Sanicula europaea L. Herb and Rhizomes with Root Extracts with Hemostatic, Wound Healing, Anti-Inflammatory and Antimicrobial Activity: Phytochemical and Pharmacological Research

1. Introduction

Among more than 440 families of flowering plants, the Apiaceae (Umbelliferae) family ranks sixth in terms of the number of taxa and seventh in terms of species diversity [1]. The highest species richness of this family is observed in the moderately warm and subtropical areas of the Northern Hemisphere, while in tropical countries, species are mainly concentrated in mountainous regions [2,3,4]. Many food, fodder, medicinal, and aromatic plants belong to the Apiaceae family and are utilised across various countries. Certain Apiaceae species play a significant role in shaping vegetation cover, acting as dominant components of herbaceous communities, particularly in mountainous and arid regions [1,5]. Of particular interest in this context are plants of the genus Sanicula, which comprises approximately 50 species, with only Sanicula europaea L. occurring in Ukraine. This species is widely distributed across forested regions of Europe, Western Asia, and Northern Africa, and is predominantly found in deciduous and mixed forests [6,7]. S. europaea has long been used in traditional medicine for stopping bleeding, wound healing, and the treatment of inflammatory and dermatological conditions [8]. However, the number of scientifically substantiated studies supporting these uses remains limited. Its broad distribution in the European region, substantial natural resources, and traditional medicinal applications make this plant and its preparations a promising candidate for integration into pharmaceutical and medical practice.

S. europaea is characterised by a chemical diversity of biologically active compounds, which underlie its broad spectrum of pharmacological and biological activities. Species of the genus Sanicula contain a wide range of bioactive constituents, including tannins, flavonoids, organic acids, saponins, polysaccharides, coumarins, and essential oil [9,10,11]. Both the underground and aboveground parts of S. europaea accumulate organic acids; triterpenoid saponins (acidic and neutral monodesmosides, saniculosides A, B, C, D); nitrogen-containing compounds; hydroxycinnamic acids; vitamins K and C; flavonoids; tannins and bitter substances; and essential oil [12,13,14].

The rhizomes with roots of S. europaea contain organic acids (malic, citric, malonic, and oxalic), triterpenoid saponins (3.3–5.5%), the nitrogen-containing compound allantoin, as well as hydroxycinnamic acids and their derivatives: chlorogenic acid (0.8%) and rosmarinic acid (1.4%). The leaves are characterised by the presence of organic acids (malic, citric, malonic, and oxalic), triterpenoid saponins (2.8–3.6%), vitamin C, hydroxycinnamic acids and their derivatives: chlorogenic acid (0.6%) and rosmarinic acid (1.7%), as well as flavonoids including quercetin, isoquercetin, rutin, and astragalin. In the flowers and fruits, organic acids (malic, citric, malonic, and oxalic), triterpenoid saponins (3.0–3.6%), and hydroxycinnamic acids with their derivatives: chlorogenic acid (0.95% and 1.05%) and rosmarinic acid (3.1% and 1.1%), respectively, have been identified [12].

In Ukraine, S. europaea is not officially recognised as a medicinal plant, yet it is used in folk medicine and homoeopathy. In traditional medicine, infusions and decoctions are used to treat various types of bleeding, bronchitis, pneumonia, and gastrointestinal disorders. Externally, the plant is used for treating wounds, burns, and frostbite, as well as for gargling in cases of stomatitis and tonsillitis. The rhizome tincture is traditionally regarded as a remedy that enhances male sexual function [15,16,17]. In folk medicine across various countries, the multifaceted use of S. europaea has been documented. In the Czech Republic, it is applied for chronic wounds; in the Caucasus region – for respiratory diseases; in Bulgaria – for dermatological conditions; in Romania – as an expectorant and sedative; in the Netherlands – for internal bleeding; and in Germany – as an astringent and antimicrobial agent [18,19,20].

Recent studies confirm the anti-inflammatory and antiviral activity of S. europaea. Austrian researchers have demonstrated that its extracts inhibit the formation of inflammatory mediators [21], while Turkish and Japanese scientists have shown that aqueous leaf extracts exhibit activity against influenza and parainfluenza viruses [22,23]

In homoeopathy, S. europaea is included in the formulation of RICURA® spag. Peka N., which is effective in the treatment of acute rhinosinusitis. In Ukraine, the company “Nutrimed” has developed the phytocomplex “Kamavit,” used for managing urological and sexual disorders in men [24]. Thus, S. europaea exhibits a broad spectrum of pharmacological properties and represents a promising candidate for further scientific investigation and integration into medical practice.

