HU195113B - Improved process for extraction of all biologically active compounds of propolis with the aim of their separation according to the effect and increasing the efficiency of the fractions - Google Patents

Abstract

A crude propolis extract or concentrated extract of propolis are extracted using solvents, leading to the sepn. of all components according to their bioactivity and enhancing the efficiency of each fraction. - Propolis or an extract of propolis is mixed with 0.1-11 pts. wt. carbohydrate based adsorbent or active charcoal and 0.1-15 pt. wt. water. Solvents are to be removed if the starting material was propolis extract. The resulting pulp is solvent extracted with single solvents or suitable mixtures in one or consecutive steps, the solvent types being: aliphatic, cycloaliphatic or aromatic hydrocarbons, ethers, chlorinated hydrocarbons, ketones, esters or alcohols, at temps. of 4 deg.C and solvent b.pt. Water or water solvent mixts. at 10-80 deg.C can beused respectively. The sequence used in extraction is optional and the final fractions can be freed from solvents if required.

Description

The present invention relates to an improved process for extracting all biologically active substances of propolis, separating them by their action and increasing the efficiency of each fraction with organic solvents, from crude or extracted propolis or from concentrated propolis extract.

Propolis is known to have a beneficial effect on anti-inflammatory, local anesthetic, bacterial and antifungal infections, and especially on chronic infections caused by Gram-positive bacteria, which is of particular importance due to the Treatment of chronic bacterial infections caused by antibiotics often results in the development of resistant strains and may cause allergic hypersensitivity, fungal infections and other side effects.

The biological efficacy of a propolis is related to the extraction method. Most of the substances present in propolis are soluble in alcohol, so alcoholic or aqueous alcoholic extraction is generally used.

One known method of extraction is to extract the propolis with 96% ethanol. The dry residue of the extract is 50%. The 10% extract of alcohol obtained from this extract with alcohol has a relatively good antifungal effect, but its effect is less than that of nystatin and its antibacterial activity is behind the oxytetracycline. Extraction does not ensure recovery of all biologically active propolis material Bekemeier H. et al., Derm.Msch. 159, 443-449 (1973).

In another known method, propolis is extensively extracted with 70% ethanol in 40% yield. Extraction does not provide recovery of all biologically active material, and the recovery rate is relatively low. Popescu et al., Dermato-Venerologia XII. (1), 57-65 (1967).

According to another known method, the propolis extract is extracted with 70% methanol, then the concentrated extract is dissolved in ether and then with 5% hydrochloric acid, then with saturated sodium bicarbonate solution, followed by 10% sodium carbonate solution and finally % shaken with caustic soda. The sodium hydroxide fraction is considered to be the most effective antimicrobial fraction, but its recovery rate is only 7.5%, according to Ghisalberti E. L. et al., Experientia 34, 157-158 (1977). This extraction method also does not provide for the recovery of all biologically active substances in propolis, and the recovery percentage of antibacterial fractions is very low. Acidic and alkaline shakes may cause decomposition or change in the propolis contents and the coloring agents will turn brown.

No. 395,087. According to the US patent, propolis is extracted with petroleum ether, chloroform and alcohol and the resulting combined extract is used to prepare the fungicidal polyphenolic substances. This process 2 also does not allow the recovery of all biologically active substances of propolis and is not suitable for isolating the active substances of propolis by action.

No. 167,862. According to the Austrian patent, propolis is extracted with ethanol followed by turpentine oil. The combined extract thus obtained is considered to have very good antiseptic activity, but due to the adverse side-effect of turpentine it is of limited therapeutic use.

No. 2935/80, No. 182800; According to the Hungarian patent, the propolis is extracted twice with ethanol, then mixed with water or aqueous ethanol and simultaneously crushed, the resulting emulsion is cleaned of mechanical impurities and separated into several fractions by sedimentation. This process is not suitable for isolating the active ingredients of propolis or for isolating the active ingredients of propolis. for the production of high-efficiency special fractions.

The object of the present invention is to provide an improved process for the extraction of all biologically active substances in propolis, to increase the efficiency of each fraction, and to produce propolis fractions of special efficiency.

The present invention is based on the discovery that the above object can be achieved by the organic solvent extraction of propolis by means of an adsorbent.

A further basis of the invention is the discovery that the biological separation of the extraction fractions can be enhanced if the organic solvent extraction of propolis is carried out in the presence of water after the addition of the adsorbent.

