RESEARCH ARTICLE

Hepatoprotective Effects of Aqueous Extract of Ventilago

madraspatana Gaertn. Root- Bark on CCl4 Induced Damage in
Rats
K Sujith1*, K Shanmuganathan2, C Ronald Darwin2, Anitha Mary Mathews1
Abstract: The root bark of V. madraspatana is used in traditional medicine for the treatment of liver disorders. In this study the
rats were divided into five groups, control group, negative control (carbontetrachloride), extract treated groups at dose of 200
mg/kg and 400 mg/kg and a standard treated group. Hepatoprotective activity of aqueous extract of Ventilago madraspatana
root- bark on CCl4 induced damage in rats was investigated by estimating the biochemical parameter in both serum and liver of
extract treated group. The extract treated groups showed significant decrease in serum glutamic oxaloacetate transaminase
(SGOT), serum glutamic pyruvate transaminase, alkaline phosphatase (ALP), total bilirubin and increase in total protein. The
liver homogenate of extract treated group showed decrease in total protein and inhibited lipid peroxidation (LPO) and increased
the levels of glutathione peroxidase(GPx), glutathione-s-transferase(GST), glutathionereductase(GRD), superoxide dismutase
(SOD), catalase (CAT) and vitamin E level. Histopathological studies of rats treated with aqueous extract of Ventilago
madraspatana root bark reveals reduced cellular damage induced by CCl4. The results of present study suggest aqueous extract
of Ventilago madraspatana root bark protects CCl4 induced chronic hepatotoxicity in rats by restoring the antioxidant status.

INTRODUCTION A voucher specimen (no: KK/P/023) is kept in the

The herbal medicines are effective in the treatment of
various ailments. Very often these drugs are
unscientifically exploited and improperly used. The
detailed investigation and documentation of plants used in
local health traditions and pharmacological evaluation of
these plants and their taxonomical relatives can lead to the
development of invaluable plant drugs for many dreaded
diseases. Ventilago madraspatana Gaertn. (Family-
Rhamnaceae), indigenous to India, is a large evergreen
woody climber with long sarmentose branches.
Traditionally, the root bark of V. madraspatana is used as a
carminative, stomachic, tonic dyspepsia, debility, skin
diseases, liver disorders and stimulant. The powdered stem
bark mixed with gingelly oil is applied externally to treat
skin diseases and itch. [1] The root bark contain
naphthalene derivatives and several naphthaquinones,
anthraquinones-islandicin, xanthorinand its 5-methyl
ether. [2] Therefore based on the above facts, as no
scientific study have been carried out regarding the
hepatoprotective activity of Ventilago madraspatana.The
present study has been undertaken to evaluate the
antioxidant and the hepatoprotective activity of aqueous
extract of Ventilago madraspatana root bark in CCl4
intoxicated liver injury in rats.
MATERIALS AND METHODS
Plant Materials
The basic plant material of Ventilago madraspatana root
bark used for the investigation was purchased from the R.
R. Herbals Chennai. The plant was identified and
authenticated by chief botanist of Captain Srinivasa Multi
Drug Research Institute for Ayurveda, Chennai, Tamil Nadu.
department for future reference.
Preparation of Aqueous Extract
The dried root bark of this plant was coarsely powdered.
The powder was then extracted with distilled water using
soxhlet extractor. The aqueous extract was dried under
reduced pressure using a rotary flash evaporator. The
extract thus obtained was used for the preliminary
phytochemical screening and pharmacological studies. The
extracts were administered to the animals as suspension in
1% aqueous gum acacia. The percentage yield of aqueous
extract was 7% w/w respectively.
Experimental Animals
Colony inbred adult wistar male rats (150-200 gms) were
used in the pharmacological studies. The animals were
maintained in well ventilated room temperatures with
natural day-night cycle in polypropylene cages. They were
fed balanced rodent pellet diet and tap water ad libitum
throughout the experimental period. The animals were
housed for one week, prior to the experiments to
acclimatize to laboratory conditions. The animals were
randomly distributed into five different groups with six
animals in each group. The institutional animal ethical
committee approved the protocol (IAEC IX-2/LBCP/03)
and care of animals was taken as per guidelines of
committee for the purpose of control and supervision in
experiments on animals (CPCSEA), representative of
animal welfare, Govt. of India.
Phytochemical Evaluation
Aqueous extract of the root bark of V. madraspatana was
studied for its phytochemical constituents. [3]

