**6. Determination of total ash**

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).

#### **7. Microbiological purity test**

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 category are as follows:


• Enterobacteria and other gram-negative bacteria—not more 103 1 g or 1 ml.

under reflux. The resulting solution was filtered through a paper filter after cooling.

*Cyanidine sample:* 0.1 g of magnesium dust, 2 ml of concentrated hydrochloric acid were added to 1 ml of extraction, heated in a water bath for 2–3 min, and after some time red-orange staining was observed; 2 drops of 2% basic lead acetate solution were added to 1 ml of the recovery, and yellow-lemon staining appeared; 1 ml of a 10% ammonia solution was added to 1 ml of the recovery, and yellow staining turned orange on heating appeared. 1 ml of a 2% solution of aluminum chloride in 96% ethyl alcohol was added to 1 ml of the recovery, and lemon-yellow

Qualitative reactions with these reagents showed the presence of flavonoid substances in the grass of the Caucasus penny, which allowed us to use the chromatography method for further analysis, which is widely used for their detection and identification. Chromatographic separation of the sum of flavonoids and xanthones was carried out in the preparation of the extracts, and ethyl alcohol of 96, 80, 60, and 40% concentration was used as the extractant. 0.05 ml of the *Hedysarum caucasicum* Bieb. extracts were applied to a 40 40 cm Whatman chromatographic paper and subjected to ascending chromatography in a solvent system: butanolglacial acetic acid-water in a ratio of 4:1:5 compared to witness substances. When viewing the chromatogram in UV light, three main spots were found in extracts of the following concentrations of ethanol 96:80:60%. First spot corresponds to mangiferin, second to hyperoside, and third to campferol. Further, chromatograms were sprayed with alcohol solution AlCl3, and a change in stain color was observed.

**9. Qualitative detection and quantification of xanthones**

glycoside-mangiferin was present in these samples (**Table 3**).

**10. Quantification of the sum of xanthones mangiferin by UV**

Thin layer chromatography was used for qualitative detection of mangiferin. Chromatography was carried out in systems: n-butanol-acetic acid-water (4:1:5); chloroform-methanol-water (13:7:2); and 15% acetic acid, on "Sorbfil PTCC-AF-A"

The development of the plates was carried out by spraying with the following reagents: a solution of iron(III) chloride of 2%, an alcoholic solution of aluminum chloride of 1%, and ammonia vapors and UV radiation (fluorescent lamp UV-A). As a result of the TLC study of the extraction of raw materials there are seventy percent ethyl alcohol compared to standard samples indicated that xanthone

The content of the sum of xanthones in the test subjects in terms of mangiferin was calculated in two ways, using the optical density of the solution of the standard sample of mangiferin and the value of the specific absorption index of mangiferin under similar conditions. *Determination of specific value of mangiferin uptake:* A precise suspension of a standard mangiferin sample (about 0.01 g) was placed in a measuring flask with a capacity of 25 ml, 20 ml of 70% ethyl alcohol was added, stirred until the standard sample was completely dissolved, and the volume of solution was adjusted to a mark in the flask. Aliquots from the resulting solution were placed in measuring flasks with a capacity of 25 ml and labeled with the same solvent. The optical density was measured on a spectrophotometer at a wavelength

Reactions were carried out with the resulting solution.

staining was observed [31].

Hedysarum *Species from Caucasus*

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

plates 10 10 cm and 10 15 cm.

**spectrophotometry**

**157**


Before preparing the dosage form, the studied vegetable raw material of the *Hedysarum caucasicum* Bieb. was tested for microbiological purity. The presented results make it possible to conclude that according to the indicator "microbiological purity," the sample of vegetable raw materials of the *Hedysarum caucasicum Bieb.*, presented for analysis, meets the requirements for medicinal vegetable raw materials "angro," used without thermal treatment.

