JP2011213675A - Method for producing 2, 6-diprenyl-4-vinylphenol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently and safely producing 2, 6-diprenyl-4-vinylphenol (DPVP) from propolis, and to provide an antimicrobial agent, an antioxidant, a cell proliferation inhibitor, and an anticancer agent each comprising DPVP as an effective component.SOLUTION: DPVP that cannot be produced by a hydrolysis reaction, is produced by subjecting a composition comprising propolis to a heat treatment in the presence of a metal salt at a temperature of 120°C or higher. The pressure condition preferably is 0.1 MPa or higher. An antimicrobial agent, an antioxidant, a cell proliferation inhibitor, and an anticancer agent each comprising DPVP as an effective component exhibit a superior action relative to conventional ones derived from propolis.

Description

  The present invention relates to a process for producing 2,6-diprenyl-4-vinylphenol from propolis. The present invention also relates to antibacterial agents, antioxidants, cell growth inhibitors and anticancer agents.

  Various biologically active compounds are indispensable in order to lead a healthy and prosperous life such as maintaining / promoting health and recovering from disease. Efficiently obtaining useful bioactive compounds is an eternal desire for mankind, and various methods have been developed so far.

  The former method is obtained from the natural world, and mainly extracts useful physiologically active compounds from plants and animals. However, this method has a problem that only natural compounds can be obtained, and that rare components are recovered in a small amount. Therefore, studies have been repeated for a long time on methods for efficiently extracting natural physiologically active compounds and novel production methods other than extraction in order to obtain non-naturally physiologically active compounds, and many techniques have been proposed to date.

  Although these techniques are so numerous that it is impossible to describe in detail, the techniques can be roughly classified conceptually. Techniques for optimizing extraction conditions (Patent Document 1), techniques for synthesizing bioactive compounds by chemical techniques (Patent Document 2), techniques for producing by fermentation using microorganisms (Patent Documents 3 and 4), enzymes Producing methods (Patent Document 5), Generating recombinant bacteria and recombinant cells using genetic recombination techniques, producing bioactive compounds (Patent Document 6), primitive methods such as sun drying, heating, For example, a basic treatment such as acid or base is performed to convert the substance. At present, we have greatly benefited from these technologies, and we have been able to achieve a high standard of living that was impossible in the past by utilizing bioactive ingredients as nutritional components, functional ingredients and pharmaceutical ingredients in foods. Yes. However, it is also true that there are still problems with either method.

  With regard to the technique for optimizing the extraction conditions, the treatment of the raw material from which the biologically active compound is derived, the device for extracting the biologically active compound, the device for the extraction method, etc. are mainly raised. However, even if all of them are optimized, it is impossible to recover more than the amount in which the target physiologically active compound is originally contained, so there is an upper limit in efficiency. Despite many examinations of extraction conditions, it is often possible to recover only a small amount of the target physiologically active compound. Furthermore, the structure of the bioactive compound may change during the extraction process, and the desired bioactivity may be attenuated or lost.

  In the case of a method of synthesizing a bioactive compound by a chemical method, there are many disadvantages such as complicated reaction steps, risky, high cost, and often the safety of the obtained compound itself. There is also one side. Although it is a fact that has been realized by the advancement of life science and organic chemistry, and is still the royal road of drug discovery methods, in fact, in order to produce practically safe biologically active compounds However, there are still problems such as significant development costs, especially safety testing costs, long development periods, and cautions when taking them.

  Production of bioactive compounds using microorganism fermentation takes time to select microorganisms and optimize culture conditions, yield is insufficient, and purification work is burdened. The challenge remains.

  Since the method of producing a physiologically active compound using an enzyme is based on the substrate specificity of the enzyme, there is a limit to the types of compounds that can be necessarily obtained.

  The method of creating bioactive compounds by creating recombinant bacteria and recombinant cells using genetic recombination technology is also the labor of recombinant production, technical limitations at present, safety issues, Issues such as limitations on types remain.

  Primitive techniques such as sun-drying and basic treatments such as heating, acid, base, etc. can also be applied to convert substances, so that only a limited reaction can occur, so that the compounds obtained are limited.

  While these approaches are effective, it can be seen that they still have many challenges. In the first place, in order to artificially produce useful biologically active compounds, it is natural that synthetic processes, production, Furthermore, it takes a lot of energy to eliminate the danger. Here, energy means physical energy such as fuel, materials, processing costs, human labor input, and intellectual labor required to meet social factors necessary to meet legal standards such as safety. Indicates. If it is easy to understand that a lot of energy is required for manufacturing, it is easy to require a lot of labor to assemble a large number of necessary parts accurately and quickly according to the design document in order to complete the object. As you can imagine. Also, even if there is a device that can complete the object efficiently and safely, it can be easily imagined that much effort is required to devise, design, and assemble the device itself.

  On the other hand, an extraction method with few artificial factors does not require energy as compared with the above-described artificial technique, but has a drawback of low recovery efficiency due to the limited amount existing in the natural world.

  If we can obtain useful materials with low energy, we can make great progress in society. Low energy means obtaining useful substances simply, at low cost, safely and efficiently. In recent years, as the development of green chemistry has been advocated from the viewpoint of protecting the global environment, technological development to efficiently obtain useful materials with low energy is also a requirement of the times.

  Now, propolis is a material containing a lot of useful physiologically active compounds, and is eaten and consumed in Japan as a supplement or food. There are countries that are used as pharmaceuticals in the world, and certain usefulness has already been recognized, and further improvement in efficacy and efficacy is expected.

  Propolis is a resinous substance that is applied to the nest to protect the nest from pathogens and viruses. Propolis contains the secretions of plants and herbs around the nest, shoots, pollen, and beeswax secreted by the honeybees that collect them. Therefore, it is known that the composition of the propolis bulk is different depending on the environment around the nest and the type of bees (see Non-Patent Document 1).

  In addition, as the Greek word "Propolis (city state) pro (front)" (protect) is the origin of "Propolis", the antibacterial power of this substance in Europe has varied since ancient Greek times. It has been reported that it has been used as a folk medicine mainly in Eastern Europe. After that, propolis is known to have antibacterial, analgesic, anti-inflammatory, antioxidant, immunity enhancement, blood purification, and other areas where it can be used externally, such as by oral administration. In addition to flavonoids with high antioxidant activity, terpenoids and polysaccharide components have been clarified as active ingredients for these actions.

  Furthermore, as mentioned above, there are different types and contents of active ingredients in propolis depending on the production area, so that there are differences in the ingredients contained in propolis depending on the production area, and it is reported that their physiological effects have various characteristics. Has been.

