JP5331046B2 - Bioactive substances derived from peanut germ - Google Patents

Description

  The present invention relates to a physiologically active substance derived from peanut germ, and a use and production method of the physiologically active substance.

  In recent years, with an increase in lifestyle-related diseases associated with aging and deterioration of the living environment, interest in health has increased, and the need for food functionality has also increased.

  Functional foods that help prevent health management and lifestyle-related diseases at the waterfront are expected to occupy as important positions as pharmaceuticals in the future, and experimental scientific evidence is essential for their development. Produces high value-added products.

  Inflammation is a biological defense reaction caused by the immune system in various tissues due to foreign body invasion, tissue damage or infections that are unfavorable to biological tissues, such as allergic diseases in which inflammation excessively damages tissues due to excessive immune reactions, or from the outside Anti-inflammatory action that suppresses inflammation such as itching, pain, swelling and tissue damage caused by inflammatory substances (antigens such as bacteria, pollen and air pollutants) entering the body is important for maintaining the quality of health Function.

  The legumes of peanuts (scientific name: Arachis hypogaea L.) consist of shells (legumes), seed coats (thin skins), and seeds (seeds) from the outermost layer to the inner layer. Peanuts generally use the seed part, but most of the germ is discarded during the processing. Peanut seed coat contains abundant proanthocyanidins, and peanut seed coat extract or proanthocyanidins isolated from this extract is known to have bone marrow cell proliferation activity, anti-HIV activity, antitumor effect, antioxidant activity, etc. (For example, see Patent Documents 1 to 3). On the other hand, germs separated from peanuts have a bitter taste and are unsuitable as foods, and have not been used as pharmaceuticals.

  The present inventors have previously reported that a hot water extract of peanut germ increases cytokine production (Non-patent Document 1).

Japanese Patent No. 3217278 (for example, claim 1) JP-A-11-246431 (for example, claim 1) Japanese Patent Laying-Open No. 2004-217558 (for example, claim 1)

The 56th Annual Meeting of the Japanese Biopharmaceutical Society (2009)

  An object of the present invention is to effectively use peanut germ.

  The present inventors conducted a search for physiologically active substances using immune responses using monocyte cells as an index for the purpose of finding out the potential of peanut embryos as functional foods or pharmaceuticals, and isolated novel compounds. The specific fraction obtained in the step of isolating and purifying the compound and the peanut germ extract from the compound and the peanut germ extract has been successfully purified, contrary to the hot water extract before the peanut germ purification, The inventors have found that the production of inflammatory cytokines is suppressed and are useful as anti-inflammatory agents, and the present invention has been completed.

で示される化合物又はその塩。 That is, the gist of the present invention is as follows.
(1) The following formula (I):

Or a salt thereof.

(2) An anti-inflammatory agent comprising the compound according to (1) or a salt thereof.
(3) An inflammatory cytokine production inhibitor containing the compound or salt thereof according to (1).
(4) The peanut germ extract is loaded onto adsorption chromatography, the fraction that has passed through the column and the fraction eluted with water are removed, and then the fraction eluted with 40-100% lower alcohol or a purified product thereof is used. Contains anti-inflammatory agents.

(5) The peanut germ extract is loaded onto adsorption chromatography, the fraction that has passed through the column and the fraction that has been eluted with water are removed, and then the fraction that has been eluted with 40 to 100% lower alcohol or a purified product thereof. An inhibitor of inflammatory cytokine production.
(6) A method for producing the compound according to (1), comprising isolating the compound according to (1) from an extract of peanut germ.

  According to the present invention, a novel compound useful as an anti-inflammatory agent and an inflammatory cytokine production inhibitor is provided using peanut germ that has been discarded so far, and a purified fraction of the compound and an extract of peanut germ The present invention can be used as an anti-inflammatory agent and an inflammatory cytokine production inhibitor.

