 3: Physico Chemical constants of antimastitis herbal formulation

215
Parameters Results
Solubility (Water) Soluble
Foreign maters Nil
Loss on drying 6.3988%
Total Ash Value 20.04%
Acid Insoluble ash 0.69%
Alcohol soluble extractive value 9.559% w/w
Water Soluble extractive value 15.956% w/w
pH 7.80

Table 4: Phyto chemical analysis of anti-mastitis herbal formulation

Components Phytochemical Phytochemical content (%)


Screening
Phenol + 14.7890.21
Tannin + 8.6510.41
Flavanoid + 16.569 0.23
Saponin + 1.256 0.61
Anthraquinone glycoside + 3.1520.58
Alkaloid + 0.7410.36
(+) indicates Presence, Values are expressed Mean Standard deviation

Table 5: TLC study of antimastitis herbal preparation

S.No Rf Values of spots - Methanolic Extract of antimastitis herbal formulation

366 nm 254 nm
1 0.26 0.47
2 0.43 0.75
3 0.61 0.90
4 0.66 -
5 0.68 -
6 0.79 -
6
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Figure 1: TLC Image: Methanolic extract of antimastitis herbal formulation

216
366 nm 254 nm

Figure 2: Powder Microscopy of antimastitis ethnoveterinary herbal preparation

7
Page

5.2 Field testing of EVM preparation against clinical mastitis, Thanjavur

217
i. California Mastitis test (CMT)
The CMT was conducted as described in standard procedures using CMT kit.
ii. Somatic cell count (SCC):
Somatic cells content in milk increases due to mammary gland infections.
SCC greater than 2, 00,000 cell/ml is a threshold level distinguishing healthy udder
from a diseased udder.The milk samples were thoroughly mixed and 10 ul of milk
was taken on the predrawn 1 cm2 marked area over a clean glass slide and uniformly
smeared using loop. The smear was dried and strained with modified Newmans
lampert stain for 2 min. The smear was washed in tap water and dried. The dried
smear was examined under oil immersion objective of microscope. The average
number of cells calculated after counting in 20 -30 different fields.
iii. pH
pH of the milk was assessed before and after treatment using pH meter
following standard procedures.
iv. Microbiological analysis of milk from udder before and after treatment was
done using standard procedures.
5.2.1 Incidence of mastitis
Out of 94 animals examined under field conditions, 67 animals were positive for
mastitis of which 7 animals could not be followed. The animals were treated for
mastitis using anti mastitis herbal preparation.
5.2.2 Anti mastitis herbal treatment
Aloe vera (250g), Curcuma longa (50g) and calcium hydroxide (10g) were ground and
made into thick paste. A handful of the paste was mixed with water to make dilute
solution.
The affected udder was stripped of the contents and washed thoroughly. The herbal
solution was applied all over the udder. The procedure was repeated every one hour till
the condition is cured.
5.2.3 Results
The effect of anti mastitis herbal therapy inflammatory reactions of the udder are
detailed in Table.1 Treatment with anti mastitis herbal preparation could effectively
eliminate intramammary infections and showed a significant decline in SCC (Fig.1) and
pH (Fig.2). The average time required for reduction in size of the udder from the
8

initiation of treatment was 4.71.15 days whereas the time required returning to normal
Page

milk yield was 5.91.39 days.

218
Fig 1. Effect of
herbal
therapy on
Somatic cell counts (SCC) before and after treatment

Fig 2. Effect of herbal therapy on pH of the milk before and after treatment
(Dr. J.Vijay Anand, faculty EVM centre, field testing the EVM herbal recipe)
9
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219
Parameter Before treatment After treatment

pH 7.40.44a 6.420.24b

SCC 5.470.78a 1.990.48b


Organisms Staphylococci E.coli
present Streptococcci
E.coli
CMT + Negative

Table.1 Effect of herbal therapy on inflammatory reactions of the udder

Dr. A. Elamurugan, faculty EVM centre field testing of EVM herbal recipe

5.3 Treatment of Sub-Clinical mastitis using Anti-mastitis fresh herbal


preparation

Total number of animals examined: 94


Total number of animals positive for subclinical mastitis: 67

5.3.1 Tests performed to diagnose subclinincal mastitis

I. White side test (WST):

WST was performed by adding one drop of 4% NaOH to 5 drops of milk on a


clean glass slide and mixed vigorously with a glass rod for 20 s. The
10

development of viscid mass was regarded as positive.


