Asian J. Pharm. Tech. 2012; Vol. 2: Issue 1, Pg 15-18 [AJPTech.

]


15


ISSN- 22315705 (Print) www.asianpharmaonline.org
ISSN- 22315713 (Online) 0974-3618


RESEARCH ARTICLE
Antioxidant and Hepatoprotective properties of Terminalia arjuna Bark on
Isoniazid Induced Toxicity in Albino rats

P. Doorika and T. Ananthi*

P.G. Department of Biochemistry, S.T.E.T Womens College, Mannargudi 614 601.
*Corresponding Author E-mail: elangani576@ gmail.com

ABSTRACT:
The aqueous extract of Terminalia arjuna bark was investigated for its hepatoprotective effect against isoniazid
induced acute liver damage on albino rats. Isoniazid (100mg/kg) significantly elevated the serum levels of biochemical
markers like SGPT, SGOT, ALP, ACP, Bilirubin, Protein and depleted antioxidant enzymes GSH and SOD upon
administration of Isoniazid (100mg/kg) to albino rats. This indicated that there the aqeous extract of bark of
Terminalia arjuna at 200mg/kg dose significantly reduced the elevated levels of biochemical markers mentioned
above. Test extract treatment also increased the level of SOD and GSH. These results suggest that aqueous extract of
Terminalia arjuna may have the potential therapeutic value in the treatment of Isoniazid induced hepatic damage and
some liver diseases. Hepatoprotective activity of the study plant may be attributed to the anti-oxidant principles in it.

KEY WORDS: Terminalia arjuna, Isoniazid, Hepatoprotective, antioxidant

INTRODUCTION:
The liver is largest glandular organ in the body and weight
1.5kg making up about 2-3% of the total body weight. It is
responsible for detoxifying the poisonous substances in the
body by transforming and removing toxins and wastes. The
liver serves a variety of functions. The most crucial is its
role in the bodys metabolism. There is no organ is more
important to healthy metabolism than the liver in many
ways (Robbins et al., 2003).

Liver disease is a collective term for a whole group of
problems that afflict the tissues, structures and cells of the
human liver. The liver performs a multitude of important
functions, so theres plenty of opportunity for something to
go wrong. One of the most common causes of liver disease
is inflammation, which often results from abuse of alcohol,
poor diet or even malnutrition (Arias et al., 1989).










Received on 30.12.2011 Accepted on 10.01.2012
Asian Pharma Press All Right Reserved
Asian J. Pharm. Tech. 2(1): Jan.-Mar. 2012; Page 15-18

Drug induced liver injury is the major health problem that
challenges not only care health professional but also the
pharmaceutical industry and drug regulatory agencies.
According to the United States Acute Liver Failure Study
Group, drug induced liver injury accounts for more than
50% of acute liver failure, including hepatotoxicity caused
by over dose of acetaminophen (39%) and idiosyncratic
liver injury triggered by other drugs (Micheale and
Cynthiya, 2006) .

Medicinal herbs are significant source of pharmaceutical
drugs. Latest trends have shown increasing demand of
phyto drugs and some medicinal herbs have proven hepato
protective potential. Medicinal herbs and extracts prepared
from them are widely used in the treatment of liver diseases
like hepatitis, cirrhosis, and loss of appetite (Nadkarni and
Nadkarni, 1954).

Terminalia arjuna is a deciduous and ever green tree,
standing 20-30m above ground level. It belongs to
Combretaceae family (Chopra and Ghosh, 1929). It is
found in Uttar Pradesh, South Bihar, Madhya Pradesh,
Delhi and Deccan region near ponds and livers. Ancient
Indian physicians used the powdered tree bark of
Terminalia arjuna for alleviating Hritshool (angina) and
other cardiovascular conditions. Its stem bark possesses
glycosides, large quantities of flavonoids, tannins and
minerals. Flavonoids have been detected to exert
antioxidant, anti inflammatory and lipid lowering effects
Asian J. Pharm. Tech. 2012; Vol. 2: Issue 1, Pg 15-18 [AJPTech.]


16
while glycosides are cardiotonic, thus making Terminalia
arjuna unique amongst currently used medicinal plants.

MATERIALS AND METHODS:
Collection of plant materials:
The plant material Terminalia arjuna was collected from
Trichy district which was carefully identified with the help
of regional floras.

