HK1011858A - Propolis extract with improved water-solubility - Google Patents

Description

Propolis extract with improved water solubility

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The present invention relates to a propolis extract having improved water solubility, and more particularly, to a propolis extract in which the water solubility of the effective components of propolis is improved by adjusting the pH of the propolis extract to 5.5-7.0, its preparation and use, and a method for improving the water solubility of the propolis extract.

Propolis is a resinous product stored in the hive by bees containing resin, beeswax, essential oils, pollen and flavonoids and has long been used in various folk remedies, as described in Propolis in Natural therapeutics (1983), second revised edition, published by Librairie, Malorie S.A. Editeur, Fragrance Journal, No.83, PP.20-28 and PP.36-43(1987) of Paris, France and Apidilogue, Vol.22, PP.155-162 (1991).

It is known that the main activities of propolis are preservative activity, antioxidant activity, anti-inflammatory activity, local anesthesia, virus growth inhibitory activity, immunoregulatory activity and macrophage activating activity, and the main components of propolis are flavonoid, aromatic carboxylic acid and aromatic aldehyde.

Before the present invention was made, the present inventors studied the effective components of propolis. As a result, it has been found that 3- [ 4-hydroxy-3, 5-bis (3-methyl-2-butenyl) phenyl ] -2-propenoic acid, also known as "Artemisillin C" (hereinafter referred to as "substance C" throughout the specification), which shows preservative activity, hair growth activity and anticancer activity, as an effective ingredient, and a related invention which identifies the use of substance C: for example, japanese patent laid-open No. 256,177/94 discloses a preservative containing substance C as an active ingredient, japanese patent laid-open No. 312,918/94 discloses a hair-mending agent containing substance C as an active ingredient, and japanese patent laid-open No. 191,015/95 discloses an anticancer agent containing substance C as an active ingredient.

As described above, the present inventors have long understood that substance C is an important component in propolis.

Since propolis is a dark brown bulky product and since it is mainly composed of water-insoluble hydrophobic products, it is not easy to use, and is often used in the form of a dissolved liquid propolis extract or propolis tincture, which is extracted with a highly concentrated organic solvent solution that is readily soluble in water, examples of which are acetone, acetic acid, and lower alcohols such as methanol, ethanol, isopropanol (or isopropyl alcohol). In general, ethanol is most suitably used.

However, when such propolis extract is used for food products such as health foods, anti-infectious agents such as therapeutic and/or preventive agents for susceptible conditions, and cosmetics such as skin cleansers and skin bleaches, there are the following drawbacks, and these drawbacks make the use of propolis in various fields difficult.

(1) Ingredients of propolis extract such as resin and wax are easily crystallized. When the propolis extract is diluted with saliva, these components are easily crystallized, and their absorption and utilization by the living body are strongly inhibited. When the propolis extract is diluted with water, these components are crystallized in a large amount, and make handling properties very difficult.

(2) The color of the propolis extract is not satisfactory and the extract easily becomes blackish brown or opaque; and is

(3) The smell and taste of propolis extract are unsatisfactory, i.e. it has a muddy taste and a pungent or sharp taste.

In order to solve these drawbacks of the conventional propolis extracts, the present inventors have continued research on the concentration of inorganic solvents that are readily soluble in water.

As a result, the inventors have identified and disclosed in Japanese patent laid-open No. 316,968/93 that a purified propolis extract, on a dry solid basis,when its absorbance at 310nm and 660nm is converted into absorbance at a concentration of propolis extract of 2 w/w%, the absorbance ratio is A_310nm/A_660nmMore than 4,000 (the term "based on dry solids" is abbreviated as "d.s.b" and the above purified propolis extract may be referred to as "propolis extract" throughout the specification), which also shows that the effective ingredients of propolis are dissolved with a readily soluble aqueous inorganic solvent at a concentration of 30-55 v/v%, and the propolis extract may be obtained from the dissolved fraction.

Through further studies on the propolis extract, the present inventors found that when diluted with water according to a conventional method, a part of the propolis extract becomes insoluble and the extract becomes a white suspension containing an insoluble propolis component. Detailed studies have shown that most of the propolis active ingredients, especially substance C, become insoluble.

Further studies revealed that the effective ingredients of propolis are easily dissolved in a readily soluble aqueous inorganic solvent having a relatively high or medium concentration, but they are hardly or substantially insoluble in the inorganic solvent when diluted with water, with serious consequences, causing a defect that the effective ingredients of propolis are prevented from exerting their activities.

The object of the present invention is to identify a propolis extract having improved water solubility, biological activity and stability and having satisfactory odor and taste, and a method for preparing the same and use thereof. In order to provide such a propolis extract, the present inventors have conducted intensive studies on the propolis extract.

As a result, the inventors have found that the water solubility of substance C as an effective ingredient of propolis can be greatly improved, and the intrinsic activity of propolis can be easily exhibited by adjusting the pH of normal pH of about 5.0 to pH5.5 or more, preferably to pH5.5-7.0, more preferably to pH5.8-6.4 by adding a pH controlling agent, and that propolis extract with improved stability, smell and taste can be obtained.

More specifically, the present inventors have found that when a propolis extract without pH control, which has a normal pH of about 5.0 and contains 10 w/w% of the propolis extract d.s.b. in a 50 v/v% aqueous ethanol solution, is diluted about 100-fold with water according to a conventional method, the substance C is dissolved in the aqueous dilute ethanol solution only at a concentration of 0.03ppm at 25 ℃, and when the pH of the aqueous dilute ethanol is increased from about 5.0 to 5.5-7.0, the solubility of the substance C in the solution is increased at least 15-fold to 100-fold, and the inherent activity of the substance C is also easily exhibited.

The present invention relates to a propolis extract having improved water solubility, and more particularly, to a propolis extract in which the water solubility of the effective components of propolis is improved by adjusting the pH of propolis or its extract to 5.5-7.0, its preparation and use, and a method for improving the water solubility of the propolis extract.

Any method can be used in the present invention as long as the propolis extract can be obtained by adjusting pH of propolis to 5.5-7.0, preferably 5.8-6.4.

