EVALUATION OF THE HEPATOPROTECTIVE EFFECT OF UVARIA AFZELII AGAINST PARACETAMOL INDUCED LIVER TOXICITY IN WISTAR RATS.HTML Full Text
EVALUATION OF THE HEPATOPROTECTIVE EFFECT OF UVARIA AFZELII AGAINST PARACETAMOL INDUCED LIVER TOXICITY IN WISTAR RATS.
SI Omoruyi *, AB Enogieru and OI Momodu
Department of anatomy, University of Benin, Benin City, PMB1154, Benin
ABSTRACT: Uvaria afzelii (UV) Sc Elliot (Annonaceae) locally is used in the treatment of kidney disorders, digestive problems, liver disorders, diabetes as well as gonorrhea. This study aims to investigate the possible hepatoprotective activities of the crude aqueous root extract of Uvaria afzelii against Paracetamol-induced hepatotoxicity with Silymarin being used as a reference standard. Twenty five (25) adult wistar rats were randomly assigned into a control group (A) and four treatment groups (B, C, D and E) each containing five rats (n=5/group). Animals in each group were allowed access to 200g/day growers’ mash and water ad libitum. Rats in the treatment groups were administered paracetamol orally at a dose of 800mg/kg body weight daily for fourteen days. Rats in group B were not pretreated while groups C, D and E rats were pretreated daily with 50mg/kg body weight of Silymarin, 250mg/kg and 500mg/kg body weight of crude aqueous extract of UV root respectively. The animals were sacrificed on the 15th day of the experiment and several serum markers, aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP) and total protein (TP) was measured to assess the effect of the extract on paracetamol -induced hepatic damage. The study included histopathological examination of liver sections. Paracetamol induced a significant rise in AST, ALT, ALP and TP. Treatment of rats with the crude UV extract significantly (P<0.05) altered serum marker enzymes levels to near normal against acetaminophen treated rats. The activity of the extract was comparable to the standard drug, silymarin (50 mg/kg.). Histopathological changes of liver tissues were compared with respective control. Results indicate that Uvaria afzelii possesses hepatoprotective effect on paracetamol-induced hepatotoxicity in rats
Liver enzymes, Paracetamol
INTRODUCTION: Uvaria afzelii (UV) Sc Elliot (Annonaceae) is a small tree or spreading shrub growing up to 5 meters tall and is used locally, being harvested from the wild for food and medicines. It is mostly found in West tropical Africa - Sierra Leone to Nigeria. It is widely distributed and grown in the south and eastern part of Nigeria, where it is known by various local names such as; “gbogbonishe” (Yoruba), “Umimiofia” (Igbo) and “Osu-umimi” (Ukwani) 1.
Locally it is used in the treatment of cough, vaginal tumour, breast aches, swollen hands and feet, diabetes as well as leucorrhoea and gonorrhea 2, 3. Previous studies carried out on the plant to ascertain the claimed uses include it’s reported bacteriocidal activity against gram positive and acid fast bacteria 4 ,5, anti-helminthic and anti-parasitic activities 6.
More so, some compounds have been isolated from the plant and they include; Syncarpic acid, Dimethoxymatteucinol, Emorydone, 2-hydroxydemethoxy matteucinol, Uvafzelic acid, syncarpurea and Afzeliindanone 7, 8. Some of these compounds have been credited with specific biological activities as Afzeliindanone and Emorydone are reported to possess potent activity against trypanosomiasis brucei. Silymarin is a standardized extract of the milk thistle plant (Silybum marianum) which majorly contains flavonoids; silybin, silybinin, silydianin and silychristin 9. The seeds of this plant have been used over time for the treatment of liver and gall bladder disorders, including hepatitis, cirrhosis and jaundice. More so, the seeds protect the liver against poisoning from chemicals, environmental toxins, snake bites, insect stings, mushroom poisoning and alcohol 10. Research overtime has demonstrated its use as a standard drug with exhibition of potent hepatoprotective activity at the dose range from 25-200 mg/kg 11, 12.
