Study of the Composition of Biologically Active Substances of Horse Chestnut Flowers by TLC

Abstract

Currently, chromatographic methods, in particular thin-layer chromatography (TLC), are one of the main tools for the pharmacopoeial analysis of medicinal plant materials (MPM). Their key advantage is their high selectivity, which allows for the reliable identification and separation of individual compounds in complex plant compositions of biologically active substances (BAS). Horse chestnut belongs to the genus Aesculus in the family Sapindaceae. Its natural habitat is limited to the mountainous regions of the Balkan Peninsula. In Russia, horse chestnut is cultivated in regions with a temperate continental climate. Horse chestnut flowers, according to literary data, exhibit pronounced pharmacological activity, including antioxidant, capillary-protective, venotonic, anti-inflammatory, and antimicrobial effects. The pharmacological activity of horse chestnut flowers is due to a complex of biologically active substances, among which the leading role belongs to polyphenolic compounds and triterpene saponins. Additional contributions are made by phytosterols, organic acids including ascorbic acid, amino acids, polysaccharides, and tannins. In this regard, determining the chromatographic profile of the biologically active substances of chestnut flowers is a pressing task, since the separation and identification of individual groups of biologically active substances by TLC allows us to obtain the data necessary for developing standardized quality assessment criteria. The results of the work may form the basis for a future pharmacopoeial monograph (PM) for this medicinal plant species. The aim of the work was to assess the geographical variability of the accumulation of various groups of biologically active substances (using anthocyanin compounds, flavonoids, amino acids, monosaccharides and saponins as examples) in horse chestnut flowers to substantiate the standard quality indicators for this type of medicinal plant material. As part of the TLC study, it was established that the qualitative composition of the main groups of biologically active compounds (BAC) in horse chestnut flowers does not change depending on the region where the raw material is grown. When analyzing the amino acid composition, ten chromatographic zones were found, of which arginine (R_f 0.10), proline (R_f 0.32), glycine (R_f 0.38), glutamic acid (R_f 0.42), methionine (R_f 0.52), leucine (R_f 0.62), and phenylalanine (R_f 0.74) were identified; three zones with R_f values of 0.15, 0.26, and 0.84 correspond to unidentified amino acid compounds. In the group of anthocyanin pigments, three zones with R_f values of 0.40, 0.60, and 0.77 were detected. Chromatographic analysis of the carbohydrate fraction of the flowers revealed five zones of simple sugars, one of which was identified as glucose (R_f 0.40). The flavonoid complex was represented by fourteen components, among which the presence of rutin (R_f 0.35), quercetin (R_f 0.78), and apigenin (R_f 0.98) have been established. The TLC profile of saponins showed the presence of three zones. The presence of beta-Escin with an R_f value of 0.45 was established. The obtained TLC profiles allowed us to recommend unified criteria for assessing the quality of this raw material and its inclusion in the future project of the Federal Standard "Horse Chestnut Flowers".