Generally, the propolis extract is obtained by the following two ways: contacting propolis with water solution of easily soluble water-soluble organic solvent without pH adjustment, dissolving and extracting effective components of propolis, adjusting pH of propolis extract to 5.5-7.0 with pH control agent, and collecting extract; or contacting propolis with an aqueous solution of a readily soluble aqueous organic solvent adjusted to pH5.5 or more, preferably to pH5.5 or more but less than 8.0, to dissolve and extract the effective components of propolis, and optionally adjusting the pH of the propolis extract to 5.5-7.0 with a pH control agent before selectively collecting the extract. At this time, the concentration of the readily soluble aqueous organic solvent in the aqueous solution is usually 70 v/v% or more. In order to obtain a purified propolis extract, according to the method of Japanese patent laid-open No. 316,968/93, an aqueous solution of 30-55 v/v% of a readily soluble aqueous organic solvent is used. The method for contacting propolis with the aqueous solution and extracting effective components of propolis comprises: the dissolving and extracting method comprises placing propolis in a container, and pouring a water-soluble organic solvent into the container. The countercurrent extraction method comprises cascade columns which are used for propolis action and are fed with the soluble aqueous organic solvent, and is suitable for industrial scale production process due to high extraction efficiency. If necessary, diluting aqueous solution of 30-55 v/v% water-soluble organic solvent containing propolis effective components with water, contacting the diluted solution with macroporous synthetic adsorbent, adsorbing effective components therein, and eluting the components from the adsorbent to obtain propolis extract with improved water solubility.

The pH of the propolis extract is adjusted to pH5.5-7.0, preferably 5.8-6.4, so as to obtain a high quality propolis extract with improved water solubility, stability, smell and taste. When the pH of the propolis extract is less than 5.5, the water solubility of the substance C is unsatisfactory. When the pH of the propolis extract exceeds 7.0, the extract is heavier in color and deteriorated, while other effective ingredients such as 4- (2-carboxyvinyl) -2- (3-methyl-2-butenyl) phenyl phenylpropionate and flavonoids become unstable. These all deteriorate the quality, smell and taste of the extract during storage.

Any PH control agent may be used in the present invention as long as the PH of the propolis extract can be controlled, and specifically those having a concentration of not less than 2 w/w% d.s.b. can be adjusted to the PH range specified in the present invention. For example, various bases such as magnesium oxide, calcium oxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and magnesium carbonate can be used. These bases may be used in combination with other metal salts such as ferrous hydroxide, zinc carbonate and copper carbonate, and may also be optionally used in combination with acids such as hydrochloric acid, phosphoric acid, acetic acid and citric acid, if desired.

Among the PH controlling agents, edible and pharmaceutically acceptable buffers can be used more satisfactorily in the present invention, and the amount is usually less than 10 w/w% of the propolis extract d.s.b. for the purpose of the present invention. After pH control, the propolis extract may be filtered and centrifuged if necessary to obtain a fraction containing propolis effective components such as substance C.

The thus obtained propolis extract is a 2-25 w/w% propolis solution d.s.b., the substance C as the propolis effective ingredient has improved water solubility, and the solution can be optionally concentrated and pulverized into a powdery propolis extract, if necessary. In addition, the propolis concentrate in liquid form may be optionally dehydrated and pulverized by mixing with anhydrous saccharides, for example, may be mixed with anhydrous maltose, anhydrous trehalose and anhydrous lactose and/or substances such as α -, β -and γ -cyclodextrin. In the pulverizing process, the water solubility of the effective ingredient of propolis contained in the final product can be further improved by using a pH controlling agent such as sodium hydrogen carbonate and magnesium carbonate.

Since the propolis extract contains an effective ingredient of propolis which can be easily absorbed and assimilated by a living body, it can be used alone or in combination with one or more other substances such as an antioxidant, a stabilizer, a taste-imparting agent, a colorant, an odor-imparting agent, a filler, an adjuvant and an excipient. The activity, effect, stability and handling of the propolis extract can be satisfactorily improved particularly when used together with one or more vitamins such as tocopherol (vitamin E), carotenoid, L-ascorbic acid (vitamin C), α -glycosyl-L-ascorbic acid, rutin and α -glycosyl rutin, organic acids and saccharides; examples of organic acids are citric acid and maleic acid; examples of sugars are glucose, maltose, trehalose, lactose and maltitol.

Since the present propolis extract has inherent activity of propolis active ingredient, it can be used alone or in combination with other one or more raw materials in compositions such as food products, cosmetics and anti-infectious agents as a health food, preservative, antioxidant, anti-inflammatory agent, local anesthetic, virus growth inhibitor, immunomodulator, macrophage activator, anticancer agent, hair healant, antialipemics, ultraviolet absorber, yellowness imparting agent, vitamin P supplement, quality improving agent or taste imparting agent supplement to maintain and promote health, prevent and/or treat diseases, recover health from disorders, and maintain and improve skin conditions.

The propolis extract can be directly used for urine therapy, which is widely known and recently prevailed, as a supplement for urine therapy to improve the effect and reduce unpleasant urine odor.

The propolis extract has a color, smell and taste preferred by people so that it is very harmonized with substances having salty, astringent, bitter or taste, and it also has a strong preservative activity. Therefore, the propolis extract can be used not only for specific health foods but also for general various compositions such as foods including seasonings, Japanese confectionery, western confectionery, water ices, ice creams, drinks, spreads, paste foods, kimchi, processed meat, processed fish meat, milk and egg products, processed vegetables, processed fruits and processed line products to improve their smell, taste, shelf life and quality stability.

The propolis extract can also be used as a preservative, an intestine-controlling agent, a vitamin P supplement, a stress-eliminating agent or a taste-improving agent for feed and pet food, for domestic animals, pigs, bees, silkworms and fish.

In addition, the present propolis extract can be used for any products in solid, paste or liquid form including cosmetics and medicines, such as tobacco, cigarettes, lozenges, multivitamins, sublingual agents, oral fresheners, chewing gum and mouth washes, nutritional products for tube feeding, natural medicines, internal medicines, injections, dentifrices, lipsticks including those for wrinkled and cracked lips, sun blocks, face soaps, shampoos, hair dyes, bath salts, anti-infectious agents, skin cleansers, skin whiteners, and hair remedies.

The term "anti-infectious agent" referred to in the present invention means an agent for preventing and/or treating an infectious disease, and the term "infectious disease" referred to in the present invention means a disease that can be prevented and/or treated by the present propolis extract: examples of such susceptible diseases are viral diseases, bacterial diseases, traumatic diseases, immunological diseases, rheumatic diseases, diabetes, circulatory diseases, malignant tumors and neurological diseases.

The agent against a susceptible disease may be prepared in a suitable form according to its end use: for example, it can be formulated into liquid form such as spray, nasal rinse, spray, mouthwash, injection; agents in paste form, such as ointments, soft drinks, creams; and agents in solid form, such as powders, granules, capsules or tablets.

The dosage of the agent can be controlled according to the content of propolis extract, administration route or administration frequency. Typically it ranges from about 0.0001 to 10 g/day per adult.

