INTRODUCTION:

A wound is defined as an injury or damage to the body's tissue, typically involving the skin or underlying structures, that disrupts the normal anatomical integrity and physiological function of the affected area^1-5.

Wounds can be broadly categorized into different types based on their underlying causes and characteristics. Common types of wounds include incisions, lacerations, abrasions, punctures, and ulcers. Each type presents unique challenges in terms of treatment and healing due to differences in depth, severity, and potential for infection^6-9. Wound healing is a complex process that involves multiple components, including soluble mediators, blood cells, extracellular matrix, and parenchymal cells. It encompasses a collection of intricate processes necessary for the restoration and repair of damaged tissue^10-14.

Naturally derived polymers find extensive application in regenerative medicine owing to their inherent biocompatibility, biodegradability, biological attributes, and structural resemblance to human tissues^15-17. In situations where the damaged tissue lacks the natural ability to regenerate, solely applying a protein wound dressing (WD) to the affected area does not facilitate tissue repair. In such cases, additional methods are employed, involving the use of various biologically active substances. These substances, which can include growth factors, angiogenic factors, cell surface receptors, as well as antimicrobial, and anti-inflammatory agents, are incorporated into the WD to enhance cellular behavior and promote tissue regeneration^18-21. Certain naturally derived materials obtained from animals or plants, typically made up of proteins or polysaccharides, replicate the fibrillar arrangement found in the original extracellular matrix and exhibit comparable architectural characteristics^22-24. In folk medicine, a wide array of plants such as Curcuma longa (L.), Terminalia arjuna, Centella asiatica, Bidens Pilosa, Aloe barbadensis, and Rauwolfia serpentine have confirmed wound healing activity and are found to be effective in the treatment of wounds^25,26. Extracts of medicinal plants exhibit wound healing effects through various mechanisms. These include modulation of the wound healing process, reduction in bacterial count, enhancement of collagen deposition, and promotion of the proliferation of fibroblasts and fibrocytes, among others. These mechanisms contribute to the overall efficacy of medicinal plants in facilitating the healing of wounds⁶. Plants belonging to the Rumex genus, which is categorized within the Polygonaceae family, have a long history of use in traditional medicine in the world. Numerous studies have highlighted the antimicrobial properties, cytotoxic effects, antioxidant activity, and wound healing potential exhibited by extracts derived from various plant species belonging to the Rumex genus^27-29. Therefore, the aim of the study was to explore the wound healing and antimicrobial properties of extracts derived from R. confertus Willd. and formulate a collagen-based WD composition integrated with the biologically active substance obtained from this plant.

MATERIALS AND METHODS:

Extraction of polyphenolic compounds from the R. confertus Willd:

When selecting the optimal conditions for the release of tannins, we studied the influence of several factors: the degree of grinding of raw materials, hydromodulus, temperature, and the dynamics of the extraction process. To determine the optimal degree of grinding of raw materials, we conducted extractions using both the root and aerial parts of the plant. These parts were crushed to different degrees: larger than 10cm, 5-10cm, 2.5-5cm, and smaller than 2.5cm. For the selection of the optimal hydromodulus, extractions were performed using raw materials with a degree of grinding of the root at 2.5-5 cm and the aerial part at 5-10cm. Different hydromodulus ratios of 1:3, 1:5, 1:7, and 1:10 were employed, taking into account the moisture absorption coefficient. The influence of temperature on the tannin extraction process was investigated at various levels: room temperature (18-20°C), 30-40°C, and 40-50°C. This temperature range allowed us to assess the impact of heat on tannin release. To study the extraction dynamics, we periodically replaced the saturated extractant until the raw material was completely depleted. This approach enabled us to monitor the progress of the extraction process over time. By systematically evaluating these factors, we aimed to identify the optimal conditions for tannin release while minimizing inefficiencies in the extraction process^30-32.

