World Journal of Pharmaceutical Research

Sathyamurthy et al. SJIF Impact

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Research
Volume 5, Issue 6, 1711-1722. Research Article ISSN 2277– 7105

RENAL PROTECTIVE ACTIVITY OF HELIOTROPIUM INDICUM

IN RIFAMPICIN INDUCED NEPHROTOXICITY RATS

Mokhalad Waheeb Abdullah1, Jaffar S. K.1, Ghousia Begum2 and

Balasubramanian Sathyamurthy3*

1
Department of Biochemistry, Acharya Nagarjuna University, Guntur, (A.P), India.
2
Department of Biochemistry, Bangalore University, Bangalore, India.
3
*Associate Professor, Department of Biochemistry, M S Ramaiah College of Arts, Science
and Commerce Bangalore – 560054.

Article Received on
11 April 2016,
Revised on 01 May 2016,
Accepted on 21 May 2016
DOI: 10.20959/wjpr20166-6393
*Corresponding Author
Prof. Balasubramanian
Sathyamurthy
Associate Professor,
Department of
Biochemistry, M S Ramaiah
College of Arts, Science and
Commerce Bangalore –
560054.
ethnolic extract of Heliotropium indicum possesses nephroprotective activity.
ABSTRACT
The nephro protective effect of whole plant of ethanolic extract of
Heliotropium indicum was confirmed by our study. In the present
study, the whole plant of ethanolic extract of Heliotropium indicum
significantly reduced the toxicant elevated levels. It was observed the
extract of Heliotropium indicum treated group animals were found to
reduce such changes in kidney histology induced by Rifampicin,
indicating nephroprotection. Heliotropium indicum contain phenols,
flavonoids, triterpenoids, alkaloids and saponins offers organ
protection by virtue of their free radical scavenging activity. Phyto
constituent analysis showed the presence of phenols, flavonoids,
triterpenoids, alkaloids and saponins as phytoconstituents. Based on
improvement in serum and urine marker levels, histopathological
studies, antioxidant enzymes and presence of phytoconstituents the

KEYWORDS: Rifampicin, nephrotoxicity, Heliotropium indicum, ethanolic extract, soxhlet

apparatus.

INTRODUCTION
Kidneys the primary organs of the urinary system, does purification of the blood by removing
wastes from it and excreting them from the body in urine. Nephrotoxic injury is damage to
one or both of the kidneys that is due an exposure to a toxic material, mostly through

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ingestion. Nephrotoxic injury can cause acute renal failure, in which the kidneys lose their
ability to function suddenly, or chronic renal failure, in which functions of kidney slowly
deteriorates. If unchecked, renal failure can also result in death. Over 50 drugs have been
reported as causes of acute interstitial nephritis (AIN), including penicillin’s, sulphonamides,
NSAID. Other drugs that are well known to cause this condition include ciprofloxacin,
Rifampicin, phenytoin and cephalosporins. The underlying Immunological mechanism
remains to be elucidated.[1]

Rifampicin a potent drug is the main drug in the multidrug therapy for lung tuberculosis. It
can induce hepatitis and nephritis in hypersensitive patient. Drug induced nephrotoxicity is
often associated with marked elevation of biochemical parameters like urea, creatinine and
uric acid in blood and urine and decrease in serum protein levels.[2]

Heliotropium indicum Linn, is also known as ‘Indian heliotrope’ is an annual plant that is a
common weed in west places and settled areas. It is native to Asia. It is very common in India
and some parts of Africa and Bangladesh, but also found in other countries.[31] Many reports
had indicated wound healing activity of Heliotropium indicum. They showed that topical
application of 10% w/v of Heliotropium indicum increased the percentage of wound
contraction and completed.[3]

The present study was conducted to demonstrate protective effect of Heliotropium indicum
against Rifampicin induced nephrotoxicity in Rats. Rifampicin, is an antibiotic used to treat
number of bacterial infections such as tuberculosis and leprosy and requires long term
treatment which induces toxic effects on kidney, liver, GIT and cardiovascular systems.
Hence treatment with such antibacterial agents requires adequate protective measures against
organ toxicity. In this study the whole plant of heliotropium indicum has been selected for
studying nephroprotective activity on experimentally induced nephrotoxity in rats.

