IDENTIFICATION AND COMPONENT ANALYSIS OF TRITERPENOIDS IN

Despite the progress in the creation of synthetic drugs, herbal medicines do not lose their relevance nowadays. Triterpenoids are quite common in the plant world and exhibit a wide range of biological activity. Medicinal plants of the Lamiaceae family can accumulate mainly pentacyclic triterpenoids which demonstrate the anti-inflammatory, antioxidant, hepatoprotective, anticancer and antimicrobial properties. Nowadays a number of new, poorly-studied species of the family have been introduced in Ukraine, including Monarda fistulosa L. and Ocimum americanum L.The aim of this study was to identify the triterpenoids and to analyze the contents of their components in ethanolic extracts of M. fistulosa and O. americanum (Lamiaceae) aerial parts under the condition of their farming in West Podillya (Ukraine).Methods: foam test, sedimentation and color reaction were used to determine the presence of triterpenoids in plant material; high-performance liquid chromatography (HPLC) method was used to determine their components' content; determination was carried out on a liquid chromatograph Shimadzu LC 20 Prominence.Results. The presence of triterpenoids in M. fistulosa and O. americanum ethanolic extracts was proved by the reactions of identification and HPLC. Effective extraction of triterpene compounds from the investigated plants was achieved using 96 % and 70 % ethanol. The comparative HPLC-analysis showed significant differences in content of ursolic, euscaphic, tormentic and oleanolic acids, betulin and lupeol in the aerial parts of investigated species. The most abundant components of M. fistulosa and O. americanum herbs were the ursolic and euscaphic acids.Conclusions: It was identified the presence and evaluated the contents of 6 triterpenoids in M. fistulosa and O. americanum aerial parts. The obtained data can be used in the planning of pharmacological studies and in the chemotaxonomy of Lamiaceae family


Introduction
Despite the progress in the creation of synthetic drugs, herbal medicines do not lose their relevance nowadays. Phytopreparations are, as a rule, multicomponent composition, and, consequently, multipurpose, as in addition to the effect on target organs, the regulatory systems of the organism are also activated [1]. Medicinal plants with essential oils are an important source of medical and prophylactic treatments from ancient times to the present day, in connection with which scientists are interested in their phytochemical research [2, 3].

Formulation of the problem in a general way, the relevance of the theme and its connection with important scientific and practical issues
Medicinal plants of the Lamiaceae family containing essential oils have long been used in scientific and folk medicine as a source of therapeutic and prophylactic medicines [3,4]. To date, a number of newly unknown species of the family [5] have been introduced in Ukraine, among them -Bee balm (Monarda fistulosa L.) and American basil (Ocimum americanum L.). The above-ground parts of these plants are used in folk medicine of different countries, but they are not used in officinal medicine [4]. It is important that, along with the therapeutic and preventive action, both plants have good spice and taste qualities, which opens the possibility of their wide application in the manufacture of galenic preparations.

Analysis of recent studies and publications in which a solution of the problem are described and to which the author refers
A rather important group of compounds of secondary synthesis, which have a wide range of biological activity, are triterpenoids. According to various authors [6][7][8], representatives of the Lamiaceae family accumulate mainly ursane and oleanane derivatives that exhibit anti-inflammatory, antioxidant, hepatoprotective, antitumor, antiviral and antimicrobial properties. It is worth noting that some of these biological properties also show other compounds of secondary synthesis, previously found in various organs of plants of this family [9][10][11].

The field of research considering the general problem, which is described in the article
An important stage in the pharmacological research of poorly-studied, but promising species of medicinal plants is the analysis of compounds of secondary synthesis, which include triterpenoids. The peculiarities of the accumulation of triterpene compounds in the herb of the non-fictional medicinal plants of the family Lamiaceae, in particular M. fistulosa and O. americanum, which have been introduced in Ukraine in recent years, remain not studied.

Formulation of goals (tasks) of article
The purpose of this work was to identify the composition and component content of triterpene compounds in the aboveground parts of M. fistulosa and O. americanum (under the condition of their cultivation on the territory of Western Podillya, Ukraine).

Presentation of the main research material (methods and objects) with the justification of the results
Extraction of triterpenic saponins from the herb of M. fistulosa and O. americanum was carried out using ethanol of various concentrations (50 %, 70 % and 96 %), which is considered an effective and environmentally justified extractive agent [5,7]. The particles of 1 mm herb of investigated plants (5.0 g) were placed in a 250 ml circular flask, and 100 ml of ethanol of appropriate concentration was added. At first, ethanol-filled raw material was infused at room temperature for 3 hours, stirring periodically. Further, extraction was performed on a water heater at boiling point of the extractive agent for 1 hour. The cooled extract was filtered through a paper filter in a 100 ml volumetric flask and, if necessary, adjusted to the label with the appropriate extractant. An aliquot of each extract was evaporated to a dry residue and used for HPLC analysis. The remains were evaporated to 1/4-1/5 of the original volume and used to identify triterpene saponins.