For cosmetic and food products, they may be used similarly to the above anti-infectious agents.

Methods for incorporating the propolis extract into the composition for use in the present invention include those capable of incorporating the propolis extract: these methods include mixing, kneading, dissolving, soaking, infiltrating, spraying, spreading, painting, and injecting.

Hereinafter, the present invention will be described in detail: experiment 1 demonstration of insoluble substance C

Breaking propolis pieces, injecting into 3 columns and adding 50 v/v% aqueous ethanol solution by countercurrent extraction method, dissolving and extracting propolis effective components. Filtering the extract to obtain a liquid propolis extract containing about 20 w/w% dry solids, the dry solids yield being about 30% of that of the raw propolis, d.s.b.

As a sample, propolis extract A_310nm/A_660nmThe absorbance ratio was 7,365. Throughout the specification, the sample was diluted with a 50 v/v% aqueous ethanol solution to a 2 w/w% solution, d.s.b., and the absorbance was measured using a 1-cm path length cell at 660 nm. Similarly, samples were diluted to 0.004 w/w% solution, d.s.b., and absorbance was measured at 310 nm. The absorbance at 310nm was multiplied by 500 times and converted to d.s.b. absorbance of a 2 w/w% solution, and then A was calculated_310nm/A_660nmThe ratio of (a) to (b).

The pH of the sample was 5.0 as measured by a pH meter. The sample was placed in a beaker and diluted 101 times with water to a dilute solution of aqueous ethanol in the form of a white suspension, which was then left at 25 ℃ for 1 hour with gentle stirring and centrifuged to obtain the supernatant. The concentration of substance C in the supernatant was quantified using High Performance Liquid Chromatography (HPLC) according to the method described in Bioscience, Biotechnology and Biochemistry, Vol.58 No.5, pp.945-946 (1994). The sample was diluted 101-fold with ethanol instead of water as a blank. At this point, the sample did not become a suspension and the dilution was treated in a similar manner to that described above for determining the concentration of substance C. Table 1 shows the results.

TABLE 1

Concentration of substance C (ppm)

Diluted supernatant with water 0.03

Dilute supernatant 46.7 (blank) diluted with ethanol

It is clear that, when the propolis extract was diluted 101 times with water to obtain a suspension containing a diluted aqueous ethanol solution, the concentration of the substance C that can be dissolved in the supernatant of the suspension was 0.03ppm as judged from the results in Table 1. The concentration thereof was only 0.06% of the substance C concentration of 46.7ppm of the blank supernatant obtained by 101-fold dilution with ethanol, which means that most of the substance C did not dissolve when diluted with water.

The precipitate collected by centrifugation was dissolved in ethanol and the amount of substance C in the precipitate was similarly determined, indicating that at least 99% of substance C in the sample was contained in the precipitate.

The results demonstrate that dilution of the propolis extract with water to a dilute aqueous ethanol solution results in insolubilization of most of the substance C. Experiment 2 improving the Water solubility of substance C

The sample of experiment 1 was mixed with an equal amount of 50 v/v% aqueous ethanol solution, with or without an appropriate amount of sodium hydroxide, and the mixture PH5.0 was adjusted to PH5.2, 5.5, 5.8, 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, 7.5 or 8.1 before preparing about 10 w/w% liquid propolis extract, d.s.b., as a sample for the following experiment. Experiment 2- (1) insolubility of propolis extract

Samples prepared in experiment 2 at different pH values were diluted 101-fold to aqueous ethanol dilute solution in the form of white suspension, and the effect of pH on propolis extract insolubility was investigated using a 1-cm path cell to measure turbidity or absorbance at 720 nm. Experiment 2- (2) Water solubility of substance C in propolis extract

In a similar manner to experiment 1, samples of different pH values were diluted 101-fold with water, the resulting white suspension was centrifuged, and the concentration of substance C in the resulting supernatant was measured to investigate the effect of pH on the water solubility of substance C in the propolis extract. Experiment 2- (3) color number

Samples of different pH values remained in solution form even after 21-fold dilution with ethanol, and the effect of pH on the color and deterioration of propolis extract was investigated using the resulting dilutions measured at 480nm using a 1-cm path cell. Experiment 2- (4) A_310nm/A_660nmAbsorption ratio of

As conditions for maintaining the quality of propolis extract stable, the pH of the sample was investigated for A_310nm/A_660nmThe influence of the absorbance ratio. Experiment 2- (5)

After standing at 25 ℃ for 1 month, the samples were subjected to the experiment in a manner similar to that of experiments 2- (1) -2- (4), and sensory evaluation was carried out by 12 criticizing groups. The data are presented in table 2.

TABLE 2

Test number	During preparation						After being placed for 1 month						General evaluation
															X	A	B	C	D	E	A	B	C	D	E	Flavor and taste**
	1	5.0	0.224	0.03	1	0.197	7,365	0.230	0.03	1	0.210	6,918															Is satisfactory in	Not good	Blank sample
2													5.2	0.202	0.06	2	0.197	7,187	0.205	0.07	2	0.213	6,729	Is satisfactory in	Not good	Blank sample
	3	5.5	0.188	0.45	15	0.198	6.514	0.188	0.45	15	0.215	6,375															Is satisfactory in	Good taste	The invention
4													5.8	0.176	2.30	77	0.204	6,434	0.180	2.38	79	0.224	6,213	Is satisfactory in	Is excellent in	The invention
	5	6.0	0.144	4.28	143	0.210	6,378	0.148	4.37	146	0.231	6,023															Is satisfactory in	Is excellent in	The invention
6													6.2	0.134	8.40	280	0.221	6,015	0.136	8.48	283	0.245	5,682	Is satisfactory in	Is excellent in	The invention
	7	6.4	0.101	13.6	453	0.248	5,659	0.102	13.8	460	0.266	5,054															Is satisfactory in	Is excellent in	The invention
8													6.8	0.078	18.7	623	0.260	5,401	0.078	18.9	630	0.282	4,868	Are mostly satisfactory	Good taste	The invention
	9	7.0	0.058	24.1	803	0.283	4,973	0.059	24.2	807	0.305	4,054															Are mostly satisfactory	Good taste	The invention
10													7.2	0.051	28.3	943	0.303	4,614	0.053	28.3	943	0.399	3,860	Slightly pungent and bad smell	Not good	Blank sample
	11	7.5	0.049	31.4	1,047	0.329	4,303	0.050	31.0	1,033	0.458	3,675															Bad pungent smell	Not good	Blank sample
12													8.1	0.041	36.4	1,213	0.464	2,935	0.044	36.2	1,207	0.588	2,320	Bad pungent smell	Not good	Blank sample

In Table 2, the symbols "X", "A", "B", "C", "D" and "E" are as follows: x: pH of propolis extract; a: haze at 720mm (A720 nm); b: concentration of substance C in the supernatant (ppm); c: the concentration ratio of the substance C in the supernatant to the substance C in test No.1, i.e., to the substance C in the aqueous ethanol solution without pH adjustment; d: chroma at 480nm (A480 nm); e: a310nm/A660nm absorbance ratio. The symbols ". star" and ". star" represent "no PH control" and "evaluation summary of 10 or more out of 12 evaluation groups", respectively.

