JP5742634B2 - 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.
The p-coumaric acid has a food experience and is a highly safe component.

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).

  It has been known for a long time that piceatannol, which is a hydroxystilbene contained in grape skin, etc., has an anticancer effect (Non-patent Document 2). In recent years, it has been reported that resveratrol contained in grape skin and the like is metabolized by the metabolic enzyme CYP1B1 in cancer cells to produce piceatannol and to act specifically on cancer cells. (Non-patent document 3), the anticancer action of piceatannol has attracted attention. Moreover, since many useful physiological functions of piceatannol have been reported, there are prior arts related to the physiological functions. For example, there are a lipoxygenase inhibitor (Patent Document 8) and a skin cosmetic (Patent Document 9).

  As described above, since many of p-coumaric acid, piceatannol, and derivatives thereof exhibit excellent utility, a technique for efficiently producing these compounds as raw materials and lead compounds is also disclosed. Yes. For example, a method for producing p-hydroxycinnamic acid (p-coumaric acid) or the like by a microorganism using a recombinant is known (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 4)
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 JP-A-7-53359 JP 2007-223919 A JP-T-2005-522180

Cancer Epidemiology, Biomarkers & Prevention, 9, 1163-1170, (2000) Journal of Natural Products, 47, 347-352 (1984). British Journal of Cancer, 86, 774-778 (2002) Journal of Dental Research, 84, 401-406 (2008)

  In light of the above-described situation regarding piceatannol and p-coumaric acid and the current situation of cancer, particularly oral cancer, the present inventors have established a search for a hydroxystilbene derivative having more potent physiological activity and a method for producing the same. As a result of intensive studies, it was surprising that piceatannol, p-coumaric acid, luteolin, and resveratrol were used in a simple and safe manner by heat-treating piceatannol and p-coumaric acid in the presence of a metal salt. The present inventors have succeeded in producing a novel hydroxystilbene derivative having an anticancer activity superior to that of ε-viniferin as a body, particularly an activity against oral cancer, and has completed the present invention.

Therefore, the present invention provides a novel hydroxystilbene derivative having stronger anticancer activity than piceatannol, p-coumaric acid, luteolin and ε-viniferin, particularly potent anticancer activity against oral cancer. An object is to provide a method for efficiently and safely producing a stilbene derivative.
Another object of the present invention is to provide an anticancer agent, food, medicine and quasi-drug, which contains the novel hydroxystilbene derivative.

The gist of the present invention is as follows:
[1] The following 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 process for producing a novel hydroxystilbene derivative as described in [1] above, wherein the desired compound is produced by heat-treating piceatannol and p-coumaric acid at 110 ° C. or higher in the presence of a metal salt. About.

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 with piceatannol, p-coumaric acid, luteolin and ε-viniferin. .
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 peak of 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.

  Therefore, as a result of intensive studies, the present inventors have found that the novel hydroxystilbene derivative can be produced efficiently and safely by heat-treating piceatannol and p-coumaric acid in the presence of a metal salt. I found it. 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, piceatannol is used as a precursor. There are structural isomers of trans form and cis form in piceatannol, but some conversion of trans form and cis form occurs by heating and ultraviolet rays. Therefore, piceatannol may be a trans isomer, a cis isomer, or a mixture of a trans isomer and a cis isomer. Piceatannol may be a naturally-derived product extracted and purified from grape skin or berries, or may be a chemically synthesized chemical product with high purity. When naturally occurring piceatannol 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 ingredients other than tananol 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 piceatannol is desirable as a raw material.
As naturally-occurring piceatannol, extracts from raw materials such as grape skins and berries, freeze-dried products thereof, 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 hydroxystilbene 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, piceatannol, p-coumaric acid, or a mixture of piceatannol and p-coumaric acid is dissolved in a suitable solvent. At this time, if the solvent is only water, the solubility of piceatannol and p-coumaric acid is remarkably low. There is no restriction | limiting in particular in the compounding ratio of water and an organic solvent, and the kind of organic solvent, Piceatannol 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. When the final purification of the post-reaction composition containing the novel hydroxystilbene derivative of the present invention is applied to food without sufficient application, ethanol or hydrous ethanol is used as a solvent for safety and legal purposes. It is desirable.
There is no limitation on the concentration of piceatannol and p-coumaric acid in the solution containing the resulting piceatannol, p-coumaric acid, or a mixture of piceatannol and p-coumaric acid. Since each concentration has a merit such that the amount of solvent used is small, the concentration of piceatannol and p-coumaric acid is close to the concentration at which piceatannol and p-coumaric acid are saturated with respect to each solvent. preferable.
Further, piceatannol and p-coumaric acid may not be completely dissolved in the solution before the formation reaction. For example, when a piceatannol-containing solution and a p-coumaric acid-containing solution are mixed, even if the piceatannol concentration and the p-coumaric acid concentration in each solution are equal to or higher than a saturated concentration, May be adjusted to be close to the saturation concentration.

