BR102012013590A2 - PROPOLIS MICROENCAPSULATES, PROCESS OF OBTAINING MICROENCAPSULES, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM, PROCESS OF OBTAINING PHARMACEUTICAL COMPOSITIONS AND USES - Google Patents

Abstract

RED PROPOLIS MICROENCAPSULATES, DEMICROENCAPSULATED GETTING PROCESS, PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME, PROCESS OF OBTAINING PHARMACEUTICAL COMPOSITIONS AND USES. Microencapsulated red propolis (MPV) are proposed, process for obtaining MPV, pharmaceutical compositions containing MPV, process for obtaining pharmaceutical compositions containing MPV and uses. The production of MPV is linked to the use of standardized hydroalcoholic tinctures of red propolis, followed by the spray-dryer technique, in the form of microparticles. In addition, the present application deals with gastro-resistant pharmaceutical compositions containing said MPV. MPV and pharmaceutical compositions containing them have applications in the pharmaceutical industry, mainly due to their antimicrobial activities.

Description

FIELD OF THE INVENTION FIELD OF THE INVENTION The present invention belongs to the most specifically natural field, chemical-pharmaceuticals containing the same field, pharmaceutical-specific compositions, pharmaceutical products and the like-chemical field. utility to public health. Red propolis (MPV) microencapsulated, process for obtaining MPV, pharmaceutical compositions containing MPV, process for obtaining pharmaceutical compositions containing MPV and uses are proposed. The production of MPV is linked to the use of standardized red propolis hydroalcoholic dyes, followed by the Spray-Dryer technique, in the form of microparticles. Additionally, the present application deals with gastroresistant pharmaceutical compositions containing said MPV. MPVs and pharmaceutical compositions containing them have application in the pharmaceutical industries, mainly due to their antimicrobial activities.

BACKGROUND OF THE INVENTION

Propolis Propolis is the generic name for a natural product with resinous, balmy, gummy and highly adherent characteristics, harvested by bees (Apis melllfera). In hives, propolis is used to close small cracks, embalm dead insects and protect them against insect and microorganism invasion.

The collecting bees, through their hind legs, collect vegetable resins from buds, fruits, flowers and exudates, which suffer from the addition of bee secretions. Because it is collected from various plant species, propolis is a substance of highly variable composition. This variable composition depends on the geographical origin in which the apiary is located and may vary due to the place of collection, botanical origin containing different species of medicinal plants, different times of the year and different geographical conditions including climatic zones.

The chemical constituents of honey and propolis have been identified worldwide (Pietta, P.; Gardana, C.; Scaglianti, M.; Simonetti, P., Journal of Pharmaceutical and. Biomedical Analysis, V. 45, p. 390 Flavonoids, phenolic acids and terpenes are the main substances found and used to track quality and, in some cases, to demonstrate the authenticity of propolis in some geographic regions (Volpi, N.; Bergonzini, 6. , Journal of Pharmaceutical and Biomedical Analysis, V. 42, pp. 354-361, 2006. Among the most commonly used flavonoids and phenolic acids as markers are quercetin, canferol, naringenin, chrysin, pinocembrine, galangin (flavonoids). ); gallic acid, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acid and derivatives (phenolic acid), clerodanes (diterpenoids) (Rosalen, PL; Castro, ML; Cury, JA, Química Nova, V. 30, N 07, 1512-1517, 2007); rán s-Barbe, F. A .; Martos, I.

Cossentini, M .; Ferreres, F., J. Agric. Food Chem. , P. 45, p. 28242829, 1997); (Yao, L.; Jiang, Y .; Singanusong, R., Datta, N.; Raymont, K., Food Chemistry, V. 86, p.169-177, 2004); (Weston, R.J., Mitchell, K.R., Allen, K.L., Food Research International, V. 37, pp. 166-174, 2004); (Mitamura, T.; Matsuno, T.; Sakamoto, S.; Maemura, M.; Kudo, H.; Suzuki, S.; Kuwa, K.; Yoshimura, S.; Sassa, S.; Nakayama, T. Nagasawa, H., Anticancer Research, V. 16, No. 5a, pp. 2669-2672, 1996).

Other markers are specific for Brazilian green propolis such as isosakuranetin, bakarina, drupanine and Artepilina-C. Artepillin C has been demonstrating excellent anti-tumor activity (Kimoto, T.; Koya-Miyata, S., Hino, K.; Micallef, MJ; Hanaya, T.; Arai, S.; Xkeda, M.; Kurimoto, M. , Virchows Arch, V. 438, pp. 259-270, 2001); (Orsolic, J.N.; Terzic, S.; Sver, L.; Basic, I., Food and Agricultural Immunology, V. 16, No. 3-4, pp. 165-179, 2005); (Li HZ, Kapur, A.; Yang, JX; Srivastava, S.; McLeod, DG; Paredes-Guzman, JF; Daugsch, A.; Park, YK; Rhim, JS, International Journal of Oncology, V. 31, No. 3, pp. 601-606, 2007). The ever-increasing popular use of propolis and its antimicrobially acting derivatives (Sforcin, JM; Fernandes Jr., A., Lopes, CAM; Bankova, V. And Funari, SRC, J. Ethnopharmacology, V. 73, p. 243-249, 1998), anti-inflammatory (Khayyal, MT; el-Ghazaly, MA; el-Khatib, AS, Drug Experimental and Clinical Research, V. 19, p. 197-203, 1993), antiviral (Vynograd, N. Vynograd, I .;

Sosnowisky, Z., Phy tomedicine, V. 7, p. 1-6, 2000), anticarcinogenic (Bazo, A. P.; Rodrigues, M. A. M .;

Sforcin, J.M .; Camargo, J.L.V., Ribeiro, J.L .; Salvador, D. M.F., Teratogenis, Carcinogenis and Mutagenis, V. 22, p. 183-194, 2002) and immunomodulatory (Sforcin, J.M., Kanemo, R. Funari, S.R.C., Journal of Venemous Animal and Toxins, V. 8, p. 19-29, 2002); (Sa-Nunes, A.; Faccioli, L.H., Sforcin, J.M., Journal of Ethnopharmacology, V.83, pp. 93-97, 2003) has been demonstrating the great therapeutic power of propolis as a substitute for conventional synthetic drugs. The characterization of all these biological activities associated with the tendency to use natural products has resulted in increased demand for propolis and propolis-containing products such as extracts, tablets, capsules, nebulisers or powders (Menezes et al., 1997).