Preparation of composition for wound dressings based on collagen:

The tendons of cattle (bovine) underwent a thorough cleaning process to remove muscles, ligaments, and other impurities. Subsequently, the cleaned tendons were crushed into pieces measuring 1-2mm in size. These crushed tendon pieces were then treated with a 0.25% trypsin solution at a temperature of 37°C. After the trypsinization procedure, the tendon fragments underwent multiple washes with distilled water to eliminate non-collagen proteins. Subsequently, they were subjected to hydrolysis in a 0.1 N acetic acid solution for 48-72hours, maintaining a ratio of 1:100 (tendon to acetic acid solution). Following the hydrolysis, the resulting gel-like substance, which contained collagen, was homogenized at 3000rpm for 5-7 minutes. This homogenization step ensured thorough mixing and uniformity of the collagen mass. Next, the collagen mass was filtered through a mesh material to remove any large pieces, which were subsequently sent for re-hydrolysis. This additional hydrolysis step aimed to extract collagen from the remaining larger pieces and maximize the yield of collagen.

Creation of WD compositions based on collagen with biologically active substances:

The method for optimizing the production of films from rat and cattle collagen has been successfully developed. A 1% glycerin was incorporated into the film composition to enhance the elasticity of the resulting films. To conduct a comparative analysis of specific activity, particularly in the context of wound healing, and assess toxicity, novel compositions of films were formulated. These compositions include: RC-Rc (This refers to a combination of rat collagen (RC) with an extract isolated from R. confertus Willd. (Rc)); CC-Rc (This denotes the combination of cow collagen (CC) with an extract isolated from R. confertus Willd. (Rc)). In this composition, the extract from R. confertus Willd. was added at a concentration of 50µg/ml.

Creation of a model of burn wounds IIIA degree:

To establish a model for the study, burn wounds were deliberately induced on outbred white rats with a weight range of 180-200g³³. The animals were placed under ether anesthesia during the experiment. Two wounds were inflicted on each animal using a specialized electrical device. The device consisted of a soldering iron with a copper plate attached to its end, with an area measuring 1.5x1.5cm. The depilated areas on the backs of the animals were subjected to the application of a soldering iron heated to 1200°C. Each animal was housed in a separate cage to ensure individual monitoring and care. On the second day of the experiment, a 30% salicylic acid ointment was applied to the wounds for a duration of 3hours. Afterward, the scabs on the wounds were gently removed using gauze. Subsequently, the wounds were cleansed using sterile saline, and the investigated wound healing-related substances, denoted as WD (with the extract), were promptly administered. The advancement of the wound healing process was evaluated based on various criteria. Clinical observations encompassed the assessment of soft tissue edema, skin hyperemia, and pain during manipulations, along with the identification of necrotic detritus and wound exudate on the wound surface. Additionally, parameters such as the resolution of perifocal inflammation, the development and maturation of granulation tissue, the initiation of epithelialization, and the timeframe for complete healing were monitored. Objective data for assessing the course of the wound process were obtained through the histomorphology of the scarred area.

To gauge the rate of wound healing, planimetric research methods were employed. A sterile cellophane or polyacrylic plate was positioned over the wound, and the contours were traced onto the plate. Subsequently, the image of the wound contours was transferred to graph paper, and the dimensions were measured on days 3, 12, and 19 to track the progress of the wound over time.

All experimental procedures adhered to the regulations outlined in the "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" (Strasbourg, 1986). Approval for these procedures was obtained from the Institutional Ethical Committee in accordance with the legislation of the Republic of Uzbekistan.

Antimicrobial Activities of Extracts:

The antimicrobial activity of the extracts was assessed using the agar well-diffusion method^34,35. The test microorganisms utilized in the study were Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. The extracts from the plants were tested in 2 concentrations (1 and 2mg per well). The Rutan (polyphenolic compounds (tannins) obtained from Rhus coriaria) was used as positive control at concentration of 1mg per well. The plates containing bacteria were incubated at 37 °C for 24hours, while plates with C. albicans were incubated at 28°C for 48hours. Then diameter of the inhibition zone was measured.

Study of acute and chronic toxicity of WD components:

The acute toxicity study of the components of the WD (with the extract) was conducted on mature white male rats. The experimental groups consisted of 6 rats each. The components were administered orally to the animals in a single dose. The doses used were as follows: 500, 1000, 2000, 3000, 4000, and 5000mg/kg. Around 3-4 hours following the administration of the components, the rats were given both natural and briquette feed. Subsequently, the animals were monitored for a duration of 14 days to assess and observe any potential adverse effects or signs of toxicity.

