JP2012062292A - Method for producing 4-vinyl catechol polymerization compound, or pharmaceutically acceptable salt thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a 4-vinyl catechol polymerization compound, or a pharmaceutically acceptable salt thereof having excellent anticancer activity and lipase inhibitory activity, a lipase inhibitor, an anti-obesity agent, skin disease therapeutic agent and anticancer agent containing the 4-vinyl catechol polymerization compound, or the pharmaceutically acceptable salt thereof, and a food, a pharmaceutical agent or a cosmetic product containing the 4-vinyl catechol polymerization compound, or the pharmaceutically acceptable salt thereof.SOLUTION: The method for producing a 4-vinyl catechol polymerization compound represented by the formula (1), or a pharmaceutically acceptable salt thereof comprises heating caffeic acid in the presence of a metal salt. Also provided are a lipase inhibitor, an anti-obesity agent, skin disease therapeutic agent and anticancer agent, and a food, a pharmaceutical agent or a cosmetic product containing the 4-vinyl catechol polymerization compound represented by the formula (1), or the pharmaceutically acceptable salt thereof.

Description

  The present invention relates to a method for producing a 4-vinylcatechol polymerization compound or a pharmaceutically acceptable salt. Further, the present invention relates to a lipase inhibitor, an anti-obesity agent, a skin disease therapeutic agent, an anticancer agent, a food, a pharmaceutical, or a cosmetic containing the 4-vinylcatechol polymerization compound or a pharmaceutically acceptable salt.

  Caffeic acid is one of the secondary metabolites of plants. For example, it is a precursor of lignin and lignan, which are the main components of trees, and also a precursor of functional components such as chlorogenic acid and rosmarinic acid. It is a component that exists relatively abundantly in nature. It is contained in coffee beans and the like as chlorogenic acid, and in Lamiaceae plants as rosmarinic acid. Moreover, caffeic acid is contained in the fruits and peels of many fruits. These are all ingredients that have dietary experience and are highly safe for humans. Although there are disclosures of usefulness of caffeic acid and useful caffeic acid derivatives, a production method for producing a 4-vinylcatechol polymerization compound from caffeic acid has not been found.

  There is prior art related to the physiological function of caffeic acid. For example, an agent for preventing and treating hypertension composed of caffeic acid and ferulic acid (Patent Document 1), an autonomic nervous function improver containing caffeic acid as an active ingredient (Patent Document 2), and a vascular endothelial function improving agent containing caffeic acid as an active ingredient (Patent Document 3), blood fluidity improver containing caffeic acid as an active ingredient (Patent Document 4), cerebral fatigue recovery agent containing caffeic acid as an active ingredient (Patent Document 5), secondary containing caffeic acid as an active ingredient A bile acid lowering agent (Patent Document 6) is known.

  There is also prior art related to caffeic acid derivatives. For example, a composition for cosmetics or an external preparation for skin containing a caffeic acid amide derivative as an active ingredient (Patent Document 7), an antimicrobial agent containing a sugar transfer product of caffeic acid as an active ingredient (Patent Document 8), a caffeic acid derivative Neurite elongation agent (Patent Document 9) containing as an active ingredient, hemodynamic improving agent containing Serotoninamide of caffeic acid or its glycoside as an active ingredient (Patent Document 10), Alzheimer containing caffeoylquinic acid as an active ingredient Disease prevention or treatment agent (Patent Document 11), method for efficiently producing caffeic acid phenethyl ester which is a minor component in propolis (Patent Document 12), enzyme synthesis method of cinnamic acid derivative (Patent Document 13), 2- An anticancer agent containing a caffeic acid derivative such as caffeic acid cyclohexaester as an active ingredient (Patent Document 14), a novel polypheno obtained by enzymatic synthesis using caffeic acid as one of the raw materials Le compound (Patent Document 15) are known.

  As described above, since many caffeic acids and caffeic acid derivatives exhibit excellent utility, a technical disclosure for efficiently producing caffeic acid as a raw material or a lead compound has been made. For example, a method of producing from coffee lees (patent document 16), a method of producing from burdock leaves (patent document 17), and a method of producing from sweet potato shochu distilled liquor (patent document 18) are known.

