JP4837686B2 - Supercritical extract, method for producing the same, method for detecting the same, food and beverage, cosmetics and pharmaceuticals containing the same - Google Patents

Description

  The present invention is a supercritical extract extracted from a propolis bulk as a raw material using a supercritical extraction method. Specifically, the supercritical extract is made of a specific alcohol. Furthermore, it is related with the manufacturing method of a supercritical extract, its detection method, the food-drinks containing it, cosmetics, and a pharmaceutical.

  Propolis is a glue-like or wax-like substance made by mixing saliva with plant exudates, shoots, and resins collected by bees for the purpose of defense and reinforcement of the nest. This propolis contains various components because bees are produced by collecting various plants around the nest box as raw materials.

  Antioxidant action and immunostimulatory action are known as main physiological activities of propolis. For this reason, propolis has long been used as a raw material for medicines and health foods in Europe, but since 1985, it has been used in many products for prevention and treatment of diseases in addition to health foods and cosmetics. Came to be used.

  Since it is difficult to ingest the propolis bulk as it is, useful components contained in the propolis bulk have been conventionally extracted by the method disclosed in Patent Document 1, for example. Specifically, an extraction method using a hydrophilic organic solvent, an extraction method using water, and a supercritical extraction method are used.

As active ingredients contained in propolis, very various kinds of active ingredients such as organic acids with high polarity, flavonoids, polyphenols, and terpenoids with low polarity have been confirmed. It is considered that the physiological activities of these various kinds of active ingredients act in a complex manner to form the excellent physiological activity of propolis. However, it is considered that the propolis bulk material contains many useful components other than the above.
JP 2003-61593 A

  In the propolis extract obtained by using a specific extraction method from the propolis bulk, the present invention has discovered a novel active ingredient that has not been previously known to be contained in the propolis bulk. Is based.

  The object of the present invention is a supercritical extract comprising a novel alcohol obtained from a propolis bulk using a supercritical extraction method, a production method thereof, a detection method thereof, and a food and drink containing the same, To provide cosmetics and medicines.

In order to achieve the above object, the supercritical extract of the invention described in claim 1 extracts a green propolis bulk, or a fraction insoluble in a hydrophilic organic solvent, water, or a mixture thereof from the propolis bulk. In a supercritical extract containing alcohols obtained by combining an extraction process using a supercritical extraction method and a process of hydrolyzing an ester bond as a raw material, the alcohols are 1-tetracosanol , 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol.

The method for producing a supercritical extract of the invention according to claim 2 is characterized in that a green propolis raw mass or a fraction insoluble in a hydrophilic organic solvent, water or a mixture thereof from the propolis raw mass is used as a raw material for extraction. A step of extracting a fatty acid ester compound using an extraction method, and then hydrolyzing the fatty acid ester compound to thereby produce 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1- It comprises a step of obtaining at least one alcohol selected from dotriacontanol, 1-tetratriacontanol, and lupeol.

According to a third aspect of the present invention, there is provided a method for producing a supercritical extract comprising a green propolis raw mass or a fraction insoluble in a hydrophilic organic solvent, water or a mixture thereof from the propolis raw mass as an extraction raw material. A step of hydrolyzing the ester bond of the ester compound contained therein, followed by extraction using a supercritical extraction method, whereby 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-tria It consists of a step of obtaining at least one alcohol selected from contanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol.

According to the method for detecting a supercritical extract of the invention described in claim 4, supercritical extraction is performed using a green propolis bulk or an insoluble fraction of the propolis bulk in a hydrophilic organic solvent, water or a mixture thereof as an extraction raw material. Extracting a component dissolved in a supercritical fluid using a method, then hydrolyzing an ester bond with respect to the extracted component, then separating using a chromatography, and 1-tetra It comprises a step of detecting at least one alcohol selected from cosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol. It is characterized by.

The method for detecting a supercritical extract of the invention according to claim 5 uses the green propolis raw mass, or a fraction insoluble in the hydrophilic organic solvent, water, or a mixture thereof from the propolis raw mass as an extraction raw material. A step of hydrolyzing the ester bond with respect to the components contained therein, then a step of extracting components dissolved in the supercritical fluid using a supercritical extraction method, and then using chromatography on the extracted components. And at least one selected from 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol It comprises a step of detecting the alcohol.

