PHYTOCHEMICAL STUDY OF SALVIA GRANDIFLORA AND SALVIA OFFICINALIS LEAVES FOR ESTABLISHING PROSPECTS FOR USE IN MEDICAL AND PHARMACEUTICAL PRACTICE

Сировинна база лікарських рослин України є достатньою тільки для половини фармакопейних видів. Велика частина ЛРС зростає у недостатній кількості та виникає потреба в їх імпортуванні. В умовах імпортозалежності та дефіциту вітчизняної рослинної сировини, пошук нових джерел біологічно активних речовин серед представників флори України є актуальним завданням сучасної фармацевтичної науки. Мета роботи – провести порівняльне фітохімічне дослідження листя S. grandiflora та S. officinalis для встановлення можливості використання нефармакопейного виду у фармацевтичній та медичній практиці. Матеріали та методи. Об’єктоми дослідження було листя S. grandiflora та S. officinalis, яке було заготовлено у ботанічному саду Львівського національного університету імені І. Франка. Дослідження макрота мікроелементного складу у листі S. officinalis та S. grandiflora проводили атомно-емісійним спектрографічним методом. Визначення якісного складу та кількісного вмісту основних груп БАР проводили методом ВЕРХ. Кількісне визначення фенольних сполук також проводили спектрофотометричним методом. Результати. В обох досліджуваних видах виявлено вміст 15 мікрота макроелементів. У листі S. officinalis та S. grandiflora було ідентифіковано 15 амінокислот та 8 сапонінів. Методом ВЕРХ було встановлено якісний склад та кількісний вміст речовин фенольної природи у листі S. officinalis та S. grandiflora (13 та 9 сполук відповідно). Обговорення. Домінуючими мікрота макроелементів у обох видах сировини були силіцій, фосфор, магній, кальцій, натрій та калій. Загальний вміст мікроелементів у листі S. grandiflora у 1,67 раз більший ніж у фармакопейному виді S. officinalis. Домінуючими амінокислотами в листі обох видів є глутамінова кислота, аспарагінова кислота, валін та лейцин. Домінуючими сапонінами у листі S. officinalis були урсолова та олеанолова кислоти, загальний вміст яких становить 75,82 %. У листі S.grandiflora домінуючими були урсолова та еускапова кислоти, загальний вміст яких становить 63,25 %. Загальний вміст флавоноїдів найбільший в листі S. officinalis та становить 4,90 мг/г. Загальний вміст гідроксикоричних кислот найбільший в листі S. grandiflora та становить 4,49 мг/г, що на 221,18 % (в 2,21 рази) більше ніж у фармакопейному виді S. officinalis (2,03мг/г). Загальний найбільший вміст похідних кавової кислоти переважає в листі Salvia officinalis (0,77 мг/г). Найбільший вміст суми всіх виявлених сполук фенольної природи характерний для листя S. officinalis та становить 6,93 мг/г. Висновки. У результаті проведеного порівняльного фітохімічного дослідження листя S. grandiflora та S. officinalis встановлено, що S. grandiflora є перспективним видом для впровадження у медичну та фармацевтичну практику саме як джерело фенольних сполук Ключові слова: рід Шавлія, нефармакопейний вид, листя, хімічний склад, фенольні сполуки


Introduction
The pharmaceutical market for synthetic drugs is showing rapid growth. At the same time, there remains high level of topicality of herbal medicines research. New drugs based on biologically active substances of plant origin are emerging every year [1,2]. The market of medicinal herbal raw materials in Ukraine is rapidly developing, as evidenced by the development and increase in the number of enterprises of all units from the cultivation and harvesting of medicinal products to the production of finished products. However, in today's conditions, the market of medicinal plants of Ukraine is dominated by imported raw materials, which exacerbates the issue of import substitution to ensure the medical safety of the country [3,4].
About 23 % of all pharmacopoeial species of Ukraine have to be imported from abroad for the needs of the domestic pharmaceutical industry. In conditions of import dependence and scarcity of domestic medicinal plant raw materials, the search for new sources of biologically active substances among the representatives of the flora of Ukraine is an urgent task of modern pharmaceutical science [1,5].
Particular attention is drawn to the study of representatives of the Salvia genus in Ukraine [6,7]. It is the largest genus in the Lamiacea family. 21 species grow in Ukraine [8][9][10]. The pharmaceutical market of Ukraine presents about 40 drugs, the components of which have biologically active substances (BAS) of Salvia leaves [11,12].
Previous chemotaxonomic studies of representatives of the Salvia in Ukraine have shown the prospect of using raw materials of S. grandiflora in the pharmaceutical industry [1,9,13]. Therefore, it is advisable to conduct a comparative study of the leaves of this species and the galenic drug on its basis in comparison with the pharmacopoeial species -leaves of S. officinalis [14,15].
The aim of the study was to conduct a comparative phytochemical study of S. grandiflora and S. officinalis leaves to determine the possibility of using nonpharmacopoeial species in pharmaceutical and medical practice.

