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60
South Indian Journal of Biological Sciences 2015; 1(2); 60-65 Online ISSN:2454-4787
Hepatoprotective activity of ethanolic extract of Alysicarpus
vaginalis against nitrobenzene-induced hepatic damage in rats
Muthaiyan Ahalliya Rathi1, Periasamy Meenakshi2,
Velliyur Kanniappan Gopalakrishnan3,*
1Department of Biochemistry, Sree Narayana Guru College, Coimbatore- 641 021, Tamil Nadu, India.
2Department of Soil Science and Agricultural Chemistry, Anbil Dharmalingum Agricultural College and Research Institute, Trichy
620009, Tamil Nadu, India.
3Department of BioChemistry, Karpagam University, Coimbatore 641 021, Tamil Nadu, India
*Corresponding Author:
V.K.Gopalakrishnan
E-mail: vkgopalakrishnan@gmail.com
Tel.: +914222611146
Fax: +9104222611043
Manuscript details Abstract
Article History:
Received 1 July 2015
Revised 19 August 2015
Accepted 20 September 2015
Published 29 September 2015
Keywords:
Alysicarpus vaginalis, Antiox-
idant enzymes,
Liver injury,
Liver marker enzymes,
Nitrobenzene.
Alysicarpus vaginalis (L.) DC commonly known as Alyce clover, belongs to the
botanical family Fabaceae. It is widely distributed in India, Pakistan, Sri Lanka ,
Africa and Australia. In India it is traditionally used by the tribals for diuretics,
leprosy, pulmonary troubles and back pain. e present study aimed to evaluate
the hepatoprotective activity of ethanol extract of A. vaginalis aerial parts in ni-
trobenzene (NB) induced hepatic injury in Wistar rats. Liver injury was induced
in rats by single oral administration of of NB (50 mg/kg bw). One days aer NB
induction, the rats were treated with ethanolic extract of A. vaginalis orally at
the doses of 200 mg/kg bw daily for 30 days. Silymarin (25 mg/kg bw orally) was
used as reference drug. Aer 30 days serum biochemical parameters, antioxi-
dant enzyme status and histopathological parameters were analyzed. e results
indicated that the ethanol extract of A. vaginalis the doses of 200 mg/kg orally
signicantly (P< 0.05) and dose-dependently reduced and normalized the serum
marker enzymes, and increased the antioxidant enzyme status as compared to
that of NB control group. Further more it was conrmed by histopathological
studies. is study concludes that A. vaginalis demonstrated promising hepato-
protectivity agent in NB induced hepatic damaged rats.
1. Introduction
Liver plays an important role in the biotransforma-
tion of foreign compounds and is particularly vulner-
able to toxic chemical assaults. ere is a wide variety
of hepatotoxins which damage liver. Nitrobenzene
(NB) is considered a hazardous air pollutant and has
proven to be an animal carcinogen. It is classied as
a group B2 chemical according to the 1986 Cancer
guide lines (Cattley et al., 1994) i.e. a likely human
carcinogen. Metabolism of NB produce intermedi-
ates such as Nitrosobenzene (NOB) and phenylhy-
droxylamine (PH) that play an important role in the
process of NB carcinogenesis (Howard et al., 1983).
Following accidental NB poisoning in humans, the
highest concentration was found in the liver, brain,
blood and stomach (International Programme on
Chemical Safety, 2003).
Several endogenous protective mechanisms have
been evolved to limit the injury caused by hepato-
toxins (Keppler et al., 1968). But endogenous pro-
tective mechanisms are unable to protect completely
from the toxic eects, hence the additional protec-
tive mechanism of dietary antioxidants may be of a
great importance, which would protect the liver from
hepatotoxins. Many natural and articial agents pos-
sessing antioxidative properties have been used to
prevent and treat hepatopathies induced by oxidative
stresses. Approximately 80% of the world population
is almost entirely dependent on traditional medicines
(Meenakshi et al., 2010; Balamurugan 2015). ese
are not only used for primary health care not just in
rural areas in developing countries, but also in de-
veloped countries as well where modern medicines
are predominantly used. Several plants are known to
exhibit potent antioxidant and hepatoprotective po-
61
tential (Senthilkumar et al., 2008; Antonisamy et al.,
2015).