Red Propolis As far as red propolis is concerned, it was classified as the 13th subtype of Brazilian propolis found in the mangroves, lagoons, rivers and beaches of northeastern Brazil between the states of Sergipe, Alagoas, Pernambuco and Para iba by researcher Park and col. (2007). The main botanical origin of red propolis is the Dalbergia ecastophyllum plant, locally known as the Howler's Tail, which has a red resinous exudate released from its sap. Red propolis can also be found in Cuba in the provinces of Pinar Del Rio (La Coloma), Villa Clara (Manicaragua) and Matanzas (Jagüey Grande) (Anna Lisa Piccinelli et al. J. Agric. Food Chem. 2005, 53, 9010-9016); (Osmany Cuesta-Rubio et al. J. Agric. Food Chem. 2007, 55, 7502-7509).

National and international researchers with expertise in propolis have been identifying the constituents of red propolis. Eleven isoflavonoids and 1 chalcone were identified from Cuban red propolis and among these substances we can mention: Formononetine (Biochanina B), Bichanina A, vestitol, ortho-methyl vestitol, medicarpine, homopterocarpine, other pterocarpane derivatives, liquiritigenin and isoliquiritigenin (chalcona) {Anna Piccinelli et al. J. Agric. Food Chem. 2005, 53, 9010-9016). Other flavonoid subclasses have also been identified, including new types of flavonoids from northeastern red propolis that we can mention: rutin, quercetin, luteolin, pinocembrin and biochanin A, formononetin, daidzein, liquiritigenin, pinobanksin, dalbergin (neoflavonoids) (Park and et al (2008) 5 (4) 435-441).

Researchers studying Marechal Deodoro-Alagoas mangrove red propolis have identified the crisino and quercetin flavonoids, new isoflavonoids in the Alagoas red propolis, vestitol, 2'4'dhydroxymethoxy flavan, isoprenylated benzophenones, and ferulic ethers. methoxyheugenol, methylhekenol, guiacol, butanedioic acid dimethyl esters, hexadecanoic acid methyl esters (Alencar et al. (2008) 5 (3) 313-316).

In addition, researchers have identified 14 substances in the Alagoas mangrove red propolis, including triterpenes (cycloartenol, lupeol, elemicin,? -Amyrin and p-amyrin), phenolic acids (anethole, eugenol and metileugenol), isoflavonoids (isosativan, medicarpine) ( Vassya Bankova et al (2006) 3 (2) 249-254).

With a view to the discovery of molecules and their therapeutic targets for cure of diseases, red propolis has been subjected to several detailed studies on its biological activities. The presence of various flavonoids and phenolic acids in propolis extracts and tinctures show that these substances act synergistically in cytostatic / cytotoxic, anti-inflammatory, healing, antimicrobial and antioxidant action. Thus, the cocktail of combined substances, even at low concentrations, will promote a potent action against pathogens.

Microencapsulation The development and production of herbal and opotherapeutic drugs have been regulated in the country with resolutions that establish quality control methodologies for these products. Phytotherapeutic and opotherapeutic standardization studies advocate trials to ensure the quality of these drug classes from collection, phytochemical screening, qualitative and quantitative determination of phytochemical markers, authenticity testing, processing, production, stability study, validation of analytical methodologies. , packaging and product quality control.

Development of new drug delivery or functional (nutraceutical) food delivery systems (Lira, CR G; et al., Rev. Bras. Farm.,. (2009) 90 (1): 45-49) in certain therapeutic targets is a need for therapeutic success or to demonstrate physiological health benefit in disease prevention, and is as important as the discovery of molecules with a particular therapeutic action. Thus, in the present invention different formulations for pharmaceutical, nutraceutical or veterinary use have been devised in the solid form for oral use.

In the development of new drug delivery systems or substances with functional activity (nutraceutical) it is extremely important to choose pharmaceutical excipients with inert properties (without pharmacological or therapeutic action), being essential in a pharmaceutical / food composition, as they have characteristics that ensure stability to composition, facilitate administration, promote the release of active substances from the chemical-pharmaceutical matrix thus promoting bioavailability and consequently pharmacological action, as well as consumer acceptability.

For the development of drug / nutraceutical release system in solid form it is necessary to have a set of excipients that will properly release the drug or active substances from a pharmaceutical or food matrix and among them are the excipients: diluents, binders, disintegrants, dyes , flavorings, sweeteners, surfactants or wetting agents, fluidizers and lubricants. From a process and production cost point of view, famacotechnical incompatibilities between active substances and excipients should be avoided. The choice and number of excipients influence drug release from the matrix and absorption into biological fluids (blood and circulatory system). Thus, the number of excipients should be reduced to the minimum possible and should contain only essential excipients (Croeley, 1999; Robertson, 1999).

Some pharmaceutical excipients may generate negative influences on the pharmaceutical or nutraceutical composition. Diluents are generally the most widely used excipients in a composition and their choice should be well defined to avoid problems of drug-excipient or excipient-excipient incompatibility, reduce the stability of the active substance (s). ), composition as well as toxicity problems. Classic examples are mannitol and sorbitol diluents which may reduce drug absorption, reduce intestinal peristalsis, cause hyperosmolarity and hypersensitivity problems (AD Adkin, SS Davis, RA Sparrow, PD Huckle, JR Wilding, Br. J. Clin. Pharmacol. 39 (1995) 381-387), (DA Adkin, SS Davis, RA Sparrow, PD Huckle, JR Wilding, J. Pharm. Sci. (1995) 84 1405-1409), (E. Jantratid, S. Prakongpan, JB Dressman, GI Amidon, E.Junginger, K.K Midha, MD Barends; Journal of Pharmaceutical Sciences, (2006) 95 974-984). Microencapsulation techniques are used by the pharmaceutical and food industries to solve technological bottlenecks among them: protecting the active substance (s) against extrinsic agents (moisture, light, oxidation) increasing the shelf life of the product , prevent loss of volatile substances, promote controlled release of active substances, as well as improve specific flow characteristics of powders during specific tablet compression processes or in the filling of gelatin capsules. The development of solid microencapsulates can be achieved by spray-drying technique and can solve some industrial problems in the pharmaceutical and food field due to the insoluble nature of some drugs or the presence of complex matrices containing various substances with different solubilizing properties. which may hinder absorption processes and consequently therapeutic effect.