Accordingly, the present invention provides an improved process for extracting all biologically active substances of propolis, separating them by their action and increasing the efficiency of each fraction with organic solvents, crude or extracted propolis or concentrated propolis extract. According to the present invention, the starting material propolis or propolis extract is mixed with 0.1-15 sr of a pharmaceutically acceptable organic adsorbent and 0.1-15 sr of water, and, when the propolis extract is used, the mixture is de-solvented and The pasty mixture thus obtained is extracted with one or more organic solvents at a temperature between + 4 ° C and the boiling point of the solvent and water at a temperature between 10 ° C and 80 ° C.

As the organic based adsorbent, it is expedient to use plant cellulose, preferably ground sunflower kernels, polysaccharides, preferably starches, and activated carbon.

Suitable organic solvents are aliphatic hydrocarbons, preferably petroleum ether, ethers, preferably diethyl ether or isopropyl ether, chlorinated hydrocarbons, preferably dichloromethane or chloroform, ketones, preferably acetone, esters, preferably ethyl-2195113.

acetate and alcohols, preferably methanol, ethanol or butanol.

Extraction can be accomplished by stirring, shaking, blending or countercurrent extraction.

The combined extracts obtained by the process of the present invention have a complex therapeutic effect and each extract, when used individually, has a special therapeutic effect. The extracts, after concentration, can be used directly or in an alcoholic solution for the preparation of drugs or cosmetics.

The main advantages of the process according to the invention are as follows.

a) Easy to implement.

b) Ensures recovery of all biologically active propolis.

c) Enables simultaneous extraction and fractionation.

d) It is more efficient than the known methods to isolate the biologically active substances of propolis by action and thus to obtain fractions of special therapeutic efficacy.

The following examples illustrate the process of the invention.

Example 1 A crude ground propolis powder was successively extracted extensively with 70% ethanol followed by 96% ethanol followed by ethyl acetate, dichloromethane and finally petroleum ether. The resulting fractions were concentrated to a syrup density and combined (stock extract).

sr basic extract is mixed with 1 sr potato starch and 1 sr water, de-solvented and successively extracted with petroleum ether (A / a), petroleum ether / chloroform 1: 1 (A / b), then chloroform (B) followed by ethyl acetate (C), 96% ethanol and finally 30% ethanol (D / a and D / b) followed by water.

The concentrated extract obtained above after concentrating, because of its complex therapeutic effect, and the concentrated fractions obtained therefrom, because of their special effect, can be used for medicinal or cosmetic purposes.

Extracts obtained with petroleum ether (A / a) and 1: 1 petroleum ether / chloroform (A / b) have very good anti-inflammatory and capillary resistance enhancing effects. The extracts significantly inhibited carrageenan-induced rat foot edema in a dose-dependent manner, 100 mg / kg i.p. dose showed a 32% anti-inflammatory effect. The 50 mg / kg i.p. Phenylbutazone administered at a dose of 29% had a 29% anti-inflammatory effect.

Extracts show a statistically significant increase in capillary resistance in a dose-dependent manner. The 100 mg / kg i.p. the capillary resistance-enhancing effect of the extract given over a period of more than 10 hours; the low vessel wall4

maximal effect of capillary resistance enhancement was 293.85% versus the control rat group. 300%. Venoruton 100 mg / kg i.p. dose, the maximal effect on capillary resistance after 24 hours was 246.6%.

The extract obtained with petroleum ether (A / a) and petroleum ether / chloroform (1/1) (A / b) has very good anti-inflammatory and capillary resistance properties in the treatment of various inflammatory diseases, e.g. for the treatment of rheumatism and arthralgia, varicose veins, and microvascular injuries.

Fraction (B) obtained with chloroform was 50 mg / kg i.p. at a dose of 43.3% and is highly effective against Gram-positive and Gram-negative bacteria and fungi.

The concentrated extract, when used at 4% by weight in vitro, exhibits the same antibacterial mite dilution as the 1% active ingredient oxytetracycline. When used in external formulations, 10% by weight of the extract has an effect over oxytetracycline. A further advantage of the extract is that it can be effective against strains resistant to oxytetracycline. A further advantage is that it also has a therapeutic effect in the treatment of co-existing fungal infections, the same effect as nystatin. Its anti-peeling effect helps to heal the infected area, and its anti-inflammatory and local anesthetic effect causes rapid resolution of clinical symptoms.

Due to the complex effect of the concentrated chloroform moiety, it can be used to prevent various inflammatory, fungal and bacterial infections, difficult to heal wounds, leg ulcers, microvascular injury, furunculosis, and acne. It can be used externally and internally.

