**11. Quantification of mangiferin in grass of** *Hedysarum* **species by capillary electrophoresis**

For the study of these species of the genus *Hedysarum* capillary electrophoresis "Kapel - 105m" (Lumex Marketing OJSC, Russia), quartz capillary (Leff/Ltol = 50/ 60 cm, ID = 75 μm) was used. The quartz capillary was previously washed successively with purified water, 1 M aqueous solutions of sodium hydroxide and hydrochloric acid.

The optical density of the prepared solution was measured on a spectrophotometer in the wavelength range of 200–500 nm. For this purpose, an aliquot of 1 ml was placed in two measuring flasks with a capacity of 25 ml. In one of the measuring flasks, the solutions were labeled with alcohol 70% ethyl, and in the other with borate buffer solution 0.01 M. As comparison solutions, ethyl alcohol 70% was used in the first case, and in the second case, borate buffer solution 0.01 M was used. A shift of the maximum light absorption of mangiferin from 365 nm to 383 nm in the borate buffer solution was observed, which may be due to the formation of a complex of mangiferin with sodium tetraborate.

Solution B with the following concentrations (mg/ml): 0.35; 0.25; 0.15; and 0.05. For this, aliquots of solution B (1; 0.7; 0.5; 0.3; and 0.1 ml) were placed in 1 ml Eppendorf tubes. 0, 0.3, 0.5, 0.7, and 0.9 ml of 70% ethyl alcohol were added. Centrifugation of the solutions was carried out for 5 min at 8000 rpm.

**Figure 5.**

**Figure 4.**

Hedysarum *Species from Caucasus*

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

**Figure 6.**

**161**

*Electrophoregram of extraction of* Hedysarum caucasicum.

*Calibration graph of peak area versus mangiferin concentration in solution.*

*Electrophoregram of extraction of* Hedysarum grandiflorum *pall.*

Capillary electrophoresis analysis was carried out at +20 kV, with capillary temperature of + 20°C, and detection was carried out spectrophotometrically at a wavelength of 383 nm, and the analysis time was 10 min. As an electrolyte, a borate buffer solution 0.01 M with a pH of 9.2 0.02 was used, prepared in accordance with GOST 4919.2-2016 "Reagents and especially pure substances. Methods for the preparation of buffer solutions." Previously the capillary was washed consistently with solutions of acid hydrochloric 1 M and sodium hydroxide 1 M.

Washing between acid and alkali solutions, as well as final washing before analysis, was carried out with purified water. Washing solutions and electrolyte solutions were filtered through a Vladipor paper filter of type with a membrane diameter of 25 mm. The buffer solutions, like the test solutions, were centrifuged at 8000 rpm for 5 min. The results were processed and a calibration plot was plotted (**Figure 4**).

The preparation of purified alcohol extracts from the raw material was carried out in accordance with the procedure developed by us to determine the sum of xanthones in terms of mangiferin by UV spectrophotometry for the samples under study (the procedure described above in the UV spectrophotometry section) (**Figures 5**–**7**). Centrifugation of solution A was carried out for 5 min at 8000 rpm.

Based on the experimental data obtained, it can be concluded that the Caucasian penny is the largest content of mangiferin among the studied species of the genus,

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

**Figure 4.** *Calibration graph of peak area versus mangiferin concentration in solution.*

**Figure 5.** *Electrophoregram of extraction of* Hedysarum caucasicum.

**Figure 6.** *Electrophoregram of extraction of* Hedysarum grandiflorum *pall.*

**11. Quantification of mangiferin in grass of** *Hedysarum* **species by**

For the study of these species of the genus *Hedysarum* capillary electrophoresis "Kapel - 105m" (Lumex Marketing OJSC, Russia), quartz capillary (Leff/Ltol = 50/ 60 cm, ID = 75 μm) was used. The quartz capillary was previously washed successively with purified water, 1 M aqueous solutions of sodium hydroxide and

**Species The content of the sum of xanthones**

*The content of the sum of xanthones in terms of mangiferin in the above-ground part of species of the genus*

*Hedysarum caucasicum* M. Bieb. 0.62 0.02 *Hedysarum grandiflorum* Pall. 0.60 0.02 *Hedysarum daghestanicum* Rupr. ex Boiss. 0.56 0.01

Hedysarum *L. by the value of the specific absorption index of mangiferin.*

**in terms of mangiferin, %**

The optical density of the prepared solution was measured on a spectrophotometer in the wavelength range of 200–500 nm. For this purpose, an aliquot of 1 ml was placed in two measuring flasks with a capacity of 25 ml. In one of the measuring flasks, the solutions were labeled with alcohol 70% ethyl, and in the other with borate buffer solution 0.01 M. As comparison solutions, ethyl alcohol 70% was used in the first case, and in the second case, borate buffer solution 0.01 M was used. A shift of the maximum light absorption of mangiferin from 365 nm to 383 nm in the borate buffer solution was observed, which may be due to the formation of a

Solution B with the following concentrations (mg/ml): 0.35; 0.25; 0.15; and 0.05.

For this, aliquots of solution B (1; 0.7; 0.5; 0.3; and 0.1 ml) were placed in 1 ml Eppendorf tubes. 0, 0.3, 0.5, 0.7, and 0.9 ml of 70% ethyl alcohol were added.

Capillary electrophoresis analysis was carried out at +20 kV, with capillary temperature of + 20°C, and detection was carried out spectrophotometrically at a wavelength of 383 nm, and the analysis time was 10 min. As an electrolyte, a borate buffer solution 0.01 M with a pH of 9.2 0.02 was used, prepared in accordance with GOST 4919.2-2016 "Reagents and especially pure substances. Methods for the preparation of buffer solutions." Previously the capillary was washed consistently

Washing between acid and alkali solutions, as well as final washing before analysis, was carried out with purified water. Washing solutions and electrolyte solutions were filtered through a Vladipor paper filter of type with a membrane diameter of 25 mm. The buffer solutions, like the test solutions, were centrifuged at 8000 rpm for 5 min. The results were processed and a calibration plot was plotted

The preparation of purified alcohol extracts from the raw material was carried out in accordance with the procedure developed by us to determine the sum of xanthones in terms of mangiferin by UV spectrophotometry for the samples under study (the procedure described above in the UV spectrophotometry section) (**Figures 5**–**7**). Centrifugation of solution A was carried out for 5 min at 8000 rpm. Based on the experimental data obtained, it can be concluded that the Caucasian penny is the largest content of mangiferin among the studied species of the genus,

Centrifugation of the solutions was carried out for 5 min at 8000 rpm.

with solutions of acid hydrochloric 1 M and sodium hydroxide 1 M.

**capillary electrophoresis**

*Legume Crops - Prospects, Production and Uses*

complex of mangiferin with sodium tetraborate.

hydrochloric acid.

**Table 6.**

(**Figure 4**).

**160**

**Figure 7.** *Electrophoregram of extraction of* Hedysarum daghestanicum *Rupr. ex Boiss.*

which confirms the assumption based on molecular genetic studies, since it is this species that belongs to the Obscura section, as well as the alpine penny used to obtain mangiferin.