For example, with regard to antioxidant activity, it has been reported that the antioxidant activity of Portuguese propolis varies depending on the region, and that of Brazilian propolis is reported to vary depending on the season as well as the region.
In addition, it has been reported that the extracts obtained by adjusting propolis from China and Brazil with fat-soluble and water-soluble solvents have large differences in antioxidant activity and component ratio, and caffeic acid contained in Brazilian propolis ( caffeic acid), Artepillin C and drupanin have superior antioxidant activity, and new flavanone compounds isolated from propolis and the flavanone compounds have superior antioxidant activity It has also been reported (see Non-Patent Documents 2, 3, 4, 5, and Patent Document 7)

Regarding antibacterial activity, Artepillin C and baccharin contained in propolis have been reported to have excellent antibacterial activity, and baccharin has also been reported to have antibacterial activity against methicillin-resistant Staphylococcus aureus. Yes.
(See Patent Documents 8 and 9)

  In addition, with regard to anticancer activity, reports that quinic acid derivatives isolated from propolis have been confirmed to have anti-proliferative, cell differentiation, and apoptosis-inducing effects on cancer cells, and Artepillin C contained in propolis have normal apoptosis. In addition to Artepillin C contained in propolis, the anticancer effect of drupanin and baccharin has also been reported. Proporin (propolin) A and propolin B It has also been reported that apoptosis is induced through the mitochondrial pathway. (See Patent Documents 10 and 11, Non-Patent Documents 6 and 7)

  As described above, propolis contains many compounds having physiological activity, and their effects vary. However, their content in propolis is very small, making it difficult to produce on an industrial scale.

  Thus, techniques for increasing the physiologically active effect of propolis and the content of physiologically active compounds are disclosed.

For example, for improving antibacterial activity, a method of performing a glycosyltransferase reaction on propolis or propolis-derived quercetin and α-glucosyl saccharified product has been reported.
In addition to antibacterial activity, an improvement in anticancer activity has also been reported in a method for obtaining a novel prenyl flavonoid by allowing prenyltransferase to act on specific flavonoids contained in propolis.
Next, for the improvement of anticancer activity, a method of blending propolis and red cedar, a method of esterifying organic acid in propolis, a propolis or Artepillin C derived from propolis is fermented with mycelia of basidiomycetes, and new Methods for obtaining cinnamic acid derivatives have been reported.
(See Patent Documents 12, 13, 14, 15, and 16)

  However, the above enzyme reaction is expensive at an industrial level, and it is difficult to control the reaction. In addition, regarding the cell culture method, the purification and isolation method of the target product after the culture is complicated. Furthermore, since other methods use chemical substances in the reaction process, there are many hurdles in applying the final product to food. Therefore, the conventional methods as described above are insufficient in terms of improving the effectiveness of propolis simply and safely.

  In addition, since the components contained in propolis are very small in content, there are some compounds whose existence has been confirmed but their actions are unknown. For example, 2,6-diprenyl-4-vinylphenol (2,6-diprenyl-4-vinylphenol, hereinafter also referred to as DPVP) was a novel compound isolated from Brazilian propolis at the time, and its simple substance. Although a method for separation and purification has been reported, detailed experimental facts and data on its action have not yet been reported, and its physiological activity has not been speculated and is unknown (patent document) 17, see Non-Patent Document 8).

Patent 4205334 Patent No. 2976003 Japanese Patent No. 3965804 Japanese Patent No. 3782837 Japanese Patent No. 2743005 Patent No. 2926990 Japanese Patent No. 4268896 Japanese Patent No. 3481269 JP-A-9-151131 JP 2006-213636 A JP-A-9-328425 Japanese Patent No. 3134233 JP 2009-46414 A JP 2003-252773 A JP 2007-53947 A JP 2008-81 A Japanese Patent Laid-Open No. 2002-255883

Nikkei Bio latest terminology dictionary 5th edition Food and Chemical Toxicology 46 3482-3485 (2008) eCAM 177 1-9 2008 Boil. Pharm. Bull. 32 (12) 1947-1951 (2009) BMC Complementry and Alternative Medicine 9: 4 1-9 (2009) Boil. Pharm. Bull. 26 (7) 1057-1059 (2003) Cancer Letters 245 218-231 (2007) Chem. Pharm. Bull. 49 (9) 1207-1209 (2001)

  In light of the above-mentioned situation regarding propolis, the present inventors have focused on DPVP, which is unknown in its action, but its action is unknown. Under the heat treatment below, a completely new phenomenon that has not been reported so far, the content of DPVP present in a minute amount is significantly increased, and the present invention has been completed.

Therefore, an object of the present invention is to provide a method for efficiently and safely producing 2,6-diprenyl-4-vinylphenol (DPVP) from propolis.
In addition, since the DPVP can be efficiently obtained by the method of the present invention, the physiological activity of DPVP that was unknown in the conventional method could be clarified for the first time. Therefore, an object of the present invention is to provide an antibacterial agent, antioxidant, cell growth inhibitor and anticancer agent containing DPVP as an active ingredient.

The gist of the present invention is as follows:
[1] A method for producing 2,6-diprenyl-4-vinylphenol, characterized by heat-treating a composition containing propolis in the presence of a metal salt at a composition temperature of 120 ° C. or higher.
[2] The method for producing 2,6-diprenyl-4-vinylphenol according to [1], wherein the pressure condition in the heat treatment is 0.1 MPa or more,
[3] The above [1] or [2], wherein the metal salt is at least one selected from magnesium salt, calcium salt, sodium salt, potassium salt, zinc salt, copper salt, mineral water and plant-derived extract A method for producing 2,6-diprenyl-4-vinylphenol,
[4] The method for producing 2,6-diprenyl-4-vinylphenol according to [3], wherein the plant-derived extract is an extract derived from coffee beans,
[5] The method for producing 2,6-diprenyl-4-vinylphenol according to any one of [1] to [4], wherein the composition containing the propolis is a propolis extract.
[6] The method for producing 2,6-diprenyl-4-vinylphenol according to any one of [1] to [5], wherein the heat treatment is an autoclave treatment,
[7] An antibacterial agent containing 2,6-diprenyl-4-vinylphenol as an active ingredient,
[8] An antioxidant containing 2,6-diprenyl-4-vinylphenol as an active ingredient,
[9] A cell growth inhibitor containing 2,6-diprenyl-4-vinylphenol as an active ingredient,
[10] An anticancer agent containing 2,6-diprenyl-4-vinylphenol as an active ingredient,
About.

  According to the present invention, the DPVP isolated yield can be improved by significantly increasing the DPVP content in the composition containing propolis. Thereby, the composition containing the propolis obtained by this invention can improve the bioactivity function and the efficacy of propolis. In addition, a drug containing DPVP as an active ingredient is superior in that it has high antibacterial activity, antioxidant activity, cell growth inhibitory activity, and anticancer agent physiological activity compared to a physiologically active substance isolated from conventional propolis. Antibacterial agents, antioxidants, cell growth inhibitors and anticancer agents can be provided.