FIG. 1 is a diagram showing a procedure for extraction and fractionation of peanut germ. FIG. 2 is a diagram showing the influence of peanut embryo hot water extract and HP-20 fraction on cytokine (TNF-α and IL-6) production.

  The compound represented by the formula (I) can be obtained by extraction and isolation from peanut germ.

  The peanut germ used in the present invention can be taken out without roasting peanut fruit shells (beans and fruits) and roasting the seed coat (thin skin), but it is easy to remove the seed coat (thin skin). In general, roasting is performed. After roasting according to a conventional method, peanuts without seed coats (thin skins) are divided into two with a normal roller, so that the halves and embryos can be obtained. Therefore, peanut germs can be easily obtained by selecting both.

Examples of the extraction solvent include water, organic solvents, and mixed solvents thereof. The organic solvent is preferably a water-miscible organic solvent such as lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol; ethers such as ethyl ether, dioxane; ketones such as acetone, and water and organic. As a mixed solvent of a solvent, a water-ethanol mixed solvent and a water-methanol mixed solvent are preferable.
Usually, 1-15 L of extraction solvent is used per 1 kg of peanut germ.

  The extraction temperature is usually within the range of the melting point of the solvent to the boiling point of the solvent, preferably 0 to 100 ° C, more preferably 5 to 95 ° C. Supercritical extraction may be performed. The extraction is usually performed under normal pressure, but may be performed under pressure or under reduced pressure. The extraction time varies depending on the extraction temperature and the like, and is usually 5 minutes to 1 day. When extracting using hot water (85-100 degreeC) as an extraction solvent, extraction time becomes like this. Preferably it is 5-60 minutes, More preferably, it is 10-30 minutes.

  The target extract can be obtained by filtering the extract obtained as described above with a cloth, stainless steel filter, filter paper or the like to remove peanut germ, impurities, and the like. Moreover, you may give processes, such as a spray-dry process, a freeze-dry process, a supercritical process, to the extract after filtration.

  The extract thus obtained is subjected to adsorption chromatography, preferably adsorption chromatography using an aromatic synthetic adsorbent, etc., and the fraction passed through the column and the fraction eluted with water are removed. A fraction eluted with -100% lower alcohol is obtained.

The aromatic synthetic adsorbent is a synthetic substance having an ion exchange group and having a porous structure, and is mainly used for separation and purification of organic substances by hydrophobic interaction. Examples thereof include those described in Japanese Patent No. 2784627, Japanese Patent No. 3527661, and Japanese Patent No. 389746. Specifically, include styrene and aromatic prepared by polymerizing divinylbenzene-based synthetic adsorbent, as commercially available products, DIAION ^TM HP20, the HP21, Sepabeads ^TM SP825L, the SP850, the SP700, the SP70 (Mitsubishi Chemical Co., Ltd. or Nippon Nensui Co., Ltd.), Amberlite ^TM XAD2, XAD4, and XAD16 (Rohm and Haas, USA) may be mentioned.

  The fraction obtained as described above can be used as an active ingredient as it is or after further purification by various purification means such as reverse phase chromatography and dialysis. At that time, it is preferable to purify using an immune response using monocyte cells as an indicator.

  The compound of the present invention can be isolated by purifying the fraction by, for example, reverse phase chromatography using an ODS column.

  Since the compound of the present invention has a carboxyl group and a phenolic hydroxyl group, it can be converted into a salt such as an alkali metal salt such as a sodium salt or a potassium salt, if necessary.

  The anti-inflammatory agent and inflammatory cytokine production inhibitor of the present invention are formulated by combining the compound represented by the formula (I) or a salt thereof, or the fraction or a purified product thereof with a known food or pharmaceutical carrier. can do. The dosage form is not particularly limited and is appropriately selected as necessary. In general, tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, elixirs, etc. It is used as oral preparations or parenteral preparations such as injections, drops, suppositories, inhalants, transdermal absorbents, transmucosal absorbents, patches, ointments and the like.

