Study of the flavonoid profile of sea buckthorn fruits of various varieties by thin layer chromatography
Abstract
Flavonoids are essential components of biologically active substances (BAS). One of the richest
sources of flavonoids is the fruit of sea buckthorn (Hippophae rhamnoides L.) Previous studies determined
that sea buckthorn fruit contains such flavonoids as quercetin, kaempferol, hesperidin, rutin, hyperoside,
isorhamnetin, myricetin, citrin, catechin, etc. The standardisation of this plant material is currently based on the content of carotenoids, while little attention has been paid to the polyphenolic compounds in it. All sea buckthorn varieties are considered acceptable during the procurement process. However, numerous studies have demonstrated that the content of biologically active substances in these varieties is not the same. It is therefore important to develop algorithms for express identification of sea buckthorn fruit varieties using thin-layer chromatography (TLC) fingerprint profiles. The aim of this paper was to study the composition of various groups of flavonoids in varieties of sea buckthorn by means of thin-layer chromatography (TLC). In our study we used the dried fruit of several varieties of sea buckthorn (Stolichnaya, Galerit, Ryabinovaya, Botanicheskaya lyubitelskaya, Botanicheskaya, Trofimovskaya, Studencheskaya, Botanicheskaya aromatnaya, Krasnokarminovaya, and Nivelena) harvested at the Botanical garden of the Faculty of Biology of Lomonosov Moscow State University in September 2018, in accordance with the standards for the procurement of medicinal plant materials of this morphological group. The extraction of flavonoids was performed by heating accurately weighed quantities of dried and grounded raw material with 70% ethanol at the ratio of 1:10. 10×10 cm Silica gel 60 F254 aluminum-backed TLC plates (Germany) were used in the experiment. The eluent was ethyl acetate - glacial acetic acid - water system (7.5:1.5:1.5). A 10% NaOH alcoholic solution was used to identify the obtained zones in visible and ultraviolet light. The TLC of the extract of dried sea buckthorn fruit of the Galerit variety showed 9 fluorescent chromatographic zones against a dark background when observed under UV light at 365 nm. After the plate was developed it showed 11 zones when observed under UV light. The optimal volume of the sample was 7 mcl of the extract. The fruit of the studied varieties contained rutin (0.54±0.02) and quercetin (0.78±0.02). The unidentified zones were also fluorescent under UV light, so they may also be flavonoids. The obtained data was used to calculate the parameters of the efficiency of the chromatographic process in a thin layer of sorbent. These parameters include values R¦ and Rs,
the distribution coefficient (К), and the selectivity coefficient (L). The applied eluent system yielded satisfactory separation of chromotographic zones of flavonoids, as the selectivity coefficient is above 1. The largest number of separated zones was observed in the Ryabinovaya variety (13 zones) and the smallest in the Stolichnaya and Trofimovskaya varieties (10 zones). Interestingly, zones with values of Rf=0.20±0.02,
0.25±0.01, 0.42±0.02, 0.86±0.02, 0.95±0.01, and 0.98±0.01 were present in all the studied varieties, and thus
can be characterised as marker zones for the whole species. The optimal conditions for chromatographic separation of flavonoid zones in the fruit of sea buckthorn were experimentally determined and theoretically substantiated. For the first time, the TLC profiles of flavonoids of 10 studied varieties of sea buckthorn were obtained and partly identified. The presence or absence of unique marker zones was determined for the studied varieties: Ryabinovaya, Botanicheskaya aromatnaya, Stolichnaya, Trofimovskaya, Nivelena, and Botanicheskaya. The TLC profile of flavonoids of sea buckthorn fruit allows for approximate identification of the variety by means of the fingerprint method. However, highly accurate methods, such as HPLC-MS, should be used for precise identification of the variety.
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