FIG. 1 is a graph showing the results of HPLC analysis performed in Example 1. The figure shows the HPLC analysis result about the post-reaction composition which heat-processed the propolis extract, the coffee extract, the mixture of a propolis extract and a coffee extract, and the mixture of a propolis extract and a coffee extract from the top, respectively. The horizontal axis indicates time, the vertical axis indicates the peak size, and the “*” mark indicates the DPVP peak. The post-reaction composition means a composition containing DPVP produced by heat-treating a composition containing propolis in the presence of a metal salt. FIG. 2 is a graph showing the results obtained from the cell growth inhibition test of Example 5. The vertical axis of FIG. 2 shows the cell viability and the horizontal axis shows four types of samples. FIG. 3 is a graph showing the results of HPLC analysis of the post-reaction composition of Example 7 and the purified DPVP. The upper figure shows the post-reaction composition obtained from Example 2, and the lower figure shows the purified DPVP. FIG. 4 is a graph showing the results obtained by the DPPH radical elimination method of Example 8. In FIG. 4, the vertical axis represents the DPPH radical residual rate, and the horizontal axis represents the concentration of each sample. FIG. 5 is a graph showing the results obtained from the cell growth inhibition test of Example 10. In FIG. 5, the vertical axis represents cell viability and the horizontal axis represents each sample. FIG. 6 shows the result of electrophoresis obtained by the DNA ladder method of Example 11.

  Hereinafter, the present invention will be described in detail.

  The present invention is a method comprising a step of heat-treating a composition containing propolis in the presence of a metal salt so that the composition temperature becomes 120 ° C. or higher. The content of DPVP in the post-reaction composition that is the treated product can be increased.

  The composition containing the above-mentioned propolis is not limited as long as it is a composition containing at least a propolis component, such as a propolis bulk or a propolis extract obtained by extracting a propolis bulk by a known method. However, from the viewpoint of DPVP production efficiency, a composition containing a high amount of propolis bulk or a propolis extract is preferably used. In particular, propolis extract is more preferably used from the viewpoint of easy DPVP formation reaction and easy handling due to the low content of other materials.

  The propolis block used to obtain the propolis extract can be from any production area such as South American countries including Brazil, Asian countries such as China and Japan, European countries, North American countries, Oceania countries, etc. . The bee that makes Brazilian propolis is a bee that is a cross between a native bee and an African bee, and it is known that the production of propolis is greater than that of the bee. Therefore, it is more preferable to use Brazilian propolis among the above when considering the problem of securing raw materials and lot differences.

  Examples of the propolis extract include a water extract of propolis, an alcohol extract, a hydrous alcohol extract, an organic solvent extract, a supercritical extract, and a micellized extract. Any extract from the above may be used, but it is preferable to use an alcohol extract or a hydrous alcohol extract that is frequently reported to have high physiological activity. In addition, as alcohol used for preparing alcohol extract or hydrous alcohol extract, lower alcohols such as methanol, ethanol, butanol, propanol, isopropanol are mainly used. Then, it is more preferable to use ethanol among the above.

  Furthermore, a component containing DPVP and a fraction containing the component may be separated from the propolis extract, and the obtained substance may be used as a composition for heat treatment. The separation method is not particularly limited, and for example, the propolis extract can be performed by appropriately combining various column chromatography such as gel filtration chromatography, high performance liquid chromatography, ion exchange chromatography, etc. In some cases, it can be carried out according to a conventional method.

  Next, the metal salt may have any shape, and examples thereof include powder, ingot, and a molded body obtained by sintering powder. Moreover, as a metal salt, you may use what belongs to any category among these, such as an inorganic metal compound, an organometallic compound, a mononuclear complex, a polynuclear complex, a hydrogen compound, and these derivatives.

  The metal salt may be any of an acidic salt, a basic salt, and a normal salt, and may be any of a single salt, a double salt, and a complex salt. Furthermore, the metal salt may be one kind or a mixture of plural kinds. As an example of the metal salt, those approved as food additives are preferable in terms of safety. For example, magnesium salt, calcium salt, sodium salt, potassium salt, zinc salt, copper salt and the like that are permitted to be added to foods can be mentioned.

  Moreover, as the mixture of the metal salts, for example, a mineral premix (a mineral mixture mainly composed of zinc gluconate, ammonium iron citrate, calcium lactate, copper gluconate, and magnesium phosphate, Tanabe Seiyaku Co., Ltd.) Examples include substances containing several kinds of metal salts. Moreover, mineral water can also be mentioned as a mixture containing a some metal salt. Furthermore, an appropriate plant-derived extract can be used as the metal salt. For example, coffee beans are suitable for use because they contain a relatively large amount of metal salt, and the extract therefore also contains a relatively large amount of metal salt. It is also possible to use an extract of a material containing a relatively large amount of such a metal salt as the metal salt. Any other metal salt may be used as long as it contains the metal salt as described above. However, an extract derived from coffee beans such as instant coffee or hard water is preferable in terms of the flavor of the composition after reaction. Among them, the coffee bean-derived extract is excellent in the effect of masking some unpleasant odors possessed by propolis, and is particularly effective in this respect as well.

  The amount of the metal salt present in the composition containing propolis is not particularly limited as long as the DPVP formation reaction proceeds, but from the standpoint of efficiency, the amount of the metal salt is 0. It is preferable to use about 01 to 60% by weight. In particular, considering the amount of DPVP produced in the reaction product after the heat treatment, it is more preferable that the metal salt is used in an amount of about 1 to 30% by weight.

  In addition, when using the composition containing a propolis original lump, it is not necessary to add a metal salt. In the first place, the propolis bulk is a mixture of various components and therefore contains some metal salt. Therefore, whether or not the metal salt is added when the propolis bulk is used may be determined based on the efficiency of the DPVP production reaction.

The heating temperature at the time of the production reaction is not limited, but a higher temperature is preferable because the amount of DPVP produced increases. The phenomenon that the amount of DPVP generated increases is that when heated during a chemical reaction, the kinetic energy of the molecule also increases, the number of molecules with energy higher than the activation energy increases, and the number of collisions between the reactive molecules increases. It is presumed that DPVP bound to the substance contained in is separated. In the present invention, DPVP produced during the reaction is remarkably increased by setting the composition temperature to 120 ° C. or higher, preferably 130 ° C. or higher by heat treatment. The upper limit of the temperature may be a temperature at which the generated DPVP is not significantly reduced due to thermal decomposition or the like, but is preferably 500 ° C. or less, more preferably 300 ° C. or less in view of safety and processing costs.
Compared with conventional methods using chemical reaction or enzymatic reaction, heat treatment is a low-energy method and can be said to be a simple method in terms of labor.

  The heating time at the time of the production reaction is not limited as with the heating temperature, and may be a time condition under which the desired reaction proceeds efficiently. In particular, the heating time depends on the heating temperature, and it is desirable to set the heating time according to the heating temperature. For example, when heating near 130 ° C., a heating time of 5 to 60 minutes is desirable.

  Although it is not always necessary to apply pressure at the time of heating, pressurizing is an effective technique and can be appropriately taken in view of reaction efficiency. The pressure conditions are not limited as long as the DPVP formation reaction proceeds efficiently. By increasing the pressure, it is possible to add more kinetic energy to the molecule and increase the DPVP generation efficiency. From this viewpoint, it is preferable to apply a pressure of 0.1 MPa or more during the heat treatment. Moreover, it is preferable to adjust to a pressure of 150 MPa or less from the viewpoint of work safety and a limit in apparatus specifications.