Extraction of plant material:
Aqueous extract was prepared according to the
methodology of Indian pharmacopoeia. The shady dried
plant materials were subjected to pulverization to get coarse
powder. The coarse powder material was subjected to sox
let extraction separately and successively with distilled
water. These extract was concentrated to dryness in flash
evaporator under reduce pressure and controlled
temperature (40 50
o
C). The aqueous extract put in air
tight containers stored in refrigeration.

Phytochemical analysis:
Phytochemical analysis for major phytoconstituents of the
plant extract was undertaken using standard methods .The
plant was screened for the presence of biologically active
compounds like sugars, amino acids, proteins, phenols,
terpenoids, etc.

Collection of animal species:
The healthy young adult female rats (120-200gm sex) were
kept separately in individual polypropylene cages with
stainless steel hopper. The females was nulliparous and non
pregnant, at the commencement of the study, the weight of
the animals were minimal and not exceed 20% of the
mean weight.

Experimental hepatotoxicity:
Isoniazid (100 mg/kg body weight) solution was prepared
separately in sterile distilled water. Rats were treated with
Isoniazid, administered for 10 days by IP route (Jing et al.,
2004).

In order to the study the effect of aqueous extract of bark of
Terminalia arjuna in rat 200mg/kg.bwt (Gupta et al., 2005).
Rats were divided into four groups.

Experimental design:
Albino rats of either sex between 120-200g were randomly
assigned into 4 groups of 6 animals each. Group-I
(Negative control) received 1ml/kg normal saline, Group-II
(Isoniazid 100mg/kg), Group-III (200mg/kg aqeous extract
only) and Group-IV (treated 200mg/kg aqeous extract after
induction with Isoniazid) were treated with respective
treatments for 20 days. The blood samples were drawn from
all the animals by puncturing retro-orbital plexus on 20
th

day of the treatment. The blood samples were centrifuged
immediately to get clear serum and subjected for estimation
of various biochemical parameters namely SGPT (Reitman
and Frankel, 1957), SGOT (King and King, 1954), ALP,
ACP (Committee on enzymes of the Scandinavian society),
and Serum protein (Lowry et al., 1951), Serum Bilirubin
(Malloy and Evelyn, 1937), SOD (Kakkar and
Viswanathan, 1998) GSH (Griffith1980).

Statistical Analysis:
The value are reported are means + SD (n=6). Experimental
results were statistically analyzed using the variance
(ANOVA) by student t test.

RESULTS AND DISCUSSION:
Preliminary phytochemical Screening:
The plant extract of Terminalia arjuna were screened for
the presence of biologically active compounds like steroids,
tannins, phenolics compound, quinone, terpinoids, sugar,
alkaloids and flavonoids (Table 1).

Table I : Preliminary phytochemical investigation in the aqueous
extract of bark of Terminalia arjuna
Phytochemical compound Result of qualitative test
Sugar +
Terpinoids +
Alkaloids +
Phenolic compounds +
Tannins +
Flavanoids +
Volatile oil -
Quinones +
Steroids +
Coumarins -
+ indicates presence; - indicates absence

Serum Protein:
A significant increase (p<0.01) in serum protein level was
noted in group III when compared to Group II. Also there
was a significant increase (p<0.05) in serum protein level in
group IV when compared to Group II. Thus the restoration
of the level of serum and tissue protein after the
administration of Terminalia arjuna confirmed the
hepatoprotective nature of it (Table II).

Previous studies on the hepatoprotective effect of
Terminalia chebula extract against antipyretic drug induced
hepatotoxicity proved that the extract exhibited
hepatoprotective nature by restoring the total serum protein
level (Gujarti et al., 2006).

Serum Bilirubin:
A significant increase (p<0.05) in serum bilirubin levels in
Group II was observed when compared to Group I. A
significant decrease (p<0.05) in serum bilirubin level was
noted in group IV when compared to Group II. But there
was no significant change when Group I was compared
with Group IV (Table II).

