Influence of exogenous cinnamic acid on the production of chlorogenic acid in Cichorium intybus L cell culture

Abstract

Cichorium intybusL(C. intybus) is a source for several important secondary metabolites (SM), including 3-O-caffeoylquinic acid (CGA). Isolation of SM from field plants is difficult due to their batch to batch variation in response to various environmental factors. The study aims to circumvent this problem in C. intybus by increasing CGA concentration through optimization of media conditions and elicitors using plant cell culture. An increased callus growth was observed in leaf explant inoculated on MS solid medium supplemented with 2 mg/l 1-napthaleneacetic acid (NAA), 2 mg/l kinetin (Kn), 30 g/l sucrose and 0.7% agar. Optimum callus growth (13 g/l) was obtained with 5% callus (w/v) sub-cultured in MS suspension medium containing 2 mg/l NAA, 2 mg/l Kn, and 50 g/l glucose at pH 5.5. After optimization of culture conditions, elicitors (cinnamic acid, jasmonic acid, chitosan and methyl jasmonate) were added towards the end of log phase to increase CGA content in callus. Among the various concentrations of elicitors, 0.5 mg/l cinnamic acid was found to reduce cell death and increase phenol content in callus as well as the culture media. These results correlated with the TLC-densitometry study, which showed cinnamic acid (0.5 mg/l) treatment to increase CGA content in callus by 0.39- fold in comparison to control callus. The elicitation was confirmed when 3T3-L1 adipocytes treated with 1 ng/ml methanol extract of callus exposed to 0.5 mg/l cinnamic acid showed a significant 0.3-fold increase in glucose uptake compared to 1 ng/ml control callus and field plant methanol extracts. The elicited callus showed a decrease in the levels of hydrogen peroxide, nitric oxide, and phenylalanine ammonia lyase activity, demonstrating the role of cinnamic acid as a precursor for CGA synthesis.