#### **8. High-quality phytochemical analysis**

*Determination of tanning substances*: About 1.0 g of raw material was poured with 100.0 g of water, heated for 20–30 min in a water bath and filtered. The following reactions were carried out with the resulting solution [15]: Several drops of iron ammonium alum were added to 2 ml of the solution, and black and green staining appeared, indicating the presence of condensed tanning agents; a few drops of a 1% solution of quinine hydrochloric acid were added to 2 ml of the solution, and opalescence appeared.

*Determination of polysaccharides*: For qualitative detection of polysaccharides, water extraction was prepared from 2.0 g of alpine penny roots in a water bath for 30 min. Then it was filtered off and the filter was washed with hot water. The recovery was evaporated to 1/5 volume and three times the volume of 96% ethanol was added thereto. As a result, a loose curd precipitate of the polysaccharide complex was formed. The precipitate was separated, dissolved in water, reprecipitated, washed with alcohol, and dried. The obtained polysaccharide complex is an amorphous mass soluble in water. In the composition of water-soluble polysaccharides of the penny, mucous substances predominate [43].

*Definition of the restoring sugars*: About 1.0 of the milled raw material was placed in a 25 ml flask, poured with 10 ml of water, and refluxed for 0.5 h. The solution was filtered through gauze and washed with water. 5 ml of the resulting solution was transferred to a tube and 15 ml of 95% ethyl alcohol was added. Precipitation of the bulk precipitate was observed. The solution was filtered, the precipitate was transferred to a test tube, 5 ml of diluted hydrochloric acid was added, boiled for several minutes, 5 ml of Feling reagent was added and boiled again, and orange-red staining was observed [14].

*Determination of free organic acids:* A 1:10 decoction was prepared from Caucasus penny grass while heating in a water bath for 1 h. Broth was filtered. Five drops of digestion were placed in the tube and adjusted to 1 ml with purified water. One drop of the methyl red indicator was added, and red staining was observed, indicating the presence of organic acids [15].

*Definition of amino acids:* For qualitative detection of amino acids, reaction with 0.1% solution of ninhydrin in n-butanol on filter paper was used, and characteristic blue-violet staining appeared in formation of Rueman complex [15].

*Determination of flavonoids and xanthones:* In order to determine flavonoids, it was necessary to obtain an alcohol extract from the raw material. Extraction was carried out with 80% ethyl alcohol. About 1 g of the feed was placed in a 25 ml flask, 10 ml of 80% ethyl alcohol was added and heated in a water bath for 10–15 min

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

• Enterobacteria and other gram-negative bacteria—not more 103 1 g or 1 ml.

Before preparing the dosage form, the studied vegetable raw material of the *Hedysarum caucasicum* Bieb. was tested for microbiological purity. The presented results make it possible to conclude that according to the indicator "microbiological purity," the sample of vegetable raw materials of the *Hedysarum caucasicum Bieb.*, presented for analysis, meets the requirements for medicinal vegetable raw

*Determination of tanning substances*: About 1.0 g of raw material was poured with 100.0 g of water, heated for 20–30 min in a water bath and filtered. The following reactions were carried out with the resulting solution [15]: Several drops of iron ammonium alum were added to 2 ml of the solution, and black and green staining appeared, indicating the presence of condensed tanning agents; a few drops of a 1% solution of quinine hydrochloric acid were added to 2 ml of the solution, and

*Determination of polysaccharides*: For qualitative detection of polysaccharides, water extraction was prepared from 2.0 g of alpine penny roots in a water bath for 30 min. Then it was filtered off and the filter was washed with hot water. The recovery was evaporated to 1/5 volume and three times the volume of 96% ethanol was added thereto. As a result, a loose curd precipitate of the polysaccharide complex was formed. The precipitate was separated, dissolved in water, reprecipitated, washed with alcohol, and dried. The obtained polysaccharide complex is an amorphous mass soluble in water. In the composition of water-soluble polysaccharides of

