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

High-performance liquid chromatography, photocolorimetry, complexonometry, and chromato spectrophotometry are most commonly used to quantify mangiferin content in plant raw materials as well as biological fluids [10–15]. The 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 in biological fluids, a sensitive reverse phase HPLC technique is proposed [17, 24, 25].

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 method [11, 26].

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 characterized by speed and quality [27–30].

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

extractant system—acetone:water in ratio 1:1 with acidification with 5% hydrochloric acid followed by chromatography in the system with 15% acetic acid. The eluation of the zones with mangiferin is preparing after viewing the chromatogram in UV light. The optical density of the solutions was measured at 372 nm. The mangiferin content was calculated from the specific absorption index.

There are a number of intense absorption bands in the UV spectrum of mangiferin. The most convenient to quantify a substance is a band with a maximum at 369 nm and a specific absorption coefficient of 295 0.92. In the field of working concentrations, the absorption of mangiferin solutions is subject to Lambert-Beer law [23].

Analysis of literary sources has shown that the genus *Hedysarum* L. combines about 285 species, which are predominantly common in Eurasia. *H. caucasicum* Bieb. is endemic, growing in the highland meadows of the Caucasus. In a literary search, it has been found that chemical study information relates mainly to *H. alpinum* and *H. flavescens*. Information on the chemical study of *H. caucasicum* is fragmented and insufficient. Plants of the genus *Hedysarum* are widely used in folk and waitinal medicine, as an antibacterial, antiviral, immunomodulatory, and antiinflammatory agent [6, 8, 17, 26, 31–39]. The following methods of analysis are used to identify and quantify the main active substances of xanthones: spectrophotometry, photocolorimetry, and chromatographic methods of analysis (TLC, HPLC, and mass spectrometry) [40–42]. The study of theoretical bases of extraction of medicinal raw materials allows to find the optimal conditions of technology for creation of medicinal forms on the basis of herbal of Caucasus.

### **4. Results**

*Morphological study*: Life-form—a perennial herbaceous plant 40–50 cm high, and underground organs reach 30 cm length. The escape is elongated, branched, straight-standing, or raised. The number of leaflets located on rachis varies from 11 to 15. The leaves have an egg shape, a rounded base, a whole edge, and a spiky top. Decaying of leaflets is insignificant. The flower is simple botriode, brush. The flower is zygomorphic. The cup consists of five cups, a moth-type crown, and the color of the petals is pink-purple. The android consists of nine converged tangles and one free. Ginecey is monocarpal. Tie the top one. Fruit: according to morphogenetic classification of fruits, it refers to monocarp; according to morphological bob, flat, oblong in shape, and consists of rounded arthropods. Number of squads range from 3 to 5.

sclerenchym of pericyclic origin. The leaflets are arranged on small cherries, which have cylindrical shape on the cross section. The covering tissue of the cherry is represented by an epidermal, under it there is a collenchym in 1–2 layers, and the main volume occupies chlorenchyma. The collateral conducting bundle surrounded by sclerenchym is located in central part of cut. The rachis at the bottom has a grooved shape with a deep horseshoe spoon on the abaxial side. Collateral

*Microscopic signs of the above-ground organs of* Hedysarum caucasicum *M. Bieb. (А)* Hedysarum daghestanicum *Rupr. ex Boiss. (B)* Hedysarum grandifloru*m Pall. (C) 1. Transversal section of the stem in the lower part; 2. transversal section of the leaf petiole in the lower part; 3. transverse slice of leaf plate; 4. the*

*adaxial epidermis of the leaf blade; and 5. the abaxial epidermis of the leaf blade.*

conducting bundles of 6–8 are arranged in an arc-like manner. It is interesting that idioblasts with yellow content are found in the phloem, in the pericyclic zone, and in the parenchyma of the core adjacent to the xylem portion of the conductive

The stem has a polyhedral shape on the cross section. A cavity may form in the central part of the stem. Under the epidermal continuous ring in 1–2 layers of cells, there is a plate-type collenchym. In the projection regions, the number of layers of collenchym cells increases to 5–7. Chlorenchym is located following collenchym in discontinuous regions in 2–3 layers of cells. The pericyclic zone is represented by sclerenchymal fibers arranged in discontinuous areas above the conducting beams. Conducting system is of bundle type. Conducting beams are of open collateral type

The cambium forms secondary conductive beams and thick-walled parenchyma, which is subsequently strongly ligated. Idioblasts are found, as in the cherry, in the phloem part of the conducting beam, in the pericyclic zone, as well as in the parenchyma of the core. The abovementioned micromorphological characteristics of the above-ground organs of the *Hedysarum caucasicum* can be used in drawing up regulatory documentation for the Hedysarum caucasicum grass as an additional raw

beam.

