SK352021A3 - Propolis extracts and method for their preparation - Google Patents

Abstract

Extracts from propolis containing dimethylsulfoxide and the method of their preparation are described.

Description

The field of technology

The solution concerns extracts from bee propolis and the method of their preparation.

Current state of the art

Almost all beekeeping products are more or less useful, and mankind has been using them practically for thousands of years. In addition to honey, which is the main bee product, wax, pollen, royal jelly, propolis, bee venom, larvae of bees and dead bodies of naturally dead bees are also used - podmor.

Propolis, or so-called propolis is a resin-based substance that bees obtain from tree buds or other plant sources. Bees use propolis to fill unwanted smaller holes in the hive and also for disinfection. The name "propolis" is of Greek origin and literally means suburb or city fortification. At room temperature, it is an amorphous, flexible, sticky mass, usually yellow to dark brown in color, which becomes brittle and crumbles when cooled. The mechanism of propolis formation is still not clearly resolved. The quality of propolis and its properties depend on which plants and under which conditions the bees collect resins and other plant components. According to some authors, bees can process the same substances brought to the hive in different ways depending on what they are to be used for in the hive. It is also reported that propolis from different locations in the hive has a different chemical composition. Since in the case of propolis it is always a mixture of substances, the content of which varies practically from sample to sample, the chemical composition of propolis cannot be uniform. The calculation of the components contained in propolis is truly impressive. In a considerable generalization, it can be stated that propolis contains 50 to 85% resin, 12 to 40% wax, 12 to 40% tannins and 18 to 20 percent macro and microelements in the ash. Propolis contains vitamins A, C, E, H and P, group B vitamins, benzyl alcohol, chrysin, acacetin, quercetin, camphorides, galantin, salinin, pectolinarigenin, pinostrobin, sakuranetin, isosakuranetin, 3,3-dimethylether quercetin, pterostiben, xanthorrhoeol, cinnamyl alcohol, vanillin and isovaniline, myristic, sorbic, cinnamic, caffeic and ferulic acids, other essential oils, flavones, flavonoids, flower pollen, from the minerals calcium, iron, copper, cobalt, nickel, zinc and silicon and also trace amounts of vanadium and titanium.

Propolis is a legendary medicinal agent containing a number of biologically active substances. Among other things, bees use propolis to "mummify" the corpses of unseen intruders (mice, lizards, insects...), which do not decompose in the hives, and the bee colony is thus protected from harmful bacteria and gases. This effect was already used by the ancient Egyptians, who used mixtures containing propolis during embalming. Propolis was also used in the treatment of gunshot wounds caused by arrows. Also during II. During World War II, the Red Army used propolis to treat wounds. Biochemists from the Max Planck Institute and Copenhagen scientist Karl Lund Augaard conducted experiments with almost twenty thousand people, confirming the antibiotic effect of propolis on a wide range of microorganisms. Propolis is one of the strongest anti-inflammatory agents, it helps with a number of diseases and health problems, it is bactericidal, it has anesthetic effects, it supports cell regeneration, as a result of which it accelerates the healing of wounds.

In addition, it has been shown that propolis activates the function of the thymus, the so-called children's gland (lat. thymus), which is an important organ in the immune and hormonal system. It is also reported to increase cell turnover and cell division up to twofold. It is also often stated that propolis is the "antibiotic of the 21st century".

In the case of propolis, it is easier to list the indications for which it is not used. The main confirmed indications for which propolis is effective include, above all, diseases in the neck area, inflammation of the oral mucosa and nasopharynx, chronic and acute rhinitis, inflammation of the frontal sinuses, inflammation of the tonsils and ears, hay fever, cough, bronchitis, bronchial asthma, digestive diseases organs, inflammation of the gallbladder, periodontal disease and toothache. It has a beneficial effect on metabolic disorders, high blood pressure, increased blood fat and cholesterol, disorders of the vascular system, atherosclerosis, weakened immunity and also as a prevention of colds. It is also effective for inflammation of the kidneys, urinary tract and bladder, prostate problems and vaginal infections. In the case of external application, beneficial effects are achieved in the treatment of skin diseases (psoriasis, various cold sores), in the treatment of external injuries, frostbite and burns of various origins. The biological effectiveness of propolis led to the emergence of a separate field of medicine - propolis therapy (Dobrovoda, I.: Bee products and health, Príroda - Bratislava, 1986, Johan Richter: Léčení včelími products, Eko-konsult, 1999, Jonáš, J. - Kuchar, J. : World of natural antibiotics - secret weapons of plants, Eminent, 2014, Chismatullinova.N.Z. : Practical apitherapy, Perm 2012, Hydra 2015). Extensive and intensive scientific research into the therapeutic possibilities of propolis is being carried out at many research facilities abroad, as well as in our country. Among several professional events, we can mention, for example, the symposium held in Bratislava and Košice on the topic "Propolis as a medicinal agent", in which more than 350 scientists from 38 countries participated. Participants

