Cinnamic acid Supplementation Regulates the Production of Licochalcone A, Liquirtigenin and Licoisoflavone B in Glycyrrhiza glabra Callus Cultures.
Keywords:Glycyrrhiza glabra, Cinnamic acid, Precursor, Callus culture, Flavonoids
Glycyrrhiza glabra is an ancient herbal medicine rich in large number of secondary metabolites which attribute to its therapeutic properties. These metabolites are usually obtained from field grown plants and their yields vary greatly on the basis of environmental conditions. Plant tissue culture can thus be a preferred method for consistent production of such metabolites. This study dealt with the enhancement of flavonoids through precursor feeding in callus cultures of G. glabra and investigated the influence of cinnamic acid on phenylalanine ammonia lyase (PAL) activity and production of licochalcone A, liquirtigenin and licoisoflavone B. Unorganized callus cultures were established from young leaf explants on Murashige and Skoog’s (MS) medium supplemented with NAA (1mg/l), BAP (0.5 mg/l) and various concentrations of cinnamic acid. Flavonoids were obtained from calli through solvent extraction and were identified and quantified through Gas-Chromatography Mass spectrometry. Cinnamic acid supplementation at appropriate concentrations (50mg/100ml for licochalcone A and liquirtigenin, and 125mg/100ml for licoisoflavone B) significantly increased their production to 1.28, 1.2 and 9.76 folds respectively. However, prolonged treatment of cinnamic acid at concentration beyond 50mg/100ml led to decrease in the production of liquirtigenin and licochalcone A , but caused fair increase in licoisoflavone B. Also cinnamic acid concentrations higher than 50mg/100ml reduced the activity of PAL enzyme due to its feedback inhibition, but at the same time might have modulated other intermediate enzymes of the pathway like chalcone isomerase favoring the formation of licoisoflavone B. Therefore, this study provides clear evidences of enzymatic regulation of phenylpropanoid pathway by cinnamic acid in G. glabra callus cultures.
. Fukai T, Marumo A, Kaitou K, Kanda T, Terada S, Nomura T. Anti Helicobacter pyroli flavonoids from licorice extract, Life Sci. 2002; 71: 1449-1463.
. Kolbe L, Immeyer J, Batzer J, Wensorra U, Tom Dieck K, Mundt C, Wolber R, Stäb, F, Schönrock U, Ceilley R.I, Wenck H. Anti-inflammatory efficacy of Licochalcone A: correlation of clinical potency and in vitro effects. Arch Dermatol Res. 2006; 298: 23-30.
. Kim YW, Zhao RJ, Park SJ, Lee JR, Cho IJ, Yang CH, Kim SJ, Kim SC. Anti inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-kappaB dependent iNOS and pro inflammatory cytokines production. J Pharmacol. 2008; 154:165–173.
. Liu Y, Sirou X, Wang Y, Luo K, Wang Y, Cai Y. Liquiritigenin Inhibits Tumor Growth and Vascularization in a Mouse Model of Hela Cells. Molecules. 2012; 17: 7206-7216.
. Vijayalakshmi U, Shourie A. Gas Chromatography-Mass Spectrometric analysis of ethanolic extracts of Glycyrrhiza glabra Linn. roots. Int J Pharm Bio Sci. 2013; 4: 741-755.
. Wang HJ, Murphy PA. Isoflavone content in commercial soybean foods. J. Agric. Food Chem. 1994; 42: 1666-1673.
. Shourie A, Tomar P, Srivastava D, Chauhan R. Enhanced biosynthesis of quercetin occurs as a photoprotective measure in Lycospersicon esculentum Mill. under acute UV-B Exposure. Braz. Arch. Biol. Technol. 2014; 57: 31-325.
. Yu O, McGonigle B. Metabolic engineering of isoflavone biosynthesis. Adv. Agron. 2005; 86: 147–190.
. Jiang H, Wood KV, John AM. Metabolic Engineering of the Phenylpropanoid Pathway in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2005; 71: 2962-2969.
. Rahimi S, Hasanloo T, Najafi F, Nejad RAK. Enhancement of silymarin accumulation using precursor feeding in Silybum marianum hairy root cultures. POJ. 2011; 4: 34-39.
. Baranek KS, Pietrosiuk A, Marcin R, Naliwajski, Kawiak A, Jeziorek M, Wyderska S, Lojkowska E, Chinou I. Effect of L-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst. In Vitro Cell.Dev.Biol.-Plant. 2012; 48: 555–564.
. Vijayalakshmi U, Shourie A. Elicitor induced flavonoid production in callus cultures of Glycyrrhiza glabra and regulation of genes encoding enzymes of phenylpropanoid pathway. Der Pharm Lettre. 2015a; 8: 156-166.
. Vijayalakshmi U, Shourie A. Evaluation of different methods for extraction of antioxidant phenolic compounds from Glycyrrhiza glabra roots. World J Pharm Res. 2015b ; 10: 1524-1537.
