Determination of phenolic acids and phenylpropanoids in propolis

The phenolic acids and phenylpropanoids have an important biological activity and are therapeutic agents of crude drugs. Development of validated analysis techniques of these phytotherapeutic agents (fingerprinting and assay procedures) is an important practice for efficacy, safety and quality control of herbal drug preparations. 18 samples of propolis were collected from different districts of Lithuania. The aim of the present work was to study chemical composition of collected samples and estimate analytical capabilities of the evaluation of selected phenolic acids and phenylpropanoids: caffeic acid, chlorogenic acid, cinnamic acid, coumaric acid, ferulic acid, gallic acid, protocatechuic (3,4-dihydroxybenzoic) acid, rosmarinic acid, vanillic acid and vanillin. Optimization and validation procedures of rapid and simple method of reversed phase high performance liquid chromatography were carried out. The mobile phase of the optimized chromatographic method consisted of methanol and 0.5 per cent acetic acid solvent in water. Under the optimal separation conditions (described in Methods), analysis was done in 27 min, with the retention times 4.04 min (gallic acid), 6.93 min (protocatechuic acid), 11.30 min (chlorogenic acid), 12.26 min (vanillic acid), 12.93 min (caffeic acid), 13.80 min (vanillin), 16.97 min (coumaric acid), 17.95 min (ferulic acid), 22.29 min (rosmarinic acid) and 26.30 min (cinnamic acid). The limits of detection and the limits of quantitation were between 0.004���0.021 and 0.014-0.07 μg/ml, respectively (Table 3). Repeatability was better than 2.6 per cent for amount. The within-day and between-day accuracy was also measured in range 95.1-106.4 per cent. Coumaric acid (4,1 – 13,27 mg/g) and ferulic acid (3,53 – 10,53 mg/g) are the dominating phenolic acids of propolis. Chlorogenic acid (0,01 – 0,22 mg/g) and gallic acid (0,09 – 0,36 mg/g) were determinated at the lowest amounts. The largest amounts of phenolic acids and phenylpropanoids were determinated in propolis from Šilalė district (total amount - 37,09 mg/g), also Kelmė district (34,75 mg/g), Klaipėda district (31,91 mg/g) and Trakai district (30,44 mg/g). The lowest amounts were determinated in propolis from Švenčioniai district (12,24 – 13,3 mg/g), Anykščiai district (15,08 mg/g), Jurbarkas district (17,04 mg/g) and Varėna district (17,98 mg/g). Recommendation would be collect propolis in Šilalė region, Kreiviai village (1,48 proc.), Kelmė region (1,39 proc.) and Klaipėda region (1,28 proc.) if the largest amounts of phenolic acids and phenylpropanoids is necessary