The aim of this study was to identify the key bioactive constituents of S. europaea herb and rhizomes with root extracts and evaluate their antimicrobial, anti-inflammatory, haemostatic, and wound-healing properties. This study presents, for the first time, specific aspects of the phytochemical composition of aqueous and aqueous-ethanolic S. europaea herb and rhizome-with-root extracts, and provides new in vitro evidence of their bacteriostatic effects, along with experimental confirmation of haemostatic and wound-healing activities. The research aims to contribute to the scientific understanding of the plant and support its potential integration into contemporary pharmaceutical and clinical applications.

2. Materials and Methods

2.1. Plant Raw Materials

The plant raw materials: herb and rhizomes with roots of S. europaea (0.5 kg of each kind) were collected in Ivano-Frankivsk region (Viktoriv village, Halych district, 49.050° N 24.628° E.) during the phases of peak flowering and senescence of the aerial parts, respectively. Standard protocols for harvesting medicinal plant raw materials were strictly followed, with particular attention paid to preserving the surrounding flora [25]. Prior to collection, the plant species was accurately identified. The identification was confirmed based on a botanical catalogue [26], with consultative support from Professor A. R. Hrytsyk of Ivano-Frankivsk National Medical University (IFNMU). The plant material was dried in the shade in a well-ventilated room, spread in a thin layer on paper. The dried raw material was stored in paper bags and used for experimental purposes within one year from the date of collection. Voucher specimens № 421–423 of S. europaea were deposited at the Department of Pharmaceutical Management, Drug Technology, and Pharmacognosy at IFNMU.

2.2. Extract Preparations

Complexes of biologically active compounds (BACs) from the herb and rhizomes with roots of S. europaea were obtained by remaceration, infusing the raw materials at a temperature of 50–60 °C with periodic stirring. The raw materials were ground with Laboratory Grinder PG500 (Labtehservice, Odesa, Ukraine) to particle sizes ranging from 1.0 to 2.5 mm. Purified water and 70% ethanol solution were used as extractants, as these solvents are most commonly employed in traditional medicine for the preparation of teas, decoctions, and tinctures, respectively.

To obtain the extract (solvent – purified water), 0.2 kg of the dried herb or 0.2 kg of the rhizomes with roots of S. europaea were infused with 2.80 L or 2.66 L of purified water, respectively. The raw material-to-solvent ratio was 1:10, taking into account the water absorption coefficient, which was 4.0 for the herb and 3.3 for the rhizomes with roots. The mixture was left to infuse for 1 hour at room temperature. After swelling, the material was extracted for 30 minutes at a temperature of 50-60 °C. The resulting extract was cooled and decanted, while the plant residue was re-extracted twice with fresh portions of solvent (2.0 L) under the same conditions. The obtained extracts (SEH0 and SEH70, respectively) were combined, allowed to settle, and filtered.

To obtain the extract (solvent – 70% ethanol), 0.2 kg of the dried herb or 0.2 kg of the rhizomes with roots of S. europaea were infused with 2.64 L or 2.44 L of 70% ethanol, respectively. The raw material-to-solvent ratio was 1:10, taking into account the ethanol absorption coefficient, which was 3.2 for the herb and 2.2 for the rhizomes with roots. The mixture was left to infuse for 1 hour at room temperature. After swelling, the material was extracted for 30 minutes at a temperature of 50-60 °C. The resulting extract was cooled and decanted, while the plant residue was re-extracted twice with fresh portions of solvent (2.0 L) under the same conditions. The obtained extracts (SER0 and SER70, respectively) were combined, allowed to settle, and filtered.

After decantation and filtration, the obtained extracts were dispensed into sterile 200 mL vials and subjected to lyophilisation. Initially, the vials were frozen in alcohol baths at a temperature not exceeding –40 °C for 30 minutes. Subsequently, the frozen extracts were tempered and stored in a refrigerator at a temperature not exceeding –30 °C for 12 hours prior to loading into the freeze-drying apparatus. Lyophilisation was performed using a KS-30 sublimation unit (Frigera plant, Brno, Czech Republic). During the initial phase of drying, the pressure in the sublimator was reduced from 1·10⁻¹ to 1·10⁻⁵ mmHg, and the temperature of the frozen extracts was lowered from –35 °C to –50 °C. After 2-2.5 hours, heating was initiated, and over the next 12-16 hours, the temperature was gradually increased from subzero to positive values. In the final stage of drying, the product temperature did not exceed +40 °C. The total duration of the drying process was 28-32 hours.

