**1. Introduction**

To improve modern pharmacy, the study of biologically active substances exhibiting specific pharmacological activity from the natural raw materials of plant origin is of obvious interest. In our time, the problem of obtaining antiviral and antibacterial agents of plant origin is especially acute. At the moment, the problem of chronic recurrent viral diseases is very relevant. According to WHO data, about 67% of the world's population suffer from diseases of various organs and systems caused by the Herpes simplex virus.

Mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetraoxyxanthone) belongs to the group of xanthones proper, is the most widespread representative of C-glycosides, and has antiviral and antibacterial properties (**Figure 1**).

In species of the genus *Hedysarum*, Fabaceae, xanthones, namely mangiferin, are the main group of biologically active substances. Russian scientists have developed an antiviral drug Alpizarin, from *H. alpinum* L. and *H. flavescens* Regel & Schmalh, the active substance of which is mangiferin, which has an antiviral activity against DNA-containing viruses (*Herpes simplex, Varicella zoster, Cytomegalovirus*), immunostimulating properties, and a bacteriostatic effect on Gram-positive and Gram-negative bacteria. VILAR (Russia) produces this in two forms of production "Alpizarin ointment" and "Alpizarin tablets." In addition to domestic drugs, the

diverse in morphological and anatomical features. Seeds are without endosperm or

The genus *Hedysarum* L. combines about 285 species, which are predominantly common in Eurasia. The genus *Hedysarum* is distributed mainly in the European part, but some species are also found in Asia. *H. caucasicum* Bieb. grows in all highland areas of the North Caucasus, including the Rocky Ridge, in the alpine belt, up to 3500 m [2]. This species is endemic, growing on the highland meadows of the Caucasus: in the pre-Caucasus, Western and Eastern Transcaucasia, in Daghestan. It is described from the Caucasus type in Leningrad [1]. C. Linnaeus (1753) and B. A. Fedchenko (1902) were engaged in cystematics of the species of *Hedysarum* L. The species *Hedysarum caucasicum* Bieb we are investigating first was described in 1808 [3]. In the flora of Caucasus in 1873, the species was classified as *H. obscurum var. caucasicum* Trautv. The origin of the name of this plant is related to the shape of the fruit. Beans in appearance resemble coin meetings, and the Latin name *Hedysarum* comes from the Greek words "hedys" aroma, which literally means pleasantly smelling. Members of this genus are perennial herbs, shrubs, or semiplants of seamless or stem-shortened plants. They grow both in forests and on raw meadows, on riverbanks, and on alpine and subalpine meadows, on rocky sprouts, and in

with scant endosperm. Spare nutrients are deposited directly in the seed.

Stems are often highly branched, branching, and sometimes completely undeveloped and the flower arrow comes out of shortened shoots developing at the neck of the rhizome; leaves are unparalleled, common: 5–9-paired, less common: 1–3-paired or even consisting of just one unparalleled leaf (**Figure 2**). The genus is characterized by simple botrioid flowers—brushes; the cup is bell-shaped, its teeth are search-and-take longer than the tube; the crown exceeds the cup; and wings are slightly or two times shorter than a boat, less often longer than it. The beans are artenic, and sometimes part of the seed does not develop and the bob consists of 1–3 artens; sprouts are flat-compressed or slightly convex, smooth, bare, or more often dried, mesh or with transverse ribs, often shrunk with short or longer bristles.

According to Flora of the USSR, in the *Hedysarum caucasicum* Bieb.—a plant 30– 60 cm high, the stems are straight or ascending, not shortened, olfactory; and the leaves are 7- are 12-pairs, elliptical or egg-like long-lasting-watt, with sharpness at the top, 12–16 mm long, 7–9 mm wide. Flowers (without brush) are longer than leaves; brushes are not very thick, of 25–35 flowers; the lower tooth of the cup is equal to the tube, the rest are shorter; and the crown is dark-magenta, 16–18 mm

steppes.

Hedysarum *Species from Caucasus*

*DOI: http://dx.doi.org/10.5772/intechopen.91741*

**Figure 2.**

**149**

*Species of* Hedysarum *from Caucasus (Russia).*

**Figure 1.** *Mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetraoxyxanthone).*

foreign antiviral drug Mangogerpin (BV Pharma, Vietnam) is known, which also has two forms of release: tablets and ointment, the active substance of which is mangiferin.