  The food / beverage product, cosmetic product or pharmaceutical product according to claim 6 contains the supercritical extract according to claim 1 as an active ingredient.

  According to the present invention, a supercritical extract comprising a novel alcohol obtained from a propolis bulk using a supercritical extraction method, a production method thereof, a detection method thereof, a food and drink containing the same, a cosmetic, and Drugs can be provided.

(First embodiment)
Hereinafter, a first embodiment embodying the supercritical extract of the present invention will be described.
Supercritical extract of the first embodiment, as the extraction raw material raw propolis mass or the like, an extraction process using supercritical extraction method, a hydrolyzing treatment of the ester bond, an alcohol obtained by combining Contains . More specifically, the alcohols include 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol. Can be mentioned. Of these, lupeol is a kind of triterpene alcohol, and other alcohols are policosanol (higher saturated monohydric alcohol having 24 to 34 carbon atoms). In propolis, many of these alcohols exist as ester compounds with fatty acids. Policosanol is ester-bonded with fatty acids such as palmitic acid, stearic acid, and oleic acid. Lupeol is ester-bonded with fatty acids such as tetracosanoic acid, hexacosanoic acid, octacosanoic acid, triacontanoic acid, and dotriacontanoic acid. The fatty acid ester compound of the alcohols present in the propolis constitutes a wax component in the propolis. The alcohols are novel active ingredients that have not been known to be contained in the propolis bulk.

  Propolis as a raw material is a glue-like or wax-like substance made by mixing saliva with plant exudates, shoots and bark etc. collected by bees such as honey bees for the purpose of defense and reinforcement of the nest. The production area of propolis used in this embodiment is not particularly limited, and uses South American countries such as China, Brazil, Argentina and Uruguay, Europe such as Hungary and Bulgaria, North America such as Canada, Oceania such as Australia and New Zealand, etc. be able to.

  The propolis bulk can be used as an extraction raw material in the form as it is. Further, an insoluble fraction (insoluble residue) of the propolis bulk with respect to the hydrophilic organic solvent, water, or a mixture thereof can be used as an extraction raw material. As the hydrophilic organic solvent used here, in addition to lower alcohols such as ethanol, methanol and isopropanol having the property of dissolving in water, ketones such as acetone and methyl ethyl ketone can be appropriately selected and used. These hydrophilic organic solvents can be used alone or in combination of two or more. Among these, ethanol, water, or a mixture thereof is most preferable in consideration of finally ingestion. When methanol other than ethanol is used as the solvent, it is preferable to completely remove the solvent in the post-treatment step.

  Moreover, when extracting using the said solvent, it is preferable to remove and grind | pulverize impurities, such as a dust mixed at the time of extraction, before an extraction process. When a mixture with ethanol or water is used as the solvent, the extraction temperature is preferably 5 to 40 ° C. When the extraction temperature is less than 5 ° C., the separation efficiency of the dissolved component and the insoluble component decreases, which is not preferable. Conversely, when the extraction temperature exceeds 40 ° C., the extraction solvent (ethanol) evaporates, leading to a reduction in extraction efficiency. Further, when water is used as the solvent, the extraction temperature is not particularly limited. In addition, what is necessary is just to perform extraction operation for about several hours, stirring at the said extraction temperature. And after extracting a melt | dissolution component on said extraction conditions, the insoluble fraction with respect to various solvents can be obtained from a propolis original lump by applying well-known solid-liquid separation methods, such as filtration and centrifugation. In the present embodiment, the insoluble fraction is extracted at a predetermined extraction temperature using various solvents as well as an insoluble fraction obtained when extraction is performed at a predetermined extraction temperature using various solvents. When the soluble fraction obtained at this time is allowed to stand at the extraction temperature or lower, or contains an insoluble component that precipitates after freezing and thawing of the solvent.

