Development of methods for purification of individual biological active substances obtained from extracts of Hedysarum neglectum

Hedysarum neglectum is a forage plant. Xanthone glycoside - mangiferin is extracted from this plant and used for medicine “Alpizarin”. In addition to substances of xanthone nature (mangiferin and isomangiferin) Hedysarum neglectum contains sugars, vitamins and provitamins, tannins; in the underground part it contains oligomeric catechins, isoflavonoids, butylphenols, alkaloids, tannins, flavonoids, saponins, coumarins, carbohydrates, vitamin C. For selecting optimal schemes of fractionization of substances, it is necessary to resort to multi-stage schemes of group-wide (preliminary) isolation and preparative accumulation. In particular cases, it is necessary to take into account the presence of concomitant substances, as well as the effectiveness and selectivity of the sorption-chromatographic technologies used. According to the results of the studies, the use of silica gel and sefadex LH-20 for the isolation of a complex of flavonoids and gallic acid is the most effective method for the selection of the optimal variant of the preparative isolation of the total amount of BAS in mcg/ml. The results of the research allowed us to identify the target biologically active substances with a degree of extraction of at least 80%: - fractions of xanthones, flavonoids, gallic acid.

Xanthone glycoside -mangiferin is extracted from this plant, from which the medicine "Alpizarin", which has an antiviral effect, was produced [4].
In the work of S. A. Kubentaev, it is indicated that in addition to substances of xanthone nature (mangiferin and isomangiferin), Hedysarum in its ground part contains: sugars, vitamins and provitamins, tannins; in the underground part it contains oligomeric catechins, isoflavonoids, butylphenols, alkaloids, tannins, flavonoids, saponins, coumarins, carbohydrates, vitamin C [3].
Due to the content of such biological active substances (BAS), the plant has antiinflammatory, antitumor, immunostimulating and tonic properties.
Plant cells and tissues, along with secondary metabolites of target substances (flavonoids, alkaloids, saponins, coumarins, etc.), contain products of general metabolism -primary metabolites: carbohydrates, organic acids, amino acids, proteins, etc., and also in significant quantities they may contain resinous substances, carotenoids, chlorophylls. Therefore, when choosing the optimal schemes of fractionation of substances, it is necessary to resort to multistage schemes of group-wide (preliminary) isolation and preparative accumulation. In particular cases, it is necessary to take into account the presence of concomitant substances, as well as the effectiveness and selectivity of the sorption-chromatographic technologies used.
The chromatographic mobility of compounds of plant origin is subject to strict laws and correlates with the chemical structure. Thus, the sorption-chromatographic parameters of one of the most common and practically significant group of biologically active compounds, flavonoids, are affected by such structural features as the variation in the number of hydroxyl groups in the molecule, the presence of methoxyl substituents, acithelination, glycosidation features (carbohydrate, the amount of sugar residues, their position), and, in fact, the ortho and vicial position of substituents.  The extraction of plant material was carried out on a water bath of the PE-4310 brand (Russia, EKROSHIM) with a reverse refrigerator. The extraction frequency is 2, the concentration of the extractant is 70%, the extraction temperature is 30 °C, the extraction exposure is 6 h.

Objects and methods
LC chromatography was used for the analysis of extracts of callus cultures of Hedysarum neglectum. The preparative isolation and accumulation of mangiferin was performed in the lowpressure chromatography mode using cross-linked agarose as a sorbent. Water-alcohol extracts were used in the examination. The extraction data after evaporation were chromatographed on CL-4 B sepharose gel (Pharmacia, Sweden) under working conditions. Gel volume was 10 ml, elution rate was 0.1-0.4 ml / min. The eluent was: deionized water and 0.01 M sodium hydroxide solution. Chromatography was performed on a BioLogic low-pressure chromatograph (BioRad).
A chromatographic column with a sorbent modified with octadodecyl and endcapped with aminophenol groups was used to identify the BAS of Hedysarum neglectum. As an eluent, a mixture of water: acetonitrile, 0.1% tributylamine 85:15 was used. Elution was performed in the isocratic mode, flow rate of 1 ml / min, elution time of 55 min. Detection was carried out using a fluorescent detector, the excitation wavelength was 350 nm, the detection wavelength was 415 nm, the collection of individual substances was carried out automatically using a fraction collector.
The selection of rational parameters for the extraction of BAS from plant extracts was primarily guided by the chemical composition, structure and properties of the target BAS and associated ballast compounds, which had to be separated from the target BAS [5,6]. Also, when selecting rational parameters, a significant factor was the quantitative content of the target BAS in the analyzed sample, which had to be taken into account at the stages of concentration [1,7].
Based on the above, the schemes of analysis, isolation, purification and preparative accumulation are unique in relation to a specific plant and the biologically active compound isolated from it, as well as individual in relation to the features of the technological process and the resulting extraction.
When developing methods for the purification of individual biologically active substances obtained from medicinal plant extracts, they were primarily guided by ensuring adequate purification from accompanying (ballast) components [8,9].

Results and discussion
The extraction of biologically active substances from the root crop extract of the Hedysarum neglectum includes the following steps: 1. The ethanol extract is dried in a vacuum on a rotary evaporator IKA RV 8 at a temperature of 450C.
3. Methanol is fed to the column for desorption of gallic acid.
4. The fractions are selected using a 300 ml fraction collector each. Thus, nine fractions are collected (fractions 1-9). From fraction 3, a crude mangiferin crystal is obtained, which is then recrystallized from the mixture: petroleum ether-acetone, in the ratio of 20:1.
The selection scheme is shown in Figure 1.   2. After that, methanol is fed to the column for desorption of gallic acid.
3. The fractions are selected using a collector of fractions of 300 ml each. Thus, nine fractions are collected (fractions 1-9). From fraction 3, a crude mangiferin crystal is obtained, which is then recrystallized from the mixture: petroleum ether-acetone, in a ratio of 20 : 1, and purified by rechromatography on sepharose CL6B (Sigma -Aldrich) on a BioLogic low-pressure chromatograph (BioRad), which allows the pure substance of mangiferin to be isolated.