METHODOLOGY
Pharmacological evaluation
Animals
Female wistar rats were procured and they were housed in microloan boxes with standard
laboratory diet and water ad libitum at temperature of 22˚ c ± 2˚ c and humidity of 45%-64%.
The study was conducted post obtaining Institutional Animal Ethical Committee Clearance.

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Nephroprotective studies
Rats were divided into five groups each group consisting of six animals.[4]

Group 1: Normal control: Administered with equivalent volumes of 5ml/kg of normal saline
(0.9%Nacl) for 2 weeks.
Group 2: Positive control: Received Rifampicin (1g/kg/bodyweight) every 72 hrs for 2 weeks
to induce nephrosis.
Group 3: Standard: Received cystone (500mg/kg/bodyweight) and simultaneously after 30
min. administered Rifampicin (1g/kg/bodyweight) every 72 hrs for 2 weeks.
Group 4: Given Ethanolic extract of Heliotropium Indicum (100mg/kg/body weight) and
simultaneously after 30 min. administered Rifampicin (1g/kg/body weight) every 72 hrs for 2
weeks.
Group 5: Given Ethanolic extract of Heliotropium Indicum (200mg/kg/body weight) and
simultaneously administered after 30 min. Rifampicin (1g/kg/body weight) every 72 hrs for 2
weeks.

The study was carried out for 2 weeks. On 15th day all the animals were slaughtered under
diethyl ether anaesthesia. Blood samples were collected, allowed to clot and serum separated
by centrifuging at 2500 rpm for 15 mins. It was then analysed for the various biochemical
parameters.

Assessment of kidney function

Biochemical parameters i.e., Estimation of urinary and Blood parameters like urea,
Creatinine, uric acid and total proteins were analyzed according to the reported photometric
methods.

The kidneys were removed, weighed and their morphological changes were observed. A 10%
of kidney homogenate was used for antioxidant studies like superoxide dismutase (SOD),
catalase, lipid peroxidation (LPO) and glutathione peroxidase (GPx).

A portion of kidney was fixed in 10% formalin for carrying out histopathological studies.

Biochemical parameters estimation

The biochemical parameters were estimated as per the standard procedure prescribed by the
manufacturer’s instruction manual provided in the standard kit using photometric systems.
1. Estimation of Urea - GLDH - Kinetic method[5]:

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2. Estimation of Creatinine – Modified Jaffe’s kinetic method[6]:

3. Estimation of Uric Acid - Uricase/ PAP method[7]:
4. Estimation of serum total protein[8, 9]:

Antioxidant studies
Renal cortex tissues were homogenized in KCl (10 Min), phosphate buffer (1.15%) with
EDTA (pH 7.4) and were centrifuged at 12000 rpm for 60 min. The supernatant was used for
assay of marker enzymes.
a. Estimation of Catalase[10, 11]:
b. Estimation of Lipid Peroxidation[12]:
c. Estimation of Glutathione peroxidase[13]:
d. Estimation of Superoxide dismutase[14]:

Histopathological studies
A histopathological study of kidneys was performed in histopathology laboratory by
consultant histopathologist.

Statistical analysis
The values were expressed as Mean ± SEM. Statistical analysis was performed by
(ANOVA), one way analysis of variance and by Dunnett’s multiple comparison test. P values
< 0.05 were considered as significant.

RESULTS
Table - 1: Data Showing Preliminary Phytochemical Screening of the ethanolic extract
of Heliotropium Indicum
Phytoconstituents Present or absent
Carbohydrates +
Glycosides +
Gums & mucilage _
Proteins & amino acids +
Saponins +
Tannins +
Steroids +
Flavonoids +
Alkaloids +
Triterpenoids +
Fixed oils and fats +

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Biochemical parameters
Table – 2: Rifampicin treated group of animals the concentration of urea, uric acid,
creatinine in urine and serum
Creatinine Total protein
Group

Urea (mg/dl) Uric acid (mg/dl)

