Bioassay-guided evaluation of hepatoprotective potential of selected indigenous medicinal plants in carbon tetrachloride intoxicated mice

Abstract

This dissertation encompasses the phytochemical and pharmacological role of Viola canescens, Ziziphus oxyphylla and Rosa webbiana. The objective of this study was to authenticate folk uses of these plants in liver diseases, by isolating active pure compounds and to test them for hepatoprotection. The crude methanolic extracts of the selected plants were fractionated into n-hexane, chloroform, ethyl acetate and water. These extracts were qualitatively screened for various chemical constituents such as alkaloids, carbohydrates, polyphenols, flavonoids, saponins, tannins, triterpenes, proteins, fixed oils, essential oils and fats. The polyphenols and flavonoids were determined quantitatively. The crude extracts were tested for acute toxicity study and hepatoprotective activity in BALB/c mice. All the plant extracts were found safe upto 2000 mg/kg body weight. Among various fractions, the ethyl acetate fraction of Viola canescens, and chloroform fraction of Ziziphus oxyphylla and Rosa webbiana exhibited more pronounced hepatoprotection. Therefore, these fractions were subjected to isolation and purification of compounds responsible for hepatoprotective activity. Structures of pure compounds were elucidated by NMR and mass spectrometric techniques and placed in group of triterpenes, flavonoid and sterol. The isolated compounds were screened for acute toxicity, anti-inflammatory and hepatoprotective activity. Antioxidant activity of solvent extracts was evaluated by DPPH and H2O2 scavenging assays. In DPPH assay, ethyl acetate subfraction (EAF + Me) of Viola canescens exhibited DPPH scavenging action (86.89 ± 0.16%, IC50=12 μg/ml), while chloroform fraction (CF) of Rosa webbiana (RW) exhibited (84.7 ± 0.3 %) activity. IX In H2O2 scavenging assay, Viola canescens EAF + Me exhibited excellent scavenging of H2O2 (IC50, 5 μg/ml), the IC50 value for CF of RW was 100 μg/ml. Hepatoprotective activity was evaluated in carbon tetrachloride (CCl4) induced hepatotoxicity by determination of hepatic biomarkers, antioxidant parameters, changes in body and liver weight and histopathology of liver. Seven days treatment with CCl4 caused elevation in ALT (195.6 ± 1.4 U/L), ALP (315.3 ± 1.7 U/L), total bilirubin (2.73 ± 0.07 mg/dl) and decline in total protein (3.91 ± 0.47 g/dl). Viola canescens (EAF + Me) reduced the levels of ALT (50.67 ± 0.67), ALP (187 ± 0.68), TB (0.7 ± 0.05) while TP was enhanced (4.78 ± 0.25). Ziziphus oxyphylla (CF) reduced ALT level to (57 ± 0.13), ALP (204 ± 0.17), TB (0.82 ± 0.18) and increased TP level (4.50 ± 0.16). Rosa webbiana (CF) reduced the level of these parameters to 97.3 ± 1.2, 204 ± 1.34, 1.01 ± 0.11 and 4.35 ± 0.21 respectively. In order to probe into mechanism of hepatoprotection, antioxidant enzymes (CAT, SOD), lipid peroxidation and phenobarbital induced sleeping time (PST) was determined. It was observed in the current study that CCl4 caused decrease in activities of CAT (15.87 ± 0.24) and SOD (18.3 ± 0.95) while MDA level was increased (43.5 ± 0.45). Viola canescens subfraction (EAF + Me) enhanced the level of CAT (40.17 ± 0.08), SOD (53.3 ± 0.28) while MDA level declined (15.1 ± 0.1). Ziziphus oxyphylla (CF) enhanced the level of CAT, SOD and MDA upto 39.8 ± 0.23, 51.7 ± 0.15 and 17.4 ± 0.24 respectively. CCl4 induced prolongation of phenobarbital sleeping time (130 ± 3.88) in comparison to normal group (85 ± 2.26); however, in Viola canescens (EAF + Me) treated animals sleeping time was reduced (96 ± 1.15 minutes). Chloroform fraction (CF) of Ziziphus oxyphylla also reduced righting reflex (94 ± 0.88 minutes). X Furthermore, the hepatoprotective