A mixture for obtaining a pharmaceutical composition containing an extract from the raw material of the reynoutria genus, a method for obtaining the composition, a use of the composition in dental and / or periodontal therapy, and a use of the mixture

Abstract

The subject of the invention is a mixture for obtaining a pharmaceutical composition containing a pharmaceutical carrier and an extract from a plant of the Reynoutria genus. The invention also relates to methods for obtaining a pharmaceutical composition, a dental and / or periodontal pharmaceutical composition containing an extract from a plant of the Reynoutria genus in dentistry and / or periodontology, and a use of the mixture for obtaining a pharmaceutical composition containing a pharmaceutical carrier and an extract from a plant of the Reynoutria genus.

Description

[0001] A mixture for obtaining a pharmaceutical composition containing an extract from the raw material of the Reynoutria genus, a method for obtaining the composition, a use of the composition in dental and / or periodontal therapy, and a use of the mixture

[0002] The subject of the invention is a mixture for obtaining a pharmaceutical composition containing a pharmaceutical carrier and an extract from a plant of the Reynoutria genus. The invention also relates to methods for obtaining a pharmaceutical composition, a dental and / or periodontal pharmaceutical composition containing an extract from a plant of the Reynoutria genus in dentistry and / or periodontology, and a use of the mixture for obtaining a pharmaceutical composition containing a pharmaceutical carrier and an extract from a plant of the Reynoutria genus.

[0003] These formulations are intended for use in dentistry, both in topical applications on mucous membranes and the tongue, as well as in periodontology, in the context of applications to periodontal pockets.

[0004] Reynoutria japonica (Japanese knotweed), known as Japanese knotweed, belongs to the Polygonaceae family, whose natural habitat spans regions of Asia and South America. In Traditional Chinese Medicine (TCM), the rhizome of R. japonica is considered a medicinal raw material known as Hu Zhang, and its analgesic, antipyretic, expectorant, and diuretic properties have been described. Since 1977, the monograph on the rhizome of R. japonica is included in the Pharmacopoeia of the People's Republic of China, and since 2017, the monograph on the rhizome of R. japonica as a plant material with medicinal properties is also included in the European Pharmacopoeia. The extract obtained from the rhizome of R. japonica is rich in substances with therapeutic potential, including procyanidins, flavan-3-ols, flavonoids, hydroxycinnamic acid derivatives, disaccharide esters of phenylpropanoids, and stilbene derivatives, mainly resveratrol (RSV) and piceid. The high resveratrol content determines its anti-inflammatory, antibacterial as well as anti-allergic and anti-mutagenic properties [Liu, S., Zhang, R., Zhang, X., Zhu, S., Liu, S., Yang, J. (...) & Hu, H. (2022). The invasive species Reynoutria japonica Houtt. as a promising natural agent for cardiovascular and digestive system illness. Frontiers in Pharmacology, 13, 863707, Bozin, B., Gavrilovic, M., Kladar, N., Rat, M., Anackov, G., & Gavaric, N. (2017). Highly invasive alien plant Reynoutria japonica Houtt. represents a novel source for pharmaceutical industry-Evidence from phenolic profile and biological activity. Journal of the Serbian Chemical Society, 82(7-8), SOB- SIB.]

[0005] The World Health Organization (WHO) estimates that oral diseases affect nearly 3.5 billion people every year. Although the quality of care for all oral disorders shows an increasing trend on a global scale, oral health conditions still rank among the top ten of all diseases globally, which has a negative impact on the global economy. Annual expenditure on the treatment thereof was estimated at USD 387 billion in direct costs and another USD 323 billion in indirect costs. The treatment of severe inflammation of the oral cavity with the use of standard therapy, i.e., antibiotic therapy or mechanical cleaning, often has limited efficacy, allowing for only a short-term reduction in the symptoms of the disease. A promising area of research is the development of unique carriers for new active compounds from plant material, i.e., plants of the Reynoutria genus with therapeutic potential for use in dental therapy. There is an ongoing search for new solutions to improve the application of the carrier to mucous membranes and the bioavailability of active substances, components of extracts obtained from plants.

[0006] Resveratrol, as an active substance with potential therapeutic properties, requires special attention in terms of its release rate. The key factor is not only the presence of resveratrol at the site of action, but also the duration for which it remains available to the body. Traditional dosage forms, such as semi-solid or liquid, often present limitations related to short retention times and difficulties in achieving prolonged release of the substance. The introduction of mucoadhesive preparations is becoming an innovative approach that can significantly improve treatment efficacy. Due to its close contact with mucous membranes and prolonged retention time, resveratrol may be released in a controlled manner, which increases its bioavailability and therapeutic efficacy. Bioadhesive properties of polymers, used as carriers, allow for increased mucoadhesion, which has a direct impact on the efficacy of active substance release. Consequently, developing new dosage forms using mucoadhesive carriers is crucial for optimizing therapy with the use of resveratrol and may contribute to achieving better clinical results. The prolonged release of the active substance at the site of action is therefore a fundamental aspect that should be taken into account in research on new pharmaceutical forms.

