Skin wounds healing patch and the method of its preparation

Abstract

The wound healing plaster and the method for its preparation are applicable in medicine. The plaster includes a polymer mixture of polyvinyl alcohol and chitosan, glycerol, and methanol extract from the plant Marrubium peregrinum. The three components are present in the following weight parts: 30-70 parts of polymer mixture, 20-60 parts of methanol extract from Marrubium peregrinum, and 3-25 parts of glycerol. The plasters containing methanol extract from Marrubium peregrinum with a concentration of 4 to 8 mg / cm2 demonstrate very well-expressed wound healing activity, as proven by histological examinations of wound surface samples from rats after euthanasia. The analysis of pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL- 10) cytokines in the tissue homogenates, as well as the level of hydroxyproline, showed increased cell proliferation and collagen synthesis compared to control samples that do not contain Marrubium peregrinum extract.

Description

[0001] SKIN WOUNDS HEALING PATCH AND THE METHOD OF ITS PREPARATION

[0002] Field of Technology

[0003] The invention relates to a plaster for healing skin wounds and a method for its preparation. The plaster can be classified within the field of medical preparations containing plant materials and will find application in medicine, particularly in the treatment of skin wounds.

[0004] Prior Art

[0005] Wound healing involves all the processes that lead to the closure of the wound. After the formation of the wound, coagulation first stops, and then the healing process begins. The body forms new tissue and builds new blood vessels. Wound healing progresses through various phases. The first phase, lasting from the first few minutes to hours, is the exudative phase, during which the blood coagulates and forms a scab that seals the wound externally and protects the injured area from the invasion of microbes and potential infections. The second phase, lasting from the first to the third day, is the resorptive phase, during which phagocytes (the "destroyer" cells) begin to remove blood and cellular debris, as well as combat potential microbes. The third phase, lasting from the fourth to the seventh day, is the proliferative phase, during which new cells are generated inside the wound, blood vessels are formed, and connective tissue is created. The final, fourth phase is the reparative phase, which continues from the eighth day and for months thereafter. During this phase, the skin begins to close the wound with new skin cells, and the wound gradually closes.

[0006] Injury and the healing process of a wound often lead to inflammation and infections, which delay the healing process. This necessitates the use of medications with antiinflammatory and regenerative effects. Commonly, oral antibiotics and intravenous injections are used, but they have a number of side effects. To facilitate wound healing and achieve rapid recovery without side effects, transdermal preparations are increasingly being applied. It has been found that preparations containing plant material are particularly suitable for this purpose, as they generally do not cause allergic reactions.

[0007] A plaster comprising plant material, known from [1], is described, which has anti-inflammatory and hemostatic effects. It contains plant material with the following weight components: from 0.25 to 1.56 mg aescin (saponins from horse chestnut), from 6 to 25 mg Herba Portulacae, and from 0.5 to 2.1 mg acorns. The method for preparing the plaster is also disclosed. The plaster with anti-inflammatory and hemostatic action, disclosed in the mentioned patent application, is safe and non-toxic and can effectively inhibit bleeding from soft tissues and lymphatic exudation, effectively suppress inflammation, and promote wound healing.

[0008] A method for producing a transdermal plaster for wound healing, known from [2], involves extracts from pumpkin plants, specifically from Momordica cymbalaria tuber. The transdermal plaster is made from a natural polymer, pectin, and M. cymbalaria, S. auriculata, and clove oil. It also includes DMSO and polysorbate-80. The plaster has been evaluated based on its organoleptic and physicochemical properties, such as thickness, weight, moisture absorption, moisture content, etc. To assess its therapeutic efficacy, an in vitro analysis of Stratch was performed. The wound-healing potential of the M. cymbalaria tuber extract was determined through a scratch test in human epidermal keratinocytes at concentrations of 25 pg / ml and 50 pg / ml.

[0009] A topical hemostatic drug, specifically a hemostatic plaster containing plant material, and a method for its preparation, are known from [3]. The hemostatic plaster consists of a plaster body and an ointment, with the ointment being prepared from the following weight parts: 9-15 parts Radix Sanguisorbae extract (root of medicinal Sanguisorba), 9-15 parts artichoke extract, 3-10 parts Radix Scutellariae extract (root of Baikal skullcap), 10-20 parts hydroxymethyl chitosan, 3-5 parts glycerol, and 0.5-1 part plant starch. The plaster is prepared by using extracts from Sanguisorba Officinalis, artichoke, and Radix Scutellariae as raw materials. These three extracts work synergistically, providing hemostatic, anti-inflammatory, and analgesic effects, tightening and activating the coagulation system, assisted by hydroxyethyl chitosan. Together, they have a synergistic effect, while the plant starch is added for the rapid absorption of water in the blood.

[0010] Plants are the most important source of phytochemicals, and most of them are widely used as natural phytomedicines with broad therapeutic applications. In the traditional medicine of many countries around the world, such as China, India, and Bulgaria, numerous medicinal plants have been used since ancient times in the form of teas, decoctions, tinctures, ointments, poultices, and more. In Bulgarian folk medicine, the following remedies are used for the healing of skin wounds: marigold, applied as an ointment; an ointment made from crushed white elecampane and lard; poultices of mullein, onion, garlic, or broadleaf plantain, applied locally to the wound; infusions for washing made from burdock, yarrow, chamomile, tormentil, wild strawberry, blue bile, walnut, or black elecampane, etc.

