INTRODUCTION:

Rubus is a genus of the Rosaceae family, which is one of the numerous species diversity and prevalence. The genus has several subgenera, including Ideaobatus (Raspberry), Eubatus (Blackberry), Chamaeborus (Cloudberry) and others.

Plants of the “Blackberry” subgenus have been known to mankind since ancient times: their fruits are eaten, the leaves are used in folk medicine, but the study of individual species is currently not complete enough. Researchers are interested in all parts of plants of this subgenus, especially fruits and leaves.

Among the wide variety of species of the subgenus Eubatus, the simplest and most common in Russia and the CIS is the European dewberry (R.caesius L.), a wild plant widespread everywhere, which is a shrub with two–year-old shoots up to 1-1.5m in length, covered with curved, downward-pointing spikes. The leaves are complex, consisting of 3 leaflets, abundantly pubescent on the underside. Petioles and rachis are covered with numerous thorns. The flowers are large, five-membered, white. The fruits are blue, black, purple aggregate-accessory fruits, covered with an intense bluish bloom ^1-4. Currently, the raw material of R.caesius is not official (fruits and leaves are used in folk medicine as an astringent, diaphoretic, for the treatment of the respiratory system and gastrointestinal tract), and therefore is of interest to researchers as a promising medicinal plant raw material for further study. Researchers have previously conducted studies on the chemical composition and pharmacological activity of fruits, leaves, stems of R. caesius. In the course of phytochemical studies of leaves, stems, flowers and fruits of European dewberry, the presence of phenolic compounds in the raw materials, presented in Table 1, was proved.

Table 1: Phenolic compounds in the raw materials of R.caesius L.

Compound	Raw materials	Link to a literary source
Rutin	Leaves, stems, flowers, fruits	5-7
Quercetin	Leaves, stems, flowers	5-8
Isoquercetin	Leaves, stems	5-6, 8
Hyperoside	Leaves, stems	5-6, 9
Luteolin	Leaves	5
Taxifolin	Leaves, stems	5
Tiliroside	Leaves, stems	5-6
Naringenin	Leaves, stems	5
Astragalin	Leaves, stems	5
Myricetin	Leaves, flowers	7
Pedunculagin	Leaves	8
Kaempferol (derivatives)	Leaves	6,8
Ellagic Acid	Leaves	6-8
Gallic acid	Leaves, fruits	6-7
Catechin	Leaves, stems, flowers, fruits	5,7
Epicatechin	Leaves, fruits	7
Fumaric acid	Leaves	6
Caffeic acid	Leaves, flowers, fruits	7
Ferulic acid	Leaves, fruits	7
Benzoic acid	Fruits	10
Salicylic acid	Fruits	10

The fruits of R.caesius are rich in organic acids, including oxalic, malic, citric, tartaric, amber, ascorbic¹⁰, sugars (glucose, fructose), pectin substances, anthocyanins¹¹.

In the course of studies, R.caesius leaves showed hypoglycemic activity^9,12, antiproliferative, antitumor effect⁶, extracts from R.caesius leaves and shoots also led to a decrease in platelet aggregation¹³, affected the fibrinolytic system of the blood¹⁴. Fruits of R.caesius have antidiarrheal activity due to astringent action¹⁵.

The aim of this study is to study the composition of phenolic compounds of R.caesius leaves, to evaluate the antioxidant, anti-inflammatory, antimicrobial activity of the infusion from raw materials.

MATERIALS AND METHODS:

The objects of the study were the leaves of the European dewberry, collected on the territory of the Moscow region, Khimki (55.96852^оN, 37.28119^оE) during florification and fructification of the source plant (July-August 2021-2022). The raw materials are subjected to shade drying.

