INTRODUCTION:

Currently, one of the priority areas of health care in Russia is the expansion of the range of medicines due to new drugs of plant origin. The studies of the creation of nootropic, anxiolytic and immunomodulatory herbal preparations are topical. It also highlightsthe importance of the development of pharmacopoeial articles on pharmaceutical substances of plant origin.¹

Common bilberry (Vaccinium myrtillus L.) is a low, deciduous shrub of the heather family (Ericaceae). It occurs in Northern and Eastern Europe, Eastern Asia and North America. As a rule, blueberries grow in deciduous and coniferous forests, in the tundra, forest tundra.^2,3

Plants of Vaccinium genus plants are important for the food and pharmaceutical industries because they accumulate biologically active substances and are valuable raw materials.^4,5Common bilberry plant raw material accumulates various groups of biologically active substances: tannins, flavonoids, organic and hydroxycinnamic acids and others.^6-12In medical practice, common bilberry shoots have a hypoglycemic, anti-inflammatory and antiseptic effect.^13,14It was also shown that the common bilberry shoots normalize metabolic processes, have a regenerative, antioxidant effect, and also improve the vascular microcirculation of the brain. Considering the revealed effects, the staff of Sechenov University and the staff of the Research Institute of Pharmacy and Regenerative Medicine named after E.D. Goldberg were developed medicinal herbal teas with nootropic and anxiolytic effects, including common bilberry shoots.^15,16For the industrial release of these teas, regulatory documentation that is up-to-date is required, including for individual components.

The purpose of research is comparativestudy of the main biologically active substances (BAS)of common bilberry (Vaccinium myrtillus L.) shoots, collectedin the Bryansk, Tver and Vladimir regions (Russia).

MATERIALS AND METHODS:

1. Plant material:

Air-drycommon bilberry (Vaccinium myrtillus L.) shoots was collected in the Bryanskand Vladimir regions (Russia) in 2016-2017 during the growing season. Raw material was collected also in Tver region in 2017.

2. Chemicals:

Alugram® XtraSILG / UV254 5× 7.5 TLC plates were used as stationary phase. Formic acid (chemically pure), ethylacetate (chemically pure) were purchased from Himmed (Russia). Referent standarts were rutin(≥97%, CN AcrosOrganics, CAS 153-18-4), arbutine(≥98%, Alfa Aesar (Lancaster; Avocado)) and laboratory prepared standards –tanninand gallic acid.

3. Determination of BAS:

The studies were conducted at the Department of Pharmaceutical Natural Science at the Sechenov University.

3.1TLC conditions:

To determine the main biologically active substances by thin-layer chromatography, water-alcohol extracts of the common bilberryshoots were prepared. The mobile phase was anhydrous formic acid - water - ethyl acetate (5:5:40). Detection was carried out in UV light at a wavelength of 254 nmafter treatment of a 3% solution of ferrum (III) chloride with heating in an oven at a temperature of 100-110°C. Water-alcohol extract (70% ethanol) was prepared from common bilberry shoots.The following solutions were used in TLC as reference standards: rutin, arbutin, tannin and gallic acid. Test solutions (20µl) and solutions ofrutin(75 µl), tannin (15 µl), gallic acid and arbutin (25 µl) were applied to TLC plates.

3.2 HPLC conditions:

UV-detection of separation and analysis of various groups of BAS collection of anxiolytic on a C18 column using gradient elution of mobile phase: acetonitrile - 0.1% orthophosphoric acid solution (30:70). Chromatographic system parameters and analysis method: Aquilon “Stayer” device (“Aquilon”, Russia), stationary phase – column Phenomenex Luna 5u C18 (2) 100A, mobile phase – acetonitrile (phase B) : 0.1% phosphoric acid (Phase A) (30:70), gradient elution, sample volume injected into the chromatograph – 50 μL, column temperature – 35 ° С, analysis time – 45 min, software – ChemStation, UV detection wavelength – 280nm.