**153**

**Figure 3.**

Hedysarum *Species from Caucasus*

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

in an amount of 14–16 (**Figure 3**).

material source of xanthone glycoside mangiferin.

The leaf is amphistomatic. The upper epidermal has weak and almost straight anticline cell walls. The mouth is abnormal, surrounded usually by 3–5 near-oral cells. Trichomes were not detected on the abaxial side of the leaf. The lower epidermal has strong anticline cell walls. An anomocytic type oyster is surrounded by usually 3–5 parotid cells. The embossing is formed by simple single-cell hairs located either in the region of the veins or on the edge of the leaf plate. When considering the sheet microreparation from the surface, rhombic calcium oxalate crystals located in large veins are found. They form a characteristic crystalline lining of the veins. On the cross section, the leaves have a characteristic dorsoventrale structure (**Figure 3**).

The palisade mesophyll localizes only under the upper epidermis in one layer, its cells are stretched, tightly pressed to each other. The mechanical fabric is a collenchym which is located both under the upper and lower epidermis in the region of the core. In the central part of the main core, there is a large collateral conducting bundle, and from the dorsal side to the conducting bundle there is a

#### **Figure 3.**

extractant system—acetone:water in ratio 1:1 with acidification with 5%

*Legume Crops - Prospects, Production and Uses*

mangiferin content was calculated from the specific absorption index.

medicinal forms on the basis of herbal of Caucasus.

law [23].

**4. Results**

range from 3 to 5.

(**Figure 3**).

**152**

There are a number of intense absorption bands in the UV spectrum of mangiferin. The most convenient to quantify a substance is a band with a maximum at 369 nm and a specific absorption coefficient of 295 0.92. In the field of working concentrations, the absorption of mangiferin solutions is subject to Lambert-Beer

hydrochloric acid followed by chromatography in the system with 15% acetic acid. The eluation of the zones with mangiferin is preparing after viewing the chromatogram in UV light. The optical density of the solutions was measured at 372 nm. The

Analysis of literary sources has shown that the genus *Hedysarum* L. combines about 285 species, which are predominantly common in Eurasia. *H. caucasicum* Bieb. is endemic, growing in the highland meadows of the Caucasus. In a literary search, it has been found that chemical study information relates mainly to *H. alpinum* and *H. flavescens*. Information on the chemical study of *H. caucasicum* is fragmented and insufficient. Plants of the genus *Hedysarum* are widely used in folk and waitinal medicine, as an antibacterial, antiviral, immunomodulatory, and antiinflammatory agent [6, 8, 17, 26, 31–39]. The following methods of analysis are used to identify and quantify the main active substances of xanthones: spectrophotometry, photocolorimetry, and chromatographic methods of analysis (TLC, HPLC, and mass spectrometry) [40–42]. The study of theoretical bases of extraction of medicinal raw materials allows to find the optimal conditions of technology for creation of

*Morphological study*: Life-form—a perennial herbaceous plant 40–50 cm high, and underground organs reach 30 cm length. The escape is elongated, branched, straight-standing, or raised. The number of leaflets located on rachis varies from 11 to 15. The leaves have an egg shape, a rounded base, a whole edge, and a spiky top. Decaying of leaflets is insignificant. The flower is simple botriode, brush. The flower is zygomorphic. The cup consists of five cups, a moth-type crown, and the color of the petals is pink-purple. The android consists of nine converged tangles and one free. Ginecey is monocarpal. Tie the top one. Fruit: according to morphogenetic classification of fruits, it refers to monocarp; according to morphological bob, flat, oblong in shape, and consists of rounded arthropods. Number of squads

The leaf is amphistomatic. The upper epidermal has weak and almost straight anticline cell walls. The mouth is abnormal, surrounded usually by 3–5 near-oral cells. Trichomes were not detected on the abaxial side of the leaf. The lower epidermal has strong anticline cell walls. An anomocytic type oyster is surrounded by usually 3–5 parotid cells. The embossing is formed by simple single-cell hairs located either in the region of the veins or on the edge of the leaf plate. When considering the sheet microreparation from the surface, rhombic calcium oxalate crystals located in large veins are found. They form a characteristic crystalline lining of the veins. On the cross section, the leaves have a characteristic dorsoventrale structure