The experiments were conducted using white outbred rats, with six rats tested for each dose. A control group of animals was included, which received water under similar experimental conditions. All experimental and control animals were kept in the same conditions and provided with a normal diet. Throughout the experiment, the animals underwent daily observations, encompassing assessments of their general condition, behavior, food and water consumption, as well as the state of their hair and mucous membranes. Following the final dose administration, blood samples were collected from all groups of animals through partial resection (0.5-1.0cm) of the tail vein. These blood samples were used to determine various hematological parameters using the BC-3000 hematological analyzer. Following the blood collection, the animals were placed under light ether anesthesia, and blood was collected through simultaneous decapitation for further biochemical studies.

Study of skin irritant action:

The study on the skin-irritating action was conducted following the guidelines outlined in GOST ISO 10993-11-2011. The test substance was applied as an application to a 2x2cm area on a clipped section of the back of the animals, which had a total area of 15x10cm. The animals were immobilized for a duration of 4 hours. Skin reactions were observed and recorded at the end of the 4-hour exposure period, as well as at 1 and 16hours after the application of the test substance. The skin reactions were evaluated using a scale of skin tests, with points assigned based on the observed indicators of the skin reaction.

Study of the local irritant effect on the eye:

To assess the local irritant effect on the eyes, rabbits were used as test subjects. The evaluation focused on determining the severity of specific eye reactions, including hyperemia (redness) and edema (swelling) of the conjunctiva, scleral vessels, as well as the condition of the cornea and iris. Additionally, the quantity and quality of discharge from the eyes were also noted during the assessment.

Statistical analysis:

Statistical analysis and exponential curve fitting were performed using Origin 8.6 software (Microcal Software Inc., Northampton, MA). Results were expressed as mean±S.E.M. To determine the statistical significance of the results One-Way ANOVA and two-tailed t-test were performed.

RESULTS AND DISCUSSION:

Technology of obtaining polyphenolic compounds from the R. confertus Willd:

The technology of obtaining polyphenolic compounds from botanical sources has garnered considerable attention in recent years due to the diverse array of bioactive molecules that can be harnessed for various applications, including pharmaceuticals, nutraceuticals, and functional food additives. Among the plant species under investigation, R. confertus Willd., a member of the Polygonaceae family, has emerged as a promising candidate rich in polyphenolic compounds. The extraction and characterization of polyphenols from this botanical source have become the focal point of scientific research aimed at unlocking the potential benefits of these compounds^36,37. In this study, we present the results obtained through our systematic investigation into the technology of obtaining polyphenolic compounds from R. confertus Willd. The results of the study indicate that the yield of tannins from roots with rhizomes varied based on the degree of grinding. When the raw materials were ground larger than 10cm, the yield was 3.7%. At a grinding size of 5-10cm, the yield increased to 4.2%, while at 2.5-5cm, it further increased to 4.5%. The highest yield of 4.7% was obtained when the raw materials were ground to less than 2.5cm. For the aerial parts, the yield of tannins was lower. Grinding larger than 10cm resulted in a yield of 1.2%, while at 5-10cm, it increased to 1.9%. Grinding at 2.5-5cm and less than 2.5cm yielded 2.1% and 2.2%, respectively. Excessive fine grinding of the raw materials complicated the filtration process and had a minimal effect on the yield of tannins from the aerial parts, making grinding sizes of 5-10cm optimal for the aerial parts and 2.5-5cm optimal for the roots.

Increasing the total hydromodulus to 1:5 significantly increased the yield of tannins. The yield increased from 5.06% to 9.78% in the roots and from 1.18% to 5.51% in the aerial parts. Further increasing the hydromodulus resulted in a slight increase in the yield of the target product to 9.96% and 6.11%, respectively, but with a significant increase in extractant consumption.

Temperature did not have a significant effect on the yield of tannins in both the roots and aerial parts of the R. confertus Willd. plant. The optimal temperature for the extraction process was observed to be 18-20°C.

The extraction dynamics study revealed that the equilibrium concentration of tannins was achieved at different contact times for each extraction phase: 8hours for the first phase, 6hours for the second, 4hours for the third, and 2hours for the fourth.

