Asian Pacific Journal of Tropical Medicine (2011)470-474 470 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document heading doi: Protective effects of Phyllanthus acidus (L.) Skeels leaf extracts on acetaminophen and thioacetamide induced hepatic injuries in Wistar rats Nilesh Kumar Jain*, Abhay K Singhai Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.)-470003, India ARTICLE INFO ABSTRACT Article history: Objective: To investigatePhyllanthus and compareacidus the hepatoprotectiveP. acidus) effects of crude ethanolic and Received 21 February 2011 aqueous extracts of (L.) Skeels ( leaves on acetaminophen (APAP) Received in revised form 11 April 2011 and thioacetamide (TAA) induced liver toxicity in wistar rats. Silymarin was the reference Accepted 15 May 2011 Methods: P. acidus hepatoprotective agent. In two different sets of experiments, the extracts Available online 20 June 2011 (200 and 400 mg/kg, body weight) and silymarin (100 mg/kg, body weight) were given orally for 7 days and a single dose of APAP (2 g/kg, per oral) or TAA (100 mg/kg, subcutaneous) were given Keywords: to rats. The level of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline Phyllanthus acidus phosphatase (ALP)Results:, total bilirubin and total protein were monitored to assess hepatotoxicity and Acetaminophen hepatoprotection. APAP or TAA administration caused severe hepatic damage in rats as AST, ALT, ALP, Thioacetamide evident from significant riseP. acidus in serum total bilirubin and concurrent depletion in Hepatoprotective total serum protein. The extracts and silymarin prevented the toxic effects of APAP or DPPH TAA on the above serum parameters indicating the hepatoprotective action. The aqueous extract was found to be more potent than the corresponding ethanolic extract against both toxicants. The phenolic and flavonoid content (175.02依4.35 and 74.68依1.28, respectively) and 2,2-diphenyl-1- (DPPH) [IC50 = (33.2依0.31) picrylhydrazil 毺g/mL] scavengingConclusions: potential was found maximum with aqueous extract as compared to ethanolic extract. The results of present study P. acidus suggests that the aqueous extract of leaves has significant hepatoprotective activity on APAP and TAA induced hepatotoxicity, which might be associate with its high phenolic and flavonoid content and antioxidant properties. 1. Introduction leaf have shown protection against carbon tetrachloride induced hepatotoxicity in rats[6]. Some important chemical constituents of the leaf include kaempferol, hypogallic acid, Phyllanthus acidus P. [5, 6] acidus (L.) Skeels (Euphorbiaceae) ( gallic acid, quercetin, and adenosine . ), commonly known as harfarauri or star gooseberry, The aim of present study was to investigate and compare is a widely distributed plant in India and other Asian the hepatoprotectiveP. acidus effects of crude ethanolic and aqueous countries. It is about 4-6 m high with obliquely ovate acute extracts of leaves on acetaminophen (APAP) and and distichous thin leaves. The leaf is analgesic, antipyretic, thioacetamide (TAA) induced acute liver toxicity in rats. antirheumatic and cures jaundice, small pox, itching and The protective effects were compared with silymarin, a well gum infection. Traditionally it is used as liver tonic and known hepatoprotective against APAP and TAA induced blood purifier[1, 2]. The leaf decoction is used by tribal hepatotoxicity[7]. healers of Chittagong Hill Tracts region of Bangladesh to [3] treat liver disease . An aqueous extract of leaf is reported 2. Material and methods to have remarkable antiviral[4] and anti cystic fibrosis [5] P. acidus properties . Studies with methanolic extract of 2.1. Chemicals and drugs *Corresponding author: Nilesh Kumar Jain, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar 470003, India. Tel: +91-9926504077 Acetaminophen, and 2,2-diphenyl-1-picrylhydrazil Fax: +91-7582-220916 E-mail: [email protected] (DPPH) were purchased from Sigma Chemical Co. (St. Nilesh Kumar Jain et al./Asian Pacific Journal of Tropical Medicine (2011)470-474 471 Louis, MO, USA). Thioacetamide was obtained from Loba standard pellet diet (Him Feed H.P., Agro industries) and Chemie, Mumbai. Silymarin was obtained as a gift sample water ad libitum. Animal studies were approved by the from Serum International Ltd., Pune, India. Aspartate Institutional Animal Ethics Committee (379/01/ab/CPCSEA) transaminase (AST), alanine transaminase (ALT), alkaline and conducted as per the regulations of Committee for the phosphatase (ALP), bilirubin and protein estimation kits Purpose of Control and Supervision of Experiments on were procured from Span Diagnostics, Surat, India. All other Animals (CPCSEA). chemicals and reagents used were of highest commercially 2.6. Acute oral toxicity studies available purity and purchased from Qualigens, India. 