[0011] In recent years, biologists in Bulgaria have focused their attention on studying the medicinal properties of other plants, one of which is the perennial plant Marrubium peregrinum (horehound). This plant is widely distributed in Europe (central and southeastern parts, Balkan Peninsula), the Caucasus, and Southwest Asia, growing in dry, grassy, and rocky places up to an altitude of about 1000 meters.

[0012] The results of a study titled "Metabolomic Profiling of Marrubium peregrinum L. and Marrubium friw aidsky anum Boiss" [4], conducted by a team of scientists from Plovdiv, reveal the potential of these plants as sources of plant-based pharmaceutical products. The publication highlights that these plants contain numerous flavonoids, such as apigenin, quercetin, rutin, and their derivatives, including large amounts of phenylethanoids and phenylpropanoid glycosides, such as forsythoside, calceolarioside, and caffeoylquinic acid, all of which have well-established antioxidant, antibacterial, anticancer, and other bioactive effects. It is also noted that samples of Marrubium peregrinum L. have been found to contain the flavonoids isovitexin, procyanidin B, and naringenin dihydrochalcone, which are known for their pharmacological properties.

[0013] The plant Marrubium peregrinum is found throughout the country, is not protected by the Biodiversity Act, and evidently contains bioactive components. This makes it particularly attractive for the development of new pharmaceutical products that include plant material.

[0014] Technical Essence of the Invention

[0015] The aim of the present invention is to create a plant-based pharmaceutical product containing an extract from the widely distributed plant Marrubium peregrinum in Bulgaria, specifically to develop a plaster for healing skin wounds with anti-inflammatory and regenerative effects, which includes an extract of Marrubium peregrinum, as well as a method for preparing the plaster. The problem is solved by creating a plaster for healing skin wounds, which includes chitosan, glycerol, and plant extract.

[0016] According to the invention, the plaster for healing skin wounds also includes polyvinyl alcohol, which, together with chitosan, forms a polymer blend. The plant extract is a methanol extract of Marrubium peregrinum, with a concentration of 4 to 8 mg / cm2. The components of the plaster are in the following weight parts: 30-70 parts polymer blend, 20-60 parts methanol extract of Marrubium peregrinum, and 3-25 parts glycerol.

[0017] According to one preferred embodiment of the plaster, the polymer blend is 50 weight parts, the Marrubium peregrinum extract is 40 weight parts with a concentration of 8 mg / cm2, and glycerol is 10 weight parts.

[0018] In another embodiment of the plaster, the concentration of chitosan and polyvinyl alcohol is in the range of 1-5%, and the ratio of weight parts of chitosan to polyvinyl alcohol in the polymer blend varies between 10:90 and 90:10.

[0019] The preferred concentration of chitosan and polyvinyl alcohol is 2%, and the ratio of weight parts of chitosan to polyvinyl alcohol in the polymer blend is 50:50.

[0020] In another variant of the plaster, the concentration of glycerol is 0.1-1%, preferably 0.2%.

[0021] The invention also includes a method for preparing plasters for healing skin wounds, which consists of the following stages:

[0022] Stage 1) Extraction of the Marrubium peregrinum plant, in which: the dry plant material, including the aerial parts of Marrubium peregrinum, is mixed with an extracting solvent - methanol, in a volume percentage of 60-80%, in a ratio of 1:8 to 1:12; the resulting mixture is placed in flasks, which are wrapped in aluminum foil and placed on a magnetic stirrer for 24 hours; a three-step ultrasonic extraction is carried out in an ultrasonic bath, including three cycles, approximately every 3-4 hours, for 15 minutes at 30°C; the obtained samples are strained through gauze, and the supernatant is centrifuged for 10 minutes at 5000 rpm to obtain a maximally purified sample, which is stored in a refrigerator; additional extracting solvent is added to the remaining plant material, and the procedure is repeated twice more for 24 hours, until a triple extract is obtained; the obtained supernatants are combined and evaporated using a vacuum evaporator to dry matter; the dry matter is dissolved in deionized water to obtain a concentration of the methanol extract from Marrubium peregrinum of 4 to 8 mg / cm2.

[0023] Stage 2) Preparation of a polymeric mixture from solutions of polyvinyl alcohol and chitosan in distilled water, with a concentration of 1 -5%, in a weight ratio between the two components from 10:90 to 90:10;

[0024] Stage 3) Preparation of a glycerol solution with a concentration of 0.1-1%;

[0025] Stage 4) Mixing 30-70 weight parts of the polymeric mixture with 20-60 weight parts of the methanol extract from Marrubium peregrinum and 3-25 weight parts of glycerol;

[0026] Stage 5) Homogenizing the solution obtained in stage 4), pouring it into Petri dishes, and drying it until a constant mass is reached for the resulting patches.

[0027] According to one variant of the method, the dry plant material from the aerial parts of Marrubium peregrinum is mixed with 70% methanol in a ratio of 1 : 10.

[0028] In another variant of the method, the solution of polyvinyl alcohol and the solution of water-soluble chitosan are both at a concentration of 2%, and the weight ratio between them is 50:50.