In conclusion, the total yield of tannins from the underground and above-ground parts was determined to be 8.3% and 5.1%, respectively. By performing a three-fold extraction of the roots with a grinding size of 2-5 cm and the aerial parts with a grinding size of 5-10 cm, at a total hydromodulus of 1:5 and room temperature, the total yield of tannins increased to 10.9% for the roots and 5.8% for the aerial parts.

Application of WD compositions based on polyphenols with collagen on wound healing:

The specific activity of wound dressings was evaluated on models of IIIA degree thermal burns in 20 rats. Two wounds per animal were created using a 1.5x1.5cm copper plate fixed on a soldering device, ensuring consistent size and depth of the burns. The resulting wounds were treated as follows: scabs were removed using 40% salicylic acid, followed by saline washing, and application of wound dressings. For comparison, a collagen-based wound dressing called NeuSkin-F, manufactured by Eucare Pharmaceuticals (P) Limited in India, was used. During the application of the dressings, it was observed that NeuSkin-F exhibited poor adhesion and congruence with the wound surface, whereas our own developments adhered immediately to the wound beds.

It was determined that wound dressings identified as RC-Rc and CC-Rc led to the healing of wounds in the specified area within 18.8-21.1 days, while NeuSkin-F took 22.1-22.9 days, and untreated wounds healed in 25.4-26.2 days. Films based on collagen isolated from cattle tendons demonstrated the highest effectiveness as regenerators. Furthermore, all collagen-based wound dressings resulted in the proper architectonics of the peri-scar and scar repair zones, as confirmed by histomorphological data (Table 1, Figure 1).

Table 1 Effect of WD on changes in wound area and healing time (М ± м, n = 10, Р < 0,05)

Animal groups

Average wound area, cm²

Terms of wound regeneration

3^rd day

9^th day

15^th day

18^th day

day

RC-Rc

2.7±

0.10

1.9±

0.07

1.1±

0.03

0.3±

0.01

20.9±

0.2

CC-Rc

2.5±

0.07

1.8±

0.01

0.8±

0.01

0.02±

0.01

18.9±

0.1

Neu Skin-F

2.9±

0.12

2.2±

0.03

1.9±

0.06

0.9±

0.09

22.5±

0.4

control

2.9±

0.11

2.3±

0.09

1.8±

0.07

1.1±

0.07

25.8±

0.4

Note: the healing time is indicated until the complete epithelialization of wounds.

As shown in Table 4, the complete epithelialization of experimental burn wounds without human intervention took 25.4-26.2 days in rats. However, wound therapy using our collagen-based WD compositions reduced the time for complete wound epithelialization to 18.8-21.1 days, accelerating the process compared to the control by approximately 1.4 times. Among the compositions, CC-Rc demonstrated the best result, promoting wound healing in 18.8-19.0 days. The introduction of exogenous collagen with bio-additives helps in reducing the phases of wound healing, facilitating the rapid closure of tissue defects and the formation of a strong scar, as supported by histomorphological data. In contrast, the commercial reference drug "NeuSkin-F" (Eucare Pharmaceuticals (P) Limited, India), consisting of collagen without drug additives, exhibited very limited wound healing properties in this experiment, with complete healing occurring only on 22.5±0.4 days. We believe that this is due to the drug's poor adhesion to the wound bed. It is known that in clinical settings, NeuSkin-F is typically fixed with additional dressings, which proved challenging to replicate in an animal experiment. In this regard, our WD, which demonstrates significantly better congruence and adhesion to the wound, yields excellent results without requiring additional fixation.

On the 20^th day, some of the animals were euthanized for morphological examination, revealing incomplete healing of specific wounds. The morphological assessment of the skin utilized a 3-point system developed by Ipsum Pathology (Tashkent, Uzbekistan), the location of the histomorphological studies. This system aimed to simplify lesion assessment as follows: point 1 indicated minor changes characterized by thinning of the epidermis, small erosions, sparse inflammatory infiltration in the dermis, and proliferation of connective tissue elements, indicating tissue regenerative activity; point 2 represented erosive changes, moderate granulation, inflammatory infiltration with slight connective tissue replacement; point 3 denoted ulcerative defects in the skin, dense lymphocytic infiltration, and pronounced granulomatosis (Figure 1).