The liver is the key organ regulating homeostasis in the body. It is involved with almost all the biochemical pathways related to growth, fight against disease, nutrient supply, energy provision and reproduction 13. More than 900 drugs have been implicated in causing liver injury 14 and it is the most common reason for a drug to be withdrawn from the market. Drug-induced liver injury is a potential complication of nearly every medication that is prescribed, because the liver is central to the metabolic disposition of virtually all drugs and foreign substances 15.
Paracetamol which is safe for use at recommended doses can also cause potential fatal liver damage in cases of acute overdose as it is it is one of the most common causes of poisoning worldwide and also the most common cause of acute liver failure in the United States and United Kingdom 16, 17. An over dosage of paracetamol is known to be the cause of acute hepatic necrosis in both experimental animals 18, 19 and humans 20, 21.
However, conventional or synthetic drugs used in the treatment of liver diseases are sometimes inadequate and can have adverse effect 22. So there is a worldwide trend to go back to the traditional medicinal plants 23, 24, 25. In view of the undesirable side effects of synthetic agents researchers have develop scientific basis to evaluate traditional herbal medicines which are claimed to possess hepatoprotective activity 26. Therefore, paracetamol mediated hepatotoxicity was chosen as the experimental model for liver injury in this study. The aim of this research was to investigate the effect of roots extracts of Uvaria afzelii on paracetamol induced liver damage and to compare the observed effects with a known hepatoprotective drug (Silymarin).
MATERIALS AND METHOD:
The roots of the plant Uvaria afzelii were gotten from a forest in Orhionmwon near Benin City during the month of July 2013. The plant was identified by Mr. Sunny Nweke, the curator of the Department of Pharmacognosy, Faculty of Pharmacy, University of Benin Edo state.
Preparation of Extract:
The roots of Uvaria afzelii were chopped into little bits and allowed to dry at room temperature for 5 weeks. The dried roots were pounded in a wooden mortar and pestle and milled into fine powder in an electric blender. 1.5kg of the powder was obtained soaked in 2litres of distilled water for 24hours. The mixture was filtered with a white filter paper and to obtain the filtrate which was evaporated at 60oC in a vacuum rotary evaporator. The residue obtained was dried and stored in a refrigerator. A measured portion of the extract was dissolved in distilled water and an appropriate dose was administered to the animals daily.
Animal Care and Management:
A total of 25 adult male wistar rats were used for this study. The animals were inbred rats obtained from the rat Colony of the Animal House, Department of Anatomy, University of Benin, Benin City. The animals were housed and maintained in accordance with the guidelines of the Research Ethics Committee of the College of Medical Sciences, University of Benin, Benin City.
The rats were randomly assigned into a control group (A) and four treatment groups (B, C, D and E) each containing five rats (n=5/group). Animals in each group were allowed access to 200g/day growers’ mash and water ad libitum. Rats in the treatment groups were administered 800mg/kg body weight of paracetamol daily for fourteen (14) days during the experimental period. Rats in group B were not pretreated while groups C, D &E rats were pretreated daily with 50mg/kg body weight of Silymarin, 250mg/kg and 500mg/kg body weight of crude aqueous extract of UV root respectively.
Sacrifice of the Animals:
The experimental period lasted for 14 days and on the 15th day animals were sacrificed by cervical dislocation. A midline incision was made through the anterior abdominal wall of the rats. 5ml blood samples were collected from the descending abdominal aorta, in heparin coated bottles for biochemical analysis and the liver were excised and fixed in 10% buffered formal saline.
Assay of Marker Enzymes of Liver Damage:
The blood samples collected were centrifuged at 3000 revolutions/minute using a table-top centrifuge (Shanghai Surgical Instrument Factory, Shanghai, China) at 37 ºC for 15 min to separate the sera. Serum alanine (ALT) and aspartate (AST) aminotransferases and alkaline phosphatase (ALP) as well as (TP) Total Protein were assayed using Randox diagnostic kits 27.