SK 35-2021 A3 symposium learned, among other things, that in Denmark they confirmed the healing effects of propolis on a set of more than 50 thousand people.

Tincture refers to an extract, or extract, usually from medicinal plants, in the preparation of which biologically effective - medicinal substances are obtained from certain parts of plants (flowers, roots, fruits, herbs, bark, wood) by the effect of a suitable extraction agent, usually concentrated alcohol. The word tincture comes from the Latin term tinctura, i.e. colored, because during preparation the extraction agent - alcohol is colored by leached substances. Two approved pharmaceutical procedures are commonly used in the preparation of tinctures: maceration and percolation. Tinctures are most often prepared from dried plants (so-called drugs), while 5 parts of the extraction liquid are usually dosed for 1 part of the drug. In the case of drugs with strong effects, they are made from 1 part of dried plants (drug) and 10 parts of extractant. The extraction liquid (extractor) is usually ethanol. Ethanol with 70% alcohol content and 30% water is mostly used. In addition to ethyl alcohol, glycerol (glycerin) and acetic acid are also sometimes used in the pharmaceutical preparation of tinctures, especially in the form of fruit vinegars (R,Beiser, H.EII-Beiserová: Tinctúry z šáručích plantán, Foni book, 2018).

Medicinal substances are isolated from propolis in the form of extracts. Currently, the literature mentions water, alcohol and ether extract of propolis. Alcoholic extract is referred to as propolis tincture, which is a popular agent used when using propolis. In addition to direct use, propolis tincture is also used to prepare a whole range of medical and cosmetic preparations, such as propolis soaps, shampoos, creams, various gels and the like.

Aqueous propolis extract is prepared either by heating the aqueous suspension of propolis in a water bath for one hour, or by adding 100 parts by weight of distilled water to 30 parts by weight of propolis, while the extraction takes place at room temperature for 4 to 5 days. After extraction, the solid undissolved residue is separated by filtration.

However, the most frequently used alcohol propolis extract - propolis tincture. This is prepared in pharmaceutical quality by first extracting propolis with ether in a Soxhlet apparatus for 6 hours. Subsequently, the ether is removed from the obtained extract by evaporation, and the thus obtained dry propolis extract is dissolved in a ratio of 1:10 in pure 96% alcohol.

At home, propolis tincture is usually prepared by mixing ground propolis with absolute alcohol in a ratio of 1:5 and letting it macerate for several days at room temperature, first with constant and then at least occasional stirring. Then the mixture is filtered.

The essence of the invention

It has now been found that dimethyl sulfoxide, CH ₃ SOCH ₃ (DMSO), can be advantageously used for the extraction of biologically active components from bee propolis. It is a very effective and relatively cheap solvent from the group of alkylsulfur compounds. It is characteristic of solvent systems in the sense of this solution that they contain either dimethyl sulfoxide (DMSO) itself,

or its mixture with some other solvent, especially with water, ethyl alcohol, isopropyl alcohol, glycerin, ether, propylene glycol, or with acetic acid.

The industrial production of dimethyl sulfoxide, CH3SOCH3 (DMSO) is closely related to the chemical processing of wood, or with the production of cellulose. During this production, sulfated lignins are demethylated with the help of anions: SH and CH3S. Demethylation of lignins has the character of nucleophilic substitution. Depending on the demetallization conditions (pressure, temperature, pH), different proportions of dimethyl sulfide, CH3-S-CH3 (DMS), methyl mercaptan, CH3SH (MM), and dimethyl disulfide, CH3-S-S-CH3 (DMDS), are formed. The most important product is dimethyl sulfide (DMS), from which dimethyl sulfoxide, CH3SOCH3 (DMSO), or dimethylsulfone - methylsulfonylmethane, CH3SO2CH3 (MSM), which, unlike liquid DMSO, is already a white crystalline substance (melting point 109 °C), soluble only in hot water and in a wide range of organic solvents.