The heat source at the time of heating is not particularly limited as long as the temperature of the composition can be heated to 120 ° C. or higher, heating an appropriate reaction vessel with direct flame, heating with microwave, heating with steam, Anything such as warming with warm water or heating with a heating wire may be used, and the container may be warmed in a hot water bath or an oil bath. In addition, when raising the reaction temperature, it is also an effective technique to use liquid oil or the like having a boiling point higher than that of water or alcohol as a solvent. The method of heating by autoclaving and industrially using a retort sterilizer for this purpose can increase the workability if conditions that increase the reaction efficiency can be set. Practical and preferred.
Among these, the heat treatment is preferably autoclaved from the viewpoint of easily heating and pressurizing the composition containing the propolis and metal salt to be treated. The autoclave apparatus to be used is not particularly limited as long as it can be adjusted to the temperature and pressure ranges during the heat treatment.

  Furthermore, the pH range of the composition containing the propolis and the metal salt at the time of the reaction is not limited, and any pH may be used as long as the DPVP generation reaction proceeds.

In order to optimize the reaction conditions for generating DPVP, the reaction conditions can be devised from time to time, depending on what state each of the composition and metal salt containing propolis is used, It is possible to further increase the DPVP generation amount.
For example, a case where an ethanol extract of propolis is used as a composition containing propolis and sodium hydrogen carbonate is used as a metal salt is listed below.
The ethanol extract of propolis needs to be diluted or concentrated in consideration of the solid content of propolis contained therein. The concentration of propolis during the formation reaction is preferably about 0.01 to 20% by weight. Actually, about 3 to 10% is more preferable because of higher reaction efficiency.
Moreover, since sodium hydrogencarbonate is water-soluble, it is more preferable to add it dissolved in water than to add it to the ethanol extract of propolis in a solid state and react it. In practice, an aqueous solution of 0.01 to 3% by weight of sodium bicarbonate is preferred, and an aqueous solution of about 0.05 to 0.3% by weight is more preferably reacted.
In the present invention, even when a composition other than the ethanol extract and a metal salt other than sodium bicarbonate are appropriately selected, appropriate conditions may be adjusted in consideration of the reaction efficiency.

  In the present invention, there are various methods for managing the temperature, pressure and time during the heat treatment, and control may be performed by any method.

  DPVP obtained by the production method of the present invention is a compound having a structure represented by the following formula [1].

  The compound of formula [1] is named 2,6-diprenyl-4-vinylphenol in the IUPAC nomenclature, and was isolated from Brazilian propolis in March 2001, and was then reported as a novel compound. Although the patent document 17 also describes effects related to aroma, detailed experimental facts and data on physiological activity have not yet been reported.

  DPVP is a compound that can significantly increase its content compared to when it is untreated by heat-treating a composition containing propolis in the presence of a metal salt so that the composition temperature is 120 ° C. or higher. It is.

  The increase in the DPVP content in the composition after the reaction can be confirmed by a known analysis method. As mentioned above, propolis, which is a natural product, is a natural product whose components vary depending on the season as well as the production area. In such cases, it is easy to cause a lot difference between the raw material and the composition after reaction. is expected. Therefore, it is considered important to establish a method for analyzing DPVP in the reactant in order to ensure the reliability of the production of DPVP in the reactant. For example, a part of the solid or solution after the reaction is collected, and after solvent extraction or pretreatment with a cartridge, instrumental analysis such as gas chromatography, HPLC, NMR, mass spectrometry and the like can be mentioned. It is possible to accurately evaluate based on the peak area value and quantitative data of the obtained data.

  The present inventors have revealed for the first time in the present invention that DPVP is excellent in antibacterial activity, antioxidant activity, cell growth inhibitory activity, and anticancer activity. For the purpose of these effects, the reaction product obtained after the heat treatment includes not only a single form but also a form of a composition such as liquid, pasty and solid foods, cosmetics and pharmaceuticals. .

  For example, in the case of food, food such as water, alcohol, starch chamber, protein, fiber, sugar, lipid, vitamin, mineral, flavoring, coloring, sweetener, seasoning, stabilizer, preservative In the case of cosmetics, the main ingredients, base materials, surfactants, foaming agents, wetting agents, thickeners, clearing agents, flavoring agents, coloring agents, and stabilizing agents As a composition with a preservative, preservative, disinfectant, etc., and in the case of a pharmaceutical product, as a composition of a carrier, excipient, diluent, stabilizer, and, if necessary, other physiologically active substances These forms are also included.

  The reaction product after the heat treatment in the present invention, that is, the composition having a high DPVP content can be used in the state of a mixture as it is, and DPVP is concentrated, purified or isolated from the composition, Such food, cosmetic or pharmaceutical forms are also possible. Depending on the application and cost, it may be determined whether to use a mixture or to obtain a purified product or an isolated product.

  The concentration and purification of DPVP can be performed by a known method. It can be extracted and concentrated by a solvent extraction method such as chloroform or ethyl acetate or a supercritical extraction method using carbon dioxide gas. It is also possible to perform concentration and purification using column chromatography. A membrane treatment method such as a recrystallization method or an ultrafiltration membrane is also applicable. It is possible to obtain a pure DPVP product by drying the purified DPVP solution under reduced pressure or freeze drying.

  When selecting a method for enriching, purifying, or isolating DPVP, there is no particular limitation, but the method procedure may include a known chemical synthesis method, but includes a chemical method. It is also possible to select one that does not exist. This method is easy to operate, inexpensive and safe. In addition, since the purified product or DPVP obtained by such a technique can be added to foods as well as pharmaceuticals, a method of purification or isolation from a composition is more preferable.

  The composition containing DPVP obtained by the method of the present invention is highly safe because the propolis as a raw material and the metal salt to be added are also used in foods, and the processing means is simple and low processing. A composition containing DPVP and DPVP that are useful at a low cost can be widely supplied to the market. The current market demands higher safety, effectiveness and lower prices, but can meet these demands.

  As described in the examples below, the results of the study by the present inventors show that the post-reaction composition obtained by the production method of the present invention and DPVP isolated therefrom have antibacterial action, antioxidant action, Data that it is very excellent in cell growth inhibitory action, anticancer action, etc. has been obtained. Therefore, the present invention provides an antibacterial agent, antioxidant, cytostatic agent and anticancer agent containing DPVP as an active ingredient. In particular, considering the physiologically active field of DPVP, it is preferable to use a composition containing DPVP in a field that is familiar and in demand such as lifestyle-related diseases.

  Further effects and efficacy of the composition and DPVP obtained in the present invention can be used within a range that can be inferred from the above data.

When DPVP is used for pharmaceutical purposes, for example, the content of DPVP as an active ingredient in 100% by weight of the composition containing DPVP can be in the range of about 1 × 10 ^{−4 to} 95% by weight. In addition, the intake of DPVP is not particularly limited as long as the desired improvement, therapeutic or preventive effect can be obtained. Usually, the mode, age, sex, constitution and other conditions of the patient, the type of the disease and its It is appropriately selected depending on the degree. Usually, DPVP as an active ingredient is preferably about 1 × 10 ^{−3 to} 85 mg / kg per day, which can be taken in 1 to 4 times a day.