  The dosage of the anti-inflammatory agent and the inflammatory cytokine production inhibitor of the present invention varies depending on the age, body weight, degree of disease, route of administration of the patient, but in oral administration, the compound represented by the above formula (I) or its The salt is usually 3 to 600 mg per day, and the fraction or purified product thereof as a dry powder is usually 50 to 3000 mg per day, and the administration frequency is usually 1 to 3 times per day for oral administration.

  Oral preparations are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.

  In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used in addition to the above-mentioned excipients.

  Specific examples of the binder include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose sodium , Ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl METAKU Rate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.

  Specific examples of disintegrants include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partially pregelatinized. Starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth can be mentioned.

  Specific examples of surfactants include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate Sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol.

  Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc, Examples thereof include magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, waxes, hydrogenated vegetable oil, and polyethylene glycol.

  Specific examples of the fluidity promoter include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

  The anti-inflammatory agent and inflammatory cytokine production inhibitor of the present invention contain a flavoring agent and a coloring agent when administered as a liquid agent such as a syrup, suspension, emulsion, elixir, or drink. May be.

  The anti-inflammatory agent and inflammatory cytokine production inhibitor of the present invention have an action of suppressing the production of inflammatory cytokines such as interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and infectious diseases It is useful as an anti-inflammatory agent that suppresses inflammation such as tissue damage caused by excessive immune reactions such as allergies. The anti-inflammatory agent and the inflammatory cytokine production inhibitor of the present invention are added to foods, chewing gums, beverages, etc., so-called foods for specific health (for example, health maintenance, prevention of lifestyle-related diseases, reduction of neutral fat, blood sugar Food for the purpose of improving the quality of food), and general foods. The peanut embryo which is a raw material for producing the anti-inflammatory agent and the inflammatory cytokine production inhibitor of the present invention is used for food as part of peanut, and its safety is established.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.

(Example 1) Preparation and fractionation of hot water extract of peanut embryo 5 ml of water was added to 1.5 kg of peanut germ, heated, and extracted at 90 ° C for 15 minutes. The obtained extract was freeze-dried, and the same operation was performed for 1.5 kg of germs separately, and a total of 3 kg of germs were combined to obtain 333 g of hot water extract. The obtained hot water extract (317 g) was passed through Diaion ^TM HP20 (Nippon Nensui Co., Ltd.) column chromatography (column capacity 1.5 L), and water, 20% methanol aqueous solution, 60% methanol aqueous solution and Elution and fractionation was performed with 4.5 L each in the order of methanol. 6 g of the 7.2 g fraction obtained by elution with 60% aqueous methanol was further subjected to reverse phase column chromatography using ODS (136 g) (water 1.1 L, 10% aqueous methanol, 50% aqueous methanol, 70% Fractions were eluted with 0.88 L each of methanol aqueous solution and methanol). A 10% methanol aqueous solution elution fraction (300 mg) was further fractionated by reverse phase column chromatography using ODS (136 g) (eluted with 1.1 L of 1% aqueous methanol solution and 1.1 L of 30% aqueous methanol solution) to give a 30% aqueous methanol solution. Fraction 110 mg obtained by elution was fractionated by reverse phase HPLC (ODS 20 × 300 mm, acetonitrile / methanol / water = 1/2/8 + 0.1% formic acid, 6 ml / min) to obtain compound A (6.4 mg). (FIG. 1).
Compound A: 2-O-β-apiofranosyl-6- (4′-carboxy-3′-hydroxy-3′-methylbutanoyl) -1-β- (4 ″ -hydroxyphenyl) ethyl glucopyranoside
IR λmax 3370, 1720, 1510, 1080 cm ^-1 ; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ1.34, 2.57, 2.62, 2.66, 2.70, 2.81, 3.29, 3.36, 3.42, 3.46, 3.57, 3.60 , 3.65, 3.68, 3.91, 3.93, 3.96, 4.18, 4.34, 4.42, 5.36, 6.69, 6.69, 7.05, 7.05; ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ27.8, 36.5, 46.0, 46.5, 64.6 , 66.2, 70.7, 71.7, 72.0, 75.1, 75.4, 78.0, 78.4, 78.6, 80.7, 103.3, 110.5, 116.2, 116.2, 130.8, 130.9, 130.9, 156.8, 172.5, 175.2; HRFABMS m / z 577.2122 [M + H ] ⁺ (C ₂₅ H ₃₇ O ₁₅ , Δ-1.0 mmu).