Dimethyl sulfoxide (DMSO) is a polar (hydrophilic) aprotic solvent, heavier than water (specific mass-density at room temperature 1.092 g/cm3), with a high boiling point at normal pressure (189 °C) and a high value of dipole moment (4.7 D). Although the solidification temperature of pure dimethyl sulfoxide is less suitable due to its use as a solvent (DMSO solidifies at a temperature close to +19 °C), by adding suitable additives, its very high phase stability can be achieved (e.g. by adding water up to -60 °C).

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The ability of DMSO to activate and penetrate membranes makes it easily pass through biological barriers, e.g. skin, cell walls, membranes of organ cells, bacterial walls, blood-encephalitic barrier, etc. A special feature of DMSO when passing through biological membranes is its ability to take other substances with it - DMSO thus serves both as a solvent and also as a "carrier" of active substances. The great advantage of using alkylsulfur compounds and especially dimethyl sulfoxide (DMSO) in the preparation of propolis extracts is the fact that these are compounds that can be considered as natural forms of organically bound sulfur that are found in nature as part of the natural sulfur cycle in nature.

Scientific studies have proven that algae and plankton in the world's seas and oceans absorb large amounts of sulfur from the water and subsequently transform it into organic forms, the basis of which are precisely alkyl sulfur compounds.

It is reported that dimethylsulfide (DMS) is primarily formed, which is gradually oxidized to dimethylsulfoxide (DMSO) and then to dimethylsulfone - methylsulfonylmethane (MSM) under the action of ultraviolet radiation. The mentioned alkylsulfur compounds are soluble in water and are easily concentrated in atmospheric water vapor. Through rain and snow, they return to the ground, where they become an important source of sulfur for plants and animals. This can explain why fresh fruit, vegetables and other foods contain a significant amount of alkylsulfur compounds (1-4 mg MSM per kg of fruit and fresh vegetables, 2-5 mg MSM in fresh unpasteurized milk). Also, in the bloodstream of an adult, the natural level of MSM is up to 0.2 mg per kg. Scientists have proven that there is a minimum concentration of alkylsulfur compounds in the blood, which is necessary to maintain basic vital functions and to protect tissues (Jacob.S.W.: Cerrent Status of MSM in Medicine, Am.Acad.Med.Prev., 1983, Jacob,S.W. -Herschler, R.J.: Introductory Remarks-Dimethylsulfoxide after Twenty Years, Ann.N.Y.Acad.Sci., 1983).

After calcium and phosphorus, sulfur is the third most abundant element in the human body (an average of 140 grams of sulfur is in the body of an adult). Sulfur in the body is used almost equally by the skin, muscle tissue and bones. Similarly, nails and hair are composed of keratin, which contains a significant proportion of sulphur. A low level of alkylsulfur compounds in the body is related to states of unspecified fatigue, depression, increased sensitivity to physical and psychological stress and several degenerative diseases (Mindell.E.L.: The MSMiracle.Enhance Your Health with Organic Sulfur.Keats Publishing, lnc.Connecticut, USA, 1997, Ley, B.M.: The Forgotten Nutient MSM-on Our Way Back to Health with Sulphur, BL Publications, California, 1998). Dr. Morton Walker also describes the beneficial effects of dimethyl sulfoxide on the body in his publication "DMSO Natures Healer".

Based on the remarkable findings of the beneficial effect of alkylsulfur compounds on living organisms, the Medical Information Foundation was established, the director of which is the doctor Paul Klein Breteler, based in the Netherlands. According to the published data of this organization, for example, the degree of toxicity of MSM is among the lowest (LD50 value for DMSO is 14,500 mg/kg, LD50 value for nicotine is 50 mg/kg, LD50 value for caffeine is 192 mg/kg, LD50 value for sodium chloride - table salt is 3,000 mg/kg). For example, the aforementioned organization (MIF) reports that MSM was administered to volunteers at a dose of 1 g per kilogram of body weight for 30 days, and no signs of toxicity were observed. Likewise, intravenous applications at a dose of 0.5 grams per kilogram of body weight, five days a week, did not cause any toxic effects in humans.

In mice, the lethal dose of MSM was determined as (DL50) 20 grams per kilogram of their body weight.