[0007] The need to develop new formulations results both from the natural limitations of the application site and from the inconveniences associated with the use of conventional carriers, such as semi-solid and liquid dosage forms. The most significant problems associated with the forms used include: insufficient retention time at the application site, difficulties in achieving prolonged release of the active substance at the site of action, and consequently often unsatisfactory therapeutic effects after application. Intensive development of locally applied mucoadhesive preparations is becoming a promising approach to overcoming these drawbacks. This dosage form ensures close contact between the medicinal substance and the application site, as well as its prolonged retention time. Additionally, it is a non-invasive method of application that does not interfere with patients' daily activities, e.g. speaking and drinking, which may result in improved compliance with therapeutic recommendations. Obtaining carriers with the aforementioned properties is possible thanks to bioadhesive properties of the polymers used, which are auxiliary substances and components of the developed formulations. The use of mucoadhesive polymer-based carriers may increase their mucoadhesion, and consequently promote more effective release of active substances at the application site, which in turn translates into higher adsorption and bioavailability, and as a result, therapeutic efficacy.

[0008] The present invention comprises the solutions specified in the claims attached hereto.

[0009] The subject of the invention is a polymer formulation that enables the incorporation of active substances extracted from plant material with potential antibacterial and anti-inflammatory properties, intended for application in inflammatory conditions of the oral cavity. They also mark the current direction in the search for new application forms for substances in the non- surgical treatment of periodontitis. Carriers with different compositions and different disintegration times have been developed, allowing for formulations tailored to the therapeutic needs, including the time they remain on the mucous membrane. Polymers were used as the basis for the film formulation: polyvinyl alcohol (PVA), which provides the appropriate mechanical properties. In addition, the film skeletons constitute semi-synthetic cellulose derivatives, including methylcellulose with low viscosity of 2% solutions in the range of 300-560 mPa*s, methylcellulose with an average viscosity of 2% solutions in the range of 1200-1800 mPa*s, and hydroxypropyl methylcellulose with a viscosity of 2% solutions in the range of 20-40 mPa*s. In order to ensure adequate mucoadhesive properties and to prolong the release time of the substances incorporated in the carrier, polyvinylpyrrolidone (PVP) and carboxymethyl cellulose sodium salt (NaCMC) were incorporated into the composition of the film formulations.

[0010] Furthermore, carrier formulations according to the invention are characterized by varying blurring times and varying half-release times of the active substance, which allows designing film formulations with desired release profiles of active substances tailored to clinical needs. Importantly, the selected flexible drug form ensures convenient application of the drug, both in the doctor's office and for self-use by the patient as part of the treatment prescribed by the doctor. The flexibility of these formulations translates into their pliability and ease of shaping, allowing them to be tailored to specific application sites, such as the cheek or tongue. Additionally, they may be easily cut to the desired shape, which allows for their placement in periodontal pockets. Such versatility in application allows patients to comfortably use the drug under home conditions, which increases treatment efficacy and improves compliance with therapeutic recommendations.

[0011] The technological process included several stages, which were optimized in order to obtain a mucoadhesive carrier with desired physicochemical and functional properties. The optimal formulation was developed based on the analysis of parameters such as flexibility, adhesive properties, and film dissolution time.

[0012] A technology was developed for obtaining polymer film formulations, as a potential mucoadhesive carrier, differing in composition, which directly affected their physicochemical properties, in particular mechanical properties and disintegration time, and ultimately 3 formulations were fabricated for incorporating an extract from the Reynoutria genus.

[0013] During the optimization of the technological process, placebo film formulations were obtained from low-viscosity polymer solutions, sequentially by solvent-casting method and their physical cross-linking.

[0014] Embodiments for carrying out the invention have been illustrated in the figures, where: Fig. 1 shows a schematic of preparation of polymer carrier formulations for testing in the form of films,

[0015] Fig. 2 shows a schematic of preparation of optimized polymer film formulations with a Reynoutria japonica extract.

[0016] Reagents:

[0017] Sigma-Aldrich: polyvinyl alcohol (PVA, Mwt: 85,000-124,000, degree of hydrolysis: 98-99%; CAS: 9002-89-5), polyvinylpyrrolidone (PVP, K90, Mwt: 40,000, CAS: 9003-39-8), methylcellulose 400 (MC 400, viscosity of 2% aqueous solution at 20°C: 300-560 cP, CAS: 9004-67-5), methylcellulose (MC, Methocel8A15C, viscosity of 2% aqueous solution at 20°C: 1200-1800 mPa*s, CAS: 9004-67-5), hydroxypropyl methylcellulose (HPMC, viscosity of 2% aqueous solution at 20°C: 40-60 cP, CAS: 9004-65-3), mucin from pig stomachs (type III, bound to sialic acid: 0.5-1.5%, CAS: 84082-64-4); VWR Chemicals: carboxymethyl cellulose sodium salt (NaCMC, viscosity of 2% aqueous solution at 25°C: 300-600 mPa*s, CAS: 9004-32-4); Fagron: glycerol (Glycerolum 85%, batch no.: 081056, CAS: 56-81-5); Honeywell: ethyl alcohol (96% v / v, CAS: 64-17-5); J. T. Baker: purified water LC-MS (CAS: 7732-18-5). Roth: resveratrol, a standard substance (>98%, serial no.: N811.3, CAS: 501-36-0).

