JP5742589B2 - New hydroxystilbene derivatives - Google Patents

Description

  The present invention relates to a novel hydroxystilbene derivative, a method for producing the novel hydroxystilbene derivative, and an anticancer agent containing the novel hydroxystilbene derivative.

p-Coumaric acid is a precursor of lignin and lignan, which are the main components of trees, and is also a precursor of functional components of stilbenes such as flavonoids and resveratrol. It is an abundant component. In addition, it is known that p-coumaric acid is contained in the fruits, peels and propolis of many fruits including plums.
Further, pterostilbene, which is a hydroxystilbene, is contained in grapes and berries, like resveratrol, and is a component that has been attracting attention as a result of various functional studies.
Both p-coumaric acid and pterostilbene have food experience and are highly safe ingredients.

Regarding the p-coumaric acid, there is a prior art related to its physiological function. For example, p-coumaric acid, anti-Helicobacter pylori agent containing cinnamic acid as an active ingredient (Patent Document 1), skin improving agent containing ferulic acid and / or p-coumaric acid as an active ingredient (Patent Document 2), Examples thereof include an angiogenesis inhibitor (Patent Document 3) containing cinnamic acid contained in a propolis extract including coumaric acid as an active ingredient.
There is also prior art related to p-coumaric acid derivatives. For example, an antibacterial agent containing a lignin chemically synthesized from coumaric acid as an active ingredient (Patent Document 4), an adiponectin production enhancer containing a caffeic acid amide derivative or an ester derivative as an active ingredient (Patent Document 5), p-coumaru A method for synthesizing a benzofurancarboxamide derivative that is an antibacterial agent using an acid dimer as a raw material (Patent Document 6) is mentioned.
Moreover, since many said p-coumaric acids and p-coumaric acid derivatives are also contained in a plant, p-coumaric acid and p-coumaric acid derivatives are used as fruit polyphenols of unripe fruits of apples, pears or peaches. And the like, anti-hypertensive agents, antimutagenic agents, allergy inhibitors, anti-cariogenic agents and deodorants (Patent Document 7). Moreover, although p-coumaric acid is weak, there exists a report that it has anticancer activity (nonpatent literature 1).

  Pterostilbene, which is a hydroxystilbene contained in grape skin, etc., also has prior art relating to its physiological function. For example, a nutritional and therapeutic composition containing an insulin sensitivity enhancer and a peptide fraction (Patent Document 8), an aminergic pharmaceutical composition and method (Patent Document 9), anticancer activity and antioxidant activity (non-patent document) Reference 2).

  Thus, since many of p-coumaric acid, pterostilbene, and these derivatives show the outstanding usefulness, the technique of manufacturing these compounds efficiently as a raw material or a lead compound is also disclosed. . For example, a method for producing p-hydroxycinnamic acid (p-coumaric acid) or the like by a microorganism using a recombinant is shown (Patent Document 10).

On the other hand, according to a survey by the Ministry of Health, Labor and Welfare, 30% of the causes of death in 2008 in Japan are malignant neoplasms or cancer. While many cancers are leveling off or decreasing due to the development of medical technology and drugs, one of the increasing cancers is oral cancer, accounting for 5% of cancer occurrences. In 2015, the number of affected cases is expected to be four times the current number.
In the current research, luteolin is known as a natural product-derived compound that can be ingested on a daily basis (Non-patent Document 3).
However, it is desired to develop a therapeutic or preventive agent for safe cancer, particularly oral cancer, which has stronger anticancer activity and can be taken on a daily basis.

JP-A-11-106335 Japanese Patent No. 3309961 JP 2007-223948 A Japanese Patent No. 4395623 JP 2007-262050 A JP 2008-189554 A Japanese Patent Laid-Open No. 2002-47196 Japanese Patent No. 4719465 Special table 2008-536866 gazette JP-T-2005-522180

Cancer Epidemiology, Biomarkers & Prevention, Vol. 9, 1163-1170, November (2000) J. et al. Agric. Food. Chem. 2002, 50, 3453-3457. Journal of Dental Research, 84, 401-406 (2008)

  In view of the above-described situation regarding pterostilbene and p-coumaric acid and the current situation of cancer, particularly oral cancer, the present inventors should search for hydroxystilbene derivatives having stronger physiological activity and establish a production method thereof. As a result of intensive studies, surprisingly, pterostilbene, p-coumaric acid, luteolin and ε -The present inventors have succeeded in producing a novel hydroxystilbene derivative having an anticancer activity superior to that of vinyliferin, in particular, an activity against oral cancer, and completed the present invention.