. Bradford MM. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248–254.
. Turgut-Kara N, Cakir O. Comparative phylogenetic analysis of phenyl propanoid metabolism genes of legume pants. POJ.2015; 8: 55-61.
. Gaspar T, Kevers C, Penel C, Greppin H, Reid DM, Thorpe TA. Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell. Dev. Biol. Plant. 1996; 32:272–289.
. Lila MA, Yousef GG, Jiang Y, Weaver CM. Flavonoid mixtures from cell cultures. J. Nutr. 2005; 135: 1231-1235.
. Konczaka I, Teraharab N, Yoshimotoc M, Nakatanid M, yoshinagac M, Yamakawa .Regulating the composition of anthocyanins and phenolic acids in sweet potato cell culture towards production of polyhenolic complex with enhanced physiological activity. Trends. Food Sci.Technol. 2005;16: 377-388.
. Arya D, Patni V. Comparative analysis of total flavonoids and quercetin content in vivo and in vitro and enhancement of quercetin via precursor feeding In pluchea lanceolata oliver & hiern. Int J Pharm Pharm Sci. 2013; 5: 617-621.
. Salvador VH, Lima RB, Dantas dos Santos W, Soares AR, Feitoza Böhm PA, Marchiosi, R, Ferrarese ML, Ferrarese-Filh. Cinnamic Acid Increases Lignin Production and Inhibits Soybean Root Growth .PLOS One. 2013; 8: 1-10.
. Ouyang J, Wang XD, Zhao B, Wang YC. Enhanced production of phenylethanoid glycosides by precursor feeding to cell culture of Cistanche deserticola.Process Biochem. 2005; 40: 480-484.
. Vasconsuelo A, Boland R. Molecular aspects of the early stages of elicitation of secondary metabolites in plants. Plant Sci. 2007; 172: 861-875.
. Hahlbrock K, Knobloch KH, Kreuzaler F, Potts JRM, Wellmann E. Coordinated induction and subsequent activity changes of two groups of metabolically interrelated enzymes. Eur J Biochem. 1976; 61: 199–206.
. Bolwell GP, Mavandad M, Miller DJ, Edwards KH, Schuch W. Dixon RA. Inhibition of mRNA levels and activities by trans-cinnamic acid in elicitor-induced bean cells. Phytochemistry. 1988; 27: 2109–2117.
. Ni W, Fahrendorf T, Balance GM, Lamb CJ, Dixon RA. Stress responses in alfalfa (Medicago sativa L.): XX.Transcriptional activation of phenylpropanoid pathway genes in elicitortreated cell suspension cultures. Plant Mol Biol. 1996; 30: 427–438.
. Dixon RA, Paiva NL. Stress-Induced Phenylpropanoid Metabolism. Plant cell. 1995; 7:1085-1097.
. Boudet AM. Evolution and current status of research in phenolic compounds. Phytochem. 2007; 68: 2722-2735.
. Ibrahim MH, Jaafar HZE. Involvement of carbohydrate protein and Phenylalanine ammonia lyase in upregulation of secondary metabolites in Labisia pumila under various CO2 and N2 levels. Molecules. 2011; 16: 4172-4190.
. Blount JW, Korth KL, Masoud SA, Rasmussen S, Lamb C, Dixon RA. Altering Expression of Cinnamic Acid 4-Hydroxylase in Transgenic Plants Provides Evidence for a Feedback Loop at the Entry Point into the Phenylpropanoid Pathway. Plant Physiol. 2000; 122:107–116.
. Lamb CJ. Regulation of enzyme levels in phenylpropanoid biosynthesis: characterization of the modulation by light and pathway intermediates. Arch Biochem Biophys. 1979; 192: 311–317.
. Mavandad M, Edwards R, Liang X, Lamb, CJ, Dixon RA. Effects of trans-cinnamic acid on expression of the bean phenylalanine ammonia- lyase gene family. Plant Physiol. 1990; 94: 671–680.
. Gerrish C, Robbins MP, Dixon RA. Trans-cinnamic acid as a modulator of chalcone isomerase in bean cell suspension cultures. Plant science. 1985; 38: 23-27.
. Ye SF, Zhou YH, Sun Y, Zou LY, Yu JQ. Cinnamic acid causes oxidative stress in cucumber roots, and promotes incidence of Fusarium wilt. Environ Exp Bot. 2006; 56: 255-262.
. Ding J, Sun Y, Xiao CL, Shi K, Zhou YH. Physiological basis of different allelopathic reactions of cucumber and fig leaf gourd plants to cinnamic acid. J Exp Bot. 2007; 58: 3765-3773.
. Yu JQ, Sun Y, Zhang Y, Ding J, Xia XJ. Selective trans-cinnamic acid uptake impairs [Ca2+] cyt homeostasis and growth in Cucumis sativus L. J Chem Ecol. 2009; 35: 1471-1477.