DEVELOPMENT OF PHENOLIC COMPOUNDS CHROMATOGRAPHIC IDENTIFICATION IN BILBERRY SHOOTS

Анотація

DEVELOPMENT OF PHENOLIC COMPOUNDS CHROMATOGRAPHIC IDENTIFICATION IN BILBERRY SHOOTS

L. V.  Vronska, M. B. Chubka, A. E.  Demyd

SHEI "Ternopil State Medical University by I. Ya. Horbachevsky of MPH Ukraine"

Introduction. Bilberry shoots have hypoglycemic properties and are used for easy forms of diabetes as in the form of plant raw materials and as part of various charges, such as Arfazetyn and others. Shoots of bilberry are considered as herbal substance that contains tannins, although their presence is not associated with mild hypoglycaemic effect. It is known that the bilberry leaves contain tannins, hydroxycinnamic acids, flavonoids, triterpene acids and vitamins. Standardization of bilberry shoots as herbal substances is still going to be important because it is still little studied and in the State Pharmacopoeia of Ukraine there is no corresponding monograph. Manufacturers of bilberry shoots officinal herbal raw material perform their identification by means of a quality reaction on tannins with ammonium iron (III) sulphate, which is a non-specific method. Moreover, taking into consideration the literature data and a various biological activity hydroxycinnamic acids and flavonoids it is necessary to study their composition for raw materials growing within theterritory ofUkraine.

         The objective of our work was investigation of bilberry shoots phenolic compounds with the help of chromatographic methods, development of methodology of their identification, selection of identification markers.

Investigation methods. The qualitative composition of four bilberry shoots samples that were harvested in summer in the following regions: 1 – Transcarpathia, 2 - Ternopil, 3 - Volyn, 4 - Ivano-Frankivsk, and five samples of "Shoots of bilberries" therapeutic agent, produced in PLC "Liktravy", Zhytomyr city was investigated.

The qualitative composition of phenolic compounds was studied by the methods of thin layer and high performance liquid chromatography.

Results and discussion. Bilberry shoots contain chlorogenic acid mostly from phenolcarboxylic acids, the caffeic acid appears on the chromatograms as a zone with weak fluorescence intensity. In both solvent systems three located next to each other flavonoids zones with an average intensity of fluorescence are shown at the top of the chromatogram; the central of these zones is identified as a quercitrin.

Isoquercitrin appears on chromatograms of all samples as a zone of very weak fluorescence and hyperoside – with an average fluorescence. Under a zone of chlorogenic acid in the first solvent system and over a zone of chlorogenic acid in the second solvent system, much more hydrophilous, flavonoid glycoside appears as the most intensive fluorescence area. Another flavonoid with a large intensity of fluorescence appears over rutin zone in both solvent systems. Flavonoids aglycones were not found in methanol extractions of bilberry shoots in the described conditions.

 The flavonoids aglycone content studying of nine bilberry shoots samples allowed to identify aglycones: quercetin as a zone of very intense orange fluorescence and kaempferol as a zone of weak yellow-blue fluorescence. In the described  sample processing conditions there are other fluorescent zones on the TLC chromatogram. Thus, the main bilberry shoots aglycone of flavonoids, which grows within theterritoryofUkraineand is industrially stored up, is quercetin.

The results of TLC about the presence of quercitrin, hyperoside, traces of rutin, as well as chlorogenic and traces of caffeic acid in significant amount in all studied samples of bilberry shoots were confirmed by HPLC analysis. HPLC investigation of bilberry shoots samples after hydrolysis confirms the presence of quercetin as a dominant aglycone and traces of kaempferol.

The difference between Ukrainian samples analysis results and data of numerous authors is obviously caused by not only differences in sample preparation and sensitivity of the methods used, but also indicates the quality different from bilberry shoots phenolic compounds that grow in different conditions.

Therefore, TLC identification method of bilberry shoots was developed for medicinal plants growing inUkraine.

Conclusions.

1. All bilberry shoots analyzed samples contain the rutin, chlorogenic acid, hyperoside, quercitrin, that have been identified with the help of TLC and HPLC methods.
2. TLC identification method of bilberry shoots was developed and these compounds were proposed as identification markers.

1. Caffeic acid and isoquercitrin are contained in the studied samples of raw materials in small quantities, so they aren't recommended as obligatory identification markers.