  Since the post-reaction composition obtained in the present invention contains DPVP having the above physiological activity as an active ingredient, it can be used for functional foods, health foods, health-oriented foods, and the like. The food may be in any form such as beverage, jelly, confectionery, etc., and among confectionery, hard candy, soft candy, gummy candy, tablet, etc. that are excellent in storage and carrying due to its capacity etc. However, there is no particular limitation. Moreover, it is also possible to use these foods in combination as a composition.

Further, when the post-reaction composition or DPVP is orally administered or ingested as a pharmaceutical or food, it may be a tablet, pill, capsule, fine granule, granule or the like based on a conventional method. If necessary, the granules of capsules containing tablets, pills, granules, granules can be sugar-coated with sugars such as sucrose, sugar alcohols such as maltitol, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. It can also be coated. Alternatively, it may be covered with a film of gastric or enteric material. Moreover, in order to improve the solubility of a formulation, a well-known solubilization process can also be performed.
Furthermore, if it is a purified DPVP, it may be used in combination with an injection or a drip in accordance with a conventional method.

  Since the pharmaceutical or food is excellent in safety, it is not only for humans, for example, non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats. It may be added to a therapeutic agent or feed for mammals such as dogs, monkeys, chimpanzees, birds, amphibians, reptiles, and the like.

<Method for producing DPVP and physiological activity of post-reaction composition>
By heat-treating a mixture of propolis extract, which is a composition containing natural polymer compounds, and coffee extract, which is a mixture of metal salts, the content of DPVP in the composition after reaction is increased, and after reaction The physiological activity of the composition can also be increased. In the following Examples 1 to 5, the heat treatment method of the propolis extract and the coffee extract, the characteristic analysis results of the obtained post-reaction composition, and further two antibacterial effects on the physiological activity of the post-reaction composition And it shows about the cell growth inhibitory action test which is one of the anticancer evaluation systems.

(Example 1: Heat treatment of propolis extract and coffee extract (instant coffee) and HPLC analysis of post-reaction composition)
Propolis extract (ethanol extract of Brazilian propolis bulk. Solid content 13%. "Propolis extract" in the following examples uses this) 1.0 g, instant coffee (Ajinomoto General Foods Co., Ltd. "Blendy" (registered trademark) In the following examples, “instant coffee” is used.) 0.1 g and 5 ml of water are mixed (pH of the mixture is 4.7), and the mixture is autoclaved (SANYO LABO AUTOCLAVE) at 130 ° C. for 20 minutes, 0. Heated at 2 MPa. The obtained post-reaction composition was diluted to 50 ml with methanol, and 10 μl of this was analyzed by HPLC.
HPLC analysis was performed under the following conditions.
Column: Column for reverse phase “Develosil (registered trademark) C-30-UG-5” (4.6 mm.d. × 250 mm)

Mobile phase: A: H ₂ O (0.1% trifluoroacetic acid (TFA)), B: Acetonitrile (0.1% TFA)
Flow rate: 1 ml / min
Injection: 10 μl
Detection: 254 nm
Gradient (% by volume): 30 minutes from 80% A / 20% B to 20% A / 80% B, 5 minutes from 20% A / 80% B to 100% B, 10 minutes at 100% B (all linear)

The obtained chromatogram is shown in FIG.
It is a HPLC analysis result about the post-reaction composition which heat-processed the propolis extract, the coffee extract, the mixture of a propolis extract and a coffee extract, and the mixture of a propolis extract and a coffee extract from the top. The post-reaction composition in which the propolis extract and the coffee extract were heat-treated had a different HPLC profile as compared with the propolis extract before the heat treatment and the mixture of the propolis extract and the coffee extract before the heat treatment. In particular, for DPVP, which is the peak indicated by “*”, it was confirmed that the content in the composition after the reaction was increased by about 10 times compared with that before the heat treatment.

(Example 2: Heat treatment 2 of propolis extract and coffee extract)
10 g of propolis extract and 10 ml of 10% (w / v) instant coffee aqueous solution were mixed (pH 5.1 of the mixture), and heated in an autoclave (SANYO LABO AUTOCLAVE) at 130 ° C. for 20 minutes at 0.2 MPa. The obtained post-reaction composition was further lyophilized to obtain a tan powder. This was used for the following experiments.

  The post-reaction composition obtained in Example 2 was tested by the paper disk method for the purpose of confirming the presence or absence of antibacterial action. The paper disk method is a known technique that can easily check the presence or absence of antibacterial activity of a test substance by creating each inhibition circle of the test substance.

(Example 3: Examination of antibacterial action by paper disk method)
Bacteria were obtained using Bacillus subtilis NBRC 3134 and Staphylococcus aureus subsp. Aureus NBRC 12732 purchased from the National Institute of Technology and Evaluation NBRC. No. 1 which is a culture solution designated by NBRC for the method of starting and culturing cells. 702 and no. 802 was prepared and used, and the culture temperature was 30 ° C. and 37 ° C., respectively, according to the temperature specified by NBRC.
The experimental method ((agar) culture medium, culture method, test preparation, test method) of the paper disc method was performed according to the BSAC standardized disc sensitivity test method (8th edition) (hereinafter, BSAC standardized disc susceptibility testing method).
The types of samples were the propolis extract and the coffee extract obtained in Example 2 after the reaction, and the propolis extract and coffee extract mixture (no heat treatment) and the propolis extract (no heat treatment), respectively. It was dissolved and adjusted in 80% ethanol so that the extract content was 150 ppm and 100 ppm. The positive control was prepared by dissolving chloramphenicol Wako 036-10571, an antibacterial component used as a pharmaceutical, in 80% ethanol, and the negative control was 80% ethanol.
The sample prepared in this manner was immersed in a paper disk and sufficiently dried, then placed on an agar medium inoculated with Bacillus subtilis and staphylococci, cultured, and after 24 hours, inhibition circles were measured. .

  The obtained results are shown in Table 1-1 and Table 1-2.

Table 1-1 shows the allocation of samples used in the following experiments.
Sample A is a propolis extract, sample B is a mixture of a propolis extract and a coffee extract, and sample C is a mixture of a propolis extract and a coffee extract, and the resulting post-reaction composition is shown. The names of these samples will be used in the following experiments as the same samples.

Table 1-2 shows the results obtained from the paper disk method of Example 3.
About Table 1-2, the presence or absence of the antibacterial activity of each sample obtained by the paper disk method was shown. The notation (+) indicates that a blocking circle has been formed, and the number indicates the size of the blocking circle. (+) Indicates that the diameter is 1 cm, (++) indicates that the diameter is 1 cm to 2 cm, and (++) indicates that the diameter is 2 cm or more.
Since the inhibition circle was not formed in 80% ethanol, and the inhibition circle size of chloramphenicol was sufficient, it corresponds to the data described in the BSAC standardized disc susceptibility testing method. The reliability of this embodiment is shown.
This result shows that all of the propolis extract, the mixture of propolis extract and coffee extract, and the composition after the heat treatment can obtain a sufficiently large inhibition circle, and thus have sufficient antibacterial activity. It was done.

