Determination of cinnamic acid and 4‐coumaric acid in alfalfa (Medicago sativa L.) cell suspension cultures by gas chromatography

Abstract

A capillary gas chromatographic (GC) method for the assay of cinnamic acid and 4-coumaric acid in alfalfa (Medicago satiua L.) cell extracts is described. These compounds were assayed as their trimethylsilyl derivatives; hydrocinnamic acid was used as the internal standard for quantifications. This method was qualitatively validated by GC/mass spectral analysis and quantitatively validated by measuring the extraction recovery from alfalfa cells, the recovery from derivatization, the stability of silylated derivatives, the limits of detection, the linearity of the detector response to increasing amounts of analytes, and the intra-and inter-assay precisions. Cinnamic acid and 4-coumaric acid were present in alfalfa cell extracts and were resolved from all other components by this method. As little as 4 pmol of each acid could be quantified, and the detector response was linear over a range covering five orders of magnitude. The cellular concentrations of free cinnamic acid and 4coumaric acid ranged from ca. 5-100 pmol/kg fresh weight tissue.

Stress Responses in Alfalfa (Medicago sativa L.) (XIV. Changes in the Levels of Phenylpropanoid Pathway Intermediates in Relation to Regulation of L-Phenylalanine Ammonia-Lyase in Elicitor-Treated Cell-Suspension Cultures)

“…3A). We have now used a high-resolution capillary GC method that resolves CA and 4CA from a11 components of the plant cell extracts (Orr et al, 1993). Determinations of caffeic and femlic acids by this method are sometimes hampered by cochromatographing material; the levels of these compounds were measured in each experiment in the present paper but are only reported when they were clearly resolved.…”

“…We have designed a high-resolution GC protocol that allows for the unequivocal determination of endogenous free CA and 4CA levels in alfalfa cells (Orr et al, 1993). We now report correlations between the levels of endogenous phenylpropanoid pathway intermediates, PAL transcript levels, and PAL enzymic activity in cells exposed to a number of different experimental manipulations of the flux through the phenylpropanoid pathway.…”

“…Furthermore, exogenous application of CA results in its conversion into many metabolites , and the use of PAL inhibitors may result in reductions in the levels of many phenylpropanoidderived compounds. Most important, the levels of free CA and its biosynthetic derivatives have not been determined in most of the previous studies that have suggested regulation by these compounds, and the methods that have been used in severa1 studies have been found to be unreliable (Orr et al, 1993).…”

“…The silyl derivatives were also hydrolyzed with dilute acid, and the resultant carboxylic acids were methylated with diazomethane and analyzed again by GC with coinjection with authentic methyl cinnamate and methyl 4-coumarate. Finally, GC-MS was used to confirm the nature and purity of the CA and 4CA peaks in separated cell extracts (Orr et al, 1993).…”

Phytochemical and pharmacological potential ofMedicago sativa: A review

“…Carotene (Gupta et al, 1981). Coumarins: myrsellinol, scopoletin, esculetin, 4-coumaric acid (Duke, 1985;El-Khrisy et al, 1994;Orr et al, 1993). Digestive enzymes (Duke, 1985).…”