The palisade mesophyll localizes only under the upper epidermis in one layer, its

cells are stretched, tightly pressed to each other. The mechanical fabric is a collenchym which is located both under the upper and lower epidermis in the region of the core. In the central part of the main core, there is a large collateral conducting bundle, and from the dorsal side to the conducting bundle there is a *Microscopic signs of the above-ground organs of* Hedysarum caucasicum *M. Bieb. (А)* Hedysarum daghestanicum *Rupr. ex Boiss. (B)* Hedysarum grandifloru*m Pall. (C) 1. Transversal section of the stem in the lower part; 2. transversal section of the leaf petiole in the lower part; 3. transverse slice of leaf plate; 4. the adaxial epidermis of the leaf blade; and 5. the abaxial epidermis of the leaf blade.*

sclerenchym of pericyclic origin. The leaflets are arranged on small cherries, which have cylindrical shape on the cross section. The covering tissue of the cherry is represented by an epidermal, under it there is a collenchym in 1–2 layers, and the main volume occupies chlorenchyma. The collateral conducting bundle surrounded by sclerenchym is located in central part of cut. The rachis at the bottom has a grooved shape with a deep horseshoe spoon on the abaxial side. Collateral conducting bundles of 6–8 are arranged in an arc-like manner. It is interesting that idioblasts with yellow content are found in the phloem, in the pericyclic zone, and in the parenchyma of the core adjacent to the xylem portion of the conductive beam.

The stem has a polyhedral shape on the cross section. A cavity may form in the central part of the stem. Under the epidermal continuous ring in 1–2 layers of cells, there is a plate-type collenchym. In the projection regions, the number of layers of collenchym cells increases to 5–7. Chlorenchym is located following collenchym in discontinuous regions in 2–3 layers of cells. The pericyclic zone is represented by sclerenchymal fibers arranged in discontinuous areas above the conducting beams. Conducting system is of bundle type. Conducting beams are of open collateral type in an amount of 14–16 (**Figure 3**).

The cambium forms secondary conductive beams and thick-walled parenchyma, which is subsequently strongly ligated. Idioblasts are found, as in the cherry, in the phloem part of the conducting beam, in the pericyclic zone, as well as in the parenchyma of the core. The abovementioned micromorphological characteristics of the above-ground organs of the *Hedysarum caucasicum* can be used in drawing up regulatory documentation for the Hedysarum caucasicum grass as an additional raw material source of xanthone glycoside mangiferin.

A comparative morphological-anatomical study of three species of the genus *Hedysarum* L., growing in the North Caucasus, was carried out. The most significant diagnostic morphological features have been established, which provide to establish the species belonging of the medicinal raw materials specified in **Table 2**.

rachis of the complex sheet is represented by three large and 2–7 small conducting

Thus, the rachis zone is usually characterized by 2–4 additional conductive beams, or they are completely absent. The number and arrangement of conducting beams seem to us most interesting and more informative from the point of view of diagnostics of this species, growing under various environmental conditions (**Figure 1**). When conducting a comprehensive morphological-anatomical study of three species of the genus *Hedysarum* L., the morphological-anatomical diagnostic

By the moisture content of the raw material, it is meant a loss in mass due to hygroscopic moisture and volatile substances, which is determined by drying to a constant mass. Medicinal plant raw materials should not contain moisture above permissible standards. Moisture content of the analyzed raw material was

determined by the method of drying by Pharmacopea XIV [14]. The weight loss on

The ash of plant raw materials refers to the residue of inorganic substances obtained after burning the raw materials and then calcining the residue to a constant weight. Plant ash (total ash) consists of a mixture of various inorganic substances in the plant itself and mineral impurities (earth, sand, dust, and stones) that can enter the raw materials when collected and dried. The common ash most commonly contains the following elements: Na, K, Ca, Mg, Fe, Si, F, P, and C, which are in the form of oxides or salts of carbonic, phosphoric, sulfuric, and other acids. Ash determination of total (x) was carried out by Pharmacopea of Russia. The

total ash content was 4.04% (average of two parallel determinations).

For research, the grass of the *Hedysarum caucasicum* Bieb. family legumes (*Fabaceae*) were collected in the flowering phase on the southeast slope of Mount Alibek (Dombay Gorge District, CHR) and used. According to amendment No. 3 to the article of Pharmacopea of the publication "Methods of microbiological control of medicines," introduced on June 19, 2003, the studied plant raw materials belong to the category 4Б—medicinal plant preparations and medicinal plant raw materials "angro," prepared without the use of boiling water. The requirements for this

• Total number of aerobic bacteria—no more than 105 in 1 g or 1 ml.

• Total number of fungi—not more than 104 in 1 g or in 1 ml.

the rib zone, also reinforced by sclerenchyma on the phloem side.

features were revealed, which are presented in this chapter.

drying was 8.60% (average of two parallel determinations).

**5. Determination of humidity**

**6. Determination of total ash**

**7. Microbiological purity test**

category are as follows:

**155**

All conducting bundles have a pronounced collateral structure, while one large, dorsal conducting bundle is located on the adaxial side, and it has a characteristic rounded or semilunar shape and is reinforced with sclerenchymal fibers on the phloem side. Two ventral conducting bundles are smaller and are usually located in

bundles (**Figure 3**).