Fraction (C) obtained with ethyl acetate shows weak antibacterial and antifungal activity, but exhibits good anti-inflammatory activity.

Extracts (D / a) and (D / b) obtained with 96% and 30% ethanol were 100 mg / kg i.p. at a dose of 44.9% with anti-inflammatory activity; this effect is better than 90 mg / kg i.p. doses of Phenylbutazone. A special advantage is that they can be used internally. Extracts show a statistically significant dose-dependent increase in capillary resistance. 100 mg / kg i.p. at the dose, the capillary resistance enhancement effect of the extracts is greater than 6 hours, with a maximum effect of 343.85% as a percentage of the control rat group. Venoruton 100 mg / kg i.p. dose at 246.6% after 6 hours.

The extracts obtained with ethyl acetate, ethanol and aqueous ethanol, and the extracts obtained with petroleum ether and a 1: 1 mixture of petroleum ether and chloroform, are very effective in the therapeutic and

-3195113 for cosmetic purposes, in particular for the treatment of inflammatory diseases, and, due to their capillary resistance-enhancing effect, for the treatment of microvascular injuries and difficult to heal wounds.

Table I shows the anti-inflammatory, capillary resistance enhancement, and the relative antibacterial activity relative to chrysin and the relative antifungal activity relative to nystatin of the base extract and its fractions.

II. Table 1 shows the various propolis samples without adsorbent and without water, respectively. shows the relative antibacterial and relative antifungal activity as determined by the medium dilution technique of the adsorbent and water fractions as compared to chrysin and nystatin based on the mean minimum inhibitory concentration in mg / ml.

In III. Table 1 illustrates the relationship between the individual extraction procedures and the yield and biological efficiency of the obtained fractions. _: The antibacterial activity was calculated as a percentage of total activity, so that the individual fractions úrtékkel reciprocal $ average minimum inhibitory concentration (, χ ^ MIC) times jellemezett activity expressed in mg in the total number of fraction. The resulting value for the base extract

The total activity of the adsorbent and water fractions as a percentage of the stock extract was calculated as 100%. The substances responsible for the antibacterial and antifungal activity were predominantly in fraction B. THE

A total activity value greater than 100% indicates the presence of an antibacterial agent.

The IV. and Tables V, Table

In the basic extract and in some fractions of the procedure of Example 1, the most important active compounds were isolated and detected.

Table I

Results of the activity studies of the propolis fractions obtained according to Examples 1 or 2

Test sample 100 g ball Anti-inflammatory in yield / inhibition 7, -a 7, /

Capillary Resistant Relative Antibacterial Boosting Activity /% of Control mu- / Chrysin = 1 / t max. effect / Gram-po- Against gram-negative bacteria

Relative antifungal activity / Cystatin = 100 /

Pet 1JB alapkívcnat 78.2 50 mg / kg i.p., 23.6 100 mg / kg ip after 8 ^h 193 * 5 34,08 27.89 9.58 Example 1 The / a 18.5 100 mg / kg i.p. . 100 mg / kg i.p. 0.93 1.00 The / b 14.7 32 83. 293.85 26.68 24.59 1.14 300 Example 1 50 mg / kg i.p. ε 43 43.3 72.62 47.78 15.17 Wand 1 c 16.8 31.15 27.89 2.06 Example 1 100 mg / kg i.p. , 100 mg / kg i.p. D / a 4 44.9 103.343.81 1.55 1.08 D / b 3 Example 2 150 mg / kg i.p. , 00 mg / kg i.p. EEPi Basic Extract 16.3 40.9 After 4 hours, 164.08 48.38 41.83 7.64 Example 2 .50 mg / kg i.p. EEPi / B 33.3 43.3 72.62 47.78 15.17

II. spreadsheet

The relative antibacterial and relative antifungal activity of propolis samples and fractions determined by culture dilution / agar dilution / + / i s-Mic y ^a pj ^an Relative antibacterial activity / Chrysin = 1 /

Fractions tested against Gram-Gram-positive bacteria

Relative antifungal activity / Nystatin = = 100 /

Propolis sample 1 Basic extract of Example 1 / without adsorbent and water / 34,08 27.89 9.58 Example 1 Fraction B / adsorbent and water purification / 72.62 47.78 15.17 2. Propolis sample Example 2 base extract / adsorbent and water-free EEPi / 48.38 41.83 7.64 Example 2 Fraction B / adsorbent and water purification / 72.62 47.78 15.17 ⁺ / x MIC = average minimum inhibitory concentration (mg / ml)