2.3. Phytochemical Analysis

The phenolic content was analyzed using a high-performance liquid chromatography system (Agilent 1200 3D LC System Technologies,Santa Clara, CA, USA), equipped with a UV-Vis diode-array detector (G1315C), a vacuum degasser (G1322A), a four-channel low-pressure gradient pump (G1311A), an autosampler (G1329A), a column thermostat (G1316A), and both diode-array (G1315C) and refractive index (G1362A) detectors. The system was operated in conjunction with a personal computer running Agilent ChemStation software. Phenolic compounds were isolated using reversed-phase chromatography with a Discovery C18 column (250 × 4.6 mm), packed with silica gel modified with octadecyl groups and a particle size of 5 μm. For the separation of hydroxycinnamic acids, flavonoids, and coumarins, the mobile phase consisted of 0.005 N orthophosphoric acid (eluent A) and acetonitrile (eluent B). For the separation of tannins, a mixture of 0.1% trifluoroacetic acid, 5% acetonitrile, and deionised water (eluent A), and 0.1% trifluoroacetic acid in acetonitrile (eluent B) was used [27,28].

The total phenolic content in the studied extracts was determined spectrophotometrically in accordance with the requirements of the State Pharmacopoeia of Ukraine, Edition 2.0 [29]. The results were expressed in terms of pyrogallol equivalents. Quantitative determination of total flavonoids was also performed spectrophotometrically using the same pharmacopoeial procedures, with rutin as the reference standard [30,31]. All analyses were conducted in triplicate to ensure statistical reliability of the results [29,32].

2.4. Pharmacological Research

The study of the specific pharmacological activity of S. europaea extracts was conducted with the consultative support of Professor O. H. Popadynets, Head of the Department of Human Anatomy, and Associate Professor V. M. Ivanochko of the same department. Biochemical and haematological parameters of the experimental animals' blood were examined at the “Bioelementology Centre” of IFNMU.

The experiments were conducted on white outbred mice, rats, and guinea pigs bred in the vivarium of IFNMU. The animals were standardised according to physiological and biochemical parameters and maintained under vivarium conditions in compliance with sanitary and hygienic regulations, housed in plastic cages, and fed a standard diet in accordance with current norms. Animal care and experimental procedures were carried out in accordance with both national and international guidelines for the humane treatment of laboratory animals, in alignment with the provisions of the “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (Strasbourg, 1986), the Law of Ukraine No. 3447-IV “On the Protection of Animals from Cruelty,” and the general ethical principles for animal experimentation adopted by the First National Congress of Ukraine on Bioethics (2001) [33,34,35]. The study was approved by the Bioethics Commission of the IFNMU (protocol 154/25 dated 22 October 2025).

2.4.1. Anti-Inflammatory Activity

To investigate the effect of S. europaea extracts on the exudative phase of inflammation, a rat paw oedema model induced by subplantar injection of a phlogogenic agent was employed. Specifically, 0.1 mL of a 1% carrageenan solution was administered beneath the aponeurosis of the hind paw [36,37]. The experiments were conducted on white outbred male rats weighing 190-230 g. The animals were divided into six groups, each consisting of six rats.

One hour before and immediately after administration of the phlogogenic agent, rats in the first and second groups received intragastric aqueous solutions of S. europaea herb extracts (solvent – purified water or 70% ethanol) at a dose of 100 mg/kg. Animals in the third and fourth groups were administered extracts from the rhizomes with roots of S. europaea (solvent:purified water or 70% ethanol) at the same dose of 100 mg/kg. The fifth group received the reference plant-based anti-inflammatory agent, quercetin (granules “Quercetin” (0500924), PJSC “Borshchahivskiy Chemical-Pharmaceutical Plant”, Kyiv, Ukraine), at a conditionally effective dose of 5 mg/kg. The sixth group served as the control.

Paw volume was measured oncometrically prior to the experiment, at 1 hour, 3 hours, and at the peak of oedema development — 5 hours after administration of the phlogogenic agent. The effect of S. europaea extracts was assessed based on their ability to suppress paw oedema in rats over time, in comparison with the control group and the reference drug. The obtained data were evaluated using statistical analysis methods.

2.4.2. Wound Healing and Haemostatic Activity

The haemostatic activity of the extracts from the herb and rhizomes with roots of S. europaea was studied in sexually mature guinea pigs bred in the vivarium of IFNMU, standardised according to physiological and biochemical parameters.

The haemostatic properties of the tested extracts were evaluated by measuring bleeding time using the Duke method, following the induction of a linear incised wound [38,39]. Bleeding duration was recorded with a stopwatch. The wound was modelled by making an incision through all layers of previously depilated skin on the lateral surface of the hind limb using a scalpel. The standard wound size was 2.5 × 0.3 cm. Prior to incision, local anaesthesia was administered using a 2% solution of novocaine [36,38,39]. The animals were divided into six groups (six individuals per group). Immediately after modelling the incised wound, animals in the first and second groups received a quadruple-folded gauze pad soaked in aqueous extracts of S. europaea herb (solvent – purified water or 70% ethanol) applied to the wound surface. Animals in the third and fourth groups were treated with aqueous extracts of S. europaea rhizomes with roots (solvent – purified water or 70% ethanol). The fifth group received the reference preparation “Liquid Extract of Water Pepper” (PJSC “Phytopharm”), and the sixth group served as the control.

