INTRODUCTION:

The genus Oxytropis DC. of the family Fabaceae includes about 580 species of mostly caudex-forming herbaceous plants, less often shrubs or semi-shrubs with compound unpaired leaves, flowers of different colors, collected in raceme-like inflorescences, two flower petals fused to form a spicule, due to which the genus got its name, fruits - oblong, sometimes bloated legumes.

Plants are distributed mainly in the cold and temperate zones of the Northern Hemisphere, in the Arctic tundra and high mountains. Also, the genus Oxytropis DC. contains endemic species that grow in West and Central Asia. On the territory of the Russian Federation there are about 200 species, which grow mainly in mountainous areas of the Urals, Altai, Transbaikalia, of which 16 species are included in the Red Book^1,2,3.

Plants of the genus Oxytropis DC. contain a rich complex of biologically active substances: phenolic compounds (flavonoids and phenolcarboxylic acids), alkaloids, coumarins, saponins^4,5,6. The diversity of chemical composition determines anti-inflammatory, choleretic, diuretic, antiarrhythmic, analgesic, hypotensive, cardiotonic, antihypoxic and other properties of species of the genus Oxytropis DC. They are also of interest as sources of anti-allergic, antimicrobial agents, as well as for the treatment of intoxications of various etiologies^5,7. Different species are used in folk medicine mainly in eastern countries (Tibetan, Mongolian, etc.)⁸. Despite such a great variety of species of the genus Oxytropis DC, none of them is used in ophicinal medicine as a medicinal product.

One of the promising species of Oxytropis DC. Oxytropis rosea (Oxytropis rosea Bunge) is one of the promising species of Oxytropis DC. O. rosea is a herbaceous stemless plant, mainly found in the wild in mountain meadows and steppes in the mountainous regions of Central Asia and Kazakhstan, on the Western Tien Shan and Karatau in Kyrgyzstan. In folk medicine of Kyrgyzstan, this species of O. rosea is used as a sedative, analgesic, and for insomnia⁹.

The aim of the research was the phytochemical study of the herb and roots of O. rosea and comparative analysis of their antioxidant activity.

MATERIALS AND METHODS:

Materials:

The object of the study were herbs and roots of O. rosea, which were harvested from wild plants during the expedition in Jalal-Abad region of Kyrgyzstan in 2022. Plant parts were collected manually. The place of harvesting of plant raw materials - Tash-Komur reservoir, Babash-Ata ridge, altitude 900-1000 m above sea level. Identification of O. rosea was carried out using plant identifier¹⁰. Plant growing conditions in this area are characterized by great diversity - moderately cold winters and hot summers, which is associated with the orographic structure and sharp changes in relative altitudes of the area. Due to the large contrasts, the average annual air temperature varies differently in different places. The average annual air temperature in July is +19.1°C, while the average annual air temperature in January is - 0.2°C. Average annual precipitation ranges from 100-120mm at the bottom of the basin to 500 mm on the eastern slopes¹¹. Samples of roots and herbs of O. rosea harvested at different periods of vegetation were dried using air-shade drying and stored in paper bags at 25°C and humidity not more than 50%. Moisture content of raw materials was determined according to GFA.1.5.3.0007.15 "Determination of moisture content of medicinal plant raw materials and medicinal plant preparations"¹². The morphological description of the plant's raw materials was carried out according to microscopic analysis^13,14.

Qualitative reactions:

For qualitative reactions we used infusion from the herb, decoction from the roots, as well as water-alcoholic extracts from the herb and roots of O. rosea, in which we performed known chemical reactions for various groups of biologically active substances: flavonoids, alkaloids, saponins, coumarins, tannins, as well as TLC for the presence of ascorbic acid^{15,16,17,18,19,20}.

Quantitative determination of ascorbic acid:

The ascorbic acid content in the studied O. rosea raw material was determined by redox titration with 0.001 M solution of 2,6-dichlorophenolindophenolate sodium^{12, 21}.