*Definition of the restoring sugars*: About 1.0 of the milled raw material was placed in a 25 ml flask, poured with 10 ml of water, and refluxed for 0.5 h. The solution was filtered through gauze and washed with water. 5 ml of the resulting solution was transferred to a tube and 15 ml of 95% ethyl alcohol was added. Precipitation of the bulk precipitate was observed. The solution was filtered, the precipitate was transferred to a test tube, 5 ml of diluted hydrochloric acid was added, boiled for several minutes, 5 ml of Feling reagent was added and boiled again, and orange-red staining

*Determination of free organic acids:* A 1:10 decoction was prepared from Caucasus penny grass while heating in a water bath for 1 h. Broth was filtered. Five drops of digestion were placed in the tube and adjusted to 1 ml with purified water. One drop of the methyl red indicator was added, and red staining was observed, indicating

*Definition of amino acids:* For qualitative detection of amino acids, reaction with 0.1% solution of ninhydrin in n-butanol on filter paper was used, and characteristic

*Determination of flavonoids and xanthones:* In order to determine flavonoids, it was necessary to obtain an alcohol extract from the raw material. Extraction was carried out with 80% ethyl alcohol. About 1 g of the feed was placed in a 25 ml flask, 10 ml of 80% ethyl alcohol was added and heated in a water bath for 10–15 min

blue-violet staining appeared in formation of Rueman complex [15].

• Absence of *Escherichia coli*—in 1 g or 1 ml.

materials "angro," used without thermal treatment.

**8. High-quality phytochemical analysis**

the penny, mucous substances predominate [43].

opalescence appeared.

was observed [14].

**156**

the presence of organic acids [15].

• Absence of *Salmonella* in 10 g or 10 ml.

*Legume Crops - Prospects, Production and Uses*

under reflux. The resulting solution was filtered through a paper filter after cooling. Reactions were carried out with the resulting solution.

*Cyanidine sample:* 0.1 g of magnesium dust, 2 ml of concentrated hydrochloric acid were added to 1 ml of extraction, heated in a water bath for 2–3 min, and after some time red-orange staining was observed; 2 drops of 2% basic lead acetate solution were added to 1 ml of the recovery, and yellow-lemon staining appeared; 1 ml of a 10% ammonia solution was added to 1 ml of the recovery, and yellow staining turned orange on heating appeared. 1 ml of a 2% solution of aluminum chloride in 96% ethyl alcohol was added to 1 ml of the recovery, and lemon-yellow staining was observed [31].

Qualitative reactions with these reagents showed the presence of flavonoid substances in the grass of the Caucasus penny, which allowed us to use the chromatography method for further analysis, which is widely used for their detection and identification. Chromatographic separation of the sum of flavonoids and xanthones was carried out in the preparation of the extracts, and ethyl alcohol of 96, 80, 60, and 40% concentration was used as the extractant. 0.05 ml of the *Hedysarum caucasicum* Bieb. extracts were applied to a 40 40 cm Whatman chromatographic paper and subjected to ascending chromatography in a solvent system: butanolglacial acetic acid-water in a ratio of 4:1:5 compared to witness substances. When viewing the chromatogram in UV light, three main spots were found in extracts of the following concentrations of ethanol 96:80:60%. First spot corresponds to mangiferin, second to hyperoside, and third to campferol. Further, chromatograms were sprayed with alcohol solution AlCl3, and a change in stain color was observed.

### **9. Qualitative detection and quantification of xanthones**

Thin layer chromatography was used for qualitative detection of mangiferin. Chromatography was carried out in systems: n-butanol-acetic acid-water (4:1:5); chloroform-methanol-water (13:7:2); and 15% acetic acid, on "Sorbfil PTCC-AF-A" plates 10 10 cm and 10 15 cm.

The development of the plates was carried out by spraying with the following reagents: a solution of iron(III) chloride of 2%, an alcoholic solution of aluminum chloride of 1%, and ammonia vapors and UV radiation (fluorescent lamp UV-A). As a result of the TLC study of the extraction of raw materials there are seventy percent ethyl alcohol compared to standard samples indicated that xanthone glycoside-mangiferin was present in these samples (**Table 3**).