  Examples of the treatment for obtaining the alcohols from the propolis bulk include an extraction treatment using a supercritical extraction method and a treatment for hydrolyzing an ester bond. Any of the extraction process using the supercritical extraction method and the process of hydrolyzing the ester bond may be performed first in the order of the process, and may be performed at least once in the process. From the viewpoint of manufacturing cost, first, a process of hydrolyzing ester bonds is performed on the components contained in the raw material, and then a process of extracting components that are dissolved in the supercritical fluid using the supercritical extraction method is performed. Is preferred.

  First, the supercritical extraction method will be described. The supercritical extraction method can be carried out by using a known supercritical fluid extraction apparatus. A predetermined component is extracted from the extraction raw material by bringing the supercritical fluid into a supercritical state under the conditions of the critical temperature or higher and the critical pressure or higher and the extraction raw material. When carbon dioxide is used as a supercritical fluid, dissolved components are extracted from the extraction raw material by carbon dioxide that has become a supercritical fluid state at a critical temperature of 31.1 ° C. or higher and a critical pressure of 72.8 atm (7.4 MPa) or higher. Extracted. In this supercritical extract using carbon dioxide, ester compounds bonded to fatty acids of the above alcohols that have not been confirmed to be contained in propolis until now (hydrolysis of the ester bond was performed first. In the case, free alcohols) are contained.

  As the supercritical fluid, ethane, propane, carbon dioxide, nitrous oxide and the like can be used in addition to carbon dioxide, but it is most preferable to use carbon dioxide. This carbon dioxide has physical and chemical properties compatible with the compound to be extracted such that the critical temperature is close to room temperature and the polarity is lower than ethanol. Carbon dioxide is not only excellent in physical properties in the extraction process in this way, but also tasteless and odorless and does not affect the taste of the supercritical extraction product, so carbon dioxide is the most supercritical fluid used in this embodiment. preferable.

  For the operation in supercritical fluid extraction, the supercritical fluid is suitable for the temperature and pressure of the treatment because it uses the property that density and solubility greatly change with a slight temperature difference and pressure difference near the critical point. A vertical width is required. The operating temperature in the case of using carbon dioxide is preferably 32 to 80 ° C, more preferably 32 to 50 ° C. The operating pressure is preferably 73 to 500 atmospheres (7.4 to 50.7 MPa), more preferably 73 to 400 atmospheres (7.4 to 40.5 MPa). The flow rate (flow rate) of carbon dioxide as a supercritical fluid is preferably 1 to 10 kg / hour, more preferably 3 to 7 kg / hour, with respect to 1 kg of the propolis raw material. The processing time varies depending on the amount and state of the propolis raw material, but can be appropriately determined by confirming the time for completing the extraction from the test or the processing results.

  And the property of the supercritical extract of propolis extracted under the above extraction conditions also changes depending on the elapsed time of extraction. Most are paste-like, but some are obtained as powder or lump solid. When ethanol is used as an entrainer, it becomes a liquid containing ethanol. The supercritical extract contains ethanol contained in the extraction raw material immediately after extraction, ethanol as an entrainer, etc., and carbon dioxide solidified by adiabatic expansion. Furthermore, since it is obtained as a heterogeneous extract with different composition depending on the elapsed time of extraction, pure supercritical extraction can be obtained by removing ethanol and carbon dioxide with uniform stirring to form a solid supercritical extract and then grinding. It becomes a product powder.

  Next, the process which hydrolyzes an ester bond is demonstrated. As described above, most of the desired policosanols and lupeol are present as ester compounds ester-linked to fatty acids in the extraction raw material. The treatment for hydrolyzing the ester bond is performed in order to hydrolyze the ester bond of the ester compound and to liberate the target alcohol. When the supercritical extraction treatment is performed first, the desired policosanols and the ester compound in which lupeol and fatty acid are bonded are first obtained from the extraction raw material by the supercritical extraction treatment. Next, the target alcohol is obtained by hydrolyzing the ester compound. Moreover, when performing the process which hydrolyzes an ester bond before a supercritical extraction process, the hydrolyzate of the ester compound (ester bond) in an extraction raw material is first obtained. Next, the intended free alcohols are obtained by extraction using a supercritical extraction method.

  A known method can be appropriately employed for the hydrolysis treatment of the ester bond. For example, the method of heat-processing by adding water is mentioned. Moreover, the method of adding an alkali and performing a saponification process, and the method of using an acid and a hydrolase as a catalyst are mentioned.