To obtain extracts, 2.0g of dry crushed raw material was poured 50ml of purified water and insisted at room temperature for 30minutes. Then the extracts were filtered into a 50ml measuring flask and brought the flask with purified water to the mark. Then 1 ml of the extract was taken, 1ml of 2% hydrochloric acid solution, 13ml of purified water was added and titrated with 0.001 M sodium 2,6-dichlorophenolindophenolate solution to pink color.

The content of ascorbic acid in terms of absolutely dry raw material in percent (X) is calculated according to the formula:

V - 0.000088 . K. 50. 100. 100

X= ---------------------------------

A .1.(100-W)

Where

0.000088 - amount of ascorbic acid corresponding to 1ml of 0.001 M solution of 2,6-dichlorophenolindophenolate sodium, g;

V- volume of 0.001 M sodium 2,6-dichlorophenolindophenolate solution used for titration, ml;

a - weight of raw material, g;

W - moisture content of raw materials, %;

K - correction factor.

Quantitative determination of tannins:

The content of tannins in the studied O. rosea raw material was determined by redox titration of 0.02 M potassium permanganate solution using indigo sulfonic acid solution as an indicator¹².

To obtain extracts, 2.0g of dry crushed raw material was poured 250ml of purified water and boiled for 30 minutes. Then the extracts were filtered into a measuring flask for 250ml and brought the flask with purified water to the mark. Then 25ml of the extract was taken, 500 ml of water, 25ml of indigo sulfonic acid solution was added and titrated with 0.02M potassium permanganate solution to golden-yellow color. A control experiment was carried out in parallel. The content of the sum of tannins in terms of tannin in absolutely dry raw material in percent (X) is calculated by the formula:

(V-V1) - 0.004157.250.100.100

X= ---------------------------------

a .25.(100- W)

where

V - volume of potassium permanganate solution 0.02 M, consumed for titration of aqueous extraction, ml;

V₁ - volume of 0.02 M potassium permanganate solution consumed for titration in the control experiment, ml;

0.004157 - amount of tannins corresponding to 1ml of potassium permanganate solution 0.02 M (in terms of tannin), g;

a - weight of raw material, g;

W - moisture content of medicinal plant raw material, %;

Quantitative determination of flavonoids:

The content of flavonoids in the studied O. rosea raw material was determined by differential spectrophotometry with complexing additive 5% alcoholic solution of aluminum chloride²².

To obtain extracts, 2.0g of dry crushed raw material was poured into 100ml of 70% ethyl alcohol and heated in a boiling water bath with a reflux condenser for 30 minutes. Then the extracts were filtered into a 100ml measuring flask and brought the flask with 70% ethyl alcohol to the mark (solution A). Then 2ml of the obtained solution was taken into a 25ml volumetric flask, added 4ml of 5% alcoholic solution of aluminum chloride, 0.1ml of 30% acetic acid solution, brought to the mark with 70% ethyl alcohol (solution B), stirred and after 30 minutes measured the optical density on a spectrophotometer "Shimadzu UV-1800" in a cuvette with a layer thickness of 1cm at a wavelength of 410 nm. The extraction acidified with 0.1ml of 30% acetic acid solution was used as a comparison solution. In parallel, an experiment with 1ml of 0.05% alcoholic solution of standard rutin sample was carried out under the same conditions. Calculation of flavonoid content in the raw material is carried out according to the formula:

D. 100.C₀ .100.100

X= ------------------------------

D₀ .2.Q.(100-W)

Where

D-optical density of the analyzed extract;

D₀ - optical density of rutin solution;

C₀ -content of rutin in the sample, g;

a-mass of raw material, g;

W-moisture of raw material, %;

V-volume of extract taken for analysis, ml.

Methods of antioxidant activity:

To study the antioxidant activity of the studied raw materials, an infusion of the herb and decoction of the roots were obtained in a ratio of 1:10 according to the GFA "Infusions and decoctions" and two methods were used ¹².

Method 1 of antioxidant activity:

Method 1 - antioxidant activity was studied using spectrophotometric technique based on the ability to inhibit adrenaline autooxidation²³.

