STUDY OF THE POLYPHENOLIC COMPOUNDS ACCUMULATION IN THYMUS SERPYLLUM L. HERB DURING THE FLOWERING PERIOD

compounds in Thymus serpyllum L. herbal raw material during the flowering period by Thin Layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC) methods

Herbal raw materials, phytopreparations and biologically active food additives of them are widely used in modern medicine for the treatment and prevention of many common human diseases. At our time, according to the message of World Health Organization, in Europe and the USA up to 35% of drugs are obtained from medicinal plants. In the next twenty years, there will be a tendency for their increase up to 60% [1,2,3].
It is important to note, that wild species of the Thymus L. genus, growing in natural steppe and forest-steppe biocenoses of Ukraine flora, have a powerful plant raw material base with hight cultivation potential for obtaining modern highly effective phytopreparations. At the same time, side toxic effects are not observed during long-term use in modern phytotherapy. They are number up to 200 species, of which only up to 50 have been found in the modern Ukraine flora [4,5].
The well-known wild species of the Thymus L. genus (Thyme) are well distributed and constantly found in the natural biocenoses of many world countries with moderate climatic conditions as a source for obtaining highly effective medicinal plant raw materials, phytopreparations and biologically active additives with pronounced antimicrobial, antiinflammatory and antioxidant activity [6,7,8] The most widespread wild species in steppe and forest-steppe biocenoses of Europian countries and Ukraine is Thymus serpyllum L. This is promising for the procurement of herbal raw materials, cultivatiоn in the conditions of specialized farms. Morphologically and phylogenetically closely related to it are the widespread species Thymus marschallianus Willd., Th. pallasianus H. Braun., Th. ovatus Mill., hybrids that have a similar chemical composition and are also widely used in modern folk medicine [1][2][3][4][5][6][7][8].
In European countries, the best-known related species of the genus are: The State Pharmacopoeia of Ukraine includes the Thymus serpyllum L. herb and a mixture of the Thymus vulgaris L. with Th. zygis L. herbs without distinctive diagnostic features of the these species herbal raw material [13].
The study of the chemical composition of Thymus serpyllum L. herbs from different countries of the world during the flowering period the presence and accumulation of essential oils, polysaccharides, tannins, fatty oils, vitamins, triterpene saponins, inorganic elements were established [14,15,16].
It is generally known that essential oils and liquid alcohol extracts from Thymus serpyllum L. herb are part of modern phytopreparations with pronounced antimicrobial, anti-inflammatory and antioxidant activity [17,18].
Pharmacological studies have established that the pronounced biological activity of the aqueous infusion (1:10) and extractable phytopreparations obtained from Thymus serpyllum L. herb is largely determined by the presence of flavonoids, hydroxycinnamic acids and tannins [19,20,21,22,23].
But at the same time, almost no studies have been conducted on the presence and content of biologically active polyphenolic compounds from the groups of flavonoids and hydroxycinnamic acids in the Thymus serpyllum L. herb during the flowering period. This is evidenced by the limited scientific information on studies of polyphenolic compounds presence in plant raw materials of Thymus L. genus species by Thin Layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC) methods.
The distribution of the wild species Thymus serpyllum L. in Ukraine, the possibility of cultivation and obtaining phytopreparations with pronounced anti-inflammatory, antimicrobial and antioxidant activity provides the prospect of standartization of herbal raw materials according to the content of polyphenolic compounds.
Aim are determination the qualitative composition and quantitative content of biologically active polyphenolic compounds in Thymus serpyllum L. herbal raw material during the flowering period by Thin Layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC) methods.

M a t e r i a l s a n d m e t h o d s
The object of research was the Thymus serpyllum L. herbal raw material, which obtained from wild plants in v. Baburka, Zaporizhzhia region (June-August 2022). It consisted of flowering upper shoots with inflorescences up to 15 cm long, individual leaves and parts of twigs (no more than 2%) and fully met the requirements of the State Pharmacopoeia of Ukraine (SPhU) [24].
The harvesting of the Thymus serpyllum L. herbal raw material was carried out according to generally accepted methods. The drying process was carried out for 24 hours in the Termolab SNOL 24/350 device at a temperature of 35 °С, up to the final water content no more than 10%.
The combination of TLC and HPLC metodsallows simultaneous separation of the studied components, their identification and quantitative content determination. At the same time, the essential advantages are the use of small samples, speed of research, good reproducibility of results and small relative error of analyses.
Methodology: about 1,0 g (exact weight) of herbal raw material was crushed to a particle diameter (d = 0.3 mm), placed in a flask with a capacity of 100 ml, 30 ml of 96% ethanol was added, and heated on the VB-4 Micromed device (t = 100 °C) for 30 min with thorough mixing. The process was repeated two more times with new volume portions of the 96% ethanol. Extracts were combined, cooled for 30 min, centrifuged on the SM-3.01. Micromed device, filtered into a flask with a capacity of 100 ml and brought to the mark. Samples were filtered through a teflon membrane filter (d = 0.45 μm) into an analysis vials. The chromatographic separation and determination of individual polyphenolic compounds were carried out in selected solvent systems.
The mobile phase was: trifluoroacetic acid 0.2%, mixture of trifluoroacetic acid 0.2% with anhydrous methanol and a mixture of trifluoroacetic acid 0.2% with methanol 70%.
The rate of supply of the mobile phase was 0.25 ml/min; the working pressure of the eluent was from 240 to 300 kPa; the temperature of the column thermostat is 32 °C; sample volume 5 μl. Definition parameters: measurement scale 1.0; scanning time 0.5 sec; λ = 190-600 nm.
The methods of standard additions samples 96 ethanol solutions of flavonoids and hydroxycinnamic acids were used.
The identification and quantitative content of polyphenolic compounds bythe HPLC method were determined by the following parameters: retention time, spectral characteristics, peaks areas of compounds in comparision with standard samples.
Statistical processing of the results was carried out using the standard statistical package of the license program «STATISTICA ® for Windows 6.1» (Stat Soft Inc., № AXXR712D833214FAN5), аs well as «SPSS 16.0» [25].

R e s u l t s a n d d i s c u s s i o n
The qualitative component composition and quantitative content of compounds in the 96% ethanol extract (1:100) from Thymus serpyllum L. herb were determined by TLC and HPLC methods. The resence of 25 compounds of the polyphenolic structure were established.
T  Up to 25 polyphenolic compounds during the flowering period were identified in the Thymus serpyllum L. herb by TLC and HPLC methods, of which 13 were assigned to flavonoids and 12 to hydroxycinnamic acids.
The obtained results indicate for perspective of further research of Thymus serpyllum L. herbal raw materialare able is the pronounced effective anti-inflammatory, antimicrobial and antioxidant activities.
C o n c l u s i o n s 1. The Thymus serpyllum L. herb was studied for the polyphenolic compounds content during flowering period. Up to 25 polyphenolic compounds, classified as 13 flavonoids and 12 hydroxycinnamic acids, were identifiedin alcoholic extracts (1:100) by TLC and HPLC methods. Most of them are well known for their pronounced anti-inflammatory, antimicrobial and antioxidant activities.
2. The simultaneous use of TLC and HPLC methods is promising for the identifications and determinations of the polyphenolic compounds quantitative content in herbal raw materials of Thymus L. species.
3. According to the content of biologically active compounds, the Thymus serpyllum L. species is promising for cultivation in Ukraine for the purpose of obtaining herbal raw materials and new complex phytopreparations.