The wound-healing activity of the extracts from the herb and rhizomes with roots of S. europaea was studied in sexually mature guinea pigs. An aseptic incised wound was modelled by making a 25 mm long and 5 mm deep incision under local aneasthesia on the depilated skin of the lateral surface of the hind limb [27,36]. The animals were divided into six groups (six individuals per group). Following the modelling of an incised wound, animals in the first and second groups received topical applications of aqueous extracts of S. europaea herb (solvent – purified water or 70% ethanol) twice daily. Animals in the third and fourth groups were treated with aqueous extracts of S. europaea rhizomes with roots (solvent – purified water or 70% ethanol) under the same regimen. The fifth group received the reference preparation “Recutan” (Liquid extract of chamomile flowers (Flores Chamomillae) in a ratio of 1:1, UA/5120/01/01, LLC “Pharmaceutical Company Zdorovya”, Ukraine), and the sixth group served as the control. Wound planimetry was performed in all experimental groups until complete healing was achieved [40].

2.4.3. Antimicrobial Activity

The antimicrobial activity of S. europaea extracts was investigated using the agar diffusion method with paper discs, following the procedure developed by A. M. Chornomyrdik [29,41]. The concentration of the active substance on the discs was 5 mg. A 5% blood agar and 24-hour culture broths prepared in 1% sugar broth were used as a universal nutrient medium, with microbial suspensions standardised to a density of 1 billion microbial cells per millilitre. For inoculation, 1 mL of the bacterial suspension was applied to the surface of the 5% blood agar and evenly rubbed in. The cultures were incubated at 37 °C for 24 to 72 hours, depending on the characteristics of the tested microorganisms. The antimicrobial activity of the extracts was evaluated using standard microbial strains: Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Proteus vulgaris ATCC 33420, Staphylococcus aureus ATCC 25923, and Staphylococcus epidermidis ATCC 12228. The activity of the herb and rhizome extracts of S. europaea was determined by measuring the diameter (in mm) of the microbial growth inhibition zones around the paper discs saturated with the test substance. For comparison of bacteriostatic activity, paper discs impregnated with standard antibiotics were used: ampicillin at 30 μg/disc and oleandomycin at 30 μg/disc.

2.5. Statistical Analysis

Data are presented as the mean ± standard deviation (SD). For phytochemical evaluations, each data point represents the average of a minimum of three independent determinations. Pharmacological assays were conducted with at least six replicates. Confidence intervals were calculated using critical values from Student’s t-distribution [32,42,43]. Statistical significance was determined through one-way ANOVA, followed by Tukey’s multiple comparison test.

3. Results

S. europaea extracts are hygroscopic powders ranging in colour from light yellow to dark brown. The loss on drying ranges from 3.80% to 4.59%, which complies with the requirements for dry extracts.

3.1. Phytochemical Research

Phenolic compounds in the dry extracts of S. europaea herb and rhizomes with roots were determined using HPLC and spectrophotometry (Table 1).

Table 1. Quantitative content of phenolic compounds in Sanicula europaea extracts.

Phenolic Compound	Content of BACs (mg/kg)
	SEH0		SEH70	SER0	SER70
Metabolites of tannins
Gallocatechin	3473±104		4236±148	3464±116	3893±325
Catechin	933±48		1104±72	325±27	435±14
Epicatechin	532±21		764±51	2123±324	2598±78
Catechin gallate	1075±54		1432±102	-	-
Epicatechin gallate	12275±422		17658±679	2015±134	2765±107
Ellagic acid	28±2		37±4	104±12	164±8
Gallic acid	-		-	68±9	109±11
Epigallocatechin	-		-	2161±275	2703±168
Flavonoids
Quercetin	132±16		196±9	-	-
Kaempferol	20±2		42±3	-	-
Apigenin	120±11		203±15	150±12	217±9
Coumarins
Coumarin		97±6	193±9	108±5	217±21
Hydroxycinnamic acids
Chlorogenic acid		108±14	142±24	425±21	612±22
Rosmarinic acid		5476±261	6024±187	3697±128	4737±308
Caffeic acid		319±41	386±24	213±13	362±21
Ferulic acid		99±11	173±9	114±9	238±17
Content of BACs groups in the extract, % (spectrophotometry)
Total polyphenols		13.54±0.24	15.02±0.19	11.08±0.15	12.24±0.24
Flavonoids		3.61±0.14	5.46±0.23	0.13±0.01	0.21±0.02

Notes: SEH0, SEH70 – herb extract (solvent – purified water and 70% ethanol respectively); SER0, SER70 – rhizome with root extracts (solvent – purified water and 70% ethanol respectively).

Table 1. Quantitative content of phenolic compounds in Sanicula europaea extracts.