Planning (methodology) of research
To achieve the aim it was necessary to solve the following problems: to analyse the macro-and micro-elemental, amino acid composition of S. grandiflora and S. officinalis leaves to study the qualitative composition and quantitative content of the main groups of biologically active substances in the leaf of the studied species to carry out a comparative analysis of the obtained results to determine the possibility of using nonpharmacopoeial species in pharmaceutical and medical practice.

Materials and methods
The object of the study was the leaves of S. grandiflora and S. officinalis, which were harvested at the Botanical Garden of Lviv National University named after Ivan Franko, under the guidance of a senior researcher, Ph.D Skibitska M. I. [16,17].
Macro-and micronutrient composition studies in the leaves of S. officinalis and S. verticillata were carried out by atomic emission spectrographic method on a DFS-8 spectrograph at State Scientific Institution "Institute for Single Crystals" of NAS of Ukraine. The AC arc was obtained with the help of the IBC-28 generator. The spectra were recorded on PFS-02 photographic plates [18,19].
The study of the amino acid composition of the plant raw materials of the studied species was performed by HPLC on a chromatograph firm Agilent Technologies (model 1100). For the chromatography we used column AA 200 × 2.1 mm and protective column; as mobile phasesolution A (20 mmM sodium acetate and 0.018 % triethylamine, adjusted to pH 7.2 with 1-2 % acetic acid, with the addition of 0.3 % tetrahydrofuran, and solution B (40 % CH 3 CN, 40 % MeOH and 20 % 100 mM sodium acetate, adjusted to pH 7.2 with 1-2 % acetic acid); volumetric flow rate -0.45 ml / min; column temperature -40 °C; detection was carried out with a UV detector after pre-column dewatering first with o-phthalic aldehyde (OPA reagent) and then with 9-fluorenylchloroformate (FMOC reagent) for for display of proline [20,21]. The identification of amino acids was performed by the retention time of the standards of the corresponding amino acids (TС 6-09-3147-83) [18,22,23].
Study of the saponin composition of the leaves of the studied species was performed by HPLC on a Shimadzu LC20 Prominence chromatograph in a modular system equipped with a four-channel pump LC20AD, thermostat STO20A columns, SIL20A automatic sampler, Bridge CST2020 diode-matrix detector and SPDM20 detector 150 mm * 4.6 mm with a grain size of 5 microns (Waters); column temperature -30 °C; detection wavelength -205 nm; the flow rate of the mobile phase is 1.0 ml / min; the volume of the sample injected is 20 μl. Mobile phase: methanol for HPLC: 0.2 % ammonium acetate solution (pH 6.75) (80:20). Elution mode: isocratic. The identification of the components was carried out by the retention time and the compliance of the UV spectra with the substance standards. The spectra of triterpene saponins have a maximum absorption at (200-210) nm, so detection of this group of compounds was carried out at 205 nm [24,25].
The study of the composition of phenolic compounds was performed by HPLC on a Shimadzu LC20 Prominence chromatograph in a modular system equipped with a four-channel pump LC20AD, thermostat STO20A column, automatic SIL20A sampler, diodematrix detector SPDM20A and ChemStation LC18 and ChemStation LC 250 mm x 4.6 mm, particle size 5 μm; column temperature -35 °C; detection wavelength -330 nm (for hydroxycinnamic acids, flavonoid glycosides), 370 nm (for flavonoid aglycones), 280 nm (for tannins), 340 nm (coumarins); the flow rate of the mobile phase is 1 ml / min; sample volume injected -5 µl; mobile phase: eluent A: 0.1 % trifluoroacetic acid solution in water; eluent B: 0.1 % trifluoroacetic acid solution in acetonitrile. The identification of the components was performed by the retention time and the compliance of the UV spectra with the substance standards [23, [26][27][28].
The quantitative determination of phenolic compounds was also performed by spectrophotometric method. The optical density was measured on an Evolution 60S spectrophotometer (USA) at the appropriate wavelength. The content of the sum of hydroxycinnamic acid derivatives was determined in terms of rosmarinic acid at 505 nm, the content of flavonoids in terms of luteolinat a wavelength of 410 nm, the content of the amount of phenolic compounds in terms of per gallic acidat 270 nm. For statistical accuracy, the experiments were performed at least five times [20, 29, 30].

The results of the study Analysis of macro-and micronutrient composition of leaves of S. officinalis and S. grandiflora.
As a result of the analysis, the content of 15 macro-and micronutrients was identified and established (Tab. 1).   The spectrophotometric method was used to determine the quantitative content of phenolic compounds in the studied objects, including derivatives of hydroxycinnamic acids, flavonoids and the sum of phenolic compounds (Table 5).