There is a need to expand the raw material base for the production of broadspectrum antiviral drugs. In this regard, a comprehensive analysis, taking into account the features of growth, biological characteristics, chemical composition, and known pharmacological properties, will make it possible to substantiate the directions of their rational use as well as the creation of additional domestic raw materials of mangiferin as a known antiviral drug based on medicinal plant raw materials. Thus, the study of species of the following genus is promising: *Hedysarum* L., *Hedysarum caucasicum* M. Bieb., *Hedysarum grandiflorum* Pall., and *Hedysarum daghestanicum* Rupr. ex Boiss., which are growing in the North Caucasus as additional raw materials for mangiferin.

The purpose of our work is to study plants of species of the genus *Hedysarum* L., including *Hedysarum caucasicum* M. Bieb., *Hedysarum daghestanicum* Rupr. ex Boiss., and *Hedysarum grandiflorum* Pall., as additional raw materials for mangiferin.

To achieve this goal, it is advisable to achieve the following objectives:


The family *Fabaceae Lindl.* (legumes) has about 650 genera and over 18,000 species, whose range of growth covers all continents of the globe. Within its range, species of the legume family are characterized by a variety of habitats. Life-form of members of the family *Fabaceae* Lindl.—perennial herbaceous plants, semiplants, and rarely trees—there are tree or herbaceous lians [1]. The leaves are complex with sills. The leaflet is next. Flowers are in finite or sinus brushes. The flowers are zygomorphic, quite large, and bright. The near-wind is double, the cup is most often actinomorphic, consists of five converged cups, and the crown consists of five petals, two of which grow into a "boat," one large—a "sail" and two small petals —"vests" remain free. The android consists of 10 stamens, nine of which usually grow together and one stamen remains free. Gynecium monocarpic tie the upper one. The fruit of legumes is monocarpium, one-, two-, or multifamily legumes, very

#### Hedysarum *Species from Caucasus DOI: http://dx.doi.org/10.5772/intechopen.91741*

diverse in morphological and anatomical features. Seeds are without endosperm or with scant endosperm. Spare nutrients are deposited directly in the seed.

The genus *Hedysarum* L. combines about 285 species, which are predominantly common in Eurasia. The genus *Hedysarum* is distributed mainly in the European part, but some species are also found in Asia. *H. caucasicum* Bieb. grows in all highland areas of the North Caucasus, including the Rocky Ridge, in the alpine belt, up to 3500 m [2]. This species is endemic, growing on the highland meadows of the Caucasus: in the pre-Caucasus, Western and Eastern Transcaucasia, in Daghestan. It is described from the Caucasus type in Leningrad [1]. C. Linnaeus (1753) and B. A. Fedchenko (1902) were engaged in cystematics of the species of *Hedysarum* L. The species *Hedysarum caucasicum* Bieb we are investigating first was described in 1808 [3]. In the flora of Caucasus in 1873, the species was classified as *H. obscurum var. caucasicum* Trautv. The origin of the name of this plant is related to the shape of the fruit. Beans in appearance resemble coin meetings, and the Latin name *Hedysarum* comes from the Greek words "hedys" aroma, which literally means pleasantly smelling. Members of this genus are perennial herbs, shrubs, or semiplants of seamless or stem-shortened plants. They grow both in forests and on raw meadows, on riverbanks, and on alpine and subalpine meadows, on rocky sprouts, and in steppes.

Stems are often highly branched, branching, and sometimes completely undeveloped and the flower arrow comes out of shortened shoots developing at the neck of the rhizome; leaves are unparalleled, common: 5–9-paired, less common: 1–3-paired or even consisting of just one unparalleled leaf (**Figure 2**). The genus is characterized by simple botrioid flowers—brushes; the cup is bell-shaped, its teeth are search-and-take longer than the tube; the crown exceeds the cup; and wings are slightly or two times shorter than a boat, less often longer than it. The beans are artenic, and sometimes part of the seed does not develop and the bob consists of 1–3 artens; sprouts are flat-compressed or slightly convex, smooth, bare, or more often dried, mesh or with transverse ribs, often shrunk with short or longer bristles.