[0018] The tests were conducted using purified water obtained by ion exchange and reverse osmosis, meeting the requirements of the Aqua purifica monograph included in FP XII. Purified water obtained by ion exchange and reverse osmosis was used, meeting the requirements of the Aqua purifica monograph published in FP XIII (04 / 2018:0008).

[0019] Test methods: a) Testing Mucoadhesive Strength

[0020] The test was performed on a 2.5 cm x 2.5 cm fragment of the film using a TA. XT Plus texture analyzer with a movable arm and a probe equipped with an A / Muc mucoadhesion measurement fixture (Stable Micro System, Godaiming, United Kingdom). In order to perform the measurement, a 13 mm diameter mucin disc, prepared by compressing 250 mg of powdered substance using a hydraulic press (Specac, 15t, UK) with a 13 mm diameter die (Specac 13mm DIE PT. No 3000. United Kingdom), under a pressure of 10 tons for 30 seconds, was attached to the movable arm with the probe using double-sided tape. Before the mucoadhesion test, the mucin disc was moistened with a 5% aqueous solution of mucin. Next, the arm with the probe and mucin disc was lowered at a speed of 1 mm / s into a solution of artificial saliva in a beaker with a film fragment installed in the A / Muc fixture. The artificial saliva solution used was prepared according to the composition listed in Table 1. The beaker with the immersed A / Muc fixture was placed on a magnetic stirrer with a heating function in order to simultaneously stir and maintain the fluid at a temperature of 37±1°C during the measurement. After the mucin disc came into contact with the film fragment, the lowered arm with the probe applied a force of 0.1 N for 30 seconds, followed by a programmed lifting of the arm at a speed of 1 mm / s with simultaneous measurement of the force required to remove the mucin disc from the film surface. The method was modified based on sources [1, 2], The test was repeated for three fragments of polymer film sampled from different areas of the dry formulation. The results were presented as the average values from three measurements of the force required to detach the mucin disc from the film surface, along with the standard deviation.

[0021] Table 1 Composition of the artificial saliva solution prepared for the mucoadhesion tests b) Test of the Blurring Time and pH Value of Extracts After Blurring

[0022] The blurring time test was conducted in a thermostatic water bath (Memmert type: WB22, Germany) at a temperature of 37±1°C with a horizontal shaking function (Memmert type: S1422, Germany). Film fragments measuring 2.5 cm x 2.5 cm were carefully weighed and placed in beakers sealed with parafilm, containing 10 ml of purified water at a temperature of 37±1°C. The beakers were then transferred to a water bath and horizontally shaken at a constant frequency of 60 cycles per minute. The blurring time was defined as the time required for the complete disintegration of the carrier, with no fragments visible to the naked eye.

[0023] The pH measurements of the extracts obtained after blurring the film fragment were performed using the potentiometric method using a pH meter with a combined electrode at a temperature of 22±1°C with an accuracy of 0.01 pH units. Before measurement, the blurred film sample was left at room temperature for 1 hour. Once the time had elapsed, the pH was measured by immersing the combined electrode. The method was modified based on sources [1, 3].The test was conducted in three repetitions for each sample. The results were presented as the average value from measurements along with the standard deviation. c) Contact Angle Measurements

[0024] Measurements of the contact angle were performed for optimized formulations of polymer films measuring 2.5 cm x 2.5 cm with a precision of 0.01° using a goniometer (Ossila, UK) with a built-in digital camera and software (Ossila Contact Angle) enabling image analysis. During the measurements, the camera captures images of the droplets and sends their digital record to a computer, which automatically analyzes the image. The calculations of the contact angle are based on the assumption that the shape of a droplet is a spherical sector, and knowing its height and volume allows for the calculation of the contact angle. In order to carry out the measurement, a single film fragment was placed on the device's stand and a single 50 pl drop of purified water was dispensed using an automatic pipette; then, using the camera and the software, the angle between the sample surface and the tangent to the liquid surface was measured. The method was modified based on sources [4, 5], The measurements were made for 3 film fragments cut from different areas of the dry formulation. The results were presented as the average value from measurements along with the standard deviation.

[0025] Example 1. Technology for obtaining a mucoadhesive carrier formulation

[0026] In the first stage of the study, aqueous solutions of selected polymers were prepared in concentrations allowing them to be mixed together and cast into polystyrene dishes with an area of 92 cm2. Table 2 lists the polymers selected for testing and the concentrations of the solutions prepared therewith, which were used in the process of determining the composition of polymer films. In the following section, compositions prepared from the polymer solutions presented in Table 2 will be described. Table 2. Polymers selected for testing and concentrations of solutions prepared therewith

[0027] PVA solutions were prepared by dissolving a weighed amount of polymer in purified water and mixing at a constant speed of 50 revolutions per minute while heating the solution at a temperature of approximately 70°C for 24 hours. PVA and pullulan solutions were prepared by dissolving a weighed amount of polymer in purified water and mixing at a constant speed of 50 revolutions per minute at room temperature for 24 hours. Meanwhile, solutions of cellulose derivatives, i.e., MC 400, MC A15C, HPMC, and NaCMC, were prepared by adding weighed amounts of polymers to purified water at room temperature and leaving them to swell in a refrigerator at 4°C for 24 hours. After this time, the solutions were mixed using a magnetic stirrer at a speed of 50 revolutions per minute for 5 hours. All prepared polymer solutions were sterilized in an autoclave (SANYO, model: MLS-3750, Japan) at 121°C for 20 minutes from the moment the indicated temperature was reached.

