Phenolic compounds from Lepidium sativum

Abstract

Lepidium sativum L. (Brassicaceae), garden cress or pepper grass, is the source of valuable biologically active compounds [1]. It is used in folk medicine for cancer, uterine tumors, polyps, and other neoplasms. It is used in medicine as the alcoholic tincture, which possesses sedative and anticonvulsive activity [2]. Herein we communicate results from investigations of phenolic compounds isolated from L. sativum seeds grown in culture by us. Ground air-dried raw material (seeds, 1.0 g) was extracted successively with hexane, CCl4, and CHCl3 (20 mL each), discarding the produced extracts. Then, the remaining raw material was extracted with boiling ethanol (70%) for 1 h. The extract was cooled, filtered through paper into a volumetric flask (100-mL), and adjusted to the mark with ethanol (70%). The resulting extract (2 mL) was placed into a volumetric flask (25-mL) and adjusted with the same solvent to the mark (stock solution). A series of reference solutions in ethanol (70%) was prepared in parallel. These included rutin, quercetin, luteolin, luteolin-7-glycoside, hyperoside, hesperidin, apigenin, vicenin, vitexin, 4-hydroxycoumarin, scopoletin, robinin, dicoumarin, coumarin, umbelliferone, dihydroquercetin, catechin, orientin, and gallic, chlorogenic, neochlorogenic, cinnamic, caffeic, ferulic, salicylic, and ellagic acids. The stock and reference solutions (20 μL) were injected into a chromatograph. The qualitative composition of the phenolic compounds from L. sativum seeds was studied on a Gilston Model 305 (France) high-performance liquid chromatograph (HPLC) with a Rheodyne 7125 (USA) manual injector with subsequent computer processing of the results using the program Multichrom for Windows. The mobile phase was CH3OH:H2O:H3PO4 (400:600:5). The analysis was performed at room temperature. The eluent flow rate was 0.5 mL/min; analysis time, 120 min. Detection used a Gilston Model 151 UV/Vis UV detector operating at 254 nm. A total of 12 compounds was isolated by the HPLC analysis of L. sativum seeds. Of these, gallic acid (9.44%, content in the isolated mixture by internal normalization method), chlorogenic acid (14.77%), ferulic acid (5.63%), neochlorogenic acid (2.22%), luteolin-7-glycoside (14.67%), dihydroquercetin (4.37%), and quercetin (3.15%) were identified. Quantitative analysis of phenolic compounds from L. sativum seeds was based on the UV absorption spectra of this group of compounds. The absorption spectra were defined using the extract (70% ethanol) of seeds and a Helios (USA) selfrecording spectrophotometer in the wavelength range 210-400 nm. According to preliminary results on the identification by HPLC of phenolic compounds from L. sativum seeds, chlorogenic acid was the predominant component. Therefore, the absorption spectrum of chlorogenic acid in ethanol (70%) was recorded in parallel as a working standard in the same wavelength range. Absorption spectra of the studied solution and the chlorogenic acid solution had a common absorption maximum at 329 ± 2 nm. This enabled us to calculate the total phenolic compounds in L. sativum seeds using chlorogenic acid as a working standard. The studied solution was prepared by the method given above (weight 1.0 g). The working standard (chlorogenic acid, 0.02 g) was placed in a volumetric flask (50-mL), treated with ethanol (70%, 30 mL), stirred until dissolved, and adjusted to the mark (solution A). Solution A (1 mL) was placed in a volumetric flask (50-mL) and adjusted with the same solvent to the mark (working standard). The optical density of the resulting solutions was measured at 329 nm relative to ethanol (70%). The contents of total phenolic compounds calculated as chlorogenic acid in L. sativum seeds was 0.85%.