Biokemistri Nigerian Society for Experimental Biology ISSN: 0795-8080 Vol. 19, Num. 1, 2007, pp. 17-21

Biokemistri, Vol. 19, No. 1, June, 2007, pp. 17-21

Hepatoprotective effects of Garcinia kola   seed against hepatotoxicity induced by carbon tetrachloride in rats

Mathew O. Wegwu¹* and Blessing C. Didia²

¹Toxicological  Unit of  Biochemistry Department, University  of  Port  Harcourt, P.M.B. 5323, Port  Harcourt, Nigeria
²Department of Anatomy, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria
E-mail: wevic2000@yahoo.com Tel: +2348068698933

Received 9February 2006

Code Number: bk07003

Abstract

The protective  effects  of  Garcinia kola against  a  dose  of  Carbon-Tetrachloride (CC1₄)-induced liver  damage  in experimental rats  were  investigated. The  CC1₄ induction  (administered  intraperitoneally, 0.5m1/kg body  weight  in olive  oil-0.5ml/kg body weight)  led to significant increases in the levels of  serum  aspartate and alanine  amino  transferases,  alkaline  phosphatase  and  lipid  peroxides  in CC1₄ intoxicated  rats.  Pretreatment with  varied    concentrations  of  Garcinia kola diets (1%, 5% and 10%)  and  vitamin  E (9%,) for  21 days  prior  to CC1₄ administration   resulted  in  significant  decreases  in Liver  marker  enzymes  and  lipid  peroxides.  These  findings  suggest  that  Garcinia kola seed  may  be  acting  as   a  natural  antioxidant  that  prevents  hepatic  oxidative  stress  induced  by  CC1₄

Keywords:Hepatoprotective; Garcinia kola; Liver marker enzymes; Lipid peroxides

INTRODUCTION

The demand for therapeutic drugs from natural products is on the increase in recent   times.  This is  traceable  to  the realization  that  plant  products  contain  active constituents that  are capable  of  curing majority  of man’s  diseases.  Indeed, drugs of natural origin are the only widely used hepatoprotectives¹.

Garcinia kola belongs  to  the Family Clusiaceae  guttiferae  and contains  a  complex  mixture of  biflavonoids, prenylated  benzophenones  and  xanthones².   The plant has shown anti-inflammatory, antimicrobial, pharmacological and antiviral properties. It has  been confirmed  that  seeds  of  Garcinia kola contain energy-yielding  nutrients (proteins, lipids,  carbohydrates) and  minute  quantities  of  Kolaviron (consisting  of  biflavonoids  GB-1, GB-2 and  Kolaflavone)³.   The  seeds  of  Garcinia kola  have  been  employed  in many  herbal  preparations  in Nigeria  for the  treatment  of  ailments   ranging  from laryngitis,  bronchitis  to liver disorders⁴.

Trichloromethyl  radicals  are  generated  from  CC1₄, in  vivo,  which  stimulate  a  sequence  of  biochemical  reactions  that  lead  to  the initiation  of  lipid  membrane  peroxidation⁵.  The  ethanol  inducible  isoform  of  the  P₄₅₀ cytochrome  is  believed  to play  an  active  role  in this  process.  Indeed, the process  of   CC1₄-induction  of  lipid  peroxidation  provides  useful  information  that  could  be  explored  in examining   antioxidant  properties  of  natural products. The  aim of  this  study was  to assess  the ability  of  Garcinia kola  seeds  to exhibit  antioxidant  actions  against  CC1₄ - induced  liver damage  in rats.

MATERIALS AND METHODS

The  seeds  of  Garcinia kola (purchased  from Choba  market  in Rivers  State, Nigeria)  were peeled,  sliced  and dried  in the air  for  5 days.  The dried,  sliced  feeds  were  ground   into  flour   with an  electric  blender  (Model   MX –795N-National)⁶. Male albino rats of the Wistar strain (140g-160g) were obtained from the University of Port Harcourt animal house.  They were housed  in  Griffin and  George  modular  cage  system and  left  to acclimatize  to laboratory  conditions  for  7 days  prior  to commencement  of  work.  The animals  were  fed  with  a commercial  pelleted  diet  (purchased  from Top  Feeds,  Nigeria  Ltd.  Port Harcourt, Nigeria) and water ad libitum.