[0028] The design of a polymer film formulation intended for application to the oral mucosa began with the preparation of individual polymer solutions, followed by the inclusion of complex mixtures containing two, three, and four polymers in the process. The figure shows a schematics of the polymer film preparation process, illustrating the individual stages of the formulation preparation process.

[0029] The formulations for casting films were prepared in a laminar flow cabinet by combining sterilized solutions of individual polymers according to the composition shown in Table 3, which were then mixed in various weight ratios.

[0030] Table 3. Formulations for film casting.

[0031] Appropriate polymer solutions were prepared, which were then placed in a bottle and mixed on a magnetic stirrer at room temperature, adding an appropriate amount of a plasticizer. After mixing thoroughly for approximately 2-3 hours, the solutions were cooled to temperatures ranging from 4° to 8°C for 24 hours.

[0032] After combining the two polymer solutions, they were mixed on a magnetic stirrer for 2-4 hours at room temperature. Next, the physical cross-linking of polymers was planned at a temperature of -18°C to -22°C, which lasted from 1 to 4 hours (the mixture of polymer solutions must be stored in a freezer until it is completely frozen). After this time, the mixture was thawed until it reached a liquid consistency, and subjected to sonication in an ultrasonic bath (Advantage-Lab GmbH, type: AL-04-12) at room temperature in order to remove air bubbles until they completely disappeared. It was then mixed for another 4 hours and cooled at a temperature of 4° to 8°C for 24 hours. After cooling, it was mixed again for 2-4 hours at room temperature until a uniform mixture was obtained, which was then subjected to physical cross-linking again at a temperature of -18°C to -22°C, lasting up to 4 hours (the mixture must be frozen). After this time, the mixture was thawed and again subjected to sonication in an ultrasonic bath (Advantage-Lab GmbH, type: AL-04-12) at room temperature in order to remove air bubbles until they completely disappeared. It was then mixed for another 4 hours at room temperature and cooled to a temperature between 4° and 8°C for 24 hours, after which a plasticizer was added while mixing on a magnetic stirrer at room temperature for 4 hours. During this process, the uniformity of the mixture and the presence of air bubbles were monitored. The formulations were subjected to five stages of physical cross-linking of polyvinyl alcohol, which involved freezing and thawing the mixtures at temperatures ranging from -18°C to -22°C. After each freezing step, the formulation solutions were mixed at room temperature to obtain a uniform polymer dispersion. The mixtures were then subjected to a sonication process in an ultrasonic bath until all air bubbles were completely removed. After this stage, they were mixed for another 4 hours at room temperature and then were cooled to a temperature of 4° to 8°C for 24 hours. After cooling, they were mixed again for 2-4 hours at room temperature until a uniform mixture was obtained. After five cycles of cross-linking and obtaining a uniform mixture, formulations were cast in amounts of 45 g into polystyrene dishes with an area of 92 cm2, and then dried in a laboratory dryer at 32±1°C for 24 to 72 hours, controlling the drying process on the dishes. The obtained film formulations, listed in Table 4, were subjected to a preliminary assessment of their film-forming properties and physicochemical properties.

[0033] Table 4. Polymer film - placebo formulation compositions

[0034] OP - film formulations without active substance

[0035] 1.1. Evaluation of Polymer Films:

[0036] Measurements of thickness and mass were carried out, as well as tests of mucoadhesion strength, wettability, and blurring time; pH value of the solution formed after blurring of the polymer film was determined, and the mechanical resistance of the film to crushing was evaluated. For this purpose, 2.5 cm x 2.5 cm fragments of films were prepared, which were randomly selected and cut out for the measurements.