In addition, the Medical Information Foundation recommends, for example, to eliminate a whole range of health problems in humans and animals to use either dimethylsulfoxide (DMSO) or, due to its lighter and more pleasant application form, dimethylsulfone - methylsulfonylmethane (MSM). In most cases, at the beginning of treatment, it is recommended to take two tablets of 500 mg MSM twice a day. Interestingly, it has been shown that the effectiveness of MSM increases and is combined with vitamin C.

Considering the mentioned facts, in the case of many applications of propolis extracts in the sense of a solution, it is not necessary to remove the used solvent from the eluate and often its presence in the extract is welcome - it positively affects its effectiveness. Moreover, the presence of the solvent in question does not prevent the biocharacter of the final product from being questioned (H.P.A. Fischer: "DMSO-handbook, instructions for use - hidden classified knowledge about natural healing", Nempe 2018).

According to the solution, propolis extracts can contain only dimethylsulfoxide alone, or DMSO with other traditionally used solvents (usually, for example, together with water, ethyl alcohol, isopropyl alcohol, dimethyl ether, so-called ether, glycerin, 1,2-propanediol, so-called propylene glycol, acetic acid, or vinegar etc.).

When compared with food-grade ethyl alcohol, which is usually used in the preparation of tinctures, the price of dimethyl sulfoxide is not burdened by a special tax and registration, which is customary in the case of alcohol. Dimethylsulfoxide contained in the extraction mixture is able to isolate polar from propolis with high efficiency

SK 35-2021 A3 and non-polar substances, while these can be hydrophilic as well as hydrophobic in nature. In addition, the prepared extracts may not contain alcohol, which may limit their use in some cases.

It has also been proven that propolis extracts in the sense of a solution can be advantageously used in the preparation of preparations usable in human and veterinary cosmetics. Also in this case, the ability of DMSO to quickly penetrate the skin and to positively influence the entry of other active substances into the body of humans and animals is also beneficial.

Part of the solution is also the method of preparation of propolis extracts according to the solution. It is characteristic of this method that propolis is leached - extracted with a reagent containing dimethyl sulfoxide, (CH3)2 SO. Pure dimethyl sulfoxide or its mixture with some other solvent can be used for extraction. Mixtures of dimethyl sulfoxide with water, ethyl alcohol, isopropyl alcohol, glycerin, ether, propylene glycol, or acetic acid are especially suitable.

The efficiency of any extraction of a solid substance with a liquid depends primarily on the solubility and speed of the transition of the substance from one phase to another. Solubility can be influenced by choosing a suitable solvent. In order to achieve the required efficiency of extraction of biologically active substances from propolis, it is suitable if the extraction agent used contains at least 40 wt. % of dimethyl sulfoxide.

The rate of transition of a substance from a solid phase to a solution is mainly determined by the rate of penetration of the liquid into the solid phase, the rate of diffusion of the substance in the liquid and the rate of removal of the substance from the contact surface. In order to achieve the highest possible efficiency of extraction - bringing it closer to the equilibrium state, it helps to increase the surface of the solid phase - propolis by treating it by crushing or grinding. Likewise, the speed and efficiency of extraction can be increased by supplying still fresh solvent to the contact surface. This can be achieved by mixing or by using a countercurrent system.

Several designations have been developed for individual types of extractions. If the solid phase is extracted with a solvent at a non-elevated temperature, we speak of maceration (from the Latin word commacerar, which means to soak).

If the solid phase is extracted with a hot solvent, we speak of percolation, sometimes also digestion. This procedure can, for example, be compared to the preparation of filtered coffee.

In chemical practice, the percolation process is used, for example, in apparatus according to Franz von Soxhlet (1879).

Each of the already described extraction methods can be used to extract biologically active substances from bee propolis. Due to its simplicity and in the interests of minimal disruption of the extracted substances, the maceration of crushed or ground propolis has proven to be the most effective. In order to speed up the extraction and achieve its higher efficiency, it is advisable to mix the maceration mixture at least occasionally.

In order to deepen the extraction, remarkable results have been achieved by using an ultrasound generator to mix the maceration mixture. Similarly, in the case of percolation, the use of microwave heating to heat the maceration mixture has been proven.

It is important that the amount of solvent used is chosen so that the propolis is perfectly wet during the entire maceration period - covered with the solvent.

