Hepatoprotective activity of methanolic extract of Malva parviflora against paracetamol-induced hepatotoxicity in mice
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, phytochemical 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.
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 resistance 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, antifertility, cytotoxic, antibiotic (Amin et al., 2012), spasmolytic, bronchodilator (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 Muthusamy, 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), OfIpomoea 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., 2005), anti-diabetic (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 hepatoprotective potential of M. parviflora.
Materials and Methods
Plant material
The plant was collected from Faisalabad and identified by Dr. Mansoor Hameed, Department of Botany, University of Agriculture, Faisalabad. The voucher specimen 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 methanolic 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 histopathological examination (Ali et al., 2013).
Phytochemical screening
The phytochemical screening of various active compounds 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 Injector Valve Bracket, pump (1500 series), Column oven and prepacked C-18 column (250 x 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 investigation as shown in Figure 1. Results of the phytochemical screening are given in Table II.
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 | ||||
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 moderate dilatation of sinusoids (D) Extract 250 mg/kg treated group, mild inflammation, ballooning and moderate sinusoidal dilatation (E) Extract 500 mg/kg treated group, moderated inflammation, mild ballooning, mild necrosis and moderate sinusoidal dilatation
Discussion
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 are required to postulate this claim.
Flavonoids are important compounds in plants and have previously reported to have hepatoprotective activity (Ali et al., 2013). In our study moderate amount of flavonoids was present in whole plant. Qualitative determination of these flavonoids by HPLC confirmed the presence of kaempferol and apigenin (Figure 2). kaempferol (Song et al., 2003) and apigenin (Oh et al., 2004) have already been reported to have hepatoprotective activity. Therefore, hepatoprotective activity of the plant may be due to these compounds.
Figure 2: HPLC chromatogram of aqeous methanolic extract of Malva parviflora
Conclusion
The aqueous methanolic extract of whole plant of M. parviflora possesses hepatoprotective activity.
Ethical Issue
Experimental protocols were approved by Ethical Review Committee of the Institute.
References
Afolayan AJ, Aboyade OM, Adedapo AA, Sofidiya MO. Anti-inflammatory and analgesic activity of the methanol extract of Malva parviflora Linn (Malvaceae) in rats. Afr J Biotech. 2010; 9: 1225-29.
Ahmad M, Mahmood Q, Gulzar K, Akhtar MS, Saleem M, Qadir MI. Antihyperlipidaemic and hepatoprotective activity of Dodonaea viscosa leaves extracts in alloxan-induced diabetic rabbits (Oryctolagus cuniculus). Paki Vet J. 2012; 32: 50-54.
Akhtar MS, Asjad HMM, Bashir S, Malik A, Khalid R, Gulzar F, Irshad N. Evaluation of antioxidant and Hepatoprotective effects of Khamira Gaozaban Ambri Jadwar Ood Saleeb Wala (KGA). Bangladesh J Pharmacol. 2013; 8: 44-48.
Ali M, Qadir MI, Saleem M, Janbaz KH, Gul H, Hussain L, Ahmed B. Hepatoprotective potential of Convolvulus arvensis against paracetamol-induced hepatotoxicity. Bangladesh J Pharmacol. 2013; 8: 300-04.
Amin N, Qadir MI, Khan TJ, Abbas G, Ahmad B, Janbaz KH, Ali M. Antibacterial activity of vacuum liquid chromatography (VLC) isolated fractions of chloroform extracts of seeds of Achyranthes aspera. J Chem Soc Pak. 2012; 34: 589-92.
Amiri MS, Joharchi MR, Yazdi MET. Ethnomedicinal plants used to cure jaundice by traditional healers of Mashhad, Iran. Iranian J Pharm Res. 2014; 13: 157-62.
Arora DS, Kaur GJ. Antibacterial activity of some Indian medicinal plants. Natl Med J. 2007; 61: 313-17.
Asif MA, Qadir MI. Molecular approaches for development of malarial vaccines. Rev Pharmacol. 2011; 4: 276-78.
Bag AK, Mumtaz SMF. Hepatoprotective and nephroprotective activity of hydroalcoholic extract OfIpomoea staphylina leaves. Bangladesh J Pharmacol. 2013; 8: 263-68.
Balamurugan G, Muthusamy P. Observation of the Hepatoprotective and antioxidant activities of Trianthema decandra Linn. (Vallai sharunnai) roots on carbon tetrachloridetreated rats. Bangladesh J Pharmacol. 2008; 3: 83-89.
Chattopadhyay RR. Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract. J Ethnopharmacol. 2003; 89: 217-19.
Ehsan O, Qadir MI, Malik SA, Abbassi WS, Ahmad B. Efficacy of nanogold-insulin as a hypoglycemic agent. J Chem Soc Pak. 2012; 34: 365-70.
Farhan H, Rammal H, Hijazi A, Badran B. Preliminary phytochemical screening and extraction of polyphenol from stems and leaves of a Lebanese plant Malva parviflora L. Int J Curr Pharma Res. 2012; 4: 55-59.
Gutierrez RMP. Evaluation of hypoglycemic activity of the leaves of Malva parviflora in streptozotocin-induced diabetic rats. Food Func. 2012; 3: 420-27.
