CHROMATO-MASS-SPECTROMETRIC RESEARCH IN SALVIA GRANDIFLORA L., SALVIA PRATENSIS L. AND SALVIA VERTICILLATA L. ABOVEGROUND ORGANS

The genus Salvia L. has more than 900 species distributed throughout the globe. 21 species are growing in Ukraine. All species of this genus have essential oils. Salvia officinalis and Salvia sclarea have been used in the culture and are widely used in medical practice. The chemical composition of other species of sage and the possibility of their use in pharmaceutical and medical practice are almost not studied. Taking into account the results of chemotaxonomic studies of species of the flora genus of Ukraine, their prevalence and prospects for introduction into the culture, for further studies were selected raw materials of S. grandiflora, S. pratensis and S. verticillata. The aim. The aim of the study was to conduct a chromato-mass spectrometric study of the aboveground organs of S. grandiflora L., S. pratensis L. and S. verticillata L. to establish the prospects for the use of raw materials of these species in medical and pharmaceutical practice. Materials and methods. The objects of the study were leaves of S. officinalis, leaves, stems and flowers of S. grandiflora, S. pratensis and S. verticillata, which were harvested on the basis of the botanical garden of Ivan Franko National University of Lviv. The research of volatile substances in the objects of the research was carried out by the method of GC-MS on the basis of the Department of Natural Sciences for Foreign Students and Toxicological Chemistry of Zaporizhia State Medical University. Results. As a result of the study, 243 substances were found in the objects of the study, of which 149 were identified. 77 substances were found in the leaves of S. officinalis, 80, 26 and 63 substances in the leaves, stems and flowers of S. grandiflora, respectively, in the leaves , stems and flowers of S. pratensis – 28, 30 and 48 substances, respectively, in leaves, stems and flowers of S. verticillata – 39, 22 and 39 substances, respectively. Dominant compounds among substances of terpenoid nature are: cyclofenchene, camphene, 1,8-cineole, α-thujone, β-thujone, camphor borneol, caryophyllene, humulene, viridiflorol, sabinene, pyranone, β-pinene, phytol, kolavenol, β-copaen, loliolide, pseudolimonene and spatulenol. Among the dominant substances, 8 were detected for the first time in these species: cyclofenchene, viridiflorol, sabinene, pyranone, phytol, kolavenol, loliolide and pseudolimonene. Conclusions. The leaves of S. officinalis, leaves, stems and flowers of S. grandiflora, S. pratensis and S. verticillata of the flora of Ukraine were studied by chromato-mass spectrometric method. As a result of the study, 243 substances were identified, of which 149 were identified. Promising raw materials containing terpene compounds for S. grandiflora there are leaves, and for S. pratensis and S. verticillata – flowers, so they are promising agents for introduction into pharmaceutical practice


Introduction
The genus Salvia L. has more than 900 species distributed throughout the globe, of which 21 species are represented in the Ukrainian flora. All species of this genus have essential oils. Sage and sage entered the culture [1,2]. The chemical composition of other species of sage and the possibility of their use in pharmaceutical and medical practice are almost not studied.
Based on the literature data and the results of our own research, a chemotaxonomic study of 17 species of the genus Sage (Salvia L.): S. officinalis, S. sclarea, S. grandiflora, S. scabiosifolia, S. glutinosa, S. aethiopis, S. pratensis, S. stepposa, S. Sibthorpii, S. illuminata, S. nemorosa, S. pendula, S. sylvestris, S. nutans, S. austriaca, S. verticillata and S. cernua to identify promising species and establish the possibility of creating new drugs based on them [21]. In the list of these species, 60 compounds of phenolic nature and 185 compounds of terpenoid nature were found [21,22]. The chemical profile of the genus is characterized by phenolic compounds: caffeic and rosmarinic acids [3], flavonoids: cynaroside, kosmosiin, hispidulin and cyrsimaritin [23-25] and terpenoids: α-and β-pinene, derivatives of camphor (camphene, camphor and borneol) [26], p-cymene, 1,8-cineole and limonene [27,28]. In addition, the amino acid and monosaccharide composition of sage leaves was studied [29]. Taking into account the results of chemotaxonomic studies and the prospects of their introduction into the culture, the raw materials of S. grandiflora, S. pratensis and S. verticillata were selected for further studies.
The aim of the research was to conduct a chromato-mass spectrometric study of the aboveground organs of S. grandiflora L., S. pratensis L. and S. verticillata L. to establish the prospects for the use of raw materials of these species in medical and pharmaceutical practice.

