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1 Hepatoprotective Activity
Pushpagiri College of Pharmacy, Tiruvalla, Kerala, India.
E-mail:sujipillai76@yahpp.co.in
The animals were divided into five groups consisting of six
*Corresponding author rats in each group. [4] Group I: Animals received single daily
dose of 1% aqueous acacia on all 5 days (1 ml/kg, p.o.) and
2
K.K College of Pharmacy, Chennai, Tamil Nadu, India. olive oil (1 ml/kg , s.c.) on days 2 and 3. Group II: Animals
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Group I II III IV V
Liver Weight 33.15r0.671 46.69r0.376a 41.53r0.442b 36.98r0.461b 35.41r0.269b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Serum (U/ml) 28.83r0.87 179.16r3.64a 122.83r2.23b 34.00r1.53b 31.33r1.28b
Liver(μmolof pyruvate liberated/mg protein/min) 30.66r0.76 171.83r0.96a 107.33r2.82b 42.20r3.39b 34.00r1.29b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Serum (U/ml) 16.66r0.61 124.33r2.16a 74.33r2.66b 24.66r0.71b 19.66r1.20b
Liver(μmolof pyruvate liberated/mg protein/min) 14.83r0.40 99.33r1.12a 67.5r1.12b 24.83r1.28b 18.5r1.18b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Serum (IU/L) 115.43r1.66 234.66r5.35a 208.21r3.01b 157.42r1.89b 146.74r2.36b
P values-a<0.001 vs group I, b<0.001 vs group II., Values are meanrSEM of 6 animals in each group
Group I II III IV V
Serum 0.307r0.01 1.919r0.12a 0.832r0.04b 0.624r0.01b 0.493r0.01b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Serum (mg/dL) 7.854r0.3 5.12r0.02a 5.506r0.02b 6.849r0.03b 7.376r0.050b
Liver (mg/g tissue) 0.7817r0.001 0.5164r0.004a 0.5748r0.003b 0.6529r0.002b 0.7290r0.004b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Liver 314.11r6.06 190.85r4.941a 238.44r10.063b 270.33r5.069b 295.12r5.796b
P values-a<0.001 vs group I, b<0.001, c<0.05 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Liver 0.334r0.001 0.223r0.008a 0.256r0.001b 0.298r0.001b 0.309r0.003b
P values-a<0.001 vs group I, b<0.001, c<0.01 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Liver 25.56r0.348 14.38r0.335a 16.16r0.187b 18.54r0.120b 20.36r0.295b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
received single daily dose of 1 % aqueous acacia (1 ml/kg, aqueous extract of Ventilago madraspatana root bark on all
p.o.) for 5 days and carbon tetra chloride (2 ml/kg, s.c.) 5 days and carbon tetrachloride solution
administered on days2 and 3. Group III: Animals were (2 ml /kg, s.c.) on day 2 and 3, 30 minutes after
treated with 200 mg/kg, p.o. of aqueous extract of administration of extract. Group V: Animals were treated
Ventilago madraspatna root bark on all 5 days and carbon with 1 ml/kg p.o. of liv-52 syrup on all 5 days and carbon
tetrachloride (2 ml/kg, s.c.) administered on days 2 and 3. tetrachloride solution (2 ml/kg, s.c.) on day 2 and 3, 30 min
Group IV: Animals were treated with 400 mg/kg, p.o. of after administration of drug. All the animals were scarified
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Group I II III IV V
Liver 9.62r0.261 5.43r0.399a 6.79r0.229c 7.50r0.227b 8.56r0.156b
P values-a<0.001 vs group I, b<0.001, c<0.01 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Liver 77.51r0.257 52.22r0.272a 60.68r0.579b 69.69r0.410b 74.29r0.389b
P values- a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Liver 5.11r0.595 13.99r0.875a 10.74r0.488b 7.91r0.430b 6.62r0.536b
P values-a<0.001 vs group I, b<0.001vs group II, Values are meanrSEM of 6 animals in each group
Group I II III IV V
Liver 3.51r0.029 1.65r0.042a 1.89r0.023b 2.19r0.017b 2.63r0.017b
P values-a<0.001 vs group I, b<0.001 vs group II, Values are meanrSEM of 6 animals in each group
(d) (e)
Figure 1: (a) Normal animal (Group I), (b) CCl4 toxic animal (Group II), (c) Aqueous extract 200 mg/kg & CCl4 treated animal (Group
III), (d) Aqueous extract 400 mg/kg CCl4 treated animal (Group IV), (e) Liv-52 syrup (1 ml/kg) & Cl4 treated animal (Group V)
by cervical decapitation under light ether anaesthesia, on homogenate was prepared in 0.1 M Tris-HCl buffer pH 7.4.