Experimental procedure

The rats were divided into six groups with each group comprising five animals. Rats  in groups  1 and  2  received  the pelleted  diet  and water, while  those  in groups 3, 4 and 5 were fed  with diet formulated  with  the flour  of  Garcinia kola  and  rat pellets  as  follows: group 3-1% flour of G.  cola; group 4-5% flour of G.  cola; group 5-10% flour of G.  cola

Also, animals in group 6 received diet compounded with vitamin E and rat pellets   (9% vitamin E). All the  rats  in  the various  groups  received  their  respective diets  and water  ad libitum  for  21 days. On the 22^nd day of the experiment, CCl₄ (0.5ml/kg body weight in 0.5 olive oil) was administered   intraperiteneally to rats in groups 2, 3, 4, 5 and 6. The  animals  were allowed  to fast  for  24 hours  after which they were anaesthetized  in a  chloroform  saturated  chamber⁷. Blood  samples  were  obtained  by cardiac   puncture  from each rat  by means  of  a  5ml  hypodermic  syringe  and  needle.  The  blood  samples  were introduced  into clean,  dry bottles  without  anticoagulants  for  serum  separation. The bottles and its contents were centrifuged at 5000g for 10 minutes (model: MSE – Minor 35 centrifuge). Serum was collected into a clean, dry sample container. The serum  levels of  L-aspartate aminotransferase  (AST), L-alanine  transferase (ALT)  and  alkaline  phosphatase (ALP) were  measured spectrophotometrically as  described  by Verly⁸. The  liver  was  excised, washed  in ice-cold  saline,  and homogenized  at  0.1 M Tris-HC1 buffer  (pH 7.4; 4⁰C) in a  homogenizer  at  600 rpm for  4 minutes  using  mortar  and  pestle⁹. The  liver  homogenate was  employed  in assaying   the activities  of  the lipid  peroxides  as  described  by Hunter  et. al.¹⁰ and  modified  by Gutteridge and  Wilkins¹¹. The  mean values  of  the various  groups  were compared  using  analysis  of  variance (ANOVA) and  the  level of  significance  was  set  at  p ≤ 0.05.

RESULTS

The effects  of  pretreatment  with  seeds of  Garcinia kola  and  vitamin E, 21  days  prior  to CC1₄  administration on liver  enzymes  and lipid  peroxides  in rats  is  shown in   figures 1 and 2.

Group  2 rats that  received  a  single  dose of  CC1₄ showed   marked  elevation in the levels  of  liver  enzymes  when  compared  with those  of  the  group 1 (control)  rats.  The pretreated  groups 3, 4, 5 and 6 rats  showed  remarkable decline  in the levels  of  AST, ALT  and  ALP  when compared  with group 2 rats  that  received  CC1₄ alone.

In the liver tissue, increased levels of lipid peroxides were recorded in the group 2 rats (figure 2).  The activities of the peroxides decreased with increase in the amount of the seeds of Garcinia kola in the feed formula. This is evidenced in the values obtained in rats in groups 3, 4, and 5.  Similarly, rats  in group 6  recorded  malondialdehyde values  close  to those  of  group  5  and  1  (control) rats.

DISCUSSION

As elicited  by the elevation in the levels  of  liver  marker  enzymes  (AST, ALT, ALP),  CC1₄ administration  resulted  in a  significant  hepatic   damage.  Obviously,  the elevated  levels of  these  biochemical  parameters  are  a  direct  reflection of  alterations  in the  hepatic  structural  integrity.  The results of the enzymes obtained in this study corroborates those of Obi, et. al.⁷ and  Reinke,  et. al.⁵ who reported  elevated  levels  in the serum  content  of hepatic  enzymes  in rats  administered  with  CC1₄. In particular, the elevation of ALT is indicative of liver damage^{12, 13}. These  enzymes are located  in the cell cytoplasm and  are emptied  into the  circulation  once  the  cellular  membrane  is damaged ^{14, 15}. There is  a  growing  consensus  among  workers  that  CC1_4- induced  liver  damage  occur by the  production of  a trichloromethyl  radical from CC1₄ when it is  reductively  dechlorinated. The trichloromethyl  radical  production  abstracts a hydrogen  atom from  fatty  acid  to form a  lipid  radical  that  reacts  with molecular oxygen. The product of such reaction is the initiation of lipid peroxidation^{16, 17}.

Since  the  above  mechanism  is  suggestive  of the process  of  oxidative  stress,  it is  true, therefore, that  any  natural  product  with antioxidant  property will  prevent  or  reverse  lipid  peroxidation; including  cell  membrane damage.  The  report  of  Iwu⁴ that  implicated  seeds  of  Garcinia kola  in folk  medicine  and  herbal  preparations  for   treatment  of  liver  disorders,  informed  the screening of  its  natural  antioxidant properties. We  also  reasoned  that  a  comparison  of the results  obtained  with those of  vitamin E  (rated as  one  of  the most  powerful antioxidants) would  positively  influence our position  on its  antioxidant status.

The findings in this study shows  that  pretreatment  of  rats  21 days  prior  to CC1₄ administration  caused  a  marked  decrease  in the levels  of  hepatospecific serum enzymes. This suggests that seeds of Garcinia kola may be protective against CC1₄- induced liver damage in rats.  This was  ascertained  by a comparative  analysis  of  the  results  obtained  in rats  pretreated  with Garcinia kola  and  vitamin  E. 