Planning (methodology) of the research
Taking into account the results of chemotaxonomic studies of sage flora of Ukraine [21], which showed the prospects of using S. grandiflora, S. pratensis and S. verticillata in medical and pharmaceutical practice, it is planned to conduct chromato-mass spectrometric study of aboveground organs of these species, to identify promising species raw materials and the possibility of its further use in pharmacy (Fig. 1).

Materials and methods
The objects of the study were leaves of S. officinalis, leaves, stems and flowers of S. grandiflora, S. pratensis and S. verticillata, which were harvested on the basis of the Botanical Garden of Ivan Franko National University of Lviv (Lviv, 44 Cheremshyny Street, 79014), under the guidance of a senior researcher, PhD Skibitska Maria Ivanovna. The studied examples of Salvia genus were identified by PhD Olga Mala from the National University of Pharmacy using a special botanical catalogue [30]. The identified samples corresponded to the moisture content of SPhU [31,32]. The voucher specimens are stored at the Department of Pharmacognosy, The National University of Pharmacy, Kharkiv, Ukraine (No. 1015-1029).
Studies of volatile substances in the objects of the research were carried out by GC-MS method [33] on the basis of the Department of Natural Sciences for Foreign Students and Toxicological Chemistry of Zaporizhia State Medical University according to the method: a sample of 2.0 (exact sample) of plant material was placed in a test tube (volume of which 20 ml), poured 10 ml of methanol (Sigma-Aldrich) and left for one day. The extract was filtered using a syringe filter, the resulting filtrate was chromatographed [34,35]. Agilent 7890B gas chromatograph with 5977B mass spectrometric detector and Gerstel CIS 4 chilled injection system. DB-5ms chromatographic column 30 m x 250 μm × 0.25 μm long. The speed of the carrier gas (helium) is 1.3 ml/min. The injection volume is 0.5 μl. Pulse injection with flow division in the ratio 1:5 was used. The temperature of the sampling unit is 200°C → 12°C/s → 265°C. Thermostat temperature: programmable, 70°C (1 min delay) → 10°C/min → 270°C (4 min delay). The total chromatography time is 25 minutes. GC/MS interface temperature -275°C; ion sources -230°C; quadrupole mass analyzer -150°C. Type of ionization: electron impact at an electron energy of 70 eV. Scanned mass range: 30-700 m/z. The NIST14 mass spectrum library was used to identify the components. Quantitative determination of the content of substances in the raw material was performed in comparison with a standard sample of menthol [36,37].

