CHEMICAL STUDY OF FLAVONS AND FLAVONOLS COMPOSITION IN PROPOLIS

Настоящая статья посвящена сравнительной оценке состава флавонов и флавонолов в различных образцах прополиса для установления возможности его адекватной стандартизации. Материалы и методы. В качестве объектов исследования взяты 6 опытных образцов прополиса из разных регионов России. Из опытных образцов готовили извлечения спиртом этиловым 80%-ным по традиционной схеме изготовления настоек в соотношении 1:10. Полученные извлечения фильтровали и использовали для непосредственного анализа. Хроматографическое разделение спиртовых извлечений прополиса проводили на жидкостном хроматографе фирмы «Agilent Technologies 1200 Infinity», США. Детекцию проводили с помощью диодно-матричного детектора Agilent 1200. Результаты и обсуждение. Используя обращенно-фазную высокоэффективную жидкостную хроматографию в градиентном режиме элюирования, удалось идентифицировать флавоны и флавонолы. Выявлено, что состав прополиса имеет стабильный состав флавонов и флавонолов, включающий такие компоненты как кверцетин, изорамнетин, 3,4’-диметоксикемпферол, рамнетин, пендулетин, кемпферол, рамноцитрин, галангин, кемпферид, хризин и метоксигалангин. Из найденных компонентов наибольшее содержание приходится на флавонолы, метоксильные производные кемпферола – рамноцитрин (22,0%) и кемферид (12,0%), из флавонов – хризин (16,0%). Методом внутренней нормализации рассчитан удельный вес каждого из компонентов внутри указанной группы. Установлено, что около 84% всех флавонолов приходится на кемпферол и его метоксильные производные. Состав флавонов и флавонолов может варьировать в зависимости от образца. При этом во всех изученных объектах присутствовал кемпферол, тогда как в отдельных пробах прополиса отсутствовали отдельные из идентифицированных компонентов. Предложена стандартизация прополиса методом высокоэффективной жидкостной хроматографии по содержанию флавоноидов в пересчёте на кемпферол, как стабильный, коммерчески наиболее доступный компонент прополиса. Используя абсолютную градуировку, определено количественное содержание кемпферола в опытных образцах прополиса, которое находилось в диапазоне 0,0141–0,0159%. Заключение. Результаты проведённых экспериментов позволяют рекомендовать проводить оценку качества прополиса по содержанию кемпферола в опытных образцах. Ключевые слова: прополис, флавонолы, обращенно-фазная высокоэффективная жидкостная хроматография, метод абсолютной градуировки

Мобильные фазы готовили, используя воду сверхчистую (для жидкостной хроматографии) и the research, 6 experimental samples of propolis were taken from different regions of Russia.Using those samples, we prepared the extracts with 80% ethanol according to traditional scheme of making tinctures in the ratio of 1:10.After that our extracts were filtered and used directly in the assessment.Chromatographic separation of spirit extracts of propolis was carried out on a liquid chromatograph of "Agilent Technologies 1200 Infinity", USA.The detection was carried out on the basis of the diode array detector "Agilent 1200".Results and discussion.Using the reversed-phase HPLC in gradient elution regime we managed to identify flavonols and flavones.It was found out that the composition of propolis has a stable composition of flavones and flavonols including quercetin, isoramnetin, 3,4'-dimethoxycempferol, ramnetin, penduletin, kaempferol, ramnocitrin, galangin, kaempherid, chrysin and methoxyhalangin.Among the identified components, the highest content is in flavonols, methoxyl derivatives ramnocitrin (22,0%), and kaempherid (12,0%); in flavones it is chrysin (16,0%).The specific gravity of each component within the specified group was calculated by the internal normalization method.It was established that about 84% of all flavonols are in kaempferol and its methoxyl derivatives.The composition of flavones and flavonols can vary depending on the sample.Hereby, kempferol was identified in all the studied samples, whereas some of the identified components were absent from separate propolis samples.Propolis standardization by method of high-performance liquid chromatography in respect of the content of flavonoids in terms of kaempferol as a stable, commercially most available component of propolis was suggested.With the use of absolute calibration, the quantitative content of kaempferol in propolis samples was determined in the range of 0.0141-0.0159%.Conclusion.The results of the carried out experiments made it possible to recommend the quality assessment of propolis according to the content of kaempferol in the experimental samples.