  On the other hand, it has been reported that by roasting coffee beans at 200 ° C. or higher, many oligomers of 4-vinylcatechol are slightly produced in coffee beans by a heating reaction at a high temperature (Non-patent Document 1). Non-Patent Document 2). However, these 4-vinylcatechol oligomers are very unstable at a pH of 4.9 to 6.5 in coffee beverages and are rare components that do not exist in nature. There is no report on the activity, and the Non-Patent Documents 1 and 2 only suggest that the compound contributes to the bitterness of coffee after roasting. Coffee drinks are safe drinks that people have enjoyed for a long time, but the abundance of 4-vinylcatechol polymerized compounds is generally so low that they must be quantified by LC / MS / MS, which is highly sensitive. Is. Therefore, it is difficult to drastically improve the production efficiency of the 4-vinylcatechol polymerized compound by the above-described method for roasting coffee beans, which requires a large amount of coffee beans and is costly. Since the temperature condition at the time requires a high temperature of 200 ° C. or higher, it is not suitable for industrialization.

  Under such circumstances, establishment of an efficient production method capable of easily industrializing 4-vinylcatechol polymerization compound, which is a useful caffeic acid-related compound, has been desired.

Japanese Patent No. 3548102 Japanese Patent No. 4077149 JP 2003-261444 A JP 2004-168749 A JP 2007-297304 A JP 2009-227609 A Japanese Patent No. 3934937 JP 2004-315386 A JP 2007-230946 A International Publication No. 2007/032551 Pamphlet International Publication No. 2007/091613 Pamphlet JP 2010-158223 A JP 2009-207492 A JP 2010-180167 A International Publication No. 2010/038842 Pamphlet JP 2009-201447 A Japanese Patent No. 435597 Japanese Patent No. 4336746

J. et al. Agric. Food Chem. 2007, 55, 1945-1954 J. et al. Agric. Food Chem. 2010, 58, 3720-3728

  In light of the above-mentioned situation regarding caffeic acid and caffeic acid derivatives, the present inventors have surprisingly studied as a result of a search for a novel caffeic acid-related compound having physiological activity and a method for producing the compound. It has been found for the first time that a 4-vinylcatechol polymerization compound, which is a caffeic acid-related compound represented by the formula (1), can be produced by heat-treating caffeic acid in the presence of a metal salt, and further represented by the formula (1). The 4-vinylcatechol polymerized compound was found to have excellent lipase inhibitory activity and anticancer activity that are not recognized by caffeic acid as a raw material, and the present invention was completed.

Accordingly, the present invention provides a method for efficiently producing the 4-vinylcatechol polymerized compound represented by the above formula (1) or a pharmaceutically acceptable salt having excellent anticancer activity and lipase inhibitory activity. Objective.
The present invention also provides a lipase inhibitor, anti-obesity agent, skin disease therapeutic agent and anti-cancer agent containing the 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, It aims at providing the foodstuff, pharmaceutical, or cosmetics containing the 4-vinyl catechol polymeric compound or its pharmaceutically acceptable salt shown by Formula (1).

The gist of the present invention is as follows:
[1] A 4-vinylcatechol polymerized compound represented by the following formula (1) is produced by heat-treating caffeic acid in the presence of a metal salt. A method for producing a catechol polymerization compound or a pharmaceutically acceptable salt.

[2] A lipase inhibitor comprising a 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
[3] An antiobesity agent comprising a 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
[4] A skin disease therapeutic agent comprising a 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
[5] An anticancer agent comprising a 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
[6] A food, drug, or cosmetic comprising the 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
About.

According to the present invention, the 4-vinylcatechol polymerization compound having excellent physiological activity and the pharmaceutically acceptable salt thereof can be efficiently and safely produced as described above.
Since the 4-vinylcatechol polymerization compound and pharmaceutically acceptable salt thereof used in the present invention have higher lipase inhibitory activity and anticancer activity than caffeic acid, they are excellent antiobesity agents and therapeutic agents for skin diseases. And anticancer agents can be provided.
Further, the 4-vinylcatechol polymerized compound and pharmaceutically acceptable salt thereof according to the present invention are excellent in safety in addition to the above physiological activity, and thus are suitable for foods, pharmaceuticals or cosmetics. Can be blended.

FIG. 1 shows the results of HPLC analysis performed in Example 1. The upper figure shows the result of the first reaction, the middle figure shows the result of the first reaction, the lower figure shows the result of the second reaction, and “B” shows the peak of the 4-vinylcatechol polymerized compound. FIG. 2 is a graph showing the results obtained from the cell growth inhibition test of Example 4. The vertical axis of FIG. 2 indicates the cell viability, and the horizontal axis indicates the concentration of each sample. FIG. 3 shows the results of electrophoresis obtained by the DNA ladder method of Example 5 and shows the ability to induce apoptosis. FIG. 4 is an electrophoretogram of the protein extract of Example 5, showing detection of activated caspase-3.