Following fixation, the tissues were dehydrated in ascending grades of alcohol (ethanol), cleared in xylene and embedded in paraffin wax. Serial sections of 5 microns thick were cut using a rotatory microtome. The sections were later de-paraffinized for routine Hematoxylin and Eosin (H and E) staining using the method of Drury et al. 28. The photomicrographs were obtained from sections using a research photographic microscope in the Department of Anatomy, School of Basic Medical Sciences, University of Benin, Benin City, Edo State, Nigeria.
The data generated were analyzed using descriptive and inferential statistics. All values were presented as mean ± Standard Error of Means (S.E.M.). All statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) (version 17). The significance of difference in the means of all parameters was determined using one way analysis of variance (ANOVA; 95% confidence interval). Least Significant Difference (LSD), post hoc test was carried out for all groups with control and comparison of all pairs of groups respectively. Values were statistically significant when probability is less than 0.05 (P < 0.05).
TABLE1: EFFECT OF CRUDE AQUEOUS ROOT EXTRACT OF UVARIA AFZELII ON SERUM LIVER ENZYMES IN PARACETAMOL INDUCED HEPATOTOXICITY IN RATS
|GroupAControl||GroupB(paracetamol 800mg/kg)||GroupC(Silymarin+paracetamol 800mg/kg)||GroupDUV(250mg/k) +paracetamol 800mg/kg)||GroupEUV(500mg/kg) +Paracetamol 800mg/kg)|
*shows significance when compared with group1
**shows significance when compared with group2
The results of ALP, AST and ALT in control rats were 27.50±1.45, 10.75±1.53 and 34.25±1.11 respectively, whereas in paracetamol treated rats, these levels were elevated to 48.00±2.89, 35.25±2.84 and 54.50±1.85 respectively. Uvaria afzelii pretreatment at the dose 250mg/kg significantly (P<0.05) prevented the paracetamol induced rise in the ALP, AST and ALT to 37.00±3.03, 27.50±3.88 and 37.75±2.50
respectively when compared with paracetamol treated group. With a higher dose of 500 mg/kg further reduction of ALP, AST and ALT to 36.00±2.27, 18.00±4.32 and 36.75±2.14 respectively were noted. Silymarin (50 mg/kg) pretreatment also prevented the paracetamol induced rise in ALP, AST and ALT to 31.75±2.87, 11.00±1.08 and 33.50±1.71 respectively. Same trend was observed in total protein (TP) values.
DISCUSSION: The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, such as those used in laboratories and industries, natural chemicals (e.g., microcystins) and herbal remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called hepatotoxins. More than 900 drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market 14.
Acetaminophen hepatotoxicity is now recognized as the most common cause of the potentially devastating clinical syndrome of acute liver failure in many western countries 29. Acetaminophen (also known as paracetamol or panadol), is reported to induce a wide spectrum of toxicities especially when taken in large single dose either alone or in combination with an equally large amount of alcohol 30, 31. These toxicities often are pansystemic, involving virtually all organs/systems in the body. For example, in the liver and kidneys, paracetamol overdose causes potentially fatal conditions known as acetaminophen-induced hepatotoxicity and acetaminophen-induced nephrotoxicity, respectively 32.
Because of its wide availability as an over-the-counter analgesic-antipyretic, acetaminophen is liable to abuse and consequent toxicities including hepatotoxicity. However, the toxic dose of acetaminophen is highly variable. For example, in adults, a single dose of above 10 grams or 150 mg/kg or chronic ingestion of doses as low as 4 g/day could induce acetaminophen hepatotoxicity 33, whereas in children, acute doses above 200 mg/kg would be required to induce same degree of hepatotoxicity as in adults 34. This higher threshold is largely due to children having larger livers relative to body size than adults, hence, being more tolerant of acetaminophen overdose than adults 31. Paracetamol is documented to mediate its toxic effect on the liver by inducing the process of lipid peroxidation which is mediated principally by the highly reactive intermediate metabolite of acetaminophen, N-acetyl-parabenzoquinonimine (NAPQI) 35.