mechanism was also investigated for the ability of extracts to stabilize erythrocyte membrane. The membrane stabilizing effect of Viola canescens EAF was (IC50 3.4 ± 0.15 mg/ml), while CF of Ziziphus oxyphylla exhibited 72.5 ± 2.04% inhibition of erythrocyte lysis as compare to sodium salicylate (76.43 ± 3.22%). Protective effect of the solvent extracts of Viola canescens on CCl4 induced DNA damage in the liver tissue of mice was evaluated by polyacrylamide gel electrophoresis. In CCl4 treated mice liver tissue, severe DNA damage was observed. Administration of Viola canescens solvent extracts prevented DNA damage as compare to silymarin. Histopathological study was performed to confirm hepatoprotective effects of extracts. Administration of CCl4 caused severe fatty changes, extensive hepatocyte necrosis, lymphocytic infiltration and sinusoidal congestion. Treatment of mice with Viola canescens (EAF + Me) exhibited significant recovery from fatty changes, necrosis, lymphocytic infiltrations and sinusoidal congestion which is comparable to normal. While CF of Ziziphus oxyphylla resulted in near to normal histomorphology of liver. In case of CF of Rosa webbiana only mild cellular infiltration was observed. Moreover, the crude extracts were screened for anti-inflammatory activity via xylene and carrageenan models in mice. VCME (400 mg/kg body weight) resulted in inhibition of ear edema (53.41 ± 0.7%) induced by xylene as compare to diclofenac sodium (73.64 ± 1.14%). Paw edema in mice caused by carrageenan was reduced upto 59.46 ± 0.29% at 400 mg/kg body weight at 4th h. The standard drug (diclofenac sodium) reduced paw edema upto 62.4 ± 0.26% at 10 mg/kg body weight. RWME (400 mg/kg body weight) inhibited xylene induced ear edema (40.02 ± 0.23%). While carrageenan induced paw edema was reduced upto 52.75 ± 0.04% at 400 mg/kg. XI Compound 6 (ARW1) at 50 mg/kg inhibited 62.9 ± 0.15% xylene induced ear edema and 66.6 ± 0.17% carrageenan induced paw edema. Compound 3 (AZO2) and 4 (AZO3) isolated from Ziziphus oxyphylla, inhibited xylene induced ear edema at 50 mg/kg 51.33% and AZO3 58.66% respectively as compare to diclofenac sodium (72.66% inhibition). In carrageenan induced paw edema, compound 3 (AZO2) and 4 (AZO3) provided 33.3 ± 0.21 and 39.21 ± 0.15% inhibition respectively at 50 mg/kg. In acetic acid induced analgesic activity, compound 3 (AZO2) and 4 (AZO3) produced 64.28% and 65.35% inhibition of writhing at 50 mg/kg. Based on folk uses, the Viola canescens extract was subjected to antipyretic and antidiarrheal activity. Subcutaneous injection of brewer yeast suspension caused marked increase of body temperature of mice which was reduced in dose dependant manner by V. canescens at 100, 200 and 400 mg/kg orally. Treatment of animals with paracetamol and VCME at 100, 200 and 400 mg/kg caused reduction of pyrexia at 1st, 2nd, 3rd and 4th h in comparison to control group. Evaluation of phytotoxic effect by percent radish seed inhibition and root length inhibition shown by VCME was 59.33 ± 0.23 and 62.43 ± 0.25 respectively at 1000 ppm with IC50 240 and 125 ppm respectively. The percent cytotoxic activity of VCME was 65 ± 2.1% at 1000 ppm. VCME showed 40% antidiarrheal activity at 300 mg/kg that was enhanced to 80% at 1000 mg/kg as compare to loperamide (100% activity). It may be concluded from our findings that Viola canescens and Ziziphus oxyphylla provided good hepatoprotection while Rosa webbiana exhibited poor hepatoprotection as compare to silymarin. Compound 3 (AZO2) and 4 (AZO3) isolated from Ziziphus oxyphylla exhibited antioxidant, analgesic, anti-inflammatory and XII hepatoprotective activity while compound 6 (ARW1) isolated from Rosa webbiana showed antioxidant, anti-inflammatory and hepatoprotective activity.