[0037] All placebo films were characterized by morphological and structural uniformity, which indicates the correct selection of the composition, appropriate technology for combining and mixing the prepared polymer mixtures, and the selection of preferable conditions for casting and drying of the formulations. The technological optimization process has shown that the degree of physical cross-linking of polymers significantly affects their structure and properties, enabling polymer films to be designed as carriers with modified disintegration times, which has an impact on the release of active substances after their incorporation into the formulation. This allows adjusting the selection of the formulations to achieve both prolonged and accelerated release of active substances, depending on therapeutic requirements. After evaluating the physicochemical properties of placebo films, i.e., the thickness uniformity of dry films (in the range of 202-256 nm), average mass (in the range of 161.76-183.45 mg), mucosal adhesion value measured in a test with mucin (in the range of 190.30-270.07 g), wettability of the material by measuring the contact angle (in the range of 50.78°-60.05°), blurring time (in the range of 2 min to 24 h), pH of the extract formed after blurring the formulation (in the pH range = 6.48-7.43). In addition, all film-based carrier formulations prepared were characterized by high folding endurance. Mechanical properties of the film formulations were determined based on the method described in the publication by Vecchi et al. [Vecchi, C. F.; Cesar, G. B.; De Souza, P. R.; Caetano, W.; Bruschi, M. L. Mucoadhesive Polymeric Films Comprising Polyvinyl Alcohol, Polyvinylpyrrolidone, and Poloxamer 407 for Pharmaceutical Applications. Pharm. Dev. Technol. 2021, 26, 138-149], The formulations of placebo polymer films listed in Table 3, developed in an optimization process involving a mixture of four polymer solutions with the addition of a plasticizer, show morphological and structural uniformity, optimal mechanical and mucoadhesive properties, and prolonged total disintegration time. The percentage by weight of polymer components used, the addition of plasticizer, and the technology and conditions used in the drying process of polymer films significantly impact the morphology and structure of the films obtained. The use of polyvinyl alcohol with a degree of hydrolysis in the range of 98-99%, as well as an increase in the content of methylcellulose derivatives, i.e., MC400, MC1500 and MCA15C, relative to the content of polyvinyl alcohol, and the introduction of a physical cross-linking process of the formulations contributes to prolonging the blurring time of the prepared films. The addition of carboxymethyl cellulose sodium salt to the formulation's composition reduces the blurring time of the polymer film, while ensuring optimal mucoadhesive properties of the prepared films. Next, formulations (0W1-0W3) were selected, which are characterized by a longer disintegration time of over 8 hours, for the incorporation of active substances extracted from the raw material of the Reynoutria genus, including R. japonica.

[0038] Example 2. Obtaining an optimized composition with R. japonica extract

[0039] 1. Preparation of polymer mixtures (recipe above)

[0040] 2. Preparation of carriers with R. japonica extract

[0041] Polymer films were prepared in a laminar chamber by casting from a solution method described above for polymer - placebo films. 3 types of formulations were prepared, the compositions of which are listed in Table 4. For this purpose, 3 ml of an aqueous-ethanol solution (with a water / ethanol ratio of 25:75 m / m) containing 100 mg of dry R. japonica extract previously micronized in an agate mortar was introduced into the prepared 50 g OP1- OP3 formulations. Before being incorporated into the formulations, the prepared aqueous- ethanolic extract solution was filtered through a syringe filter with a pore diameter of 0.22 pm (PTFE; Millex Samplicity8Filters). After adding the extract solution, the formulations were mixed using a magnetic stirrer with a simultaneous heating function (Labinco LD846, Labinco BV, Netherlands), adjusting the stirrer's rotational speed to the viscosity of the polymer mixture, continuing the mixing process for 12 hours. The films were prepared in a laminar flow cabinet by preparing sterilized solutions of individual polymers according to the composition shown in Table 2, which were then mixed in various weight ratios.

[0042] Next, the formulations were subjected to five stages of physical cross-linking of polyvinyl alcohol, which involved freezing and thawing the mixtures at temperatures ranging from -18°C to -22°C. After each freezing stage, the formulation solutions were mixed for 15 minutes to achieve a uniform dispersion of the polymers. Next, the prepared mixtures were subjected to a sonication process in an ultrasonic bath (Advantage-Lab GmbH, type: AL-04-12) for 30 minutes to remove air bubbles. The prepared liquid formulations were cast in amounts of 45 g into polystyrene dishes with an area of 92 cm2and dried in a laboratory dryer (Pol-Eko- Aparatura Sp. j. type: SLN 115 simple, Poland) at a temperature of 32±1°C for 48 hours. After drying and removal from the dishes, the films were stored in sterile zip-lock bags (Chemivet) at room temperature for further testing. A schematic illustrating the formulation stage is shown in Fig. 1.

[0043] Table 5. Composition of formulations with Reynoutria japonica extract

[0044] OW - film formulations with R. japonica extract

[0045] 2.1. Evaluation of Polymer Films with Reynoutria japonica Extract:

[0046] All the prepared polymer film formulations: OW1, OW2, and OW3 with Reynoutria japonica extract were characterized by morphological and structural uniformity. All films were characterized by flexibility, ease of removal from the mold, and a smooth surface. Carriers with incorporated Reynoutria japonica extract showed a light brown color. Infrared Fourier transform spectroscopy analysis of the PVA component spectrum revealed a characteristic band recorded in the range of 3000-3500 cm1(approximately 3295 cm'1), corresponding to the stretching vibrations of -OH hydroxyl groups, forming intra- and intermolecular hydrogen bonds. The PVP spectrum revealed a band with a registered wavenumber of 1654 cm , whose presence is associated with the stretching vibrations of the C=O bonds, present in the pyrrolidone ring. Additionally, a broad absorption band in the range of 1050-1430 cm1was observed, characteristic of the stretching vibrations in C-0 bonds and the bending ones of - CH2 and -CH groups, prominent in the spectra of MC, NaCMC, and HPMC. No additional bands were found in the FTIR spectrum of the tested carriers containing R. japonica extract, except for those characteristic of the components of the preparation and the extract. Thickness uniformity of dried films is directly related to the correctness of the film preparation process. All prepared formulations were characterized by uniform thickness, with the standard deviation of results for each formulation ranging from ± 4.47 nm to ± 5.47 nm and being lower than the accuracy of the measuring instrument used, which proves the repeatability of the polymer film preparation method. The results are shown in Table 6.