Control: The integumentary epithelium is replaced by granulation tissue, resulting in an ulcerative defect. This condition corresponds to 3 points on the assessment scale. The visualization was achieved using GE coloring at a magnification of 10x10.

CC-Rc: The epidermis consists of stratified squamous keratinized epithelium. The papillary and reticular layers of the dermis are clearly observable. Signs of compensation are evident, characterized by the growth of collagen fibers. This condition corresponds to 1 point on the assessment scale. The visualization was achieved using GE coloring at a magnification of 10x10.

RC-Rc: The dermis is detached, resulting in an ulcerative defect of the integumentary layer. This condition corresponds to 2 points on the assessment scale. The visualization was achieved using GE coloring at a magnification of 10x10.

«NeuSkin-F» The reference drug exhibits detachment of the dermis and an ulcerative defect of the integumentary layer. This condition corresponds to 3 points on the assessment scale. The visualization was achieved using GE coloring at a magnification of 10x10.

Normal: The epidermis is composed of stratified squamous keratinized epithelium. The papillary layer transitions smoothly into the reticular layer of the dermis. Hair follicles are observed in the reticular dermis. The hypodermis appears well developed. The visualization was achieved using GE coloring at a magnification of 10x10.

As depicted in Figure 1, the CC-Rc wound dressings demonstrate the most favorable effect, exhibiting advanced epithelialization at the time of removal compared to the other groups, where wound healing is delayed. Therefore, the research successfully yielded a dry extract of polyphenolic compounds isolated from the ground and root parts of the R. confertus Willd. plant.

Moreover, the investigation into the antimicrobial activity of the plant substances against the test cultures revealed a certain level of activity, as shown in Table 1. The results of the study demonstrated that dry extract from the roots and aerial parts of the R. confertus exhibited the antibacterial activities with a inhibition zone ranging from 12.35 ± 0.14 mm to 27.2 ± 0.2 mm. The samples did not show any activities against Candida albicans (Table 2).

Toxicity of extract of polyphenolic compounds from the R. confertus Willd:

A new medicinal component, a dry extract isolated from the ground and root parts of the R. confertus Willd. plant, was obtained. In this study, an assessment of the toxicity and specific activity of this extract was conducted, and active doses were determined. Understanding the potential toxicity of the extract is crucial for assessing its safety and determining its suitability for various applications^38,39.

The acute toxicity of the medicinal component, a dry extract isolated from the ground and root parts of the R. confertus Willd. plant, was investigated to determine lethal and maximum allowable doses. The study involved sexually mature white male rats with an initial body weight of 150-180g. The experimental groups consisted of 6 rats each. The dry extract, dissolved in water, was administered orally to the animals once, using doses of 500, 1000, 2000, 3000, 4000, and 5000 mg/kg. Three to four hours after the administration of the extract, the animals were provided with regular and briquette feed. The experimental animals were observed for a period of 14 days. No symptoms of intoxication were observed in any of the groups, and no deaths occurred. Therefore, at this stage of the research, it was not possible to establish the LD₅₀ (lethal dose 50%) of the dry extract isolated from the ground and root parts of the R. confertus Willd. plant.

A study was conducted to investigate the local action of a dry extract isolated from the ground and root parts of the R. confertus Willd. plant on the skin and mucous membranes of the eyes, as well as its ability to penetrate intact skin. The research was performed on white rats. In the study, an aqueous solution of the dry extract was applied to the skin of the experimental animals at a rate of 10mg/cm². The skin reaction was observed and recorded at the end of the 4-hour exposure, as well as after 1 and 16hours following a single application. The findings revealed that the R. confertus Willd. extract did not induce skin irritation.

The skin-resorptive effect of the R. confertus Willd. extract was further investigated by subjecting the skin of white rats to repeated exposure (20 skin applications) at a rate of 10mg/cm2. Throughout the entire duration of the experiment, no deaths of animals or clinical signs of intoxication were observed. Consequently, it can be concluded that the studied extract does not exhibit an irritating effect on the skin upon repeated exposure.

