Process for obtaining standardized dry propolis extract without the addition of polymers, excipients, starches, carbohydrates or additives, products and uses

Abstract

The present application relates to a process for obtaining standardized dry propolis extract without the addition of polymers, excipients, vehicles and additives, carbohydrates or starches, in which the main areas of use include the pharmaceutical, cosmetic, food, veterinary, personal hygiene, agricultural and biotechnology industries. This innovative technology provides several benefits to users of the dry propolis extract without the addition of any ingredients other than propolis itself: a minimally processed product, free of preservatives, colorants, additives, pesticides, herbicides, heavy metals, and agrochemical contaminants, with excellent bioavailability, containing bioactive compounds such as polyphenols and flavonoids, and offering ease of use in various pharmaceutical forms, dietary supplements, nutritional products and foods, while complying with the regulatory guidelines issued by official bodies such as MAPA and ANVISA.

Description

PROCESS FOR OBTAINING STANDARDIZED DRY PROPOLIS EXTRACT WITHOUT THE ADDITION OF POLYMERS, EXCIPIENTS, STARCHES, CARBOHYDRATES AND ADDITIVES, PRODUCTS AND USES Field of Application

[0001] This application relates to the use of dry propolis extract, whose main areas of application include the pharmaceutical, cosmetic, food, veterinary, personal hygiene, agricultural and biotechnology industries. State of the Art

[0002] Propolis is a resinous substance collected by bees, scientifically known as Apis mellifera, from various botanical sources, primarily for the purpose of protecting the hive from invaders and maintaining the temperature and asepsis of the hive.

[0003] Propolis has been used in traditional medicine by the Egyptians, Greeks, and many other peoples since more than 300 BC. The ancient Jews cited tzori, the Hebrew name for propolis, as a remedy.

[0004] The Egyptian people, guided by bees and learning from their behavior in the hive, particularly from how they covered invaders (who were killed inside the hive and could not be removed), used propolis and wax for embalming. Through this observation, they were inspired to create techniques for embalming human bodies.

[0005] Ancient Jews described propolis as "tzori." Biblical citations mention a balm known as "Balm of Gilead" (tzori Gilead in Hebrew), whose descriptions cite characteristics very similar to propolis. In the Judean region, near the Dead Sea, the Balm of Gilead was prepared and gained a good reputation for its medicinal properties and pleasant aroma.

[0006] Ancient Jews called modern-day propolis "tzori." The Bible mentions a balm known as "Balm of Gilead" (tzori Gilead in Hebrew), which has very similar characteristics to propolis. This balm was known as the gift that the Queen of Sheba offered to King Solomon. For approximately 1,500 years, in the Judean region near the Dead Sea, the Balm of Gilead was cultivated and gained a reputation for its medicinal properties and pleasant aroma. The production of this balm was made from the resin of several species including *P. balsamifera*, *P. nigra*, and *P. gileadensis*.

[0007] There are citations made by the Romans, in Pliny's renowned work, Natural History, describing the use of propolis to relieve tendon pain, heal ulcers, and disperse tumors.

[0008] There are reports of the use of propolis in traditional medicine, with anticancer, antibiotic, antifungal, anti-inflammatory, and antioxidant activities, to promote health and prevent diseases such as inflammatory processes, diabetes, and heart disease.

[0009] Traditional knowledge of the medicinal properties of propolis was preserved in folk medicine and continued to be widely used in apitherapy in regions of Eastern Europe, commonly referred to as "Russian penicillin".

[0010] Recently, there has been great interest in scientific studies, therapeutic properties, biological activities, pharmacology, and applications of propolis in the treatment of diseases and various clinical conditions of health imbalance, using this product produced by nature and collected by bees, with reports dating back to 300 BC. There are more than 4600 publications indexed in PubMed, demonstrating the chemical compositions of various propolis, their therapeutic, pharmacological, and biochemical properties, and their applications in the treatment of various diseases and the protection of health.

