ANTI HEPATOTOXICITY STUDIES OF CRUDE EXTRACT OF FERONIA LIMONIA IN CCL4 INDUCED TOXICITY

www.wjpps.com Vol 3, Issue 7, 2014.

1536

Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences

ANTI HEPATOTOXICITY STUDIES OF CRUDE EXTRACT OF

FERONIA LIMONIA IN CCL4 INDUCED TOXICITY

Jayashree V Hanchinalmath and Ramesh Londonkar*

Dept of Biotechnology, Gulbarga University, Gulbarga - 585106, Karnataka, India.

ABSTRACT

The antioxidant activities of Feronia limonia fruit pulp methanolic

extract (MEFL) was investigated by and in vivo assay. The effect of

the extract in reducing carbon tetrachloride induced (CCl4) oxidative

stress in rats was evaluated. Hepatoprotective activity of MEFL was

studied by estimating serum enzyme activities of aspartate

aminotransferase (AST), alanine aminotransferase (ALT), alkaline

phosphatase (ALP), Serum Lactate Dehydrogenase (LDH), total

protein (TP) and total bilirubin (TB). The treatment with MEFL

showed a dose-dependent reduction in CCl4 induced elevated serum

levels of enzyme activities with parallel increase in total protein and bilirubin content

indicating the extract could preserve the normal functional status of the liver. MEFL at the

doses of 200 and 400 mg/kg body weight significantly increased the glutathione (GSH), super

oxide dismutase (SOD) and catalase (CAT) activities in a dose dependant manner. Therefore,

the results of this study illustrate that the Feronia limonia methanol extract can shield the

liver against CCl4-induced oxidative damage in rats.

Keywords: Feronia limonia, antioxidant activity, phenolic content, CCl4.

INTRODUCTION

Feronia limonia commonly known as wood apple is an important plant in Ayurveda, the

traditional system of Indian medicine. This fruit is recommended for the treatment of tumors,

asthma, wounds, diarrhea, dysentery, cardiac dysfunction, hepatitis, sore throat and is

considered as tonic, astringent (when unripe), antiscorbutic, and alexiformic agent [1,2] .

Feronia limonia fruit has also been reported to possess hypoglycemic and hypolipidaemic

and wound healing properties[3,4].

WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS SSJJIIFF IImmppaacctt FFaaccttoorr 22..778866

VVoolluummee 33,, IIssssuuee 77,, 11553366--11554466.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357

Article Received on 8 May 2014, Revised on 04 June 2014, Accepted on 24 June 2014

*Correspondence for Author

Prof. Ramesh Londonkar

Dept of Biotechnology,

Gulbarga University, Gulbarga

- 585106, Karnataka, India.


1537


Antioxidant activity is a fundamental property important for life. Many of the biological

functions, such as antimutagenicity, anticarcinogenicity, and antiaging, among others,

originate from this property[5] Antioxidants are compounds that can delay or inhibit the

oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative

chain reactions [6]. It has been suggested that natural antioxidants like L-ascorbic acid are

more safe and healthy than synthetic antioxidants such as butylated hydroxytoluene (BHT)

[7]. Hence, there is need for safe, cost effective and less toxic antioxidants with high activity

from natural sources to replace the synthetic chemicals. The importance of the antioxidant

constituents of plant materials in maintaining health and protection from coronary heart

disease. Cancer is another deadly disease raising interest among scientists and food

manufacturers towards the search of functional food with specific health effects [8]. Potential

sources of antioxidant compounds have been searched in several types of plant materials such

as fruits, leaves, seeds, barks, roots and crude plant drugs [9]. Crude extracts of plant

materials rich in phenolics are increasingly of interest in the food industry because they retard

oxidative degradation of lipids and thereby improve the quality and nutritional value of food.

The antioxidative effect is mainly due to flavonoids and phenolic acids present in the plants.

Many of the natural antioxidants, especially phenolics, exhibit a wide range of biological

effects including antibacterial, antiviral, anti-inflammatory, antiallergic, antithrombotic, and

vasodilatory actions [10].