Drug treatment (mg/dl) (mg/dl)
Serum Urine Serum Urine Serum Urine Serum
39.37 29.28 0.841 25.75 0.96 0.14 6.60
1 5ml/kg NS
±0.44 ±0.58 ±0.33 ±0.49 ±0.03 ±0.06 ±0.2
95.23 74.52 1.93 50.23 2.29 1.96 4.37
2 1g / kg Rifampicin
±0.38 ±0.49 ±0.05 ±0.32 ±0.08 ±0.31 ±0.33
500mg / kgCystone + 30 30.85 0.95 34.54 1.47 0.61 6.16
3
1g/kg Rifampicin ±0.57* ± 0.50* ±0.05* ± 0.34* ±0.05* ±0.21* ±0.28*
100mg/kgHIEA + 1g/kg 30.6 35.53 0.71 37.25 0.71 0.54 6.26
4
Rifampicin ±0.95* ± 0.32* ±0.05* ±0.51* ±0.05* ±0.04* ±0.21*
200mg/kgHIEA+1g/kg 27.16 33.92 0.25 26.09 0.76 0.35 6.8
5
Rifampicin ±0.94* ± 0.44* ±0.39* ±0.41* ±0.04* ±0.03* ±0.36*
All values are expressed as Mean ± SEM, N = 6 animals in a group. One way analysis of
variance (ANOVA) followed by multiple comparison Dunnett’s test *P<0.001 as compared
to positive control group.

Fig. 1: The concentration of urea in serum and urine in Rifampicin treated group of
animals.

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Fig. 2: The concentration of uric acid in serum and urine in Rifampicin treated group of
animals.

Fig. 3: The concentration of creatinine in serum and urine in Rifampicin treated group
of animals.

Fig. 4: The concentration of Total Protein in serum in Rifampicin treated group of

animals.

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From the Table – 2, Fig.1, Fig.2, Fig.3 and Fig.4 the following observations were found.
In Rifampicin treated group of animals the concentration of urea, uric acid, creatinine in urine
and serum were considerably increased than the normal animals (group 1).

Total protein in serum was decreased compared to normal group which indicates severe
nephrotoxicity.

Treating (group 4 & 5) with ethanolic extract of HIEA showed significant decrease (p<0.001)
in concentration of urea, uric acid, creatinine in blood and urine and significant increase of
total protein in blood compared to Rifampicin treated group 2.

Antioxidant parameters
Table - 3: Antioxidant parameter values in Rifampicin treated group of animals
Catalase Lipid peroxidise Glutathion peroxidise Superoxide
Group

(μmole of H2O2/ Sec / (nmol MDA/ (μ mole of oxidized GSH / dismutase

mg protein/ml) min × mg protein) min ×mg protein) (U/mg protein)
1 16.34 ± 1.623 9.83 ± 0.214 35.09 ± 1.976 19.94 ± 0.751
2 7.48 ± 0.953 20.57 ± 0.482 20.56 ± 1.169 7.66 ± 0.41
3 15.28 ±1.423* 14.86 ± 0.404* 29.71 ± 0.639* 17.23 ± 0.623*
NS NS NS
4 10.48 ± 0.515 18.73 ± 0.487 24.31 ± 0.963 11.71 ± 0.656*
5 14.46 ±1.526* 15.97 ± 0.818* 27.88 ± 0.995* 15.91 ± 0.79*
All values are expressed as Mean ± SEM, N=6 animals in a group. One way analysis of
variance (ANOVA) followed by multiple comparison Dunnett’s test.
Ns; indicates no significant.*P<0.05.

Fig. 5: Catalase Concentration in Rifampicin treated group of animals

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Fig. 6: Lipid peroxidise Concentration in Rifampicin treated group of animals

Fig. 7: Glutathione peroxidise Concentration in Rifampicin treated group of animals

Fig. 8: Superoxide Dismutase Concentration in Rifampicin treated group of animals

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From the Table – 3, Fig.5, Fig.6, Fig.7 and Fig.8 the following observations were found
Considerably decrease in activity of Catalase, SOD and glutathione peroxidase in rifampicin
treated animals (group 2) when compared to normal animals (group 1) was observed.

Treatment with heliotropium extract significantly prevented decrease in the level of catalase,
SOD, GPx activity compared to rifampicin treated rats (group 2).

Nevertheless considerable increase in activity of lipid peroxidase in rifampicin treated

animals (group 1) was observed.

Treatment with ethanolic extract of heliotropium significantly prevented increase in the level
of lipid peroxidase. Thus strongly inhibit lipid peroxidation in isolated tissue via its
antioxidant activity.