[0011] The Greeks, in their cultural practices, incorporated propolis as an essential ingredient in the creation of polyanthus, an elaborate perfume that combined propolis with other elements such as frankincense, styrax, and aromatic herbs.

[0012] Even in the Middle Ages, some sources from the 12th century mention the existence of medicinal preparations containing beeswax, which were used to treat infections in the oral and pharyngeal regions, as well as dental caries problems.

[0013] In this context, current literature describes at least 13 types of propolis, each from a different plant origin and with different properties. These include:

[0014] Green propolis, which originates from Baccharis dracunculifolia, known as field rosemary, is predominantly found in Brazil, especially in Minas Gerais. This type of propolis is rich in Artepillin C, phenolic compounds, and possesses antibacterial, antifungal, anti-inflammatory, and antitumor properties. It is commonly used in supplements to strengthen the immune system and prevent disease.

[0015] Red propolis, which comes from Dalbergia ecastophyllum, known as rabo-de-bugio, is native to the coastal areas of Northeast Brazil. Containing flavonoids and isoflavones, red propolis is known for its antioxidant, antimicrobial, and antitumor properties. It is used in health products to protect against infections and support cellular health.

[0016] Brown propolis, which is derived from various plants, including Populus spp. and other tree resins, is widely distributed, being found in Europe, North America, Asia, Oceania, and Brazil. Its composition varies, but it is generally rich in flavonoids and phenolic acids, providing antimicrobial, anti-inflammatory, and other properties. It is an antioxidant. It is commonly used in extracts and ointments for the treatment of wounds and infections.

[0017] Black propolis, which is extracted mainly from Populus nigra (black poplar) and is found in Europe and North America. Rich in flavonoids, phenolic acids, and terpenes, black propolis has antimicrobial and anti-inflammatory properties, and is frequently used in cosmetics and skincare products.

[0018] Bulgarian propolis is derived from Populus nigra and other local plants in Bulgaria. Rich in flavonoids and phenolic esters, this propolis is recognized for its antibacterial and antioxidant properties. It is used in dietary supplements and products to strengthen the immune system.

[0019] Each type of propolis offers a range of medicinal benefits, depending on its specific chemical composition. These natural compounds are widely used in health products, dietary supplements, and cosmetics to take advantage of their therapeutic properties.

[0020] The context goes back to the use of propolis in more traditional ways, such as in liquid form; however, nowadays it is possible to use it in a more modern way, such as in the form of dry extract, which allows the use of propolis through hard or soft gelatin capsules, tablets, pills, powders, candies and various others.

[0021] Dry propolis extract is a concentrated form of the product and has been increasingly studied and used in modern medicine and the dietary supplement industry. This format facilitates dosage standardization and incorporation into various products, as mentioned above.

[0022] Contemporary research has demonstrated a variety of benefits of dry propolis extract, ranging from supporting the immune system. even its antioxidant activity demonstrates its potential in both preventive and therapeutic applications.

[0023] In this context, document CN110664762A describes a solid preparation containing propolis extract. The extract preparation process involves freezing raw propolis at a specific temperature, grinding it, and then extracting it using absolute ethyl alcohol and petroleum ether. After filtration and concentration, the extract is freeze-dried and ground to obtain particles of a specific size. In the formulation of the solid preparation, the propolis extract is combined with microcrystalline cellulose, a solid dispersant (such as poloxamer or polyethylene glycol), a diluent (such as mannitol or lactose), a disintegrant (such as crospovidone), and a lubricant (such as magnesium stearate). The exact proportions of each component vary depending on the desired formulation.

[0024] Alternatively, document BR102017000088A2 describes a process for obtaining microencapsulated red propolis caseinates for immediate release, composed of a standardized hydroalcoholic tincture of red propolis, solubilized using solvents such as distilled water and ethanol, a protein encapsulant, a disintegrant, and a flow promoter. The production process involves the spray-dryer technique. The final composition is available in solid forms, such as powder and hard-release gelatin capsules, with micrometric-sized microcapsules and an immediate dissolution profile between 70 and 110% in 2 hours. Uses include nutraceutical supplements, food powders such as chocolate and coffee powders, and pharmaceutical capsules.