The results in Table 2 clearly show that an increase in the pH (pH5.0) of the propolis extract from 5.0 to 5.5-7.0, particularly preferably to 5.8-6.4, results in a high quality propolis extract with improved water solubility of substance C and satisfactory stability, flavor and taste.

Experiment 2- (1) shows that as the pH of the samples increased, their turbidity decreased, the water solubility increased, in particular up to a pH of about 7.0, the turbidity decreased and the water solubility improved, and that at a pH above about 7.0 the turbidity hardly decreased, i.e. substantially no further increase in water solubility was observed. Experiment 2- (2) showed that when the pH was adjusted to exceed 5.5, the water solubility of substance C, an effective ingredient of propolis, was greatly increased. Experiment 2- (3) shows that the sample is darker in color especially at a pH exceeding 7, and this tendency is more pronounced during storage, causing the sample to deteriorate. It also shows that the samples emit pungent, unpleasant and/or deteriorated flavors and tastes when stored at a PH above 7.

Experiment 2- (4) shows that the samples must be stored at pH7.0 or less so that they remain of high quality or A even after one month of storage_310nm/A_660nmThe ratio exceeds 4,000.

As described above, by adjusting the PH of the propolis extract to 5.5 to 7.0, more preferably to 5.8 to 6.4 by adding a PH controlling agent, satisfactory propolis extract can be easily produced, and also stability, flavor and taste with satisfactory effects, in which substance C has improved water solubility as an effective ingredient of propolis. Experiment 3 acute toxicity test

The lyophilized preparation of liquid propolis extract having pH6.4 obtained by the method of example A-1 was orally administered to mice for acute toxicity test using seven-week-old dd-bred mice. As a result, no mice died when the dose reached 2.5 tg/kg. This indicates that the preparation has very low toxicity. Similar tests were carried out using the liquid propolis extract freeze-dried product obtained in the method of example A-4, having a pH of 6.2, with similar results, which means that the product is also extremely low in toxicity.

The following examples a and B illustrate the propolis extract with improved water solubility of the present invention and compositions containing such propolis extract as an effective ingredient: example A-1

Soaking propolis in 95 v/v% aqueous ethanol solution and extracting in conventional manner to obtain extract, washing the precipitate with small amount of water to extract precipitate, collecting the extracts into crude propolis extract containing about 20 w/w% dry solids in 80 v/v% aqueous ethanol solution, and mixing with waterTo reduce the ethanol concentration to 50 v/v%. The diluted solution was allowed to stand at 50 deg.C for one hour to separate into two layers, the upper layer having the effective components of propolis, and the remaining lower layer having a viscous sediment. The two layers were allowed to stand at room temperature overnight, and the upper layer was collected and mixed with sodium bicarbonate to adjust the pH to 6.4, followed by recovery of a gel having 8,425A_310nm/A_660nmAn absorbance ratio and an improved water-soluble liquid propolis extract yield of about 45%, d.s.b. of the crude propolis extract.

The product containing the substance C having improved water solubility and satisfactory stability, flavor and taste can be used alone or in combination with other materials for foods, anti-infectious diseases and cosmetics as an adjuvant health food, antiseptic, antioxidant, immunomodulator, macrophage activating factor, antitumor agent or hair repair agent. Example A-2

Crushing propolis pieces, injecting into two columns, adding 50 v/v% aqueous ethanol solution for dissolving, and extracting propolis effective components by countercurrent extraction method. The resulting extract was filtered and mixed with sodium carbonate to adjust the pH to 6.2. Thus, a polycarbonate having a molecular weight of 6,015A was obtained_310nm/A_660nmAn absorbance ratio and an improved water-soluble liquid propolis extract yield of about 32%, d.s.b. of raw propolis.

Similar to the preparation of example A-1, the product contained substance C with improved water solubility, with satisfactory stability, flavour and taste. Therefore, the product can be used alone or in combination with other materials for food, anti-infectious diseases and cosmetics, as adjuvant health food, antiseptic, antioxidant, immunomodulator, macrophage activating factor, antitumor agent or hair repairing agent. Examples A to 3

Breaking propolis pieces, injecting into three columns, and dissolving in 50 v/v% aqueous ethanol solution adjusted to pH 7.8 with sodium carbonate to extract propolis effective components by countercurrent extraction method. Adjusting the pH of the extract to 6.0 with a pH control agent, and filtering to obtain a filtrate having a pH of 6,438A_310nm/A_660nmAbsorbency ratio and improved water solubilityA sexual liquid propolis extract. The yield is about 32 percent of the raw material propolis, and d.s.b.

Similar to the preparation of example A-1, the product contained improved water-soluble substance C with satisfactory stability, flavour and taste. Therefore, the product can be used alone or in combination with other materials for food, anti-infectious diseases and cosmetics, as adjuvant health food, antiseptic, antioxidant, immunomodulator, macrophage activating factor, antitumor agent or hair repairing agent. Examples A to 4

A crude propolis extract was obtained by the method of example A-1, and the ethanol concentration of an 80 v/v% aqueous ethanol solution containing the crude propolis extract was reduced to 45 v/v% according to the method of example A-1 to be separated into two layers, i.e., an upper layer and a lower layer, which were then allowed to stand at room temperature for 5 hours. The upper layer was collected and diluted with an equal amount of water and the dilution was sent to a container filled with "AMBERLITE XAD-7" (a Rohm from Philadelphia, USA)&Synthetic macroporous resin commercially available from Haas corporation), washed with water, and fed into a 95 v/v% aqueous ethanol solution to elute the effective components of propolis. Mixing the eluate with sodium bicarbonate, adjusting pH to 6.2 to obtain a gel having 21,892A_310nm/A_660nmAn absorbance ratio and a liquid propolis extract with improved water solubility. The yield is about 22% of the crude propolis extract, d.s.b.