the fifth day for estimation of biochemical parameters and The homogenate was centrifuged at 3000 rpm for 10
for histopathological studies. The liver was collected and minutes and the supernatant was used for the assay of
wet weight of liver was noted as mg weight of liver/gm marker enzymes. The following biochemical assays were
body weight. carried out in serum, serum glutamic oxaloacetate
transaminase (SGOT), serum glutamic pyruvate
Biochemical Study transaminase (SGPT), [5] alkaline phosphatase (ALP), [6]
Blood was collected from jugular veins, and allowed to clot total bilirubin, [7] total protein. [8] The supernatant liver
for 30-40 min. Serum was separated by centrifuging at homogenate was used for the assay of the following
3000 rpm for 10 minutes. Immediately after sacrifice, the enzymes and non-enzymes, glutamic oxaloacetate
liver was dissected out, washed in ice cold saline, and a transaminase (GOT), glutamic pyruvate transaminase
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(GPT), total protein, glutathione peroxidase(GPx), [9] bark treated rats showed significant decrease (p<0.001) in
glutathione-s-transferaseGST). [10] Glutathionereductase total bilirubin when compared to CCl4 treated rats. The Liv-
(GRD), [11] superoxide dismutase (SOD), [12] catalase (CAT), 52 (1 ml/kg) treated animal (group V) also showed
[13] lipid peroxidation (LPO), [14] vitamin E. [15] All the significant (p<0.001) decrease of total bilirubin level when
enzymes assay were undertaken at particular nm using compared to group II animal.
shimadzu make spectophotometer, UV–1601 model.
Total Protein
Histopathological Studies A significant (p<0.001) reduction in the total protein of
Small pieces of liver tissues were collected in 10% serum and liver was observed in CCl 4 treated rats (group
formalin solution for preparation of sections by using of II) when compared to control (group I) (Table 6).
microtome. [16] Treatment with aqueous extract (200 mg/kg and 400
mg/kg showed a significant (p<0.001) increase of protein
Statistical Analysis level, when compared to (group II) animals. The Liv-52 (1
The data are expressed as mean±SEM. Statistical analysis ml/kg) treated animals (group V) also showed significant
was done using one way analysis of variance (ANOVA) increase in protein level when compared to CCl 4
followed by dunnet test. [17] challenged (group II) rats.
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extract significantly increased (p<0.001) the CAT level aqueous extract of Ventilago madraspatana. Alkaline
when compared to group II animals. Liv-52 (1 ml/kg) phosphtase (ALP) is a membrane bound glycoprotein
treated group also showed significant (p<0.001) increase of enzyme, with high concentration is sinusoids and
catalase level when compared to group II. endothelium. ALP reaches the liver mainly from bone. It is
excreted into the bile so its elevation in serum occurs in
Lipid Peroxidation (LPO) hepatobiliary diseases. [21] The aqueous extract of Ventilaga
The lipid peroxide of liver homogenate was significantly madraspatana root bark probably stabilised the hepatic
increased (p<0.001) in CCl4 challenged rats (group II) when plasma membrane from CCl4 induced liver damage. The
compared to control rats (group I) (Table 12). Treatment serum albumin level is low in hepatic diseases. The present
with aqueous (200 mg/kg and 400 mg/kg) extract showed results reveal that when animals pretreated with Ventilago
significant decrease in LPO level when compared with CCl4 madraspatana extract prior to the challenge with CCl4, the
treated (group II) animals. The Liv-52 (1 ml/kg) treated liver biosynthesis of protein continues to be unaffected. The
group also showed a significant (p<0.001) decline in the metabolic transformation of aminoacids occurs in liver by,
LPO level when compared to group II animals. transamination protein metabolism may be impaired due
the escape of both non proteins and protein nitrogenous
Vitamin E substances from injured liver cells as evidenced by raise in
Vitamin E activity of liver was significantly (p<0.001) the serum enzyme levels of GOT, GPT and ALP. Glutathione
reduced in CC14 treated animals (group II), when peroxidase (GPx) plays a pivotal role in H2O2 catabolism [22]
compared to control animals (group I) (Table 13). The and the detoxification of endogenous metabolic peroxides
aqueous (200 mg/kg and 400 mg/kg) extract showed and hydroperoxides which catalyses GSH. [23] GPx activity