[0047] Table 6. Results of thickness and mass measurements of optimized polymer films.

[0048] The results of mucoadhesion force measurements are presented in Table 7. The highest average mucoadhesion strength values were obtained for the OW2 formulation, while the lowest values were recorded for the OW3 formulation.

[0049] Table 7. Results of mucoadhesion measurements of optimized polymer films containing the extract from Reynoutria japonica species.

[0050] Table 8 shows the average values of the contact angle measurements for three film formulations containing plant extract from Reynoutria japonica species.

[0051] Table 8. Results of contact angle measurements of optimized polymer films

[0052] An important parameter used to evaluate the effectiveness of the developed carrier is the study of the release profile of the active substance contained therein. The results of the study of the release of active substances from the optimized OW2 series film with Reynoutria japonica extract are presented as the percentage of resveratrol or piceid released, measured at 1, 3, 5, 7, 12, and 24 hours of the study. The concentrations of the substances were determined based on a calibration curve prepared using standards, pure resveratrol, and piceid of analytical-grade. Graph 2 shows the correlation between the total percentage of resveratrol released over time, while Graph 4 shows the correlation between the total percentage of piceid released over time during the study. The results of the study confirmed the release of at least 25% of the resveratrol and piceid dose from the selected carrier after 3 hours of testing, ater which a characteristic decrease in the intensity of the release process was observed, followed by a renewed increase in the released dose, until more than 50% of the dose was released after 24 hours of the study. For the optimized OW2 carrier with Reynoutria japonica extract, a test of the content of the extract components, resveratrol and piceid, respectively, in the extract after blurring a film fragment was conducted.

[0053] As a result, the formulation preparation method with incorporated extract was modified, because in the case of plant compounds, it was necessary to adapt the technology in order to improve the release of components from extracts of the Reynoutria genus. Another very important issue analyzed was the order od addition of the polymer solutions and the stage of adding the extract to the polymer mixture in order to ultimately obtain a film with the incorporated extract.

[0054] Example 3. Modification of the technology of the optimized films with R. japonica extract (Fig. 2).

[0055] 3 ml of a 25% aqueous-ethanol solution (with a water:96% ethanol ratio of 25:75 m / m) containing 200 mg of dry R. japonica extract previously micronized in an agate mortar was introduced into the prepared 30 g FR1-FR3 formulations. Before being incorporated into the formulations, the prepared aqueous-ethanolic extract solution was filtered through a syringe filter with a pore diameter of 0.22 pm (PTFE; Millex Samplicity8Filters). After mixing PVA and PVP, the mixture was cooled at a temperature of 4-8°C for 12-18 hours, followed by the addition of a 25% solution of R. japonica extract.

[0056] After the addition of the extract solution, the formulations were mixed using a magnetic stirrer with a simultaneous heating function, adjusting the stirring speed to the viscosity of the polymer mixture, continuing the mixing process for 2 hours.

[0057] After mixing and cooling at a temperature of 4-8 °C for 12-19 hours, the mixture was subjected to sonication. It was then frozen at a temperature of -18°C to -22°C for 12-19 hours, after which it was thawed again until reaching a liquid consistency and mixed for 1.5 hours. The next stage was adding a cellulose derivative (MC 400, MC A15C, or HPMC) to the mixture of the R. japonica extract, after which the whole mixture was mixed and cooled at 4°-8 °C for 12-19 hours. This was followed by the addition of glycerol, mixing for over 3 hours, and cooling again for over 12 hours. The entire mixture was then frozen and thawed five times, storing it in the freezer until completely frozen (-18°C to -22°C). After each freezing, the mixture was thawed until it reached a liquid consistency and thoroughly mixed until a uniform consistency was obtained. Finally, it was subjected to ultrasounds and cooled to a temperature of 4-8 °C. Next, amounts of 30 g were cast into polystyrene dishes with an area of 92 cm2and dried in a laboratory dryer (Pol-Eko-Aparatura Sp. j. type: SLN 115 simple, Poland) at a temperature of 32±1°C for 24-72 hours. After drying and removal from the plates, the films were stored in sterile zip-lock bags (Chemivet) at room temperature for use in further testing. The operations were performed in accordance with the schedule in Table 9.