Phenolic Compound	Content of BACs (mg/kg)
	SEH0		SEH70	SER0	SER70
Metabolites of tannins
Gallocatechin	3473±104		4236±148	3464±116	3893±325
Catechin	933±48		1104±72	325±27	435±14
Epicatechin	532±21		764±51	2123±324	2598±78
Catechin gallate	1075±54		1432±102	-	-
Epicatechin gallate	12275±422		17658±679	2015±134	2765±107
Ellagic acid	28±2		37±4	104±12	164±8
Gallic acid	-		-	68±9	109±11
Epigallocatechin	-		-	2161±275	2703±168
Flavonoids
Quercetin	132±16		196±9	-	-
Kaempferol	20±2		42±3	-	-
Apigenin	120±11		203±15	150±12	217±9
Coumarins
Coumarin		97±6	193±9	108±5	217±21
Hydroxycinnamic acids
Chlorogenic acid		108±14	142±24	425±21	612±22
Rosmarinic acid		5476±261	6024±187	3697±128	4737±308
Caffeic acid		319±41	386±24	213±13	362±21
Ferulic acid		99±11	173±9	114±9	238±17
Content of BACs groups in the extract, % (spectrophotometry)
Total polyphenols		13.54±0.24	15.02±0.19	11.08±0.15	12.24±0.24
Flavonoids		3.61±0.14	5.46±0.23	0.13±0.01	0.21±0.02

Notes: SEH0, SEH70 – herb extract (solvent – purified water and 70% ethanol respectively); SER0, SER70 – rhizome with root extracts (solvent – purified water and 70% ethanol respectively).

3.2. Anti-Inflammatory Activity

The anti-inflammatory and anti-exudative activities of the extracts SEH0, SEH70, SER0, and SER70 were studied using white non-linear rats bred in the vivarium of IFNMU and standardised according to physiological and biochemical parameters. The increase in paw volume and the anti-exudative activity of S. europaea extracts are presented in Table 2.

Table 2. Anti-exudative activity of Sanicula europaea extracts.

Animal group	Investigated sample	Dose mg/kg	$\mathbf{Paw}\mathbf{volume}\mathbf{increase},\overline{\mathit{х}}\pm \mathit{\Delta }\overline{\mathit{х}}$, n=6 (Inflammatory response inhibition index, %)
			1 hour	3 hours	5 hours
І	SEH0	100	9.26 ± 0.16*	27.46 ± 0.16*	29.73 ± 0.23*
			(10.70)	(26.97)	(36.40)
ІІ	SEH70	100	9.03 ± 0.23*	27.52 ± 0.35*	31.56 ± 0.35*
			(12.92)	(26.80)	(32.62)
ІІІ	SER0	100	9.51 ± 0.18*	29.96 ± 0.18*	32.99± 0.25*
			(8.29)	(20.31)	(29.56)
IV	SER70	100	9.93 ± 0.29*	30.61 ± 0.29*	34.02 ± 0.24*
			(4.24)	(18.59)	(27.40(
V	Quercetin	5	9.42 ± 0.21*	27.56 ± 1.13*	33.07 ± 1.11*
			(9.16)	(26.70)	(29.40)
VI	-	-	10.37 ± 0.63	37.60 ± 0.91	46.84 ± 1.01

Notes: * statistically significant difference compared to the pathological control group (p ≤ 0.05); n – number of animals in the group.

Table 2. Anti-exudative activity of Sanicula europaea extracts.

Animal group	Investigated sample	Dose mg/kg	$\mathbf{Paw}\mathbf{volume}\mathbf{increase},\overline{\mathit{х}}\pm \mathit{\Delta }\overline{\mathit{х}}$, n=6 (Inflammatory response inhibition index, %)
			1 hour	3 hours	5 hours
І	SEH0	100	9.26 ± 0.16*	27.46 ± 0.16*	29.73 ± 0.23*
			(10.70)	(26.97)	(36.40)
ІІ	SEH70	100	9.03 ± 0.23*	27.52 ± 0.35*	31.56 ± 0.35*
			(12.92)	(26.80)	(32.62)
ІІІ	SER0	100	9.51 ± 0.18*	29.96 ± 0.18*	32.99± 0.25*
			(8.29)	(20.31)	(29.56)
IV	SER70	100	9.93 ± 0.29*	30.61 ± 0.29*	34.02 ± 0.24*
			(4.24)	(18.59)	(27.40(
V	Quercetin	5	9.42 ± 0.21*	27.56 ± 1.13*	33.07 ± 1.11*
			(9.16)	(26.70)	(29.40)
VI	-	-	10.37 ± 0.63	37.60 ± 0.91	46.84 ± 1.01

Notes: * statistically significant difference compared to the pathological control group (p ≤ 0.05); n – number of animals in the group.