Similar to the preparation of example A-1, the product contained improved water-soluble substance C with satisfactory stability, flavour and taste. Therefore, the product can be used alone or in combination with other materials for food, anti-infectious diseases and cosmetics, as adjuvant health food, antiseptic, antioxidant, immunomodulator, macrophage activating factor, antitumor agent or hair repairing agent. Example B-1 gummy candy

One hundred parts by weight of "MABIT " (hydrogenated malt syrup commercially available from Okayama, Hayshibara Shoji Co., Japan) and 50 parts by weight of "TREHOSE^TM"(high purity aqueous trehalose crystals commercially available from Okayama, Hayshibaashoji Co., Japan) was heated and concentrated under reduced pressure to have a crystalline structure havingA concentrate of about 15 w/w% moisture content. The concentrate was mixed with 13 parts by weight of gelatin dissolved in 18 parts by weight of water, one part by weight of the liquid propolis extract obtained by the method of example a-1, 2 parts by weight of sodium citrate and appropriate amounts of coloring agent and flavor in a conventional manner. The mixture thus obtained is shaped and packaged into the desired product.

The product is a gummy candy having satisfactory texture and flavor and causing no caries. Example B-2 chewing Gum

Melting three parts by weight of gum base by heating until it softens, mixing the obtained mixture with 4 parts by weight of sucrose and 3 parts by weight of "TREHOSE^TM", 0.02 part by weight of the liquid propolis extract obtained by the method of example A-1 and an appropriate amount of a coloring agent were mixed, the mixture was kneaded by means of a roller, formed and packaged to obtain a desired product.

The product is a chewing gum having satisfactory texture, flavor and taste and causing no dental caries due to the effective ingredient of propolis. Example B-3Gyuhi (starch paste)

Mixing a part by weight of glutinous rice starch with 1.2 parts by weight of water, and gelatinizing the mixture by heating, and further mixing with 1.5 parts by weight of "TREHOSE^TM", 0.7 parts by weight of" SUNMALT  "(aqueous maltose crystals commercially available from Okayama, Hayshibara Shoji Co., Japan), 0.3 parts by weight of starch hydride and 0.02 parts by weight of the liquid propolis extract obtained by the method of example A-2. The mixture thus obtained is shaped in a conventional manner and packaged into the desired product.

The product is a Japanese confectionery having a natural flavor and taste, and further has satisfactory chewing characteristics, relatively long shelf life and stability due to the effective ingredient of propolis. Example B-4 creamy confection filling

One thousand parts by weight of "FINETOSE " (anhydrous crystalline maltose commercially available from hayshibaashoji, Okayama, japan), 200 parts by weight of dehydrated crystalline trehalose, 1,000 parts by weight of shortening, 50 parts by weight of cacao mass, 3 parts by weight of the liquid propolis extract obtained by the method of example a-3, and one part by weight of lecithin were mixed in a conventional manner to obtain a cream paste filling.

The product, which has satisfactory stability and shelf life due to the propolis active ingredient, has desirable chewing characteristics and palatable meltability. Example B-5 cream cake

Fifty parts by weight of unsalted cream, 50 parts by weight of shortening, 50 parts by weight of honey and 130 parts by weight of sugar were thoroughly mixed, and further mixed and stirred with 150 parts by weight of eggs. The resulting mixture was mixed with 135 parts by weight of wheat flour, 75 parts by weight of milk, 4 parts by weight of sodium bicarbonate and an appropriate amount of vanilla flavor. The mixture thus obtained is poured into moulds, baked and cooled to room temperature in a conventional manner. A slurry prepared by mixing 1.5 parts by weight of the liquid propolis extract obtained by the method of example A-1, 20 parts by weight of Japanese apricot liqueur and 20 parts by weight of Connectran was applied with a brush to the surface of the above-mentioned baked product to obtain the desired product.

The product having satisfactory flavor and taste and containing propolis effective component can be used arbitrarily for maintaining and/or promoting health and preventing and/or treating diseases, and also for promoting recovery from diseases. Example B-6 Ice cream

Twenty-three hundred parts by weight of milk are heated to about 60 ℃ and mixed at this temperature with 200 parts by weight of egg yolk, 50 parts by weight of egg, 420 parts by weight of fructose, 30 parts by weight of starch hydride, 200 parts by weight of fresh milk fat, 20 parts by weight of evaporated milk, 3 parts by weight of the liquid propolis extract obtained by the method of example a-2 and one part by weight of gelatin powder. The mixture was heat-sterilized at 75 ℃ for 15 minutes, cooled, mixed with 20 parts by weight of Japanese apricot liqueur under stirring, poured into a container and frozen to obtain the desired product.

The product containing propolis effective component can be used optionally for maintaining and/or promoting health and preventing and/or treating diseases, and also for promoting health recovery from diseases. Example B-7 powdered propolis extract

Five parts by weight of the liquid propolis extract obtained by the method of example A-2 and 7 parts by weight of dehydrated crystalline maltose were mixed, and the mixture was dried by heating to 40 ℃ for 1 hour, and further mixed uniformly with 3 parts by weight of dehydrated crystalline trehalose and 0.05 part by weight of sodium bicarbonate to obtain a powder propolis extract. 1g aliquots of powder were distributed in layered aluminum containers.

Since the product is easily dispersed and dissolved in water and has an advantage of satisfactory taste, it can be used alone as a health food, a preservative, a drug, a flavor imparting agent, a deodorant and an agent for urinary therapy, and can also be used as a raw material for other compositions. The product can also be used for maintaining and/or promoting health and preventing and/or treating diseases, and can also promote recovery from diseases.

When used as a health food, the product can be used alone or after being dispersed and dissolved in a liquid such as tea, milk or fruit juice in an amount of 0.2-1g per 200ml of the liquid. The product can also be dissolved in fresh urine in an amount of 0.5-2g per 100ml urine for use in urotherapy. Example B-8 powdered propolis extract

To 5 parts by weight of the liquid propolis extract obtained by the method of example A-4, 2 parts by weight of gamma-cyclodextrin and 5 parts by weight of dehydrated crystalline trehalose were added. The mixture was dried by heating to 40 ℃ for one hour in air, and then mixed with 7 parts by weight of dehydrated crystalline maltose powder until uniform to obtain a powdery propolis extract. 1g aliquots of powder were distributed in layered aluminum containers.