  The obtained free alcohols may be applied as they are in each field, and only alcohols may be further removed using known chromatography, such as gas chromatography, liquid chromatography, supercritical fluid chromatography, and thin layer chromatography. It may be used after separation and purification.

  The supercritical extract composed of specific alcohols obtained as described above has not been confirmed to be contained in propolis until now and can be applied to various uses. The field of application of the supercritical extract of the present embodiment is not particularly limited, and is used, for example, by being blended in foods and drinks such as health foods, pharmaceuticals (including quasi-drugs), cosmetics, and the like. For example, when it is used in health foods, the excipient, brightener, gelling agent, thickener, sweetener, emulsifier, flavoring agent, pigment, pH within the range that does not impair the physiological activity of the ingredients. A regulator or the like may be added. The supercritical extract may be applied as it is as a liquid product such as a drink or tincture. Moreover, after mixing excipient | fillers, such as starch and dextrin, you may make it into a powder form by hot-air drying or freeze-drying. It may be powdered and supplied in a dosage form such as a powder or granule, supplied as a tablet in which powder or granule is compression-molded, or supplied as a capsule filled in a gelatin capsule. Moreover, in order to further improve the solubility or powder property of the alcohols in water, basic amino acids such as arginine and lysine may be blended and applied to various applications.

  As the alcohols, for example, 1-octacosanol is known to have an endurance improving action, a cholesterol lowering action, an antitumor action and the like during exercise. Ingestion of 1-octacosanol is expected to exert their actions and effects. Further, 1-triacontanol is known to have a fatty acid metabolism improving action, a vascular function improving action, an arteriosclerosis preventing action and the like. Ingestion of 1-triacontanol is expected to exert their actions and effects. Therefore, the supercritical extract of the present embodiment can be preferably applied to foods and drinks, pharmaceuticals and the like for the purpose of exerting their action and efficacy. Lupeol as triterpene alcohol is known to have antitumor action, anti-inflammatory action, antiviral action, antibacterial action, melanin production promoting action and the like. Therefore, the supercritical extract of the present embodiment can be preferably applied to foods and drinks, cosmetics, pharmaceuticals and the like for the purpose of exerting their action and efficacy.

According to the supercritical extract of the first embodiment, the following effects can be obtained.
(1) In this embodiment, by using a propolis bulk as a raw material, the combination of an extraction process using a supercritical extraction method and a process of hydrolyzing an ester bond has been included in the propolis so far. Alcohols that were not known were obtained. Therefore, supercritical extracts composed of these alcohols can be easily applied to various fields such as foods and drinks, cosmetics, and pharmaceuticals.

  (2) In the present embodiment, an insoluble fraction (insoluble residue) in the hydrophilic organic solvent of propolis bulk, water or a mixture thereof may be used as an extraction raw material as an extraction raw material. The insoluble fraction of the propolis bulk insoluble in the hydrophilic organic solvent or the like also contains the fatty acid ester compound comprising the alcohol and the fatty acid of the present embodiment. Therefore, an extract with less impurities can be obtained by removing the impurities to some extent and then performing the extraction treatment.

(3) In the present embodiment, carbon dioxide can be preferably used as the supercritical fluid. Therefore, the extract can be safely applied to the living body.
(4) In the present embodiment, a supercritical extract composed of specific alcohols such as 1-tetracosanol was obtained by combining supercritical extraction from a propolis bulk and a process of hydrolyzing ester bonds. . Therefore, it can be obtained more efficiently than other extraction methods.

  (5) In the present embodiment, a supercritical extract composed of specific alcohols such as 1-tetracosanol was obtained by combining supercritical extraction from a propolis bulk with a process of hydrolyzing an ester bond. . Therefore, the taste derived from the wax component can be suppressed, and the taste of the propolis extract can be improved.

(6) In the present embodiment, when an enzyme is used as a catalyst for the ester bond hydrolysis treatment, it can be more safely applied to food and drink.
(7) Since the alcohols obtained by the hydrolysis treatment are hydrolyzed as compared with the fatty acid ester compound that is ester-bonded with the fatty acid, the absorbability to the living body can be further improved.