III. Spreadsheet

Relative antibacterial and relative antifungal activity and total activity of the propolis fractions obtained according to Example 1 or 2 as a percentage of the total activity of the stock extract by medium dilution / agar dilution / technique

Test substance

Yield Yield Relative antibacterial activity / g / /7.1 Rial activity / Chrysin = 1 /

Gram- Gram positive for negative bacteria

Total Activity /% /

Gram- Gram positive for negative bacteria

Relative antifungal activity / Nystatin = 100 /

Example 1

raw extract 78.2 100 34,08 27.89 100 100 9.58 Example 1 A / a 14.45 18.5 0.93 1.0 0.47 0.23 Example 1 A / b 11.5 14.7 26.68 24.59 12.81 22.06 1.14 Example 1 B 33.63 43 72.62 47.78 91.63 73.68 15.17 Example 1 C 13.14 16.8 31.15 27.89 15.36 16.8 2.06 Example 1 D / a 3.13 4 5.0 3.38 0.59 0.48 1.32 Example 1 D / b 2.35 3 1.55 1.08 0.14 0.12 78.2 1Ó0 121.0 113.4 Example 2 EEPi 16.3 48.38 41.83 23.14 24.46 7.64 Example 2 EEPj / B 33.3 72.62 47.78 50.04 38.07 15.17

-5195113

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-8195113

If all the procedures described in Example 1 are followed, but without the addition of adsorbent and water, the fractionation of the base extract can be accomplished in several steps, both by chemical separation and by biological activity.

Example 2

No. 2935/80. No. 182,800. According to Hungarian patent application, EEP extract with 96% ethanol is concentrated. 1 sr of the concentrated extract was dissolved in a 1: 1 mixture of ethanol: chloroform (96%), mixed with 1 sr of activated carbon, then de-solvented and 0.1 sr of water was added. The pulp thus obtained is successively extracted extensively with petroleum ether / diethyl ether 1: 1 (A), diethyl ether (Β), 70% ethanol (C) and finally water (D). The fractions thus obtained, after concentration, are used separately for pharmaceutical or cosmetic purposes as described in Example 1.

Example 3 This crude ground propolis is extensively extracted successively with 96% and 70% ethanol at room temperature, followed by 70% ethanol and water at 60 ° C. The residue was then extracted with ethyl acetate and finally with diethyl ether. The extracts obtained are concentrated to a syrup density and combined (base extract).

sr basic extract was mixed with 1 sr of milled sunflower kernel pellet, and after the solvent was removed, the residue was mixed with 1 sr of water to a pulp and then extracted extensively with petroleum ether / dichloromethane 6: 4 (A), dichloromethane (B), 70%. ethanol (C) and finally water (D). The resulting extracts, after concentration, are used for therapeutic or cosmetic purposes as described in Example 1.

EXAMPLE 4 Crude ground propolis was stirred at room temperature with 30% ethanol for 4 hours, then the aqueous ethanol portion (D / a) was decanted and the residue was stirred with 60 ° C water (D / b) for 4 hours. After separation of the aqueous portion, an equal weight of cellulose powder was added to the remaining propolis and mixed with 0.1 l of water to a pulp, followed by a 1: 1 mixture of petroleum ether and chloroform (A), chloroform (B), 96% and extensively with ethanol (C) and water (D). The resulting extracts, after concentration, are used for therapeutic or cosmetic purposes as described in Example 1.

EXAMPLE 5 Crude ground propolis powder was mixed with 15 parts potato starch and 8 parts water, and the resulting pulp was successively extracted extensively with dichloromethane, 96% ethanol and finally water. After concentration, each fraction 16 is applied individually or together for therapeutic or cosmetic purposes as described in Example 1.

The biological efficiency of the extract obtained with dichloromethane can be further increased by concentrating the extract, dissolving it in 90% ethanol, extracting it three times with petroleum ether, and concentrating the fractions.

EXAMPLE 6 Mixed sr of crude propolis, 1 sr of milled sunflower kernel and 1 sr of water were successively extracted with 6: 4 petroleum ether / diethyl ether (A), diethyl ether (Β), 96% alcohol (C) and finally water (D). The fractions were concentrated and used for pharmaceutical or cosmetic purposes as described in Example 1.