The findings presented in Table 2 indicate that paw oedema in experimental groups increased during the first 3 hours following administration of the phlogogenic agent, whereas in the untreated control group, oedema continued to progress for up to 5 hours. Maximum inflammation suppression was observed in treated animals at the 5-hour mark, although the anti-exudative activity varied depending on the type of administered extract. The anti-exudative effects of the S. europaea extracts and quercetin were evident as early as 1 hour after treatment initiation, compared to the control group. Throughout the 5-hour experiment, the most pronounced activity was demonstrated by the extracts from the herb of S. europaea. At the 3-hour time point, the anti-exudative effects of SEH0, SEH70 and the reference drug were comparable. However, by hour 5, the activity of the reference drug slightly declined relative to the tested extracts. The rhizomes with root extracts also exhibited anti-exudative effects compared to the control group, although the activity of SER70 was somewhat lower, and SER0 was comparable to that of quercetin.

3.3. Wound Healing and Hemostatic Activity

The haemostatic properties of the tested extracts were evaluated by measuring bleeding time using the Duke method [38]. A linear incised wound was applied to the animals, and the duration of bleeding was recorded using a stopwatch. The results of the study are presented in Table 3.

Table 3. Effect of Sanicula europaea extracts on bleeding time.

Animal group, n=6	Bleeding time, s	Reduction in bleeding time vs. control group, %	Reduction in bleeding time vs. reference drug, %
SEH0	113.33±1.45*/**	49.56	34.37
SEH70	116.17±2.89*/**	48.29	32.72
SER0	179.50±2.42*	20.11	-
SER70	193.50±2.42*	13.87	-
Reference drug: Liquid extract of water pepper	172.67±2.90	23.15	-
Control group: untreated animals (pathological control)	224.67±4.45	-	-

Notes: n — number of animals in the group; * — statistically significant difference compared to the control group (p ≤ 0.05); ** — statistically significant difference compared to the reference drug (p ≤ 0.05).

Table 3. Effect of Sanicula europaea extracts on bleeding time.

Animal group, n=6	Bleeding time, s	Reduction in bleeding time vs. control group, %	Reduction in bleeding time vs. reference drug, %
SEH0	113.33±1.45*/**	49.56	34.37
SEH70	116.17±2.89*/**	48.29	32.72
SER0	179.50±2.42*	20.11	-
SER70	193.50±2.42*	13.87	-
Reference drug: Liquid extract of water pepper	172.67±2.90	23.15	-
Control group: untreated animals (pathological control)	224.67±4.45	-	-

Notes: n — number of animals in the group; * — statistically significant difference compared to the control group (p ≤ 0.05); ** — statistically significant difference compared to the reference drug (p ≤ 0.05).

As shown in Table 3, topical application of the reference drug, liquid extract of water pepper, reduced bleeding time by 23.15% compared to the control group. The application of S. europaea herb extracts resulted in a more pronounced reduction in bleeding time by 49.56% and 48.29%, respectively, relative to the control group, and by 34.37% and 32.72% compared to the reference drug. The rhizomes with root extracts also demonstrated haemostatic activity, reducing bleeding time by 20.11% and 13.87%, respectively, compared to the control group. Thus, it can be concluded that application of the herb extracts (SEH0 and SEH70) to the wound accelerated haemostasis by 1.98 and 1.93 times compared to the control group, and by 1.52 and 1.48 times compared to the reference drug, respectively. The reference drug itself shortened bleeding time by 1.3 times relative to the control. In contrast, the rhizomes with root extracts (SER0 and SER70) exhibited lower haemostatic activity than the reference drug, but still reduced bleeding time by 1.25 and 1.16 times, respectively, compared to the control group. These findings indicate the presence of local haemostatic activity in the herb extracts of S. europaea.

Over a 16-day period, wound planimetry was performed in conjunction with assessments of the animals' general condition and the rate of wound healing. The dynamics of the wound healing process are presented in Table 4.

Table 4. Wound healing dynamics following application of Sanicula europaea herb and rhizomes with roots extracts.

Animal group, n=6	Wound healing area, %
	Day: 2	4	6	8	10	12	14	16
SEH0	4.69	15.48	48.87	80.58	100	100	100	100
SEH70	4.37	14.36	43.71	76.55	100	100	100	100
SER0	3.80	10.71	32.00	54.33	74.33	100	100	100
SER70	3.83	10.04	31.02	53.39	68.16	95.47	100	100
Reference drug: Recutan	5.33	17.34	39.63	80.69	98.76	100	100	100
Control group: untreated animals (pathological control)	3.40	8.25	30.90	51.80	68.91	79.33	94.98	100

Table 4. Wound healing dynamics following application of Sanicula europaea herb and rhizomes with roots extracts.