  Hereinafter, the present invention will be described in detail.

  The 4-vinylcatechol polymerization compound which is an active ingredient in the lipase inhibitor, anti-obesity agent, skin disease therapeutic agent and anticancer agent of the present invention is represented by the formula (1):

The structural formula is represented by:

  The 4-vinylcatechol polymerization compound represented by the formula (1) may be a pharmaceutically acceptable salt. Examples of the pharmaceutically acceptable salt include alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt, calcium salt and barium salt; aluminum salt; aluminum hydroxide salt and the like Metal amine salts of; amine salts such as alkylamine salts, dialkylamine salts, trialkylamine salts, alkylenediamine salts, cycloalkylamine salts, arylamine salts, aralkylamine salts, heterocyclic amine salts; α-amino acid salts And amino acid salts such as ω-amino acid salts; peptide salts or primary, secondary, tertiary or quaternary amine salts derived therefrom. These pharmacologically acceptable salts can be used alone or in admixture of two or more.

  The 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt (hereinafter abbreviated as 4-vinylcatechol polymerization compound which is an active ingredient in the product of the present invention) is a precursor. It is a compound having strong lipase inhibitory activity and anticancer activity not found in some caffeic acids.

  In the present invention, the 4-vinylcatechol polymerization compound can be produced by heat-treating caffeic acid in the presence of a metal salt.

  As shown in Non-Patent Document 1, the 4-vinylcatechol polymerization compound can be obtained by heating reaction from roasted coffee beans or the like. However, the content is very small, and there is a great cost obstacle in industrialization, such as the need for a highly sensitive analytical instrument such as LC / MS / MS to confirm the content. On the other hand, the production method of the present invention has a step of heat-treating caffeic acid, which can be obtained at a relatively low cost, in the presence of a metal salt, and does not require harmful reagents or dangerous steps. It is a safe and safe manufacturing method.

  In the production method of the present invention, caffeic acid is required as a precursor of the 4-vinylcatechol polymerization compound. Caffeic acid may be naturally derived or may be a chemically synthesized chemical product with high purity. When natural caffeic acid is used, it does not need to be completely purified, and the desired reaction proceeds thereafter to finally obtain a 4-vinylcatechol polymerization compound. . However, from the viewpoint of the recovered amount, a mixture containing 5% by weight or more of caffeic acid is desirable as a raw material. Such raw materials include various fruits and juices, concentrated fruit juices, extracts of peels that are often discarded, or culture solutions containing caffeic acid by microbial fermentation as shown in the prior art or after enzyme reaction. Examples include caffeic acid-containing solutions.

  A pure product of caffeic acid or a mixture containing caffeic acid is dissolved in a suitable solvent. At this time, since the solubility of caffeic acid is remarkably low if the solvent is only water, it may be dissolved in a mixture of water and an organic solvent or only in an organic solvent. There is no restriction | limiting in particular in the compounding ratio of water and an organic solvent, and the kind of organic solvent, Caffeic acid should just fully melt | dissolve. Desirably, it is desirable to use only methanol or ethanol, or a mixture of water and methanol or water and ethanol from the viewpoint of safety and cost. When using it for foods without fully applying final purification, it is desirable to use ethanol or hydrous ethanol for safety and legal reasons.

  The metal salt may be any of an acid 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.

  In the present invention, the caffeic acid-containing solution containing the metal salt is heated. In order to efficiently advance the desired reaction, the heating temperature needs to be 90 ° C. or higher. Considering the boiling point of the solvent, pressure heating is desirable. Solution temperature at least partially, such as heating caffeic acid-containing solution in an open container and heating the container at a high temperature, heating caffeic acid-containing solution in a sealed container, heating with pressure using a retort device or autoclave, etc. Needs to reach 90 ° C. or higher. From the viewpoint of recovery efficiency, it is more preferable that the solution temperature be uniformly 90 ° C to 150 ° C. If it exceeds 150 ° C., the recovery efficiency is low and not suitable. The heating time is not limited as in the case of the heating temperature, and may be a time condition in which the target reaction efficiently proceeds. 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 minutes to 300 minutes is desirable. Further, the heating reaction may be performed once, or may be repeated repeatedly in a plurality of times. Judging from the efficiency side.