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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 RESEARCH ARTICLE

Table 1: Weight of Liver (mg/g Body Weight)

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

Table 2: Glutamate Oxaloacetate Transaminase (GOT / AST)

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

Table 3: Glutamate Pyruvate Transaminase (GPT / ALT)

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

Table 4: Alkaline Phosphatase (ALP)

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

Table 5: Total Bilirubin (mg/dL)

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

Table 6: Total Protein

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

Table 7: Glutathione Peroxidase (GPx) (n Moles of GSH Oxidised/min/mg Protein)

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

Table 8: Glutathione-S-transferase (GST) (Units/mg Protein)

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

Table 9: Glutathione Reductase (GRD)(n Moles of GSSG Utilised/min/mg Protein)

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,
p.o.) for 5 days and carbon tetra chloride (2 ml/kg, s.c.)
administered on days2 and 3. Group III: Animals were
treated with 200 mg/kg, p.o. of aqueous extract of
Ventilago madraspatna root bark on all 5 days and carbon
tetrachloride (2 ml/kg, s.c.) administered on days 2 and 3.
Group IV: Animals were treated with 400 mg/kg, p.o. of
aqueous extract of Ventilago madraspatana root bark on all
5 days and carbon tetrachloride solution
(2 ml /kg, s.c.) on day 2 and 3, 30 minutes after
administration of extract. Group V: Animals were treated
with 1 ml/kg p.o. of liv-52 syrup on all 5 days and carbon
tetrachloride solution (2 ml/kg, s.c.) on day 2 and 3, 30 min
after administration of drug. All the animals were scarified

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 RESEARCH ARTICLE

Table 10: Superoxide Dismutase (SOD) (Units/mg Protein)

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

Table 11: Catalase (CAT) (n Moles of H2O2 Decomposed / min / mg Protein)

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

Table 12: Lipid Peroxide (LPO) (n Moles of MDA/mg Protein)

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

Table 13: Vitamin E (N moles / g Wet Tissue)

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

(a) (b) (c)

(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
the fifth day for estimation of biochemical parameters and
for histopathological studies. The liver was collected and
wet weight of liver was noted as mg weight of liver/gm
body weight.
Biochemical Study
Blood was collected from jugular veins, and allowed to clot
for 30-40 min. Serum was separated by centrifuging at
3000 rpm for 10 minutes. Immediately after sacrifice, the
liver was dissected out, washed in ice cold saline, and a
homogenate was prepared in 0.1 M Tris-HCl buffer pH 7.4.
The homogenate was centrifuged at 3000 rpm for 10
minutes and the supernatant was used for the assay of
marker enzymes. The following biochemical assays were
carried out in serum, serum glutamic oxaloacetate
transaminase (SGOT), serum glutamic pyruvate
transaminase (SGPT), [5] alkaline phosphatase (ALP), [6]
total bilirubin, [7] total protein. [8] The supernatant liver
homogenate was used for the assay of the following
enzymes and non-enzymes, glutamic oxaloacetate
transaminase (GOT), glutamic pyruvate transaminase