e leaf juice were used for the improvement of
eye sight and earache (Tirkey, 2006).e entire plant
was used for the treatment of renal calculi (Ediri-
weera, 2007). Root of this plant is widely used in
kidneys, diuretics; leprosy and pulmonary troubles
(Burkil, 1985). In view of this consideration, it seems
reasonable to hypothesize that the functional eects
of A. vaginalis may be particularly important in pre-
venting and diminishing NB -induced hepatotoxici-
ty. As such, this study was focused on evaluating the
protective potentials of aerial part of A. vaginalis in
NB-induced hepatotoxicity.
2. Materials and methods
2.1. Plant material and extraction
e aerial part of A. vaginalis were collected in De-
cember 2008 from the foot hill of Kanniyakumari,
Tamil Nadu India. e materials were identied
and authenticated by Dr. G.V.S. Moorthy, Botani-
cal Survey of India Coimbatore. Voucher No: (BSI/
SC/5/23/09-10/Tech.1604). e collected materials
were thoroughly washed in water, chopped, air dried
at 35–40ºC for a week and pulverized in electric
grinder. 1 g of powder was then extracted in 5 ml of
ethanol. e ethanol extract was then made to pow-
der with the help of rotary evaporator under reduced
pressure.
2.2. Experimental animals
Wistar albino rats of both sex, weighing between 160
and 180 g were used for the study. e animals were
housed in standard polypropylene spacious rat cag-
es with stainless steel top grill, under hygienic con-
ditions. Animals were provided standard pellet food
manufactured by Hindustan Lever Ltd., India and
Aqua gaurd ST 2000 ltered well water ad libitum.
e animals were quarantined for 1 week, prior to the
experiments to acclimatize to laboratory conditions.
e study protocol was approved by the IAEC (Insti-
tutional Animal Ethics Committee, Govt. of India).
2.3. Toxicity studies
e acute toxicity study was performed for ethanolic
extract of aerial parts of A. vaginalis were performed
using wistar strain of albino rats. e animals were
kept fasting for overnight providing only water, aer
which the extracts were administered orally in in-
creasing dose (100-2000 mg/kg bw) and found safe
up to 2000 mg/kg bw. One-tenth of the maximum
dose of the extract tested for acute toxicity was se-
lected for evaluation of hepatoprotective activity, i.e.,
200 mg/kg (Handa and Anupama 1990).
2.4. Treatment of animals
e rats were divided in to four groups, each group
consisting of six animals. Group I served as the con-
trol; in group II, rats received NB (50 mg/kg bw) oral-
ly as a single dose in group III, rats were subjected
to induction and treatment, NB was injected orally
and treatment with ethanolic extract (200 mg/kg bw)
orally was started a day aer the injection for a peri-
od of 30 days. In group IV, rats were treated with pos-
itive control silymarin (25 mg/kg bw) orally for 30
days. In group V, rats were treated with the ethanolic
extract alone orally for 30 days.
2.5. Preparation of the samples for biochemical
studies
e animals were anesthetized using chloroform
and sacriced. Blood was collected directly from the
heart of each animal and the clot was centrifuged for
5 min minutes at 1500 rpm to separate serum and
for biochemical analysis. Liver tissues were removed
immediately and washed in ice-cold saline, and 10%
homogenate was prepared using 0.1 M Tris–HCl, pH
7.4, a part of liver sections were dissected out for his-
topathological examinations.
2.6. Biochemical estimations
e liver marker enzymes like alanine transam-
inase (ALT) and aspartate transaminase (AST)
(King 1965), alkaline phosphatase (ALP) (King and
Armstrong 1980) total bilirubin (TB) (Gupta et al.,
2007). e liver supernatant was used for the esti-
mation of superoxide dismutase (SOD) (Misra and
Fridovich 1972). e unit of enzyme activity is de-
ned as the enzyme required to give 50% inhibition
of epinephrine to adenochrome. Catalase (CAT)
(Sinha 1972). In this method, dichromate in acetic
acid was reduced to chromic acetate. is was mea-
sured colorimetrically at 610 nm, glutathione perox-
idase (GPx) (Rotruck et al., 1973) here the reaction
between glutathione remaining aer the action of
GPx and 5,5’-dithiobis-(2-nitrobenzoic acid) to form
a complex that absorbs at 412 nm. Lipid peroxida-
tion (LPO) (Hogberg et al., 1974). Malondialdehyde
(MDA), formed as an end product of the peroxida-
tion of lipids served as an index of oxidative stress.