  Moreover, in this invention, it is preferable to adjust the pH of the caffeic acid containing solution containing the said metal salt to the range below 9. As described in Non-Patent Document 2, the oligomer of 4-vinylcatechol was originally known to be very unstable at a pH of about 4.9 to 6.5. By heating in the presence of a salt, a 4-vinylcatechol polymerization compound or a pharmaceutically acceptable salt can be produced stably even when the pH is in the range of about 4.9 to 6.5.

  Caffeic acid reacts by heat treatment in the presence of the metal salt to obtain a mixture containing a 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt. . When it is obtained in a process using only safe raw materials, it can be used in a mixture state. For example, when naturally occurring caffeic acid is dissolved in an ethanol solvent, and a mineral mix or mineral water is added and reacted by heating, the mixture can be used as one of food ingredients.

  When improvement in flavor and further enhancement of functionality are desired, the 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt is concentrated to increase the concentration, or purified and purified. Goods can be obtained. Concentration and purification can be performed by a known method. For example, it can be extracted and concentrated by a solvent extraction method such as chloroform, ethyl acetate, ethanol or methanol, a supercritical extraction method using carbon dioxide gas, or the like. 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 can also be applied. Finally, when the solvent is removed by drying under reduced pressure or lyophilization, a pure 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt can be obtained.

  The 4-vinylcatechol polymerization compound or pharmaceutically acceptable salt represented by the formula (1) has excellent lipase inhibitory activity and anticancer activity as described later.

Conventionally, hyperlipidemia, which is caused by abnormal fat deposition in the body adipose tissue and various organs, resulting in obesity or abnormally high serum lipids, is caused by hypertension, arteriosclerosis, diabetes It is known to be closely involved in the development of various lifestyle-related diseases such as. For example, dietary fats eaten by humans are broken down by pancreatic lipase and absorbed into the body through the small intestine, so lipase inhibitors can be used to prevent obesity, improve hyperlipidemia, It is possible to treat skin diseases such as acne, and its correlation has also been confirmed (for example, Japanese Patent No. 3689099, Japanese Patent No. 3826698).
Therefore, it can be used as an antiobesity agent, a skin disease therapeutic agent and an anticancer agent containing the 4-vinylcatechol polymerized compound represented by the formula (1) or a pharmaceutically acceptable salt as an active ingredient. The anti-obesity agent, skin disease treatment agent and anti-cancer agent may contain other active ingredients.

  The 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt is a liquid, paste, gel for the purpose of the anti-obesity effect, skin disease treatment effect or anti-cancer effect. And solid foods, pharmaceuticals, cosmetics and the like.

  For example, in the case of food, such as water, alcohol, starch, protein, fiber, sugar, lipid, vitamin, mineral, flavoring, coloring, sweetener, seasoning, stabilizer, preservative, etc. 4-vinylcatechol polymerization represented by the above formula (1) in combination with raw materials or materials usually blended in foods, and in the case of pharmaceuticals, in combination with carriers, excipients, diluents, stabilizers, etc. A compound or pharmaceutically acceptable salt can be used. In particular, taking into account the physiological activity of the 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt, prevention of lifestyle-related diseases such as metabolic syndrome due to anti-obesity effect, cancer prevention / It is preferably used in health maintenance and promotion such as cancer treatment, and in the field of disease healing. It is also desirable to use it in cosmetics in the beauty field for the purpose of acne treatment and prevention.

  The further effect and efficacy possessed by the 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt can be used within a range that can be estimated from the obtained physiological activity data.

  When the 4-vinylcatechol polymerized compound represented by the formula (1) or a pharmaceutically acceptable salt is used for a pharmaceutical use, for example, the 4-vinylcatechol polymerized compound represented by the formula (1) or the pharmaceutical The acceptable intake of salt is not particularly limited as long as the desired improvement, therapeutic or preventive effect can be obtained, and is usually limited in its form, patient age, sex, constitution and other conditions, disease It is appropriately selected according to the type and the degree thereof. About 0.1 mg to about 1,000 mg per day is preferable, and this can be taken in 1 to 4 times a day.