Accordingly, the present invention provides a novel hydroxystilbene derivative having stronger anticancer activity than pterostilbene, p-coumaric acid, luteolin and ε-viniferin, particularly potent anticancer activity against oral cancer. It is an object to provide a method for efficiently and safely producing a derivative.
Another object of the present invention is to provide an anticancer agent, a food, and a pharmaceutical comprising the novel hydroxystilbene derivative.

The gist of the present invention is as follows:
[1] Formula (1):

Or a pharmaceutically acceptable salt thereof (hereinafter abbreviated as a novel hydroxystilbene derivative),
[2] An anticancer agent containing the novel hydroxystilbene derivative according to [1],
[3] An anticancer agent for oral cancer cells containing the novel hydroxystilbene derivative according to [1],
[4] A food containing the novel hydroxystilbene derivative according to [1] ,
[5] A pharmaceutical or quasi-drug containing the novel hydroxystilbene derivative according to [1],
[ 6 ] A method for producing a novel hydroxystilbene derivative as described in [1] above, wherein the desired compound is produced by heat-treating pterostilbene and p-coumaric acid at 110 ° C. or higher in the presence of a metal salt. .

The novel hydroxystilbene derivative of the present invention is useful as a novel anticancer agent because it is superior in anticancer activity, particularly anti-oral cancer activity, compared to conventional pterostilbene, p-coumaric acid, luteolin and ε-viniferin. is there.
Moreover, since the novel hydroxystilbene derivative of the present invention is excellent in safety in addition to being excellent in physiological activity as described above, it can be incorporated into foods, pharmaceuticals and quasi drugs.

FIG. 1 shows the chromatogram obtained in Example 1. From the top, chromatogram before reaction, (1) Chromatogram using mineral water as metal salt, (2) Chromatogram using mineral premix, (3) Chromatogram using magnesium phosphate trihydrate Shows gram. In the figure, the peak A indicates the novel hydroxystilbene derivative of the present invention.

Hereinafter, the present invention will be described in detail.
The novel hydroxystilbene derivative of the present invention has the formula (1):

Or a pharmaceutically acceptable salt thereof.

  In the novel hydroxystilbene derivative, the carbon-carbon double bond may be trans or cis, and includes a mixture of a cis isomer and a trans isomer.

  Examples of the pharmaceutically acceptable salt of the novel hydroxystilbene derivative 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 Metal hydroxide salts such as aluminum hydroxide salts; amines such as alkylamine salts, dialkylamine salts, trialkylamine salts, alkylenediamine salts, cycloalkylamine salts, arylamine salts, aralkylamine salts, and heterocyclic amine salts Salts; amino acid salts such as α-amino acid salts and 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.

  As a result of intensive studies, the present inventors have found that a novel hydroxystilbene derivative can be produced efficiently and safely by heat-treating pterostilbene and p-coumaric acid in the presence of a metal salt. . Below, the manufacturing method (henceforth the manufacturing method of this invention) of the novel hydroxystilbene derivative of this invention is demonstrated concretely.

In the production method of the present invention, pterostilbene is used as a precursor. Pterostilbene has structural isomers of trans form and cis form, but some conversion of trans form and cis form occurs by heating or ultraviolet rays. Therefore, pterostilbene may be a trans isomer, a cis isomer, or a mixture of a trans isomer and a cis isomer. Pterostilbene may be of natural origin extracted and purified from grape skins or berries, or may be a chemically synthesized chemical product with high purity. When natural pterostilbene is used, it does not need to be completely purified, and the desired production reaction proceeds as described later, and finally the novel hydrostilbene derivative of the present invention is obtained. Mixtures containing other ingredients can also be used.
However, from the viewpoint of the recovery rate of the novel hydroxystilbene derivative, a mixture containing 1% by weight or more in terms of pterostilbene is desirable as a raw material.
As naturally occurring pterostilbene, extracts from raw materials such as grape skins and berries, freeze-dried products, and the like may be used.

