Bangladesh Journal of Pharmacology Research Article Hepatoprotective activity of methanol- ic extract of Malva parviflora against paracetamol-induced hepatotoxicity in mice BJP Introduction Almost 80% people all over the word depend upon traditional medicines to cure their major and minor ailments. Plants are always been an inspirational source for many researcher in medicine. The problem of resis- tance and tolerance to the existing drugs has created a decreased efficacy of these drugs in use. This problem has been tried to be overcome by increasing the drug delivery to the target site by the use of polymers (Khalid et al., 2009; Hussain et al., 2011) or through nanotechnology (Naz et al., 2012; Ehsan et al., 2012), synthesis of new drugs, either by the use of proteomics (Qadir, 2011), or synthesis from lactic acid bacteria (Masood et al., 2011), or marine microorganisms (Javed et al., 2011). However, now a days, the trend is also being changed to the use of herbal products or extracts to control the diseases. The plant kingdom still holds many species containing substances of medicinal value which have yet to be discovered: Large numbers of plants are constantly being screened for their possible pharmacological value particularly for their anti- inflammatory (Qadir, 2009), hypotensive (Qadir, 2010), hypoglycemic, amoebicidal, anti-fertility, cytotoxic, antibiotic (Amin et al., 2012), spasmolytic, bronchodila- tor (Bashir et al., 2013), antioxidant (Janbaz et al., 2012) and hepatoprotective properties (Ahmad et al., 2012). Many plants have been identified as hepatoprotective like Trianthema decandra (Balamurugan and Muthu- samy, 2008), Cocculus hirsutus (Thakare et al., 2009), Carica papaya (Sadeque and Begum, 2010), Carissa spinarum (Hegde and Joshi, 2010), Convolvulus arvensis (Ali et al., 2013), Dodonaea viscosa (Khan et al., 2013), Trichodesma sedgwickianum (Saboo et al., 2013), OfIpo- moea staphylina (Bag and Mumtaz, 2013), Khamira Gaozaban Ambri Jadwar Ood Saleeb Wala (Akhtar et al., 2013), Cestrum nocturnum (Qadir et al., 2014a), Viola odorata (Qadir et al., 2014b), Chenopodium murale (Saleem et al., 2014) and Morus nigra (Mallhi et al., 2014). Malva parviflora L. (family Malvaceae) is herb native to Africa, Asia and Europe. It common name is cheese- weed and locally known as Sonchal. Pharmacologically, it has been reported to be antibacterial (Shale et al., A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2014; 9: 342-346 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract Malva parviflora (cheeseweed) is traditionally used as hepatoprotective. The current study was conducted to determine its hepatoprotective activity of aqueous methanolic extract of whole plant. Two doses of plant (250 and 500 mg/kg) were administered in paracetamol intoxicated mice and results were compared with silymarin. Observational parameters were ALT, AST, ALP and total bilirubin. The results showed that the extract of M. parviflora produced significant (p<0.001) reduction in liver enzymes and total bilirubin. Results were supported by histopathological investigation, phyto-chemical screening and detection of hepatoprotective constituents (kaempferol and apigenin) by HPLC. So, the current study showed that aqueous methanolic extract of M. parviflora possesses hepatoprotective activity. Article Info Received: 10 June 2014 Accepted: 19 July 2014 Available Online: 2 August 2014 DOI: 10.3329/bjp.v9i3.19105 Cite this article: Mallhi TH, Abbas K, Ali M, Qadir MI, Saleem M, Khan YH. Hepatoprotec- tive activity of methanolic extract of Malva parviflora against paracetamol- induced hepatotoxicity in mice. Bangladesh J Pharmacol. 2014; 9: 342- 46. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Hepatoprotective activity of methanolic extract of Malva parviflora against paracetamol-induced hepatotoxicity in mice Tauqeer Hussain Mallhi1, Khizar Abbas2, Muhammad Ali3, Muhammad Imran Qadir3, Mohammad Saleem2 and Yusra Habib Khan1 1School of Pharmaceutical Sciences, University Sains Malaysia, Palau Penang, Malaysia; 2College of Pharmacy, Government College University Faisalabad, Faisalabad, Pakistan; 3Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan. 2005), antidiabetic (Gutierrez, 2012), antifungal (Wang et al., 2001). Traditionally M. parviflora is used for the treatment of inflammation, pain and liver injuries (Afolayan et al., 2010). The plant contains phenolic and flavonoid compounds (Farhan et al., 2012). Traditional importance and phytochemical profile of the plant appeal its use in liver injury. Therefore, the current study was conducted to scientifically determine hepato- protective potential of M. parviflora. Materials and Methods Plant material The plant was collected from Faisalabad and identified by Dr. Mansoor Hameed, Department of Botany, Uni- versity of Agriculture, Faisalabad. The voucher speci- men was deposited in the herbarium of College of Pharmacy, for future reference. Powdered plant was soaked in aqueous methanol (70:30). After 7-10 days solution was filtered and the filtrate was evaporated with the help of rotary evaporator to get the extract. Estimation of hepatoprotective activity Swiss albino mice of both sexes weighing 22-35 g were kept under standard laboratory conditions (25 ± 2°C with dark and light cycle of 12/12 hours). These were fed with standardized pellet diet and water ad libitum. All the animals were divided into five groups having 5 animals each. Group I was Control, receiving distilled water only. Group