In the microencapsulation process the pharmaceutical excipients are suitably chosen to provide a suitable proportion stable coating wall that promotes the encapsulation of the substances of interest in a coating matrix forming, depending on particle size, microparticles or even nanoparticles.

A polymeric nanoparticles formulation of propolis (nanofood propolis) was developed using cross-linked micellar aggregates of N-isopropylacrylamide (NIPAAm) with N-vinyl-2-pyrrolidone (VP) and poly (ethylene glycol) monoacrylate (PEG-A) (Kim, Dong-Myung, Lee, Gee-Dong, Aum, Seung-Hyun, Kim, Ho-Jun, Biological & Pharmaceutical Bulletin, (2008) 31, 1704-1710). It is important to highlight that the use of the matrix system proposed by Kim and collaborators for the formation of propolis microencapsulates presents as a major disadvantage the high cost for the acquisition of excipients. In contrast, the invention proposed herein utilizes low cost excipients, making microencapsulates and pharmaceutical compositions using them more economically accessible. Chinese patent CN101869234 demonstrates the preparation of propolis nanoemulsions containing ethyl acetate and tween. The preparation of microencapsulates using the CN101869234 matrix system has as a major disadvantage the high toxicity of ethyl acetate for human consumption. If ingested, ethyl acetate presents a potential risk of intoxication such as loss of meaning, nausea, vomiting, diarrhea, dizziness and drowsiness. KR20100078349 discloses propolis nano emulsion preparations using β-cyclodextrin excipient. Importantly, the use of the matrix system with? -Cyclodextrin for formation of propolis microencapsulates makes the product costly, which is disadvantageous in market terms. This disadvantage is potentiated when comparing the use of excipients such as starch, gelatin and aerosil as proposed herein. KR20090075395 claims propolis nanoparticles for parenteral use containing the excipients N-isopropylacrylamide, N-vinyl-2-pyrrolidinone (VP) and poly (ethylene glycol) acrylate (PEG-A). It is important to highlight that the use of excipients proposed by the coherent patent in question for the formation of propolis microencapsulates presents as a major disadvantage the high cost for the acquisition of these products.

Additionally, KR20080089723 discloses the preparation of gelatin-containing propolis powder for preparing toothpaste and floss. The use of propolis for dental purposes is widely described in the prior art. The matrix system proposed by the present document is different from that proposed by the present invention, which deals with the microencapsulated matrix system for use in gastroresistant capsules / tablets.

Propolis microencapsulates containing a modified starch (starch octenyl succinate) and gum arabic were obtained by Spray-Dryer technique (Silva FC, Thomazini M1, Alencar SM and Favaro-Trinity CS, XVII th International Conference on Bioencapsulation, Groningen, Netherlands ; September 24-26, 2009). The use of this matrix system to form propolis microencapsules presents as a major disadvantage the high cost for the acquisition of starch octenyl cuccinate in relation to the pregelatinized starch proposed in this document.

Nayan Roy's research (Colloids and Surfaces B: Biointerfaces 76 (2010) 317–325) has incorporated extracts of Indian propolis into a matrix containing gold and silver. Such products have high added value, making the value of products derived from this type of microencapsulated more expensive.

Process of obtaining propolis components in the dry state using gelatin containing propolis solution from the city of Maringá (Paraná) were prepared by spray-drying. Optimization of spray-drying conditions and proportions of gelatin and mannitol have been investigated (M.L. Bruschi, M.L.C. Cardoso, M.B. Lucch.esi, M.P.D. Gremião, International Journal of Pharmaceutics (2003) 264, 45-55). It is important to highlight that the use of different types of excipients allows different mechanisms of release of the active substances of the pharmaceutical matrix (microencapsulated), besides different physiological actions. Some pharmaceutical excipients may generate negative influences on the pharmaceutical or nutraceutical composition. Classic examples are mannitol and sorbitol diluents which may reduce drug absorption, reduce intestinal peristalsis, cause hyperosmolarity and hypersensitivity problems. Thus, it is evident that the microencapsulates proposed in the present patent document have outstanding advantages over the state of the art, by the use of suitable excipients for the release of the active elements contained in propolis. Brazilian patent application PI05001757 uses only two excipients (gelatin and mannitol) and only demonstrates the process of preparing an intermediate matrix containing propolis extracts without disclosing the origin of the propolis used. Importantly, the MPV claimed in this document presents innovative characteristics in relation to the microencapsulates of PI0500175-7, presenting a higher percentage of propolis extract in the composition (25 to 55%) compared to that document, which presents only 10 to 12% This reduces the cost of producing microencapsulates with lower amounts of excipients in the composition, as well as developing compositions containing a wide range of standard red dye concentration. Additionally, the MPV production process proposed in the present patent document can be cold prepared by the sol-gel dispersion method, containing a ternary system with three excipients (gelatin, pregelatinized starch and aerosil), having dispersing properties and also nonstick. The emulsification preparation process was carried out at a controlled temperature of 37 ° C to avoid problems of structural polymorphic changes of gelatin with loss of emulsifier functionality.

None of the disclosed patent documents disclose a dissolution method for assessing the degree of release of the active substances in the compositions. In addition to the characteristics already described, comparing the microencapsulated obtained in the state of the art with those proposed by the present patent document, no microencapsulated quoted in the state of the art present as a bioactive substance the red propolis of the mangroves of Alagoas.

Accordingly, the embodiments of the invention described herein have considerable advantages over the state of the art. The invention proposed herein seeks to fill a technological gap in the application of red propolis as an active element of pharmaceutical compositions for the treatment and prevention of diseases. Thus, we propose microencapsulates of red propolis, compositions using MPV, processes for obtaining MPV and uses of these products to prepare cytostatic / cytotoxic, anti-inflammatory, scarring, antimicrobial and antioxidant drugs.