2.7. Histopathological examination
Aer weighed, part of the hepatic tissue was imme-
diately collected from the same lobe of the liver and
xed in a 10% (v/v) neutral formalin solution for 24
h. Subsequently, hepatic tissue was dehydrated in a
series of ethanol solutions from 75% to 100%. Aer
dehydration, the tissue was embedded in paran, cut
into 5 µm sections, stained with the haematoxylin-eo-
sin dye and observed under a photomicroscope.
2.8. Statistical analysis
e values were expressed as mean±SD. e statis-
tical analysis was carried out by oneway analysis of
variance using SPSS (version 10) statistical analysis
program. Statistical signicance was considered at
p<0.05.
3. Results
e ethanolic extract of A. vaginalis did not show any
sign and symptoms of toxicity and mortality up to
2000 mg/kg. e activity of serum marker enzymes
such as alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP)
and total bilirubin (TB) analyzed in serum samples of
62
dierent groups of rats are shown in Table 1. In group
II there was a signicant increase (p<0.05) in serum
levels of ALT, AST, ALP and TB. But when the ethan-
olic extract of aerial part of A. vaginalis was given to
group III, there was a signicant decrease in the val-
ue, which tends to reach the normal values. In group
IV, when treated with positive control silymarin the
levels of liver marker enzymes get normalized. In
group V, when the plant extract alone was given, the
level approached the normal values.
e activity of antioxidant enzymes such as super-
oxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx) and lipid peroxidation analysed
in liver homogenate of dierent groups of rats are
shown in Table 2. Antioxidant enzymes such as SOD,
CAT, GPx were analyzed in dierent groups of rat.
In group II, there was a signicant decrease (p<0.05)
in the antioxidant enzyme and the lipid peroxidation
levels were found to be increased. But when the eth-
anolic extract of A. vaginalis was given to group III,
there was a signicant increase in the antioxidant
levels and lipid peroxidation levels were decreased.
In group IV, when treated with silymarin the anti-
oxidant enzyme levels get increased and lipid per-
oxidation levels get reduced. In group IV, when the
plant extract alone was given, the level approached
the normal values.
e hepato protective eect of A. vaginalis was
conrmed by histopathological examination of liver
section of control and treated animals. e histo-
pathological observation were explored in Figure 1.
In group I (normal control) rats, liver showed nor-
mal histological architecture (a). In group II (NB
control) showed marked inammatory changes as-
sociated with fatty changes and conuent hepatic
necrosis (b). Group III (NB+Alysicarpus vaginalis)
which is NB damaged and treated with the ethano-
lic extract showed almost normal architecture with
lesser degree of inammation (c). Group V (NB+ Si-
lymarin) when treated with positive control showed
normal architecture with lesser degree of inamma-
tion showing its potent hepatoprotective eects (d).
Group V (Alysicarpus vaginalis only) showed normal
liver (e).