3.3 Total tannins, flavonoids, phenolic glycosides content:

Determination of total tannins was carried out according tothe methodology of SP RF XIV ed., Vol. 2, p. 2365 “Determination of the content of tannins in medicinal plantraw materials and herbal preparations” (GPM.1.5.3.0008.18) method 1 (permanganatometric titration).¹⁷Determination ofthe total flavonoids content was carried out by differentialspectrophotometry according to the adapted methodologyof SP RF XIV ed., Vol. 4, p. 6074 “St. John's wort Herb” (PM2.5.0015.15).¹⁷ Determination of total phenolic glycosidescontent was carried out by spectrophotometry methodaccording to the Nagaslayeva L.N. author's technique. Total phenolic glycosides content by Nagaslayeva L.N.: 0.5 g (accurate sample) of the crushed CHD sieved through a laboratory sieve (hole size 1 mm) was placed in a 250 ml flask, 125 ml of 70% ethanol was added, weighed inaccurately ± 0.01 g, then connected to a reflux condenser and heated in a water bath at 50 °C for 1 hour with constant stirring. After cooling to room temperature, the flask was weighed and recovered to its original mass by 70% ethanol. The extract was filtered through a cotton swab into a dry flask, discarding the first 15 ml of the filtrate. In parallel with this operation, a column with alumina was prepared. 0.5 cm cotton swab was placed and wetted with water in a 25 cm long, 1.5 cm diameter column (a porous filter No. 2 was soldered into the bottom of the column). 2 g of alumina was placed in a 25 ml beaker, 10 ml of water was added, mixed and poured into a column with an open tap. To the remaining sorbent in the glass, another 10 ml of water was poured, mixed and poured into a column. Above the sorbent, a small cotton swab was placed and washed with 5 ml of 70% ethanol. 2 ml of filtrate was applied to a column filled with alumina and eluted with 25 ml of 70% ethanol at a rate of 4 ml/min. The solution was collected in a 25 ml volumetric flask, adjusted to label by a 70% ethanol and mixed. The column was used once. The resulting eluate was used for analysis, the optical density was determined at 286 nm, 70% ethanol was used as the reference solution.^18,19The spectrophotometric study was performed at Agilent VarianCARY 4000 UV-Vis Spectrophotometer. The measurements were carried out in the wavelength range from 600 to 200 nm. Results were visualized using the CaryWin UV software.

4. Antiradical activity:

Evaluation of antiradical activity was carried out by amethod based on the inhibition of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). As a result of the reaction, theintense violet color decreases and the optical density of theDPPH solution also decreases. The reaction mixture-4 M DPPH (Sigma - Aldrich) in96% ethanol diluted 10 times to obtain a working solution, 2ml of the test sample (antioxidant). A series of dilutions water-alcohol extract (70% ethanol) from common bilberry shoots was used. Samples with DPPH were transferred to a darkplace for 30 minutes, after which the optical density wasdetermined by spectrophotometry (at = 517 nm). Ethanol was used as a comparison solution. The antioxidant activity(AOA) of the test substances was calculated by the formula:

AOA = [(A₀-A₁)/A₀]×100%,

where: A0 - the antioxidant absorption of the DPPH solution (control); A1- the antioxidant activity of the test sample with DPPH. To calculate the IC50 – the concentration of the substrate, at which 50% of the radicals binds to the test sample, we chose the segment on the straight line plot of the dose-effect graph. The smaller the EC50 parameter value, the greater the antiradical activity of a substance.²⁰

5. Assessment of biological activity (membrane stabilizing, antioxidant and antitoxic activity):

The ability of plant BAS to increase the tolerance of Paramecium caudatum to cellular poisons may indicate a certain pharmacological effect at the cellular leveland in the macroorganism. To study the membrane stabilizing and antioxidant activity, a 3% solution of hydrogen peroxide was used, and the antitoxic activity was 2.5% sodium hydroxide solution, 10% sodium chloride (in the study of membrane stabilizing activity). Culture of ciliates in the stationary growth phase (4.5 ml) were placed to several test tubes. Distilled water (0.5 ml) was added to the first (control) tube and mixed. In the second test tube, 0.5 ml of common bilberry shoots decoction was added and mixed. Then, 0.5 ml of liquid was transferred from the second tube to the third, from the third to the fourth, etc. A rack with test tubes was thermostated for 24 hours at 25 ° C. Then, 0.05 ml of liquid was taken from the test tubes onto a glass slide, 0.05 ml of a solution of the resolving factor was added there, and the lifespan was measured until the death of 100% of the cells. The observation of motor reactions and lysis of Paramecium caudatum was carried out under a binocular microscope at room temperature, in a place protected from direct sunlight. With a value of the biological activity index of 1.10, an object is considered biologically inactive, with a value of >1.10, the object increases cell viability, with a value of <1.10, the object reduces cell viability.^21-23