[0029] In another variant of the method, the concentration of glycerol is 0.2%. According to a particularly preferred embodiment of the method, the polymeric mixture consists of 50 weight parts, the Marrubium peregrinum extract is 40 weight parts and has a concentration of 8 mg / cm2, and glycerol is 10 weight parts.

[0030] The patches containing the methanol extract of Marrubium peregrinum, with a concentration of 4 to 8 mg / cm2, exhibit very good wound-healing activity, as demonstrated by histological examinations of wound surface samples from rats after euthanasia. The analysis of inflammatory (TNF-a and IL-6) and anti-inflammatory (IL- 10) cytokines in tissue homogenates, as well as the level of hydroxyproline, shows increased cellular proliferation and collagen synthesis compared to control samples that do not contain the Marrubium peregrinum extract.

[0031] Description of the attached figures

[0032] Figure 1 shows microphotographs of three types of polyvinyl alcohol (PVA) / chitosan patches in a 50:50 weight ratio, which are as follows: a) the control; b) with the inclusion of Marrubium peregrinum extract at a concentration of 4 mg / cm2; c) with the inclusion of Marrubium peregrinum extract at a concentration of 8 mg / cm2.

[0033] Figure 2 graphically presents the water vapor transmission rate for the three types of patches.

[0034] Figure 3 graphically presents the vapor permeability of the three types of patches.

[0035] Figure 4 graphically presents the water capacity of the three patches at different relative humidity levels.

[0036] Figures 5 and 6 show graphs of the release of the methanol extract from Marrubium peregrinum, at concentrations of 4 mg / cm2and 8 mg / cm2, respectively, for patches with different ratios of PVA to chitosan.

[0037] Figure 7 shows graphs of the release of the methanol extract from Marrubium peregrinum at a concentration of 8 mg / cm2, for patches with different glycerol concentrations.

[0038] Figure 8 presents histological results from the four studied groups: control group (A); group treated with cream (B); group treated with a low dose of plant extract (C); and group treated with a high dose of plant extract (D). Figure 9 graphically presents the change in the wound healing process over days for the four groups.

[0039] Figure 10 presents photographic material showing the changes in wound healing over days for the four groups.

[0040] Figure 11 graphically shows the effect of the plant extract on the level of TNF-a. Significant differences compared to group 1 are marked with . The results are presented as mean ± SD (mean ± standard deviation).

[0041] Figure 12 graphically shows the effect of the plant extract on the level of IL-6. Significant differences compared to group 1 are marked with . The results are presented as mean ± SD.

[0042] Figure 13 graphically shows the effect of the plant extract on the level of IL- 10. Significant differences compared to group 1 are marked with , significant differences compared to group 2 are marked with #, and significant differences compared to group 3 are marked with . The results are presented as mean ± SD.

[0043] Figure 14 graphically shows the effect of the plant extract on the level of OH-proline. The results are presented as mean ± SD. Examples of Embodiments of the Invention

[0044] The present invention is illustrated without being limited to the following examples:

[0045] Example 1

[0046] 1.1. Preparation of methanol extracts from the Marrubium peregrinum plant

[0047] The dry plant material, including the aerial parts of the Marrubium peregrinum plant, is mixed with the extracting solvent methanol at a volume percentage of 70%, in a ratio of 1 : 10 w / v. The resulting mixture is placed in flasks. The flasks are wrapped in aluminum foil and placed on a magnetic stirrer for 24 hours. During this period, a three-step ultrasonic extraction is performed in an ultrasonic bath, comprising three cycles (approximately every 3-4 hours), each lasting 15 minutes at 30°C. After this period, the samples are strained through gauze, and the undissolved liquid (UDL) is centrifuged (10 minutes at 5000 rpm) and filtered through filter paper to obtain a maximally purified (clear) sample.

[0048] The obtained sample is stored in a refrigerator, and the remaining plant material is mixed with extracting solvent, and the extraction procedure is repeated two more times for 24 hours (resulting in a triple extract). The obtained undissolved liquid (UDL) is combined and evaporated using a vacuum evaporator to a dry substance at a temperature of 50°C. The mass of each flask is measured before evaporation (ml), and the UDL is added in portions (if the flask volume is 100 mL, no more than 50 mL is added). After evaporation, the mass of the flask is measured again (m2), and the difference (m2 - ml) indicates the extraction yield. Based on the obtained mass, the dry substance is dissolved in deionized water to the desired concentration. Water is added in small portions to ensure maximum dissolution and removal of the dry substance from the flask.

[0049] The following two types of methanol extracts from Marrubium peregrinum are prepared:

[0050] - Methanol extract with a concentration of 4 mg / cm2(low concentration);

[0051] - Methanol extract with a concentration of 8 mg / cm2(high concentration).

[0052] 1.2. Preparation of two types of patches containing methanol extract from the Marrubium peregrinum plant, with concentrations of 4 mg / cm2and 8 mg / cm2

[0053] Solutions of polyvinyl alcohol (PVA) and water-soluble chitosan in distilled water are prepared, each at a concentration of 2%. The two solutions are mixed in a 50:50 ratio to obtain a polymeric mixture.