To 1ml of bicarbonate buffer (pH=11) was added 0.1ml of 0.1% solution of adrenaline hydrochloride and the optical density of the resulting solution was measured after 20min at a wavelength of 347nm in a 10mm cuvette on a spectrophotometer "Shimadzu UV-1800". Bicarbonate buffer (A₁ ) was used as the reference solution. Then to 1ml of bicarbonate buffer was added 0.02ml of the studied extract and 0.1ml of 0.1% solution of adrenaline hydrochloride and after 20 minutes under the same conditions the optical density was determined (A₂ ). Comparison solution - 1ml of bicarbonate buffer and 0.02 ml of the studied extraction.

The antioxidant activity index was calculated according to the formula:

(A₁ –A₂) . 100

AOA = -------------------

A₁

Where

And₁ is the optical density of the adrenaline solution;

A₂ -optical density of the adrenaline solution in the presence of the test extract.

Method 2 of antioxidant activity:

Method 2 - antioxidant activity was studied using redox titration method when 8 ml of purified water, 1 ml of 20% sulfuric acid solution, 1 ml of 0.05 N potassium parchment solution were added to a 50 ml flask, stirred and titrated with the solution under study until pink color disappears. For calculations, 0.05% alcohol solution of quercetin is used. The concentration of biologically active substances of reducing character of the studied raw material is calculated by the formula .²⁴

C_K .V_K-V_O

B= ----------------------

Vx .m

Where

B - concentration of biologically active substances of restorative character of the object under study, consumed for titration of 1ml of 0.05 N potassium parchment solution, mg/g;

Cc - concentration of quercetin in the solution consumed for titration of 1ml of 0.05 N potassium parchment solution,mg/mL;

Vo - volume of the tested solution, ml;

Vk - volume of quercetin solution consumed for titration of 1ml of 0.05 N potassium parchment solution, ml;

Vx - volume of the solution of the object under study, consumed for titration of 1ml of 0.05 N potassium permanganate solution, ml;

m- mass of raw material, g.

Statistical processing:

All studies were carried out in 5 repetitions and for statistical processing of the conducted studies we used the parametric Student's criterion according to GFA 1.1.0013.15 "Statistical processing of chemical experiment results" and Pearson's coefficient was determined to determine the correlation^12,25.

RESULT:

Morphological features of raw materials:

Macroscopic analysis of parts of O. rosea showed that the grass is represented by numerous paired pinnately pinnately compound leaves 10-25cm long, with hairy petioles and long rachis, consisting of 10-15 pairs; leaflets are oblong-lanceolate or lanceolate, light green, covered with dense hairs over the entire surface, evenly arranged on both sides of the leaflet, leaflet margins are entire, veining is pinnatly. Bracts are 8-18mm long, fused with petiole only one-third, hairy. Inflorescence is a raceme, 8-20cm long, many-flowered, with spreading flowers, pedicels are pressed-hairy and equal to leaves. Flowers are pink-purple, flag 10-15mm long, oval or ovoid at the bend, usually emarginate; wings are almost equal to the flag, boat equal to the wings, with a pointed tip 1.5-2mm long, calyx bell-shaped, 5-10mm long, with teeth shorter or equal to the tube, whitish-mossy. Fruits are oblong yellowish-green legumes, 15mm long, 3-4 mm wide, with 2-3mm spout, compressed from dorsum to abdomen, almost double-nested, hairy, deeply grooved along the abdomen, sitting on a stalk about 1 mm long. Seeds are brown, covered with dark dots, angular bud-shaped, appressed, 2-2.5mm long. The underground organs of O. rosea consist of the main root, on the ground surface of which forms a branching, resembling a caudex. The roots are 15-40cm long, 5-15 mm in diameter and have a longitudinally wrinkled surface of gray-brown color with a yellowish fibrous fracture.

Qualitative analysis:

Preliminary phytochemical studies have shown that the herb and roots of O. rosea contain various groups of biologically active substances - flavonoids, coumarins, tannins, ascorbic acid, alkaloids, saponins (Table 1)²⁶.