Bilirubin concentrations have been used to evaluate
chemically induced hepatic injury (Giuseppe cooper et al.,
2004). Treatment with Terminalia arjuna extract reduced
the elevated level of bilirubin. This is probably due to the
presence of highest content of phenolic derivatives that
exert bile flow and liver protection. Thus Terminalia arjuna
has cholertic activity.
Asian J. Pharm. Tech. 2012; Vol. 2: Issue 1, Pg 15-18 [AJPTech.]


17
Table II : Estimation of Serum Proteins and Bilirubin
S. No Groups Proteins (mg/g) Bilirubin (m/mg)
1. Group I 0.910.4 1.590.91
2. Group II 0.610.3 4.231.24
3. Group III 0.790.9 1.880.45
4. Group IV 0.880.1 2.021.31
Values are means SD from 6 rats in each observation.
*<0.05; **<0.01; as compared with Group II. +<0.05,
as compared with normal group.

Serum ALP:
Serum level of ALP were increased significantly in group II
rat (p<0.05) when compared to Group I. A significant
decrease in ALP levels were observed in Group III (p<0.01)
and Group IV (p<0.05). (Table III). Elevated levels of
serum and tissue alkaline phosphates are indicative of
cellular leakage and loss of functional integrity of cell
membrane in liver cells (Sethu mathavan et al., 2007).
Damage to liver cells cause leakage of cellular enzymes
into serum.

In the present study the ALP levels returned to normal by
administration of Terminalia arjuna with healing of
parenchyma and regeneration of hepatocytes. Previous
study on Solanum nigrum reduced the elevated level of
ALP in CCl
4
included hepatotoxicity (Ruiz et al., 2008).

Serum ACP:
A significant increase (p<0.05) in the levels of serum acid
phosphatase level was observed. A significant decrease
(p<0.01) in serum acid phosphatase level was seen in Group
III when compared to Group II. Similarly, a significant
decrease (p<0.05) in ACP levels was noted in Group IV
when compared to Group II. But no significant changes
were observed between Group I and Group IV (Table III).

The abnormal higher level of marker enzymes such as ACP
and bilirubin in the serum of CCl4 treated rat indicate
damage to hepatic cells (Venukumar and Latha, 2002).In
the present study, treatment with Terminalia arjuna extract
reduced the elevated levels of ACP . Glycosmis arborea
extract overcome the toxic effects of hepatotoxic agents by
the lowering the levels of ACP (Gomes et al., 2003).

Table III : Estimation of Serum ALP and ACP
S.
No
Groups Serum ALP
(KA Units)
Serum ACP(KA
Units)
1. Group I 13.960.78 5.680.29
2. Group II 22.920.16 11.820.31
3. Group III 15.280.98 5.930.11
4. Group IV 19.960.21 9.861.05
Values are mean SD from 6 rats in each observation. *<0.05;
**<0.01; as compared with Group II.
+<0.05, as compared with normal group

SGOT:
A significant increase in SGOT activity (p<0.05) was noted
in Group II when compared to Group I. A significant
decrease (P<0.05) in serum SGOT level was observed in
Group III when compared to Group II. Also a significant
decrease (P<0.05) in serum SGOT level in Group IV was
noted when compared to Group II (Table IV).
The Isoniazid has got hepatotoxic potentical (Yew et al.,
2006). Isoniazid metabolite hydrazine plays an important
role in inducing hepatotoxicity (Al-Howiriny et al., 2004).
Isoniazid hepatotoxicity results in hepatocellular damage,
thus variety of enzymes normally located in the cytosol are
released into blood stream. The elevated level of SGOT due
to Isoniazid hepatotoxicity was normalized by treatment
with Terminalia arjuna. This is probably because of free
radical scavenging activity of flavonoids and polyphenols
present in Terminalia arjuna.

SGPT:
An increased level of SGPT (p<0.05) was found in Group II
rat when compared with Group I. A significant decrease
(p<0.01) in serum SGPT level was noted in Group III when
compared to Group II. Also there was a significant decrease
(p<0.05) in serum SGPT level in Group IV when compared
to Group II. But there was no significant change between
Group I and Group IV (Table IV).

The normalization of plasma alanine amino transferase
(SGPT) has been proved to be a strategy for preventing the
development of hepatocellular carcinoma in hepatitis C
virus (HCV) infection. In the present study treatment with
Terminalia arjuna extract normalized the elevated SGPT
levels. Osthole, a simple coumarin caused strong reduction
of plasma SGPT and exhibited hepatoprotective nature
(Okamoto et al., 2005).