[0054] A glycerol solution is also prepared, with a concentration of 0.2%.

[0055] The patches are obtained using the casting method. To prepare the first type, 50 weight parts of the polymeric mixture, 40 weight parts of methanol extract from Marrubium peregrinum with a concentration of 4 mg / cm2, and 10 weight parts of glycerol are mixed. Similarly, the second type is prepared, where the methanol extract from Marrubium peregrinum has a concentration of 8 mg / cm2.

[0056] After homogenizing the two obtained solutions, they are poured into Petri dishes and dried until a constant mass is reached. It is preferred that the drying process takes place over 12 hours at a temperature of 35 °C. The prepared patches are stored under standard conditions, i.e., in desiccators, at a temperature of 20°C and relative humidity (RH) of 54%.

[0057] 1.3. Analysis of the characteristics of patches containing Marrubium peregrinum extract To assess the physical and physicochemical properties of patches containing Marrubium peregrinum extract, the following three types of patches were studied:

[0058] • Control - patch containing physiological saline;

[0059] • Patch containing 4 mg / cm2extract;

[0060] • Patch containing 8 mg / cm2extract.

[0061] The patches were characterized in terms of their morphology, mechanical properties, vapor permeability, and swelling. The obtained physical and physicochemical parameters of the patches containing Marrubium peregrinum extracts were compared with those of patches with the same polymer composition but without the added plant extract (control).

[0062] 1.3.1. Morphology

[0063] The morphology of the patches was studied using a scanning electron microscope Prisma E SEM, Thermo Scientific, Waltham, MA, USA.

[0064] Figure 1 shows three microphotographs of patches containing PVA and chitosan in a 50:50 weight ratio, as follows: a) the control; b) with 4 mg / cm2extract; c) with 8 mg / cm2extract.

[0065] It is clearly observed that while the control patch is characterized by a homogeneous dense structure, the patches containing the extract are heterogeneous, with the presence of micron-sized morphological units, the concentration and size of which depend on the extract concentration.

[0066] 1.3.2. Mechanical Properties

[0067] The mechanical tests include a rupture test conducted in uniaxial tensile deformation mode using a universal testing machine LSI (Lloyd Instruments) (ISO 527 / 2012). The deformation rate of the sample (the speed of movement of the movable holder) is 1 mm / s. Ten samples of each type of patch were measured to ensure good statistical accuracy. The rupture test allows for determining the rupture point, which is characterized by the rupture stress and the relative deformation at rupture, as well as the Young’s modulus, which is calculated as the slope (first derivative) of the linear portion of the stress-strain curve.

[0068] The calculated mechanical parameters — rupture stress, relative deformation at rupture, and elastic modulus (Young's modulus) — are presented in Table 1.

[0069] Table 1

[0070] It is evident that the inclusion of Marrubium peregrinum extract in the patches leads to a deterioration in their mechanical properties and a decrease in their elasticity. At the higher extract concentration, there is a sharp decline in the values of all the investigated parameters.

[0071] 1.3.3. Vapor Permeability and Water Capacity

[0072] The rate of water vapor transmission through the patches is graphically presented in Figure 2, and the vapor permeability is shown in Figure 3.

[0073] From both figures, it is clear that the addition of the plant extract leads to an increase in vapor permeability and the rate of water vapor transmission due to the enhanced hydrophilicity of the structure. A similar behavior is observed in the investigation of the water capacity of the patches, which is graphically shown in Figure 4.

[0074] Example 2

[0075] 2.1. Preparation of ten types of patches containing Marrubium peregrinum methanol extract with concentrations of 4 mg / cm2and 8 mg / cm2, at different ratios of chitosan and PVA components

[0076] Using the method described in Section 1.1 of Example 1, two types of Marrubium peregrinum methanol extracts are prepared: with concentrations of 4 mg / cm2and 8 mg / cm2.

[0077] A 0.2% glycerol solution is also prepared.

[0078] The following five types of polymer mixtures are prepared, with different PVA-to- chitosan ratios: PVA: chitosan - 100:0; PVA: chitosan - 0:100; PVA: chitosan - 1:1; PVA: chitosan - 2:1; PVA: chitosan - 1 :2

[0079] Using a method similar to that described in Section 1.2 of Example 1 , the following 10 types of patches are prepared by combining the three components: methanol extract, polymer mixture, and glycerol:

[0080] Patch containing 4 mg / cm2extract and a PVA: chitosan ratio of 100:0 (PVA solution);

[0081] Patch containing 4 mg / cm2extract and a PVA: chitosan ratio of 0:100 (chitosan solution);

[0082] Patch containing 4 mg / cm2extract and a PVA: chitosan ratio of 1 : 1 ;

[0083] Patch containing 4 mg / cm2extract and a PVA: chitosan ratio of 2: 1 ;

[0084] Patch containing 4 mg / cm2extract and a PVA: chitosan ratio of 1 :2;

[0085] Patch containing 8 mg / cm2extract and a PVA: chitosan ratio of 100:0 (PVA solution);

[0086] Patch containing 8 mg / cm2extract and a PVA: chitosan ratio of 0:100 (chitosan solution);

[0087] Patch containing 8 mg / cm2extract and a PVA: chitosan ratio of 1 : 1 ;

[0088] Patch containing 8 mg / cm2extract and a PVA: chitosan ratio of 2: 1 ;

[0089] Patch containing 8 mg / cm2extract and a PVA: chitosan ratio of 1 :2;

[0090] 2.2. Kinetics of extract release from the patch

[0091] Release of the extract was performed in PBS (phosphate-buffered saline) over a period of 24 hours.