  The 4-vinylcatechol polymerization compound represented by the formula (1) or a pharmaceutically acceptable salt can be used for foods such as functional foods, health foods, health-oriented foods, and the like. The form of the food may be any form such as a beverage, alcoholic beverage, jelly, confectionery, etc. For example, among confectionery, hard candy, soft candy, gummy that is excellent in storage and carrying due to its capacity, etc. Examples include candy and tablets, but there is no particular limitation.

  In addition, when the 4-vinylcatechol polymerized compound represented by the formula (1) or a pharmaceutically acceptable salt is orally administered or ingested as a pharmaceutical or a food, it is based on a conventional method, tablets and pills , Capsules, fine granules, granules and the like. 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. Based on a conventional method, it may be used in an injection or a drip.

  Examples of the cosmetics include lotions, emulsions, creams, packs, finished cosmetics, hair cosmetics, facial cleansers, bath agents, and antiperspirants. These cosmetics are expected to be particularly effective for acne healing because of their lipase inhibitory effects, and can be used for the purpose of acne prevention and healing.

  Since the above-mentioned pharmaceuticals or foods are excellent in safety, 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 and chimpanzees, birds, amphibians and reptiles.

  EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example.

(Example 1: Production of 4-vinylcatechol polymerization compound)
1 g of caffeic acid (manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 20 ml of ethanol, and a mixed solution (pH 5.0) containing 20 ml of mineral water (trade name “Gerol Steiner” manufactured by Sapporo Beverage Co., Ltd.) is added to an autoclave (product). (SANYO LABO AUTOCLAVE, manufactured by Sanyo Electric Co., Ltd.), and heated at 130 ° C. for 40 minutes (first heat treatment). 1 ml of the obtained composition after the reaction was made up to 50 ml with methanol, and 10 μl of this was analyzed by HPLC.
Furthermore, 10 ml of ethanol and 10 ml of mineral water were added (pH 5.4), and the mixture was again heated in an autoclave at 130 ° C. for 40 minutes (second heat treatment), and similarly diluted and 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. From the top, the chromatograms of caffeic acid, first heating, and second heating are shown. In the figure above, the caffeic acid peak is shown. Then, 4-vinylcatechol shown by the peak of A (middle figure) is increased by the first heating, and the caffeic acid and A peaks are decreased by the second heating, and how many peaks are increased. It was confirmed that a plurality of compounds were produced. Especially, it turns out that the compound shown with the peak of the largest B is produced | generated from caffeic acid through 4-vinylcatechol.

(Example 2: Isolation and structure determination of 4-vinylcatechol polymerization compound)
Of the reaction product obtained in Example 1, the compound contained in the peak indicated by B in FIG. 1 was isolated by preparative HPLC. When dried according to a conventional method, 30 mg of brown powdery 4-vinylcatechol polymerization compound (hereinafter, UHA6005) was obtained.

Next, when the molecular weight of the UHA6005 was measured by high resolution electron ionization-mass spectrometry, the measured value was 272.2955, and the following molecular formula was obtained from comparison with the theoretical value.
Theoretical value C16H16O4 (M ⁺ ): 272.2958
Molecular formula C ₁₆ H ₁₆ O ₄

  Next, the UHA6005 is subjected to nuclear magnetic resonance (NMR) measurement, and it is confirmed from analysis of 1H-NMR, 13C-NMR and various two-dimensional NMR data that the UHA6005 has a structure represented by the formula (1). did. From this, it was shown that the 4-vinylcatechol polymerization compound represented by the formula (1) can be efficiently produced by the method of the present invention.

  In addition, about the NMR measurement value, each site | part of UHA6005 represented by Formula (1) is shown.

1H nuclear magnetic resonance spectrum and 13C nuclear magnetic resonance spectrum are shown in Table 1, respectively.
Values are δ and ppm, measured with methanol-d3.

Moreover, the physicochemical properties of UHA6005 were as follows.
(Properties)
Brown powder (soluble)
Water: Insoluble methanol: Soluble ethanol: Soluble DMSO: Soluble chloroform: Soluble ethyl acetate: Soluble

(Example 3: Lipase inhibitory action of UHA6005)
In order to see the inhibitory action of each compound on lipase, an inhibitory action test using rat intestinal lipase was performed.

  The lipase was prepared by suspending 100 mg of rat intestinal acetone powder (manufactured by Sigma Aldrich Japan) in 1 ml of 100 mM citrate buffer (pH 6.0) and stirring at 4 ° C. for 1 hour, followed by centrifugation (15000 rpm, 45 minutes, A supernatant obtained by diluting the supernatant (4 ° C.) 400 times was used as a lipase solution.

