STUDY OF THE COMPONENT COMPOSITION OF POLYPHENOLES OF THE KURIL TEA PLANT PENTAPHYLLOIDES FRUTICOSA

Курильский чай является популярным растением народной медицины Китая и Монголии. Имеющиеся научные сведения свидетельствуют о наличии у растения выраженных антиоксидантных свойств, сопряжённых с наличием полифенолов. Также у растения установлен ряд других фармакологических эффектов, в частности антибактериальный, фунгицидный, гипогликемический, противовоспалительный, противоязвенный. Однако содержание полифенолов в составе растения сильно варьирует в зависимости от геологической зоны, в которой произрастало растение, что отражается на его терапевтической активности. Поэтому целью настоящего исследования явилось изучение качественного состава полифенолов травы P. fruticosa L. для дальнейшей стандартизации сырья. Материалы и методы. В качестве объекта исследования взят образец травы P. fruticosa L., собранный на территории Алтайского края. Анализ проводился с помощью высокоэффективной жидкостной хроматографии в обращённо-фазном варианте. Результаты и обсуждение. Установлен химический состав растения, который включает 18 соединений полифенольной природы, представленный гликозидами и агликонами флавонолов, проантоцианидинами, оксикоричными кислотами, эллаготаннинами. Методом внутренней нормализации рассчитано относительное содержание каждого компонента в сумме. В результате установлено, что преобладающими являются гликозиды кверцетина. Определён состав агликонов флавонолов после кислотного гидролиза, включающий кверцетин, кемпферол и рамнетин с преобладанием первого. Одним из компонентов кислотного гидролиза оказался цианидин, что является веским доказательством наличия в траве растения проантоцианидинов. Характерно достаточно высокое содержание проантоцианидинов, составившее около 24% от общей суммы полифенолов. Заключение. Полученные результаты позволяют утверждать, что стандартизацию исследуемого объекта можно проводить в пересчёте на кверцетин, как доминирующего компонента суммы полифенолов после предварительного гидролиза. Ключевые слова: курильский чай, высокоэффективная жидкостная хроматография, флавоноиды, проантоцианидины

qualitative composition of polyphenols in P. fruticosa L. for further standardization of its raw materials.Materials and methods.As a research object, a sample of P. fruticosa L. herb collected in the Altai Territory was taken.The analysis was carried out using high-performance liquid chromatography in the reversed-phase version.Results and discussion.The chemical composition of the plant is has been established.It includes 18 compounds of polyphenolic nature, represented by glycosides and aglyconsfl avonols, proanthocyanidins, oxycinnamic acids, ellagatannins.By the method of internal normalization, the relative content of each component in the sum is has been calculated.As a result, glycosides of quercetin have been found to prevail.After acid hydrolysis the composition of aglyconfl avonols was determined.It included quercetin, kaempferol and ramnetin, with predominance of quercetin.One of the components of acid hydrolysis was cyanidine, which is a strong proof of the presence of proanthocyanidins in the plant.A fairly high content of proanthocyanidins amounting to about 24% of the total amount of polyphenols is typical of it.Conclusion.The obtained results make it possible to assert that standardization of the investigated object can be carried out in terms of quercetin as the dominant component of the sum of polyphenols after preliminary hydrolysis.