In the production method of the present invention, p-coumaric acid is also required as a precursor. The p-coumaric acid may be naturally derived or may be a chemically synthesized chemical product with high purity. When natural p-coumaric acid is used, it does not need to be completely purified, and the desired production reaction proceeds as described later, and finally the novel hydroxystilbene derivative of the present invention is obtained. A mixture containing components other than coumaric acid can also be used.
However, from the viewpoint of the recovered amount of the novel pterostilbene derivative, a mixture containing 1% by weight or more of p-coumaric acid is desirable as a raw material. Examples of such raw materials include various fruits and juices, concentrated fruit juices, or frequently-discarded fruit skin extracts, propolis or extracts thereof, or the prior art described in Patent Document 10 above. And p-coumaric acid-containing culture solution produced by simple microbial fermentation.

In the production method of the present invention, pterostilbene, p-coumaric acid, or a mixture of pterostilbene and p-coumaric acid is dissolved in a suitable solvent. At this time, if the solvent is only water, the solubility of pterostilbene and p-coumaric acid is remarkably low, so that the solvent may be dissolved only in a mixed solution of water and an organic solvent or 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, Pterostilbene and p-coumaric acid should just fully melt | dissolve. Among them, it is preferable from the viewpoint of safety and cost to use a solvent containing only methanol or ethanol, or a mixed solution of water and methanol or water and ethanol. It is desirable to use ethanol or hydrous ethanol as a solvent from the viewpoint of safety and legal regulations when using it for food without fully applying final purification to the post-reaction composition containing a novel hydroxystilbene derivative. .
There is no restriction on the concentration of pterostilbene and p-coumaric acid in the solution containing the resulting pterostilbene, p-coumaric acid, or a mixture of pterostilbene and p-coumaric acid. Since the higher the respective concentrations are, the smaller the amount of the solvent used, and the like, there are advantages such as the use of pterostilbene and p-coumaric acid.
Pterostilbene and p-coumaric acid may not be completely dissolved in the solution before the formation reaction. For example, when mixing a pterostilbene-containing solution and a p-coumaric acid-containing solution, even if the pterostilbene concentration and p-coumaric acid concentration in each solution are equal to or higher than the saturated concentration, It may be adjusted so that it is close to the saturated concentration.

  Next, it is preferable to adjust the pH of the solution containing pterostilbene and p-coumaric acid (hereinafter, pterostilbene and p-coumaric acid-containing solution) to less than 8. As an adjustment method, for example, after preparing a solution containing pterostilbene and p-coumaric acid, the pH may be adjusted by adding a pH adjuster, or the pH of the solvent is adjusted in advance when preparing the solution. Also good. If the pH at the start of the reaction of the pterostilbene and p-coumaric acid-containing solution is 8.0 or more, other reactions and decomposition of the target compound also occur on the other hand, so the final recovered amount of the new hydroxystilbene derivative decreases. To do. Therefore, the pH at the start of the reaction is desirably 3 or more and less than 8.

In the production method of the present invention, a metal salt is added to the pterostilbene and p-coumaric acid-containing solution. 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.
In addition, as a mixture of the metal salts, for example, a mineral premix (Tanabe Seiyaku Co., Ltd., a mineral mixture mainly composed of zinc gluconate, ammonium iron citrate, calcium lactate, copper gluconate, and magnesium phosphate) In addition, substances containing several kinds of metal salts are listed. Moreover, mineral water can also be mentioned as a mixture containing a some metal salt.
In addition, as content of the said metal salt, what is necessary is just the quantity which can produce | generate a novel hydroxystilbene derivative, and there is no limitation in particular.

  Next, the pterostilbene and p-coumaric acid-containing solution is heat-treated in the presence of a metal salt. By this heat treatment, a formation reaction of a novel hydroxystilbene derivative is performed. In order to advance the production reaction efficiently, it is preferable to adjust the heating temperature of the pterostilbene and p-coumaric acid-containing solution to 110 ° C. or higher. Further, considering the boiling point of the solvent to be used, pressure heating is desirable. For example, put pterostilbene and p-coumaric acid-containing solution in an open container and heat the container at a high temperature exceeding the boiling point of the solvent. Put pterostilbene and p-coumaric acid-containing solution in a sealed container and add the container. It is preferable to heat the solution so that the solution temperature reaches 110 ° C. or higher at least partially, such as heating, pressurizing and heating using a retort device or an autoclave. From the viewpoint of recovery efficiency, it is more preferable that the solution temperature be 110 ° C. to 150 ° C. uniformly. 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 20 minutes to 500 minutes is desirable. Further, the heating may be performed once or may be repeated repeatedly in a plurality of times. When heating in multiple steps, it is preferable to add a new solvent.