4. Discussion
Liver has a great capacity to detoxicate toxic sub-
stances and synthesize useful metabolites.18 Serum
levels of AST and ALT are the quite sensitivity indi-
cators to evaluate the degree of hepatic damage. e
Table 1. Eects of A. vaginalis on serum marker enzymes in control and experimental groups of rats
Groups Treatment AST (U/L) ALT (U/L) ALP (U/L) TB (mg/dL)
I Normal 50.64±1.43 59.20±1.54 130.53±1.75 1.23±0.1
II NB control 149.51±5.33a* 105.95±3.72a* 189.65±3.54a* 6.59±0.2 a*
II NB+Ethanolic
extract 102.32±4.24b* 83.36±3.64b* 163.45±4.45b* 3.94±0.1 b*
IV NB+ Silymarin 76.34 ±2.32c* 71.34±2.45c* 153.54±2.43c* 2.32±0.43c*
IV Ethanolic extract 48.42±1.23dNs 58.56±1.53d Ns 133.56±1.57d Ns 1.81±0.1d Ns
a –Group -I compared with Group- II; b - Group- III compared with Group –II; c- Group-IV compared with Group II;
d- Group V compared with Group I
*p< 0.05
Table 2. Eect of A. vaginalis on liver antioxidant status of control and experimental
SOD CAT GPx LPO
I Normal 32.25±0.37 59.56± 4.63 35.45±1.26 0.99± 0.03
II NB control 12.32±0.43a* 23.39± 2.39a* 18.54±0.59a* 4.54 ±0.52a*
III NB+Ethanolic extract 21.34±0.85b* 38.56± 2.54b* 25.65±0.65b* 2.12± 0.56b*
IV NB+ Silymarin 28.36±0.24c* 43.69±2.46c* 30.48±0.48c* 1.21±0.35c*
V Ethanolic extract 30.72±0.45d NS 57.54± 3.57d NS 27.52±0.63d NS 1.08± 0.03dNS
a –Group -I compared with Group- II; b - Group- III compared with Group –II; c- Group-IV compared with Group
II; d- Group V compared with Group I
*p< 0.05
e values are expressed as units per milligram of protein
63
level of AST is an indicator of mitochondrial damage,
because mitochondria contain 80% of the enzyme
(Tang et al., 2006)
As NB causes hepatotoxic eects, there was a sig-
nicant increase in the activities of serum liver mark-
er enzymes and bilirubin in NB-intoxicated (group
II) animals when compared to control rats (Table
1). On the other hand, the activities of these marker
enzymes were signicantly decreased in rats treat-
ed with ethanolic extract of A.vaginalis (group III)
when compared to group II animals. Estimating the
activities of serum marker enzymes, like aspartate
transaminase, alanine transaminase, alkaline phos-
phatase can make assessment of liver function. When
liver plasma membrane is damaged, a variety of en-
zymes normally located in the cytosol are released in
the blood stream. eir estimation in the serum is
a useful quantitative marker and type of hepatocel-
lular damage (Mitra et al., 1998). e serum marker
enzymes (aspartate transaminase, alanine transami-
nase, and alkaline phosphatase, and were markedly
increased in NB (50mg/kg bw) - administered rats
(Rajalakshmy et al., 2010). Simultaneously, the hepa-
toprotective eects of ethanolic extract of A. vagina-
lis were compared with those treated with silymarin,
which is an active constituent of the fruit of the milk
thistle (Silybum marianum, compositae)
Free radicals are regularly produced in vivo as a re-
sult of carcinogen treatment causing oxidative stress
that leads to damage of nucleic acids, proteins, and
lipids, which play an important mechanistic role in
the development of cancer (Waris and Ahsan 2006).
Natural antioxidants are capable of inhibiting ROS
production, and thereby, it reduces the intracellular
oxidative stress (Feng et al., 2001) e antioxidant
systems are major cell defenses, which protect mem-
branes and cytosolic components against damage
induced by free radicals under diseased conditions
(Janani et al, 2008).
A major defense mechanism involves the antioxi-
dant enzymes, including SOD, CAT and GPx, which
convert active oxygen molecules into non- toxic com-
pounds (Jain et al., 2008). In our study, the liver SOD,
CAT, and GPx activities were signicantly decreased
where as lipid peroxidation levels were increased in
NB –intoxicated animals, which were brought back
to near normal in ethanolic extract of A. vaginalis
treated animals with NB administration (Table 2).
SOD is an endogenous enzymatic scavenger which
can counterbalance the oxidative destruction of free
radicals. Most of the SOD in tissues of cytoplasmic
origin and contains Cu and Zn an essential prosthetic
groups (Rathi et al., 2009). SOD catalyzes the break-
down of O2. to O2 and H2O2 and prevents the for-
mation of OH-. us, SOD has been implicated as
playing an essential defensive role against potential
oxygen toxicity.