2.2. Plant material P. acidus The acute oral toxicity studies of extracts were OECD [9] P. acidus carried out as per guidelines . On the basis of these 200 400 The fresh leaves of were collected in May-June, studies, the oralin vivodoses of and mg/kg, bw were 2008 from University (Dr. H. S. Gour University) campus, selected for the experiments. Sagar (India) and authenticated in Botany Department of 2.7. Total phenol and flavonoid content determination University, where a voucher specimen (No.Bot/H/4322) has been preserved for future reference. The leaves were shade dried, coarsely powdered and stored for further use. The total phenol content of plant extracts was determined [10] 2.3. Preparation of extracts by Folin-Ciocalteau method . The total phenolic content was expressed as milligrams of gallic acid equivalents/g GAE 2.3.1. Preparation of ethanolic extract extract (mg /g of dry mass). The flavonoid content of extracts was estimated as The leaf powder (400 g) was sequentially extracted with quercetin equivalent[11]. The total flavonoid content was petroleum ether (60-80 曟) and ethanol (95% v/v) till expressed in milligrams of quercetin equivalents/g of extract complete exhaustion using Soxhlet apparatus. The ethanolic (mg QE/g of dry mass). extracts were filtered and the filtrate was evaporated 2.8. DPPH scavenging assay to dryness at 50 曟 under reduced pressure in a rotary evaporator. About 14 g of dried ethanolic extract was obtained from 400 g of dried leaf powder (3.5% w/w). The The free radial scavenging (antioxidant) property of extracts extract was uniformly suspended in 2% aqueous gum acacia was determined as bleaching of the stable DPPH radical[12]. prior to experiment. Ascorbic acid, a well known antioxidant was used as a 2.3.2. Preparation of aqueous extract positive control. 2.9. APAP- induced hepatotoxicity 500 g leaf powder was subjected to hot water extraction for 4 h at 80 曟 and then filtered. The filtrate was evaporated to dryness to get the water extract. The yield of extract was 33 g The experiment was conducted according to the method from 500 g of leaf powder (6.6% w/w). This crude extract was described previously[13]. Rats were randomly divided into 7 re-suspended in water prior to experiment. groups, each consisting of 6 rats. Group 栺 served as normal 1 2.4. Preliminary phytochemical screening control and received distilled water ( mL/kg, po.) daily for 7 days and 50% aqueous sucrose (10 mL/kg, po.) on days 5. Group 栻 served as hepatotoxicity (APAP) control and Preliminary phytochemical analysis on plant extracts was received distilled water (1 mL/kg, po.) daily for 7 days and performed using the following chemicals and reagents: received APAP (2 g/kg, po.) as suspension in 50% aqueous HCl 3 flavonoids (Mg metal and’ ), phenolics (FeCl ), protein sucrose solution on days 5. Group 栿 was treated with and amino acid (Millon s’ and Ninhydrin reagent), alkaloids silymarin (100 mg/kg, po.) daily for 7 days and received (Mayer and Dragendorff s reagent), saponins (Foam test), APAP (2 g/kg, po.) on days 5, 30 min after the administration of - - phytosterols and triterpenoids (Liebermann-’ Burchard Test) silymarin. Groups 桇 桋 and 桍 桏 were treated with ethanolic and glycosides (NaCl and Fehling s solution A and B) [8]. and aqueous extracts at two doses (200 and 400 mg/kg, po.), 7 APAP 2 2.5. Animals respectively, for days and received ( g/kg, po.) on days 5, 30 min after administration of extracts. 2.10. TAA- induced hepatotoxicity Wistar albino rats (200-220 g) and Swiss albino mice (20- 25 g) of either sex were used for the studies. The animals were grouped and housed in polyacrylic cages with not more Rats were randomly divided into 7 groups, each consisting than six per cage and maintained under standard laboratory of 6 rats. Group 栺 served as normal control and received conditions of temperature [(25依2) 曟] and relative humidity distilled water (1 mL/kg, po.) daily for 7 days. Group 栻 [(55依5) %] with dark and light cycle (12/12 hours). They served as hepatotoxicity (TAA) control and received distilled were acclimatized to laboratory conditions for 7 days before water (1 mL/kg, po.) daily for 7 days and received TAA (100 commencing the experiment and allowed for free access to mg/kg, sc.) as 2% w/v solution in distilled water on days 6[14]. Nilesh Kumar Jain et al./Asian Pacific Journal of Tropical Medicine (2011)470-474 472 P. acidus Group 栿 was treated with silymarin (100 mg/kg, po.) daily In acute oral toxicity studies, the extracts did not for 7 days and received TAA (100 mg/kg, sc.) on days 6, 30 show any sign and symptoms of toxicity and mortality up to min after administration of silymarin. Groups 桇-桋 and 桍 2 000 mg/kg dose, considered relatively safe. - 桏 were treated with ethanolic and water extracts at two 3.3. Total phenolic and flavonoid content doses of 200 and 400 (mg/kg, po.), respectively, for 7 days and received TAA (100 mg/kg, sc.) on days 6, 30 min after the administration of extracts.