[0025] Similarly, document 102013010639-9 A2 describes a process for obtaining active components from dry propolis extract and / or hydroalcoholic propolis extracts for use as an active component in the preparation of pharmaceutical compositions with antifungal activity. used in the treatment of fungal infections and compounds containing the active ingredients of dry propolis extract and / or hydroalcoholic extracts of propolis and honey.

[0026] Alternatively, document 102015033039-1 A2 describes a process for obtaining propolis matrix microparticles, resulting in a dry extract with micrometric particles, and includes the obtained microparticles and the formulations containing them. The process involves grinding the propolis, macerating it with an extraction solvent, percolating the solution using the ground propolis as a filtering material, preparing mixtures of encapsulating agents with the obtained extract, and finally, spray drying the mixtures. This method results in micrometric-sized propolis matrix microparticles. In its formulation, the product contains between 20% and 40% of a mixture of polyethylene glycols (such as polyethylene glycol 400, polyethylene glycol 4000 or other PEGs, isolated or combined), in addition to between 0.1% and 10% of glycerin, propylene glycol or another humectant.

[0027] Another example is described in patent application PI 1004808-1 A2, which describes a process for obtaining propolis nanoparticles that can be combined with substances of interest, such as active ingredients and adjuvants. The process involves dissolving propolis extract in an organic solvent (fraction A) and preparing an aqueous phase that may or may not contain a solution with various types of polymers, such as acrylic acid polymers, alginates, various gums (biosynthetic, natural), starches and derivatives, esters and cellulose compounds, gelatins, hydrocolloids, hyaluronic acid, glycols, polyacrylates, polyethylene oxides, vinyl derivatives, among many others (fraction B). The fractions are mixed by dropwise mixing and homogenized by mechanical stirring, forming nanoparticles from 1 to 1000 nm. Optionally, the organic solvent can be removed and the nanodispersion dried. The resulting compositions have applications in fields such as agrochemical, pharmaceutical, cosmetic, and chemical.

[0028] Thus, it can be observed that all the cited documents use some type of solvent such as ethanol, propylene glycol and water, excipients which are defined, according to ANVISA (Brazilian Health Regulatory Agency), as "any component, other than an active substance, intentionally added to the formulation of a pharmaceutical form" and additives, which are "substances intentionally added to food with the aim of modifying its physical, chemical, biological or sensory characteristics, during the stages of manufacture, processing, preparation, treatment, packaging, storage, transport or handling".

[0029] Therefore, hydroalcoholic extracts of propolis, which contain alcohol as the main vehicle, present drawbacks to consumption and adherence to use, and are not recommended for several population groups, such as children, people with alcohol dependence, and individuals of various religions that prohibit alcohol consumption.

[0030] Furthermore, other extracts, such as propolis glycolic extract, use propylene glycol as a solvent, a chemical that can cause adverse effects, hypersensitivity, and toxicity, as reported in various literature, including "The Extra Pharmacopeia Martindale" edited by James E.F. Reynolds and studies by Kelsey and Seidel on propylene oxide-derived glycols.

[0031] Besides the drawbacks of the solvents used in the extraction process, many of these solvents remain in high concentrations in the formulations, as is the case with alcoholic propolis extract. Another problem is the unpleasant palatability, due to the pungent taste, which is not well tolerated by a considerable portion of the population. This prevents many from benefiting from the functional and therapeutic effects of this excellent natural product used for millennia.

[0032] Alternatively, in the literature and in the market for propolis extract in general, one can still find other excipients such as starch, maltodextrin, and microcrystalline cellulose, which also have several disadvantages.