  From these results, in order to examine the antibacterial activity of each sample in more detail, an antibacterial activity test by the agar dilution method was performed. The agar dilution method is an experimental method in which a sample is suspended in an agar medium in advance and the ability to inhibit the growth of assay bacteria is evaluated.

(Example 4: Antibacterial activity assay by agar dilution method)
The bacterial cells were used using the same Bacillus subtilis as in Example 3, and the experimental method was performed according to the BSAC standardized disc susceptibility testing method.
For sample preparation, the lyophilized product of the post-reaction composition obtained in (Example 2) was dissolved in DMSO and suspended in an agar culture so as to be 200-fold diluted to prepare an agar medium. In addition to the post-reaction composition of the propolis extract and coffee extract obtained in Example 2, the sample type is a mixture of propolis extract and coffee extract (no heat treatment), propolis extract (no heat treatment), and positive control. As a negative control, DMSO was used as a negative control.
Each cell was measured for cell number using a hemocytometer, adjusted to 1 × 10 ⁶ cells / ml, and 28 spots were prepared on each agar medium at 5 μl / spot. This was cultured, and after 24 hours, the number of spots in which the growth of bacterial cells occurred was counted.

  The obtained results are shown in Table 2.

Table 2 shows the results obtained from the agar dilution method of Example 4.
The numerical value is the number of spots where Bacillus subtilis has occurred at each concentration. Since all 28 spots are generated in DMSO, and the chloramphenicol spot generation inhibitory concentration corresponds to the data described in the BSAC standardized disc susceptibility testing method, the reliability of this example is shown. Yes.

Comparing the test results of each sample conducted at 100 ppm, the post-reaction composition (sample C) is a propolis extract before heat treatment (sample A), and a mixture of propolis extract and coffee extract before heat treatment (sample). The number of spots was smaller than in B), and the result was that it had a stronger antibacterial action. That is, it was clarified that the antibacterial activity of the post-reaction composition obtained by heat-treating the propolis extract and the coffee extract was enhanced as compared with the extract before the heat-treatment.
Therefore, it can be seen that a composition containing DPVP as an active ingredient such as the post-reaction composition can be used as an antibacterial agent.

  Next, in order to see the effect of the post-reaction composition on cancer cells, the cancer cell proliferation inhibitory effect using HL-60 cells (Human promyelocytic leokemiacells) was tested.

(Example 5: Cancer cell proliferation inhibitory effect)
As the culture of the cells, the one obtained by adding 4 mM glutamine (L-Glutamine SIGMA G8540-100G), 10% FBS (Foetal Bovine Serum Biological industries 04-001-1A) to high nutrient medium RPMI-1690 (SIGMA R0883). Subculture was performed as a culture solution. The test used a 96-well plate for cell culture (corning 3595). On the day of the test, the number of HL-60 cells was adjusted to 1.5 × 10 ⁵ cells / ml, and seeded at 100 μl per well in a 96-well plate.
For sample preparation, the sample obtained in Example 2 was dissolved in DMSO (dimethyl sulfoxide, Wako 046-21981) at 20 mg / ml, and this was diluted 200 times (100 μg / ml in the experimental system). And suspended in the culture medium of HL-60 cells. In addition to the post-reaction composition of the propolis extract and coffee extract obtained from Example 2, the sample type is a mixture of propolis extract and coffee extract (no heat treatment), propolis extract (no heat treatment), and negative. DMSO was used as a control.
The cell growth inhibitory effect was detected using cell counting kit-8 (DOJINDO 347-07621). After 24 hours from the start of the test, 10 μl of the cell counting kit-8 detection solution was added to each well and stirred well. Thereafter, a light-shielding reaction was performed, absorbance was measured at 450 nm using a plate reader (BIO-RAD Model 680), and the obtained data was processed.

The obtained results are shown in FIG.
In FIG. 2, the vertical axis represents the cell viability and the horizontal axis represents each sample. The cell viability is a value obtained by calculating the relative number of viable cells in each sample at 100 μg / ml, assuming that the viable cell number of cells treated with a culture solution added with DMSO is 100%. is there.
The post-reaction composition (C in the figure) obtained by heat-treating the mixture of the propolis extract and the coffee extract is composed of the propolis extract before the heat treatment (A in the figure) and the mixture of the propolis extract and the coffee extract before the heat treatment (C). In the figure, it is suggested that the cell viability is lower and the effect of suppressing the growth of HL-60 cells is higher than in B).
As a result of the Tukey HSD (honest significant difference) test on the results obtained from these results, the significant difference was 0.006 between ACs and 0.035 between BCs, and this data was sufficiently significant. The difference is gained.

<Isolation of physiologically active compound DPVP and its physiological activity>
From the results of Examples 1 to 5, it is shown that the DPVP content is particularly increased in the post-reaction composition by the heat treatment, and the physiological activity of the post-reaction composition is compared with that before the heat treatment. It was shown to have even better efficacy. Therefore, DPVP was purified as a compound having a particularly increased content, and its physiological activity was verified. Hereinafter, the method for purifying DPVP from the post-reaction composition and the physiological activity (antioxidant action, antibacterial action, anticancer action) of DPVP will be described in Example 6 and later.

(Example 6: Purification method of DPVP)
The post-reaction composition obtained in Example 2 was fractionated and purified using a preparative column under the following conditions.
Column: Develosil® C-30 (20 mm.d. × 250 mm)
Mobile phase: A ... H ₂ O, B ... Acetonitrile flow rate: 10 ml / min
Injection: 1ml
Detection: 254 nm (UV)
Gradient (volume%): 50% A / 50% B to 20% A / 80% B for 30 minutes, 20% A / 80% B to 100% B for 5 minutes, 100% B for 10 minutes (all linear)

(Example 7: Method for analyzing DPVP)
The purified product obtained in Example 6 was subjected to HPLC analysis in the same manner as in Example 1, and it was confirmed that high-purity DPVP was obtained. This was also confirmed by NMR analysis. The result is shown in FIG. In addition, the upper figure of FIG. 3 is the post-reaction composition obtained from Example 2, and the lower figure is the purified DPVP. From the HPLC result, it is shown that DPVP indicated by “*” is isolated.
After the reaction, 40.8 mg of DPVP was recovered from about 10 g of propolis extract (solid content: 13.0%). This corresponds to recovery of 40.8 mg of DPVP per 1.3 g of propolis (31.3 mg of DPVP was recovered from 1 g of propolis solids).
On the other hand, in the purification of DPVP reported so far, for example, in Patent Document 17, 550 g of propolis bulk is subjected to steam distillation treatment at 120 ° C. for 5 hours to recover 27 mg of DPVP from the obtained essential oil. . This means that 0.049 mg of DPVP was recovered from 1 g of propolis solids.
Therefore, in the method of the present invention, the yield of DPVP obtained from propolis is more than 600 times that of the conventional method. From this result, it can be seen that the method of the present invention enables the production of DPVP, which has heretofore been difficult to industrially produce.
In the steam distillation treatment, the heating temperature of the aqueous solvent used in the reaction system is limited to about 100 ° C., so the heating temperature of the composition containing DPVP is substantially about 100 ° C. Yes. Moreover, although the method of extracting a propolis raw mass with a heating solvent is also known, it is common to process by heating a solvent to the temperature to 90 degreeC. Thus, in the conventional method, since the temperature at which the composition containing propolis is heated was 100 ° C. or less, it can be considered that the extremely small amount of DPVP preliminarily present in propolis was merely purified.