According to Flora of the USSR, in the *Hedysarum caucasicum* Bieb.—a plant 30– 60 cm high, the stems are straight or ascending, not shortened, olfactory; and the leaves are 7- are 12-pairs, elliptical or egg-like long-lasting-watt, with sharpness at the top, 12–16 mm long, 7–9 mm wide. Flowers (without brush) are longer than leaves; brushes are not very thick, of 25–35 flowers; the lower tooth of the cup is equal to the tube, the rest are shorter; and the crown is dark-magenta, 16–18 mm

**Figure 2.** *Species of* Hedysarum *from Caucasus (Russia).*

foreign antiviral drug Mangogerpin (BV Pharma, Vietnam) is known, which also has two forms of release: tablets and ointment, the active substance of which is

There is a need to expand the raw material base for the production of broadspectrum antiviral drugs. In this regard, a comprehensive analysis, taking into account the features of growth, biological characteristics, chemical composition, and known pharmacological properties, will make it possible to substantiate the directions of their rational use as well as the creation of additional domestic raw materials of mangiferin as a known antiviral drug based on medicinal plant raw materials. Thus, the study of species of the following genus is promising: *Hedysarum* L., *Hedysarum caucasicum* M. Bieb., *Hedysarum grandiflorum* Pall., and *Hedysarum daghestanicum* Rupr. ex Boiss., which are growing in the North Caucasus as addi-

The purpose of our work is to study plants of species of the genus *Hedysarum* L.,

• Conduct a comparative morphological-anatomical study of these species of the

*daghestanicum Rupr. ex Boiss.*, and *Hedysarum grandiflorum* Pall.) and identify

• Develop an effective procedure for quantifying the sum of xanthones in terms of mangiferin from these species of the genus *Hedysarum* L., by methods of UV spectrophotometry, capillary electrophoresis, and high-performance liquid

The family *Fabaceae Lindl.* (legumes) has about 650 genera and over 18,000 species, whose range of growth covers all continents of the globe. Within its range, species of the legume family are characterized by a variety of habitats. Life-form of members of the family *Fabaceae* Lindl.—perennial herbaceous plants, semiplants, and rarely trees—there are tree or herbaceous lians [1]. The leaves are complex with sills. The leaflet is next. Flowers are in finite or sinus brushes. The flowers are zygomorphic, quite large, and bright. The near-wind is double, the cup is most often actinomorphic, consists of five converged cups, and the crown consists of five petals, two of which grow into a "boat," one large—a "sail" and two small petals —"vests" remain free. The android consists of 10 stamens, nine of which usually grow together and one stamen remains free. Gynecium monocarpic tie the upper one. The fruit of legumes is monocarpium, one-, two-, or multifamily legumes, very

including *Hedysarum caucasicum* M. Bieb., *Hedysarum daghestanicum* Rupr. ex Boiss., and *Hedysarum grandiflorum* Pall., as additional raw materials for

To achieve this goal, it is advisable to achieve the following objectives:

genus *Hedysarum L.* (*Hedysarum caucasicum M. Bieb.*, *Hedysarum*

the main morphological-anatomical diagnostic features.

mangiferin.

**Figure 1.**

mangiferin.

**148**

tional raw materials for mangiferin.

*Mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetraoxyxanthone).*

*Legume Crops - Prospects, Production and Uses*

chromatography.

long. The fruit is a bob of 3–6 arthropods, naked or desiccated; the mushroom of the bean is oblong-elliptical, and its edge is not wide.

The microstructure of the above-ground parts of some members of the *Hedysarum* was studied by Ladygina et al. [4]. The features of the anatomical structure of leaves of five species of *Hedysarum* from the section *Gamotion* Basin were studied. These species containing mangiferin have been found to have bright golden yellow fluorescence of epidermis cells, which can be used to detect mangiferin directly in plant material [5].

We did not meet the information on the study of anatomical features of the structure of the organs in the accessible literature. The onthogenesis of species *Hedysarum* usually includes four periods and 10 age states. In the example of *H. austrosibiricum*, age spectra can be divided into four types, which probably reflect ecological-phytocenotic growth conditions [6].