To investigate the skin-resorptive impact of the R. confertus Willd. extract on white rats, the animals were securely held in specialized apparatus. The tails of the experimental animals were immersed in test tubes containing R. confertus Willd. extract (10mg/ml) for a duration of 4 hours at a temperature of 36-37°C. Following the conclusion of the experiment, the skin of the tails was thoroughly washed with soap and water. Throughout the three-week observation period of the experimental animals, no signs of intoxication or deaths were observed.

The effect of solutions containing the aforementioned extract on the mucous membrane of the eyes was examined. For this purpose, two drops of an aqueous solution of the R. confertus Willd. extract (10μg/ml) were instilled once into the conjunctival sac of the rabbit's right eye, while the left eye served as a control and received drops of a saline solution. Under the influence of the studied extract, lacrimation was observed within 2 minutes of instillation. However, this phenomenon completely ceased after 3minutes. The aqueous solution of the R. confertus Willd. extract (10 µg/ml) can be classified as having weak irritant properties on the eyes. Based on the severity of its irritant effects, it can be categorized as a 4^th class irritant.

Based on the results of the studies, it can be concluded that the R. confertus Willd. extract does not exhibit a local skin or skin-resorptive effect at various doses. Furthermore, it does not have an irritating effect on the mucous membrane of the eyes.

The subchronic toxicity of a dry extract isolated from the ground and root parts of the R. confertus Willd. was investigated in this study. Rats were used as the animal model, and the extract was administered orally to the rats' stomachs on a daily basis for a duration of one month. The doses administered were 50mg/kg, 100 mg/kg, and 500mg/kg, with six rats in each dose group. A control group of animals was also included in the study, which received water under similar conditions. All experimental and control animals were kept under the same conditions and provided with a normal diet.

Throughout the experiment, the animals underwent daily monitoring, including assessments of their general condition, behavior, food and water consumption, as well as the state of their hair and mucous membranes. Following the final administration of extracts in all groups of animals, blood samples were obtained from the tail vein through partial resection (0.5-1.0cm) to determine various parameters using a hematological analyzer. Subsequently, under light ether anesthesia, the animals were euthanized, and blood samples were collected for biochemical analysis (refer to Table 3). Additionally, internal organs were extracted for morphological studies (Figure 2).

Table 3: Blood parameters of rats treated with R. confertus Willd. extract for 30 days at three concentrations

Blood parameters	Animal groups
Blood parameters	Control (intact animals)	50 mg/kg	100 mg/kg	500 mg/kg
Leukocytes 10 ⁹/l	14,45±1,09	14.18±1,09	14,12±1,07	14,45±1,09
Absolute content of lymphocytes, 10 ⁹/l	6,43±0,73	6,40±0,66	6,40±0,67	6,43±0,73
Absolute content of a mixture of monocytes, 10 ⁹/l	2,65±0,28	2,55±0,34	2,47±0,33	2,65±0,28
Quantity in granulocytes, 10 ⁹/l	5,15±0,45	5,40±0,35	5,65±0,46	5,15±0,45
Hemoglobin, g/l	138.5±5,52	134,50±4,59	133.67±5,02	138.5±5,52
Erythrocytes, g/l RBC	6,22±0,44	6,16±0,35	6,32±0,31	6,22±0,44
Hematocrit % HCT	36,90±1,49	36,98±0,92	35,47±1,15	36,90±1,49
The average concentration of hemoglobin in the erythrocyte g/l	365.83±4,98	366,17±4,63	367,67±5	365.83±4,98
Platelets in absolute numbers, 10 ⁹/l	602,17±52,32	600.67±42,01	600,33±49,43	602,17±52,32
Thrombocrit, %	0.540±0.06	0.570±0.05	0.550±0.04	0.540±0.06
АLТ, U/L at 37 °C	64,80±3,44	66,43±2,58	67,03±3,20	70,13±4,63
АSТ, U/L at 37 °C	231,67±12,67	247,17±11,71	242,33±11,33	249,17±11,21
АLP, U/L at 37 °C	593,83±31,08	549,02±20,64	603,67±34,42	582,03±27,72
γ GT, U/L at 37 °C	4,67±0,75	4,50±0,76	4,50±0,82	5,17±0,76
Cholesterol mM/L	81,77±3,80	76,90±3,39	74,92±4,63	73,92±3,13
Total protein, g/dL	92,83±10,54	99,60±4,54	94,48±4,14	94,28±3,24

Note: P≥0.05 compared to control

A: The capsule of the spleen is observed, with extending trabeculae. Well-defined central arteries can be seen in the white pulp, along with trabecular veins. The specimen was stained with GE coloring and viewed under a light microscope at a magnification of 10 x 10.