This intermediate metabolite covalently binds to hepatocyte intracellular and membranal macromolecules to cause cell death and consequent liberation of intracellular contents including the cytosolic liver enzymes.
This present study was designed to evaluate the hepatoprotective activity of Uvaria afzelii in paracetamol induced liver damage in rats. Damage to the liver or hepatotoxicity does not result from paracetamol itself, but from one of its metabolites, N-acetyl-p-bezoquinoneimine (NAPQI) 35. NAPQI depletes the liver's natural antioxidant gluthathione and directly damages cells in the liver, leading to liver failure. In the assessment of liver damage by paracetamol, the determination of enzyme levels such as ALP, AST and ALT is largely used.
The increased levels of ALP, AST and ALT in serum are indicative of cellular liver leakage and loss of functional integrity in the cell membrane of the liver 36. However, administration of the crude aqueous UV extract at various dose levels mediated a reduction in the levels of these enzymes towards the normal value. This indicates a stabilization of plasma membrane as well as repair of hepatic tissue damage caused by paracetamol. This finding is in consonance with the common view that serum levels of transaminase return to normal following healing of liver parenchyma and regeneration of hepatocytes 37.
Histopathological observations showed severe vacoulations; which were original site of lipid deposit and necrosis of hepatocytes in the paracetamol treated group indicative of liver toxicity. However, on administration of Silymarin (50 mg/kg of body weight) and the crude aqueous UV extract (250mg/kg and 500mg/kg), the severity of these lesions were reduced. These observations suggested the possibility of the plant extract being able to condition the hepatic cells to a state of accelerated regeneration thus reducing the leakage of ALT, AST and ALP into circulation.
Several investigations have shown that silymarin improved liver function related to hepatocellular necrosis and increased membrane permeability through its antioxidant capacity 38. The protective effect of silymarin observed in our study was attributed to its antioxidant and free radical scavenging properties as reported in earlier studies 39.
The mechanism by which Uvaria afzelii exerts its protective action against paracetamol induced damage in the liver may be due to the antioxidative effect of the plant extract. These antioxidative effects might be due to the phytoconstituents such as flavonoids, triterpenoids and phenols present in the plant material as they have been reported to exhibit hepatoprotective activity 40, 41.
CONCLUSION: The findings from the present investigation demonstrate the efficacy of Uvaria afzelii plant aqueous extract against experimental hepatic damage induced by paracetamol and these findings were similar to that produced by silymarin. Thus providing preclinical evidence and therapeutic rationale requiring further studies on the above mentioned hepatoprotective activity of the plant extract in attenuation of hepatic insufficiencies, in men as well.
ACKNOWLEDGEMENTS: We wish to thank the laboratory staff of the Department of Anatomy, University of Benin for their assistance during the period of this research.
- Odugbemi T: A Textbook of Medicinal Plants from Nigeria. Lagos University Press, Lagos Nigeria, 2008: 467.
- Verger PF: Ewe: The use of plants in Yoruba society. Editorial Schwerc; Sao Paulo, 1995: 774.
- Kayode J, Ige OE, Adejogo TA., Igbakin AA: Conservation and biodiversity erosion in Ondo State, Nigeria (3): Survey of plant barks used in native pharmaceutical extraction in Akoko region. Ethnobotanical leaflet 1995; 13: 655 – 667.
- Okoli AS: Evaluation of extracts of Anthocleista djalonensis, Nauclea latifolia and Uvaria afzelii for activity against bacterial isolates from non-gonococcal urethritis. J Ethnopharmacol. 2004; 92: 235 –244.
- Lawal TO, Adeniyi BA, Wan B, Franzblau SG, Mahady GB: In vitro susceptibility of Mycobaeterium tuberculosis to extracts of Uvaria afzelii Scott Eliot and Tetracera alnifolia Afri Jour of Biomed Res 2011; 14: 17 – 21.