  (8) Alcohols obtained by the hydrolysis treatment can significantly improve water dispersibility as compared with fatty acid ester compounds ester-bonded to fatty acids. Therefore, it can be preferably applied to products provided in the form of aqueous solutions such as beverages.

  (9) In the present embodiment, the supercritical extract composed of specific alcohols is obtained as a raw material of propolis bulk that is a natural material. Therefore, it can be safely applied to various uses.

  (10) When 1-octacosanol is applied as the alcohol constituting the supercritical extract, it can be expected to exhibit endurance-improving action, cholesterol-lowering action, antitumor action, etc. during exercise by ingestion.

  (11) When 1-triacontanol is applied as the alcohol constituting the supercritical extract, it can be expected to exhibit fatty acid metabolism improving action, vascular function improving action, arteriosclerosis preventing action and the like by ingestion.

  (12) When lupeol is applied as the alcohol constituting the supercritical extract, it is known to have antitumor action, anti-inflammatory action, antiviral action, antibacterial action, melanin production promoting action, etc. by ingestion etc. Yes.

In addition, you may change the said embodiment as follows.
-The supercritical extract in the above embodiment can be applied not only to foods and drinks and medicines consumed by humans, but also to supplemented animals such as livestock as supplements, dietary supplements, medicines, etc. May be.

  -As the extraction raw material, either the propolis bulk or the fraction insoluble in the hydrophilic organic solvent, water, or a mixture thereof from the propolis bulk may be used, or a mixture thereof may be used. Good.

(Second Embodiment)
Hereinafter, a second embodiment of the supercritical extract detection method of the present invention will be described.
In the method for detecting a supercritical extract of the second embodiment, first, a process of obtaining free alcohols by combining a supercritical extraction process and a process of hydrolyzing an ester bond from a propolis bulk is performed. Any of the extraction process using the supercritical extraction method and the process of hydrolyzing the ester bond may be performed first in the order of the process, and may be performed at least once in the process. From the viewpoint of production cost, first, a step of hydrolyzing the ester bond is performed on the components contained in the raw material, and then a processing step of extracting components that are dissolved in the supercritical fluid using the supercritical extraction method. It is preferable to be performed.

  When the supercritical extraction process is first performed, first, components that are dissolved in the supercritical fluid are extracted from the extraction raw material using the supercritical extraction method. Next, hydrolysis of an ester bond is performed on the extracted component, followed by separation of the component using chromatography, and detection of each alcohol described in the first embodiment. Done.

  When the process which hydrolyzes an ester bond first is performed, the process which hydrolyzes an ester bond is first performed about the component contained in an extraction raw material. Next, a process of extracting a component dissolved in the supercritical fluid using a supercritical extraction method is performed, and then a process of separating the extracted component using chromatography is performed. Each alcohol described in the embodiment is detected.

  The supercritical extraction method is performed to extract components dissolved in the supercritical fluid from the raw material, and the supercritical extraction method described in the first embodiment can be appropriately employed. The treatment for hydrolyzing the ester bond is carried out to cleave the ester bond of the fatty acid ester compound contained in the raw material or the fatty acid ester compound extracted using the supercritical extraction method to liberate alcohols. For the treatment for hydrolyzing the ester bond, the method described in the first embodiment can be appropriately employed.

  As the chromatography used for separating the components, a known chromatography capable of separating and detecting alcohols can be appropriately employed. Specifically, gas chromatography, liquid chromatography, supercritical fluid chromatography, and thin layer chromatography can be used. Etc. As a method of using each chromatography, a known method can be appropriately employed. For example, in thin layer chromatography, for example, sulfuric acid, dragendorf, triterpene coloring reagent, ferric chloride, bromocresol green, etc. can be used as the coloring reagent. Alcohols separated by each chromatography can be detected by comparing with a standard product of each compound or by determining the structure using a nuclear magnetic resonance apparatus or the like.

According to the supercritical extract detection method of the second embodiment, the following effects can be obtained.
(1) The detection method of the supercritical extract of this embodiment confirms the component after performing the extraction process which used the supercritical extraction method from the extraction raw material derived from propolis, and the process which hydrolyzes an ester bond. Therefore, the alcohol obtained in the first embodiment is used as an index.