Hegde K, Joshi AB. Hepatoprotective and antioxidant effect of Carissa spinarum root extract against CCl4 and paracetamol-induced hepatic damage in rats. Bangladesh J Pharmacol. 2010; 5: 73-76.
Hussain A, Khalid SH, Qadir MI, Massud A, Ali M, Khan IU, Saleem M, Iqbal MS, Asghar S, Gul H. Water Uptake and Drug Release Behaviour of Methyl Methacrylate-co-itaconic acid [P(MMA/IA)] Hydrogels Cross-linked with Methylene Bis-acrylamide. J Drug Deliv Sci Tech. 2011; 21: 249-55.
Janbaz KH, Nisar U, Ashraf M, Qadir MI. Spasmolytic, bronchodilator and antioxidant activities of Erythrina superosa Roxb. Acta Pol Pharm. 2012; 69: 1111-17.
Janbaz KH, Saeed SA, Gilani AH. Studies on the protective effects of caffeic acid and quercetin on chemical-induced hepatotoxicity in rodents. Phytomedicine 2004; 11: 424-30.
Javed F, Qadir MI, Janbaz KH, Ali M. Novel drugs from marine microorganisms. Cr Rev Microbiol. 2011; 37: 245-49.
Khalid SH, Qadir MI, Massud A, Ali M, Rasool MH. Effect of degree of cross-linking on swelling and drug release behaviour of poly (methyl methacrylate-co-itaconic acid) [P(MMA/IA)] hydrogels for site specific drug delivery. J Drug Deliv Sci Tech. 2009; 19: 413-18.
Khan AZ, Mohammad A, Iqbal Z, Anis I, Shah MR, Nadeem S, Rabnawaz M, Shahidullah A, Khan H, Khan I. Molecular docking of viscosine as a new lipoxygenase inhibitor isolated from Dodonaea viscose. Bangladesh J Pharmacol. 2013; 8: 36-39.
Khattak SG, Gilani SN, Ikram M. Antipyretic studies on some indigenous Pakistani medicinal plants. J Ethnopharmacol. 1985; 14: 45-51.
Koochek M, Pipelzadeh M, Mardani H. The effectiveness of Viola odorata in the prevention and treatment of formalin induced lung damage in the rat. J Herbs Spices Med Plants. 2003; 10: 95-103.
Masood MI, Qadir MI, Shirazi JH, Khan IU. Beneficial effects of lactic acid bacteria on human beings. Crit Rev Microbiol. 2011; 37: 91-98.
Naz S, Qadir MI, Ali M, Janbaz KH. Nanotechnology for imaging and drug delivery in cancer. J Chem Soc Pak. 2011; 34: 107-111.
Oh H, Kim DH, Cho JH, Kim YC. Hepatoprotective and free radical scavenging activities of phenolic petrosins and flavonoids isolated from Equisetum arvense. J Ethnopharmacol. 2004; 95: 421-24.
Qadir MI, Ali M, Ali M, Saleem M, Hanif M. Hepatoprotective activity of aqueous methanolic extract of Viola odorata against paracetamol-induced liver injury in mice. Bangladesh J Pharmacol. 2014b; 9: 198-202.
Qadir MI, Malik SA. Anti-diabetic activity of inorganic metals Eugenia jambolana Lam. (Myrtaceae) flowers. Pharmacology-online 2010; 2: 979-85.
Qadir MI, Malik SA. Effect of Eugenia jambolana leaves extracts on blood glucose levels of experimental diabetic rabbits. Pharmacologyonline 2009; 3: 829-35.
Qadir MI, Murad MSA, Ali M, Saleem M, Farooqi AA. Hepatoprotective effect of leaves of aqueous ethanol extract of Cestrum nocturnum against paracetamol-induced hepatotoxicity. Bangladesh J Pharmacol. 2014a; 9: 167-70.
Qadir MI. Medicinal and cosmetological importance of Aloe vera. Int J Nat Ther. 2009; 2: 21-26.
Qadir MI. Medicinal values of ginger. Int J Nat Ther. 2009; 3: 19-22.
Qadir MI. Qadirvirtide. Pak J Pharm Sci. 2011; 24: 593-95.
Saleem M, Ahmed B, Qadir MI, Mahrukh, Rafiq M, Ahmad M, Ahmad B. Hepatoprotective effect of Chenopodium murale in mice. Bangladesh J Pharmacol. 2014; 9: 124-28.
Shale TL, Stirk WA, Van Staden J. Variation in antibacterial and anti-inflammatory activity of different growth forms of Malva parviflora and evidence for synergism of the anti-inflammatory compounds. J Ethnopharmacol. 2005; 96: 325-30.
Song EK, Kim JH, Kim JS, Cho H, Nan JX, Sohn DH, Ko GI, Oh H, Kim YC. Hepatoprotective phenolic constituents of Rhodiola sachalinensis on tacrineâ€induced cytotoxicity in Hep G2 cells. Phytother Res. 2003; 17: 563-65.
Wang X, Bunkers GJ, Walters MR, Thoma RS. Purification and characterization of three antifungal proteins from cheese-weed (Malva parviflora). Biochem Biophys Res Commun. 2001; 282: 1224-28.