Example 7

Preparation of an antifungal ointment Melted g of lanalkol, 30 g of cholesterol petroleum jelly, 3 g of yellow beeswax, 6 g of cetyl alcohol and 30 g of linseed oil in a water bath, stir until cooled, and then emulsify 20 g of water. Then, 6 g of the mixture of steps 1-5 are added. Examples are prepared from concentrated extract of chloroform, dichloromethane or diethyl ether (B) according to any one of Examples 1 to 4.

With the resulting ointment, rubbing the infected area several times a day will alleviate the clinical symptoms of the fungal infection within a day or two. It also shows good efficacy against other treatments that are unresponsive or resistant, especially stubborn infections.

Example 8

Preparation of Rheumatic Ointment Gram cetacea, 15 g yellow beeswax, 25 g cetyl alcohol, 35 g cholesterol vaseline and 65 g paraffin oil are melted in a water bath and stirred until cooled. A solution of 2.5 g of ascorbic acid in 60 g of water at the same temperature was emulsified, followed by 10 g of oleyl oleate, 3.5 g of eucalyptus oil, 2.5 g of peppermint oil, 0.5 g of lavender oil, 0.6 g of potassium oil, 0.6 g. g of menthol and 0.3 g of camphor. Finally, sections 1-5. 4g of the combined burglary extract prepared according to any one of Examples 1 to 4 are mixed into the ointment and homogenized.

By rubbing the painful, inflamed area with the ointment three times a day, the pain disappears within a short time, and the inflammation subsides after a day or two. It is very effective in stubborn cases that do not respond to other ointment treatments.

Example 9

Preparation of a wound healing ointment

1-5. 6 g or 4 g of the chloroform, dichloromethane or diethyl ether extract of the combined concentrated extract prepared according to any one of Examples 1 to 4 is mixed with 10 g of linseed oil and 90 g of cholesterol vaseline, followed by 2 g of honey, 0.5 g of

-917 clove oil, 0.1 g cinnamon oil and 1 g ascorbic acid dissolved in 5 g water.

Apply the ointment to the wound or ulcer three times a day. The peeling begins within a short time, the infection decreases, and the ulcer begins to heal.

Example 10

Production of anti-inflammatory ointment

1-5. 4 g of the combined concentrated extract prepared according to any one of Examples 1 to 5 or 3 g of the concentrated extract A obtained according to any of Examples 1-5 and 3 g of the combined concentrated extracts C and D are mixed with 5 g of oily ointment (Ph. Hg. VI, Volume III, page 1444) and 45 g of a non-ionic hydrophilic ointment (Ph.Hg.VIII, Volume III, page 1441) followed by 25 g of water, 25 g of glycerol and 0, 1 g of fragrance is emulsified in it.

The resulting ointment is applied to the inflamed area three times a day, and the inflammation is reduced after a day or two.

Example 11

Preparation of Skin Regenerating Face Cream Melted g of stearin, 5 g of glycerol monostearate, 1 g of cetyl alcohol and 50 g of vegetable oil in a water-bath, and stirring, in small portions, 5 g of glycerol and 5 g of sorboxetine stearate at the same temperature. After cooling, steps 1-5. 2 g of the combined concentrated extract prepared according to any one of Examples 1 to 5 or 1 to 5. 3 g of extract A and 3 g of the combined extracts C and D are mixed into the ointment and homogenized.

Example 12

For making a beauty mask

Add 100 g of vegetable oil to two egg yolks and add 2 g of honey, 5 g of pollen and IQ g of lemon juice to the resulting emulsion, then add 2 g of the yeasts 1 to 5. or 3 g of extract A prepared according to any one of Examples 1-5 and 3 g of the combined extract C and D.

The facial skin is flushed with chamomile and then lubricated with the above emulsion which dries for 15-30 minutes and smoothes the skin. Wash face with lukewarm water.

Claims (1)

Improved process for extraction of all biologically active substances in propolis 20, and to increase the efficiency of each fraction with organic solvents, crude or extracted from propolis or concentrated propolis extract, characterized in that The propolis powder or propolis extract used is mixed with 0.1-15 sr carbohydrate-based adsorbent or activated carbon and 0.1-15 sr water, the mixture obtained is removed from the organic solvents using propolis extract and the resulting pasty mixture one or more organic solvents of the aliphatic, cycloaliphatic or aromatic hydrocarbon, ether, chlorinated hydrocarbon, ketone, ester or alcohol and / or mixtures thereof Extracted in any order at a temperature between 35 ± 4 ° C and the solvent boiling point and at a temperature between 10 ° C and 80 ^° C with water or an aqueous organic solvent and in the eset40 fractions were freed from solvent is required.