  The completion of the formation reaction of the novel hydroxystilbene derivative by the heat treatment may be judged by, for example, confirming the production amount of the novel hydroxystilbene derivative by component analysis by HPLC.

The resulting reaction solution contains the novel hydroxystilbene derivative of the present invention.
In addition, when a novel hydroxystilbene derivative is produced by a process using only safe raw materials, it can be used for foods, pharmaceuticals or quasi drugs in a mixture state containing the novel hydroxystilbene derivative. For example, when natural pterostilbene and p-coumaric acid are dissolved in a water-containing ethanol solvent and heated with mineral water or mineral premix, the resulting reaction solution should be used as one of the food ingredients. Is possible.

  Also, if you want to improve the flavor and further enhance the functionality, concentrate the reaction solution to increase the concentration of the new hydroxystilbene derivative, or purify the reaction solution to obtain a pure product of the new hydroxystilbene derivative. Can do. Concentration and purification can be performed by a known method. For example, the novel pterostilbene derivative can be concentrated by extraction using a solvent extraction method such as chloroform, ethyl acetate, ethanol, or methanol, 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 can also be applied.

  In addition, when separating and recovering the novel hydroxystilbene derivative represented by the formula (1) from the reaction solution, column chromatography, HPLC or the like may be used.

  If necessary, the concentrated or purified product can be dried under reduced pressure or lyophilized to remove the solvent to obtain a powdered novel hydroxystilbene derivative.

  The novel hydroxystilbene derivative of the present invention obtained as described above has extremely excellent anticancer activity as compared with pterostilbene, p-coumaric acid, luterion and ε-viniferin. Therefore, an anticancer agent containing a novel hydroxystilbene derivative as an active ingredient can be provided.

  In addition, the further effect efficacy which the novel hydroxystilbene derivative obtained by this invention has can be used in the range which can be estimated from the obtained physiological activity data.

  Since the safety of pterostilbene and p-coumaric acid as raw materials has been confirmed, it is considered that the safety of the novel hydroxystilbene derivative of the present invention is also excellent.

  Moreover, since the novel hydroxystilbene derivative of the present invention exhibits the physiological activity as described above, it can be used in foods, pharmaceuticals, quasi drugs and the like.

  The food may be in any form such as beverage, alcoholic beverage, jelly, confectionery, etc., and among confectionery, hard candy, soft candy, gummy candy, tablet that is excellent in storage and carrying due to its capacity etc. There are no particular limitations. Further, by adding a novel hydroxystilbene derivative to wine, it is possible to obtain a new wine that further enhances the health function effect of the wine. Like this new wine, food and drink with both palatability and health function effects is a field with very high social needs and can respond to this. In addition, since the novel hydroxystilbene derivative has an excellent anticancer activity against cancer, particularly oral cancer, as described later, it can be easily ingested for the purpose of preventing oral cancer, which is currently a problem. It is also useful as a candy or tablet. The food includes functional food, health food, health-oriented food, and the like.

  Examples of the pharmaceutical include solid preparations such as powders, tablets, pills, capsules, fine granules and granules, liquids such as liquids, suspensions and emulsions, gels 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 quasi drugs include quasi drugs used in the oral cavity, such as toothpaste, mouthwash, and mouth rinse.

When preparing foods, pharmaceuticals or quasi-drugs using the novel hydroxystilbene derivatives of the present invention, any components that are usually used in foods, pharmaceuticals or quasi-drugs are arbitrarily selected within a range that does not impair the effects of the present invention. Can be blended.
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 Can be combined with raw materials or materials usually blended in
In the case of quasi-drugs, combinations of main ingredients, base materials, surfactants, foaming agents, wetting agents, thickeners, clearing agents, flavoring agents, coloring agents, stabilizers, preservatives, bactericides, etc. Based on the law, it can be made into a liquid, ointment-like or sprayable final form.
In particular, considering the physiologically active field of the novel hydroxystilbene derivative of the present invention, it is preferably used in the field of health maintenance and promotion such as cancer prevention and cancer treatment, and further in the field of disease healing.

