International Journal of Biomedical Research EFFECTS OF THE METHANOLIC LEAF EXTRACT OF ADENODOLICHOS PANICULATUS (HUA) ON RAT LIVER FUNCTION Sani, M. B.*1, Anuka, J. A.1, Zezi, A. U.1, Abubakar, A.1, Yaro, A. H.3, Magaji, M. G.1 and Sani, Y. M.2 1 Department of pharmacology and therapeutics, Ahmadu Bello University, Zaria 2 Department of pharmaceutical and medicinal chemistry, Ahmadu Bello University, Zaria 3 Department of pharmacology, faculty of medicine, Bayero University Kano *Corresponding Author: bashir_sani@yahoo.com This article is available online at www.ssjournals.com ABSTRACT In present study acute oral toxicity value and the effect of the methanolic leaf extract of the Adenodolichos paniculatus (MAP) on function status of the liver were investigated in rats after two weeks of daily oral administration of graded doses ( 250, 500 and 1000 mg/kg body weight) of the extract. The preliminary phytochemical screening of the plant revealed the presence of flavonoids, tannins, steroids, carbohydrates, glycosides, alkaloids, anthraquinones and cardiac glycosides. The oral LD50 of the extract in rats was found to be greater than 5000 mg/kg body weight suggest that the extract is relatively non-toxic and safe. Results obtained from the study showed that all values for biochemical parameters (ALT, ALT, TB and CB) for the control and experiment groups were statistically insignificant (P < 0.05) in all the graded doses used (250, 500, 1000 mg/kg body weight) except for AST which is statistically significant (P > 0.05) when compared with control group in all the graded doses used (250, 500, 1000 mg/kg body weight). The extract does not relatively show a dose dependent alteration in liver enzymes functions. It could be suggest that the methanolic leaf extract of Adenodolichos paniculatus had no adverse effects on the functional status of the liver at doses tested and for period of the study. Sub chronic and chronic toxicity study is required to ascertain this claim. Keywords: Adenodolichos paniculatus, LD50, Phytochemistry and liver Function INTRODUCTION Liver is an organ in the body where metabolism of drugs and other xenobiotics take place. Amongst other functions, the enzyme systems involved in biotransformation are localized primarily in the liver, and the active metabolites can cause liver damage. So it has a significant role in the maintenance, performance and IJBR 1[5] [2010]194‐200 regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fights against disease(s), supplies nutrients, provides energy and helps in reproduction1. The major functions of the liver are carbohydrate, protein and fat metabolism, detoxification of poisons, secretion of bile Sani et al Research Article and storage of vitamins. Thus, to maintain a the sub-acute toxicity of the methanolic leaf liver is a crucial factor for overall health extract of Adenodolichos paniculatus on and the liver in experimental or laboratory well being. continuously However, liver is exposed to environmental animals. toxins, and abused by poor drug habits, and over-the-counter drugs which can MATERIALS AND METHODS: Plant collection: The leaves of Adenodolichos paniculatus eventually lead to various liver ailment like were collected from Kundun village, Birnin hepatitis, cirrhosis and alcoholic liver Gwari, Kaduna State in June, 2009. The disease 2,3. leaves were identified and authenticated by Thus liver diseases are some of the fatal Mallam Umar S. Gallah of the Herbarium diseases in the world today. They pose a section, Department of Biological Sciences, serious challenge to international public Ahmadu health. Modern medicines have little to voucher specimen (Number 3107) was offer for alleviation of hepatic diseases and deposited at the herbarium for future it is chiefly the plant based preparations reference. which are employed for their treatment of dried and crushed into powder with pestle liver disorders. But there are not much drug and mortar. 200 g of the powdered leaf available for the treatment of liver disorders was cold macerated at room temperature 4,5 with 70% methanol for 48 hours with alcohol users and the misused of prescribed . Adenodolichos paniculatus (HUA) Bello University, Zaria. A The leaves were cleaned, air occasional shaking. 