e ROS scavenging activity of SOD is eective
only when it is followed by the actions of CAT and
GPx, because the dismutase activity of SOD generates
H2O2, which needs to be further scavenged by CAT
and GPx (Ju et al., 2004). LPO, is through metal che-
lation at the initiation level and also as a chain break-
er (Tripathi et al., 1996). Peroxy radicals are import-
ant agents that mediate lipid peroxidation there by
damaging cell membrane. Scavenging of free radicals
is one of the major antioxidant mechanisms to inhib-
it the chain reaction of lipid peroxidation. Reduced
lipid peroxidation was revealed by signicant de-
crease in MDA and hydroperoxidase level in extract
groups. Simultaneously signicant increase in SOD
and CAT content of liver suggested antioxidant activ-
ity of A. vaginalis and silymarin. Administration with
NB caused a signicant increase in MDA concentra-
tion when compared with normal group (Rathi et al,
2010a). NB carcinogenicity is considered to correlate
with its metabolic activation. It forms a number of
phenolic compounds by oxidation and nitroxides by
reduction (Holder 1999). Reduction of nitro group
plays a more potent role in NB carcinogenicity. Ni-
troreduction, which is driven by microsomal P-450s
and NAD(P)H, can produce reactive nitroxide inter-
mediates aromatic nitroso- and hydroxylamine com-
pounds, e.g. NOB and PH, associated with their reac-
tive free radicals, e.g., the nitroanion free radical and
superoxide free radical (Mason and Holtzman 1975).
In our laboratory, previously it was investigated that
induction of nitrobenzene cause hepatic damage,
which was treated with ethanolic extract of Cayratia
trifolia (Guru Kumar et al., 2011).
Fig. 1: Histopathological observation (x100 magnica-
tion)
(a) In normal control rats,showing hepatic cells with
normal nuclei and cytoplasm; (b) Section of NB- treated
(Nitrobenzene control group) rat liver, showing loss of
nuclei, kuper cells and vacuolization. (c) Section of NB
(50 mg/kg bw) + ethanolic extract of A. vaginalis treated
rat liver, showing marked improvement over NB control
group. (d) Section of silymarin (25 mg/kg bw) + NB
treated rat liver showing almost normal architecture of
liver. (e) Alysicarpus vaginalis alone treated rats showed
normal architecture of liver.
64
In our previous study, the ethanolic extract of A.
vaginalis revealed the presence of alkaloids, avo-
noids, sterols, tannins, polyphenols, and triter-
penoids (Rathi et al., 2010b). Alkaloids are plant-de-
rived compounds with physiological activity, contain
nitrogen in a hetrocyclic ring with complex structure
which possesses potent antioxidant activity (Chung
and Shin 2007). e total phenolic content of A. vag-
inalis was found to be 2.7 mg gallic acid equivalent
per gram dried plant, it also has strong free radical
scavenging activity and showed signicant antili-
pid peroxidant eect in vitro. It also exhibited anti
proliferative activity against ovarian cancer cell lines
(Rathi et al., 2010a).
e present results revealed that the extract of ae-
rial part of A. vaginalis was able to protect the mem-
brane integrity of hepatocyte against NB induced
release of marker enzymes in the blood circulation.
Preventing liver lesions from progressing to brosis
and cirrhosis, and repairing parenchymal cell dam-
age by stimulating liver regeneration are important
mechanism for hepatoprotection. Perhaps the alka-
loids, avonoids, sterols, tannins, polyphenols, and
triterpenoids present in A. vaginalis are responsible
for the marked hepatoprotective eects, observed in
the present study. Hence, it will be of great interest to
isolate the active constituents of A. vaginalis. Further
studies will be needed to identify the active com-
pounds that confer the hepatoprotective protection
of the A. vaginalis extract.
5. Conclusion
In conclusion, the result of this study seems to con-
rm that the ethanolic extract of A. vaginalis has a
potent hepatoprotective action upon nitrobenzene –
induced hepatic damage in rats and possess antilipid
peroxidative and free radical scavenging activities.
e present study thus justies the traditional use of
A. vaginalis in the treatment of liver disease and also
point out that A. vaginalis warrants future detailed
investigation as a promising hepatoprotective agent.
Conict of interest statement
We declare that we have no conict of interest.
Acknowledgements
We, the authors are thankful to our Chancellor, Advi-
sor, Vice Chancellor and Registrar of Karpagam Uni-
versity for providing facilities and encouragement.