DISCUSSION
In the present study nephrotoxicity in rats was induced by administration of drug Rifampicin.
This toxicity characterized by marked elevation in the circulating levels of urea, creatinine
and uric acid in blood and urine and decrease in total protein levels and histological features
of interstitial nephritis in group 2 rats when compared to untreated (group 1) rats. However
these changes were attributed by pre-treatment with oral administration of ethanolic extract of
Heliotropium indicum for 2 weeks.

Oral administration of plant extract significantly decreased the urea, uric acid and creatinine
but the total protein levels were elevated in both treatment group compared to toxicant group.
In renal diseases, the serum urea accumulates as the rate of serum urea production is greater
than the rate of its clearance. The elevations of urea and creatinine levels in serum were taken
as the index of nephrotoxicity. Creatinine is often found from endogenous sources by tissue
creatinine breakdown. Therefore, serum urea concentration is mostly considered as a reliable
renal function prediction than serum creatinine. It was established that rifampicin-induced
Acute tubular interstitial nephritis have demonstrated the occurrence of circulating
antirifampicin antibodies and immunoglobulin G (IgG) deposits along the tubular basement
membrane as well as containing immunoglobulin light chains in tubular lumens.

Rifampicin is known to decrease the activities of catalase, glutathione peroxidase and the
reduced level of glutathione. Therefore it is no doubt to assume that the nephroprotection
showed by Heliotropium ethanolic extract is mediated through its potent antioxidant effect. A

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relation between the oxidative stress and nephrotoxicity has been well studied in many
experimental animal models.

In Rifampicin treated rats there was a significant increase in lipid peroxidation products
(MDA) indicating the presence of oxidative stress. In histopathological study of saline treated
group shows some blood vessels are dilated as well as congested within interstitium with few
scattered mononuclear inflammatory infiltration. Rifampicin treated group showed diffuse
glomerular congestion, Tubular casts, Peritubular congestion, epithelial desquamation, and
Blood vessel congestion. While treatment group (100 mg/kg) showed Focal glomerular
congestion, Peritubular congestion, Focal hydrophic degeneration of tubular epithelial cells
and treatment group (200 mg/kg) shows only some of the blood vessels are dilated and
congested within the interstitium. Also few scattered mononuclear inflammatory infiltration
is seen within the interstitium.

From histopatological results Heliotropium ethanolic extract at dose of 100 mg/kg have
partial protective effect while Heliotropium ethanolic extract at dose of 200 mg/kg have
protective effect on Rifampicin induced nephrotoxicity. The findings suggest the potential
uses of ethanolic extract of Heliotropium indicum are therapeutically useful as
Nephroprotective agent. Further studies on this will help to explain their mechanisms of
action should be conducted in order to discover new therapeutic agents for renal disease
treatment.

CONCLUSION
The nephroprotective effect of whole plant of ethanolic extract of Heliotropium indicum was
confirmed by the following measures: In Rifampicin treated group rise in urea, uric acid and
creatinine in blood and urine and decrease in the level of protein. The same is observed in
kidney diseases in clinical practice and hence are having diagnostic importance in the
assessment of kidney function.

In the present study, the whole plant of ethanolic extract of Heliotropium indicum
significantly reduced the toxicant elevated levels of above mentioned parameters and increase
in the levels of protein. Hence, at this point it is concluded that the extract of Heliotropium
indicum offers nephroprotection.

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In Rifampicin treated animal’s glomerular, peritubular and blood vessel congestion and result
in presence of inflammatory cells in kidney sections. In the present study, the extract of
Heliotropium indicum treated group animals were found to reduce such changes in kidney
histology induced by Rifampicin, indicating nephroprotection.

Further documented reports reveal that, plant material containing phenols, flavonoids,
triterpenoids, alkaloids and saponins offers organ protection by virtue of their free radical
scavenging activity. Phytochemical analysis showed the presence of phenols, flavonoids,
triterpenoids, alkaloids and saponins as phytoconstituents. Hence, the role of these
phytoconstituents as free radical scavengers and consequent nephroprotection cannot be ruled
out.

Based on improvement in serum and urine marker levels, histopathological studies, level of
antioxidant enzymes and presence of phytoconstituents, it is concluded that the ethnolic
extract of Heliotropium indicum possesses nephroprotective activity and thus supports the
traditional application of the same under the light of modern science.

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