According to literature data, antioxidant activity is mainly possessed by such natural compounds as flavonoids, tannins, ascorbic acid^27,28,29,30. Therefore, we were interested in the concentration of these groups of compounds in the studied raw material of O. rosea, the dynamics of their accumulation depending on the vegetation phase of the plant and the presence of a relationship with the accumulation of these groups and the value of antioxidant activity.

Determination of ascorbic acid content:

During the study of ascorbic acid content in the herb and roots of O. rosea, it was found that the concentration of vitamin C in the underground organs is higher than in the herb (Table 2). Also comparing the content of ascorbic acid in raw materials harvested in different periods of vegetation, it can be concluded that ascorbic acid is maximally accumulated during the vegetation period.

Table 2 - Ascorbic acid content in herb and roots of O. rosea, % (n=5)

Raw material procurement period	*O. rosea* herb	*O. rosea* roots
Growing period	0,023±0,0008	0,028±0,0009
Flowering period	0,018±0,0007	0,025±0,0008
Fruiting period	0,015±0,0005	0,021±0,0006

Determination of tannins content:

When studying the content of tannins in the herb and roots of O. rosea, it was found that their content is also higher in the herb than in the underground organs (Table 3). Also comparing the content of tannins in raw materials harvested in different periods of vegetation, it can be concluded that tannins are maximally accumulated in the grass during the fruiting period.

Table 3: Tannin content in herb and roots of O. rosea, % (n=5)

Raw material procurement period	*O. rosea* herb	*O. rosea* roots
Growing period	1,65±0,021	1,54±0,054
Flowering period	1,98±0,034	1,86±0,083
Fruiting period	2,21±0,042	1,94±0,087

Determination of flavonoids content:

During the study of flavonoid content in the herb and roots of O. rosea, it was found that the concentration of flavonoids is also higher in the herb than in the roots (Table 4). Comparing the content of flavonoids in raw materials harvested in different periods of vegetation, it can be concluded that flavonoids are maximally accumulated in the herb during flowering.

Table 4: Flavonoid content in herb and roots of O. rosea, % (n=5)

Raw material procurement period	*O. rosea* herb	*O. rosea* roots
Growing period	1,78±0,15	0,68±0,03
Flowering period	2,21±0,13	1,05±0,06
Fruiting period	2,04±0,12	0,97±0,04

Determination of antioxidant activity:

Oxidative stress underlies most pathological processes developing in the human body. Therefore, much attention is paid to the use of antioxidants for the prevention and treatment of a variety of inflammatory, cardiovascular and other diseases^{31,32,33,34,35,36}.

When the antioxidant activity of O. rosea herb and roots were investigated using method 1, the following results were obtained (Table 5).

Table 5: Antioxidant activity of O. rosea herb and roots, % (n=5) - method 1

Raw material procurement period	O. rosea herb	*O. rosea* roots
Growing period	20,11±0,63˟	15,65±0,46 ˟
Flowering period	24,03±0,39 ˟	18,51±0,58 ˟
Fruiting period	22,34±0,55 ˟	17,54±0, 77 ˟
5% ascorbic acid solution (comparison preparation)	67,77±3,54

Note: ˟ p>0.05 - compared to the comparison drug, n=5.

According to this methodology for determining antioxidant activity, substances have antioxidant activity if the obtained value is more than 10%. Consequently, the studied samples of O. rosea herb and roots have antioxidant activity, and the value of antioxidant activity of O. rosea herb is higher than that of the roots. However, comparing the obtained data with the comparison preparation, we can say that the antioxidant activity of O. rosea herbs and roots is inferior to the comparison preparation - 5% ascorbic acid solution.

When the antioxidant activity of O. rosea herb and roots were investigated using method 1, the following results were obtained (Table 6).