References
1. Antonisamy P, Duraipandiyan V, Ignacimuthu S, Kim JH.
(2015). Anti-diarrhoeal activity of friedelin isolated from Azima
tetracantha Lam. in wistar rats. South Indian Journal of Biologi-
cal Sciences, 1(1), 34–37.
2. Balamurugan R. (2015). Smilax chinensis Linn. (Liliaceae)
root attenuates insulin resistance and ameliorate obesity in high
diet induced obese rat. South Indian Journal of Biological Scienc-
es, 1(1), 47–51.
3. Burkil HM. (1985). Useful plants of west tropical Africa, Vol. 1:
A-D. Royal Botanic Gardens, Kew, 960
4. Cattley RC, Everitt JI, Gross EA. (1994). Carcinogenicity and
toxicity of inhaled Nitrobenzene in B6C3F1 mice and F344 and
CD rats. Toxicological Sciences, 22, 328–340.
5. Chung SH, Shin CJ. (2007). Characterization of antioxidant
alkaloids and phenolics acids from anthocyanin-pigmented rice
(Oryza sativa cv. Heugjinjubyeo). Food Chemistry, 104, 1670–1677.
6. Ediriweera ERHSS. (2007). A review on medicinal uses of
weeds in Sri Lanka. Tropical Agricultural Research and Exten-
sion, 10, 11-16.
7. Feng Q, Kumagai T, Torii Y, Nakamura Y, Osawa T, Uchida
K. (2001). Anticarcinogenic antioxidants as inhibitors against in-
tracellular oxidative stress. Free Radical Research, 35, 779–788.
8. Gupta M, Mazumder UK, Tamilselvan V, Manikandan L,
Senthilkumar GP, Suresh R, Kakotti BK (2007): Potential hepa-
toprotective eect and antioxidant role of methanol extract of
Oldenlandia umbellate in carbon tetra chloride induced hepa-
totoxicity in Wistar rats. Iranian Journal of Pharmacology and
erapeutics, 6, 5-9.
9. Guru Kumar D, Sonumol M, Rathi MA, irumoorthi L,
Meenakshi P, Gopalakrishnan VK. (2011). Hepatoprotective ac-
tivity of C ayratia trifolia (L.) Domin against nitrobenzene induced
hepatotoxicity. Latin American Journal of pharmacy, 546-549.
10. Handa S, Anupama S. (1990). Hepatoprotective activity of
andrographolide from Andrographis paniculata against carbon
tetrachloride. Indian Jounal of Medical Research, 92, 276-283.
11. Hogberg J, Larson RE, Kristoferson A, Orrhenices S. (1974).
NADPH dependent reductase solubilised from microsomes by
peroxidation and its activity. Biochemical and Biophysical Re-
search Communications, 56, 836–842.
12. Holder JW. (1999). Nitrobenzene potential human cancer
risk based on animal studies. Toxicology and Industrial Health,
15, 458–463.
13. Howard PC, Beland FA, Cerniglia CE. (1983). Reduction of
the carcinogen 1-nitropyrene to1-aminopyrene by the rat intes-
tinal bacteria. Carcinogenesis, 4, 985–990.
14. International Programme on Chemical Safety (IPCS). Nitro-
benzene environmental health criteria 230. Geneva: WHO; 2003
15. Jain A, Soni M, Deb L, Jain A, Rout SP, Gupta VB, Krishna
KL. (2008). Antioxidant and hepatoprotective activity of ethan-
olic and aqueous extract of Momordica dioica Roxb. leaves. Jour-
nal of Ethanopharmacology, 115, 61-66.
16. Janani P, Sivakumari K, Parthasarathy C. (2008). Hepatoprotec-
tive activity of bacoside A against N-nitrosodiethylamine-induced
liver toxicity in adult rats. Cell Biology and Toxicology, 24, 471–474.
17. Ju EM, Lee SE, Hwang HJ, Kim JH. (2004). Antioxidant and
anticancer activity of extract from Betula platyphylla var. japoni-
ca. Life Science, 74, 1013-1026.
18. Keppler D, Lesch R, Reutter W, Decker K. (1968). Experi-
mental hepatisis induced by D-galactosamine. Experimental and
Molecular Pathology, 9,279–290.