Liver diseases, especially viral hepatitis occurs principally with an enormous impact on

public health. Carbon tetrachloride (CCl4) is widely used in animal models to induce acute

liver injury [11]. It is commonly believed that the toxicity of CCl4 results from its reductive

dehalogenation by the cytochrome P450 enzyme system into the highly reactive free radical

trichloromethyl radical [12-14]. Antioxidant action has been reported to play a crucial role in

the hepatoprotection [15]. In the present study, an attempt has been made to explore the effect

of MEFL in CCl4 induced hepatic damage pertaining to biochemical marker enzymes &

histopathology. The result of this In Vivo study will support the plant as a good herbal

antioxidant agent.

MATERIALS AND METHODS

Chemicals

CCl4 were purchased from Sigma-Aldrich (Mumbai). Liv 52 from Himalaya drugs Ltd. All

other chemicals and reagents were of analytical grade, and they were used as received.


1538


Plant material and extraction

Fruits of Feronia limonia L were collected from local market Bellary, Karnataka, India

during the month of March, 2012. It was authenticated by the Department of P.G studies and

Research in Botany, Gulbarga University, Gulbarga. Crude fruit pulp extract was prepared by

Soxhlet extraction method. About 50gm of powdered plant material was packed in a thimble

and extracted successively with 350ml of petroleum ether, chloroform, and methanol. The

process of extraction is carried out until the solvent in siphon tube of an extractor become

colorless. The extract was taken in a petriplates and kept in hot air oven and heated at 30-

40ºC till the solvent got evaporated. Solvent free methanolic extract dissolved in 1%

Dimethyl sulphoxide (DMSO) was used for the In Vivo studies.

Animals

Wistar strain Albino rats of inbred colony weighing about 150 – 175 g were used. The

protocol was approved by the Institute’s Animal Ethical Committee (IAEC Reg No. 34800/

CPCSEA Dated: 19.08.2001). Animals were kept in animal house at an ambient temperature

of 250C and 45 – 55% relative humidity, with 12 h each of dark and light cycles. They were

fed with a balanced diet as described by Central Food and Technological Research Institute

(CFTRI, Mysore) and water ad libitum [16].

Acute toxicity experiment

Albino rats were divided into control and test groups (6 animals each). Control group

received the vehicle (3% Tween 80) while the test groups got graded doses (100–4000

mg/kg) of MEFL orally and were observed for mortality till 48 h and the LD50 was

calculated.

Induction of experimental hepatotoxicity

30% CCl4 was prepared in olive oil. Animals of group 2, 3, 4 and 5 were given single dose of

CCl4 at 1ml/kg body weight (bw) intraperitoneally (i.p). Methanolic extract of Feronia

limonia at the dose level of 200 mg/kg bw and 400 mg/kg bw as low dose and high dose were

administered to animals of group 4 and 5 orally with the aid of an intragastric catheter for 7

days. Liv 52 (50 mg/kg bw) was used orally as a standard drug to group 3 at a single dose.

Rats were divided into five groups as following protocol.

GROUP 1: Normal control (n=6, the animals were given saline at 1ml/kg body weight)

GROUP 2: Hepatotoxic control (n=6, the animals were given CCl4 at 1ml/kg body weight)


1539


GROUP 3: Positive control (n=6, the animals were given CCl4 + Liv 52 for 7 days)

GROUP 4: Treatment group (n=6, the animals were given CCl4 + MEFL low dose for 7 days)

GROUP 5: Treatment group (n=6, the animals were given CCl4 + MEFL high dose for 7

days)

At the end of the experimental period, blood sample from each rat (2 ml) was withdrawn by

cardiac puncture and collected in previously labeled centrifuge tubes and allowed to clot for

30 min at room temperature. Serum was separated by centrifugation at 10,000 rpm for 5 min.

Assessment of hepatotoxicity

Liver functions were evaluated by measuring the serum activity of ALT and AST following

the method of [17] while the activities of ALP and LDH were estimated. The serum

concentrations of TB estimated according to [20] and TP as described by [21].

Assessment of oxidative stress

Liver tissue was homogenized in 10 volume of 100 mM KH2PO4 buffer containing 1 mM

EDTA (pH 7.4) and centrifuged at 12,000 rpm for 30 min at 40C. The activities of the

antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were assayed

in the hepatic tissue homogenate of the control and experimental rats according to the

methods of [22,23]. GSH tissue content was also measured [24].