  Next, it is preferable to adjust the pH of the solution containing piceatannol and p-coumaric acid (hereinafter referred to as piceatannol and p-coumaric acid-containing solution) to less than 8. As an adjustment method, for example, after preparing a piceatannol or p-coumaric acid-containing solution, a pH adjusting agent may be added to adjust the pH, or the pH of the solvent may be adjusted in advance when preparing the solution. May be. If the pH at the start of the reaction of the piceatannol and p-coumaric acid-containing solution is 8.0 or more, other reactions and decomposition of the target compound may occur on the other hand, so that the final recovered amount of the hydroxystilbene derivative is small. descend. 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 piceatannol 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 piceatannol and p-coumaric acid-containing solution is heat-treated in the presence of a metal salt. By this heat treatment, the production reaction of the novel hydroxystilbene derivative of the present invention is carried out. In order to advance the production reaction efficiently, it is preferable to adjust the heating temperature of the piceatannol 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 piceatannol 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 the piceatannol and p-coumaric acid-containing solution in a sealed container. It is preferable to heat the solution so that the solution temperature of the solution containing piceatannol and p-coumaric acid reaches 110 ° C. or higher at least partially, such as by heating with a retort apparatus or autoclave. In view of the recovery efficiency of the novel hydroxystilbene derivative, 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 reaction solution obtained as described above 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 naturally occurring piceatannol and p-coumaric acid are dissolved in a water-containing ethanol solvent and heated using mineral water or mineral premix, the resulting reaction solution is used as one of the food ingredients. It 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 hydroxystilbene derivative can be concentrated by extraction using a solvent extraction method such as chloroform, ethyl acetate, ethanol, methanol, or the like, 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 piceatannol, p-coumaric acid, luterion and ε-biniferin. 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 piceatannol 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 and safety as described above, it can be used by blending 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 field of physiological activity 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)
100 mg of piceatannol (manufactured by Tokyo Chemical Industry Co., Ltd.) and 100 mg of p-coumaric acid (manufactured by Wako Pure Chemical Industries, Ltd.) are 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 In addition, three types of piceatannol and p-coumaric acid-containing solutions (pH: (1) 5.0, (2) 5.7, (3) 5.2) were obtained. These three types of piceatannol 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 taken out and made up 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, before the reaction, the chromatograms of the reaction solutions (1), (2), and (3) are shown. After the reaction, peaks other than piceatannol 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 in the production amount due to the difference in the type of metal salt.

(Example 2: Mass production of a novel hydroxystilbene derivative)
500 mg of piceatannol and 500 mg of p-coumaric acid were dissolved in 10 mL of ethanol, and 10 mL of mineral water was added to obtain a piceatannol and p-coumaric acid-containing solution (pH = 5.0). This piceatannol and p-coumaric acid-containing solution was heated in an autoclave at 130 ° C. for 90 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 90 mg of a new compound (hereinafter UHA7034). The isolated and purified UHA7034 became a brown powdery substance.

Subsequently, when the molecular weight of UHA7034 was measured by high resolution electron ionization-mass spectrometry, the measured value was 364.3917, and the following molecular formula was obtained from comparison with the theoretical value.
Theoretical value C22H20O5 (M ⁺ ): 364.3912
Molecular formula C ₂₂ H ₂₀ O ₅

  Next, the UHA7034 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 UHA7034 has a structure represented by the formula (1). did. It was shown that the novel hydroxystilbene derivative represented by the formula (1) can be efficiently produced by the method of the present invention.

  For NMR measurements, UHA7034

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 .

UHA7034 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 UHA7034 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 piceatannol, p-coumaric acid, luteolin (manufactured by Wako Pure Chemical Industries, Ltd.) which is a natural product having anticancer activity against SCC-4 cells, and resveratrol derivative ε-viniferin (Wako Pure). Five types of pharmaceutical products (manufactured by Yakuhin Kogyo Co., Ltd.) and UHA7034 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 UHA7034 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 piceatannol, luteolin and ε-viniferin. Therefore, the high significance which converts piceatannol and p-coumaric acid into a novel hydroxystilbene derivative was shown.

(Example 5: Difference in production amount of UHA7034 depending on heating temperature)
A mixed solution of 100 mg of piceatannol, 100 mg of p-coumaric acid, 2 mL of ethanol, and 2 mL of mineral water (pH = 5.0) in an autoclave at 70 ° C., 90 ° C., 110 ° C., and 130 ° C. for 50 minutes. Heated. 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, generation of UHA7034 was confirmed at 110 ° C. or higher. The production ratio (% by weight) from the total amount of piceatannol and p-coumaric acid is 70 ° C., 90 ° C. is not produced, 110 ° C. is extremely small, 130 ° C. is 9.0%, and heating at 130 ° C. However, UHA7034 was most frequently generated.

(Example 6: Preparation of UHA7034-containing extract)
A mixed solution prepared by adding 10 g of berry extract powder (piceatannol 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 resulting reaction solution was dried under reduced pressure to obtain 13 g of UHA7034-containing extract. When 13 g of the obtained UHA7034 extract was confirmed by the same method as in Example 3, 0.012 g of UHA7034 was contained. This work was repeated as necessary.

(Example 7: Food containing UHA7034)
1 g of UHA7034-containing extract obtained in Example 6 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 8: Drug containing UHA7034)
UHA7034 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 UHA7034, 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 9: Quasi-drug containing UHA7034)
1.2 g of UHA7034 obtained by the method of Examples 2 and 3 was dissolved in 10 mL of ethanol, and 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 salt thereof according to claim 1, wherein the desired compound is produced by heat-treating piceatannol and p-coumaric acid at 110 ° C or higher in the presence of a metal salt. Possible salt production methods.

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