ISOLATION OF BIOACTIVE SECONDARY METABOLITES AND PHARMACOLOGICAL STUDIES OF VIOLA SERPENS WALL

Abstract

Viola serpens which belongs to the family Violaceae has been reported to have many folkloric uses including use as antipyretic, laxative, emollient, expectorant, purgative, diuretic, demulcent, diaphoretic, anti-asthmatic and anti-cancer. The current investigation was carried out to evaluate the crude methanolic extract and various fractions of Viola serpens for its antioxidant, antimicrobial, enzyme inhibiting potential, larvicidal activities using using in vitro assays and for antinociceptive, anti-inflammatory activities, hepatoprotective and nephroprotective effects using in-vivo studies. Furthermore, bioactive secondary metabolites were also isolated and characterized. The results showed that the crude methanolic extract and various fractions including chloroform, ethyl acetate fraction, n-hexane fraction and aqueous fractions possessed significant antimicrobial activities against S. typhi and E. coli, B. subtilis, S. flexeneri and P. aerogenes. The compounds (1-6) isolated from the plant were also tested for activity against different spp. of Gram positive and Gram negative bacteria except the compound commulin-C. Against S. typhi, sideroxyline showed maximum zone of inhibition (18 mm) followed by the other tested compounds, each with 17 mm zone of inhibition. Against the P. aerogenes, tectochrysine and cearoin proved more effective followed by sideroxylin and commulin-B. S. flexeneri proved more susceptible to the compounds tectochrysine and cearoin (17 mm) followed by commulin-A, commulin-B but sideroxylin was inactive against it. S. aureus was susceptible to all four mentioned isolated compounds except commulin-B and cearoin. Commulin-B and tectochrysine were effective against E. coli. Viola serpens crude extract, fractions and pure compounds also showed significant free radical scavenging activity in DPPH free radical scavenging assays. Concentration-dependent scavenging effect is showed by the crude methanolic extract, n-hexane and chloroform soluble fractions against DPPH with maximum activity of 67.99%, 75.98 % and 79.00 %. In pure form the isolated compounds commulin-A, commulin-B and commulin-C showed effective scavenging activity with the percent values 78.05 %, 89.45% and 78.05% with IC50 value 201 ppm, 98.15 ppm and 168 ppm respectively. Larvicidal effect of the plant was tested against the A. aegypti and C. quinquefasciatus species. The ethyl acetate soluble fraction caused maximum percent inhibition followed by the chloroform and the crude methanolic extract at 600 ppm with values of 89.91 %, 85.21 % and 59.67 % respectively. Acetyl cholinesterase assay was conducted by using three different concentrations of 250, 500 and 1000 ppm. The chloroform soluble fraction caused maximum percent inhibition followed by the ethyl acetate soluble fraction, crude extract and the aqueous soluble fraction with 89, 70.5, 68.55 and 50.75 % at 1000 ppm and IC50 values of 149, 156, 245 and 989 ppm respectively. The crude extract along with the subsequent fractions were found safe in the in vivo acute toxicity screening. Viola serpens as an effective hepatoprotective and nephroprotective plant, was proved by evaluating various blood and urine parameters (AST, ALP, ALT, urine clearance, urea creatinine and serum creatinine) against the Paracetamol (PCM) induced toxicity and as evident by histopathological studies. Being a safe drug, V. serpnse proved to be significant antinociceptive and anti-inflammatory agents by using two different protocols for analgesia (Acetic acid induced writhing test and formalin induced nociception test) and three for anti- inflammation (carrageenan and histamine induced paw edema and xylene induced ear edema). The plant extract and fractions were used at three different doses (100, 200 and 300 mg/kg). The plant proved to be an effective antinociceptive and anti-inflammatory drug following the peripheral pathway for these activities mostly in a dose dependent manner. The plant extract and the different fractions with the decreasing polarity produced more attenuated antinociceptive effect in a dose dependent manner. The effect is more significant in the crude extract followed by the n-hexane, ethyl acetate, chloroform and aqueous soluble fractions. Peripheral pathway is followed by either due to inhibiting/reducing the release of cyclooxygenase or lipoxygenase enzymes or may involve the release of certain mediators. The anti-inflammatory effect of the plant was measured by using the protocols of carrageenan and histamine induced paw edemas and xylene induced ear edema. In histamine induced paw edema the crude extract at doses of 200 and 300 mg/kg showed more pronounced anti-inflammatory effects that reached the maximum in the 3rdh. The n-hexane soluble fraction at a dose of 200 mg/kg in the 3rd h of histamine induction showed maximum anti-inflammatory effect. The chloroform and aqueous soluble fractions showed significant effects at the doses of 200 and 300 mg/kg on the 3rd h of histamine induced edema. Different percent inhibition values were obtained in case of each fraction. In xylene induced ear edema the crude extract and n-hexane fraction showed a dose dependent significance (Max. values at 300 mg/kg). This was followed by the chloroform, ethyl acetate and aqueous fraction at doses of 200 and 300 mg/kg with the maximum inhibition values of 51, 49 and 48.5 % respectively. The chloroform and ethyl acetate soluble fractions were analyzed by column chromatography which resulted in the isolation of six pure compounds. Among the six compounds, three were new (not reported before) and the other three were already reported from the other sources but obtained for the first time from this plant (V. serpens) source. Commulin-A, Commulin-B and Commulin-C were the new compounds whereas, tectochrysine, Sideroxyline and Cearoin were the already reported compounds. 1H-NMR, 13C-NMR, COSY, NOESY, HMBC, IR, UV, E1-MS and HRE1-MS were the different techniques used for elucidating the structures of the new compounds. In conclusion, V. serpens showed significant antimicrobial, antioxidant activities, antinociceptive, anti-inflammatory, hepatoprotective and nephroprotective activities. The marked pharmacological and phytochemical studies suggested further detailed studies to confirm their folk uses and isolation of compounds that can act as potent drug in future.