  In general, the confirmation of the extract extracted using, for example, a hydrophilic organic solvent, water, or a mixture thereof is difficult or cumbersome because it is difficult or complicated to separate, qualitatively and quantitatively extract all extracted components. Confirmation has been made by detecting the compound using the component p-coumaric acid, quercetin, or cinnamic acid derivative as an index. Conventionally, for example, p-coumaric acid and cinnamic acid derivatives extracted using a hydrophilic organic solvent or water are used as an index compound for an extract extracted from a propolis bulk using a supercritical extraction method. Was. Therefore, the confirmation work of the extract obtained by the supercritical extraction method has never been sufficient from the viewpoint of accuracy. That is, in order to improve the accuracy of the confirmation work of the extract, it is preferable to use a component specific to the extract as an index. In the present embodiment, for the supercritical extract obtained by combining the supercritical extraction treatment and the hydrolysis treatment, the alcohols described in the first embodiment that cannot be obtained only by applying the supercritical extraction method are used. By using the index for confirmation, the accuracy of the confirmation work of the final product can be improved.

In addition, you may change the said embodiment as follows.
-Each chromatography used in the said embodiment may be implemented combining 2 or more types. Thereby, the accuracy of the alcohol detection operation can be further improved.

  -In the said embodiment, the insoluble fraction with respect to the hydrophilic organic solvent of the propolis original lump described in 1st Embodiment, water, or those mixtures may be used for the extraction raw material. Such an insoluble fraction also contains a fatty acid ester compound.

  -In the said embodiment, you may use each alcohol isolate | separated by chromatography as a main component of a supercritical extract, for example also for confirmation of quantitative analysis and extraction efficiency.

Examples and Comparative Examples are given below to describe the above embodiments more specifically, but the present invention is not limited to these.
(Test Example 1: Production of supercritical extraction propolis hydrolyzed)
(A) Supercritical extraction After 6 kg of Brazilian green propolis bulk was pulverized with a pulverizer, 30 liters of 95% by volume ethanol was added and the mixture was stirred and extracted at room temperature for 24 hours. Next, the extract containing the propolis pulverized product was subjected to solid-liquid separation by centrifugation to obtain 4.2 kg of ethanol-insoluble fraction (solid content: 50% by weight). This insoluble fraction was dried under reduced pressure and then ground in a mortar to obtain a powder. 1 kg of the powder was subjected to supercritical fluid treatment for 2 hours with a supercritical fluid treatment apparatus (manufactured by Mitsubishi Chemical Corporation). At this time, carbon dioxide was used as a supercritical gas, and extraction was performed under conditions of a flow rate of 28 L / hour, a maximum pressure of 345 atm (35.0 MPa), and a temperature of 50 ° C. By uniformly mixing the extract, 114 g of a supercritical extract (hereinafter referred to as “supercritical propolis”) was obtained.
(B) Examination of hydrolysis process of supercritical propolis 50 g of 1 mol / L sodium hydroxide to which ethanol of 0%, 25% by volume, 50% by volume and 75% by volume is added to 1 g of the above supercritical propolis, The ester bond was hydrolyzed by subjecting the flask equipped with a reflux condenser to a saponification treatment by gently heating in a water bath for 2 hours. After cooling, the solution was neutralized with 3 mol / L hydrochloric acid solution and filtered. As a result, the filterability was best when the ethanol addition amount was 0%, and the filterability was poor when the ethanol addition amount was 50% by volume and 75% by volume. In addition, when the confirmation test of policosanols and lupeol was conducted by TLC, the spots of policosanols and lupeol were not confirmed at 0% ethanol addition, and the spots of policosanols and lupeol were confirmed in any case where ethanol was added. did it. From the above results, it was found that the amount of alcohol added in the hydrolysis treatment is preferably 25% by volume.
(C) Hydrolysis treatment of supercritical propolis 1 L of 1 mol / L sodium hydroxide 25% by volume ethanol solution is added to 100 g of supercritical propolis, and the flask equipped with a reflux condenser is gently heated in a water bath for 2 hours for saponification treatment. Thus, hydrolysis treatment of the ester bond was performed. After cooling, neutralized with a 3 mol / L hydrochloric acid solution, filtered and hydrolyzed to obtain 70 g of supercritical extraction propolis.