Table 6: Antioxidant activity of O. rosea herb and roots, mg/mL (n=5) - method 2

Raw material procurement period	*O. rosea* herb	*O. rosea* roots
Growing period	0,49±0,02 ˟	0,27±0,01 ˟
Flowering period	0,59±0,03 ˟	0,45±0,02 ˟
Fruiting period	0,53±0,02 ˟	0,39±0,02 ˟
0.2% ascorbic acid solution (comparison preparation)	2,67±0,13

Note: ˟ p>0.05 - compared to the comparison drug, n=5.

According to the methodology used, the highest antioxidant activity is also observed in O. rosea herb harvested in the flowering period. However, comparing the obtained data with the comparison preparation, we can say that the antioxidant activity of O. rosea herb and roots is inferior to the comparison preparation - 0.2% ascorbic acid solution.

DISCUSSION:

To determine the correlation between the concentration of biologically active substances and the value of antioxidant activity, Pearson correlation coefficient was calculated (Figure 1). According to the obtained data, the average correlation is observed between the content of tannins and the value of antioxidant activity ³⁷. Strong correlation is observed between flavonoid content and antioxidant activity value, especially when using technique 1^38,39,40. There is no correlation between ascorbic acid content and antioxidant activity value. When comparing the methods among themselves, we can say that there is a strong correlation between them (r=0.996), which indicates the objectivity of both methods for the screening study of antioxidant activity of plant objects.

Figure 1: Pearson correlation coefficient value (ASC+M1 - ascorbic acid concentration and antioxidant activity value determined by method 1, ASC+M2 - ascorbic acid concentration and antioxidant activity value determined by method 2, FL+M1 - flavonoids concentration and antioxidant activity value determined by method 1, FL+M2 - concentration of flavonoids and value of antioxidant activity determined by method 2, T+M1 - concentration of tannins and value of antioxidant activity determined by method 1, T+M2 - concentration of tannins and value of antioxidant activity determined by method 2, M1+M2 - value of antioxidant activity determined by method 1 and value of antioxidant activity determined by method 2).

CONCLUSION:

Thus, the obtained data on phytochemical analysis of herbs and roots of O. rosea, growing in the Kyrgyz Republic, confirms the value of this species as a valuable source of natural antioxidants - various groups of phenolic compounds (flavonoids and tannins) and ascorbic acid, the content of which in O. rosea herb is higher than in O. rosea roots. It was found that during the whole seasonal development of O. rosea the level of flavonoids and tannins prevails in the terrestrial part, which indicates the possibility of harvesting O. rosea herb as a source of phenolic compounds. It was revealed that the value of antioxidant activity correlates with the content of flavonoids in the studied O. rosea raw material. Consequently, the grass of the plant during flowering can serve as an alternative source of phenolic compounds, which significantly facilitates the harvesting of plant raw material without digging and allows to preserve the original specimen due to annual renewal from root growth points.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors are thankful to federal program of strategic academic leadership "Priority 2030".

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Received on 22.03.2024 Revised on 14.09.2024

Accepted on 27.12.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1528-1534.

DOI: 10.52711/0974-360X.2025.00219

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Group of substances	Reagent	Result (+/-), color of solution or precipitate
1.	Flavonoids	(A) Cyanidine test	+ pink staining (flavones, flavonols, flavonones)
1.	Flavonoids	B) 5% alcoholic solution of aluminum chloride	+ yellow-green coloring (oxyflavans, oxyflavones).
2.	Coumarins	10% alcoholic solution of potassium hydroxide	+ red-cherry staining, when fluorescence is examined in UV light, extraction from grass - yellowish green fluorescence, extraction from roots - green fluorescence
2.	Coumarins	Sublimation reaction	+ yellow drops and yellow crystals
3.	Saponins	Reaction to foaming	+ in the grass, persistent, abundant foam + in the roots, the lather is less persistent.
3.	Saponins	10% lead acetate solution	+ white residue
5.	Tannins	5% solution of iron-ammonium alum	+ black-green coloration (condensed group tannins).
6	Ascorbic acid	Thin-layer chromatography - developer - sodium 2,6-dichlorophenolindophenolate solution	+ white spot on a pink background.
7	Alkaloids	Dragendorf's reagent Picric acid solution	+ brown sludge + yellow precipitate