During the study, the following reagents were used: reference sample of isorhamnetin (Vestenbergsgreuth, Germany, batch 100004356, an isorhamnetin content of 99.06%, valid until 09.2022), reference sample of luteolin (Sigma-Aldrich, USA, batch 0000084920, CAS No.491-70-3, a luteolin content of ⩾ 98.0%, valid until 12.2022), reference sample of luteolin-7-O-β-D-glucoside CO (Sigma-Aldrich, USA, CAS No.5373-11-5, a luteolin-7-0-D-glucoside content of ⩾ 98.0%, valid until 12.2022), reference sample of kaempferol (Vestenbergsgreuth, Germany, batch 97791112, a kaempferol content of 99.67%, valid until 01.2023), reference sample of rutin trihydrate (Sigma-Aldrich, USA, CAS No. 250249-75-3, a rutin trihydrate content of ⩾ 94.0%, valid until 02.2023), reference sample of kemferol-3-β-D-glucopyranoside (Sigma-Aldrich, USA, CAS No. 480-10-4, a kaempferol-3- β-D-glucopyranoside content of ⩾ 97.0%, valid until 12.2023), reference sample of gallic acid monohydrate (Sigma-Aldrich, USA, CAS No. 5995-86-8, a gallic acid content of ⩾ 98.0%, valid until 05.2022), as well as DPPH, free radicals (Sigma-Aldrich, USA, CAS No. 1898-66-4, valid until 06.2022), ethanol (GOST R 51652-2000), deionized water, acetonitrile for high-performance Liquid chromatography (HPLC) (gradient class, ≥ 99.9%, CAS No.75-05-8).

Sample preparation:

To study the composition of phenolic compounds by HPLC, extracts from raw materials are prepared in accordance with the method of quantitative determination described in the pharmacopoeia monograph "PM.2.5.0069.18. Polygoni avicularis herba" of the State Pharmacopoeia of the Russian Federation. To do this, the analytical sample of raw materials was crushed to the size of particles passing through a sieve with holes of 0.5mm. About 1.0g (exact weight) of crushed raw materials were placed in a 150ml flask, 30 ml of extraction solvent was added (ethanol served as an extraction solvent in concentrations of 50%, 70% and 96%.), the flask was attached to a reverse refrigerator and heated in a boiling water bath for 30minutes. Then the flask was cooled to room temperature under a stream of cold water and filtered through a paper filter into a 100ml volumetric flask. Extraction was repeated 2 more times by the above method. Extracts were filtered through the same filter into the same measuring flask, the filter was washed with ethanol, the volume of the filtrate was brought to the mark with the same solvent and mixed.

Solutions of reference samples were prepared by dissolving an exact sample in 96% ethanol (class chemically pure).

Preparation of the infusion:

Water extraction from raw materials (infusion) was prepared in a ratio of 1:10 according to the general rules for the preparation of infusions and decoctions (GPM.1.4.1.0018.15 of the Russian Pharmacopoeia of XIV edition): an exact weight (10.0g) of leaves crushed to a size of 5mm was placed in a perforated infuser glass, and then in a pot infuser preheated in a boiling water bath for 15minutes, filled with water at room temperature, taken into account the corresponding water absorption coefficient given in the GPM.1.5.3.0012.15 "Determination of water absorption coefficient and consumption coefficient of medicinal plant raw materials", the value of which was 4.4, closed with a lid and insisted on a boiling water bath. Then the pot infuser was removed from the water bath, kept for 45 minutes at room temperature, after which it was filtered, squeezing out the rest of the medicinal plant raw materials, and 100 ml of water was added.

Study of antioxidant activity:

To evaluate the antioxidant activity of infusions from raw materials, a reaction with the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used. 2ml of the infusion in various dilutions was added to 2ml of the DPPH solution (5mg per 100ml)^16-20. The obtained samples were kept in a dark place for 30minutes, after which the absorbance was removed relative to the purified water at a wavelength of 517nm. The antioxidant activity (AOA) of the studied samples was calculated by the formula 1:

Ao – A

AoA = ---------------- x 100 %

Where Ao - Absorbance of the DPPH solution (control), A – absorbance of the analyzed sample with DPPH.

As a comparative drug, a solution of ascorbic acid powder from the company “Meligen” (Russia), prepared in concentrations from 1 to 100µg/ml, was subjected to similar tests.