It should be noted that the use of red propolis is a key feature in innovation in terms of propolis technologies, since red propolis has a characteristic composition, which will play a major role in the activity of microencapsulates and other pharmaceutical compositions, for prevention and treatment. of diseases. The main advantage of the microencapsulates and pharmaceutical composition proposed in the present invention, compared to the state of the art, is the use of compounds in an intermediate bioactivating coating matrix containing stable, inexpensive, non-toxic inert pharmaceutical excipients with rheological characteristic for solids. gastro-resistant release agents and an external matrix containing usual pharmaceutical excipients. Thus, the composition disclosed herein can be used to solve semi-industrial and industrial technical problems in the food and pharmaceutical fields, as well as some therapeutic applications.

SUMMARY OF THE INVENTION The present invention provides red propolis microencapsulates composed of red propolis tincture, diluent, flow promoter and dispersant or emulsifier. The invention also deals with the process of obtaining the microencapsulated by the Spray-Dryer technique. Additionally, the present invention provides a composition comprising the microencapsules in solid formulations, preferably in the form of gastroresistant gelatin capsules. The disclosed composition comprises i) a core containing red propolis active substance (s) combined with pharmaceutical excipients; ii) an intermediate layer of core coating that may retard the release of the active substance (s) contained in the core and which is pH dependent; iii) an outer layer responsible for the lubrication, dilution, flowability, wettability and breakdown / dissolution processes of the intermediate layer and inner core. Further, the invention addresses the process of preparing the composition by performing physical mixtures with the red propolis microencapsulates to ensure content and uniformity of content and encapsulation. Finally, uses are presented for the microencapsulates and the composition in question.

BRIEF DESCRIPTION OF THE DRAWINGS / DRAWINGS The embodiment of the invention, together with further advantages thereof can be further explained and understood by reference to the accompanying drawings and the following description: The attached Figure 1 presents a simplified flowchart of the main steps of the process of obtaining MPV. The attached Figure 2 shows a simplified flowchart of the main steps in the process of obtaining MPV-containing pharmaceutical compositions. The attached Figure 3 presents the Electron Scan Photomicroscopy of the Microencapsulated A, B, C and D of Alagoas red propolis. Photomicrographs Al, Bl, Cl and Dl 500-fold magnification (50gm scale); A2, B2, C2 and D2 1000x magnification (10pm scale) and A3, B3, C3 and D3 2000x magnification (10pm scale}).

DETAILED DESCRIPTION OF THE INVENTION

The products, processes, and uses described in the present invention may be further detailed and understood by reference to the figures herein and the following description: Red Propolis Microencapsulates (MPV) The microencapsulates disclosed herein differ from those already described in the art. technique because the concentrations of flavonoids, isoflavonoids, chalcones and phenolic acids are differentiated due to the specificity of red propolis.

The MPV, in addition to tincture with the active principles of interest, obtained from red propolis, present in their formulation the usual pharmaceutical excipients with different functionalities: active compound (tincture), diluents, flow promoter / non-stick, dispersant / emulsifier.

In one embodiment of the present invention, a microencapsulated MPV A, has a composition of: standardized red propolis hydroalcoholic tincture from the bioactive extract in a proportion of 5 to 95%, preferably 20 to 60%; diluent in a proportion between 5 and 95%; dispersant in a proportion between 5 and 95%; and flow promoter in a ratio of 0.01 to 5%.

Another embodiment of the proposed invention, a microencapsulated MPV D, has the following composition: standardized hydroalcoholic tincture from bioactive extract in a proportion of 5 to 95%, preferably 20 to 60%; diluent in a proportion between 5 and 50%; emulsifier in a proportion from 5 to 60%, preferably from 20 to 45%; and a flow promoter of between 0.01 and 5%.

In both embodiments of the invention, in both MPV A and MPV D, the dispersing system is characterized by being a ternary system composed of gelatin, pregelatinized starch and aerosil.

Red propolis microencapsulates (MPV A and MPV D), as they consist of aerosil excipients in the composition, exhibit non-stick properties that reduce contact of the intermediate coating matrix with the preparation vessel walls avoiding aggregate formation of gelatin and starch excipients and consequently avoiding variations in content uniformity of the MPV obtained.

Also, it is important to highlight that the microencapsules proposed in this document are innovative because they have a higher percentage of propolis extract in the composition (25 to 55%), compared to the state of the art, which has lower concentrations. This reduces production costs with fewer excipients in the composition, as well as developing compositions containing a wide concentration range of the standard red propolis tincture. MPV Obtaining Process

The steps for obtaining MPV include preparation of crude extract from red propolis, preparation of standardized hydroalcoholic tincture from crude extract of red propolis, preparation of sol-gel and emulsifier containing red propolis tincture systems, and drying of the systems. sol-gel and emulsifier containing red propolis tincture, forming the MPV (Figure 1). - Preparation of Red Propolis Crude Extract A 100 g sample of propolis is subjected to the maceration extraction process in 400 to 750 liters of alcohol 60 to 90 ° GL, preferably 70 to 80 ° GL, for a period of 12 to 48 hours. hours The extraction process is repeated until complete extraction of the active substances. Alternatively, percolation extraction method may be used in place of maceration. Optionally to accelerate the extraction, the material can be placed in an ultrasonic bath for complete extraction of the active substances in a period of 30 minutes to 3 hours, or it can be subjected to reflux extraction process in a soxhlet apparatus for a period of 2 to 10 hours.

After complete extraction of the active substances, the material is concentrated in a rotary evaporator, coupled to a vacuum pump, using a rotation speed of 20 to 120 rpm, in a water bath in a temperature range of 35 to 55 ° C and pressure between 400 to 700 ranHg. Alternatively, it may be concentrated using a water bath at 37 ° C to 42 ° C.