Hedysarum *Species from Caucasus*

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

A comparative analysis of the anatomical structure of the stem revealed common features, which include the multifaceted shape of the stem on the cross section, and the pubescence is represented by simple bicellular hairs, which have a narrowed base and an expanded structure, a characteristic feature of the hairs is the wariness of the cuticle (**Figure 3**).

In a comparative study of the anatomical structure of the caulifoliar system, the main attention was drawn to the anatomical structure of the petiole and rachis of a complex leaf. The following differences were revealed—the shape on the cross section of the petiole of a complex sheet changes from a clearly saddle-shaped to a rounded-triangular shape, the number of ribs on the abaxial side changes.

Differences were identified regarding the structure and location of collenchyma, sclerenchyma, and the conducting system. The conductive system of the petiole and


**Table 2.**

*Comparative morphological characterization of* Hedysarum *L. species growing in the North Caucasus.*

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

A comparative morphological-anatomical study of three species of the genus *Hedysarum* L., growing in the North Caucasus, was carried out. The most significant diagnostic morphological features have been established, which provide to establish

A comparative analysis of the anatomical structure of the stem revealed common features, which include the multifaceted shape of the stem on the cross section, and the pubescence is represented by simple bicellular hairs, which have a narrowed base and an expanded structure, a characteristic feature of the hairs is the wariness

In a comparative study of the anatomical structure of the caulifoliar system, the main attention was drawn to the anatomical structure of the petiole and rachis of a complex leaf. The following differences were revealed—the shape on the cross section of the petiole of a complex sheet changes from a clearly saddle-shaped to a

Differences were identified regarding the structure and location of collenchyma, sclerenchyma, and the conducting system. The conductive system of the petiole and

> The brushes on long legs, 11/2–2 times longer than the leaves, are not very thick. The lower tooth of the calyx is equal to the tube, the rest are shorter. Dark-purple petals

The brushes are multi-flowered, with deflected flowers. Bracts are lanceolate, light brown, hairy, bellshaped calyx, its teeth are linearawl-shaped, densely thawedhairy, Corolla yellow

The brushes are small-flowered, thick. The flowers are large, creamy white or purple. The calyx is four times shorter than the corolla. The boat is shorter than the flag and two times longer than the wings. The flowers are large, cream-white

The segments are not large, smooth, or slightly toothed

Beans 2–5 segmented, segments rounded, densely whitehaired, meshribbed, along the edges with spines bent inside

Beans from 2 to 4 segments. Lentilshaped, warty segments

the species belonging of the medicinal raw materials specified in **Table 2**.

rounded-triangular shape, the number of ribs on the abaxial side changes.

**Species Life-form Leaves Flowers Fruits**

The leaves are scattered throughout the stem, from 7 to 12 pairs of elliptical or ovate oblong leaflets with a pointed tip at the apex. Pubescence is not developed

The leaves are petiole, short pressed-hairy and, long spaced pubescent. Leaflets 1–4-paired, ovate, or broadly elliptical, and large, slightly hairy above, less often glabrous, densely silvery-silky below

Ten leaves with 3–4 pairs of leaflets, leaflets oblong, up to 18 mm long and up to 8 mm wide. The apical leaf is larger. Accrete stipules

*Comparative morphological characterization of* Hedysarum *L. species growing in the North Caucasus.*

of the cuticle (**Figure 3**).

*Legume Crops - Prospects, Production and Uses*

*Hedysarum caucasicum* M. Bieb.

*Hedysarum grandiflorum* Pall.

*Hedysarum daghestanicum* Rupr. ex Boiss.

**Table 2.**

**154**

High plant, 30– 50 cm tall. The lower internodes are not shortened

Perennial 20–40 cm tall. Stemless or almost stemless. Stipules are large, membranous, fused, brown, and scattered-hairy

Stem-free rod-root perennial. Peduncles with brushes 10–25 cm long. All parts of the plant have a grayish plaque

rachis of the complex sheet is represented by three large and 2–7 small conducting bundles (**Figure 3**).

All conducting bundles have a pronounced collateral structure, while one large, dorsal conducting bundle is located on the adaxial side, and it has a characteristic rounded or semilunar shape and is reinforced with sclerenchymal fibers on the phloem side. Two ventral conducting bundles are smaller and are usually located in the rib zone, also reinforced by sclerenchyma on the phloem side.

Thus, the rachis zone is usually characterized by 2–4 additional conductive beams, or they are completely absent. The number and arrangement of conducting beams seem to us most interesting and more informative from the point of view of diagnostics of this species, growing under various environmental conditions (**Figure 1**). When conducting a comprehensive morphological-anatomical study of three species of the genus *Hedysarum* L., the morphological-anatomical diagnostic features were revealed, which are presented in this chapter.