(Example 8: Antioxidative action of DPVP)
The antioxidant effect was tested using DPVP purified as described above. The test was conducted using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging method. The methods are listed below.
As for the main experimental method of DPPH radical scavenging method, the test proceeded with reference to the method described in “Food Function Research Method” (published by Mitsuaki Co., Ltd., Kazuo Shinohara, Keno Suzuki, Shuichi Uenogawa, 2000) . Samples were prepared by dissolving DPVP and α-tocopherol (Wako 209-01791) obtained from Artepillin C (Wako 016-19131) and (Example 6) in 75% EtOH, and adjusting each concentration. The DPPH reaction solution was adjusted to be 400 μM DPPH (SIGMA D9132), 50 mM MES (2-Morpholinoethanesulphonic acid DOJINDO 345-01625), and 75% EtOH. The reaction was carried out in a 96-well plate (As One 1-6766-03), 100 μl each of the sample and DPPH reaction solution were added, mixed and allowed to stand for 20 minutes, and each absorbance at 520 nm was measured with a plate reader.

The obtained results are shown in FIG.
Artepillin C is a physiologically active compound contained in propolis, and has already been reported to exhibit various physiological activities such as antioxidant, antibacterial and anticancer effects. As can be seen from FIG. 4, DPVP has a lower radical residual rate than Artepillin C and has an excellent antioxidant action. Further, as shown in Table 3, the DPVP IC50 (50% inhibition concentration: 100 μM) was lower than Artepillin C 180 μM. In other words, it was revealed that DPVP has higher antioxidant capacity than Artepillin C. The DPPH radical residual rate is a value calculated as a relative value of the DPPH radical residual amount in each sample, assuming that the DPPH radical residual amount detected when 75% EtOH is used for the sample is 100%.

  In Table 3, each value is a value indicating IC50 obtained by the DPPH radical elimination method. IC50 is the concentration of each sample considered to have the ability to scavenge DPPH radicals by 50%.

  As described above, the results of FIG. 4 and Table 3 suggest that DPVP has a lower IC50 than Artepillin C, and thus has superior antioxidant ability. Therefore, it is thought that the antioxidant which uses DPVP as an active ingredient has a more remarkable effect than the conventional one.

(Example 9: Antimicrobial action of DPVP)
Next, the antibacterial activity of DPVP was evaluated by an antibacterial activity test by the agar dilution method. As a sample, DPVP obtained from Artepillin C and Example 6 was used, and the bacterial cells were used for Bacillus subtilis in the same manner as in Example 4. The concentration of each sample was set as shown in Table 4.

The results obtained are shown in Table 4.
In the results obtained for DPVP and Artepillin C at 250 mM each, colonies were confirmed in all 28 spots in Artepillin C, whereas no spots were generated in DPVP, that is, it had antibacterial activity against Bacillus subtilis. It was suggested that DPVP has stronger antibacterial activity than Artepillin C. Therefore, it is considered that the antibacterial agent containing DPVP as an active ingredient has a more remarkable effect than the conventional one.

(Example 10: Anticancer effect of DPVP)
Next, in order to examine the anticancer effect of DPVP, the cancer cell proliferation inhibitory effect of cell counting kit-8 was evaluated. The sample was Artepillin C and DPVP obtained from (Example 6), the cells were HL-60, and the experimental method was the same as in (Example 5).

The obtained results are shown in FIG.
FIG. 5 suggests that DPVP has a lower cell viability than Artepillin C and a higher effect of suppressing the growth of HL-60 cells.
That is, it was shown that DPVP has a stronger cancer cell growth inhibitory effect than Artepillin C. Therefore, it is considered that a cell growth inhibitor containing DPVP as an active ingredient has a more remarkable effect than conventional ones.
The cell viability is a value obtained by calculating the relative number of cells surviving in each sample with the number of cells surviving treatment in a culture medium added with DMSO being 100%.

  The cell counting kit-8 which has detected this cancer cell growth inhibitory action is characterized by the principle of detecting a value corresponding to the number of living cells. Since DPVP has the effect of suppressing cancer cell growth and its effect is concentration-dependent, and Artepillin C has been reported to have anticancer activity, DPVP was evaluated in more detail for its anticancer activity. . As a further evaluation of anticancer activity, the detection of apoptosis by DNA ladder method was examined as an index.

(Example 11: Anticancer effect of DPVP)
Cells were performed using HL-60, and subculture of cells was performed in the same manner as in Example 5. On the day of the test, HL-60 cells were seeded in a 96-well plate at 5.0 × 10 ⁵ cells / ml, and DPVP obtained from Artepillin C and (Example 6) was adjusted with DMSO to obtain the target concentration. So that it was diluted 200-fold. Incubate for 24 hours, collect cells, and use PBS (Dulbecco's P
Cells were washed with BS (−) Wako 041-2021), and DNA was collected from the cells using Quick Apoptotic DNA Ladder Detection Kit (BioVision Research Products K120-50). The obtained sample was subjected to electrophoresis for 4 hours at 25 V using 1% agarose gel (Takara agarose L03 5003), and staining reaction was performed using Ethidium Bromide Solution BIO-RAD 161-0433. .

The obtained result is shown in FIG.
FIG. 6 is an image of an electrophoretogram of the DNA extract of each sample, with current flowing from top to bottom. From left lane, normal cultured cells (1st lane), DMSO treatment (2nd lane), DPVP 100 μM treatment (3rd lane), Artepillin C 100 μM treatment (4th lane), Artepillin C 300 μM treatment (5th lane), marker λ / Pst (6th lane) was passed. The reliability of this experiment can be confirmed from the fact that DNA ladder is not confirmed in normal cultured cells and DMSO-treated cells.
In addition, DNA ladder was confirmed in cells treated with DPVP at 100 μM, whereas in Artepillin C, DNA ladder was not confirmed at the same concentration of 100 μM, but could be confirmed at 300 μM. This indicates that DPVP has an effect of inducing apoptosis, and its potency is higher than that of Artepillin C.
Therefore, it is thought that the anticancer agent which uses DPVP as an active ingredient has a more remarkable effect than the conventional one.

<Examination of optimal conditions for this reaction>
Next, more detailed conditions for efficiently increasing DPVP having various excellent physiological activities were examined.

(Example 12: Influence of heating temperature)
The heating time is fixed at 20 minutes, 1.0 g of propolis extract, 7.8 mg of magnesium phosphate as a metal salt and 5 ml of water are mixed, and the heating temperature is 100 ° C., 110 ° C., 120 ° C., 130 ° C. in an autoclave. The reaction was allowed to proceed under conditions. The amount of DPVP in the obtained post-reaction composition was subjected to HPLC analysis by the same method as in Example 1 and measured for DPVP.