  Samples are caffeic acid (manufactured by Wako Pure Chemical Industries, Ltd.), UHA6005, which is the product of the present invention, and epigallocatechin gallate (EGCg, Wako Pure Chemical Industries, Ltd.), a green tea component that is considered to have a higher lipase inhibitory action than before. ) Manufactured) was used. For sample preparation, each compound was dissolved in DMSO (dimethyl sulfoxide, manufactured by Wako Pure Chemical Industries, Ltd.) and prepared to 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM.

For the activity measurement, “Lipase Kit S” (trade name, manufactured by Dainippon Pharmaceutical Co., Ltd.) was used. First, a color developing solution was prepared according to the preparation method described in the catalog of the lipase kit S. Prepare a reaction solution by mixing 70 μl of color developing solution, 2 μl of esterase inhibitor, 10 μl of lipase solution, and 10 μl of sample (final concentrations: 10 μM, 50 μM, 100 μM, 200 μM, 400 μM), and after pre-incubating at 30 ° C. for 5 minutes, substrate The reaction was started by adding 8 μl of the solution. After the reaction for 10 minutes, 150 μl of a reaction stop solution prepared according to the preparation method described in the lipase kit S catalog was added to stop the reaction. The absorbance was measured at a measurement wavelength of 415 nm. A reaction solution to which only DMSO as a sample solvent was added was used as a positive control, and 10 μl of 100 mM citrate buffer (pH 6.0) was added as a negative control instead of the lipase solution. From the relationship between the lipase inhibition rate calculated based on the data obtained from these and the concentration of each compound, the concentration IC ₅₀ (50% inhibition concentration: half maximum inhibitory concentration) that inhibits lipase activity by 50% was calculated (Table 2). . From these results, high lipase inhibitory activity was observed in UHA6005. This effect was not observed with caffeic acid, and since it had higher activity than epigallocatechin gallate, the significance of converting caffeic acid to a 4-vinylcatechol polymerized compound was strongly suggested.

  Therefore, UHA6005 has an excellent lipase inhibitory action, and thus is considered useful as an anti-obesity agent and further as a metabolic syndrome preventive agent. Moreover, since lipase inhibition in the skin is effective for preventing and curing acne, it is considered useful as a therapeutic agent for skin diseases such as acne prevention and healing.

(Example 4: Anticancer activity 1 of UHA6005)
Next, in order to see the effect of each compound on cancer cells, the cancer cell proliferation inhibitory action using HL-60 cells (Human proneolytic leukemia cells: human myeloid leukemia cells) was tested.

For the culture of HL-60 cells, a high nutrient medium RPMI-1690 (Sigma Aldrich) containing 4 mM glutamine (L-Glutamine, manufactured by Sigma Aldrich Japan Co., Ltd.), 10% FBS (Foetal Bovine Serum, manufactured by Biological Industries). Japan Co., Ltd.) was used. In the test, a 96-well plate for cell culture (manufactured by Corning Japan Co., Ltd.) was used, and HL-60 cells, the number of cells of which was adjusted to 5 × 10 ⁵ cells / ml, were seeded at 100 μl per well.

  Two types of samples, caffeic acid and UHA6005, which is the product of the present invention, were used. For sample preparation, each compound was dissolved in DMSO and prepared so that the final concentrations in HL-60 cell culture were 6.3 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, respectively. Started.

The number of viable cells was quantified by the MTT method using “Cell counting kit-8” (trade name, manufactured by Dojin Dou Molecular Technology Co., Ltd.). After 24 hours from the start of the test, 10 μl of Cell counting kit-8 solution was added to each well and stirred well. After a light-shielding reaction for 1 hour, the absorbance at a measurement wavelength of 450 nm is measured using a plate reader (“BIO-RAD Model 680”, manufactured by Bio-Rad Laboratories), and the cell viability is calculated based on the obtained data. Calculated (FIG. 2). The cell viability is a value calculated by setting the number of viable cells in a culture solution to which only DMSO as a solvent is added as 100% and the number of viable cells in each compound concentration as a relative value. From the relationship between the concentration of each compound and the cell viability, a concentration IC ₅₀ (50% inhibitory concentration: half maximum inhibitory concentration) that suppresses cell proliferation by 50% was calculated (Table 3). From these results, it was confirmed that UHA6005 has a strong ability to suppress cancer cell growth. This effect was not observed at all for caffeic acid, and the significance of converting caffeic acid into a 4-vinylcatechol polymerization compound was strongly suggested.