A dark solid mass will be obtained with a solvent percentage of 5 to 35%, being called crude red propolis extract. The crude extract preferably should have a solvent percentage of 5 to 12%. - Obtaining the standard hydroalcoholic tincture The bioactive extract of red propolis, obtained in the previous step, is subjected to washing with absolute ethanol, followed by evaporation of this solvent 3 times in order to eliminate the presence of toxic solvent in the formulation. Red propolis crude extract (50 to 75g) with the minimum percentage of solvent (5 to 12%) is used for preparation of standardized red propolis hydroalcoholic tincture in a concentration between 5% to 30% (w / v), preferably between 10 to 30% (w / v) and more preferably between 15 and 25% (w / v) using ethanol: distilled water (60:40, v / v) or (70:30, v / v) solvent system or (80:20, v / v) or (90:10, v / v), preferably between (70:30, v / v) or (80:20, v / v), with the aid of shaking in an ultrasonic bath for 20 minutes until complete solubilization. No precipitation is observed after more than 24 hours.

Optionally, standardized red propolis hydroalcoholic tincture at a concentration preferably between 20% and 30% (w / v) may be subjected to hexane partitioning, followed by the addition of chloroform to remove some interferents, including waxes and fatty material and This procedure will depend on the initial quality of red propolis obtained in natura. The chloroform extract obtained in this partitioning process (50g to 60g) is concentrated in rotary evaporator and used to obtain enriched chloroform extract of red propolis, which can also be called bioactive extract of red propolis. The bioactive extract of red propolis, obtained in the previous step, is subjected to washing with absolute ethanol 3 times, followed by evaporation of this solvent in order to eliminate the presence of the toxic solvent in the hydroalcoholic dye of red propolis. Standardized red propolis hydroalcoholic dyes are then prepared in a concentration preferably between 15% to 25% (w / v) to obtain MPV. - Preparation of sol-gel and emulsifier systems containing red propolis tincture For preparation of sol-gel and emulsifier systems containing red propolis tincture the usual pharmaceutical excipients with different functionalities are used: active compound (standardized hydroalcoholic tincture), diluents, flow promoter / non-stick, dispersant / emulsifier.

For preparation of sol-gel and emulsifier systems containing red propolis tincture, a proportion between 5 and 30% of the standard red propolis hydroalcoholic tincture, preferably between 15 and 25% is used. This tincture is reserved for later incorporation into the dispersed sol-gel system and the emulsion system.

The sol-gel A system preparation adjuvants containing tincture of red propolis are added in a container containing 450 mL of one pot mixture. Next, the standard red propolis hydroalcoholic tincture (150 mL) is incorporated into the sol-gel dispersed system with ethanol: water (10:90, v / v) or (20:80, v / v) or ( 30:70, v / v} or (40:60, v / v) or (50:50, v / v) or (60: 40, v / v) or (70:30, v / v) or ( 80:20, v / v), preferably between 20 and 45 ° GL and more preferably 35 ° GL containing the adjuvants at room temperature between 25 and 30 ° C. The dispersing system is comprised of ternary system (gelatin, pre-starch). gelatinized and aerosyl) which remains as an unstable dispersed system which was decanting in the absence of agitation.The system remains under constant agitation to maintain a gel state, as when the agitation process is interrupted or remains at rest, the system is converted to a sol dispersion.This property was obtained thanks to the thixotropic properties of the dispersant (aerosil) .In addition, aerosil promoted better dispersion of the standardized hydroalcoholic dye. during preparation of the microencapsulates. Another advantage of these aerosil containing compositions (MPV A and MPV D) is their non-stick property preventing loss of excipients that were deposited on the walls of the preparation vessel during agitation.

The MPV D preparation adjuvants are prepared in another container with 550 mL of ethanol: water (30:70, v / v) or (20:80, v / v) or (10:90, v / v ) or (02:98, v / v), preferably between (02:98, v / v) to (10:90, v / v) with the aid of a stirrer. Gelatin is added and solubilized, followed by the addition of pregelatinized starch and finally aerosil was added. Then the standardized hydroalcoholic tincture (150 mL) is slowly incorporated into the emulsifier system containing ethanol: water (10:90, v / v) or (15:85, v / v) or (20:80, v / v) or (30:70, v / v) or (40:60, v / v) or (50:50, v / v) and preferably between 10 and 40 ° GL containing adjuvants and more preferably 15 ° GL under preferably a temperature between 30 ° C and 45 ° C and more preferably at 37 ° C. The emulsifier system is composed of a ternary system (gelatin, pregelatinized starch and aerosil) which remains as a stable emulsion under agitation or at rest. - Drying of sol-gel and emulsifier systems containing red propolis tincture using Spray-Dryer Sol-gel and emulsifier systems containing red propolis tincture are then subjected to spray-drying process under constant agitation using a needle-injecting needle. 1 mm. Spray-Dryer has the following drying conditions: inlet temperature 120 to 180 ° C, outlet temperature 90 to 130 ° C, pumping flow 0.3 L / hr and 4.50 liter air flow. Thus, sol-gel and emulsifier powders containing red propolis tincture are obtained and these powders are microencapsulated from red propolis. The sol-gel system containing the red propolis tincture after the Spray-Dryer generates MPV A and the emulsifying system containing the red propolis tincture after the Spray-Dryer generates the MPV D. It is important to highlight that, The process proposed herein for the preparation of the microencapsulates may be carried out at room temperature by the sol-gel dispersion method. In addition, the emulsification preparation process was prepared at a controlled temperature of 37 ° C to avoid problems of structural polymorphic gelatin modifications with loss of emulsifier functionality.

MPV-containing pharmaceutical compositions

The MPV obtained can be used in various pharmaceutical compositions, preferably as solid formulations such as powders, tablets, gelatin capsules, among others.

The pharmaceutical compositions proposed herein consist of compositions, preferably in the form of hard capsules or tablets, which contain (i) a core containing red propolis active substance (s) combined with pharmaceutical excipients; ii) an intermediate layer of core coating that may retard the release of the active substance (s) contained in the core and which is pH dependent; (iii) an outer layer responsible for the lubrication, dilution, flowability, wettability and disintegration / dissolution processes of the intermediate layer and inner core. It is important to highlight that in the pharmaceutical compositions proposed here the MPVs constitute the core (i), with the active elements of the red propolis tincture, and the intermediate layer (ii), composed of the MPV constituent ternary systems.