1000C (Leguminosae) is a shrub that grow up to evaporated 4m high in percentage yield of the concentrated crude savanna region of Guinea, Northern Nigeria and across to Sudan. In Nigeria, the leaf is applied topically with at The solvent was to obtain the extract. The percentage yield of the extract is 8.45% butter oil as a dressing for burns6. The leaf has been used in toothache7 and Central Africa Republic, the root –decoction has been used for blenorrhoea and liver 7 trouble . This study is therefore aimed at evaluating IJBR 1[5] [2010]194‐200 Experimental animals: Swiss albino mice (18 - 30 g) and Wistar rats (160 - 250 g) of both sexes were obtained from the Animal House Facility of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Sani et al Zaria, Nigeria. The rats were maintained on rodent feed and water ad libitum, and housed in propylene cages at room temperature throughout the study. All experimental protocols were approved by the University Animal Ethics Committee. Research Article Sub acute toxicity study: Twenty four (24) Wister albino rats weighing between (160 – 250 g) were used for this study. The rats were randomly divided into four (4) groups of six (6) rats each. The first group served as control was administered Phytochemical analysis: Adenodolichos paniculatus was subjected to phytochemical screening using standard 8 protocol . saline (10ml/kg), while the second, third, and fourth groups were administered with 250, 500, 1000 mg/kg of the MAP orally respectively on daily basis for two weeks, after which the Acute Toxicity (LD50) determination: Oral acute toxicity in rats: The acute toxicity study of the methanolic extract was determined orally in rats using 9 the method described by Lorke (1983). The animals were fasted overnight and the LD50 evaluation was carried out in two phases. In the first phase, three (3) groups of three (3) rats each were treated with the MAP at doses of 10, 100 and 1000 mg/kg body weight orally respectively. The rats were monitored for 24 hours for signs and symptoms of toxicity, such as paw licking, salivation, normal sedation convulsion and mortality. In the second phase, four groups of one (1) rat each were further treated with animals were sacrificed after 24hrs, on the fifteenth (15th) day. Sample collection and preparation: Rats were anaesthetized in light chloroform and blood samples collected by cardiac puncture into clean, dry centrifuge tubes. Blood samples which were processed individually were allowed to stand for 10mins at room temperature and then centrifuged at 1000 rpm for 15mins on laboratory centrifuge (SM 800B, Surgifriend Medicals, England) and the supernatant (serum) carefully removed with Pasteur pipette and stored frozen until required for enzyme analysis. the MAP at doses of 1600, 2900 and 5000 mg/kg body weight respectively. The rats were also observed for signs and symptoms of toxicity and mortality. IJBR 1[5] [2010]194‐200 Enzyme assays and clinical chemistry: Alkaline phosphatase activity was assayed using the method described by10.The Sani et al Research Article procedure described by 11 was employed for serum liver enzymes at doses tested (250, the assay of aspartate aminotransferase 500 (AST) and alanine aminotransferase (ALT). respectively. and 1000 mg/kg body weight) Total bilirubin (Randox assay kit) was 12 determined by the methods of and 13 DISCUSSION The preliminary phytochemical screening revealed Statistical analysis: The data were expressed as mean ± SEM and analyzed statistically using student’s ttest. P values less than 0.05 (P < 0.05) were the presence of flavonoids, glycosides, tannins, saponins, steroids, anthraquinones and cardiac glycosides. The oral LD50 of the MAP was found to be greater than 5000 mg/kg which is relatively considered to be statistically significant. safe because no death was recorded even at RESULTS The phytochemical screening of methanolic the dose of 5000 mg/kg. The Organization for Economic Cooperation and leaf extract of Adenodolichos paniculatus Development (OECD, Paris, France)14 revealed (Walum, 1998) recommended chemical the presence of flavonoids, tannins, steroids, carbohydrates, glycosides, labeling