Study of anti-inflammatory activity:

The parameters of acute toxicity of the infusion were determined by the Kerber method²¹. An experiment to study anti-inflammatory activity was conducted in vivo experiments using 90 nonlinear male mice weighing 18-22g. The animals were kept in the vivarium of the VILAR State Medical University on a standard diet. The study was approved by the Bioethical Commission of the VILAR State Medical University (Record No. 84 of 09/26/2022). Studies in vivo experiments were carried out in accordance with the Decision of the EEC Council dated 03.11.2016 No. 81 "On approval of the Rules of Good Laboratory Practice of the EEU", the National Standard of the Russian Federation GOST 33044-2014 "Principles of good Laboratory Practice". The anti-inflammatory activity of R.caesius leaf infusion was studied on a model of formalin edema of mouse paws. As a comparison drug, an infusion of Calendula officinalis flowers was used, prepared according to the instructions of the raw material manufacturer (Krasnogorsklekarstva, Russia). The object of the study and the comparison drug were administered to mice at a dose of 17ml/kg intragastrically according to the instructions and the dose conversion factor²¹. Formalin edema was caused by a single injection of 0.05ml of 1% formalin under the aponeurosis of the right hind leg of the mouse an hour after the last administration of the drugs. The animals were divided into 3 groups of 10 individuals (Table 2). The substances were administered within 4 days before the introduction of formalin. 3 hours after the introduction of formalin, the antiexudative effect was determined using formula 2:

P_c – P₀

AoA = ---------------- x 100 %

P_c

where: Pc is the difference in the masses of paws with edema and without edema in animals of the control group; Po is the difference in the masses of paws with edema and without edema in the experimental group.

Table 2: Groups of studied animals in the study of the anti-inflammatory activity of R.caesius leaf infusion

Groups, n=10		Drug
No. 1	Control	Purified water
No. 2	Experimental	Rubus caesius leaf infusion
No. 3	Experimental	Calendula officinalis flowers infusion

To assess the significance of the differences in the three experimental groups, one-way ANOVA was used followed by post-hoc analysis. The significance of the differences with the control was considered at p<0.05. Statistical data were processed using the licensed program "Statistica version 13" (TIBCO Software Inc, USA).

When determining the bacteriostatic and fungistatic activity of the studied samples in in vitro experiments, the method of double serial dilutions of samples in liquid nutrient media was used. Pathogenic gram-positive bacteria Staphylococcus aureus 209-P (ATCC 6538), gram-negative bacteria Escherichia coli ATCC 25922, Proteus vulgaris ATCC 6896 and Pseudomonas aeruginosa ATCC 9027, yeast-like fungi Candida albicans ATCC 10231 and mycelial fungi Microsporum canis 352 were used as test microorganisms.

Meat-peptide broth (MPB) was used as a nutrient medium for determining bacteriostatic activity, and Saburo liquid medium was used for determining fungistatic activity^22-27.

Study of bacteriostatic activity:

2ml of the studied extract was placed in the first tube, 2 ml of the culture medium was added, creating a concentration of the infusion in a dilution of 1:2. A number of experimental tubes with 2ml of the culture medium (BCH) were prepared. Then, by sequentially diluting the samples in the culture medium by 2 times, a number of decreasing dilutions were prepared. The last test tube with a clean medium (without the addition of a solution) served as a control. After that, all test tubes (experimental and control) were seeded with cultures of microorganisms.

Suspensions of gram-positive and gram-negative bacteria were prepared in isotonic sodium chloride solution according to the bacterial turbidity standard CCA 42-28-85-2022 (10 IU) (109 microbial bodies/ml). A number of decreasing concentrations of microorganisms were prepared from the first test tube containing 109 microbial bodies/ml by tenfold dilutions in isotonic sodium chloride solution: 108, 107, 106, 105, 104. Then, 0.2ml of suspension containing 104 microbial bodies/ml (working microbial load) was introduced into each test tube. The crops were incubated in a thermostat at a temperature of 37°C for 24hours. The experiments were carried out in three repetitions.