(Test Example 2: Analysis of policosanols and lupeol)
(A) Analysis of hydrolyzed supercritical propolis 15 g of hexane was added to 1 g of hydrolyzed supercritical propolis and stirred, followed by centrifugation to separate the hexane layer. Hexane was added to the hexane layer obtained by performing this operation three times, and the volume was adjusted to 50 mL. 2 mL of this solution was taken, an internal standard solution (heptacosanol hexane solution) was added, and hexane was distilled off. Next, 0.2 mL of TMSI-H was added to prepare TMS, and an analysis sample was prepared, and measurement was performed with a gas chromatography mass spectrometer (GC-MS; Automass system II, manufactured by JOEL). The measurement conditions are column (DB-5: (5% Phenyl) -methylpolysiloxane, inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm), conditions (initial 200 ° C. (3 minutes hold) → 5 ° C./min. Temperature → 300 ° C. (17 minutes hold), 40 minutes measurement).

As a result, molecular ion peaks corresponding to tetracosanol, hexacosanol, octacosanol, triacontanol, dotriacontanol, tetratriacontanol and lupeol were observed. In addition, in comparison with a commercially available standard product, the retention time and the cleavage pattern coincided, and quantitative calculation was performed using the peak area as an index. Tetracosanol was 7.5%, hexacosanol was 7.6%, It was found to contain 8.1% octacosanolic acid, 12.6% triacontanol and 5.4% lupeol.
(B) Analysis of supercritical propolis The policosanols were analyzed on the supercritical propolis not hydrolyzed under the above-described analysis conditions. As a result, policosanols and lupeol were not detected.
(C) Analysis of alcohol-extracted propolis The alcohol-extracted propolis was analyzed for policosanols and lupeol under the aforementioned analysis conditions. As a result, policosanols and lupeol were not detected. Further, alcohol extracted propolis was hydrolyzed and analyzed in the same manner, but policosanols and lupeol were not detected. The results are shown in Table 1.

表１に示される結果から、加水分解処理した超臨界プロポリスにのみポリコサノール類及びルペオールが含有されることが判明した。

From the results shown in Table 1, it was found that policosanols and lupeol were contained only in the hydrocritically treated supercritical propolis.

  In addition, in the production method of the supercritical extraction propolis subjected to the hydrolysis treatment, when the hydrolysis treatment is performed first and then the supercritical extraction treatment is performed, the policosanols and lupeol are detected in the same manner as described above. I have confirmed.

(Test Example 3: Water dispersibility of hydrolyzed supercritical propolis)
30 mg of the supercritical propolis hydrolyzed was dispersed in 50 mL of water, allowed to stand overnight, and the water dispersibility was visually confirmed. As a result, the water-dispersibility of the hydrocritically treated supercritical propolis was good, and no difference was observed in the dispersibility immediately after dispersion and after standing overnight. Since supercritical propolis that has not been hydrolyzed does not disperse in water, it is possible to impart water dispersibility to the supercritical propolis by hydrolyzing it. Was found to be suitable for uses such as beverages.

(Test Example 4: Anti-inflammatory test (carrageenan paw edema test))
One group of 5 Wistar / ST rats (male), control group (distilled water), hydrolyzed supercritical proporolis 300 mg / kg administration group, supercritical propolis 300 mg / kg administration group, indomethacin 30 mg / kg as a positive control The test was conducted in 3 groups of administration groups. In addition, the sample of each administration group was prepared by suspending in 5% gum arabic.

  Each test sample was orally administered once using rats fasted 24 hours before the start of the test. One hour after oral administration, 0.1 mL of 1% λ-carrageenan solution was subcutaneously injected into the right hind limb of each group of rats to induce edema, and 1, 2, 3, 4 and 5 hours after the carrageenin injection Was measured, and edema rate and edema suppression rate were calculated, and anti-inflammatory action was evaluated.