Histopathological study

Liver was dissected out and divided into two parts. One part was kept in liquid nitrogen for

determination of antioxidant status and the other part was immediately fixed in buffered

formalin 10% and was used for histopathological examination using the standard micro

technique [25].

Statistical analysis of data

Data were presented as means ± SD of three experiments. Analysis of variance was

performed on the data obtained. Significance of differences between means was determined

by least significant differences (LSD) at P ≤ 0.05.

RESULTS

Acute toxicity study

The dose selection for MEFL was based on the acute toxicity study. The study did not show

any adverse effect of doses up to 4000 mg/kg. Accordingly, experimental oral doses of 200


1540


and 400 mg/ kg equal to one-twentieth and one-tenth of the feasible dose of the extract that

did not cause mortality in rats were selected.

Assessment of hepatotoxicity

The effects of methanol extract of Feronia limonia at dose levels of (200 and 400 mg/kg bw)

on serum marker enzymes are shown in Table 2. Hepatic injury induced by CCl4 has caused

significant rise in marker enzymes such as ALT, AST, ALP activities and decrease in serum

TP, TB levels (P < 0.01). Administration of methanol extract of Feronia limonia at two

different dose levels attenuated the increased levels of the serum enzymes, produced by CCl4,

and caused a subsequent recovery towards normalization almost like that of Liv 52 treatment.

Table 1 Effect of methanol extract of Feronia limonia on serum marker enzymes, total

protein and total bilirubin

Group ALT(IU/L) AST (IU/L) ALP (IU/L) TP (µmol/L) TB (µmol/L) NC 64.18 ± 0.23 81.56 ± 5.54 107.69 ± 1.33 8.14 ± 0.37 0.64 ± 0.04

HC 170.22 ± 0.14 b

134.91 ± 7.33 b

152.56 ± 1.07 b 4.11 ± 0.3 b 1.81 ± 0.09 b

CCl4+ Liv 52 (50mg/kg bw) 91.31 ± 0.42 d 96.07 ± 4.38

d 120.01 ± 1.25

d 7.00 ± 0.24 c 0.72 ± 0.05 c

CCl4+ MEFL (200mg/kg bw)

142.10 ± 0.25 d

126.72 ± 7.16 d

144.30 ± 1.66 d 5.01 ± 0.35 d 1.43 ± 0.06 d

CCl4+ MEFL (400mg/kg bw

117.35 ± 0.31 d

115.80 ± 7.43 d

132.78 ± 2.01 d 6.25 ± 0.65 d 1.09 ± 0.05 d

Results are expressed as mean ± S.E.M; b P < 0.01, compared with NC group; c P < 0.05, d P

< 0.01, compared with CCl4-treated group.

Note: NC (normal control), HC (hepatotoxic control).

Assessment of oxidative stress

CCl4 treatment also resulted in the depletion (P<0.01) of the hepatic antioxidant enzymes.

The activities of GSH, SOD, CAT were depleted to 1.01 ± 0.12, 17 ± 0.17 and 23.16 ± 1.27

respectively of the hepatotoxic control (Table 3). The decline in the activities were noticeably

attenuated (P<0.01) by administration of 200 mg/kg bw and 400 mg/kg bw in MEFL treated

rats. Treatment with MEFL enhanced liver antioxidant enzymes yet after CCl4 treatment.


1541


Table 2 Effect of methanol extract of Feronia limonia on GSH levels, CAT and SOD

activities

Group GSH (mg/g pro)

CAT (U/g prot) SOD (U/g prot)

NC 1.47 ± 0.43 45.02 ± 1.33 26.54 ± 0.33 HC 1.01 ± 0.12 b 23.16 ± 1.27 b 17.00 ± 0.17 b CCl4+ Liv 52 (50mg/kg bw) 1.32 ± 0.27 d 39.58 ± 1.07 d 22.03 ± 0.25 c

CCl4+ MEFL (200mg/kg bw)

1.16 ± 0.22 d 27.20 ± 1.74 c 18.21 ± 0.66 d

CCl4+ MEFL (400mg/kg bw

1.24 ± 0.26 d 34.10 ± 1.45 d 20.95 ± 0.21 d

Results are expressed as mean ± S.E.M; b P < 0.01, compared with NC group; c P < 0.05, d P

< 0.01, compared with CCl4-treated group.