[0033] Starch, although common, can compromise the stability and preservation of the product due to its tendency to absorb moisture, resulting in faster degradation. Furthermore, its limited solubility in cold water makes it difficult to prepare a homogeneous formulation, impacting bioavailability and efficacy. Starch can also interact negatively with other components, affecting the texture and effectiveness of the final product.

[0034] Cellulose, in turn, has limited solubility and can impact the texture of the product, making it more fibrous or granular, which is undesirable, especially in oral formulations. Furthermore, cellulose can interact with other ingredients, compromising the stability and quality of the product. Its ability to absorb moisture can lead to stability problems in humid environments, and complex processing can increase production costs.

[0035] Maltodextrin is highly hygroscopic, absorbing moisture from the environment and affecting the stability and quality of the product over time. Its variable solubility can make formulation homogeneity difficult. Furthermore, it can interact with other components, altering the texture, flavor, and color of the product. Maltodextrin also has a high glycemic index, which may be a concern for people with diabetes or those monitoring their sugar intake, limiting its use by a wider audience.

[0036] These disadvantages can be mitigated by choosing other, more suitable excipients that ensure the solubility, stability, efficacy, and acceptability of the propolis extract formulation, providing a better user experience.

[0037] Therefore, this application differs from the documents cited because it is a dry propolis extract that does not contain any type of excipient such as starches, maltodextrin, other added industrial polymers or additives in its production and formulation process, thus avoiding the problems mentioned above. Detailed description of the technology

[0038] The process for obtaining standardized dry propolis extracts, produced by bees, without the addition of polymers, excipients, starches, carbohydrates, and additives, is an innovative technology that follows the process described below:

[0039] Firstly, propolis producers are qualified through the selection and training of beekeepers, using documented theoretical and practical procedures, in accordance with our policy of guaranteeing and continuously improving quality. This aims to receive increasingly higher quality propolis and meet the requirements of our certifications and policies regarding quality, standardization, reproducibility, safety, and optimal results from its use.

[0040] The receipt of raw propolis from qualified beekeepers and verification of the general conditions of storage, packaging, hygiene, identification, and business compliance, prior to acceptance and forwarding for the initial selection process, which includes evaluating sensory and organoleptic characteristics such as color, odor, freshness, malleability, flavor, and foreign contaminants.

[0041] To verify and subsequently validate the results obtained in the initial selection process, we sent these samples for analytical quality control, using physicochemical and microbiological methodologies, tests to verify botanical origin, chemical markers, and other verification methods. of any undesirable contamination originating from the environment or from producer handling. Tables 1 to 6 list all the tests and parameters described and validated in our quality assurance manual, which is periodically audited and internationally certified ISO 9001 and GMP.

[0042] Following biochemical evaluations and approvals, the selected propolis is used in the production process. This standardization allows for the reproduction of similar batches, with chemical markers of botanical origin, sensory characteristics, concentration, and purity determined by the company, according to the tests described previously.

[0043] The process of standardization and reproducibility then begins sequentially, verifying through quality control methods that production was properly executed according to the standards defined for this purpose, using homogenizing equipment, reactors, and pharmaceutical-grade filters, to guarantee maximum safety and efficacy.

[0044] In a GMP-certified environment, propolis is macerated in aqueous, alcoholic, or hydroethanolic solutions at concentrations ranging from 1 to 99% w / w for 48 hours in stainless steel reactors.

[0045] The extraction process of bioactive compounds from propolis begins in a stainless steel reactor with a mechanical homogenization system, rotating at 10-50 rpm for a period of 2 to 12 days. All compounds from the propolis will be used in the composition of the final product.

[0046] Next comes the filtration process, followed by storage at a temperature of -20°C to 0°C, for preservation and subsequent removal of greasy materials, such as beeswax.