Identification of triterpenoids (according to [12])
Foam test. Approximately 1 ml of extract for identification was diluted to 5 ml with purified water, closed with a stopper and vigorously shaken for 1 min. The formation of stable foam indicated the presence of triterpene saponins.
Sedimentation reaction. To 1 ml of the extract for identification, 3-4 drops of 10 % lead (II) acetate solution were added. The appearance of sediment indicated the presence of triterpene compounds.
Colour reaction. To 1 ml of the extract for identification, 1 drop of 10 % solution of copper (II) sulphate and 1 ml of concentrated sulfuric acid solution were added, after which the mixture was carefully heated. The appearance of a blue-green colour indicated the presence of triterpene saponins.
HPLC analysis of triterpenoids (in accordance with [13,14]) Investigation of the qualitative composition and quantitative content of triterpene compounds by the HPLC method was carried out on a liquid chromatograph Shimadzu LC 20 Prominence in a modular system equipped with a four-channel LC 20AD pump, a thermostat of columns 20A, an automatic sampler SIL 20A, SPDM 20A diode-matrix detector and ChemStation LC 20 A X-Bridge C18 column of 150 mm × 4.6 mm, a particle size of 5 μm (Waters, Ireland) was used; column temperature -30 ºС; detection wavelength -205 nm; flow rate of the mobile phase -1.0 ml / min; volume of the injected sample -20 μl.
Moving phase: methanol and 0.2 % ammonium acetate solution (pH=6.75) in the ratio 80:20; elution mode isocratic. Identification of the components was carried out in accordance with the time of maintenance and compliance with the UV spectra of the substancesstandards (oleanolic, ursolic, tormentic, and euscaphic acids, betulin and lupeol), which were determined in the range 190-800 nm.
Results and discussion. In the extracts of M. fistulosa and O. americanum raw materials obtained on the basis of high concentrations of ethanol (70 % and 96 %), a positive result was found regarding the presence of triterpene compounds in the use of the foaming test, as well as the colour and sedimentation reactions.
Based on the data of the analysis of UV spectra of standard models of triterpene compounds and investigated extracts, absorption peaks were found in the range of wavelengths of 200-210 nm, so their HPLC analysis was carried out at 205 nm. The results of the HPLC analysis of triterpenoids in the herb of the investigated plants are presented in Table 1 and on Fig. 1, 2; 6 compounds were identified.
As can be seen from the results obtained (Table 1, Fig. 1, 2), effective extractants for extracting triterpene compounds from the herbs of the studied plants were 96 % and 70 % ethanol; its 50 % concentration was less effective.  Ursolic and euscaphic acids (α-amine derivatives based on the ursane skeleton - Fig. 3) are the dominant components in 96 % and 70 % of ethanol extracts of both studied plants. As can be seen from Table 1, 96 % ethanol is the optimal extractant for extracting ursolic acid from the herbs of M. fistulosa and O. americanum (0.22 % and 0.19 %, respectively), while 70 % ethanoleuscaphic one (0.32 % and 0.31 %, respectively). The obtained results correlate with the literature data for other species of the Lamiaceae family with respect to the domination of the ursane derivatives. Thus, leaves of 8 representatives of the Ocimum genus, which grew on the territory of Brazil [7], accumulated ursolic acid in the range of 0.29-2.02 %; the above-ground bodies of 4 species of the Ocimum genus, harvested in India, accumulated ursolic acid in the range of 0.008-0.296 % [15,16]. The content of this compound in the aboveground part of 5 species of plants of the Rosmarinus, Salvia and Satureja genus ranges from 0.09 to 1.6 % [17]. Ursolic acid, which is a fairly common substance in the raw materials of the Lamiaceae family, has antiinflammatory, antioxidant, hepatoprotective, antiviral, antimicrobial and cytostatic properties [6,8]. The content of tormentic acid in the raw materials of the studied plants is low (Table 1, Fig. 1, 2). Oleanolic acid (a representative of the β-aminine group, which is based on the oleanane skeleton - Fig. 3), is usually identified in parallel with ursolic acid in the raw material of many plants of the Lamiaceae family [6,14]. These triterpenoids are found both in the form of aglycones and in the form of glycosides, and have similar biological properties [6,8]. The content of oleanolic acid is higher in the raw material of O. americanum in comparison with M. fistulosa, regardless of the ethanol concentration used (see Table 1). The obtained results correlate with the data of the literature, according to which [14] the above-ground part of Ocimum gratissimum accumulates 0.14 % of oleanolic acid; the content of this compound in the above-ground parts of the plants of the Rosmarinus, Salvia and Satureja genus ranges from 0.09 to 0.9 % [17].
The best solvent for extraction of betulin (a derivative of a lupane -see Figure 3) from the herb of both plants is 70 % ethanol (see Table 1). Another representative of the lupane grouplupeolwas found in a small amount in only 96 % ethanol extract of the O. americanum herb.
It is known that the content of different groups of biologically active substances in plants has a significant impact on the peculiarities of the climate and soils of the region of growth, the presence of chemotypes, peculiarities of care and preparation, selection of extraction and analysis conditions. It should be noted that the use of HPLC analysis to determine the qualitative composition and quantitative content of triterpene saponins in plants is quite common [13,15]; some scientists also use the method of gas chromatography after precolomn derivatization [17,18]. Obviously, all these factors have a certain effect on the content of triterpene compounds in the raw material of O. americanum та M. fistulosa. 7. Conclusions 1. It was identified the triterpenoids in ethanoic extracts obtained from herbs of M. fistulosa and O. americanum; the components of triterpenoids were determined by the HPLC method.
2. It has been determined that high concentrations of ethanol (96 % and 70 %) contribute to the more efficient extraction of triterpene compounds from the raw materials of the studied species than 50 % ethanol.
3. The peculiarities of the accumulation of 6 triterpene compounds (ursolic, oleanolic, euscaphic and tormentic acids, as well as betulin and lupeol) in the herb of M. fistulosa and O. americanum were established under cultivation of plants in the West Podillya (Ukraine). Ursolic and euscaphic acid are the dominant triterpene compounds in the herb of both species.