As far as the core of these pharmaceutical compositions is concerned, it is formed by active substances from the standardized red propolis tincture, which are present in MPV. The intermediate core coating layer consists of a natural hydrophilic polymer or combination of more than one natural hydrophilic polymer (s). In a distinct embodiment of the invention, the intermediate layer may also be prepared by conjugating one or more natural hydrophilic polymer (s) to one or more synthetic hydrophilic polymer (s). Auxiliary pharmaceutical excipients, such as flowability and anti-sticking agents, binders and diluents, used in the intermediate layer, should be in the intermediate layer with varying percentages between 0.05% and 95% according to their functionality in the matrix. Pharmaceutical In terms of proportion of the intermediate layer in the final pharmaceutical composition, the core coating intermediate layer should have percentages between 5% and 95%, and preferably between 30 and 75% of the total weight of the composition. The outer layer of the composition, on the other hand, consists of pharmaceutical excipients with various functionalities, including diluents, lubricants, flow-promoting agents, wetting, disintegrating, super-disintegrating agents, whose dissolution of the composition is promoted by the pH change after the passage of particulate material through pylorus. The gastroresistant release mechanism occurs when the composition containing natural or synthetic polymers with weak or esterified acid-insoluble gastric (stomach) function with a pH <4.0 leave the stomach and enter the duodenum through the pylorus to reach pH> 6.0 dissolve. As it travels along the small intestine where the pH rises to 7 to 8. In terms of percentages of pharmaceutical excipients in the outer layer, these may represent between 0.05% and 100% of the total weight of the outer layer, according to its functionality in the pharmaceutical matrix. In terms of proportion of the outer layer in the final pharmaceutical composition, the outer layer of the composition should have percentages between 0.01% and 50%, preferably between 1 and 15% of the total weight of the composition. The pharmaceutical composition disclosed herein may or may not have a gelatin shell, forming hard or soft capsules. Thus, in one embodiment of the invention proposed herein, gelatin shell capsules of size 000 to 03 can be obtained with dark coloration.

Further, the pharmaceutical excipients used in the composition may be of various types and in various formulations, with compositions varying between the various types of excipients. Excipients used in the compositions include natural or synthetic hydrophilic polymers such as gelatin, cornstarch, pregelatinized starch, xanthan gums, guar gum, arabic gum, magnesium stearate, talc, titanium dioxide, polyvinylpyrrolidone, carbomers, polyvinyl alcohol, polaxamers, colloidal silicon dioxide, stearic acid, sodium starch glycolate (explosol), sodium lauryl sulfate, cellulose, microcrystalline cellulose, and cellulose derivatives such as CMC, methylcellulose and hydroxypropylethylcellulose.

Process of Obtaining MPV-containing Pharmaceutical Compositions

The steps for obtaining MPV-containing pharmaceutical compositions can be identified by the following major steps: obtaining red propolis microencapsulates, performing physical mixtures to ensure content and uniformity of contents and preparing the red propolis capsules in hard gelatin shell (Figure 2 ).

The MPV obtained can be used in various pharmaceutical compositions, preferably as solid formulations such as powders, tablets, gelatin capsules, among others.

Preferably, for the preparation of hard gelatin capsules containing MPV, the microencapsulates are subjected to mixing by the geometric dilution method with the disintegrant or super disintegrant type excipients in a ratio of 0.5 to 15% (preferably from 1 to 10%, and most preferably from 2 to 7%) in a time from 5 to 120 minutes, more preferably from 25 to 65 minutes. Next, the microencapsulates are mixed with wetting agent type excipients in a ratio of 0.5 to 10%, preferably from 1 to 5% and more preferably from 2 to 3.5%, over a period of 5 to 120 minutes, more preferably. between 25 and 65 minutes. The mixture was finalized by adding a lubricant-like excipient in a ratio of 0.1 to 6%, preferably 0.3 to 4%; more preferably between 0.75 and 2% over a period of 2 to 10 minutes, more preferably between 3 and 7 minutes. Encapsulation can be performed on hand instruments (encapsulation board), semi-industrial and industrial instruments, adjusting the MPV mass in terms of the standard red propolis tincture. Variations between 90 and 110% of the standard tincture content of red propolis. Variations between 85 and 115% in terms of content uniformity of standardized red propolis tincture. Variations between 75 and 115% of dissolution percentage for the major flavonoids of the standard red propolis tincture.

Uses of MPV and Pharmaceutical Compositions Containing them In the face of such biological activities as red propolis microencapsulates, they can be used in a wide range of industrial sectors such as cosmetics, pharmaceuticals and food. The use of propolis as an active ingredient in the prevention and treatment of various diseases is already widely described. However, red propolis has a different composition from other propolis already widely described in the state of the art, which indicates varied physiological activities. Red propolis has antibacterial biological activities for the bacterial strains of Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC27853, with inhibition of bacterial growth when submitted to both red propolis and microencapsulated dyes.

Due to their proven antibacterial biological characteristics, microencapsulated and microencapsulated pharmaceutical compositions obtained by the proposed processes can be used for: treatment of broad spectrum bacterial infections; treatment of inflammatory processes of bacterial origin; treatment of decubitus wounds in patients with diabetic foot; treatment of inflammatory and infectious processes in general.

The uses described here are new to the state of the art, in terms of activities and actions of red propolis.

Thus, the embodiments of the invention described herein present a breakthrough in the state of the art as they allow the production of MPV and pharmaceutical compositions containing them in an economically viable manner. Thus, MPV obtained through the proposed process can be used, alone or in composition with other products, in various industrial sectors, such as food, cosmetics and pharmaceuticals.

EXAMPLES

The MPV evaluations obtained using the proposed process demonstrated the obtention of an adequate, pure and preserved activity product. The results obtained are represented in the tables and figures indicated. EXAMPLE 1 - Microencapsulated Scanning Electron Microscopy Assays (MPV A, MPV B, MPV C and MPV D) The scanning electron microscopy assay was used to prove that the particles obtained by Spray-Dryer are microparticulate. , also called microspheres or also called microencapsulated. The results show that the particles have size between 50 pm and 10 pm. The morphology of the Red Propolis Spray-Dryer (MPV) microencapsulates were evaluated with a scanning electron microscope (SEM). The powders were attached to a 40 mA gold-coated double-sided adhesive tape under vacuum and analyzed with a JEOL JSM-6460 scanning electron microscope (Tokyo, Japan). SEM was operated at 15 kV with increases of 100, 200, 500, 1000, 2000 and 5000 times (Figure 3).