[0092] Figure 5 shows the release profiles of the methanol extract from Marrubium peregrinum at a concentration of 4 mg / cm2for the five types of patches described above in section 2.1, with different ratios of PVA to chitosan.

[0093] Figure 6 shows the release profiles of the methanol extract from Marrubium peregrinum at a concentration of 8 mg / cm2for the five types of patches described above in section 2.1, with different ratios of PVA to chitosan.

[0094] It can be seen that the release of the methanol extract, within 24 hours, is complete for all patches.

[0095] Example 2 tracks the influence of the ratio between PVA and chitosan in the polymer mixture included in the patch on the release kinetics of the extract, with an optimal ratio of 1 : 1 or 50:50 being determined.

[0096] Example 3

[0097] 3.1. Preparation of five types of patches containing methanol extract from Marrubium peregrinum with a concentration of 8 mg / cm2at different glycerol concentrations

[0098] Using the method described in section 1.1. of Example 1 , the methanol extract of Marrubium peregrinum with a concentration of 8 mg / cm2is prepared. Solutions of polyvinyl alcohol (PVA) and water-soluble chitosan in distilled water, each with a concentration of 2%, are prepared. The two solutions are mixed in a 50:50 ratio to obtain a polymer blend.

[0099] Five glycerol solutions in distilled water are prepared with the following concentrations: 0.2%, 0.4%, 0.6%, 0.8%, and 1%.

[0100] Using the method described in section 1.2 of Example 1, patches are prepared by combining the three components — the methanol extract, the polymer blend, and glycerol with the five different concentrations.

[0101] 3.2. Kinetics of extract release from the patch

[0102] Figure 7 shows the release profiles of the methanol extract from Marrubium peregrinum at a concentration of 8 mg / cm2for the patches with different glycerol concentrations.

[0103] Example 3 tracks the influence of the glycerol concentration included in the patch on the release kinetics of the extract, with an optimal concentration determined to be 0.2%.

[0104] Example 4

[0105] 4.1. Study of the action of patches with Marrubium peregrinum extract - experimental model of skin wounds in rats treated with patches containing methanol extract of Marrubium peregrinum

[0106] After obtaining permission from the Bulgarian Food Safety Agency (BFSA) for the use of animals in experiments (Permit No. 352, valid until 14.06.2028), an eleven-day experimental model was conducted to study the regenerative effects of patches with Marrubium peregrinum extracts on skin lesions.

[0107] A total of 28 male Wistar rats were used, divided into 4 groups of 7 animals each. Each animal was housed in an individual cage.

[0108] Group one (control) - treatment of the wound with a patch containing saline solution.

[0109] Group two (positive control) - treatment of the wound with a commercial cream containing plant extracts.

[0110] Group three - Treatment of the wound with a patch containing a low dose of methanol extract (100 mg / kg) from Marrubium peregrinum. Treatment is achieved by applying patches containing Marrubium peregrinum extract at a concentration of 4 mg / cm2.

[0111] Group four - Treatment of the wound with a patch containing a high dose of methanol extract (200 mg / kg) from Marrubium peregrinum. Treatment is achieved by applying patches containing Marrubium peregrinum extract at a concentration of 8 mg / cm2.

[0112] Design of the expetiment.

[0113] The animals are subjected to general anesthesia by intramuscular injection of 80 mg / kg Zoletil®. After anesthesia is induced, skin lesions are created on the dorsal area using a punch.

[0114] The resulting lesions are disinfected and covered with the studied patches containing plant extracts or a reference substance. An adhesive patch is placed on the wounds of the animals in Group 2 to prevent contamination and removal by the animal. To prevent postoperative pain and discomfort, Meloxidyl® is administered orally at a dose of 2 mg / kg on the first day following the intervention, and 1 mg / kg daily for the next 4 days. The patches are changed every 24 hours, and before applying a new patch, the lesions are photographed with a camera and a ruler placed next to the lesion, which must be visible in the photo. The photographs are taken on days 1, 3, 5, 7, 9, and 11, with the camera held perpendicular to the wound surface at a distance of 25 cm. Before applying the new patch, animals in Group 2 receive the commercial cream from the pharmacy. On the 11th day of the experiment, the animals are euthanized, and skin samples and wound tissue are collected for histological analysis.

[0115] Necessary Materials:

[0116] Shaving tools, disinfectant, cotton, punches, plant extract cream, standard adhesive patches, Meloxidyl®, Zoletil®, camera, ruler.

[0117] 4.2. Results

[0118] The wound healing effect of the patches was demonstrated through histological analysis of tissue samples from the wound surface of the rats after euthanasia, as well as by measuring pro-inflammatory (TNF-a and IL-6) and anti-inflammatory (IL- 10) cytokines in tissue homogenates, additionally, the level of hydroxyproline, indicating increased cellular proliferation and collagen synthesis, was assessed.