Animal group, n=6	Wound healing area, %
	Day: 2	4	6	8	10	12	14	16
SEH0	4.69	15.48	48.87	80.58	100	100	100	100
SEH70	4.37	14.36	43.71	76.55	100	100	100	100
SER0	3.80	10.71	32.00	54.33	74.33	100	100	100
SER70	3.83	10.04	31.02	53.39	68.16	95.47	100	100
Reference drug: Recutan	5.33	17.34	39.63	80.69	98.76	100	100	100
Control group: untreated animals (pathological control)	3.40	8.25	30.90	51.80	68.91	79.33	94.98	100

The results of the study indicate that the application of S. europaea herb extracts (SEH0 and SEH70) accelerated the wound healing process by 5.34 and 4.84 days, respectively, compared to the control group, and by 1.5 and 1.0 days relative to the reference drug Recutan. In contrast, the rhizomes with root extracts (SER0 and SER70) shortened the healing period by 2.83 and 2.01 days, respectively, compared to the control group, but were less effective than the reference drug. Thus, S. europaea herb extracts exhibit wound-healing activity, positively influence the wound repair process, and demonstrate therapeutic potential for topical treatment of skin injuries.

3.4. Antimicrobial Activity

The antibacterial activity of S. europaea raw material extracts was evaluated at the bacteriological laboratory of Military Unit A4520 (Lviv) using the agar diffusion method with paper discs. The results of the study are presented in Table 5.

Table 5. Antimicrobial activity of Sanicula europaea extracts.

Investigated sample	$\mathbf{Inhibition}\mathbf{zone}\mathbf{diameter},\mathbf{mm},\overline{\mathit{х}}\pm \mathit{\Delta }\overline{\mathit{х}}$
	Pseudomonas aeruginosa АТСС 27853	Escherichia coli АТСС 25922	Proteus vulgaris АТСС 33420	Staphylococcus aureus АТСС 25923	Staphylococcus epidermidis АТСС 12228
SEH0	12±0.38	11±0.34	10±0.36	11±0.37	12±0.38
SEH70	-	15±0.41	-	15±0.40	8±0.29
SER0	15±0.39	9±0.28	7±0.25	7±0.31	11±0.34
SER70	10±0.34	10±0.33	9±0.27	9±0.29	9±0.30

Table 5. Antimicrobial activity of Sanicula europaea extracts.

Investigated sample	$\mathbf{Inhibition}\mathbf{zone}\mathbf{diameter},\mathbf{mm},\overline{\mathit{х}}\pm \mathit{\Delta }\overline{\mathit{х}}$
	Pseudomonas aeruginosa АТСС 27853	Escherichia coli АТСС 25922	Proteus vulgaris АТСС 33420	Staphylococcus aureus АТСС 25923	Staphylococcus epidermidis АТСС 12228
SEH0	12±0.38	11±0.34	10±0.36	11±0.37	12±0.38
SEH70	-	15±0.41	-	15±0.40	8±0.29
SER0	15±0.39	9±0.28	7±0.25	7±0.31	11±0.34
SER70	10±0.34	10±0.33	9±0.27	9±0.29	9±0.30

The results of the study demonstrate that both herb and rhizomes with roots extracts of S. europaea inhibit the growth of rod-shaped and coccoid microorganisms. Extracts SEH0, SER0, and SER70 exhibited bacteriostatic activity against all tested microbial strains. In contrast, the SEH70 extract showed no activity against P. aeruginosa and P. vulgaris.

4. Discussion

A total of sixteen phenolic constituents were detected in the dry extracts of S. europaea herb and rhizomes with roots. These included one coumarin, four hydroxycinnamic acids, three flavonoids, and eight metabolites derived from tannins. Rosmarinic acid was the most abundant among the hydroxycinnamic acids. Quercetin and apigenin emerged as the leading flavonoids, and epicatechin gallate and gallocatechin were identified as the predominant tannin-related compounds. Rosmarinic and chlorogenic acids have previously been reported in the extracts of S. europaea herb [44]. Among the identified constituents, epigallocatechin gallate (EGCG) [45], caffeic, chlorogenic and rosmarinic acids, quercetin, apigenin, and kaempferol demonstrated the strongest anti-inflammatory potential. The biological activity is largely attributed to their ability to modulate key inflammatory signalling cascades, such as NF-κB, suppress pro-inflammatory cytokine production, and downregulate enzymes involved in inflammation. Specifically, chlorogenic acid is known to inhibit NF-κB activation, a central regulator of inflammatory gene expression, thereby decreasing levels of TNF-α, IL-6, and IL-1β, reducing oxidative stress, and limiting the expression of COX-2 and iNOS [46].