Table IV: Estimation of Serum SGOT and SGPT
S.
No
Groups Serum SGOT
(IU/L)
Serum SGPT
(IU/L)
1. Group I 16.282.09 6.960.75
2. Group II 32.541.28 12.330.58
3. Group III 17.291.33 6.990.55
4. Group IV 19.362.92 7.381.29
Values are mean SD from 6 rats in each observation. *<0.05;
**<0.01; as compared with Group II. +<0.05, as compared with normal
group.

SOD:
There was a significant decrease (p<0.05) in SOD levels in
Group II when compared to Group I. A significant increase
(p<0.01) in serum SOD level was noted in Group III when
compared to Group II. Also there was a significant decrease
(p<0.05) in serum level was noted in Group IV when
compared to group II. But there was no significant change
between Group I and Group IV (Table V).

SOD is very effective antioxidant enzyme and responsible
for catalytic disputation of highly reactive and potentially
toxic superoxide radicals to H2O2.The H2O2 is further
metabolized either by catalase or peroxidase. The non
enzymatic antioxidants such as GSH, Vit.E and Vit.C act as
scavengers (Foyer et al., 1993).


GSH:
There was a significant decrease (p<0.05) in level of
Reduced Glutathione in Group II when compared to Group
I. A significant increase (p<0.01) in level of Reduced
Asian J. Pharm. Tech. 2012; Vol. 2: Issue 1, Pg 15-18 [AJPTech.]


18
Glutathione was noted in Group III when compared to
Group II. Also there was a significant increase (p<0.05) in
level of reduced glutathione in Group IV. When compared
to Group II. But there was no significant change between
Group I and Group IV.

GSH in the cytosolic pool consists of 85% hepatocellular
GSH and 15% mitochondrial GSH. Hepatic GSH depletion
or even extra hepatic GSH depletion can provide useful
information on the protective role of GSH against toxic
foreign compounds. Thus GSH, be regarded as an
endogenous protective agent against drugs (Chattopadhyay,
1992).

Table V : Estimation of Serum SOD and GSH
S. No Groups Serum SOD (mg/dl) Serum GSH (mg/dl)
1. Group I 5.330.97 4.380.22
2. Group II 4.140.28 2.230.24
3. Group III 5.300.21 4.780.45
4. Group IV 5.162.01 5.151.31
Values are mean SD from 6 rats in each observation. *<0.05;
**<0.01; as compared with Group II. +<0.05, as compared with normal
group.

CONCLUSION:
The present findings demonstrated the hepatoprotective and
antioxidant effect of Terminalia arjuna aqueous bark
extract against Isoniazid induced hepatotoxicity. According
to traditional indigenous medicinal systems of India this
plant has got several medicinal effects without producing
any severe side effects. This plant could be very well used
as hepato protectant. However this is only a tentative
research and more work is to be done to identify the extract
phytochemical responsible for this curative effect.

REFERENCES:

1. Al-Howiriny TA, Al-Sohaibani MO, Al-Said MS, Al-Yahya
MA., Tahir KH. and Rafatullah S. Hepatoprotective properties of
Commiphora opobalsamum (Balessan), a traditional medicinal
plant of Saudi Arabia. J. Drugs-Exp-Clin-Res., 30 (5-6); 2004:
213-20.
2. Arias PS, Bessey AA, Lowery DM and Brock M. Lipid
peroxidation Biochemistry measurement and significance in liver
cell injury. Indian. J. Exp. Biol., 45; 1989: 87-89.
3. Chattopadhyay RR, Sarkar SK, Ganguly S, Medda C and Basu
TK. Hepatoprotective activity of Ocimum sanctum leaf extract
against paracetamol induced hepatic damage in rats. J.
Ethnopharmacol., 24; 1992:163.
4. Chopra RN and Ghosh S. Terminalia arjuna: its chemistry,
pharmacology and therapeutic action. Indian Medical Gazette,
64; 1929: 70-73.
5. Foyer CH, Alscher RG, Hess JL. Antioxidants in higher plants.
Boca Raton CRC Press 1993; 3158.
6. Gilan S, Deduce C., Singh J, Hussain Janbaz MS. Evaluation of
the protection potential of Agave Americana extract on
acetaminophen induced liver injury.2002.
7. Giuseppe Cooper JR, Bloom F.E. and Roth R.H..
Hepatopharmacological study of Agave Americana linn in
thioacetamide induced liver damage in rats. Indian Medicinal
Plants. 2004: 10-12.
8. Gomes M, Das P, Besra SE, Chakravorty A, Das B, Ganguly DK
and Vedasiromoni JR. Glycosmis arborea extract as a
hepatoprotective agent. Phytother. Res., 17(5); 2003: 571-74.
9. Griffith O. Determination of glutathione and glutathione disulfide
using glutathione reductase and 2-vinyl pyridine. Anal Biochem.,
106; 1980: 207.
10. Gujarti S, Nigerians K, Pandima Devi M, Sreepriya K and.
Balakrishnan K. Protective activity Tylophora indica extract on
paracetamol induced liver damage. I. J. of Ethnopharmacology,
3; 2006: 95-100.
11. Gupta RK, Kesari AN, Murthy PS, Chandran R, Tandon V and
Wata G. Antihyperglycemic activity of Ficus deltoidea ethanolic
extract in normal rats. J. Ethnopharmacol., 99(1); 2005: 75-81.
12. Jing TUE, Ren-xiu ENG, Jing YANG, Rui. CYP2E1 isoniazid
induced hepatotoxicity in rats. Acta Pharmacol Sin., 25(5);
2004: 699-704.
13. Kakkar P, Das B, Viswanathan PN. A modified spectrophoto
metric assay of superoxide dismutase. Indian J. Biochem.
Biophys 21; 1998: 130132.
14. King PRN and King EJJ. Estimation of plasma phosphatase by
determination of hydrolyzed phenol with aminopyrines. J. Clin.
Pathol., 7; 1954: 332.
15. Lowry OH, Rosebrough NJ, Farr A and Randall RJ. Protein
measurement with the Folin phenol reagent, J. Biol
Che.,193;1951:265.
16. Malloy HT, Evelyn KA. The determination of bilirubin with the
photometric colorimeter. J. Biol. Chem., 119; 1937: 481.
17. Micheale P, Cynthiya Ju. Mechanism of Drug induced liver
injury. The AAPS Journal., 1; 2006: 48-54.
18. Nadkarni AK, Nadkarni KM. Indian Materia Medica. First ed.
Popular Book Deport. Bomaby India, 1954:1198.
19. Okamoto Nadro, Rall, Eteng Mu, Hepatoprotective effect of
Balamites agyptiaca against the liver damage. I. J. of
Pharmacol. Sci, 30; 2005: 148-151.
20. Reitman S, Frankel S. Colorimetric method for the determination
of serum glutamatic oxaloacetic acid and glutamic pyruvic
transaminases. Am. J. Clin. Pathol., 28; 1957:56.
21. Robbins J, Fleurentin C, Hefler A. Hepatoprotective properties of
crepisrueppelli and anisotes Trisules. Journal of
ethanopharmacology., 76; 2003: 105-111.
22. Ruiz-Term F, Merdrano-Martinez A and Navaroo Ocana.
Antioxidant and free radical scavenging activities of plant extract
used in traditional medicine in mexico. African Journal of
Biotechnology. 7(12): 2008; 1886.
23. Sethumadhavan, S., Theruvathil, S., Rangaswamy, A.,
Hepatoprotective activity of chitosan against isoniazid and
rifampicin-induced toxicity in experimental rats. European J.
Pharmacol. Xx; 2007: XXX-XXX.
24. The committee on enzymes of the Scandinavian society for
clinical chemistry and clinical Physiology, Recommended
method for determination of fourenzymes in blood, J Clin. Lab.
Invest. 33; 1974: 291-98.
25. Venukumar MR. and Latha MS. Antioxidant activity of Ballota
glandulosissima in CCl4 induced hepatopathy in rats. I. J. of
Clin. Biochemi., 17; 2002: 80-87.
26. Yew AH, Gillani AH, Janbaz KH. Protective effect of Artemisia
scoparia extract against carbon tetra chloride induced
hepatotoxicity. Gen Pharmacol., 24(6); 2006:455-58.