Similar to the product of example B-7, this product was easily dispersed and dissolved in water and had the advantage of a pleasant taste. Therefore, the product can be used alone as a health food, a preservative, a drug, a flavor imparting agent, a deodorant and an agent for urinary therapy, and can also be used as a raw material of other compositions. The product can also be used for maintaining and/or promoting health and preventing and/or treating diseases, and can also promote recovery from diseases. Example B-9 Compound feed

A compound feed was prepared by mixing 30 parts by weight of wheat bran powder, 30 parts by weight of the powdered propolis extract obtained by the method of example B-7, 10 parts by weight of skim milk, 10 parts by weight of a milk sucrose-containing powder, 10 parts by weight of multivitamins, 5 parts by weight of fish meal, 5 parts by weight of calcium hydrogen phosphate, 3 parts by weight of liquid oils and fats, 3 parts by weight of calcium carbonate, 2 parts by weight of salt and 2 parts by weight of minerals.

The product exhibits satisfactory growth-promoting activity against bifidobacteria in addition to the activity of the effective ingredient of propolis, and it can be used arbitrarily to prevent infectious diseases and diarrhea of domestic animals, promote their appetite and growth, and prevent the unpleasant odor of their feces. If desired, the product can be used in combination with other feed materials such as concentrates of grains, wheat flour, starch, oil meal and wine lees or meal, and materials for coarse cellulosic foods such as straw, hay, bagasse, corn cobs and silage thereof. Example B-10 tablets

30 parts by weight of the powdery propolis extract obtained by the method of example B-7, 20 parts by weight of "NYUKAOLIGO  LS-55P" (a milk sucrose-containing powder commercially available from Okayama, Hayshibara Shoji Co., Japan), 10 parts by weight of calcium lactate, 5 parts by weight of L-ascorbic acid, 5 parts by weight of pullulan, 1 part by weight of α -glycosyl rutin, 0.2 parts by weight of magnesium carbonate, 1 part by weight of calcium tetraphosphate, 1 part by weight of sugar ester and a suitable amount of powdery flavor were mixed to be uniform. The mixture was tabletted by a tabletting machine with 20R perforations and a diameter of 12mm to give tablets. Using a sugar coating machine, heating to 55 deg.C with a sugar coating machine containing 55 w/w% "TREHOSE^TM"and 0.2 w/w% pullulan in water, and dried. The coating step was repeated to obtain sugar coated tablets with a smooth surface containing crystalline aqueous crystalline trehalose.

Generally, this product, which does not break and has satisfactory stability and swallowing properties, is orally administered to humans at a dose of about 1-40 tablets/day/adult, preferably at a dose of about 2-20 tablets/day/adult. Since the product has the activity of the effective ingredient of propolis and is rich in lactosucrose and pullulan, it shows strong intestine-controlling activity. The product is therefore satisfactory for use as an orally administrable preparation for the prevention of colon cancer. Since the product is rich in calcium salts, it can be suitably used as an orally administrable preparation for the prevention and/or treatment of osteoporosis, and since the product is rich in L-ascorbic acid and alpha-glycosylrutin, it can also be satisfactorily used as an orally administrable preparation for the promotion of health and the prevention of aging. Example B-11 tablets

10 parts by weight of the powdery propolis extract obtained by the method of example B-8, 70 parts by weight of trehalose, 62 parts by weight of corn starch and 8 parts by weight of sucrose fatty acid ester were mixed until uniform, and tabletted in a similar manner to example B-10 to obtain tablets. To make tablets that are readily dissolvable in the stomach, the product is sugar coated after precoating with 5 w/w% carboxypropylmethylcellulose, while the product is coated with 10 w/w% carboxypropylmethylcellulose to make enteric coated tablets.

These tablets can be suitably used as agents for the treatment of human malignancies such as gastric cancer, lung cancer, liver cancer, uterine cancer, breast cancer, colon cancer and melanoma. Example B-12 pharmaceutical preparations for external use

To 300 parts by weight of maltose powder and 200 parts by weight of trehalose, 20 parts by weight of the propolis effective component-containing upper layer solution obtained by the method of example A-4 and 33 parts by weight of methanol were added, and further 200 parts by weight of 10 w/w% pullulan solution was added to obtain an ointment for external wounds having suitable extensibility and stickiness.

The product exhibits satisfactory antiseptic, anti-inflammatory, anti-tumor and local anesthetic activities due to the propolis effective ingredient, and exhibits an activity of supplementing energy to active cells due to maltose and trehalose. Due to these activities, the product shortens the healing time and satisfactorily allows the injured skin tissue to heal. Examples B-13 injection solutions

The liquid propolis extract obtained by the method of example a-4 was dissolved in 30 v/v% aqueous ethanol, the resulting solution was subjected to membrane filtration in a conventional manner to obtain a solution containing no pyrogen, which was then distributed to 20ml ampoules to obtain a propolis extract of 5 mg/ampoule, and d.s.b., which was lyophilized and sealed to obtain the desired product.

The product may be used alone or in combination with other vitamins and/or minerals prior to intramuscular or intravenous administration to a human. The product can be used as desired to facilitate the treatment of diseases such as immune diseases, circulatory diseases, neurological diseases and malignancies, and is often used to restore health from these diseases. Examples B to 14 injection solutions

Six parts by weight of sodium chloride, 0.3 part by weight of potassium chloride, 0.2 part by weight of calcium chloride, 3.1 parts by weight of sodium lactate, 45 parts by weight of trehalose and 0.1 part by weight of the liquid propolis extract obtained by the method of example A-4 were dissolved in 1,000 parts by weight of a 2 v/v% aqueous ethanol solution. The resulting solution was subjected to membrane filtration in a conventional manner to give a solution free of pyrogen, which was aliquoted in 25ml portions into plastic containers to give the desired product.

The product can be used as vitamin P supplement, energy supplement or mineral supplement to promote the treatment of disease and to promote the recovery of health from disease. Example B-15 cannula-feed reagents

A composition was prepared by mixing 20 parts by weight of dehydrated trehalose, 1.1 parts by weight of glycine, 0.18 parts by weight of sodium glutamate, 1.2 parts by weight of salt, one part by weight of sodium citrate, 0.4 parts by weight of calcium lactate, 0.1 parts by weight of magnesium carbonate, 0.1 parts by weight of powdered propolis extract obtained by the method of example B-7, 0.01 parts by weight of thiamine, and 0.01 parts by weight of riboflavin. A 24g aliquot of the composition was dispensed into a multi-layer aluminum pouch and the pouch was heat sealed to give a reagent for cannula feeding.

The product can be used as an energy supplement solution, and a bag of the product dissolved in about 300-500ml of water can be administered orally or through a cannula for parenteral administration to the nasal cavity, stomach or intestine. Example B-16 bath salt

21 parts by weight of DL-sodium lactate, 8 parts by weight of sodium pyruvate, 3 parts by weight of trehalose, 5 parts by weight of the liquid propolis extract obtained by the method of example A-3, 37 parts by weight of ethanol and 26 parts by weight of purified water, and a suitable amount of a coloring agent and a suitable essence were mixed to obtain a bath salt.