Similarly, caffeic acid exerts anti-inflammatory effects by blocking the synthesis of inflammatory mediators and enzymes, primarily through the inhibition of NF-κB signalling and oxidative pathways [47,48]. EGCG is well known for its strong anti-inflammatory properties, largely due to its influence on critical cellular signalling mechanisms. Numerous in vitro and in vivo experiments have demonstrated that EGCG can effectively inhibit the activation of NF-κB, prevent its movement into the nucleus, and interfere with AP-1 transcriptional activity [45,49]. Some flavonoids detected in these dry extracts demonstrate significant anti-inflammatory effects through various molecular pathways. Quercetin, in particular, has been found to reduce the expression of major pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, as well as enzymes such as COX-2. Its mechanism of action primarily involves the inhibition of the JAK1/STAT3/HIF-1α axis and the NF-κB signalling cascade [50,51].

Thus, in the experiment, the studied extracts of S. europaea exhibit a pronounced anti-inflammatory effect in the carrageenan-induced oedema model. The SEH0 extract suppresses the inflammatory response more effectively, reducing oedema by 36.40% at the 5th hour of the experiment (compared to quercetin — 29.40%). The anti-inflammatory effect may be associated with the ability of flavonoids and phenolic acids to inhibit the synthesis of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and the activity of COX-2 enzymes, as well as to suppress the NF-kB and JAK/STAT signalling cascades, leading to reduced exudation and oedema [45,47,51]. In previous scientific literature, dichloromethane and methanol extracts of S. europaea root have been shown to exhibit moderate to strong anti-inflammatory effects in vitro on endothelial cells, reducing the expression of inflammatory mediators IL-8 and E-selectin following stimulation with TNF-α and the bacterial product lipopolysaccharide (LPS) [21,52].

Topical application of herb extracts (SEH0 and SEH70) resulted in bleeding cessation that was 1.52 and 1.48 times faster, respectively, compared to the reference drug, and 1.98 and 1.93 times faster relative to the control group. The reference drug, liquid extract of water pepper, reduced bleeding time only by 1.3 times compared to the control group. These findings indicate the presence of local haemostatic activity in the herb extracts of S. europaea. This effect may be attributed to tannins and phenolic compounds, which stimulate plasma protein coagulation and contraction of small blood vessels, thereby accelerating thrombus formation [53]. The roots of S. europaea were previously used in Austrian traditional medicine as a haemostatic agent [21,44,52], however, scientific confirmation through experimental studies has been obtained for the herb extracts for the first time.

It was established that complete wound healing occurred on days 8-9 and 9-10 following the application of SEH0 and SEH70 herb extracts, respectively. In comparison, the reference drug Recutan achieved healing on days 10–11, while in the control group, wound closure was observed only on days 15–16. The most pronounced wound-healing effect and favourable healing dynamics were recorded with the SEH0 extract. This effect is attributed to the combined action of its constituents: flavonoids and phenolic acids stimulate fibroblast proliferation and epithelialization, while tannins promote the formation of a robust granulation layer and vasoconstriction. To date, the wound-healing properties of S. europaea have been primarily documented in ethnobotanical sources and phytochemical reviews, with limited experimental validation [44,52]. Our study is the first to demonstrate complete wound closure within 8–10 days following topical application of SEH0 and SEH70 herb extracts.

It was found that S. europaea extracts inhibit the growth of rod-shaped and coccoid microflora and exhibit bacteriostatic activity against P. aeruginosa ATCC 27853, E. coli ATCC 25922, P. vulgaris ATCC 33420, S. aureus ATCC 25923, and S. epidermidis ATCC 12228. This activity is attributed to the presence of a wide range of BACs in the extracts, including tannins, flavonoids, and phenolic substances [54]. Tannins are capable of binding to bacterial cell wall proteins, disrupting membrane integrity and limiting microbial growth. Flavonoids, such as quercetin and rutin, inhibit bacterial enzymes and nucleic acid synthesis in pathogens, thereby enhancing the antibacterial efficacy of the extracts [55,56]. The observed bacteriostatic activity of S. europaea herb extracts against both Gram-negative (P. aeruginosa, E. coli, P. vulgaris) and Gram-positive (S. aureus, S. epidermidis) strains aligns with earlier phytochemical reports highlighting the antimicrobial potential of the genus Sanicula in traditional folk medicine [44,52]. However, this is the first report demonstrating direct in vitro bacteriostatic effects of S. europaea herb extracts against standard ATCC strains, thereby expanding the pharmacological evidence base for this species.

5. Conclusions

Herb extracts of S. europaea exhibit a broad spectrum of pharmacological effects. They demonstrate bacteriostatic activity against both rod-shaped and coccoid microorganisms, accelerate bleeding cessation, promote wound healing, and possess anti-inflammatory properties. The rhizomes with root extracts exhibited less pronounced activity across the evaluated parameters. The most pronounced effects were observed with the aqueous extract, indicating its potential as a natural agent with haemostatic, wound-healing, and anti-inflammatory action.