Statistical processing of the research results was carried out in accordance with the requirements of SP RF XIV ed., Vol. 1, p. 289 GPM.1.1.0013.15 “Statistical processing of experimental results” using the program Microsoft Office Excel 2017.¹⁷

RESULTS AND DISCUSSION:

1. Qualitative analysis of phenolic compounds

During the TLC separation of BAS at UV light at a wavelength of 254 nm of common bilberry shoots water-alcohol extracts, 4 adsorption zones were found on the plates (Tabl. 1.):

Table 1:Chromatographic profiles of V. myrtillus shoots extracts (70% ethanol) determined by TLC

Standard	UV light, 254 nm(4 adsorption zones)
rutin	Rf = 0,49; green
gallic acid	Rf = 0,96; dark blue
arbutin	Rf = 0,75; blue
tannin	Rf = 0,79; black blue

According to the results of chromatographic separation in comparison with standard samples in the studied extracts of common bilberry shoots confirmed the presence of rutin, gallic acid, arbutin, tannin.

The HPLC technique of gradient elution with UV detection made it possible to separate and simultaneously detect a number of compounds belonging to different chemical groups (Tabl.2)

Table 2: Chromatographic profiles of V. myrtillus shoots extracts (70% ethanol) determined by HPLC-UV

Substance		Retention time, min
1	Chlorogenic acid	14.12
2	Caffeic acid	15.50
3	Luteolin 7-glucoside	18.25
4	Hyperoside	18.70
5	Ferulic acid	19.49
6	Quercetine	25.06

2. Determination of biologically active substances content

The determination of extractive substances extracted by water, full amount of tannins in terms of tannin, full amount of flavonoids in terms of rutin, full amount of phenolic glycosides in terms of arbutin was carried out.

2.1 Determination of total tannin content

2.2

The results of quantitative determination of the amount of tannins in terms of tannin are presented in the table 3.

Table 3: The content of tannins in terms of tannin in the shoots of common bilberry (n = 6, f = 5, P = 95%, T (f, P) = 2.57)

CHD	`X	ΔХ	S	E, %
Common bilberry shoots VR, 2016	8.85	0.58	0.208	2.94
Common bilberry shoots BR, 2016	7.61	0.46	0.164	2.68
Common bilberry shoots VR, 2017	8.88	0.45	0.163	2.28
Common bilberry shoots BR, 2017	7.74	0.38	0.137	2.20
Common bilberry shoots TR, 2017	8.83	0.20	0.164	2.31

The content of tannins in terms of tannin is 8.85 ± 0.58% for raw materials harvested in the Vladimir region in 2016, and 8.88 ± 0.45% for raw materials harvested in 2017.In the shoots of common bilberry ordinary harvested in the Bryansk region in 2016 the content of tannins in terms of tannin is 7.61 ± 0.46%, in 2017–7.74± 0.38%, which is slightly less than the values in raw materials harvested in the Vladimir region. The content of tannins in terms of tannin is 8.83±0.20% for raw materials harvested in Tver region in 2017.

2.3 Determination of the content of the amount of totalflavonoids content:

A study of the spectral characteristics of Common bilberry shoots water-alcohol extracts (70% ethanol) and the spectrum of rutinwith aluminum chloride showed their similarity (Fig. 1). It makes possible to recalculate the total flavonoids content in terms of rutin. The results are shown in Table 4.

Table 4: The total flavonoids content in terms of rutin in Common bilberry shoots(n = 6, f = 5, P = 95%, T (f, P) = 2.57)

CHD	`X	ΔХ	S	E,%
Common bilberry shoots VR, 2016	2.79	0.10	0.037	1.67
Common bilberry shootsBR, 2016	2.47	0.10	0.034	1.72
Common bilberry shootsTR, 2017	2.76	0.06	0.049	2.19
Common bilberry shoots VR, 2017	2.88	0.08	0.028	1.24
Common bilberry shootsBR, 2017	2.69	0.09	0.031	1.44
Common bilberry shootsTR, 2017	2.79	0.07	0.060	2.68

Thus, in the shoots of common bilberry, harvested in the Vladimir region in 2016 and in 2017, the content of the sum of flavonoids in terms of rutin is 2.79±0.10% and 2.88±0.08%, respectively. In raw materials harvested in the Bryansk region in 2016, and in 2017, the content of flavonoids in terms of rutin is 2.47 ± 0.10% and 2.69 ± 0.09%, respectively. In raw materials harvested in the Tver region in 2016 the content of flavonoids in terms of rutin is 2.76 ± 0.06%, and 2.79 ± 0.07% – in 2017.