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220
Grading of the milk on the basis of precipitation

Grade Description
Mixture remains opaque and free of particles (-) Negative
Fine dispersed particles on close inspection (+/-) Trace
A definite thickening and mixture separates into milk (+)Distinct
whey and white particles positive
Mixture thickens immediately and follows glass rod (++) strong
positive

II. Somatic cell count (SCC):


Somatic cells content in milk increases due to mammary gland infections.
SCC greater than 2, 00,000 cell/ml is a threshold level distinguishing healthy
udder from a diseased udder.
The milk samples were thoroughly mixed and 10 ul of milk was taken on the
predrawn 1 cm2 marked area over a clean glass slide and uniformly smeared
using loop. The smear was dried and strained with modified Newmans lampert
stain for 2 min. The smear was washed in tap water and dried. The dried smear
was examined under oil immersion objective of microscope. The average
number of cells calculated after counting in 20 -30 different fields.
III. Reverse pharmacology studies on EVM herbal recipe.

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221
with

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222
Conventional Hydrogen bond

Carbon Hydrogen bond

Electrostatic bond

Hydrophobic bond

5.3.2 Conclusion

Staphylococcus aureus is a major pathogen in the pathogenesis of bovine mastitis.


Biotin protein ligase, Penicillin binding protein, opuCB, sirA, sortase A and DNA
gyrase of S. aureus were found to be potential drug targets as they are necessary for the
survival and pathogenesis of the organism.
The molecular docking study revealed that the active ingredients from aloe vera and
turmeric interact with the proteins those play crucial role in Staphylococcus aureus.
Hence the mixture of aloevera, turmeric and lime can be used to treat bovine mastitis.

5.4Adoption of Ethnoveterinary herbal preparation for mastitis treatment by various


organizations and their success stories
EVM training in Mastitis at Sabarkantha District co-operative milk producers Union
LTD, Gujarat, May 2016.

No of Recovery status
Type of mastitis animals Not
treated Complete % Partial %
recovered
Acute 223 199 89 18 8 6
Chronic 10 4 40 6 60 0
Sub clinical 8 6 75 2
13

*Incompletely treated cases were not considered


Page

223
Use of fresh herbal EVM recipe-Data from Sabar Dairy, Gujarat
Sample recording of milk data before and after EVM treatment done by sabra
Dairy, Gujarat June July 2016

14
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224
Sabar dairy has planted 5000 seedlings of Aloe vera for supply to the farmers to
use in mastitis in dairy animals. February 2017

5.5 Economic impact of using EVM for mastitis in organized Dairy

Use of Ethno-veterinary medicine (EVM) in mastitis treatment at Sabar dairy


More than 5000 cases of mastitis treated with 92% success through EVM alone
The number of visits decreased by 32,000 in 6 months as compared to the same
period from the previous year
Supply 5000 Aloe vera plants to sabar farmers
Establishment of ethno-veterinary demo plot at Sabar dairy
Training of 15 veterinarians on EVM is completed
Based on the success training of all Veterinarians (no.144) in the union on EVM
is to be taken up

15
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225
5.6 Treatment of mastitis using Anti-mastitis fresh herbal preparation under field
conditions
5.6.1 The benefits of EVM treatment in the long run

Increased
productio
n
Better
milk Reduction
quality in mastitis
incidence
Benefits
of treating
Reduction mastitis savings in
in milk with EVM treatment
spoilage costs

No
Reductio
n in SCC antibiotic
residues

The above observations are based on the field studies conducted across the country
through training of veterinarians and farmers

6. Deliverables
a. Training and Guest Lectures
Sl.No Kind of activity No.of
Beneficiaries
Men
Women
1 Training on Ethno-veterinary Practices for Cattle, 1517 965
Sheep/ goat and Poultry to Farmers at the EVM centre
16