Study of fungistatic activity:

The samples were prepared in the same way as for the determination of bacteriostatic activity, but using a Saburo culture medium. A number of test tubes with 2 ml of culture medium were prepared. By sequentially diluting the infusion samples in a culture medium, a number of decreasing concentrations were prepared 2 times. The last test tube with a clean medium served as a control. After that, all test tubes (experimental and control) were seeded with cultures of microorganisms. Microsporum canis 352 culture was previously ground in a sterile porcelain mortar, washed into a sterile test tube with an isotonic 2-3ml of sodium chloride solution, for which a suspension of fungi was prepared from the resulting thick mass in an isotonic sodium chloride solution according to the bacterial turbidity standard. Then the resulting suspension was diluted with isotonic sodium chloride solution 20 times. 0.2ml of the resulting suspension was seeded into experimental and control tubes. The crops were incubated at a temperature of 30-32^oC: yeast–like fungi – for 48hours, mycelial fungi - for 10-14 days. The experiments were carried out in three repetitions.

The bacteriostatic and fungistatic effect was determined by the minimal, suppressing growth of bacteria and fungi dilution of the studied sample, in which the growth of microorganisms was not visually observed.

Equipment:

Chromatographic separation and detection were carried out on a high-performance liquid chromatograph Nexerai LC -2040 (Shimadzu Corporation, Japan), equipped with a column thermostat and samples, a degasser, an autosampler and an ultraviolet detector. Raw data was processed using LabSolutions Single LC software (Shimadzu Corporation, Japan).

The antioxidant activity was determined using the spectrophotometer СФ-2000 (“Spectrum”, Russia) with a spectral range of 190-1100 nm. Statistical processing of the obtained data was carried out in the Excel program (ver. 2016, Microsoft, USA).

Chromatographic separation and detection conditions:

· Chromatographic column: Grace HPLC Column Platinum C8-EPS, 250×4.6mm, 5mm (Grace, USA).

· Precolumn: Phenomenex SecurityGuardTM Cartridges Widepore C18, 4×3.0mm.

· Thermostat temperature: 27 °C.

· Mobile phase: 0.1% solution of formic acid water (by volume) (eluent A); 0.1% solution of formic acid in acetonitrile (by volume) (eluent B).

· Flow rate of the mobile phase: 1.0ml/min.

· Injection volume: 10μl.

· Retention time: 40min.

· Detection: UV detector with a dynamic change in the absorption wavelength during analysis from 365±2 nm to 254±2nm. (table 3)

Table 3: Elution’s gradient

Time, min.	А, %	В,%	Detection wavelength
0	90	10	365 nm
10	70	30	365 nm
15	70	30	254 nm
20	50	50
30	30	70
35	90	10	365 nm
40	90	10	365 nm

RESULTS AND DISCUSSION:

During the study, a chromatogram of phenolic compounds in the studied raw materials was obtained, which is shown in Figure 1.

Figure 1: Chromatogram of phenolic compounds in ethanol extracts from European dewberry leaves (R. caesius L.): 1-extraction with 50% ethanol, 2-extraction with 96% ethanol, 3- extraction with 70% ethanol.

The quantitative content of these components in the leaves of the European dewberry in terms of dry raw materials when extracted with ethanol of various concentrations is presented in Table 4.

Table 4: Content (%) of phenolic compounds in the leaves of the European dewberry (R. caesius L.)

Compound	Retention time, min	Content, %
Compound	Retention time, min	50% ethanol	70% ethanol	96% ethanol
Gallic acid	7,9	0,002 ± 0,00004	0,0133 ± 0,0002	0,0126 ± 0,0002
Rutin	10,6	0,010 ± 0,0002	0,114 ± 0,003	0,029 ± 0,0015
Cynaroside	11,2	0,005 ± 0,0001	0,021 ± 0,002	0,009 ± 0,0002
Narcissin	12,1	0,006 ± 0,0002	0,008 ± 0,0002	0,007 ± 0,0002
Astragalin	12,3	0,006 ± 0,0002	0,02 ± 0,0005	0,007 ± 0,0001
Kaempferol	20,6	0,016 ± 0,0008	0,015 ± 0,0004	0,013 ± 0,0003
Isorhamnetin	21,0	0,035 ± 0,0012	0,041 ± 0,0003	0,030 ± 0,001

The results of the study of bacteriostatic and fungistatic activity are presented in Table 9.