[0119] 4.2.1. Histological Analysis of Wound Surface Samples from Rats

[0120] Seven rats from each of the four study groups were euthanized. The entire wound, along with a margin of approximately 5 mm of surrounding uninjured skin, was excised. All samples were routinely fixed in 10% buffered formalin and embedded in paraffin for histopathological evaluation. Tissue sections with a thickness of 5 microns, taken from blocks 1 to 10 of each paraffin block, were stained with hematoxylin-eosin (H&E) and examined retrospectively by two independent pathologists.

[0121] The healing status was evaluated using the histological parameters (1-12) described in [5], as follows:

[0122] Absent (1-3):

[0123] Absent / minimal presence of fibroblasts

[0124] Absent granulation tissue

[0125] Absent epithelial migration

[0126] Poor (4-6):

[0127] Thin, immature granulation tissue, mainly consisting of inflammatory cells and few fibroblasts, capillaries, and collagen deposits

[0128] Minimal epithelial migration

[0129] Satisfactory (7-9):

[0130] Moderately thickened granulation tissue, mainly composed of inflammatory cells, more fibroblasts, and collagen deposits Marked neovascularization

[0131] Minimal / moderately expressed epithelial migration

[0132] Good (10-12):

[0133] Thickened, vascular granulation tissue, primarily composed of fibroblasts and collagen deposits

[0134] - Presence of partial or complete epithelial coverage

[0135] The healing status was categorized as: Good 4 (10-12), Satisfactory 3 (7-9), Poor 2 (4-6), Absent 1 (1-3).

[0136] Group 1 - 5 cases (absent) and 2 cases (poor)

[0137] Group II: 4 cases (poor) and 3 cases (satisfactory)

[0138] Group III: 4 cases (satisfactory) and 3 cases (good)

[0139] Group IV : 6 cases (good) and 1 case (satisfactory)

[0140] The heterogeneous results, represented by the presence of both immature and mature granulation tissue in wound healing, are due to the individual response of each organism to the pathological factors. Among all the groups studied, complete healing with mature granulation tissue was observed in the last group, treated with the high dose of the extract (in 6 out of 7 rats). The histological results from the four groups studied (H&E) are shown in Figure 8.

[0141] Control group (A): Both the absence of granulation tissue and the lack of epithelial coverage are observed.

[0142] Group treated with cream (B): Immature granulation tissue is found, primarily consisting of individual inflammatory cells and a few fibroblasts, with minimal epithelial migration.

[0143] Group treated with low-dose plant extract (C): Moderately thickened granulation tissue is observed, mainly consisting of inflammatory cells, fibroblasts, and marked neovascularization, as well as moderately expressed epithelial migration.

[0144] Group treated with high-dose plant extract (D): Thickened granulation tissue is found, primarily composed of fibroblasts and collagen deposits, with complete epithelial coverage.

[0145] Figure 9 shows the graphical representation of the change in wound healing percentage over the days for each group. Higher values indicate better wound healing activity.

[0146] The exact values for the changes in wound healing percentages over the days are provided in Table 2. In the early phase (days 3-5) of wound healing, the patches demonstrate better effects compared to the cream available on the market. In the later phase (days 9-11), the effect of the high- dose extract remains strong, but a significant effect is also observed in the group treated with the cream. The cream shows better results in the later phase of healing.

[0147] Table 2. Change in the percentage of wound healing over days

[0148] Figure 10 shows images of the wound surfaces, illustrating the changes in wound healing over the days.

[0149] 4.2.2. Biochemical Analysis of Pro-inflammatory (TNF-a and IL-6) and Antiinflammatory (IL-10) Cytokines, and Hydroxyproline Levels in Tissue Homogenates

[0150] The comparative analysis of the obtained results was conducted using the statistical program SPSS, One-way ANOVA, and Post Hoc - LSD. The results are presented as mean ± SD.

[0151] TNF-a:

[0152] Group 1 vs. Group 4; p = 0.017

[0153] Figure 11 presents graphically the effect of the plant extract on TNF-a levels. Statistically significant differences compared to Group 1 are marked with an asterisk (*). The results are presented as mean ± SD.

[0154] The statistical analysis showed a significant decrease in TNF-a levels in Group 4, treated with a 200 mg / kg body weight extract (0.46 ± 0.11 pg / mg), compared to the control group with the empty film (0.9 ± 0.45 pg / mg; p = 0.017). No significant differences were found in Group 3, treated with a 100 mg / kg body weight extract (0.71 ± 0.30), compared to Group 1 (empty film). This indicates a better effect of the higher dose of the extract compared to the lower dose.

[0155] This suggests a statistically significant difference between Group 1 and Group 4 regarding the levels of TNF-a, with the statistical significance indicated by p < 0.05. The analysis likely continued for other cytokines and parameters, but this part of the text focuses on the comparison of TNF-a levels.

[0156] IL-6:

[0157] Group 1 vs. Group 2; p = 0.034

[0158] Group 1 vs. Group 4; p = 0.003

[0159] Figure 12 presents graphically the effect of the plant extract on IL-6 levels. Statistically significant differences compared to Group 1 are marked with an asterisk (*). The results are presented as mean ± SD.