The results obtained are shown in Table 5. Table 5 shows a comparison result of the DPVP production amount depending on the heating temperature, and the area value indicates the numerical value of the DPVP peak area obtained by the analysis by HPLC. It is shown that the higher the heating temperature, the larger the DPVP peak area.
From this result, it was confirmed that the higher the heating temperature, the more DPVP was produced, and the DPVP increased to a concentration of 10 times or more with respect to the unheated control product in the heating time of 120 ° C. It was. Therefore, it was revealed that the heating temperature is preferably 120 ° C. or higher.

(Example 13: Effect of heating time)
Next, 1.0 g of propolis extract, 0.1 g of instant coffee as a metal salt, and 5 ml of water are mixed, the heating temperature is fixed at 130 ° C. in an autoclave, and the heating time is 10 minutes, 20 minutes, and 30 minutes. The reaction was allowed to proceed. The amount of DPVP in the obtained post-reaction composition was subjected to HPLC analysis by the same method as in Example 1 and measured for DPVP.

  The results obtained are shown in Table 6. Table 6 shows a comparison result of the DPVP generation amount according to the heating time, and the area value is calculated by performing data processing in the same manner as in Table 5. It is shown that the DPVP peak area increases as the heating time increases. In particular, at a heating time of 10 minutes, it was confirmed that DPVP increased to a concentration 10 times or more that of the unheated control product. Therefore, it was revealed that the heating time is preferably 10 minutes or more.

(Example 14: Influence of composition during heating)
Next, the heating conditions by the autoclave are fixed at a heating temperature of 130 ° C. and a heating time of 20 minutes, the composition used for the reaction is fixed based on 1.0 g of propolis extract and 0.1 g of instant coffee as a metal salt, and added water. The amounts were 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml, respectively, and the reaction was allowed to proceed under each condition. The amount of DPVP in the obtained post-reaction composition was subjected to HPLC analysis by the same method as in Example 1 and measured for DPVP.

  The results obtained are shown in Table 7. Table 7 shows a comparison result of the amount of DPVP produced by the composition during the heat treatment. The calculation of the area value is shown by performing data processing as in Table 5. It is shown that the larger the amount of water added, the smaller the peak area of DPVP. In particular, it was confirmed that DPVP increased to a concentration about 1.7 times that of the composition added with 1 ml of water compared to the composition added with 5 ml of water. Although there was no restriction | limiting in particular as a composition at the time of reaction, it turned out that the efficiency of reaction which produces | generates DPVP is so high that a solution concentration is deep.

(Example 15: Effect of pH of composition during heating)
Next, the heating conditions by the autoclave were fixed at a heating temperature of 130 ° C. and a heating time of 20 minutes, and the composition solution used for the reaction was fixed based on 2.0 g of propolis extract and 9 ml of water to be added. And for adjusting pH, the pH of the composition before the start of the reaction is set to 5, 6, 7, 8, 9, 10, 11, 12, 13 and the reaction is allowed to proceed under each condition. It was. The amount of DPVP in the obtained post-reaction composition was subjected to HPLC analysis by the same method as in Example 1 and measured for DPVP.

Table 8 shows the obtained results. Table 8 shows a comparison result of the DPVP production amount depending on the pH of the composition. The calculation of the area value is shown by performing data processing as in Table 5.
As a result, the production amount of DPVP was different depending on the pH, and it was found that there was a preferable composition pH. Specifically, as the pH of the composition liquid, the peak area of DPVP was large in 7-11. Considering the efficiency of producing DPVP, it was considered that the pH of the composition solution before the reaction when sodium hydroxide is used as the metal salt is preferably 7 to 11. However, when sodium hydrogen carbonate is used as the metal salt, it is considered that the pH of the composition solution changes while the reaction is in progress, and the optimum conditions for the pH of the composition before the reaction cannot generally be shown. Furthermore, a mixture of metal salts such as an extract derived from coffee beans used in the reaction as the metal salt in the above-described Examples 1, 2, and 13 similarly has substances contained in the mixture during the reaction. The effects are varied and the reaction is considered to be complex. That is, it is considered that the optimum pH of the pre-reaction composition liquid varies depending on the type of metal salt added.

(Example 16: Effect of metal salt type)
Next, the heating conditions by the autoclave were fixed at a heating temperature of 130 ° C. and a heating time of 20 minutes, and the composition used for the reaction was fixed based on 1.0 g of propolis extract and 5 ml of water to be added, and various metal salts and acids. The effect of increasing DPVP amount was investigated. The results are shown in Table 9.

From the results shown in Table 9, as a single metal salt, any of the magnesium salt, calcium salt, sodium salt, potassium salt, copper salt, and zinc salt showed an effect of increasing the DPVP amount. As a mixture of metal salts, instant coffee and mineral water were found to increase DPVP.
All of the above metal salts are appropriately selected from those that can be added to food, and other metal salts are considered to have a similar DPVP increase effect.
Among the metal salts in Table 9, magnesium phosphate is magnesium phosphate, calcium salt is calcium lactate, calcium carbonate, and sodium salt is sodium bicarbonate, sodium carbonate, sodium glutamate, and potassium salt is carbonate. Potassium, dipotassium hydrogen phosphate, zinc salt as zinc gluconate, copper salt as copper gluconate, iron salt as ammonium iron citrate, metal salt mixture as mineral premix, hard water, instant coffee There was an effect of addition.
In addition, among the obtained post-reaction compositions containing DPVP, those using hard water and instant coffee are preferable in terms of flavor, and especially those using instant coffee have the highest DPVP masking effect. It was.
It has been clarified that acids such as lactic acid, glutamic acid, and gluconic acid that do not form a salt with a metal cation are very weak and need to exist as metal salts.

Claims (10)

  A method for producing 2,6-diprenyl-4-vinylphenol, comprising heat-treating a composition containing propolis at a composition temperature of 120 ° C. or higher in the presence of a metal salt.   The method for producing 2,6-diprenyl-4-vinylphenol according to claim 1, wherein a pressure condition in the heat treatment is 0.1 MPa or more.   The said metal salt is 1 or 2 types chosen from magnesium salt, calcium salt, sodium salt, potassium salt, zinc salt, copper salt, mineral water, and plant-derived extract, 2,6 of Claim 1 or 2 -Production method of diprenyl-4-vinylphenol.   The method for producing 2,6-diprenyl-4-vinylphenol according to claim 3, wherein the plant-derived extract is an extract derived from coffee beans.   The method for producing 2,6-diprenyl-4-vinylphenol according to claim 1, wherein the composition containing propolis is a propolis extract.   The method for producing 2,6-diprenyl-4-vinylphenol according to any one of claims 1 to 5, wherein the heat treatment is an autoclave treatment.   An antibacterial agent containing 2,6-diprenyl-4-vinylphenol as an active ingredient.   An antioxidant containing 2,6-diprenyl-4-vinylphenol as an active ingredient.   A cell growth inhibitor containing 2,6-diprenyl-4-vinylphenol as an active ingredient.   An anticancer agent containing 2,6-diprenyl-4-vinylphenol as an active ingredient.

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