[0047] This extract is then sent back to the microbiological, physicochemical, high-performance liquid chromatography, and contaminant testing quality control laboratory for standardization verification and validation of all quality and safety parameters as presented in the [reference to documentation / documents]. Tables 1 to 5. Only after approval of all tests established in our quality management and assurance system does the subsequent step proceed. Table 1. Physicochemical analysis of raw propolis. Table 2. High-performance liquid chromatography analysis of raw propolis. Table 3. Undesirable contaminants in raw propolis (antibiotics) Table 4. Microbiological control in raw propolis. Table 5. Undesirable contaminants in raw propolis (heavy metals).

[0048] The standardized extract is concentrated to remove hydroethanolic vehicles to the limit of their structural bonding, or as needed for the desired final product concentration. This process is carried out in proprietary equipment that provides negative pressure in the manufacturing process, in a vacuum system, with variable temperature. 30°C to 75°C, and a vacuum of 300 to 800 mmHg, until standardization of the markers present is achieved.

[0049] The final step in obtaining the novel process and product consists of micronizing the concentrated product as described above, without the addition of any ingredients, such as the excipients described in other patents, as is common in the market. These products often contain various ingredients such as starch, maltodextrin, microcrystalline cellulose, corn and cassava starch, gelatin, various polymers, and others. In contrast, our product, through our innovative process, uses only structurally suitable propolis to contain its active ingredients.

[0050] Thus, the present innovation presents a process that consists of using only the propolis itself to produce its dry extract, preserving its entire composition and optimally its activities, although it can be in varying concentrations according to the final application needs of the dry extract.

[0051] In this invention, the dry propolis extract can be standardized in concentrations ranging from 1 to 100% w / w of active compounds extracted from both aqueous and alcoholic or hydroethanolic fractions.

[0052] At the end of the process, the solvents are properly removed to preserve quality and sensory characteristics, without the addition of any other ingredients. The product is then sent back for physicochemical and microbiological quality control, high-performance liquid chromatography, and UV-Vis chromatography for phenolic compounds and flavonoid compounds, as specified in Tables 6 and 7. Table 6: Dry extracts of propolis and their specifications Table 7: Microbiological control of dry propolis extracts

[0053] This innovative invention provides several benefits to users of dry propolis extract without the addition of any ingredients other than propolis itself. Propolis: a minimally processed product, with no ingredients other than propolis, free of genetically modified substances as is the case with most corn-derived starches, without dyes, free of microbiological contaminants, heavy metals, agrochemical contaminants such as fungicides, insecticides and herbicides, without preservatives, rich in flavonoid polyphenols, with excellent bioavailability, easy to use in various pharmaceutical forms, food and nutritional supplements, and foods, complying with the regulatory guidelines of official regulatory bodies such as MAPA and ANVISA. Designs

[0054] Figure 1 shows the botanical origin of propolis, with (A1) being the chromatogram of the standard solution of Baccharis dracunculifolia and (A2) the chromatogram of the raw propolis sample of Baccharis dracunculifolia, batch PG0220.2, visualized at a wavelength of 336nm. In (B1) is shown the chromatogram of the standard solution of Baccharis dracunculifolia and (B2) the chromatogram of the raw propolis sample of Baccharis dracunculifolia, batch PG0220.2, visualized at a wavelength of 254nm.