The time of extraction - maceration depends on specific conditions (composition of the solvent extraction agent, quality and granulometry of propolis, used ratio of extraction agent to propolis, temperature, nature and intensity of mixing of the maceration mixture, etc.). If propolis maceration is carried out at normal room temperature (approx. 20 °C) and the maceration mixture is thoroughly homogenized at least once a day, a quasi-equilibrium state can usually be reached within a few days, usually within two weeks. The end of the maceration can be roughly determined based on the color of the liquid phase (the intensity of the yellow-brown color no longer changes clearly). By warming it slightly (to a temperature of 30-40 °C), the process of releasing substances can be supported and accelerated. One of the physico-chemical parameters of the solution phase (specific gravity - density, electrical conductivity, vapor, etc.) can be used to accurately indicate the achievement of the equilibrium state.

After the end of the maceration, the maceration mixture always has a suspension character, since propolis always contains insoluble substances. Depending on the subsequent use of the extract, the macerate can either be homogenized and stabilized, or the solid residue is separated after extraction.

If the suspension nature of the extract will not be a problem during further use, it is advisable to stabilize the maceration mixture after thorough homogenization by adding a suitable thickener (xanthan gum, starch, agar, gelatin, etc.), which inhibits the sedimentation process of solid particles.

If a solution form of the extract is required as the final product, it is necessary to separate the solid part from the maceration mixture. This separation can be done by filtration, sedimentation, or centrifugation.

In order to ensure the long-term microbial and chemical stability of propolis extracts in the sense of a solution, in some cases it is advisable to use some of the antioxidant substances and preservatives (acid, ascorbic, vitamin E, sorbic acid, benzoic acid, colloidal silver, thymol, etc.).

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Examples of implementation of the invention

The following examples clarify and document the solution in question, but in no way limit the claims for its protection.

Example 1

In order to prepare the propolis extract according to the solution, the raw propolis stored in a closed glass container was stored in the freezer (- 20 °C) until the next day. The cooled, brittle propolis was briefly ground using a kitchen mixer.

20 g of ground propolis and 100 g of chemically pure dimethyl sulfoxide - (CH3)2SO (DMSO) were weighed into the flask. The flask was placed in a tempering water bath, in which it was heated to about 50 °C for about 5 hours, with occasional stirring. The undissolved - solid part was separated from the brown extract of propolis in terms of solution by vacuum filtration through ordinary filter paper.

Example 2

When obtaining the yield by extracting propolis at room temperature according to the solution, the following composition of the maceration mixture was used:

material % ground propolis, wt. % of dimethyl sulfoxide, (CH3)2 SO and wt. % of water.

The extraction was carried out at room temperature for 5 days. The solid part was separated from the mixture after maceration by filtration through a filter used in the preparation of coffee.

Example 3

In order to prepare an extract from propolis, in terms of the solution, an extraction agent consisting of:

material % of demineralized water, wt. % of dimethyl sulfoxide, (CH3hSO and wt. % of glycerol (glycerin).

Propolis extraction was carried out at room temperature (approx. 25 °C). We worked with a mass ratio of propolis to extraction agent of 1:8. Mixing of the mixture was ensured using an ultrasonic generator. The maceration mixture was divided by filtration through a textile filer.

Example 3

In order to prepare propolis extract according to the solution, the same recipe as in example 2 was used. However, in this preparation, the product did not separate after maceration, but after its disintegration using a kitchen knife mixer, the suspension thus prepared was stabilized by the addition of potassium benzoate and xanthan gum.

Industrial applicability

It is characteristic of propolis extracts, in terms of solution, that they contain dimethyl sulfoxide (DMSO) CH3SOCH3. The method of preparing these extracts is also part of the solution. Its basis is the extraction of propolis with a reagent containing dimethylsulfoxide. The mixture after extraction can either be homogenized and stabilized, or the solid residue can be separated by sedimentation, filtration, or centrifugation.

The process of obtaining propolis extracts is undemanding, the extraction agent used has a synergistic effect on the effectiveness of the prepared extract, and its implementation does not require the use of special equipment.

Claims (2)

1. Extracts from propolis, characterized by the fact that they contain dimethyl sulfoxide - DMSO, (CHahSO. 2. The method of preparing extracts from propolis according to claim 1, characterized in that propolis is 5 is extracted with a reagent containing dimethylsulfoxide, (CHahSO), whereby the obtained macerate is either homogenized and stabilized, or the solid residue is separated after extraction.