INTRODUCTION.
Propolis is a sticky resinous substance collected by bees from treekidneys and modified by their enzymes [1].Propolis can have different colors from dark green to yellow and red; its taste is bitter and peppery, the odour is resinous [2].According to its physicochemical properties, propolis is a unique natural product, the composition of which includes more than 800 substances [3].Propolis includes a range of valuable biologically active components, and therefore has a variety of pharmacological activities.Primarily propolis is characterized by strong antibacterial, anti-inflammatory, immunomodulating and antitumoral properties [4].Propolis contains a number of microelements, nitrogen compounds -amides, amines, amino acids.In trace amounts, vitamins are found.However, polyphenols represented by flavonoids, phenolic acids and oxycinnamic acids are of the greatest pharmacological value.Flavonoids of propolis have a variety of pharmacological activities: choleretic, diuretic, capillary-strengthening and anti-inflammatory effects [3,5,6,7,8,9].Among all the propolis flavonoids it's possible to extract the flavonols, which are the most wide-spread natural representatives of flavonoids and have a broad therapeutic effect [10,11].In particular, quercetin has an anti-inflammatory effect, reduces risk factors for a cardiovascular disease and is positioned as an agent that suppresses atherogenesis [12].Isoramnetin protects cardiomyocytes from active forms of oxygen, suppressing apoptosis, has anti-carcinogenic properties, inducing apoptosis of myeloid erythroleukemia cells [13].3,4'-dimethoxycempferol, ramnetin, andpenduletin show anti-inflammatory activity [14].Galangin is able to suppress the factors determining the resistance of bacteria to antibiotics [15].Kaempferol starts the process of apoptosis of ovarian cancer cells, has strong antioxidant properties, which are manifested in the suppression of oxidation stress [16,17].It is assumed that kaempferol has anti-inflammatory and anti-allergic action, prevents the development of atherosclerosis [18,19,20].It also has a neuroprotective effect, preventing the development of Parkinson's disease [21].Kaempferol prevents the destruction of pancreatic cells caused by chronic hyperglycemia [22].In experiments in vivo, kaempferol is able to regulate the differentiation of proadipocytes, which is manifested in a decrease in the growth of adipose tissue [23].
Since the composition of propolis flavonols varies considerably in different literary sources, and the problem according to what group of substances to standardize this object remains unsolved, the AIM of this study was an objective comparative assessment of flavones and flavanols composition in various propolis samples in order to establish the possibility of its adequate standardization in these groups of substances.
MATERIALS AND METHODS.To carry out the research, 6 experimental samples of propolis were taken from different regions of Russia.Using those samples, Фармация и фармакология Т. 6 № 3, 2018

Фармацевтическая и токсикологическая химия Pharmaceutical and toxicological chemistry
we prepared the extracts with 80% ethanol according to traditional scheme of making tinctures in the ratio of 1:10.The obtained extracts were filtered and used directly in the assessment.
Chromatographic separation of propolis spirit extracts was carried out on a liquid chromatograph of "Agilent Technologies 1200 Infinity", USA.The detection was carried out on the basis of the diode array detector "Agilent 1200".
The mobile phases were prepared with the use of ultra-pure water (for liquid chromatography) and ethyl alcohol (according to GOST R 51652).As an acid modifier, formic acid was used (qualificationchemically pure).
The number of theoretical plates characterizing the efficiency of the column [24] was calculated according to Formula 1: (1 where t is retention time for the analyzed component; µ 0.5 is a peak width at the half-height, mm.As the optimal criterion, the value not less than 5000 was taken.
The peak separation coefficient R s was calculated according to Formula 2: (2) Where Δl is the distance between the peaks of two adjacent peaks, mm; µ 0.5(1) , µ 0.5 (2) is the peak width at the half-height of the two components, mm.Separation coefficient R s should not be less than 1,5.
The peak asymmetry coefficient (Т f ) is calculated according to Formula 3: (3) where µ 0,05 is the peak width at 5,0% height above the base line, mm; ƒ is the distance from the beginning of the peak at 5.0% height above the baseline to the perpendicular drawn from its apex, mm.
The asymmetry coefficient Т f should be not less than 2.
The components were identified by the retention time, by comparison with the corresponding standard samples and by the results of the detection.The relative composition of individual components in sum was determined using of the internal normalization (Formula 4): (4) where Si is the average area of the analyte peak on the chromatograms of the sum; ∑S is the average value of the sum of peak areas on chromatograms.