Claims

PROCESS FOR OBTAINING STANDARDIZED DRY PROPOLIS EXTRACT WITHOUT THE ADDITION OF POLYMERS, EXCIPIENTS, STARCHES, CARBOHYDRATES AND ADDITIVES, characterized by comprising the following steps: (a) to carry out an evaluation of the sensory and organoleptic characteristics of propolis, such as color, odor, freshness, malleability, taste and foreign contaminants; (b) to verify and subsequently validate the result obtained in the process described in step “a”, perform tests on the samples for analytical quality controls, with physicochemical and microbiological methodologies, tests to verify the botanical origin, chemical markers, verification of any undesirable contaminations, originating from the environment, or from the handling by the producers; (c) Macerate the propolis approved in steps “a” and “b” in aqueous, alcoholic or hydroethanolic solution; (d) perform the extraction of the bioactive compounds from the propolis extracted in step “c” in a reactor with a mechanical homogenization system; (e) filter the product obtained in step “d” and store the extract for preservation and subsequent removal of fatty materials; (f) perform microbiological, physicochemical, high-performance liquid chromatography and contaminant quality control; (g) Concentrate the standardized extract obtained in step “f” under vacuum in a negative pressure system, and (h) perform micronization of the concentrated product without the addition of any ingredient, and the resulting dry extract presents the following marker components in mg / 100g: 0.015 to 840 Coumaric Acid, 0.03 to 150 Rutin, 0.005 to 270 Pinobanksin, 0.002 to 130 Quercetin, 0.010 to 540 Kaempferol, 0.008 to 4 Apigenin, 0.002 to 140 Pinocembrim, 0.011 to 5500 Pinobanksin-3-acetate, 0.004 to 230 Chrysin, 0.001 to 60 Galangin, 0.011 to 580 Tectocrisin, 0.021 to 10500 Artepillin C, 0.0078 to 3900 Bacarin, 500 to 25000 Compounds Phenols, 250 to 25000 of Flavonoid Compounds and 0.0018 to 12800 derivatives of Caffeoylquinic Acid.

2. PROCESS FOR OBTAINING STANDARDIZED DRY PROPOLIS EXTRACT, according to claim 1, characterized in that the maceration of step “c” is carried out in aqueous, alcoholic or hydroethanolic solution in concentrations ranging from 1 to 99% w / w, for a period of 4 to 48 hours.

3. PROCESS FOR OBTAINING STANDARDIZED DRY PROPOLIS EXTRACT, according to claim 1, characterized by the homogenization of step “d” being carried out at a rotation speed of 10 to 50 rpm, for a period of 2 to 12 days.

4. PROCESS FOR OBTAINING STANDARDIZED DRY PROPOLIS EXTRACT, according to claim 1, characterized in that the storage of step “e” is carried out at a temperature of -20°C to 0°C, for preservation and subsequent removal of fatty materials, such as beeswax.

5. PROCESS FOR OBTAINING A STANDARDIZED DRY PROPOLIS EXTRACT PRODUCT, according to claim 1, characterized by the concentration of step “g” being carried out in equipment that provides negative pressure, in a vacuum system, with a temperature varying from 30°C to 75°C and a vacuum of 300 to 500 mmHg until the hydroethanolic vehicles at the limit of their structural bonding are removed.

6. STANDARDIZED DRY PROPOLIS EXTRACT, according to the process defined in claims 1 to 5, characterized by not containing added polymers, excipients, additives, starches, carbohydrates and presenting the following marker components in mg / 100g: 0.015 to 840 of Coumaric Acid, 0.03 to 150 of Rutin, 0.005 to 270 of Pinobanksin, 0.002 to 130 of Quercetin, 0.010 to 540 of Kaempferol, 0.008 to 4 of Apigenin, 0.002 to 140 of Pinocembrim, 0.011 to 5500 of Pinobanksin-3-acetate, 0.004 to 230 of Chrysin, 0.001 to 60 of Galangin, 0.011 to 580 of Tectocrisin, 0.021 to 10500 of Artepillin C, 0.0078 to 3900 mg of Bacarin, 500 to 25000 mg of Phenolic Compounds, 250 to 25000 mg of Flavonoid Compounds, and 0.0018 to 12800 mg of Caffeoylquinic Acid derivatives.

7. USE OF PROPOLIS DRY EXTRACT PRODUCT STANDARDIZED, according to claim 6 and as per the process defined in any of claims 1 to 5, characterized by being used in the pharmaceutical, cosmetic, food, veterinary, personal hygiene, agricultural and biotechnology industries, having antiviral, antioxidant, antibacterial, antifungal, hepatoprotective, anti-inflammatory, antitumor, immunomodulatory, neuroprotective and healing properties, in humans or for veterinary use.

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