[0160] The statistical analysis showed a significant decrease in IL-6 levels in the group treated with the high dose of the extract (0.08 ± 0.05 pg / mg), compared to the control group with the empty film (0.16 ± 0.05 pg / mg; p = 0.003). A significant decrease in the levels of IL-6 was also observed in Group 2 (0.11 ± 0.04 pg / mg), compared to the control group (0.16 ± 0.05 pg / mg; p = 0.034).

[0161] The results suggest that treatment of skin wounds with an extract at a dose of 200 mg / kg leads to a significant reduction in IL-6 levels compared to the control group. This indicates a better effect of the higher dose of the extract compared to the lower dose.

[0162] IL-6 plays a role in the first phase of wound healing, which is the inflammatory phase. It is one of the main mediators of the acute-phase response. IL-6 levels may persist or increase again during the next two phases of the healing process — granulation tissue formation and remodeling [6].

[0163] Increased expression of TNF-a and IL -6 has a negative effect on wound healing, related to a delayed onset of the healing phase due to the induction of proteolytic enzymes. Moreover, high levels of pro-inflammatory cytokines lead to increased fibrosis and scarring. Lowering the levels of these mediators could stimulate the process of skin wound healing, thus making them a good therapeutic target [7].

[0164] IL-10:

[0165] 1 vs. Group 4; p = 0.003

[0166] 2 vs. Group 4; p = 0.020

[0167] 3 vs. Group 4; p = 0.042

[0168] Figure 13 shows graphically the effect of the plant extract on the level of IL- 10. The symbol * indicates significant differences compared to Group 1 ; # indicates significant differences compared to Group 2; and . indicates significant differences compared to Group 3. The results are presented as mean ± SD.

[0169] A statistically significant decrease in IL- 10 levels is observed in Group 4 (1.15 ± 0.28 pg / mg) compared to Group 1 (1.88 ± 0.56 pg / mg; p = 0.003), compared to Group 2 (1.69 ± 0.36 pg / mg; p = 0.020), and compared to Group 3 (1.61 ± 0.36 pg / mg; p = 0.042).

[0170] IL- 10 is an anti-inflammatory cytokine with a primary role in preventing scarring. It is secreted by various cells to suppress the action of pro-inflammatory cytokines. IL- 10 also facilitates the transition from the inflammatory to the proliferative phase in wound healing by regulating the type and number of immune cells migrating to the site of injury, and by reducing the expression of pro- inflammatory cytokines [8].

[0171] OH-proline: Figure 14 graphically presents the effect of the plant extract on the level of OH-proline. The results are presented as mean ± SD.

[0172] Despite the lack of significant intergroup differences, the data suggest increased levels of the marker in the group treated with a high dose of the plant extract (Group 4 - 0.53 ± 0.26 pg / mg) compared to the control group (0.37 ± 0.18 pg / mg).

[0173] Hydroxyproline is an amino acid that is part of the structure of collagen fibers in granulation tissue. The current study shows increased levels of OH-proline concentration in skin homogenate from animals treated with a high dose of plant extract compared to the control group. This indicates a positive effect of the extract on enhanced cellular proliferation and collagen synthesis. Similar data on the effects of plant extracts are also reported in [7],

[0174] 4.3.Conclusion

[0175] According to the histological results conducted at the end of the experiment, the best wound healing was observed in the fourth group of animals treated with patches containing the highest amount of extract (200 mg / kg). When monitoring wound healing dynamically by days, the most pronounced effect on the wound surface was observed in the early phase (days 3-5) in the third group of animals treated with patches containing a lower amount of plant extract (100 mg / kg). The cytokine data indicate a significant reduction in the levels of TNF-a in group 4 compared to the control group with a blank film (p = 0.017). A similar effect of the high dose of extract was also observed regarding IL-6 (Group 4 vs. Group 1, p = 0.003). This suggests a better effect of the higher dose of extract compared to the lower dose (100 mg / kg). Regarding IL- 10 levels, a significant decrease was observed in the fourth group compared to all other groups (Group 4 vs. Groups 1 , 2, and 3; p = 0.003, p = 0.020, p = 0.042, respectively). This indicates a transition from the inflammatory phase to the remodeling phase. Regarding OH-proline, the data suggest increased levels of the marker in groups treated with the plant extract compared to the control group, although statistical significance was not reached. Furthermore, the group treated with the higher concentration of extract (Group 4, 200 mg / kg body weight) exhibited the highest levels of OH-proline compared to all other groups.