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 RESEARCH ARTICLE
(GPT), total protein, glutathione peroxidase(GPx), [9]
glutathione-s-transferaseGST). [10] Glutathionereductase
(GRD), [11] superoxide dismutase (SOD), [12] catalase (CAT),
[13] lipid peroxidation (LPO), [14] vitamin E. [15] All the
enzymes assay were undertaken at particular nm using
shimadzu make spectophotometer, UV–1601 model.
Histopathological Studies
Small pieces of liver tissues were collected in 10%
formalin solution for preparation of sections by using of
microtome. [16]
Statistical Analysis
The data are expressed as mean±SEM. Statistical analysis
was done using one way analysis of variance (ANOVA)
followed by dunnet test. [17]
RESULTS
Preliminary Phytochemical Screening
The aqueous extract of V. madraspatana root bark showed
the presence of alkaloids, carbohydrates, phenols,
flavonoids, gums and mucilage, saponins and terpenes.
Hepatoprotective Study
Weight of Liver
CCl4 treated animals (group II) showed a significant
increase (p<0.001) in wet weight of the liver compared to
control (Table 1). There was a significant decrease in liver
wet weight of animals treated with 200 mg/kg and 400
mg/kg of aqueous extract respectively, when compared
with group II.
Glutamate Oxaloacetate Transminase (GOT) and
Glutamate Pyruvate Transaminase (GPT)
GOT and GPT levels of serum and liver homogenate were
significantly increased (p<0.001) in group II animals
challenged with CCl4, when compared to control (Table 2
and 3). A dose dependent reduction of (p<0.001) GOT and
GPT levels were observed in animals treated with aqueous
extract (200 mg/kg and 400 mg/kg) when compared to
group II. Liv-52 (group V) produced a significant reduction
(p<0.001) at the dose of 1 ml/kg body weight/p.o. when
compared to group II.
Serum Alkaline Phosphatase (ALP)
The serum ALP level was significantly increased (p<0.001)
in CCl4 challenged rats (group II) when compared to control
rats (group I) (Table 4). Treatment with aqueous extract
(200 mg/kg and 400 mg/kg) showed a significant
reduction in ALP level when compared to group II animals.
The Liv-52 (1 ml/kg) treated group also showed a
significant decline of ALP (p<0.001) when compared to
group II animals.
Total Bilirubin
There was a significant increase (p<0.001) in the level of
total serum bilirubin in CCl4 treated animal (group II),
when compared to group I (Table 5). Aqueous extract (200
mg/kg and 400 mg/kg) of Ventilago madraspatana root
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bark treated rats showed significant decrease (p<0.001) in
total bilirubin when compared to CCl4 treated rats. The Liv-
52 (1 ml/kg) treated animal (group V) also showed
significant (p<0.001) decrease of total bilirubin level when
compared to group II animal.
Total Protein
A significant (p<0.001) reduction in the total protein of
serum and liver was observed in CCl 4 treated rats (group
II) when compared to control (group I) (Table 6).
Treatment with aqueous extract (200 mg/kg and 400
mg/kg showed a significant (p<0.001) increase of protein
level, when compared to (group II) animals. The Liv-52 (1
ml/kg) treated animals (group V) also showed significant
increase in protein level when compared to CCl 4
challenged (group II) rats.
Glutathione Peroxidase (GPx)
Liver Glutathione peroxidase activity was significantly
(p<0.001) reduced in CCl4 treated animals (group II) when
compared to control (group I) (Table 7). The aqueous
extract (200 mg/kg and 400 mg/kg dose levels)
significantly increased (p<0.001) the GPx levels, when
compared to group II. Liv-52 (1 ml/kg) treated animals also
showed significant (p<0.001) increase of GPx level in the
liver homogenate compared with group II animals.
Glutathione-S-transferase (GST)
Liver Glutathione-S-transferase level was significantly
reduced (p<0.001) in CCl4 treated animals when compared
with normal animals (Table 8). Treatment with aqueous
extract of Ventilago madraspatana root bark at 200 mg/kg
and 400 mg/kg dose levels showed significant increase
(p<0.001) in GST level when compared to CCl4 treated
group. Liv-52 (1 ml/kg) treated animals also showed
significant increase of GST level when compared to group II.
Glutatione Reductase (GRD)