INTRODUCTION. Pentaphylloides fruticosa L.
(Potentilla fruticosa L.) -Kuril tea -is a plant from the Rosaceae family.The plant used to be referred to as the genus Potentilla, but now it is considered a representative of the genus Pentaphylloides [1].This is a popular plant in folk medicine in China, Tibet, Mongolia.Its name came from the fact that its dried leaves and fl owers were used as tea by the peoples living in the Ural and the Kuril Islands [2,3].In China, this plant is used for diseases of the gastrointestinal tract, as a means to regulate metabolism, menstruation.In Mongolian folk medicine it is used for diarrhea as a hemostatic [4,5,6].This plant, as well as other members of the genus Potentilla, is characterized by a number of important pharmacological properties, such as antioxidant, antimicrobial, antiviral, hypoglycemic, antiinfl ammatory, antitumor and antiultserogennye [4,7,8,9,10,11,12].The current research indicates a signifi cant antioxidant capacity in vitro and protective effect against Escherichia coli at Oxidative stress produced by phenolic components P. fruticosa L., primarily hyperoside, ellagic acid and (+)-catechin have [13,14].A comparative phytochemical study of three types of Potentilla (Potentilla fruticosa, Potentilla glabra and Potentilla parvifolia) revealed the presence of hyperoside, catechin, caffeic and ferulic acid, rutin and ellagic acid in all of these objects.All of these species exhibited antioxidant and antimicrobial properties against Gram-positive bacteria, Pseudomonas aeruginosa and Candida albicans [15].
A signifi cant increase in the antioxidant activity of P. fruticosa L. leaves extracts in combination with green tea polyphenols at the ratio 3:1 respectively has also been established [16].
Chemical and pharmacological studies have shown that polyphenols of several classes, namely fl avonoids, derivatives of quercetin and kaempferol, and tannins are responsible for high antioxidant activity of P. fruticosa L. [17].However, the content of pharmacologically signifi cant components in the plant composition can vary depending on the ecological and cenotic conditions of the growing region.This factor is refl ected in the amount of biologically active compounds in the plant and, as a con-sequence, on its therapeutic activity [18,19,20,21,22,23].Accordingly, the evaluation of the qualitative composition and their quantitative contents in the P. fruticosa L. herb is an actual problem.
In view of the foregoing, THE AIM of this study was to study the qualitative composition of polyphenols in P. fruticosa L. for standardizing its raw materials.The study of this plant has been carried out within the framework of the development of a new scientifi c direction called "Pharmaceutical remake" [24].
MATERIALS AND METHODS.As a research object, a sample of P. fruticosa L. herb collected in the Altai Territory was taken.The polyphenol complex from P. fruticosa L. herb was extracted with 70% ethyl alcohol at the ratio of 1:50 of raw materials to the extractant.The resulting extraction was chromatographed under the following conditions.
The analysis was carried out using high-performance liquid chromatography in the reversed-phase version.
For this purpose the chromatograph produced by «Agilent Technologies 1200 Infi nity» was used.
As the mobile phase, the HPLC qualifi cation water and ethyl alcohol (according to GOST R 51652) were used, acid formic acid served as an acid modifi er.
The calculation of the number of theoretical plates separating the capabilities of the chromatographic system -the separation coeffi cient R s and the asymmetry of the chromatographic peak -the asymmetry coeffi cient -was calculated according to the European Pharmacopoeia.The adequate values of the number of theoretical values are accepted not less than 5000, the separation factor R s should be not more than -1.5, and the asymmetry coeffi cient T f should be less than 2 [25].
The polyphenol complex was chromatographed under the following conditions: the mobile phase speed was 0,5 ml/min.; the column thermostat temperature was +35 о С; the sample volume was 1 μl.
The gradient elution was carried out under the conditions indicated in Table 1.
The component composition was identifi ed by the correspondence of the retention times of the analytes with standard samples, as well as by the results of diode-matrix detection.
The relative content of individual components was determined by the internal normalization method, the calculation was carried out by formula: where Si is the average value of the peak area of the component on the chromatograms of the sum; ∑S is the mean value of the sum of all peak areas on chromatograms.
For the objective assessment of the content of fl avonoids aglucones, the acid hydrolysis of P. fruticosa L polyphenols was carried out.The hydrolysis was carried out with a 2M solution of hydrochloric acid for 60 minutes.The resulting hydrolysate was fi ltered and chromatographed.
RESULTS AND DISCUSSION.The chromatogram of the separation of alcohol extract from P. fruticosa L. herb is shown in Figure 1.