alkaloids, systemic toxicity based on oral LD50 values anthraquinones and cardiac and classification of acute glycosides. as: very toxic, < 5 mg/kg; toxic, > 5 < 50 Acute toxicity study: The median oral mg/kg; harmful, > 50 < 500 mg/kg; and not lethal dose of Adenodolichos paniculatus in rats was found to be greater than 5000 mg/kg, toxic or harmful, > 500 < 2,000 mg/kg. Based on this classification, the oral LD50 up to 5,000 mg/kg established for rats Enzyme Assays and Clinical Chemistry indicated relative oral safety. According to The effects of administration of MAP to the toxicity scale15, any compound with an rats on the phosphatase, activities aspartate of alkaline aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin and conjugated bilirubin respectively are shown in Tables 1. The administration of MAP for 14 days did not produce significant increase (P > 0.05) in IJBR 1[5] [2010]194‐200 oral LD50 of between 500 – 2000 mg/kg should be considered practically non toxic. This also agrees with the findings of16 .WHO ranks all plants whose LD50 figures are less than 25 mg/kg in “very toxic group”, between 25 and 200 mg/kg in Sani et al Research Article “toxic group” and from 200 to 2000 mg/kg In this study, as shown in table 1, the in be MAP did not altered any significant (P < orally 0.05) changes in all the indices of liver administered drugs and compounds do function (AST, ALT, ALP, TB, and CB). undergo some events that potentially There decrease the amount reaching systemic between treated groups (ALT, ALP, TB, circulation for pharmacological effects 17 and CB). (P < 0.05) when compared with In the assessment of liver damage by drugs the control except for AST where there was or any other hepatotoxin, the determination significant difference (P > 0.05) in all doses of enzyme levels such as ALT and AST is used (250, 500, 1000 mg/kg) when "noxious attributed group”. to largely used damage the 18 This fact could that . Necrosis or membrane releases significant difference compared with the control. Thus, these findings provide evidence for the clinical circulation; therefore, it can be measured in safety of the plant. Conjugating ability of serum. High levels of AST indicate liver the liver was intact as revealed by total and damage, such as that due to viral hepatitis, conjugated bilirubin levels. The plasma cardiac infarction and muscle injury. The ALT and AST activities are markers of ratio of AST to ALT is sometimes used in hepatocellular damage; their levels in the differentiating causes of liver damage19. rat were not affected by the extract intake. Elevated AST are not specific for liver It is probable that the presence of damage, and AST has also been used as Flavonoids which are hepatoprotectives21. cardiac the Therefore, the observed antioxidant and conversion of alanine to pyruvate and hepatoprotective activity of Adenodolichos glutamate, and is released in a similar paniculatus may be due to the presence of manner. Therefore, ALT is more specific to flavonoids. the liver, and is thus a better parameter for In conclusion, the presence study suggest detecting liver injury19. Serum ALP level that all indices for liver functions were not on the other hand, is related to the function affect by extract intake for a period of of hepatic cell. Increase in serum level of fourteen (14) days also established that the ALP is due to increased synthesis of the extract has no adverse effect on the liver enzyme, in presence of increasing biliary functions and support ethno medicinal uses ALT 20 pressure . 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Effects of MAP on the functional status of the liver after 14 days treatment in Rats MEAN ± SEM Treatment Dose mg/kg AST 255.3 ± 22.3 163.2 ± 21.3 163.17 7.50 200.8 24.17 ALT ALP TB 14.5 ± 3.74 26.7 ± 3.37 22.7 ± 1.22 24.5 ± 5.61 119.33 ± 7.07 25.5 ± 0.66 25.5 ± 5.99 122.5 ± 6.42 24.83 ± 1.1 32.33 ± 5.9 1 All values for AST, ALT, ALP, TB and CB are statistically insignificant at (P < compared with control using student’s t-test. n = 6 Normal saline Extract Extract Extract 10 250 500 1000 IJBR 1[5] [2010]194‐200 123.8 ± 3.74 111.3 ± 134.16 CB 17.30 ± 3.21 13.33 ± 3.30 13.33 ± 2.61 16.00 ± 2.86 0.05)