Figure 1: Absorption spectra: A – V. myrtillus shoots flavonoid complex with a solution of aluminum chloride; B – rutin with a solution of aluminum chloride

2.3 Determination of total phenolic glycosides content

The results of determining the amount ofphenolic glycosides in terms of arbutinare presented in Table 5, Absorption spectra in Fig. 2.

Table 5:The content of the amount of phenolic glycosides in terms of arbutin in Common bilberry shoots (n = 6, f = 5, P = 95%, T (f, P) = 2.57)

CHD	`X	ΔХ	S	E, %
Common bilberry shoots VR, 2016	1.54	0.08	0.027	2.24
Common bilberry shootsBR, 2016	1.62	0.02	0.008	0.77
Common bilberry shoots VR, 2017	1.39	0.07	0.024	2.18
Common bilberry shootsBR, 2017	1.59	0.07	0.025	2.00
Common bilberry shootsTR, 2017	1.62	0.04	0.035	2.71

The content of the amount of phenolic glycosides in terms of arbutin in the common bilberry shoots harvested in the Vladimir region in 2016 was 1.54 ± 0.08%, in 2017–1.39± 0.07%. The content of the amount of phenolic glycosides in terms of arbutin in the common bilberry shoots harvested in the Bryansk region in 2016 was 1.62±0.02%, in 2017 –1.59±0.07%. In raw materials harvested in the Tver region in 2016 the content of content of the amount of phenolic glycosides in terms of arbutin is 1.62 ± 0.04% in 2017.

Figure 2: Absorption spectra: A – purified V. mirtillus shoots water-alcohol extract; B – arbutin water-alcohol solution

2.4. Antiradical activity:

Antiradical activity of the water-alcohol common bilberry shootsextract (70% ethanol) was confirmed. This is due to the content of natural antioxidants, namely phenolic compounds. IC50 value of 6.231±0.211 mg/ml was established. The AOA of the initial sample was 41.7%, and for a dilution of 1:10 and 1:100, it was 71.63% and 70.51%, respectively. This indicates a sufficiently high antiradical activity even with large dilutions of the extracts.

2.5. Assessment of biological activity (membranestabilizing, antioxidant and antitoxic activity):

Analyzing the data obtained, it can be noted that the decoction of common common bilberry shoots has the highest activity in the highest concentrations at a dilution of 1·10^-2 and 1·10^-3, then the activity decreases (Tab. 6, Fig. 3).

Table 6: Membrane-stabilizing, antioxidant and antitoxic effects of Common bilberry decoction

No.	Dilution degree of common bilberry shoots decoction	Biological activity index (BAI)× 3% Н2О2, 0.05 ml	Biological activity index (BAI) × 0.1 н NaOH, 0.05 ml	Biological activity index (BAI) × 10% NaСl, 0.05 ml
1	1	1	1	1
2	10^-1	1.39	1.29	1.12
3	10^-2	1.36	1.50	1.30
4	10^-3	1.17	1.61	1.16
5	10^-4	1.29	1.59	1.39
6	10^-5	1.04	1.41	1.27
7	10^-6	1.07	1.27	1.10
8	10^-7	1.06	1.19	1.00
9	10^-8	1.03	1.00	0.99
10	10^-9	0.98	1.09	0.94

Figure 3: Membrane-stabilizing, antioxidant and antitoxic effects of Common bilberry decoction

CONCLUSION:

Thus, the study of the composition of phenolic compounds of common bilberry shoots (Vaccinium myrtillus L.) was carried out. The presence of arbutin, rutin, tannin, and gallic acid in CHD was found. A quantitative analysis of the common bilberry shoots, harvested in the Bryansk and Vladimir regions in 2016-2017, and the Tver region in 2017 showed similar results. The obtained data can be used in the development of regulatory documentation for common bilberry shoots. It is shown, that common bilberry shoots have antiradical, membrane stabilizing, antioxidant, and antitoxic activity.The resulting data can be used for standardization purposes.^24-32

CONFLICTS OF INTEREST:

None.

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Received on 20.02.2022 Modified on 31.01.2023

Research J. Pharm. and Tech 2023; 16(11):5426-5431.

DOI: 10.52711/0974-360X.2023.00879

*Corresponding Author E-mail: kovaleva_t_yu@staff.sechenov.ru