Photomicrographs of MPV B which contained only a single adjuvant revealed large microparticles,> 50pm (Figure 3B2), an irregular and non-uniform surface for MPV B. MPV C had a spherical, smooth, agglomerated, non-uniform surface with brittle appearance. microparticles with size between 50pm and 10pm (Figure 3C1 and 3C2).

The microphotographs of the MPV A and D microparticles showed small particles with a spherical and very smooth, non-agglomerated, uniform surface. Photomicrographs revealed spherical particles with particle size between 50 and 10pm. Microencapsulates can be classified as red propolis microspheres, regardless of the method of preparation (dispersion or emulsification), in these proportions and type of adjuvants used in the composition of MPV. EXAMPLE 2 - CIM Antimicrobial Assay for Red Propolis Dye and Microencapsulated For the analyzes the following bacterial strains were used: BAC 1 - Staphylococcus aureus ATCC 25923; BAC 2 - Pseudomonas aeruginosa ATCC27853, by the diffusion method on Agar.

The Minimum Inhibitory Concentration (MIC) was determined for the standard red propolis tincture (Table 1) and for the MPV A and D after spray-drying process in order to verify if there was loss of biological activity after processing (Tables 2 and 3).

Table 1 - Inhibitory Concentration Minimal standardized hydroalcoholic tincture of red propolis using agar diffusion method.

Table 2 - Minimum inhibitory concentration of MPV A containing standardized red propolis tincture using agar diffusion method. Table 3 - Minimum inhibitory concentration of MPV D containing standardized red propolis tincture using agar diffusion method.

Claims (35)