Note: NC (normal control), HC (hepatotoxic control)

Histopathology

The histopathological examination displayed significant recovery of hepatocytes in the

standard drug and MEFL treated animals, which is again correlated with the biochemical

parameters. The results of the liver histopathological studies (Figs 5a-5e) showed hepatocytes

swelling and necrosis in CCl4-treated rats (Fig 5b) in comparison with normal control rats

(Fig 5a). Treatment with MEFL exhibited a significant protection against hepatocytes injury

and showed complete normalization of the tissues where no fatty accumulation or necrosis

was seen (Fig 5d, 5e). The central vein appeared clearly indicating a potent anti-hepatotoxic

activity. MEFL was found to exhibit a potent anti-hepatotoxicity compared with standard

drug Liv 52 (Fig 5c). The liver section showed the structure of the portal triad and a normal

liver parenchyma, and the central vein appeared clearly. There was no lymphocytic

infiltration and fatty deposition representing a potent anti-hepatotoxicity for the MEFL under

study.


1542


Fig 5a

Fig 5b Fig 5c

Fig 5d Fig 5e

Fig 5: Light microphotographs (20x) of liver cells of rats a. Normal control b. CCl4

induced c. CCl4 + Liv 52 d. CCl4 + MEFL(200 mg/kg bw) e. CCl4 + MEFL (400 mg/kg

bw)

DISCUSSION

Free radicals are known to play a definite role in a wide variety of pathological

manifestations. Antioxidants due to their scavenging activity are useful for the management


1543


of these diseases. They exert their action either by scavenging the reactive oxygen species or

protecting the antioxidant defense mechanisms [26]. The present study suggests that Feronia

limonia can be used as a source of antioxidants for pharmacological preparations. Non-

phenolic compounds of the plants such as trace elements may decrease the antioxidant

activity of the phenolic compounds [27].

Liver injury caused by CCl4 in rats was first reported [28] and is extensively and effectively

used by many investigators. Carbon tetrachloride is metabolized by cytochrome P-450 in

endoplasmic reticulum and mitochondria with the formation of CCl3O-, a reactive oxidative

free radical, which initiates lipid peroxidation. In the presence of oxidative stress more of

lipid peroxidation products are formed due to cell damage. In this study, carbon tetrachloride

damage to erythrocytes was confirmed by the increase in SOD, GSH and CAT activities, and

decrease in membrane fluidity. SOD is one of the crucial components in the antioxidant

defense system for the reduction of reactive oxygen species (ROS) and peroxides produced in

the living organism and in detoxification of compounds of exogenous origin, thus playing a

primary role in the maintenance of a balanced redox status[29]. The increase of SOD activity

suggests that the MEFL has an efficient protective mechanism in response to ROS. Catalase

is a very important component of the antioxidant defense system. The increased SOD activity

resulted in the accumulation of hydrogen peroxide, which stimulated increases in CAT

activity. The MEFL increased the activities of Catalase in CCl4 induced liver damage rats to

prevent the accumulation of excessive free radicals and thus protects the liver from

intoxication. GSH is a naturally occurring substance abundant in many living creatures; GSH

depletion increases the sensitivity of cells to various aggressions leading to tissue disorder

and injury[30]. In the present study we demonstrated the efficiency of the extract by using

CCl4 induced rats and found that exogenous MEFL supplementation elevated GSH levels in

rats with CCl4 treatment and thus might provide a mean of recovering reduced GSH levels to

prevent tissue injury. Treatment of experimental animals with the MEFL exhibited an

improved free radical scavenging ensuring decrease in activities of enzymes towards normal.

The field of dietary modification and chemoprevention show considerable effective approach

against oxidative stress and are the focus of research these days. In the assessment of liver

damage by CCl4, the determination of enzyme levels such as AST, ALP and ALT is largely

used. Bilirubin concentration has been used to evaluate chemically induced hepatic injury.