The product can be dissolved in a bath table (tab) by hot water to be diluted by 10,000 times by 100 times, and can be used as a skin cleaning agent or a skin whitening agent. Similarly, the product may be diluted in a wash solution or a wash solution prior to use. Examples B-17 washing milk

Half part by weight of polyoxyethylene behenyl ether, one part by weight of polyoxyethylene sorbitol tetraoleate, one part by weight of oil-soluble glycerol monostearate, 0.5 part by weight of pyruvic acid, 0.5 part by weight of behenyl alcohol, one part by weight of avocado oil, one part by weight of the liquid propolis extract obtained by the method of example a-2, and an appropriate amount of vitamin E and preservative were dissolved by heating in a conventional manner. With 0.5 part by weight of sodium L-lactate, 0.5 part by weight of trehalose, 5 parts by weight of 1, 3-butenediol, 0.1 part by weight of a carbonyl vinyl polymer, and 84.3 parts by weight of purified water and the above mixture, blending was conducted, and emulsification was conducted by a homogenizer. Mixing the emulsion with appropriate amount of perfume to obtain washing milk.

The product can be used as sunscreen, skin cleanser or skin whitening agent. Example B-18 emulsifiable concentrate

Two parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glycerin monostearate, 2 parts by weight of the liquid propolis extract obtained by the method of example a-3, one part by weight of liquid paraffin, 10 parts by weight of glyceryl tricaprylate, and a suitable amount of preservative. The mixture was dissolved by heating, and then mixed with 2 parts by weight of trehalose, 5 parts by weight of 1, 3-butenediol and 66 parts by weight of refined water, followed by emulsifying the mixture by a homogenizer and blending with an appropriate amount of a perfume to obtain an emulsifiable concentrate.

The product can be used as sun-shading cream, skin cleaning agent or skin whitening agent. Example B-19 shampoos

A shampoo was prepared by dissolving by heating 1.0 part by weight of the liquid propolis extract obtained by the method of example a-4, 0.2 part by weight of an alkyldiaminoethyl glycerol hydrochloride solution, 20.0 parts by weight of lauryl dimethyl glycine betaine, 25.0 parts by weight of lauroyl methyl tauride (tauride), and 52.0 parts by weight of refined water, appropriate amounts of a preservative and a perfume, by heating and dissolving to obtain a shampoo.

Since the product has satisfactory shampoo properties and less damage to hair, it can be used arbitrarily to promote the growth of hair or hair of animals including humans, and to treat and/or prevent dandruff, tinea capitis, and alopecia. Example B-20 damping

Two half parts by weight of the liquid propolis extract obtained by the method of example A-1, 2.0 parts by weight of distearyldimethylammonium chloride, 2.0 parts by weight of cetyl alcohol, 2.0 parts by weight of silicone oil, 1.0 part by weight of polyoxyethylene oleyl alcohol ether and an appropriate amount of perfume were dissolved by heating. A mixture consisting of 3.0 parts by weight of 1, 3-butenediol, 89.0 parts by weight of purified water and an appropriate amount of a preservative was mixed with the above mixture under stirring, and the resulting mixture was allowed to cool and stand to obtain a touch-down.

The product is effective for rinsing hair and can be used optionally for promoting hair growth of animal including human, and treating and/or preventing dandruff, tinea capitis and alopecia. Examples B-21 Hair Reinforcement

Two parts by weight of "α G RUTIN" (α -glycosylrutin commercially available from Tokyo, Toyo Sugar Refining co., ltd.) and 20.0 parts by weight of glycerin were mixed and dissolved with 550 parts by weight of purified water heated to 60 ℃, and the mixture was mixed with a solution prepared by dissolving 0.05 parts by weight of "KANKOSO 301(6- [2- [ 5-bromo-2-pyridyl) amine ] vinyl ] -1-ethyl-2-methylpyridinium iodide" (cyanine dye commercially available from Okayama, photosensifying Dyes co., ltd.) in 440 parts by weight of ethanol and 10 parts by weight of the liquid propolis extract obtained by the method of example a-2, and a solution prepared by dissolving 2.0 parts by weight of 1-menthol in 10 parts by weight of ethanol. Filtering the obtained mixture and bottling to obtain the hair strengthening agent.

The product is effective in promoting hair growth and treating and/or preventing alopecia and alopecia, and it can be satisfactorily used for preventing dandruff and tinea capitis, and also as a therapeutic agent for trauma due to its satisfactory antiseptic and anti-inflammatory activities. Example B-22 shampoo

A composition was prepared by mixing 55.0 parts by weight of ethanol, 20.0 parts by weight of polyoxypropylene (40) butyl ether, and 13.0 parts by weight of purified water and 1.0 part by weight of the liquid propolis extract obtained by the method of example A-1, and 3.0 parts by weight of 2-0-alpha-D-glucopyranosyl-L-ascorbic acid. The composition is mixed with appropriate amounts of a pH controlling agent, a fragrance and a preservative in a conventional manner to obtain a shampoo.

The product is effective in promoting hair growth and treating and/or preventing alopecia and alopecia, and it can be satisfactorily used for preventing dandruff and tinea capitis, and also as a therapeutic agent for trauma due to its satisfactory antiseptic and anti-inflammatory activities. Example B-23 shampoo

A composition was prepared by mixing 3.0 parts by weight of beeswax, 15.0 parts by weight of vaseline, 42.0 parts by weight of liquid paraffin, 3.0 parts by weight of polyoxyethylene (5) stearate, 2.0 parts by weight of polyoxyethylene (6) oleyl ether, 1.0 part by weight of polyoxyethylene (6) cetyl ether, 1.0 part by weight of the liquid propolis extract obtained by the method of example A-3, 0.3 part by weight of 2-0-alpha-D-glucopyranosyl-L-ascorbic acid and 32 parts by weight of purified water. The shampoo is obtained by mixing the composition with appropriate amounts of pH controlling agent, perfume and antiseptic in a conventional manner.

The product can be used for promoting hair growth and treating and/or preventing alopecia and alopecia, dandruff and tinea capitis. Example B-24 toothpaste

A toothpaste was prepared consisting of the following ingredients. The product with preservative activity can be satisfactorily used for preventing halitosis and dental caries. Composition comprising a metal oxide and a metal oxide

Divalent 45.0 parts by weight of calcium phosphate

2.94 parts by weight of pullulan

Sodium lauryl sulfate 1.5 weight parts

20.0 parts by weight of glycerin

Polyoxyethylene sorbitan lauryl ester 0.5 part by weight

0.05 part by weight of preservative

12.0 parts by weight of the liquid propolis extract obtained by the method of example A-3

Maltitol 5.0 parts by weight

13.0 parts by weight of water

As described above, the present invention relates to a propolis extract having an improved water-soluble propolis effective ingredient, particularly, substance C, and provides a propolis extract which easily exerts the activity of the propolis effective ingredient and has satisfactory stability, flavor and taste.