(Example 5: Anti-cancer effect 2 of UHA6005)
Subsequently, the test of the anticancer activity was performed using HL-60 cells. Caffeic acid prepared by seeding HL-60 cells in a 100 mm standard dish (“BD Falcon”, manufactured by Nippon Becton Dickinson Co., Ltd.) to 5.0 × 10 ⁵ cells / ml and adjusting with DMSO UHA6005 was added to 37.5 μM, 75 μM, and 100 μM, respectively. The cells were cultured for 24 hours, and the collected cells were washed with PBS (DulBecco's PBS (−), manufactured by Wako Pure Chemical Industries, Ltd.), and DNA was extracted from the cells using a known DNA extraction method. The obtained DNA sample was applied to a 1% agarose gel (“Agarose TAKARA”, manufactured by Takara Bio Inc.) at 200 ng / well. Electrophoresis was performed and the staining reaction was performed using ethidium bromide (Ethidium Bromide Solution, manufactured by Bio-Rad Laboratories Co., Ltd.). Moreover, protein was extracted from the cell using the known protein extraction method from the cell cultured on the same conditions. The obtained protein sample was applied to a polyacrylamide gel (“READY GRLS J”, manufactured by Bio-Rad Laboratories Co., Ltd.) at 200 ng / well. Electrophoresis was performed, and the protein was transferred to a PVDF membrane (“Immobilon-PSQ” trade name, manufactured by Nippon Millipore). As the detection antibody, a caspase-3 rabbit polyclonal antibody (“Caspase-3 Rabbit AB” trade name, manufactured by Cell Signaling Technology Japan Co., Ltd.) is used as a primary antibody, and an HRP-linked rabbit IgG polyclonal antibody (“Anti” is used as a secondary antibody). -Rabbit IgG HRP-linked AntiBody "trade name, manufactured by Cell Signaling Technology Japan Co., Ltd.) was used. The detection reaction was performed using a tetramethylbenzidine solution (“3,3 ′, 5,5′-tetramethylbenzidine TMB Stabilized Substrate for HRP”, trade name, manufactured by Promega Corporation).

The obtained results are shown in FIGS.
FIG. 3 is an electrophoretogram of the DNA extract of each sample, with current flowing from top to bottom. From left lane, DNA molecular weight marker λ / Pst (first lane), normal cultured cells (second lane), DMSO treatment (third lane), caffeic acid 37.5 μM treatment (fourth lane), 75 μM treatment (first lane) 5 lanes), 100 μM treatment (6th lane), UHA6005 37.5 μM treatment (7th lane), 75 μM treatment (8th lane), and 100 μM treatment (9th lane). Since the DNA ladder is not confirmed in the additive-free cultured cells (second lane) and DMSO-treated cells (third lane), the reliability of this experiment can be confirmed.
In addition, DNA laddering can be observed in cells treated with UHA6005 at 37.5 μM or more (7th, 8th and 9th lanes), whereas DNA laddering is observed at the same concentration of caffeic acid. None (3rd, 4th, 5th lane).

FIG. 4 is an electrophoretogram of the protein extract of each sample, with current flowing from top to bottom. From the left lane, protein molecular mass marker ("Precision Plus Protein ™ Standards", trade name, manufactured by Bio-Rad Laboratories Co., Ltd., 1st lane), the following lanes are according to the lanes of the electrophoresis photograph of the previous DNA extract Yes. Since no activated caspase 3 was detected in the vicinity of 17 kDa in the additive-free cultured cells (second lane) and DMSO-treated cells (third lane), the reliability of this experiment can be confirmed.
In addition, caspase 3 activated in cells treated with UHA6005 at 37.5 μM or more (7th, 8th and 9th lanes) can be observed in the vicinity of 17 kDa, whereas caspase 3 at the same concentration of caffeic acid. Activation was not observed (3rd, 4th, 5th lane). This result shows the same tendency as the DNA ladder shown above. From this, it was shown that UHA6005 has a high effect of inducing apoptosis, and its potency is significantly higher than caffeic acid.

  Therefore, since UHA6005 has an excellent anticancer effect, it is considered useful as an anticancer agent and further as a cancer preventive agent.