The data showed that the control group demonstrated a normal range of AST, ALP, ALT,

protein and bilirubin levels, while the CCl4-treated group showed elevated levels confirming


1544


that CCl4 caused liver injury, altered membrane integrity and as a result enzymes in

hepatocytes leak out [31]. However, after treatment with MEFL, the increase in ALT, AST

and ALP were significantly restored. Increase in the level of TP by MEFL indicates

hepatoprotective activity, as stimulation of protein synthesis accelerates the regeneration

process and production of liver cells. These results indicate that the extract has the ability to

protect against CCl4-induced hepatocyte injury, which is in agreement with the previous

study of [32] that reported the protective consequence of polyphenolic compounds against

CCl4-induced liver cirrhosis. Therefore, it is valid to consider that MEFL, possess antioxidant

property which is capable of protecting the hepatic tissue from CCl4-induced injury and

inflammatory changes might be due to the presence of phenolics.

CONCLUSION

Our investigations reveals that MEFL hold potent antioxidant activity. The current study

demonstrates that MEFL exerts effective protection against acute chemical induced hepatic

damage suppressing liver enzyme activities and thus preventing cell membrane damage.

Histopathological study revealed morphological evidence and bioassays revealed functional

evidence. MEFL can thus be proposed to protect the liver against CCl4‐induced oxidative

damage in rats. The in vivo assays indicate that this plant extract is a significant source of

natural antioxidant, which might help prevent the progress of oxidative stress. However, the

components responsible for the antioxidant activity are currently unclear. Efforts are in

progress in our laboratory to isolate and purify the active principle of this medicinal plant.

REFERENCES

1. Kirtikar K.R, Basu BD.(1989) Indian Medicinal Plants. Lalit Mohan Basu Publications.,

Allahabad, India.

2. Morton J F., (1987).Fruits of warm climates. Indiana.USA: Centre for New crops & plant

products. Purdue University.

3. Kangralkar V.A., Patil S.D., Bandivadekar R.M., Nandagaon V.S., Burli S.C. (2010)

Hypoglycemic and hypolipidaemic effect of methanolic extract of Feronia elephantum

fruits in alloxan diabetic rats. Int. J. Pharm. Sci. Rev. Res .4(1):64-66.

4. Ilango K., Chitra V.(20120) Wound healing and antioxidant activities of the fruit pulp of

Limonia acidissima Linn (Rutaceae) in rats. Trop. J. Pharm. Res.9(3):223-230.


1545


5. Huang M.T., Ho C.T., Lee C.Y.(1992) Phenolic Compounds in Food and Their Effects on

Health. II. Antioxidants and Cancer Prevention. ACS Symposium Series 507, American

Chemical Society., Washington.

6. Sun., Chu., Wu., Liu.(2002) Antioxidant and antiproliferative activities of common fruits.

J. of Agri. and Food Chem .50:7449-7454 .

7. Galati E.M., Mondello M.R., Lauriano E.R., Taviano M.F., Galluzzo., Miceli N.O.(2005)

Fruit juice protects liver from carbon tetrachloride-induced injury. Phytother. Res.

19(9):796-800.

8. Lo-liger J.(1991).The use of antioxidants in food. In Free Radicals and Food Additives.

Taylor and Francis., London.

9. Ramarathnam N., Ochi H., Takeuchi M .(1997) Antioxidant defense system in vegetable

extracts. In Shahidi F (ed) Natural Antioxidants: Chemistry, Health Effects, and

Applications. AOCS Press., Champaign.

10. Pietta P.G.(1998) Flavonoids in medicinal plants. In CA Rice- Evans & L Packer (Eds)

Flavonoids in health and disease. New York: Dekker., New York.

11. Cook N.C., Samman S.(1996) Flavonoids-Chemistry, metabolism, cardioprotective

effects and dietary sources. Nutr. Biochem . 7: 66-76.

12. Mizuoka H., Shikata N., Yang J., Takasu M., Inoue K., Tsubura A.(1999). Biphasic effect

of colchicines on acute liver injury induced by carbon tetrachloride or by

dimethylnitrosamine in mice. J Hepatol.1:825-833.