During 3 years of life, alpine individuals undergo the following age conditions: in the first year—seedlings, juvenile, immature, and more than 60% of individuals are adult vegetative; in the second year, more than 90% of individuals enter a young reproductive state; and in the third year, all individuals transition to a reproductive state. Seeds of tested plants need pre-sowing treatment. Mechanical scarification and treatment with concentrated sulfuric acid are the best ways to disrupt the rest of both species [6–8].

**3. Methods for quantitative analysis of mangiferin**

**coordinates**

32,21″,

49,1"

15,7″,

45,8"

38,2″,

27,2"

32,9″,

46,9"

32,21″,

49,1"

*List of objects of study with indication of ecological and biological features of growth.*

5. N—42°19<sup>0</sup>

6. N—42°38<sup>0</sup>

7. N—42°41<sup>0</sup>

8. N—42°59<sup>0</sup>

9. N—42°19<sup>0</sup>

E—47°09<sup>0</sup>

E—46°09<sup>0</sup>

E—46°14<sup>0</sup>

E—46°54<sup>0</sup>

E—47°09<sup>0</sup>

**Species № Geographical**

*DOI: http://dx.doi.org/10.5772/intechopen.91741*

Hedysarum *Species from Caucasus*

*Hedysarum daghestanicum* Rupr.

ex. Boiss.

**Table 1.**

content in plant raw materials as well as biological fluids [10–15]. The

High-performance liquid chromatography, photocolorimetry, complexonometry, and chromato spectrophotometry are most commonly used to quantify mangiferin

**Altitude above sea level**

**Ecological and biological features**

**of growth**

1250 m Deep location of river valleys

1100 m The slope of the southeast

1250 m Deep location of river valleys

between mountain spurs

exposition, steepness 30°

between mountain spurs

460 m Steepened rocky-fine-crushed steep section at the top of the slope

850 m Rocky-fine-brimmed steep slope of northeastern exposition

photocolorimetric definition of mangiferin in both the raw material and crystalline powder has a number of advantages, such as shortening the analysis steps and replacing the deficient vegetable, tetrahydrofuran, with dioxane. The method is based on mangiferin's ability to produce a complex compound with chlorine iron. Mangiferin was determined from a calibration plot of the solution optical density versus mangiferin concentration. When comparing spectrophotometric and

photocolorimetric methods, almost the same results were obtained [5, 10, 12, 16, 17]. As for the current method of high-performance liquid chromatography, according to literature, separation of xanthone glycosides by HPLC method could not be achieved on sorbents containing amino and cyano groups [7, 18–23]. The best results were obtained on reverse phase С18 sorbents, and methanol-water, ethanol-water, and acetonitrile-acetic acid were used as the mobile phase. In aqueous systems, the shape of the mangiferin peak deteriorated. To quantify mangiferin

The mobile phase was acetonitrile and a 3% CH3COOH solution at a ratio of 16:84 was chromatographed at a wavelength of 254 nm using an external standard

Furthermore, in the quantitative determination of mangiferin by HPLC, a system consisting of acetonitrile, water, and phosphoric acid was used as the mobile phase. The selected conditions allowed to achieve a clear separation of mangiferin and isomangiferin peaks on chromatogram. In addition to the previous systems, methanol, tetrahydrofuran, or acetonitrile, an aqueous solution of phosphoric acid in various ratios are used as the mobile phase in HPLC gradient elution. Of the latest techniques for the quantitative determination of mangiferin, liquid chromatography followed by mass spectrometric determination was used, and the method is

The chromate-spectrophotometric examination is based on sequential chromatography and spectrophotometry. The raw material is treated with the following

in biological fluids, a sensitive reverse phase HPLC technique is proposed

[17, 24, 25].

**151**

method [11, 26].

characterized by speed and quality [27–30].

The flowering phase of the *Hedysarum caucasicum*—the beginning and the middle of July, and the fruiting phase—the end of July—and the beginning of August [1]. For the genus *Hedysarum*, the following set of chromosomes is characteristic: 14, 16, and 48. For *Hedysarum caucasicum*, the chromosome number is 14 [9].