Liver GRD activity was significantly (p<0.001) reduced in
CC14 treated animals (group II), when compare to control
(group I) (Table 9). The aqueous extract (200 mg/kg and
400 mg/kg) showed significant increase in GRD level, when
compared to group II animals. Liv-52 (1 ml/kg) treated
group also showed significant (p<0.001) increase of GRD
level when compare to group II.
Superoxide Dismutase (SOD)
Liver superoxide dismutase level was significantly
reduced (p<0.001) in CCl4 treated animals when compared
with normal animal (Table 10). The aqueous extract (200
mg/kg and 400 mg/kg) extract showed significant increase
in SOD levels, when Liv-52 (1 ml/kg) treated animals also
showed significant (p<0.001) increase of SOD level when
compared to group II.
Catalase (CAT)
Liver catalase activity was significantly (p<0.001) reduced
in CCl4 treated (group II), when compared to control (group
I) (Table 11). The aqueous (200 mg/kg and 400 mg/kg)
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 RESEARCH ARTICLE
extract significantly increased (p<0.001) the CAT level
when compared to group II animals. Liv-52 (1 ml/kg)
treated group also showed significant (p<0.001) increase of
catalase level when compared to group II.
Lipid Peroxidation (LPO)
The lipid peroxide of liver homogenate was significantly
increased (p<0.001) in CCl4 challenged rats (group II) when
compared to control rats (group I) (Table 12). Treatment
with aqueous (200 mg/kg and 400 mg/kg) extract showed
significant decrease in LPO level when compared with CCl4
treated (group II) animals. The Liv-52 (1 ml/kg) treated
group also showed a significant (p<0.001) decline in the
LPO level when compared to group II animals.
Vitamin E
Vitamin E activity of liver was significantly (p<0.001)
reduced in CC14 treated animals (group II), when
compared to control animals (group I) (Table 13). The
aqueous (200 mg/kg and 400 mg/kg) extract showed
significant increase (p<0.001) of Vitamin E level in dose
dependent manner when compared to group II animals.
Liv-52 (1 ml/kg) treated group also showed significant
(p<0.001) increase in Vitamin E level when compared to
group II.
Histopathological Studies
Group I treated animals showed dilated central vein,
normal hepatocytes. Group II treated animals showed
perivenular inflammatory infiltration and hepatocytic fatty
change, diffuse mild hepatocellular vacuolation. Group III
treated animals showed change central vein, mild fatty
change. Group IV treated animals showed dilated central
vein, mild sinusoidal dilation and no hepatocellular
damage. Group V treated animals showed normal central
vein and mild hepatocytic fatty change. This result
correlates well with the biochemical findings. From the
results of the histopathological studies, administration of
aqueous extract of Ventilago madraspatana root bark
reveals reduced cellular damage induced by CCl4, the
widely used hepatotoxicant (Figure 1).
DISCUSSION
The present study has demonstrated hepatoprotective
activity with increase in in-vivo antioxidant status of aqueous
extract of Ventilago madraspatana root bark in CCl4 induced
liver injury in rats. Damage to the structural integrity of liver
is reflected by an increase in the levels of serum transminase
[18] because these are cytoplasmic in location and released
into circulation after cellular damage. [19] It is generally
accepted that hepatotoxicity of carbon tetrachloride is
attributes to trichloromethyl free radical, and this free
radical reacts rapidly with oxygen to form a
trichloromethylperoxy radical, which may contribute to the
hepatotoxicity and subsequent increase in hepatic enzymes.
[20] In this context we have observed a rise in the levels of
GOT and GPT in carbon tetrachloride treated rats due to
toxic compounds affecting liver and the reversal of the
elevated enzymatic levels to normal after the treatment with
Inventi Rapid: Ethnopharmacology Vol. 2013, Issue 808
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aqueous extract of Ventilago madraspatana. Alkaline
phosphtase (ALP) is a membrane bound glycoprotein
enzyme, with high concentration is sinusoids and
endothelium. ALP reaches the liver mainly from bone. It is