19. King EJ, Armstrong AR. (1980). Calcium, magnesium, phos-
phorus and phosphatase. In:Varley B,Gowenlock AH, Bell M,
editors. Practical clinical biochemistry, London: Heinemann, 1,
850–852.
20. King EJ. (1965). Practical clinical Enzymol. London: D. Van
Nostrand Co, 83-93.
21. Mason RP, Holtzman JL. (1975). e role of catalytic super-
oxide formation in O2 inhibition of nitroreductase. Biochemical
and Biophysical Research Communications, 67, 1267–1274.
22. Meenakshi P, Bhuvaneshwari R, Rathi MA, irumoorthi
L, Chinnaguravaiah D, Jiji MJ, Gopalakrishnan VK. (2010).
Antidiabetic activity of ethanolic extract of Zaleya decandra in
65
alloxan-induced diabetic rats. Applied Biochemistry and Biotech-
nolo g y, 162, 1153- 1159.
23. Misra HP, Fridovich I. (1972). e role of superoxide anion in
the antioxidation of epinephrine and simple assay of superoxide
dismutase. e Journal of Biological Chemistry, 247, 3170–3175.
24. Mitra SK, Venkataranganna MV, Sundaram R, Gopumadha-
van S. (1998). Protective eect of HD-03, a herbal formulation,
against various hepatotoxic agents in rats. Journal of Ethanophar-
macology, 63, 181-186.
25. Rajalakshmy MB, Rathi MA, irumoorthi L, Gopalakrish-
nan VK. (2010). Potential eect of Bacopa monnieri on nitro-
benzene induced liver damage in rats. Indian Journal of Clinical
Biochemistry, 25, 401–404
26. Rathi MA, Meenakshi P, Guru kumar D, Arul Raj C, iru-
moorthi L, Gopalakrishnan VK. (2010a). Potential antioxidant
and antiproliferative activities of Alysicarpus vaginalis (L.) DC.
Journal of Pharmacy Research, 3, 2375-2377.
27. Rathi MA, irumoorthi L, Sunitha M, Meenakshi P, Guru-
kumar D, GopalakrishnanVK. (2010b). Hepatoprotective activity
of spermacoce hispida linn. extract against nitrobenzene induced
hepatotoxicity in rats. Journal of Herbal Medicine and Toxicology, 4,
201- 205.
28. Rathi MA, Bala Pearl DL,. Sasikumar JM, Gopalakrish-
nan VK. (2009). Hepatoprotective activity of ethanolic extract of
Hedyotis corymbosa on perchloroethylene induced rats. Pharma-
cologyonline, 3, 230-239.
29. Rotruck JT, Pope AL, Ganther HE. (1973). Selenium, bio-
chemical role as a component of glutathione peroxidase puri-
cation and assay. Science, 179, 588–590.
30. Senthilkumar N, Badami S, Dongre SH, Bhojraj S. (2008).
Antioxidant and hepatoprotective activity of the methanol ex-
tract of Careya arborea bark in ehrlich ascites carcinoma- bear-
ing mice. Journal of Natural Medicines, 62,336-339.
31. Sinha AK. (1972). Colorimetric assay of catalase. Analytical
Biochemistry, 47, 389–394.
32. Tang X, Gao J, Wang Y, Fan YM, Xu LZ, Zhao XN, Xu Q, Qian
ZM. (2006). Eective protection of Terminalia catappa L. leaves
from damage induced by carbon tetrachloride in liver mitochon-
dria. e Journal of Nutritional Biochemistry, 17,177–182.
33. Tirkey A. (2006). Some ethanobotanical plants of family- Faba-
ceae of Chhattisgarh state. Journal of Traditional Knowledge, 5,
551-553.
34. Tripathi YB, Chaurasia S, Tripathi E, Upadhyay A, Dubey GP.
(1996). Bacopa monniera Linn. As an antioxidant: mechanism of
action. Indian Journal of Experimental Biology, 34, 523–526.
35. Waris G, Ahsan H. (2006) Reactive oxygen species: role in the
development of cancer and various chronic conditions. Journal
of Carcinogenesis, 5, 14