Discussion of research results
The obtained data from the study of macro and microelement (tab. 1) indicate a significant content in both types of micro elements such as: silicon (330-1040 mg / 100 g), phosphorus (145-220 mg / 100 g), magnesium (290-390 mg / 100 g), calcium (730-1300 mg / 100 g), sodium (290-117 mg / 100g) and potassium (2050-3250 mg / 100 g). The total micro element content of S. grandiflora leaves is 1.67 times greater than that of the S. officinalis pharmacopoeial species. The content of toxic elements such as cobalt, cadmium, arsenic and mercury, lead and molybdenum are within the maximum permissible concentrations for raw materials and foodstuffs.
In the S. officinalis leaf, 15 amino acids were identified (tab. 2). The dominant ones are glutamic acid, aspartic acid, valine and leucine, with a total content of 43.07 %. Among the identified amino acids 8 are irreplaceable, their content is 47.93 % of the total number of amino acids. In the letter of S. grandiflora, 15 amino acids were also identified. Glutamic acid is dominant, aspartic acid, valine and leucine have a total content of 43.66 %. Among the identified amino acids 8 are indispensable. Their content is 48.33 % of the total number of amino acids.
In S. officinalis leaves, 8 saponins were identified (tab. 3). Ursolic and oleanolic acids were dominant, with a total content of 75.82 %. In S. grandiflora's leaves, 8 saponins were identified. Ursolic and euscapic acids were dominant, with a total content of 63.25 %.
The total content of flavonoids is highest in leaves of S. officinalis and is 4.90 mg / g. The total hydroxycinnamic acid content is highest in the leaves of S. grandiflora and is 4.49 mg / g, which is 221.18 % (2.21 times) more than in the pharmacopoeial species of S. officinalis (2.03 mg / g). The overall highest content of coffee acid derivatives is dominated by leaves of S. officinalis (0.77 mg / g).
The highest content of the sum of all detected compounds of phenolic nature is characteristic of leaves of S. officinalis and is 6.93 mg / g.
According to the spectrophotometric study of the content of phenolic compounds (tab. 5) in the leaf of the studied species of the genus Salvia, it was found that the highest content of hydroxycinnamic acid derivatives is specific for S. grandiflora leaves, the highest content of flavonoid compounds, and the total content of phenolic compounds is specific for S. officinalis.
The content of phenolic compounds in the two species studied is practically at the same level, except for the hydroxycinnamic acid content. In the leaf of the nonpharmacopoeial species S. grandiflora, the content of the amount of hydroxycinnamic acids is 2.21 times higher. Particular attention is drawn to the high content of rosemary acid in leaves of S. grandiflora 4.18 times more than in leaves of S. officinalis. The results of qualitative and quantitative analysis of BAS in the leaf of the nonpharmacopoeial species of S. grandiflora indicate the prospect and possibility of its use in medical and pharmaceutical practice as a source of phenolic compounds, in particular hydroxycinnamic acids.
Study limitations. For the statistical significance of the study, it would be advisable to investigate, even wild samples of raw materials from different regions of Ukraine, and not only cultivated and harvested in the Botanical Garden of the National University of Lviv named after Ivan Franko. It was advisable to compare not only the chemical composition of the raw material, but also to compare the pharmacological activity of galenic and neogalenic agents from these raw materials.
Prospects for further research. According to the results of the studies, further screening of pharmacological studies, analysis of terpenoid composition and development of parameters for standardization of S. grandiflora leaves are planned.

Conclusions
Comparative pharmacognostic and pharmacological studies of S. grandiflora and S. officinalis leaves revealed that S. grandiflora is a promising species for introduction into medical and pharmaceutical practice as a source of phenolic compounds, in particular hydroxycinnamic acids.
In both studied species, was found the presence of 15 micro and macronutrients, of which dominant are silicon, phosphorus, magnesium, calcium, sodium and potassium. The total trace element content of S. grandiflora leaves is 1.67 times greater than that of the pharmacopoeial species of S. officinalis. In the leaves of S. officinalis and S. verticillata, 15 amino acids and 8 saponins were identified. HPLC determined the qualitative composition and quantitative content of phenolic substances in leaves of S. officinalis and S. grandiflora (13 and 9 compounds, respectively). The total content of flavonoids is highest in leaves of S. officinalis and is 4.90 mg / g. The total hydroxycinnamic acid content is highest in the leaf of S. grandiflora and is 4.49 mg / g, which is 221.18 % (2.21 times) more than in the pharmacopoeial species of S. officinalis (2.03 mg / g). The overall highest content of coffee acid derivatives is found in leaves of S. officinalis (0.77 mg / g). The content of phenolic compounds in the two studied species is practically at the same level, except for the hydroxycinnamic acid content. In the leaf of the non-pharmacopoeial species S. grandiflora, the content of the amount of hydroxycoric acids is 2.21 times higher. Particular attention is drawn to the high content of rosmarinic acid in leaves of S. grandiflora, which is 4.18 times more than in leaves of S. officinalis.
The results of the comparative phytochemical and pharmacological study of S. officinalis leaves and S. grandiflora leaves significantly extend the data on nonpharmacopoeial species and indicate the undoubted prospect of using S. grandiflora leaves in pharmaceutical and medical practice.

Conflict of interests
There are no conflicts of interest regarding this study.