#### **2. Material and methods**

The object of the study was the grass of *Hedysarum caucasicum* Bieb. family legumes (Fabaceae), which were collected in the flowering phase on the southeast slope of Mount Alibek at an altitude of 2200 m (Dombay District, Russia).

Freshly harvested and dried samples of raw materials of the species of the genus *Hedysarum* L., including *H. caucasicum* M. Bieb., *H. daghestanicum* Rupr. ex Boiss., and *H. grandiflorum* Pall. were obtained (**Table 1**). The collected test raw materials were dried without direct sunlight in the air.

**Research methods:** comparative morphological, ecological-geographical, molecular-genetic, morphological-anatomical and histochemical, and phytochemical (UV-spectrophotometry, HPLC, chromato-mass spectrometry, capillary electrophoresis).



**Table 1.**

long. The fruit is a bob of 3–6 arthropods, naked or desiccated; the mushroom of the

We did not meet the information on the study of anatomical features of the structure of the organs in the accessible literature. The onthogenesis of species *Hedysarum* usually includes four periods and 10 age states. In the example of *H. austrosibiricum*, age spectra can be divided into four types, which probably

During 3 years of life, alpine individuals undergo the following age conditions: in the first year—seedlings, juvenile, immature, and more than 60% of individuals are adult vegetative; in the second year, more than 90% of individuals enter a young reproductive state; and in the third year, all individuals transition to a reproductive state. Seeds of tested plants need pre-sowing treatment. Mechanical scarification and treatment with concentrated sulfuric acid are the best ways to disrupt the rest

The flowering phase of the *Hedysarum caucasicum*—the beginning and the middle of July, and the fruiting phase—the end of July—and the beginning of August [1]. For the genus *Hedysarum*, the following set of chromosomes is characteristic: 14, 16, and 48. For *Hedysarum caucasicum*, the chromosome number is 14 [9].

The object of the study was the grass of *Hedysarum caucasicum* Bieb. family legumes (Fabaceae), which were collected in the flowering phase on the southeast

**Research methods:** comparative morphological, ecological-geographical, molecular-genetic, morphological-anatomical and histochemical, and phytochemical (UV-spectrophotometry, HPLC, chromato-mass spectrometry, capillary

Freshly harvested and dried samples of raw materials of the species of the genus *Hedysarum* L., including *H. caucasicum* M. Bieb., *H. daghestanicum* Rupr. ex Boiss., and *H. grandiflorum* Pall. were obtained (**Table 1**). The collected test raw materials

> **Altitude above sea level**

**Ecological and biological features**

**of growth**

2100 m Open moistened areas among large boulders

2200 m Southeastern slope of Alibek, gorge of Mount Alibek

between mountain spurs

between mountain spurs

1250 m Deep location of river valleys

200 m Deep location of river valleys

slope of Mount Alibek at an altitude of 2200 m (Dombay District, Russia).

The microstructure of the above-ground parts of some members of the *Hedysarum* was studied by Ladygina et al. [4]. The features of the anatomical structure of leaves of five species of *Hedysarum* from the section *Gamotion* Basin were studied. These species containing mangiferin have been found to have bright

golden yellow fluorescence of epidermis cells, which can be used to detect

bean is oblong-elliptical, and its edge is not wide.

reflect ecological-phytocenotic growth conditions [6].

mangiferin directly in plant material [5].

*Legume Crops - Prospects, Production and Uses*

of both species [6–8].

**2. Material and methods**

electrophoresis).

M. Bieb.

*Hedysarum grandiflorum* Pall.

**150**

*Hedysarum caucasicum*

were dried without direct sunlight in the air.

**Species № Geographical**

**coordinates**

<sup>46</sup>″,

17″,

32,21″,

49,1"

56″,

16"

0"

65"

1. N—43°43<sup>0</sup>

2. N—40°30<sup>0</sup>

3. N—42°19<sup>0</sup>

4. N—49°45<sup>0</sup>

E—42°53<sup>0</sup>

E—44°40<sup>0</sup>

E—47°09<sup>0</sup>

E—44°16<sup>0</sup>

*List of objects of study with indication of ecological and biological features of growth.*