- Okpekon T, Yolou S, Gleye C, Roglot F, Loiseau P, Bories C, Grellier P, Frapier F, Lauren A, Hocquemiller R: Antiparasitic activities of medicinal plants used in Ivory Coast. Journal of Ethnopharmacology 2004; 90(1): 91 – 97.
- Hufford CD, Oguntimein B, Martin M, Clardy J: Syncarpurea; a novel metabolite from afzelii. Tetrahedron Letters 1984; 25(4): 371-374.
- Okpekon T, Millot M, Champy P, Gleye C, Yolou S., Bories C., Loiseau P., Lauren A., Hocquemiller R. (2009) A Novel I-indanone isolated from Uvaria afzelii roots. Nat. Prod. Res.; 23(10): 909 – 15.
- Flora K., Hahn M, Rosen H: Milk thistle (Silybum marianum) for the Therapy of Liver Disease. American Journal of Gastroenterology 1996; 93: 139 - 143.
- Kren V, Walterová D: Silybin and silymarin-new effects and applications. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2005; 149(1): 29-41.
- Wills PJ, Asha VV: Protective effect of Lygodium flexuosum (L.) Sw. extract against carbon tetrachloride-induced acute liver injury in rats. Journal of Ethnopharmacology 2006; 108: 320 - 326.
- Salam OM, Sleem AA, Omara EA: Effect of ribavirin alone or combined with silymarin on carbon tetrachloride induced hepatic damage in rats. Drug Target Insights 2007; 2: 19 - 27
- Ward FM, Daly MJ: Hepatic disease in Clinical Pharmacy and Therpeutics. Walker R, Edward C, Eds Churchill Livingston, New York, 1999: 195-212.
- Friedman SE, Grendell JH, McQuaid KR:Current diagnosis & treatment in gastroenterology. New York, Lang Medical Books/McGraw-Hill, 2003: 664–679.
- Ostapowicz G, Fontana RJ, Schiodt FV: "Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States". Intern. Med 2002; 137(12): 947–54.
- Ryder SD, Beckingham IJ: ABC of diseases of liver, pancreas, and biliary system. Other causes of parenchymal liver disease. BMJ 2001; 322 (7281): 290–2.
- Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, Reisch JS, Schiødt FV, Ostapowicz G, Shakil AO, Lee WM: Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology 2005, 42 (6): 1364–72.
- Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR, Brodie BB: Acetaminophen induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 1973; 187: 185-194.
- Lim SP, Andrews FJ, O’Brien PE: Misoprostol protection against acetaminophen induced hepatotoxicity in the rat. Dig.Dis. Sci. 1994; 39: 1249-1256.
- McJunkin RP, Barwick KW, Little WC, Winfield JB: Fatal massive hepatic necrosis following acetaminophen overdose. JAMA 1976; 236: 1874-1875.
- Golden DP, Mosby EL, Smith DJ, Markercher P (1981). Acetaminophen toxicity. Reports of two cases. Oral Surg. Oral Med. Oral Patho 1981; 51: 385-389.
- Guntupalli M, Chandana V, Pushpangadan P, Shirwaikar A: Hepatoprotective effects of rubiadin, a major constituent of Rubia cordifolia J. Ethnopharmacol 2006; 103: 484–490.
- Dhuley JN, Naik SR: Protective effect of Rhinax, a herbal formulation against CCl4 -induced liver injury and survival in rats. J. Ethnopharmacol 1997; 56: 159-164
- Venkateswaran S, Pari L, Viswanathan P, Menon VP: Protective effect of Livex, a herbal formulation against erythromycin estolate induced hepatotoxicity in rats. J. Ethnopharmacol 1997; 57: 161-167.
- Mitra SK, Seshadri SJ, Venkantaranganna MV, Gopumadhavan S, Venkatesh UU, Sarma DN: Effect of HD-03 -a herbal formulation in galactosamine-induced hepatopathy rats. Indian J. Physiol. Pharmacol 2000; 44: 82-86.
