Screening and Purification of Resveratrol from Endophytic Fungi

Abstract

Resveratrol is a phenolic compound naturally produced during pathogenic attack by plants defense system. It is being exploited as potent antioxidant, anti-cancer, anti-inflammatory and anti- aging agent. There exists an urgent need for exploration of alternative natural sources of reseveratrol production which can contribute in meeting the increasing demand of this valuable drug and preserve the biodiversity of nature also. Endophytic fungi exist within the tissues of host plants in symbiotically fashion without exerting any negative effects to the host plant. The endopytic fungi are believed to be “Gold mines” of novel pharmacologically important bioactive compounds. The present study reports the exploration of endophytic fungi isolated from various medicinally important plants viz. Aegle marmelos, Cinnamomum malabaricum, C. camphora, Vitis vinifera, Catharanthus roseus and Raulwofia serpentina for their potential to produce resveratrol. In the present study cuture filtrates of endophytic fungi obtained from czapeck dox broth were screened for their potential to produce resveratrol extracellularly. Libermann and Acetic anhydride tests were used for confiriming the presence of resveratrol initially. After the preliminary screening 9 endophytic isolates were further subjected for mass production of secondary metabolites on CDB. These were subsequently extracted with solvents and tested for their presence of resveratrol. Out of selected 9 cultures, the crude EA extract of #5VVSTL and #16CRLPAL were found to be potent producers of Resveratrol. The resveratrol content in ethyl acetate extract of #5VVSTL was found to be maximum. Hence, Ethyl acetate extract of #5VVSTL was separated into 7 fractions by using Chloroform:Ethyl acetate:Formic acid in ratio10:2.4:0.4 as solvent system. The fungal resveretrol was purified by scrapping off TLC plates 40 mg of resveratrol was optained by this method. The purity and concentration of TLC Purified Reservertrol was then determined by using HPLC using C18 reverse phase discovery column the mobile phase combined of H3PO4 (0.1%): Acetonitrile (10-70%). The purified fungal Resveratrol and standard resveratrol exhibited same retention time of 35.2 min and visualized at violet color spot on TLC plate with Rf value of 0.63. The potent resrveratrol producing endophytic isolate, #5VVSTL was identified as Quambalaria cyanescens by using morphological and molecular taxonomic techniques.