В: The stroma of the specimen is characterized by loose connective tissue. Within the parenchyma, the renal tubules are observed. The collecting tubes do not show any notable modifications. The specimen was stained with GE coloring and viewed under a light microscope at a magnification of 10 x 10.

С: The liver capsule is composed of loose connective tissue. Within the parenchyma, the lobules are poorly distinguishable. Each lobule contains a central vein located at its center. Radial strands, known as hepatic beams, are formed by two rows of epithelial cells called hepatocytes. The hepatocytes have monomorphic nuclei and light cytoplasm. The specimen was stained with GE coloring and viewed under a light microscope at a magnification of 10 x 10.

D: Crypts of various sizes and Brunner's glands are observed in the specimen. The specimen was stained with GE coloring and viewed under a light microscope at a magnification of 10 x 10.

E: The histological preparation reveals the large intestine with short villi and a moderate lymphocytic infiltration in the stroma. The specimen was stained with GE coloring and viewed under a light microscope at a magnification of 10 x 10.

F: The mucous membrane of the stomach is covered by a single-layered columnar glandular epithelium. The mucous membrane contains simple, unbranched glands. The muscular layer of the stomach consists of three layers: an inner circular layer, an outer circular layer, and a middle longitudinal layer. The submucosa is composed of loose fibrous connective tissue. The specimen was stained with GE coloring and viewed under a light microscope at a magnification of 10 x 10.

Figure 2: Histological sections of the organs from animals that received a dose of 500 mg/kg of R. confertus Willd. extract for a duration of one month are shown in the following order: A – Spleen; B – Kidney; C – Liver; D - Small intestine; E - Large intestine; F – Stomach.

As can be seen from tables 3, and figure 2, hematological, biochemical and histomorphological parameters of tissues and fluids of animals treated with R. confertus extract are within the normal range, which indicates the absence of toxicity of this extract containing polyphenolic compounds.

CONCLUSION:

The composition developed using the polyphenolic extract of R. confertus Willd. in combination with collagen isolated from the tendons of rats and cattle, and WD films, exhibited superior wound healing activity compared to the commercial reference drug "NeuSkin-F" from Eucare Pharmaceuticals (P) Limited, India. Furthermore, the R. confertus Willd. extract demonstrated significant antibacterial activity against the tested bacteria. Moreover, it has been determined that the intragastric administration of the R. confertus Willd. extract did not result in toxicity effects or mortality in experimental animals. Based on its safety profile, it can be classified as a 5^th hazard class substance, practically non-toxic. Additionally, the extract did not exhibit local skin or skin-resorptive effects, and it did not cause irritation to the mucous membrane of the eye.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

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Received on 26.09.2023 Modified on 07.11.2023

Research J. Pharm. and Tech 2024; 17(5):2256-2264.

DOI: 10.52711/0974-360X.2024.00355

No.	Inhibition Zone (mm, ±SE, p ≤ 0.05)
No.	*Bacillus subtilis*	*Escherichia coli*	*Pseudomonas aeruginosa*	*Staphylococcus aureus*	*Candida albicans*
Control - Rutan	28.4±0.25	25.15 ± 012	28.25 ± 0.15	30.2 ± 0.25	11.5 ± 0.13
Dry extract from the roots and aerial parts of the R. confertus, 1 mg per well	23.2±0.41	NA	18.1 ± 0.25	12.35 ± 0.14	NA
Dry extract from the roots and aerial parts of the R. confertus, 2 mg per well	25.35±0.2	25.1 ± 0.15	27.2 ± 0.2	18.19 ± 0.11	NA