The propolis extract is not limited to use in conventional highly irritant and water-soluble organic solvent high-concentration solutions, but it may be used in lower-concentration solutions of these organic solvents as desired. Therefore, the propolis extract can be arbitrarily used in compositions such as food products, agents against susceptible diseases, and cosmetics.

Has a molecular weight of more than 4,000A_310nm/A_660nmThe present propolis composition, which has an absorbance ratio and satisfactory color, flavor and taste, will exert a profound influence on the fields of food products, cosmetics and pharmaceuticals.

While there have been described what are believed to be the preferred embodiments of the invention, it will be apparent that various modifications may be made thereto, and it is intended to cover in the appended claims all such modifications as fall within the true spirit and scope of the invention.

Claims (22)

1. In the propolis extract, the improvement wherein said extract has a pH of 5.5-7.0 to improve the water solubility of the propolis effective ingredient.

2. The propolis extract as claimed in claim 1, which is in the form of an aqueous ethanol solution having a concentration of about 0.5 v/v% ethanol and containing at least 0.45ppm of 3- [ 4-hydroxy-3, 5-bis (3-methyl-2-butenyl) phenyl ] -2-acrylic acid as an effective ingredient of said propolis at 25 ℃.

3. The propolis extract as claimed in claim 1, which has an A of more than 4,000 when the absorbance at 310nm and 660nm is converted into the absorbance of the propolis extract at a concentration of 2 w/w%_310nm/A_660nmAbsorbance ratio, wherein the concentration is based on dry solids.

4. The propolis extract of claim 1, which is prepared by using an organic solvent that is easily soluble in water, the organic solvent being selected from the group consisting of acetone, acetic acid, methanol, ethanol, isopropanol and a mixture thereof.

5. The propolis extract of claim 1, wherein the pH adjustment of said propolis extract is achieved by using a pH controlling agent selected from the group consisting of magnesium oxide, calcium oxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate and magnesium carbonate and mixtures thereof.

6. A method for producing a propolis extract, comprising (i) contacting propolis with an aqueous solution of a readily water-soluble organic solvent without pH adjustment, dissolving and extracting an effective ingredient of propolis, adjusting the pH of the obtained propolis extract to 5.5-7.0 by adding a pH controlling agent, and collecting the propolis extract; or (ii) contacting propolis with an aqueous solution of an organic solvent which is easily soluble in water and has a pH adjusted to 5.5 or higher by adding a pH controlling agent, dissolving and extracting the effective components of propolis, optionally adjusting the pH of the resultant extract to 5.5-7.0, and collecting the propolis extract having a pH of 5.5-7.0.

7. The method of claim 6, wherein said propolis is dissolved and extracted by a mode selected from the group consisting of a soaking extraction mode and a counter-current extraction mode.

8. The method of claim 6, wherein said propolis extract is in the form of an aqueous ethanol solution having a concentration of less than 1 v/v% ethanol and containing at least 0.45ppm of 3- [ 4-hydroxy-3, 5-bis (3-methyl-2-butenyl) phenyl ] -2-acrylic acid as an effective ingredient of said propolis at 25 ℃.

9. The method as claimed in claim 6, wherein the propolis extract has an A of more than 4,000 when the absorbance at 310nm and 660nm is converted into the absorbance of the propolis extract at a concentration of 2 w/w%_310nm/A_660nmAbsorbance ratio, wherein the concentration is based on dry solids.

10. The method of claim 6, wherein said water-miscible organic solvent is selected from the group consisting of acetone, acetic acid, methanol, ethanol, isopropanol, and mixtures thereof.

11. The process of claim 6 wherein said pH controlling agent is selected from the group consisting of magnesium oxide, calcium oxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate and magnesium carbonate and mixtures thereof.

12. A composition comprising propolis extract as an active ingredient at pH of 5.5-7.0, wherein the composition is prepared by adjusting pH of propolis to 5.5-7.0 to improve water solubility of said propolis active ingredient.

13. The composition of claim 12, wherein said propolis extract is an aqueous ethanol solution having a concentration of less than 1 v/v% ethanol and containing at least 0.45ppm of 3- [ 4-hydroxy-3, 5-bis (3-methyl-2-butenyl) phenyl ] -2-acrylic acid as an effective ingredient of said propolis at 25 ℃.

14. The composition as claimed in claim 12, wherein the propolis extract has an a of more than 4,000 when the absorbance at 310nm and 660nm is converted into the absorbance of the propolis extract at a concentration of 2 w/w%_310nm/A_660nmAbsorbance ratio, wherein the concentration is based on dry solids.

15. The composition of claim 12, which is in the form of a food, cosmetic or pharmaceutical product.

16. The composition of claim 12, wherein said propolis extract is prepared by using an organic solvent readily soluble in water selected from the group consisting of acetone, acetic acid, methanol, ethanol, isopropanol and mixtures thereof.

17. The composition of claim 12 wherein the PH adjustment of said propolis extract is achieved by the use of a PH control agent selected from the group consisting of magnesium oxide, calcium oxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate and magnesium carbonate and mixtures thereof.

18. A method for improving the water solubility of the effective ingredients of propolis comprises a step of adjusting the pH of said propolis to 5.5-7.0 by adding a pH controlling agent.

19. The method of claim 18, dissolving at least 0.45ppm of 3- [ 4-hydroxy-3, 5-bis (3-methyl-2-butenyl) phenyl ] -2-acrylic acid as an effective ingredient of said propolis in an aqueous ethanol solution at a concentration of less than 1 v/v% at 25 ℃.

20. The method as claimed in claim 18, wherein the absorbance at 310nm and 660nm is converted to the absorbance of the propolis extract at a concentration of 2 w/w%, so that the propolis extract gives an A of more than 4,000_310nm/A_660nmAbsorbance ratio, wherein the concentration is based on dry solids.

21. The method of claim 18, wherein said PH adjustment of said propolis extract is effected in a readily water soluble organic solvent system selected from the group consisting of acetone, acetic acid, methanol, ethanol, isopropanol and mixtures thereof.

22. The process of claim 18 wherein said PH control agent is selected from the group consisting of magnesium oxide, calcium oxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and magnesium carbonate, and mixtures thereof.