Malodialdehyde (MDA) is a product of lipid peroxidation⁹. An increase in the liver MDA levels is an indication of elevated level of lipid peroxidation¹⁷. Extensive lipid peroxidation  leads  to disorganization  of  membrane  by peroxidation  of  unsaturated fatty  acids  which also  alters  the ratio of  poly-unsaturated  to other  fatty acids.  This would lead to a decrease in the membrane fluidity and the death of cell⁹.

CONCLUSION

The  marked  decrease  in the levels  of  lipid  peroxides  recorded  in rats  pretreated  with  Garcinia kola seeds  suggests  that  the seed  may  possess  the natural  antioxidants  necessary  for protection against  free radical  damage  induced  by CC1₄ in rat  liver.

REFERENCES

1. Hukkeri, V. I., Jaiprakash, B., Lavhale, M. S., Karadi, R. V. and Kuppast, I. J. (2003) Hepatoprotective activity of Ailanthus excelsa  Roxb. Leaf extract on experimental liver damage in rats.  Pharmacognosy 11:1-2.
2. Terashima,  K., Takawa,  Y. and Niwa, M. (2002) Powerful antioxidative agents based  on  Garcinonic  acid  from  Garcinia kola.  Bio. Org. Med. Chm. 10:1619-1625.
3. Iwu, M.M., Igboko, O. A., Onwuchekwu, U. and Okunji, C. O (1987) Evaluation of the antihepatoxicity of the biflavonoids-Garcinia Kola seeds. J. Ethnopharmacol. 21:127-142.
4. Iwu, M. M. (1982) Traditional Igbo Medicine. Institute of African studies, University of Nigeria, Nsukka, p.104.
5. Reinke L. A., Lai, E.K. and McCay, P. B. (1988) Ethanol feeding stimulates trichloromethyl radical formation from carbon tetrachloride in liver. Xenobiotics 18:1311-1318.
6. Onyeike, E. N. and Omubo-dede, T.T. (2002) Effects of heat treatment on the proximate composition, energy values, and levels of some toxicants in African  yam bean (Sphenostylis stencocarpa) seed varieties. Plant foods for human nutrition 57:223-231.
7. Obi, F.O., Usenu, L.A., and Osayande, J.O. (1998) Prevention of carbon tertrachloride induced hepatotocixity in the rat by H.rosainensis anthocyanin extract administered in ethanol. Toxicology 131:93-98.
8. Verly, H. (1967) Practical clinical Biochemistry, 4^th ed. Heinemann, London, Pp. 891-921.
9. Devaki, T., Raghavendran, H.R.B. and Sathivel, A. (2004) Hepatoprotective nature of seaweed alcoholic extract on acetaminophen-induced hepatic oxidative stress. J. Hlth. Sci. 50:42 – 46.
10. Hunter, F.E., Gebicki, J. M., Hoffstein, P.E., Weinstein J., and Scolt, A. (1963) Swelling and Lysis of rat liver mitochondria induced by ferrous ions. J. Biol. Chem. 238:828-835.
11. Gutteridge, J.M.C. and Wilkins, C. (1982) Copper dependent hydroxyl radical damage to ascorbic acid: Formation of a thiobarbituric acid reactive products. FEBS Lett: 137:327-340.
12. Lin, J. K. and Wang, C. J. (1986) Protection of crocein dyes in the acute hepatic damage induced by aflatoxin B1 and dimethylnitrosamine in rats. Carcinogenesis 7: 595-599.
13. Ngaha, E.O., Akanji, M. A. and Madusuolunmo, M.A. (1989) Studies on correlation between chloroquine-induced tissue damage and serum enzyme changes in rat. Experimentia 45:143-146.
14. Mohan-Rao, G.M., Morghmom, L.O., Kabur, M.N., Benmohamud, B.M. and Ashibanic, K.C. (1989) Serum glutamic, oxaloacetic transaminase (GOT) and glutamic pyruvic transminase (GPT) levels in diabetes mellitus. Int. J. Med. Sci. 5:188-192.
15. Lin, S. C., Chung, T. C., Ueng, T.H., Linn, Y.H., Hsu, S.H., Chiang, C.L. and Lin, C.C. (2000) The hepatoprotective effects of Solnum alatam moench on acetaminophen0induced hepatotoxicity in mice. Am. J. Clin. Med. 28:105-114.
16. Sipes, I. G., Krishna, G. and Gillette, J. R. (1977) Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P₄₅₀. Life Sci. 20:1541-1548.
17. Trible, D. L., AW, T.Y. and Jones, D.P. (1987) The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology 7:377-387.

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