  When the novel hydroxystilbene derivative of the present invention is added to food, it is usually preferable to add 0.001 to 20% by weight based on the food.

  When the novel hydroxystilbene derivative of the present invention is used for pharmaceutical purposes, for example, the intake thereof is not particularly limited as long as the desired improvement, therapeutic or preventive effect can be obtained. It is appropriately selected according to age, sex, constitution and other conditions, the type and degree of disease. 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.

  When the novel hydroxystilbene derivative of the present invention is added to a quasi drug, it is usually preferably added in an amount of 0.001 to 30% by weight in the quasi drug.

  In addition, since the novel hydroxystilbene derivative of the present invention 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, dogs, monkeys, chimpanzees and other mammals, birds, amphibians, reptiles, etc. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for eel, Thailand, yellowtail, shrimp, etc., dogs, Pet foods used for cats, small birds, squirrels, etc. are listed.

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

(Example 1: Study on production method of novel hydroxystilbene derivative)
Pterostilbene (manufactured by Tokyo Chemical Industry Co., Ltd.) 100 mg and p-coumaric acid (manufactured by Wako Pure Chemical Industries, Ltd.) 100 mg were dissolved in 2 mL of ethanol, and (1) mineral water (trade name “Gerol Steiner” Sapporo Beverage Co., Ltd.) )) 2 mL, (2) mineral premix 100 mg, water 2 mL, (3) magnesium phosphate trihydrate (manufactured by Wako Pure Chemical Industries, Ltd., main component of mineral premix) 100 mg, water 2 mL, respectively In addition, three types of pterostilbene and p-coumaric acid-containing solutions (pH: (1) 5.0, (2) 5.7, (3) 5.2) were obtained. The three types of pterostilbene and p-coumaric acid-containing solutions were heated in an autoclave (SANYO LABO AUTOCLAVE) at 130 ° C. for 60 minutes. 1 mL of the obtained reaction solution 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 (volume%): 100% A / 0% B to 0% A / 100% B for 33 minutes, 100% B for 7 minutes (all linear)

The obtained chromatogram is shown in FIG. From the top, the chromatograms of the reaction solutions (1), (2), and (3) are shown before the reaction. After the reaction, peaks other than pterostilbene and p-coumaric acid were detected, and it was confirmed that a plurality of compounds were produced.
In addition, the peak of A in the figure is a peak newly appearing in the reaction, and there was no difference between the metal salts.

(Example 2: Mass production of a novel hydroxystilbene derivative)
1 g of pterostilbene and 1 g of p-coumaric acid were dissolved in 20 mL of ethanol, and 20 mL of mineral water was added to obtain a pterostilbene and p-coumaric acid-containing solution (pH = 5.0). This pterostilbene and p-coumaric acid-containing solution was heated in an autoclave at 130 ° C. for 180 minutes. When 1 mL of the obtained reaction solution was made up to 50 mL with methanol and analyzed by HPLC in the same manner as in Example 1, the same chromatogram as in Example 1 was confirmed.

(Example 3: Isolation and structure determination of a novel hydroxystilbene derivative)
Among the reactants obtained in Example 2, the compound contained in the peak indicated by A in FIG. 1 was isolated by preparative HPLC and dried by a conventional method to obtain 188 mg of a new compound (hereinafter referred to as UHA7033). The isolated and purified UHA7033 became a brown powdery substance.

Subsequently, when the molecular weight of the UHA7033 was measured by high resolution electron ionization-mass spectrometry, the measured value was 376.4447, and the following molecular formula was obtained from comparison with the theoretical value.
Theoretical value C24H24O4 (M ⁺ ): 376.4450
Molecular formula C ₂₄ H ₂₄ O ₄

  Next, the UHA7033 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 UHA7033 has a structure represented by the formula (1). did. It was shown that the novel pterostilbene derivative represented by the formula (1) can be efficiently produced by the method of the present invention.

  For NMR measurements, UHA7033

Table 1 shows each ¹ H nuclear magnetic resonance spectrum and ¹³ C nuclear magnetic resonance spectrum.
The values were δ and ppm, and the solvent was measured with methanol-d ₃ .

