Investigation of the composition of flavonoids of horse chestnut flowers in various growing regions
Abstract
Horse chestnut (Aesculus hippocastanum L.), a plant of the horse chestnut (Aesculus L.) species from the horse-chestnut family (Hippocastanaceae A.Rich.), is a promising medicinal plant material (MPM), widely used for various vascular diseases in official and traditional medicine in many countries. According to previous studies, its flowers are a promising source of biologically active substances (BAS), in particular substances with a flavonoid nature. Due to the high set of horse chestnut, the collection of flowers does not affect the procurement of seeds, the main pharmacopeial raw material. This fact contributes to the relevance of further research of this MPM. The goal of the work was to separate and comparatively study the flavonoids of horse chestnut flowers from different regions of growth using TLC.
During the study, we established the range of variation of the quantitative content of this group of BAS in the raw materials (in terms of rutin) using the method of differential spectrophotometry (from 3.30 to 4.55%). Optimal chromatography conditions were also found: 10 μl of the extract were applied to a Silica gel 60 F254 plate sized 10×15 cm; eluent: ethyl acetate – glacial acetic acid – water (5: 1: 1); detecting agent: 5% alcoholic solution of aluminium chloride or 5% alcoholic solution of sodium hydroxide under UV light (365 nm). At the same time, 5 μl of 0.1% standard solutions of rutin, quercetin, and apigenin were chromatographed. To analyse the chromatograms, visual assessment and processing methods were applied using the Sorbfil Densitometer software. In these conditions, it was possible to achieve optimal separation of 14 zones of flavonoid BAS, among which we identified rutin, quercetin, and apigenin. The obtained data showed that when flowers were harvested from the horse chestnut growing in different ecological and geographical conditions, the qualitative composition of flavonoids did not change, although it differed significantly in the quantitative content of individual components in total, which could be traced by the size of individual zones and the intensity of their fluorescence. The selected TLC method for separating flavonoids and identifying the studied raw materials by the type of chromatographic profile showed the reproducibility of the Rf values of flavonoid zones on the tracks of extracts from flowers harvested in different regions of the Russian Federation.
The research objects were studied by IR spectroscopy using the VERTEX 70 FTIR spectrometer with an ATR unit and subsequently processed with the OMNIC or GRAMS 4/32 software.
Regardless of the region of procurement of horse chestnut flowers, the IR spectra had similar areas of absorption bands by position, which indicated the reproducibility of the spectrum type. We found no relationship between the type of IR spectrum of flowers and the intensity of individual absorption bands with the presence of certain flavonoids in the raw materials.
Based on the conducted studies, unified criteria can be recommended for assessing the quality of this raw material for the future project of the “Common Horse Chestnut Flowers” pharmacopeial item: according to the “Quantitative determination” indicator of the amount of flavonoids in terms of rutin was not less than 2%. “Determination of the main groups of biologically active substances. Thin-layer chromatography”: the chromatogram of the tested solution should show greenish-blue adsorption zones at the level of the adsorption zones of standard samples of rutin and quercetin; other adsorption zones of flavonoids can also be detected.
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References
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