Figure 1 -Chromatograms of alcohol extract from herb P. fruticosa L. (diode-matrix detection, λ = 365 nm)
To assess the effectiveness of the separation of the polyphenols sum in the matched chromatographic system, the eligibility criteria have been calculated, the results of which are presented in Table 2.

is the peak separation factor, T f is an asymmetry coeffi cient, W b is a baseline width
Based on the results obtained in Table 2, the efficiency criteria fi t into the referenced values proposed by the European Pharmacopoeia.Therefore, this chromatographic system can be considered effective.Decoding of the results of diode-matrix detection of P. fruticosa L. polyphenols is refl ected in Figure 2. As follows from the data shown in Figure 2, the polyphenolic composition of P. fruticosa L. herb is represented by catechines, ellagatannins and ellagic acid, fl avonoids, quercetin glycogenides, ramnetin and kaempferol, chlorogenic acid.The percentage distribution of polyphenols in the polyphenolic complex of P. fruticosa L. is shown in Figure 3.

-Percentage distribution of polyphenols in the P. fruticosa L polyphenol complex
As it is seen in the fi gure, the main mass of P. fruticosa L. polyphenols is represented by glycosides of quercetin, catechins and chlorogenic acid.In smaller quantities there are glycosides of kaempferol and ramnetin.
A characteristic feature of the products of acid hydrolysis of P. fruticosa L. polyphenols was that they had a bright red color, indicating the presence of anthocyanins.The hydrolyzate was further chromatographed, chromatographic results are shown in Figures 4 and 5.The results of decoding the results of acid hydrolysis of P. fruticosa L. polyphenols are shown in Figure 6.Thus, aglycones of fl avonols with a signifi cant predominance of quercetin have been found in hydrolysis products.Cyanidin has also been identifi ed, which confi rms the presence of proanthocyanidins in the polyphenols of the plant under study.
In the course of this study, it has been established that Kuril tea contains fl avonoids represented by glycosides of three aglycons -quercetin, kaempferol and ramnetin, with predominance of quercetin.The presence of fl avonoids in the object, as a rule, causes a variety of diverse pharmacological effects, including capillarystrengthening, antioxidant, antitumor.

As a result of chromatography of products of acid hydrolysis of polyphenols of the investigated object
As a result of chromatography of products of acid hydrolysis of the investigated object's polyphenols, it was found out that the plant contains a large number of proanthocyanidins -the groups of compounds characterized by a pronounced antioxidant effect.This, of course, determines the prospects of the plant as a source of this valuable group of substances.In addition, the presence of ellagic acid has been established in the products of hydrolysis, which indicates the nature of tannins of the plant -ellagatannins.Elvolatannins are Фармация и фармакология Т. 6 № 2, 2018 Фармакогнозия, ботаника Pharmacognosy, Botany known as anti-infl ammatory, hemostatic, antitoxic agents.Consequently, the principle of Kuril tea (P.fruticosa L.) is three classes of compounds -fl avonoids, proanthocyanidins and elligotannins.The presence of such a spectrum of valuable polyphenolic components in the plant makes it possible to assert that the studied object seems promising in medical practice.Therefore, it can be recommended for further pharmacological testing and for the development of medicines based on its polyphenolic complex.
CONCLUSION.As a result of the conducted studies it was established that Kuril tea (P.fruticosa L.) contains about 18 polyphenol compounds, represented by glycosides and aglyconsfl avonols, proanthocyanidins, oxycinnamic acids, ellagatannins.About 46% of the total amount of polyphenols are fl avonoids, 24% are proanthocyanidins, 18% are oxycinnamic acids and 7% are ellagatannins.The results obtained make it possible to assert that the standardization of the object under study can be carried out on the sum of polyphenolic compounds in terms of quercetin as the dominant component of the sum of polyphenols after pre-hydrolysis or on prolanthocyanidins.

5 *Figure 6 -
Figure 6 -Composition of products of hydrolysis of P. fruticosa L. polyphenols

Table 2 -Results of calculations of effi ciency criteria of the chromatographic system for determination of P. fruticosa L. polyphenols
where t R is the component retention time, N is the number of theoretical plates, R s