II served as paracetamol control, receiving paracetamol p.o. 250 mg/kg dissolved in water for 7 days. Group III, silymarin control in which silymarin was given as reference drug 50 mg/kg daily for 7 days and paracetamol was administered 3 hours after silymarin. Group IV received aqueous methanolic (70:30) extract of M. parviflora at doses 250 mg/kg p.o. for 7 days and received paracetamol 250 mg/kg 3 hours after extract dose. Group V received aqueous metha- nolic extract of M. parviflora at doses 500 mg/kg p.o. for 7 days and received paracetamol 250 mg/kg 3 hours after extract dose. At the 8th day, blood samples were collected to estimate ALT, AST, ALP and total bilirubin; and livers from animals were separated for histo- pathological examination (Ali et al., 2013). Phytochemical screening The phytochemical screening of various active com- pounds in the extract was accomplished by methods used by Farhan et al., 2012. HPLC analysis For qualitative separation of compounds, SYKAM HPLC system was used equipped with S-1122 Solvent Delivery System, S-3210 UV/VIS Detector, S-5111 Injec- tor Valve Bracket, pump (1500 series), Column oven and pre-packed C-18 column (250 × 4.5 mm, 5 um particle size). Data was analyzed by using Sample- Clarity Light software. Acetonitrile-water (1:1) with few drops of acetic acid (1%) was used as mobile phase with flow rate of 1 mL/min; and compounds were detected at 254 nm (Saddique et al., 2011). Statistical analysis All the data were subjected to one-way ANOVA by SPSS for statistical analysis. Results were represented as mean ± SE. Results Effect of aqueous methanolic extract of M. parviflora (AMMP) on liver enzymes and total bilirubin is given in Table I. Aqueous methanolic extract of M. parviflora with 250 mg/kg reduces elevated ALT by 53% (p<0.001)), AST by 53% (p<0.01), ALP by 27% (p<0.001) and TBR by 53% (p<0.001) as compared to paracetamol control. At 500 mg/kg dose, elevated ALT reduced by 55% (p<0.001), AST by 57% (p<0.001), ALP by 30% (p<0.001) and TBR by 53% (p<0.001) as compared to paracetamol control. There is insignificant (p>0.05) difference between two doses with exception of AST whose reduction is higher with 500 mg/kg (p<0.001) as compared to 250 mg/kg (p<0.01). These results are also comparable to that of silymarin (p<0.001, as compared to paracetamol control). Histopathological examination of liver sections also supported biochemical investiga- tion as shown in Figure 1. Results of the phytochemical screening are given in Table II. Bangladesh J Pharmacol 2014; 9: 342-346 343 Table I Effect of aqueous methanolic extract of Malva parviflora on liver enzymes and total bilirubin Treatment groups ALT (IU/L) AST (IU/L) ALP (IU/L) TBR (g/dL) Normal (D/W) 32.8 ± 02.0 36.5 ± 03.6 216.2 ± 09.9 0.8 ± 0.0 Paracetamol Control (250 mg/kg) 112.1 ± 4.1 101.2 ± 09.1 413.4 ± 21.7 2.0 ± 0.2 Silymarin (50 mg/kg) + Paracetamol 41.6 ± 03.0b 39.8 ± 07.8b 266.4 ± 32.6a 0.9 ± 0.1a Extract (250 mg/kg) + Paracetamol 52.6 ± 04.4b 47.6 ± 04.9a 300.6 ± 10.4a 0.9 ± 0.0a Extract (500 mg/kg) + Paracetamol 51.0 ± 05.6b 43.8 ± 03.2b 287.6 ± 09.8a 0.9 ± 0.0a ap<0.01, bp<0.001 Discussion M. parviflora is an endangered species of Malvaceae in Pakistan. It has traditional use for hepatitis and jaundice in rural areas of Central Punjab, Pakistan. Its leaves are also edible and cooked with leafy vegetables for its taste, in other countries it is also used for salad dressing. The number of plants of this family has been reported to have hepatoprotective activity. Depending pon its broad use in medicine, hepatoprotective traditional use, edible importance, phytochemistry and relevancy with other hepatoprotective plants of family, this plant was evaluated for its hepatoprotective use. Aqueous methanolic extract of M. parviflora reduces elevated ALT by 53 and 55% (p<0.001), AST by 53 and 57% (p<0.01, p<0.001; 250 and 500 mg/kg respectively), ALP by 27 and 30% (p<0.001) and TBR by 53% (p<0.001) with 250 and 500 mg/kg doses respectively in hepatotoxic mice and results are significant when compared to paracetamol control. There is insignificant (p<0.9) difference between two doses with exception of AST whose reduction is higher with 500 mg/kg (p<0.001) as compared to 250 mg/kg (p<0.01). These results are also comparable to that of silymarin (p<0.001). It seems high dose provides better results but not in all observational parameters so further studies 344 Bangladesh J Pharmacol 2014; 9: 342-346 Table II Phytochemical screening of whole plant of Malva parviflora Constituents Inferences Constituents Inferences Constituents Inferences Phenols ++ Alkaloids + Coumarines - Flavonoids ++ Resins + Volatile Oils - Saponins + Tannins + Terpinoids - +++ = high amount after added of reagent immediately; ++ = moderate amount after 5 min of reagent added; + = low amount after 10 min of reagent added and - = absent of active compound after 20 min Figure 1: Histopathological pictures of (A) Normal hepatocytes (B) Paracetamol treated group, marked inflammation, necrosis, sinusoidal constrictions and ballooning (C) Silymarin treated group, improvement in necrosis, inflammation, ballooning and mod- erate dilatation of sinusoids (D) Extract 250 mg/kg treated group, mild inflammation, ballooning and moderate sinusoidal dilata- tion (E) Extract 500 mg/kg treated group, moderated inflammation, mild ballooning, mild necrosis and moderate sinusoidal dila- tation are required to postulate this claim. 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