1. Microencapsulated characterized by comprising active ingredients derived from red propolis; Microencapsulated according to claim 1, characterized in that the active ingredients are derived from red propolis extracts and have a final percentage of red propolis extract between 25 and 55%; Microencapsulated according to claim 1, characterized in that it comprises: a) standardized hydroalcoholic tincture of red propolis from the bioactive extract in a proportion from 5 to 95%, preferably from 20 to 60%; b) diluent in a proportion between 5 and 95%; c) dispersant in a proportion between 5 and 95%; d) and flow promoter between 0.01 and 5%; Microencapsulated according to claim 1, characterized in that it comprises: a) standardized hydroalcoholic tincture from the bioactive extract in a proportion from 5 to 95%, preferably from 20 to 60%; b) diluent in a proportion between 5 and 50%; c) emulsifier in a proportion from 5 to 60%, preferably from 20 to 45%; d} and a flow promoter of between 0.01 and 5%; Microencapsulated according to Claims 3 and 4, characterized in that it comprises a ternary system dispersing system, preferably composed of gelatin, pregelatinized starch and aerosil; Microencapsulated according to claims 1 to 5, characterized in that it has non-stick properties; Microencapsulated according to claims 1 to 6, characterized in that it contains, among its components, compounds derived from red propolis, preferably standard red propolis tincture, for use in the treatment of bacterial infections and inflammatory processes of bacterial origin; Microencapsulated according to Claim 7, characterized in that it contains, among its components, compounds derived from red propolis, preferably standard red propolis tincture, for use in the treatment of bacterial infections and inflammatory processes, caused preferably by Staphylococcus aureus and Pseudomonas aeruginosa. ; Process for obtaining microencapsulated, comprising the following steps: a) Preparation of the crude extract from red propolis; b) Preparation of standardized hydroalcoholic tincture from the raw extract of red propolis; c) Preparation of sol-gel and emulsifier systems containing red propolis tincture; d) Drying of sol-gel and emulsifier systems and formation of red propolis microencapsulates; Process for obtaining microencapsulated according to claim 9, characterized in that, in step a), a sample of propolis is subjected to complete extraction of the active substances with alcohol 60 to 90 ° GL, preferably 70 to 80 ° GL. either by maceration or percolation, with subsequent concentration in rotary evaporator or water bath and obtaining material with solvent percentage between 5 and 35%, preferably between 5 and 12%; Process for obtaining microencapsulated according to claim 9, characterized in that, in step b), the crude extract is subjected to solvent washing, preferably absolute ethanol, with subsequent total evaporation of the solvent and solubilized in solvent ethanol: distilled water. (60:40, v / v) or (70:30, v / v) or (80:20, v / v) or (90:10, v / v), preferably between (70:30, v / v) ) or (80:20, v / v), to obtain standardized propolis red hydroalcoholic tincture in a concentration between 5% to 30% (w / v), preferably between 10 and 30% (w / v) and more preferably between 15 and 25% (w / v); Process for obtaining microencapsulated according to claim 9, characterized in that, in step c), for the preparation of ternary systems containing red propolis tincture: i) active compound (standardized hydroalcoholic tincture), ii) diluents iii) flow promoter / nonstick and iv) dispersant / emulsifier; Process for obtaining microencapsulated according to claim 9, characterized in that, in step c), for preparation of the sol-gel A system, one pot mixture is incorporated into the ternary dispersant system, gelatin, starch. pre-gelatinized and aerosil, at a temperature of 25 to 30 ° C, 450 mL of distilled water and 150 mL of standardized red propolis hydroalcoholic tincture with ethanol-water solvent (10:90, v / v) or (20:80, v / v) either (30:70, v / v) or (40:60, v / v) or (50:50, v / v) or (60:40, v / v) or (70:30, v / v) v) or (80:20, v / v), preferably between 20 to 45 ° GL and more preferably 35 ° GL; Process for obtaining microencapsulated according to claim 9, characterized in that, in step c), for preparation of emulsifying system D, the ternary dispersing system, gelatin, pregelatinized starch and aerosil are added under stirring. mL of ethanol: water (30:70, v / v) or (20:80, v / v) or (10:90, v / v) or (02:98, v / v), preferably between (02: 98, v / v) at (10:90, v / v), 150 mL of the standard hydroalcoholic tincture containing ethanol: water (10:90, v / v) or (15:85, v / v) or (20:80, v / v) or (30:70, v / v) or (40:60, v / v) or (50:50, v / v) and preferably between 10 and 40 ° GL under temperature preferably between 30 ° C and 45 ° C and more preferably at 37 ° C; Process for obtaining microencapsulated according to claim 9, characterized in that, in step d), sol-gel and emulsifier systems containing red propolis tincture are subjected to spray-drying to obtain microencapsulates. the drying conditions being preferably constant agitation, 1 mm injection needle, inlet temperature from 120 to 160 ° C, outlet temperature from 90 to 130 ° C, pumping flow 0.3 L / h and air flow 4.50 Liters / Composition, characterized in that it comprises microencapsulates of red propolis as active ingredient, alone or in conjunction with other actives; Composition according to Claim 16, characterized in that it is preferably solid formulations such as powders, tablets, capsules, among others and more preferably in the form of gelatin capsules; Composition according to Claim 16, characterized in that it comprises i) a core containing red propolis active substance (s) combined with pharmaceutical excipients; ii) an intermediate layer of core coating that may retard the release of the active substance (s) contained in the core and which is pH dependent; iii) an outer layer responsible for the lubrication, dilution, flowability, wettability and breakdown / dissolution processes of the intermediate layer and inner core; Composition according to Claim 18, characterized in that it has a gastroresistance characteristic; Composition according to Claim 18, characterized in that it has a gelatin shell or not; Composition according to Claim 18, characterized in that it comprises a core (i) composed of the active substances of red propolis, derived from the standard dye of red propolis and pharmaceutical excipients; Composition according to Claim 18, characterized in that it comprises an intermediate layer (ii) composed of pharmaceutical excipients, preferably flow-promoting and non-sticking agents, binders and diluents, in a percentage between 0.05% and 95% of the total weight of the intermediate layer. according to the functionality of the pharmaceutical excipient in the pharmaceutical matrix and having a ratio of 5% to 95%, preferably 30 to 75% of the total weight of the composition; Composition according to Claim 18, characterized in that it comprises an outer layer (iii) whose dissolution is promoted by pH change, comprising pharmaceutical excipients with various functionalities, preferably diluents, lubricants, flow-promoting agents, wetting, disintegrating, super-active agents. - disintegrants, in the percentage between 0.05% and 100% of the total weight of the outer layer, according to the functionality of the pharmaceutical excipient in the pharmaceutical matrix, and present a proportion between 0.01% and 50%, preferably between 1 and 15%. relative to the total weight of the composition; Composition according to Claims 18, 21 to 23, characterized in that it comprises pharmaceutical excipients of various types and in various formulations; Composition according to Claim 24, characterized in that it comprises a natural hydrophilic polymer or combination of more than one natural hydrophilic polymer (s) or a combination of one or more natural hydrophilic polymer (s). ) and one or more synthetic hydrophilic polymer (s) such as gelatin, maize starch, pregelatinized starch, xanthan gums, guar gum, arabic gum, magnesium stearate, talc, titanium dioxide, polyvinylpyrrolidone, carbomers, polyvinyl alcohol, polaxamers, colloidal silicon dioxide, stearic acid, sodium starch glycolate (explosol), sodium lauryl sulphate, cellulose, microcrystalline cellulose, and cellulose derivatives such as CMC, methylcellulose and hydroxypropylethylcellulose; Composition according to Claims 16 to 25, characterized in that it contains, among its components, compounds derived from red propolis, preferably microencapsulated from red propolis, for use in the treatment of bacterial infections and inflammatory processes of bacterial origin; Composition according to Claim 26, characterized in that it contains, among its components, compounds derived from red propolis, preferably microencapsulated from red propolis, for use in the treatment of bacterial infections and inflammatory processes, caused preferably by Staphylococcus aureus and Pseudomonas aeruginosa; A composition preparation process comprising the following steps: (a) making physical mixtures with the red propolis microencapsulates to ensure content and content uniformity; (a) physical mixtures by the geometric dilution method; The. 2) Physical mixtures for incorporating wetting and disintegrating excipients; a * 3) Physical mixtures for incorporating lubricating excipients; b) Preparation of red propolis capsules; Composition preparation process according to Claim 28, characterized in that, in step a) the microencapsulates of red propolis are subjected to physical mixtures, preferably for the preparation of gelatin capsules; Composition preparation process according to Claim 28, characterized in that, in step (al), the microencapsulates are subjected to mixing by the geometric dilution method with disintegrant or super-disintegrant type excipients in a ratio of 0.5 to 15%. %, preferably from 1 to 10%, and more preferably from 2 to 7%, in a time from 5 to 120 minutes, more preferably from 25 to 65 minutes; Composition preparation process according to Claim 28, characterized in that in step a.2) the microencapsulates are mixed with wetting agent-type excipients in a ratio of 0.5 to 10%, preferably 1 to 5%. and most preferably between 2 and 3.5% over a period of 5 to 120 minutes, more preferably between 25 and 65 minutes; Composition preparation process according to Claim 28, characterized in that, in step a.3) the microencapsulates are subjected to a lubricant-type excipient in a ratio of 0.1 to 6%, preferably from 0.3 to 4%. more preferably between 0.75 and 2% over a period of 2 to 10 minutes, more preferably between 3 and 7 minutes; Composition preparation process according to Claim 28, characterized in that, in step b) the encapsulation is performed by adjusting the mass of red propolis microencapsulates in terms of the standard red propolis tincture with the following variations; 90 to 110% of standard propolis red dye content, 85 to 115% in terms of uniformity of standard red propolis dye content, 75 to 115% of dissolution percentage for major flavonoids of standard propolis dye red, being encapsulation by hand or semi-industrial or industrial instruments; Use of the microencapsulate according to claims 1 to 8, characterized in that it is isolated or as an adjuvant to fight infections and inflammatory processes of bacterial origin; Use of the composition according to claims 16 to 27, characterized in that it is isolated or as an adjuvant to combat infections and inflammatory processes of bacterial origin.