UHA7033 has the same physicochemical properties as follows.
(Properties)
Brown powder (soluble)
Water: Slightly soluble methanol: Dissolved ethanol: Dissolved DMSO: Dissolved chloroform: Dissolved ethyl acetate: Dissolved

(Example 4: Anticancer effect of UHA7033 on human myeloid leukemia cells)
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-1640” (Sigma) containing 4 mM glutamine (manufactured by L-Glutamine Sigma-Aldrich Japan), 10% fetal bovine serum (manufactured by Foetal Bovine Serum: FBS Biological industries). Aldrich Japan) was used. For the test, a 96-well plate for cell culture (manufactured by Corning Japan Co., Ltd.) was used and seeded with 100 μL per well of HL-60 cells adjusted to a cell number of 5 × 10 ⁵ cells / mL. Used for.

Three types of samples were used: pterostilbene, p-coumaric acid, and UHA7033 which is the product of the present invention. In the sample preparation, each compound was dissolved in dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.), and the final concentrations in the HL-60 cell culture solution were 6.3 μM, 12.5 μM, The test was started under the culture conditions of 37 ° C. and 5% CO ₂ by adding 25 μM, 50 μM, and 100 μM. A negative control was prepared by adding the same amount of DMSO as a solvent.

The number of viable cells was quantified by the MTT method using “Cell counting kit-8” (manufactured by Dojin Chemical Laboratory). That is, 24 hours after the start of the test, 10 μL of the Cell counting kit-8 solution was added to each well and stirred well. The light-shielding reaction was performed for 1 hour at 37 ° C. and 5% CO ₂ . Thereafter, absorbance at a measurement wavelength of 450 nm was measured using a plate reader (“BIO-RAD Model 680”, manufactured by Bio-Rad Laboratories), and the cell viability was calculated based on the obtained data. 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 (half maximum inhibitory concentration: 50% inhibitory concentration) that suppresses cell proliferation by 50% was calculated (Table 2). From these results, cancer cell growth suppressing ability superior to UHA7033 was recognized. This effect was not observed at all for p-coumaric acid, and showed higher activity than pterostilbene. Therefore, the high significance which converts pterostilbene and p-coumaric acid into a novel hydroxystilbene derivative was shown.

(Example 5: Anticancer effect of UHA7033 on human oral cancer cells)
Next, in order to see the effect of each compound on cancer cells, the cancer cell proliferation inhibitory action using SCC-4 cells (human tongue squamous cell carcinoma cells, manufactured by ATCC) which are human oral cancer cells was tested.

For the culture of SCC-4 cells, 400 ng / mL hydrocortisone (Hydrocortisone, manufactured by Sigma-Aldrich Japan), 1% antibiotic-antimycotic (manufactured by Antibiotic-Antilytic, Gibco (GIBCO)), 10% FBS DMEM / F-12 (1: 1) medium (Gibco) containing (ATCC) was used. For the test, a collagen I-coated 96-well plate for cell culture (manufactured by BD Japan) was used, and 100 μL of SCC-4 cells adjusted to have a cell number of 5 × 10 ⁵ cells / mL were seeded per well. This was cultured for 24 hours under conditions of 37 ° C. and 5% CO ₂ , and used for testing in a state of 80% confluence or higher.

Samples include pterostilbene, p-coumaric acid, luteolin (manufactured by Wako Pure Chemical Industries, Ltd.), which is a natural product having anti-cancer activity against SCC-4 cells, and ε-viniferin, a resveratrol derivative (Wako Pure Chemical). Kogyo Co., Ltd.) and UHA7033 which is the product of the present invention were used. Samples were prepared by dissolving each compound in DMSO to 0.63 mM, 1.25 mM, 2.5 mM, 5 mM, and 10 mM. This was added so that the final concentrations in the SCC-4 cell culture medium were 6.3 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, respectively, and the test was started under 37 ° C. and 5% CO ₂ culture conditions. . A negative control was prepared by adding the same amount of DMSO as a solvent.

