Hepatoprotective activity of methanolic extract of Malva parviflora against paracetamol-induced hepatotoxicity in mice

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 mg/kg 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.


Malva parviflora L. (family Malvaceae
) is herb native to Africa, Asia and Europe.It common name is cheeseweed and locally known as Sonchal.Pharmacologically, it has been reported to be antibacterial (Shale et al., 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 hepatoprotective potential of M. parviflora.

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 8 th 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 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 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 investigation as shown in Figure 1.Results of the phytochemical screening are given in Table II.(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  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 1 :
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