excreted into the bile so its elevation in serum occurs in
hepatobiliary diseases. [21] The aqueous extract of Ventilaga
madraspatana root bark probably stabilised the hepatic
plasma membrane from CCl4 induced liver damage. The
serum albumin level is low in hepatic diseases. The present
results reveal that when animals pretreated with Ventilago
madraspatana extract prior to the challenge with CCl4, the
liver biosynthesis of protein continues to be unaffected. The
metabolic transformation of aminoacids occurs in liver by,
transamination protein metabolism may be impaired due
the escape of both non proteins and protein nitrogenous
substances from injured liver cells as evidenced by raise in
the serum enzyme levels of GOT, GPT and ALP. Glutathione
peroxidase (GPx) plays a pivotal role in H2O2 catabolism [22]
and the detoxification of endogenous metabolic peroxides
and hydroperoxides which catalyses GSH. [23] GPx activity
was significantly reduced in the CCl4 treatment when
compared to control. The reversal of the GPx activity to
normal after pretreatment with the plant extract exhibits the
antioxidant activity of the extract in scavenging or
detoxifying the endogenous metabolic peroxides generated
after CCl4 injury in the tissues. Many investigators have
suggested that Glutathione-S-transferase (GST) offers
protection against LPO by promoting the conjugation of toxic
electrophiles with reduced glutathione (GSH). [24] GST plays a
physiological role in initiating the detoxification of potential
alkalating agents. Chemicals like chloroform, CCl4 etc. alter
the hepatic Glutathione-S-transferase activity. [25] GST level
was significantly reduced in CCl4 treated animals and
upward reversal was observed after the treatment with
aqueous extract of the plant. The present study reveals the
extract along with other protective mechanism also increase
the autoprotection of the liver function by the GRD level. In
the present study the superoxide dismutase activity is
significantly reduced in CCl4 intoxicated rats. The SOD
activity was brought to near normal after treatment with the
extract in CCl4 intoxicated rats. Decreased activity of catalase
was observed in group II animals treated with CCl4.
Presumably a decrease in catalase activity could be
attributed to cross linking and inactivation of the enzyme
protein in the lipid peroxides. Decreased catalase activity is
linked up to exhaustion of the enzyme as a result of oxidative
stress caused by CCl4. The catalase activity was restored to
normal after treatment with extract evidently shows that
antioxidative property of the extract against oxygen free
radical. Carbon tetrachloride treatment may elevate the level
of malondialdehyde (MDA) a product of lipid peroxidation.
Therefore an increase in the liver MDA level indicates an
increase in the degree of lipid peroxidation, a well known
biochemical mechanism of liver damage. [26] A significant
decrease in the levels of lipid peroxides in aqueous extract of
the Ventilago madraspatana root bark extract pre-treated
rats suggests that the extract may have the ability to protect
the liver from free radical injury induced by
carbontetrachloride. The level of Vitamin E was significantly
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 RESEARCH ARTICLE
depleted in carbon tetrachloride intoxicated rats. The
Ventilago madraspatana root bark aqueous extract pre
treated rats showed an improvement in the levels of vitamin
E. The hepatoprotective nature of aqueous extract of
Ventilago madraspatana root bark against CCl4 induced
hepatic oxidative stress may be attributed to the presence of
phenolic compounds. Further studies are required to isolate
the active principle present in Ventilago madraspatana root
bark and evaluating its pharmacological activity which may
give potential hepatoprotective drug molecule.
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Cite this article as: K Sujith, K Shanmuganathan, C
Ronald Darwin et al., Hepatoprotective Effects of
Aqueous Extract of Ventilago madraspatana Gaertn.
Root- Bark on CCl4 Induced Damage in Rats. Inventi
Rapid: Ethnopharmacology, 2013(2):1-6, 2013.
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Published on Web 06/03/2013, www.inventi.in