The number of viable cells was quantified by the MTT method using “Cell counting kit-8” in the same manner as in Example 4. That is, 48 hours after the start of the test, 10 μL of the Cell counting kit-8 solution was added to each well and well stirred. After a light-shielding reaction at 37 ° C. and 5% CO ₂ for 1 hour, the absorbance at a measurement wavelength of 450 nm was measured using a plate reader, and the cell viability was calculated based on the obtained data. From the relationship between the concentration of each compound and the cell viability, the concentration IC ₅₀ that suppresses cell proliferation by 50% was calculated (Table 3). From these results, since the IC ₅₀ of UHA7033 it had the lowest, a strong oral cancer cell growth suppressing ability was observed. This effect was not observed at all for p-coumaric acid, and further showed higher activity than pterostilbene, luteolin and ε-viniferin. Therefore, the high significance which converts pterostilbene and p-coumaric acid into a novel hydroxystilbene derivative was shown.

(Example 6: Difference in production amount of UHA7033 depending on heating temperature)
A mixed solution of 100 mg pterostilbene, 100 mg p-coumaric acid, 2 mL ethanol, and 2 mL mineral water (pH = 5.0) is heated in an autoclave at 70 ° C., 90 ° C., 110 ° C., and 130 ° C. for 50 minutes. did. 1 mL of the post-reaction composition obtained under each temperature condition was diluted to 50 mL with methanol and analyzed by HPLC in the same manner as in Example 1.

  As a result, the formation of UHA7033 was confirmed at 110 ° C. or higher. The production ratio (% by weight) from the total amount of pterostilbene and p-coumaric acid is 70 ° C., 90 ° C. is not produced, 110 ° C. is extremely small, 130 ° C. is 9.4%, and heating at 130 ° C. Mostly UHA7033 was generated.

(Example 7: Preparation of UHA7033-containing extract)
A mixed solution prepared by adding 10 g of berry extract powder (pterostilbene raw material), 10 g of propolis extract (p-coumaric acid raw material), 10 mL of ethanol and 10 mL of mineral water was heated at 130 ° C. for 180 minutes in an autoclave. The obtained reaction solution was heated under reduced pressure to dryness to obtain 13 g of UHA7033-containing extract. When 13 g of the obtained UHA7033 extract was confirmed by the same method as in Example 3, 0.012 g of UHA7033 was contained. This work was repeated as necessary.

(Example 8: Food containing UHA7033)
1 g of UHA7033-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 a condition of 115 ° C. to obtain a milk hard candy having a moisture value of 3.0% by weight. This milk hard candy is easy to eat as a confectionery, as well as reducing the risk of cancer spread in cancer patients, reducing the risk of developing cancer, and as a functional food that is expected to prevent cancer. Can also be used.

(Example 9: Drug containing UHA7033)
UHA7033 obtained by the same method as in Examples 2 and 3 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 UHA7033, 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 medicine for cancer healing.

(Example 10: Quasi-drug containing UHA7033)
1.2 g of UHA7033 obtained by the method of Examples 2 and 3 was dissolved in 10 mL of ethanol, 20 g of taurine, 0.12 g of vitamin B1 nitrate, 0.6 g of sodium benzoate, 4 g of citric acid, 60 g of sugar, and 10 g of polyvinylpyrrolidone. All were dissolved in purified water and made up to 1000 mL. The pH was adjusted to 3.2 using dilute hydrochloric acid. 50 ml of 1000 ml of the obtained solution was filled in a glass bottle and sterilized at 80 ° C. for 30 minutes to complete a quasi-drug drink. In addition to the purpose of nutritional supplementation, this drink is effective as a quasi-drug for the purpose of reducing the risk of cancer spread in cancer patients, reducing the risk of developing cancer, and preventing cancer. Available.

Claims (6)

Following formula (1):

Or a pharmaceutically acceptable salt thereof.   An anticancer agent comprising the novel hydroxystilbene derivative according to claim 1 or a pharmaceutically acceptable salt thereof.   An anticancer agent for oral cancer cells comprising the novel hydroxystilbene derivative according to claim 1 or a pharmaceutically acceptable salt thereof. Claim 1 new hydroxystilbenes derivative according or a pharmaceutically foods containing acceptable salts. A pharmaceutical product or quasi-drug containing the novel hydroxystilbene derivative or pharmaceutically acceptable salt thereof according to claim 1. The novel hydroxystilbene derivative or a pharmaceutically acceptable product thereof according to claim 1, wherein the target compound is produced by heat-treating pterostilbene and p-coumaric acid to 110 ° C or higher in the presence of a metal salt. Method for producing salt.

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