RESULTS AND DISCUSSION.
In the course of chromatographic separation it was found out that 12 components of flavone and flavonol nature are present in propolis.The chromatogram of separation of spirit extraction from propolis, recorded at λ = 375 nm of diode-matrix detection, is shown in Fig. 1.
As seen in Figure 1, most of the components present on the chromatogram are separated along the baseline.That indicates good selectivity of the chromatographic conditions used.
The efficiency of the chromatographic system has been confirmed by calculating suitability criteria.The results are shown in Table 2.

Fig. 1 -Chromatogram of propolis registered atλ = 375 nm with diode-matrix detection
The results of calculating suitability criteria presented in Table 2 (N> 5000, Rs> 1.5, Tf<2) generally correspond to the referenced values.Consequently, the present chromatographic system can be recognized as effective for the determination of flavones and flavonols in propolis.
The peak areas of flavones and flavonols obtained during chromatography were used to calculate the relative content of each component by the internal normalization method.
The results of the detection of components of flavones and flavonols and their composition in sum are given in Table 3.The percentage distribution of flavones and flavonols within the group is shown in Figure 2.
However, the composition of flavones and flavonols can vary depending on the sample.Hereby, kempferol was identified in all the studied samples, whereas some of the identified components were absent from separate propolis samples.Therefore, we proposed the standardization of propolis according to the composition of flavonoids in terms of kaempferol as a stable and commercially most available component of propolis.For the quantitative determination of flavonols in propolis, the method of absolute calibration in terms of kaempferol was used.For this purpose, a calibration curve prepared from a calibration standard sample of kaempferol was prepared in advance.The range of concentrations of calibration solutions was 0.004-0.2%.
A calibration graph showing the peak area (S) as a function of the concentration of the inserted substance (C%) is shown in Figure 3.

Fig. 3 -Calibration graphic of peak area versus concentration of kaempferol
In the indicated range of concentrations, the calibration dependence had a rectilinear dependence and the equation had the form: y = 33267x R 2 , a correlation coefficient, equals to 0,999.That shows the linear dependence between the concentration of the standard sample of kaempferol and its peak square.
The results of the calculation of the kaempferol composition in propolis different test samples are shown in Table 4.
Фармация и фармакология Т. 6 № 3, 2018 252 The data presented in Table 4 indicates that the content of kaempferol in the pilot propolis samples was in the range 0.0141-0.0159%.

Фармацевтическая и токсикологическая химия Pharmaceutical and toxicological chemistry
Propolis is a concentrate of polyphenolic compounds, due to which it has a wide spectrum of biological activity.The carriers of propolis pharmacological properties are primarily flavonoids, represented exclusively by aglikones.
According to the data available in the literature and in the course of the research of our own, it has been established that in propolis aglikones occur in the range of 10-15, which significantly complicates the standardization of this object.However, there are flavonoid aglycons, which are present in almost all propolis samples in major concentrations.Such a basic flavonoid is primarily kaempferol.The economic availability and chemical stability of this component makes it a convenient means for standardizing propolis.These reasons allowed us to propose a novel standardization of propolis according to the composition of kaempferol.The developed technique with the use of absolute calibration has linearity, reproducibility and can be recommended as one of the options for standardization of propolis.
CONCLUSION.Thus, in the course of the carried out research, it has been established that the composition of the propolis samples is characterized by a fairly stable, same-type set of flavonols, including: quercetin, isoramnetin, ramnetin, penduletin, kempferol derivatives, galangin, methoxyhalangin.All the components are well separated under the conditions of gradient chromatography.The dominant propolis flavonols are ramnocitrine, kaempferol, camphoride, the derivatives of caffefer occupy an average of about 84% of the total amount of flavonols.By method of absolute calibration, it was possible to estimate the quantitative composition of kaempferol in the investigated propolis samples.The reproducible composition of flavnols in propolis, their good chromatographic behavior under RP HPLC conditions allow us to recommend the standardization of propolis in terms of kaempferol.

Table 4 -
The content of kaempferol in propolis