DISCUSSION:

As a result of HPLC-UV analysis of ethanol extracts from the leaves of the European dewberry, 7 compounds of phenolic nature were identified from 31 detected components: rutin, isorhamnetin, kaempferol, cynaroside, astragalin, gallic acid and narcissin It was found that the predominant component of phenolic compounds in the leaves of European dewberry is rutin. In almost all measurements, a higher content of phenolic compounds is observed when ethanol extracts 70% of the concentration.

The relative error of a single measurement according to the results of statistical processing of measurements does not exceed 2.94% (Table 5).

At the first stage of pharmacological studies, we studied the parameters of acute toxicity of R.caesius leaf infusion using the Kerber method and found that mice of all groups had no clinical signs of intoxication during the entire follow-up period (14 days). The clinical and functional status of all animals had no deviations from the physiological state inherent in mice of this age group. During the experimental period, there were no deaths of mice in the experimental groups. The animals were active, willingly ate food, adequately reacted to external stimuli. The control group animals also remained clinically healthy.

Thus, when studying the acute toxicity of the infusion of R.caesius leaves, LD50 indicators were not established, since the doses of the studied extract into the stomach of mice did not cause the death of animals. In accordance with the classification of chemical toxicity, the infusion of R.caesius leaves presented for research is a low-toxic substance ^28-29.

As can be seen from Table 6, infusion of R.caesius leaves with four-day administration had an anti-inflammatory effect: formalin edema decreased by 21% compared to the control group of animals, while infusion of Callendula officinalis flowers reduced formalin edema by 22% compared to the control group. It is obvious that under the influence of biologically active substances, primarily of a phenolic nature, contained in the infusion of R.caesius leaves, its anti-inflammatory activity is manifested, which is confirmed by literary data ⁶.

Based on this dependence, the IC50 value (the concentration of the infusion at which 50% of the free radical of DPPH binds) was calculated, which was 43.6 ± 0.4 µg/ml, which is only 1.5 times lower than the antioxidant activity of the ascorbic acid solution (comparison drug) (Table 8).

The antioxidant activity of the infusion of R.caesius leaves is estimated as high even with large dilutions, which indicates a high content of phenolic compounds in the raw materials (flavonoids, tannins, phenolic carboxylic acids), vitamins (vitamin C) ⁵.

Our results are consistent with the literature data, given that the authors studied the antioxidant activity of R.caesius collected at different times ⁵.

As a result of studying the bacteriostatic activity, it was found that the infusion of R.caesius leaves had a moderate bacteriostatic effect against gram-positive bacteria Staphylococcus aureus 209-P and gram-negative bacteria Proteus vulgaris ATCC in 1:16 dilution, against gram-negative bacteria Escherichia coli ATCC 25922 in 1:8 dilution and Pseudomonas aeruginosa ATCC 9027 in 1:64 dilution.

When studying the fungistatic effect of R.caesius leaf infusion, it was found that it had moderate fungistatic activity against the mycelial fungus Microsporum canis 352 – at a dilution of 1:16 and weak activity against the yeast-like fungus Candida albicans ATCC 10231 at a dilution of 1:4.

The data obtained indicate that the infusion of R.caesius leaves has both bacteriostatic activity against pathogenic gram-positive and gram-negative bacteria in a dilution of 1:8-1:64, and fungistatic activity against mycelial and yeast-like fungi in a dilution of 1:4-1:16.