[0176] LITERATURE ) CN113069479 (A) ) CN110269891 (Al) ) AU2021103381 (A4) ) Donika Gyuzeleva; Maria Benina; Valentina Ivanova; Emil Vatov; Saleh Alseekh; Tsvetelina Mladenova; Rumen Mladenov; Krasimir Todorov; Anelia Bivolarska; Plamen Stoyanov, Metabolome Profiling of Marrubium peregrinum L. and Marrubium friwaldskyanum Boiss Reveals Their Potential as Sources of Plant-Based Pharmaceuticals, International Journal of Molecular Sciences, 2023-12-01, Journal article, https: / / www.mdpi.com / 1422-0067 / 24 / 23 / 17035 ) Xiao-bo Wu, Xian-qun Luo, Shu-ying Gu, Jia-hong Xu. The effects of Polygonum cuspidatum extract on -wound healing in rats. Journal of Ethnopharmacology. 141(2012):934-937 ) Grellner W. Time-dependent immunohistochemical detection of proinflammatory cytokines (IL- 1 beta, IL-6, TNF-alpha) in human skin wounds. Forensic Sci Int. 2002 Dec 4;130(2-3):90-6. doi: 10.1016 / s0379-0738(02)00342-0. PMID: 12477628. ) Gunasekaran, S., Nayagam, A. A. J. & Natarajan, R. Wound healing potentials of herbal ointment containing Calendula officinalis Linn, on the alteration of immunological markers and biochemical parameters in excision wounded animals. Clin Phytosci 6, 77 (2020). https: / / doi.org / l 0.1186 / s40816-020-00215-7. ) Singampalli KL, Balaji S, Wang X, Parikh UM, Kaul A, Gilley J, Birla RK, Bollyky PL, Keswani SG. The Role of an IL-10 / Hyaluronan Axis in Dermal Wound Healing. Front Cell Dev Biol. 2020 Jul 17;8:636. doi: 10.3389 / fcell.2020.00636. PMID: 32850791; PMCID: PMC7396613.

Claims

PATENT CLAIMS1. Skin wounds healing patch, comprising chitosan, glycerol, and a plant extract, characterized in that it further includes polyvinyl alcohol, which, together with chitosan, forms a polymeric mixture, and the plant extract is a methanol extract from Marrubium peregrinum, with a concentration of 4 to 8 mg / cm2, wherein the components are in the following weight parts: 30-70 parts of the polymeric mixture, 20-60 parts of the methanol extract of Marrubium peregrinum, and 3-25 parts of glycerol.

2. Skin wounds healing patch according to claim 1, characterized in that the polymeric mixture consists of 50 weight parts, the Marrubium peregrinum extract consists of 40 weight parts and has a concentration of 8 mg / cm2, and glycerol consists of 10 weight parts.

3. Skin wounds healing patch according to claim 1 or 2, characterized in that the concentration of chitosan and polyvinyl alcohol is 1-5%, and the ratio of the weight parts of chitosan to polyvinyl alcohol in the polymeric mixture varies from 10:90 to 90:10.

4. Skin wounds healing patch according to any of claims 1 to 3, characterized in that the concentration of chitosan and polyvinyl alcohol is 2%, and the ratio of the weight parts of chitosan to polyvinyl alcohol in the polymeric mixture is 50:50.

5. Skin wounds healing patch according to any of claims 1 to 4, characterized in that the concentration of glycerol is 0.1-1%, preferably 0.2%.

6. Method for preparing skin wounds healing patches, characterized by the following stages: stage (1) preparation of extract from the plant Marrubium peregrinum, wherein: the dry drug, including the aerial parts of Marrubium peregrinum, is mixed with an extractant - methanol, in a volume percentage of 60-80%, in a ratio of 1:8 to 1:12; the obtained mixture is placed in flasks, which are wrapped with aluminum foil and placed on a magnetic stirrer for 24 hours; a three-step ultrasonic extraction is performed in an ultrasonic bath, consisting of three cycles, approximately every 3-4 hours, each lasting 15 minutes at 30°C; the resulting samples are filtered through gauze, and the filtrate (extract) is centrifuged for 10 minutes at 5000 rpm to obtain the purest possible sample, which is stored in the refrigerator; the remaining drug is added to an extractant, and the procedure is repeated two more times for 24 hours, until a threefold extract is obtained; the obtained extracts are mixed and evaporated using a vacuum evaporator until dry; the dry residue is dissolved in deionized water to obtain a concentration of the methanol extract from Marrubium peregrinum of 4 to 8 mg / cm2; stage (2) preparation of a polymeric mixture from solutions of polyvinyl alcohol and chitosan in distilled water, with a concentration of 1 -5%, in a weight ratio between the two ingredients from 10:90 to 90: 10; stage (3) preparation of a glycerol solution with a concentration of 0.1-1%; stage (4) mixing of 30-70 weight parts of the polymeric mixture with 20-60 weight parts of the methanol extract from Marrubium peregrinum and 3-25 weight parts of glycerol; stage (5) homogenization of the solution obtained in stage 4, pouring it into petri dishes, and drying until a constant weight of the obtained patches is reached.

7. Method for preparing skin wounds healing patches, according to claim 6, characterized in that the dry drug from the aerial parts of Marrubium peregrinum is mixed with 70% methanol in a ratio of 1 : 10.

8. Method for preparing skin wounds healing patches, according to claim 6 or 7, characterized by the fact that the solution of polyvinyl alcohol and the solution of water-soluble chitosan have a concentration of 2%, and the weight ratio between them is 50:50.

9. Method for preparing skin wounds healing patches, according to any of claims 6 to 8, characterized by the fact that the concentration of glycerol is 0.2%.

10. Method for preparing skin wounds healing patches, according to any of claims 6 to 9, characterized by the fact that the polymeric mixture consists of 50 weight parts, the extract of Marrubium peregrinum consists of 40 weight parts with a concentration of 8 mg / cm2, and glycerol consists of 10 weight parts.

Images