(Example 6: Difference in production amount of UHA6005 depending on heating temperature)
A mixed solution (pH = 5.0) of 100 mg of caffeic acid, 1 ml of ethanol and 1 ml of mineral water was heated in an autoclave at 70 ° C., 90 ° C., 110 ° C., and 130 ° C. for 20 minutes. 1 ml of the post-reaction composition obtained under each temperature condition was made up to 50 ml with methanol and analyzed by HPLC in the same manner as in Example 1.

  As a result, the formation of UHA6005 was confirmed under conditions other than 70 ° C. The production ratio of UHA6005 from caffeic acid was non-produced at 70 ° C., trace amount at 90 ° C., 0.5% by weight at 110 ° C., and 3% by weight at 130 ° C. That is, heating at 130 ° C. was the most efficient.

(Example 7: Preparation of UHA6005-containing extract)
A mixed solution prepared by adding 10 g of kiwifruit juice concentrate, 10 ml of ethanol and 10 ml of mineral water was heated in an autoclave at 130 ° C. for 60 minutes. The obtained reaction solution was heated under reduced pressure to dryness to obtain 10 g of UHA6005-containing extract. In 10 g of the obtained UHA6005 extract, it was confirmed by the same method as in Example 6 that 0.015 g of UHA6005 was contained. This work was repeated as necessary.

(Example 8: Food containing UHA6005)
1 g of UHA6005-containing extract obtained in Example 7 was dissolved in 100 mL of ethanol in advance, 500 g of sugar and 400 g of starch syrup were mixed and dissolved therein, and after mixing 100 g of fresh cream, 20 g of butter, 70 g of condensed milk, and 1.0 g of emulsifier, The pressure was reduced by −550 mmHg in a vacuum kettle and concentrated under the condition of 115 ° C. to obtain a milk hard candy having a moisture value of 3.0 wt%. This milk hard candy is not only easy to eat as a confectionery, but also improves obesity, prevents obesity, reduces the risk of cancer spreading in cancer patients, reduces the risk of developing cancer. It can also be used as a functional food that is expected to prevent cancer.

(Example 9: Drug containing UHA6005)
UHA6005 obtained by the same method as in Examples 1 and 2 was dissolved in ethanol, adsorbed onto microcrystalline cellulose, and then dried under reduced pressure. This was tableted according to a conventional method. The formulation is 10 parts by weight of UHA6005, 23 parts by weight of corn starch, 12 parts by weight of lactose, 8 parts by weight of carboxymethyl cellulose, 32 parts by weight of microcrystalline cellulose, 4 parts by weight of polyvinylpyrrolidone, 3 parts by weight of magnesium stearate, 8 parts by weight of talc. Street. This tableted product can be effectively used as a pharmaceutical for the purpose of improving obesity, preventing obesity, and curing cancer.

(Example 10: Cosmetics containing UHA6005)
1 part by weight of polyoxyethylene sorbit tetraoleate, 0.5 part by weight of polyoxyethylene stearyl ether, 1 part by weight of glyceryl monostearate, 0.5 part by weight of pyruvic acid, 0.5 part by weight of stearyl alcohol, avocado 1 part by weight of oil and 0.1 part by weight of UHA6005 obtained in the same manner as in Examples 1 and 2 were dissolved according to a conventional method, and 1 part by weight of sodium lactate, 5 parts by weight of propylene glycol, and carboxyvinyl polymer were dissolved therein. 0.1 part by weight, a very small amount of perfume and 89.3 parts by weight of purified water were added and emulsified with a homogenizer to obtain an emulsion. This milky lotion can be effectively used as a medicinal cosmetic product having an effect of treating and preventing skin diseases such as acne.

Claims (6)

The 4-vinylcatechol polymerization compound represented by the formula (1) is characterized in that a 4-vinylcatechol polymerization compound represented by the following formula (1) is produced by heat-treating caffeic acid in the presence of a metal salt. Alternatively, a method for producing a pharmaceutically acceptable salt.
A lipase inhibitor comprising a 4-vinylcatechol polymerization compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
An anti-obesity agent comprising a 4-vinylcatechol polymerization compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
A skin disease therapeutic agent comprising a 4-vinylcatechol polymerization compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
An anticancer agent comprising a 4-vinylcatechol polymerization compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
A food, pharmaceutical or cosmetic comprising a 4-vinylcatechol polymerization compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.