- Subramoniam A, Evans DA, Rajasekaran SP: Hepatoprotective activity of Trichopus zeylanicus extract against paracetamol induced damage in rats. Ind J Expt Biol 1998; 36: 385-389.
- Reitman S, Frankel SA: Colorimetric method for the determination of serum levels of glutamic oxaloacetic acid and pyruvic acid transaminases. Am J Clin Pathol 1957; 10: 394-9.
- Drury RAB, Wallington EA, Cameron RC: Histological techniques: 4th ed., Oxford University Press, NY. U.S.A, 1976: 279-280
- Schiodt FV, Rochling FA, Casey DL, Lee WM: Acetaminophen toxicity in an urban county hospital. New England Journal of Medicine 1997; 337: 1112 – 1117.
- Cohen SD, Hoivik DJ, Kairallah EA: Acetaminophen-induced hepatotoxicity. In: Plaa GL, Hewitt WR, editors: Toxicology of the Liver. Philadelphia: Taylor and Francis, 1998: 159 - 186
- Tenenbein M: Acetaminophen: the 150 mg/kg myth. Journal of Toxicology and Clinical Toxicology 2004; 42(2): 145 – 148
- Jackson-Roberts II L, Morrow JD: Analgesic-antipyretic and anti-inflammatory agents and drugs employed in the treatment of gout. In: Hardman JG, Limbird LE, Gilman AG, editors: Goodman and Gilman’s The Pharmacological Basis of Therapeutics. 10th edition. New York: McGraw-Hill Medical Publishing Division, 2001: 687-732
- Dart RC, Erdman AR, Olson KR, Christainson G, Manoguerra AS, Chyka PA, Caravati EM, Wax PM, Keyes DC, Woolf AD, Scharman EJ, Booze LL, Troutman WG: Acetaminophen poisoning: an evidence-based concensus guideline for out-of-hospital management. Clinical Toxicology 2006; 44(1): 1 – 18.
- Rumack B, Matthew H: Acetaminophen poisoning and toxicity. Pediatrics 1975; 55(6): 871 – 876.
- Muriel P, Garciapina T, Perez-Alverez V, Mourelle M. (1992). Silymarin protects against paracetamol-induced liver peroxidation and liver damage. Journal of Applied Toxicology 1992; 12: 439 - 442.
- Drotmann RB, Lowghorn GT: Serum enzymes as indicators of chemical induced Liver damage. Drug Chem. Toxico 1978; 1: 163 – 171
- Thabrew MI, Joice PD, Rajatissa WA: Comparative study of the efficacy of Paetta Indica and Osbeckia octandra in the treatment of liver dysfunction. Planta med 1987; 53: 239 – 241.
- Ramadan LA., Roushdy HM, Senna GMA: Radioprotective effect of silymarin against radiation induced hepatotoxicity. Pharmacological Research 2002; 45(6): 447 - 454
- Horvath ME, Gonzalez-Cabello R, Blazovics A, Van der Looij M, Barta I, Muzes G, Gergely P, Feher J: Effect of silibinin and vitamin E on restoration of cellular response after partial hepatectomy 2001; 77(2-3): 227-232
- Pietta P: Flavonoids as anti-oxidants. J of Nat Prod 2000; 63(7): 1037 –1047.
- Jovanovic S, Steekens S, Mihaylo T, Marjanovic B, Simic M: Flavonoids as anti-oxidants. J Amer. Chem. Society 1994; 116(11):4846 –4831.
How to cite this article:
Omoruyi SI, Enogieru AB and Momodu OI: Evaluation of the Hepatoprotective Effect of Uvaria Afzelii Against Paracetamol Induced Liver Toxicity In Wistar Rats. Int J Pharm Sci Res 2015; 6(7): 2734-40.doi: 10.13040/IJPSR.0975-8232.6(7).2734-40.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
SI Omoruyi *, AB Enogieru and OI Momodu
Department of anatomy, University of Benin, Benin City, PMB1154, Benin
07 November, 2014
25 February, 2015
17 May, 2015
01 July, 2015