13. Rao P.S., Mangipudy R., Mehendale M.H.(1997) Tissue injury and repair as parallel and

opposing responses to CCl4 hepatotoxicity: A novel dose-response. Toxicology. 118:

181-193.

14. Czaja M.J., Xu J., Alt E.(1995) Prevention of carbon tetrachloride induced rat liver injury

by soluble tumor necrosis factor receptor. Gastroenterology.108:849-854.

15. Recknagel R.O., Glende E.A., Dolak J.A., Waller R.L. (1989).Mechanisms of carbon

tetrachloride Toxicity. Pharmacol. Ther. 43:139-154.

16. OECD (2008).Organisation for economic co-operation and development ,OECD

guidelines for the testing of chemicals 425.

17. Reitman S., Frankel S.(1957)Colorimetric methods for aspartate and alanine

monotransferases. Am. J. Clin. Pathol.:55–60.

18. Babson L.A., Greeley S.J., Coleman, C.M., Phyllips G.D.(1996) Phenolphthalein

Monophosphate as a substrate for serum alkaline phosphatase Clin. Chem. 12:482–490.


1546


19. King J.(1965) The hydrolase and alkaline phosphatase. In: Practical Clinical

Enzymology. Nostrand Co. Ltd, London.

20. Walter M., Gerarade H.(1970) Ultramicro method for the determination of conjugated

and total bilirubin in serum or plasma. Microche. J.15:231

21. Henary R.J., Cannon D.C., Winkleman J.W.(1974).Clinical Chemistry Principles and

Techniques. In : Harper and Roe (second ed).

22. Sun M., Zigman S. (1978). An improved spectrophotometric assay for superoxide

dismutase based on epinephrine autoxidation. Anal. Bioch.247:81–89

23. Chance B., Maehley A.(1995).Assay of catalases and peroxidases. Meth. Enzymol .2: 764

24. Moron M.J., Diperre J.W., Mannerv K.B(1979). Levels of glutathione, glutathione

reductase and glutathione-S-transferase activities in rat lungs and liver. Biochem.

Biophys. Acta.582:67–71

25. Luna L.G.(1968) Manual of histology, staining methods of armed forces. In Mc Graw-

Hill Book Co., New York.

26. Umamaheswari M., Chatterjee T.K.(2008). In vitro antioxidant activities of the fractions

of Coccinnia grandis L. leaf extract. Afr. J. Trad. Compl. Altern. Med.5:61–73.

27. Vinson J.A., Hao Y., Su X., Zubik L.(1998). Phenol antioxidant quantity and quality in

foods: vegetables. J. Agric. Food. Chem.46:3630-3634.

28. Cameron G.R., Thomas J.C., Karunarathe.(1936). WAE: The pathogenesis of liver injury

in carbon tetrachloride and thioacetamide poisioning. J. Path. Bact.41:297.

29. Alia M., Horcajo C., Bravo L., Goya L.(2003). Effect of grape antioxidant dietary fiber

on the total antioxidant capacity and the activity of liver antioxidant enzymes in rats.

Nutr. Res.23:1251–1267.

30. Jollow D.J.(1980).Glutathione thresholds in reactive metabolite toxicity. Arch. Toxicol.

Suppl.3:95–110.

31. Cheng H.L., Hu Y.Y., Wang R.P., Liu C., Liu P., Zhu D.Y.(200).Protective actions of

salvianolic acid on hepatocyte injured by peroxidation in vitro. World. J.

Gastroenterol.6:402–404.

32. Xiao-hui H., Liang-qi C., Xi-ling C., Kai S., Yun-jian L., Long-juan Z.(2007).Polyphenol

epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon

tetrachloride–induced hepatic fibrosis. Nutr. Biochem.3:511–51.

ANTI HEPATOTOXICITY STUDIES OF CRUDE EXTRACT OF FERONIA LIMONIA IN CCL4 INDUCED TOXICITY

Documents

Transcript of ANTI HEPATOTOXICITY STUDIES OF CRUDE EXTRACT OF FERONIA LIMONIA IN CCL4 INDUCED TOXICITY