Thanjavur.
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226
2 Guest Lectures
Ethnoveterinary practices for Primary healthcare of 200 farmers
livestock at 14 th National conference on Traditional
Medicine
Phytoproducts as an alternative to antibiotics for 250 students and
Livestock health and production at World Veterinary faculty
day celebration 2016, VCRI ,Namakkal-one health
concept
Ethnovetrinary practices in dairy farming at Vizhaga 50 vets
dairy Farmers, Vizhakapattam, Andhra Pradesh
Ethnovetrinary practices in dairy farming at Vizhaga 100 farmers
dairy for Veterinarians Vizhakapattam, Andhra
Pradesh
Ethnovetrinary practices in dairy farming and field 100 vets
demonstaration at Sabar dairy farmers, Anand, Gujarat.
Ethnoveterinary Practices for sustainable organic 40 NDDB
livestock farming and antibiotic free milk production, officers vets
Meeting with NDDB officers, Anand, Gujarat.
Ethnoveterinary Practices Refresher course for 850 farmers
farmers on organic farming, Erode, Tamil nadu
Ethnoveterinary Practices for primary health care of 150 farmers
livestock, Trichy, Tamil nadu
Ethnoveterinary Practices-Sri SS Seva organizations , 50 farmers
Coimbatore, Tamil nadu
Ethnoveterinary Practices, Thanjavur, Tamil nadu 50 farmers
Ethnoveterinary Practices-Annual Siddha Festival, Dr 250 public
MGR Janaki Arts College, Chennai. Tamil nadu
Ethnoveterinary Practices-Refresher training on 24 vets
Bovine breeding, Erode, Tamil nadu

Ethnoveterinary Practices-Refresher training on 24 vets


17

Bovine breeding, Erode, Tamil nadu


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227
Ethnoveterinary Practices-NDDB training for 24 vets
Veterinarians Coimbatore, Tamil nadu
Ethnoveterinary Practices-Third anniversary of 200 farmers
Nammalwar, Karur, Tamil nadu
Ethnoveterinary Practices Peruntholuvu, Tamil nadu 100 farmers
b. Conference /Seminar-Paper/Abstract Presentation and Awards
Sl.N Conference /Seminar Title/lecture Awards
1. 9 th Joint natural products A decade of clinical Invited lecture
conference, Cpenhagen, Research and
Denmark July 2016 applications of
Ethnoveterinary
knowledge in India-The
pragmatic way of
facilitating Medicinal
Plants to replace
synthetics in animal
Health and Production
2 National Symposium on A lead paper on Dr.N.Punniamur
Anilmal Health and Ethnopharmacology thy
Production-Challenges and Awarded
Oppurtunities for Fellow of
Veterinary Pharmacology Indian Society
and Toxi (ISVPT) at of Veterinary
Navsari, Gujarat Nov, 2016 Pahrmacology
and
Toxicology
3 7 th World Ayurveda 39 Abstracts on EVM
congress and Arogya Expo from PGDEVP from
at Kolkata on Dec 2016. the centre
4 National Symposium, Two Abstracts and One
Indian society for Lead paper.
18

Veterinary Medicine, at
Page

228
VCRI, Tirunelveli, Tamil
nadu Feb 2017
5 International conference on Lead paper on Dr.N.Punniamur
Herbal and Natural Reverse thy
components as the future of pharmacological Awarded
pharmacology and 7 th validation of a Fellow of
Annual meet of national clinically successful National
society for Ethnoveterinary Society
Ethnopharmacology at formulation For
Avinasilingam University, Ethnopharmacol
Coimbatore, Tamil nadu ogy
Feb and Mar 2017.
6 National symposium on Treatment of clinical Awarded third
Innovative Techniques, mastitis in cattle under prize in oral
Emerging Issues and field conditions using presentation on
advancement in Vetrinary fresh EVM herbal Alternate
medicine to meet the preparations medicine
challenges : Present and the session.
future, Feb 2017

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229