Results of the research
Determination of the qualitative composition and quantitative content of volatile substances in the study objects was performed by GC-MS method using an Agilent 7890B chromatograph. The results of the study of volatile substances in the aboveground organs Step 1 •Harvesting and identification of S. officinalis, S. grandiflora, S. pratensis and S. verticillata raw materials Step 2 •Chromato-mass spectrometric study of raw materials Step 3 •Analysis of obtained results Step 4 •Determination of promising raw materials of the studied species Step 5 •Identification of promising areas of use of raw materials and further research of the studied species of the genus Salvia are given in Table 1. In Fig. 2 Table 1   1   2   3  4  5  6  7  8  9  10  11  12   136 6a, 14a-methanopicene, perhydro-1,2,4a, 6b, 9,9,12a-hexamethyl-10-hydroxy-   As a result of the study, 243 substances were found in the objects of the study, of which 149 were identified.
In the leaves of S. pratensis 28 substances were found, pyranone and phytol were dominant. 30 substances were found in the stem of S. pratensis, pyranone and α-amyrin were dominant. 48 substances were found in S. pratensis flowers, pyranone, caryophyllene, phytol and kolavenol were dominant.
In the leaves of S. verticillata 39 substances were found, the dominant ones were caryophyllene, β-copaen, loliolide and phytol. 22 substances were found in the stem of S. verticillata. In S. verticillata flowers 39 substances were found, the dominant ones were cyclofenchene, pseudolimonene, pyranone, caryophyllene, spatulenol and phytol.
The raw materials of all three studied species S. grandiflora, S. pratensis and S. verticillata are rich in caryophyllene, so they could be potential sources of it. The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). Number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states [38]. More importantly, BCP inhibited EtOH-CPP acquisition and exacerbated LORR duration. Interestingly, these effects were abrogated when mice were pre-injected with a selective CB2 receptor antagonist, AM630. Overall, the CB2 receptor system appears to be involved in alcohol dependence and sensitivity and may represent a potential pharmacological target for the treatment of alcoholism [39]. Moreover, oral administration of BCP effectively rescued β-cells by mitigating hyperglycemia through enhancing insulin release and also averted oxidative/inflammatory stress in pancreatic tissue of diabetic rats. The efficacy of BCP was compared with glibenclamide, a standard antidiabetic drug [40]. The characterization tests indicated that βCP were efficiently incorporated into βCD. The oral treatment with βCP-βCD, at all doses tested, produced a significant (p<0.05) reduction on mechanical hyperalgesia and a significant (p<0.05) increase in muscle withdrawal thresholds, without produce any alteration in force. In addition, βCP-βCD was able to significantly (p<0.05) decrease Fos expression in the superficial dorsal horn. Thus, βCP-βCD attenuates the noninflammatory chronic muscle pain in mice and inhibits the Fos expression in the lumbar spinal cord [41].
In addition, all three studied species S. grandiflora, S. pratensis and S. verticillata are rich in phytol and its derivatives, so they could be potential sources of it. In the pharma-medico viewpoint, PYT and its derivatives have been evident to have antimicrobial, cytotoxic, antitumorous, antimutagenic, anti-teratogenic, antibioticchemotherapeutic, antidiabetic, lipid lowering, antispasmodic, anticonvulsant, antinociceptive, antioxi-dant, antiinflammatory, anxiolytic, antidepressant, immunoadjuvancy, hair growth facilitator, hair fall defense and antidandruff activities. Otherwise, the important biometebolite of PYT is phytanic acid (PA). Evidence shows PA to have cytotoxic, anticancer, antidiabetic, lipid lowering and aniteratogenic activities. In addition, it may be considered as an important biomarker for some diseases such as Refsum's Disease (RD), Sjögren Larsson syndrome (SLS), rhizomelic chondrodysplasia punctata (RZCP), chronic polyneuropathy (CP), Zellweger's disease hyperpipecolic academia (ZDHA) and related diseases. Thus, phytol may be considered as a new drug candidate [42,43].
The raw materials of the studied species S. grandiflora, S. pratensis and S. verticillata are rich in γ-Sitosterol, so they could be potential sources of it. The study demon-strates in vitro results, which support the ethnomedical use of γ-Sitosterol against cancer. Experimental results of this study suggest that γ-Sitosterol exerts potential anticancer activity through the growth inhibition, cell cycle arrest and the apoptosis on cancer cells [44]. γ-sitosterol was screened for its antidiabetic property in streptozotocin (STZ) induced diabetic rats. Furthermore γ-sitosterol showed antihyperlipidemic activity as evidenced by significant decrease in serum total cholesterol, triglycerides and very low density lipoprotein-cholesterol levels coupled with elevation of high density lipoprotein-cholesterol levels in treated rats. A significant decrease in the activities of alanine aminotransaminase, aspartate aminotransaminase, alkaline phosphatase and acid phosphatase in γ-sitosterol treated rats when compared to diabetic control rats indicated its protective role against liver damage. γ-Sitosterol increased insulin secretion in response to glucose. Immunohistochemical study of pancreas also confirmed the biochemical findings. These results indicated that γ-sitosterol possessed antihyperglycemic activity [45].
Given the qualitative composition and quantitative content of terpene substances, promising raw materials for S. grandiflora are leaves, and for S. pratensis and S. verticillataflowers, so they are promising agents for introduction into pharmaceutical practice.
Study limitations. Not all substances could be identified during the GC MS study of raw materials, as they were not available in the library that was used.
The prospects for the further research. To create new drugs, it is advisable to conduct a more thorough phytochemical study of raw materials of the studied species of sage, to standardize these raw materials and obtain galenic drugs based on it.
A promising raw material containing terpene compounds for S. grandiflora are leaves, and for S. pratensis and S. verticillataflowers, so they are promising agents for introduction into pharmaceutical practice.

Conflict of interests.
Authors declare there is no conflict of interest.

Funding.
The research was funded by the Ministry of Health Care of Ukraine at the expense of the State Budget in the framework No. 2301020 "Scientific and scientific-technical activity in the field of health protection" on the topic "Modern approaches to the creation of new medicines for a correction of metabolic syndrome".