The antimicrobial activity we have established is confirmed by other researchers. So, for example, Hering A. and co-authors showed the greatest growth-inhibiting activity of aqueous extracts of stems and leaves of R.caesius against gram-positive bacteria: Clostridium sporogenes and Clostridium bifermentans in the range from 0.5 to 0.0156 mg/ml and Enterococus faecalis in the range from 1.25 to 0.625 mg/ml (with the exception of the aqueous extract of stems of R.caesius). At the same time, the inhibitory activity of the studied aqueous extracts against gram-negative rods was 5 mg/ml or higher. The minimum bactericidal concentration (MBC 0.5 mg/ml) was established for an aqueous extract of R.caesius stems against Enterococcus faecalis, Escherichia coli and Salmonella enterica bacteria ⁵.

The results obtained by us and other authors indicate that the leaves of R.caesius are a promising source of biologically active substances of antimicrobial action.

CONCLUSION:

As a result of the study, the HPLC-UV analysis of the leaves of the European dewberry (Rubus caesius L.) was carried out, and the antioxidant activity of the infusion from this raw material was evaluated. The presence of 7 phenolic compounds (gallic acid, rutin, cynaroside, narcissin, astragalin, kaempferol, isoramnetin) has been proven, among which rutin is predominant. It is shown that ethanol at a concentration of 70% is the optimal extraction solvent for better extraction of this group of compounds. Based on the studies conducted in vivo and in vitro experiments, it was found that the infusion of Rubus caesius leaves is low-toxic, has high antioxidant, anti-inflammatory and antimicrobial activity. Thus, the studied raw materials - Rubus caesius leaves and medicines based on it are promising for further research and use in medical practice.

The data obtained regarding the identification of the studied species^31-32, chemical composition^{33, 35-38}and pharmacological activity^5,34,39are in most cases comparable with previous studies.

CONFLICT OF INTEREST:

The authors declare that they have no obvious and potential conflicts of interest related to the publication of this article.

ABBREVIATIONS:

HPLC - high-performance liquid chromatography, DPPH - 2,2-diphenyl-1-picrylhydrazyl, IC50 - half-maximal inhibitory concentration

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Received on 30.11.2023 Revised on 14.06.2025

Accepted on 16.10.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2179-2186.

DOI: 10.52711/0974-360X.2026.00314

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

Compound	x̅, %	s²_x	s_x	s_x̅	∆х	ε,%
Gallic acid	0,0133	4,264 x 10^-8	2,06 x 10^-4	9,23 x 10^-5	0,0002	1,36
Rutin	0,114	1,137 x 10^-5	3,37 x 10^-3	1,51 x 10^-3	0,003	2,59
Cynaroside	0,021	5,2 x 10^-8	2,28 x 10^-4	1,02 x 10^-4	0,0002	0,97
Narcissin	0,008	5,754 x 10^-8	2,40 x 10^-4	1,07 x 10^-4	0,0002	2,53
Astragalin	0,024	3,175 x 10^-7	5,64 x 10^-4	2,52 x 10^-4	0,0005	2,07
Kaempferol	0,015	2,622 x 10^-7	5,12 x 10^-4	2,29 x 10^-4	0,0004	2,94
Isorhamnetin	0,041	9,026 x 10^-8	3,00 x 10^-4	1,34 x 10^-4	0,0003	0,64

Groups of animals, n=10	Anti-inflammatory effect	% suppression of edema
1. Control	114,5 ± 5,1	-
2. Infusion of Rubus caesius leaves	90,6 ± 3,3^*	21
3. Infusion of Calendula officinalis flowers	89,2 ± 1,8^*	22

Delution*	Concentration of the infusion, µg/ml	% of binding of the DPPH radical	IC50, µg/ml
1:160	82,5	17,9 ± 0,28	43,6 ± 0,4
1:320	41,2	26,8 ± 3,05
1:640	20,6	42,3 ± 0,37
1:800	16,5	47,0 ± 0,3
1:4000	3,3	77,3 ± 0,46

Concentration, µg/ml	% of binding of the DPPH radical, %	IC50, µg/ml
100	93,2 ± 1,8	29,1 ± 0,64
50	76,0 ± 1,0
10	49,1 ± 3,1
5	33,3 ± 2,3
1	13,0 ± 0,9