[0058] Table 9. Table 10. Quantitative composition of the poured gel formulation with Reynoutria japonica extract for obtaining polymer films

[0059] 2.2. Evaluation of Polymer Films with R. japonica Extract:

[0060] Measurements of thickness and mass were carried out, as well as tests of mucoadhesion strength, wettability, and blurring time; pH of the solution formed after blurring of the polymer film was determined, and the mechanical resistance of the film to crushing was evaluated. Said films were analyzed using Fourier transform infrared spectroscopy with OMNIC data analysis software and an ATR fixture used to obtain FTIR-ATR spectra. An evaluation was also carried out of the release profile of the active substances being the components of the extract from R. japonica, i.e., resveratrol and piceid. The content of active substances in carriers was evaluated. For this purpose, 2.5 cm x 2.5 cm fragments of films were prepared, which were randomly selected and cut out for the evaluation.

[0061] All films with R. japonica extract were characterized by morphological and structural uniformity, which indicates the correct selection of the composition, appropriate technology for combining and mixing the prepared polymer mixtures, and the preferable conditions for casting and drying of the formulations. After evaluating the physicochemical properties of films, i.e., the thickness uniformity of dried films (in the range of 209-258 nm), average mass (in the range of 138-184.23 mg), mucosal adhesion value measured in a test with mucin (in the range of 188.90-299.43 g), wettability of the material by measuring the contact angle (in the range of 48.87°-61.84°), blurring time (up to 24 h), pH of the extract formed after blurring the formulation (in the pH range = 7.15-7.65). The prepared film formulations were characterized by high folding endurance, i.e., they do not break or become damaged. The endurance test was performed by repeatedly folding at an angle of 180° until breaking or tearing, or until 300 folds were made without the film carrier breaking.

[0062] The conducted FT-IR spectroscopy analysis confirmed the presence of characteristic bands both for individual active substances of extract components and for the auxiliary extract components. When evaluating the release of active substances, extract components, into water, the highest percentage of resveratrol was released from formulation 1R, reaching a value of 87.84%. In the case of using phosphate buffer as a release medium, the highest release was observed for formulation R2, amounting to 84.12%. Similarly, this formulation yielded the highest release of resveratrol into the artificial saliva solution, amounting to 49.74%. In the case of formulation R3, 48.53% of resveratrol was released into the phosphate buffer within 24 hours.

[0063] The release process was carried out at a temperature of 37±1 °C. A stirrer was placed in the diffusion chamber, whose speed was adjusted to a frequency of 60 cycles per minute, which corresponded to the shaking conditions used in the traditional method. One randomly selected film fragment from each of the formulations FR1, FR2, FR3, FE1, and FE2 was used for the test. Formulation FE3 was rejected due to the lack of uniformity in the distribution of the active substance. For each of the formulations listed, round film fragments with a diameter of 1.5 cm were used. The test was conducted in two types of solvents: water and phosphate buffer with a pH of 6.8. The test lasted 24 hours, and samples were collected automatically at: 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, and 24 hours after the start of release. The device automatically collected the medium from the chamber, simultaneously replenishing the collected amount (1.5 ml) with new medium. After the test was completed, samples were collected and then filtered through PTFE syringe filters with a diameter of 0.22 pm. From the solution obtained, 100 pl was collected, diluted with 80% (m / m) aqueous methanol solution to a volume of 1 ml, and subjected to quantitative and qualitative analysis using high-performance liquid chromatography (HPLC). The analysis was performed using a method developed by Nawrot- Hadzik (Nawrot-Hadzik et al., Phytochemical Diversity in Rhizomes of Three Reynoutria Species and their Antioxidant Activity Correlations Elucidated by LC-ESI-MS / MS Analysis. Molecules. 2019 Mar 21;24(6):1136. doi: 10.3390 / molecules24061136. PMID: 30901974; PMCID: PMC6470775). Table 11. Comparison of the properties of polymer films containing Reynoutria japonica extract (FR1-FR3) and polymer films enriched with resveratrol (FRSV1-FRSV3).

[0064] Polymer films containing resveratrol (RSV) were prepared analogously to polymer films containing Reynoutria japonica extract. For their production, an RSV solution with a concentration of 0.395 mg in 3 ml of 25% ethanol (EtOH) was introduced at the same stage of the technological process as the R. japonica extract. Literature:

[0065] 1. Vecchi, C. F.; Cesar, G. B.; De Souza, P. R.; Caetano,W.; Bruschi, M. L. Mucoadhesive Polymeric Films Comprising Polyvinyl Alcohol, Polyvinylpyrrolidone, and Poloxamer 407 for Pharmaceutical Applications. Pharm. Dev. Technol. 2021, 26, 138-149. 2. Ptaczek M., Sznitowska M.: Zjawisko mukoadhezji i jego znaczenie w aplikacji leku. [The mucoadhesion phenomena and importance in drug application] Polimery Med., 2009, 2, 49-64.

[0066] 3. Garsuch V., Breitkreutz J.: Comparative investigations on different polymers for the preparation of fast dissolving oral films. J. Pharm. Pharmacol., 2010, 62, 539-545. 4. Yuan Y., Lee T. R.: Contact angle and wetting properties. Surf. Sci. Tech., 2013, 51, 3-

[0067] 34.

[0068] 5. Morales J. O., McConville J. T.: Manufacture and characterization of mucoadhesive buccal films. Eur. J. Pharm. Biopharm., 2011, 77, 187-199.