significant increase (p<0.001) of Vitamin E level in dose was significantly reduced in the CCl4 treatment when
dependent manner when compared to group II animals. compared to control. The reversal of the GPx activity to
Liv-52 (1 ml/kg) treated group also showed significant normal after pretreatment with the plant extract exhibits the
(p<0.001) increase in Vitamin E level when compared to antioxidant activity of the extract in scavenging or
group II. detoxifying the endogenous metabolic peroxides generated
after CCl4 injury in the tissues. Many investigators have
Histopathological Studies suggested that Glutathione-S-transferase (GST) offers
Group I treated animals showed dilated central vein, protection against LPO by promoting the conjugation of toxic
normal hepatocytes. Group II treated animals showed electrophiles with reduced glutathione (GSH). [24] GST plays a
perivenular inflammatory infiltration and hepatocytic fatty physiological role in initiating the detoxification of potential
change, diffuse mild hepatocellular vacuolation. Group III alkalating agents. Chemicals like chloroform, CCl4 etc. alter
treated animals showed change central vein, mild fatty the hepatic Glutathione-S-transferase activity. [25] GST level
change. Group IV treated animals showed dilated central was significantly reduced in CCl4 treated animals and
vein, mild sinusoidal dilation and no hepatocellular upward reversal was observed after the treatment with
damage. Group V treated animals showed normal central aqueous extract of the plant. The present study reveals the
vein and mild hepatocytic fatty change. This result extract along with other protective mechanism also increase
correlates well with the biochemical findings. From the the autoprotection of the liver function by the GRD level. In
results of the histopathological studies, administration of the present study the superoxide dismutase activity is
aqueous extract of Ventilago madraspatana root bark significantly reduced in CCl4 intoxicated rats. The SOD
reveals reduced cellular damage induced by CCl4, the activity was brought to near normal after treatment with the
widely used hepatotoxicant (Figure 1). extract in CCl4 intoxicated rats. Decreased activity of catalase
was observed in group II animals treated with CCl4.
DISCUSSION Presumably a decrease in catalase activity could be
The present study has demonstrated hepatoprotective attributed to cross linking and inactivation of the enzyme
activity with increase in in-vivo antioxidant status of aqueous protein in the lipid peroxides. Decreased catalase activity is
extract of Ventilago madraspatana root bark in CCl4 induced linked up to exhaustion of the enzyme as a result of oxidative
liver injury in rats. Damage to the structural integrity of liver stress caused by CCl4. The catalase activity was restored to
is reflected by an increase in the levels of serum transminase normal after treatment with extract evidently shows that
[18] because these are cytoplasmic in location and released antioxidative property of the extract against oxygen free
into circulation after cellular damage. [19] It is generally radical. Carbon tetrachloride treatment may elevate the level
accepted that hepatotoxicity of carbon tetrachloride is of malondialdehyde (MDA) a product of lipid peroxidation.
attributes to trichloromethyl free radical, and this free Therefore an increase in the liver MDA level indicates an
radical reacts rapidly with oxygen to form a increase in the degree of lipid peroxidation, a well known
trichloromethylperoxy radical, which may contribute to the biochemical mechanism of liver damage. [26] A significant
hepatotoxicity and subsequent increase in hepatic enzymes. decrease in the levels of lipid peroxides in aqueous extract of
[20] In this context we have observed a rise in the levels of the Ventilago madraspatana root bark extract pre-treated
GOT and GPT in carbon tetrachloride treated rats due to rats suggests that the extract may have the ability to protect
toxic compounds affecting liver and the reversal of the the liver from free radical injury induced by
elevated enzymatic levels to normal after the treatment with carbontetrachloride. The level of Vitamin E was significantly
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depleted in carbon tetrachloride intoxicated rats. The 12. Misra H P, Fridovich I. The role of superoxide anion in the auto
Ventilago madraspatana root bark aqueous extract pre oxidation of epinephrine and a simple assay for superoxide
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