  As a result, both the hydrolyzed supercritical propolis 300 mg / kg administration group and the supercritical propolis 300 mg / kg administration group showed a significant difference in edema inhibition rate at 1, 2, 3 and 4 hours, and the hydrolyzed supercritical propolis 300 mg / kg administration group. In the critical propolis administration group, a significant difference was observed even at 5 hours. Subsequently, the edema suppression rate 4 hours after subcutaneous injection in which the foot edema becomes the maximum was 38.5% in the hydrolyzed supercritical propolis 300 mg / kg administration group and 32.1% in the supercritical propolis 300 mg / kg administration group. And 74.1% in the indomethacin 30 mg / kg administration group. From this test result, it was confirmed that the supercritical propolis hydrolyzed has an anti-inflammatory effect superior to that of the supercritical propolis. This is thought to be due to the release of lupeol as an alcohol by the hydrolysis treatment.

(Test Example 5: Endurance improvement test (mouse forced swimming test))
The test was conducted in three groups of six 10-week-old ddy mice (male) as one group, a control group (distilled water), a supercritical propolis 300 mg / kg administration group, and a hydrolyzed supercritical proporris 300 mg / kg administration group. went. In addition, the sample of each administration group was prepared by suspending in 5% gum arabic.

  Each mouse before sample administration was loaded with a weight of about 7% per body weight and forced to swim, and the swimming time (time until the nose tip completely submerged in water) was measured. Each test sample was orally administered once, and the swimming time was measured 20 minutes after the administration in the same manner as before the administration. The difference in swimming time before and after administration was calculated, and the endurance improvement effect was evaluated.

  As a result, in the supercritical propolis 300 mg / kg administration group, there was no significant difference, but in the supercritical propolis 300 mg / kg administration group, the swimming time was extended as compared with the control group and further hydrolyzed. Compared with the control group, swimming time was significantly extended (p <0.05) (data not attached).

  From the above results, it was confirmed that the hydrocritically treated supercritical propolis has an excellent endurance improving effect as compared with the supercritical propolis. This is thought to be due to the release of 1-octacosanol as an alcohol by hydrolysis.

Claims (6)

An extraction process using a supercritical extraction method, and a process of hydrolyzing an ester bond, using an insoluble fraction of the green propolis bulk or the propolis bulk insoluble in a hydrophilic organic solvent, water or a mixture thereof as a raw material for extraction In the supercritical extract containing alcohols obtained by combining, 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacone A supercritical extract, wherein the extract is at least one selected from tanol, 1-tetratriacontanol, and lupeol. A step of extracting a fatty acid ester compound using a supercritical extraction method using a green propolis raw mass or a fraction insoluble in a hydrophilic organic solvent, water or a mixture thereof from the propolis raw mass as a raw material for extraction, and then the fatty acid It is selected from 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol by hydrolyzing the ester compound. A method for producing a supercritical extract comprising a step of obtaining at least one alcohol. A step of hydrolyzing an ester bond of an ester compound contained in the raw material using an insoluble fraction of the green propolis raw material or a hydrophilic organic solvent, water or a mixture thereof from the propolis raw material as an extraction raw material; In addition, 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, by extraction using a supercritical extraction method, And a method for producing a supercritical extract comprising a step of obtaining at least one alcohol selected from lupeol. In the method for detecting a supercritical extract,
A step of extracting a component dissolved in a supercritical fluid using a supercritical extraction method using an insoluble fraction in a hydrophilic organic solvent, water or a mixture thereof as an extraction raw material from the green propolis bulk or the propolis bulk,
Next, a step of hydrolyzing an ester bond with respect to the extracted component,
Next, a step of separating using chromatography, and
A step of detecting at least one alcohol selected from 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol. A method for detecting a supercritical extract, comprising: In the method for detecting a supercritical extract,
A step of hydrolyzing an ester bond for a component contained in the raw material of green propolis, or a fraction insoluble in a hydrophilic organic solvent, water or a mixture thereof from the propolis raw material,
Next, a step of extracting components dissolved in the supercritical fluid using the supercritical extraction method,
Next, the step of separating the extracted components using chromatography, and
A step of detecting at least one alcohol selected from 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol, 1-dotriacontanol, 1-tetratriacontanol, and lupeol. A method for detecting a supercritical extract, comprising:   A food, beverage, cosmetic or pharmaceutical comprising the supercritical extract according to claim 1 as an active ingredient.