Claims

Claims1. A mixture for obtaining a pharmaceutical composition containing a pharmaceutical carrier and an extract from a plant of the Reynoutria genus, characterised in that the carrier comprises, in percentage by weight of the composition: a) an aqueous solution of polyvinyl alcohol with a degree of hydrolysis of 98% to 99% at a concentration of 2.5% in an amount from 11% to 14.31%, b) a 5% w / w aqueous solution of polyvinylpyrrolidone in an amount of 6.5% to 10%, c) an aqueous solution of a cellulose derivative selected from the group comprising: methylcellulose with a viscosity of a 2% aqueous solution at 20°C in the range of 300-560 mPa-s, hydroxypropyl methylcellulose with a viscosity of a 2% aqueous solution at 20°C in the range of 20-40 mPa-s, methylcellulose with a viscosity of a 2% aqueous solution at 20°C in the range of 1200-1800 mPa-s, in an amount of 17.5% to 22.76%, d) a 4% w / w aqueous solution of carboxycellulose sodium salt in an amount of 15% to 19.6%, e) a 85% w / w solution of glycerine in an amount of 1.5% to 12%, which constitutes a plasticizer, and f) and a water and ethanol mixture in an amount from 3%, in which the ethanol content constitutes 75% w / w of the water and ethanol mixture, wherein the watenethanol mixture contains a Reynoutria japonica extract, g) purified water up to 100% of the composition weight.

2. The mixture according to claim 1, characterised in that the cellulose derivative is selected from the group comprising: methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 300-560 mPa-s, hydroxypropyl methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 20-40 mPa-s or methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 1200- 1800 mPa-s.

3. The mixture according to claim 1 or 2, characterized in that the aqueous solution of methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 300- 560 mPa-s has a 5% w / w concentration.

4. The mixture according to claim 1 or 2, characterized in that the aqueous solution of hydroxypropyl methylcellulose has a 5% w / w concentration.

5. The mixture according to claim 1, characterized in that the aqueous solution of carboxymethyl cellulose sodium salt has a 5% w / w concentration.

6. The mixture according to claim 1 or 2, characterized in that the aqueous solution of methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 1200- 1800 mPa-s has a 3% w / w concentration.

7. The mixture according to claim 1, characterized in that the aqueous solution of polyvinylpyrrolidone has a 4% w / w concentration.

8. A method for obtaining a pharmaceutical composition containing an extract from a plant of the Reynoutria genus, comprising preparing an extract from raw material of the Reynoutria genus and a solution containing the extract, and preparing aqueous solutions of the carrier composition components, as defined in claim 1, characterized in that it comprises the steps of: a) combining an aqueous solution of polyvinyl alcohol with an aqueous solution of polyvinylpyrrolidone, cooling and mixing the mixture, b) adding a solution of Reynoutria japonica extract and mixing, c) subjecting the mixture to at least one cycle of freezing and thawing, d) adding a selected cellulose derivative, cooling and mixing, e) adding glycerin, cooling and mixing, f) subjecting the mixture to multiple cycles of freezing and thawing, g) casting the mixture into a mold and subjecting it to drying.

9. The method according to claim 10, characterised in that the cellulose derivative is selected from the group comprising: methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 300-560 mPa-s, hydroxypropyl methylcellulose or methylcellulose with a viscosity of 2% aqueous solution at 20°C in the range of 1200- 1800 mPa-s.

10. The method according to claim 8, characterised in that after stage b) the mixture is frozen at a temperature of -18°C to -22°C for 12 to 19 hours, thawed to roomtemperature and mixed for 1.5 hours before adding the cellulose derivative in step d), and after the final mixing in step f), the mixture is subjected to ultrasounds.

11. The method according to claim 8, characterised in that after stage b) the mixture is subjected to ultrasounds, followed by freezing at a temperature of -18°C to -22°C for 12 to 19 hours, after which it is thawed and mixed for 1.5 hours before adding the cellulose derivative in step d).

12. A dental and / or periodontal pharmaceutical composition containing an extract from raw material of the Reynoutria genus, characterized in that it is obtained according to the method defined in claim 8 or from the mixture defined in claim 1.

13. The dental and / or periodontal pharmaceutical composition according to claim 14, characterized in that the mucoadhesion value measured in the test with mucin ranges from 180 g to 300 g.

14. The dental and / or periodontal pharmaceutical composition according to claim 12, characterized in that the blurring time ranges from 2 min to 24 h.

15. The dental and / or periodontal pharmaceutical composition according to claim 12, characterized in that the pH of the extract formed after blurring of the formulation ranges from 6.48 to 7.43.

16. The pharmaceutical composition according to claim 12, characterized in that the contact angle measurement ranges from 58° to 62°.

17. A pharmaceutical composition containing an extract from a plant of the Reynoutria genus as defined in claim 12, obtained by the method defined in claim 8 or from the mixture defined in claim 1, for use in dental and / or periodontal therapy.

18. A use of the mixture according to claim 1 for obtaining a dental and / or periodontal composition.

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