(Example 2) Evaluation method of anti-inflammatory activity using THP-1 cells Human monocytic cell line THP-1 (Dainippon Pharmaceutical Co., Ltd.) was cultured according to information, and cells in the logarithmic growth phase were used. . Specifically, THP-1 cells were prepared by suspending them in RPMI-1640 medium (with 5% fetal calf serum) so as to have a concentration of 1.0 × 10 ⁵ cells / well, and 225 μL was added to each 96-well plate. Pre-cultured for hours. To this was added 25 μL of lipopolysaccharide (LPS) diluted with a basic medium so that the final concentration was 100 ng / mL. To this, a solution in which peanut embryo extract, fraction or isolated component was dissolved in various concentrations in a 0.3% DMSO / PBS solution was added, and the condition of 37 ° C. in an incubator in the presence of 5% CO ₂ was added. The cells were cultured for 48 hours.

  After completion of the culture, the culture supernatant containing the cells was collected from the 96-well plate in an Eppendorf tube, and centrifuged to collect the supernatant. These supernatants were stored at −80 ° C. until the inflammatory cytokine, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) concentrations in the culture supernatant were measured.

  TNF-α and IL-6 contained in the culture solution were measured by ELISA kit (Cytoscreen) manufactured by Biosource according to the operation manual. Culture was performed in 4 wells per sample, and the average value (pg / mL) ± standard deviation was displayed. Statistical significance of data is analyzed using analysis of variance, and if equal variance, the significance of the population is compared with the Dunnett's method against the population data, and statistical comparison is performed on the comparison population with a risk rate of less than 5%. It was determined that there was a significant difference.

  In a preliminary test in which cells were allowed to act at a concentration of 100 μg / mL, peanut embryo hot water extract significantly increased TNF-α 48 hours after LPS stimulation compared to the control group (sample non-added control group; the same applies hereinafter). Production was increased, and the water elution fraction (1a) and the 20% aqueous methanol solution elution fraction (1b) significantly increased IL-6 production as compared to the control group 48 hours after LPS stimulation. On the other hand, the 60% methanol aqueous solution elution fraction (1c) and the methanol elution fraction (1d) showed a tendency to suppress the production of TNF-α as compared to the control group 48 hours after LPS stimulation.

  In a preliminary test conducted at a concentration of 100 μg / mL, 48 hours when the change in cytokine production was large was determined as the culture time, and the same test was performed while changing the sample concentration. The results are shown in FIG. The 60% methanol aqueous solution elution fraction (1c) significantly suppressed the production of TNF-α at a concentration of 10 μg / mL compared to the control group (p <0.05), and the methanol elution fraction (1d) was 100 μg. The production of TNF-α was significantly suppressed at a concentration of / mL compared to the control group (p <0.01).

  Compound A significantly suppresses the production of TNF-α at concentrations of 0.5 and 5 μg / mL (p <0.01) and exhibits an effect of moderately suppressing the production of IL-6. It became clear that it was one of the anti-inflammatory components of the germ (without squares in FIG. 2).

Extract Peanut germ hot water extract 1a Water elution fraction 1b 20% methanol aqueous solution elution fraction 1c 60% methanol aqueous solution elution fraction 1d Methanol elution fraction A Compound A

Claims (7)

Formula (I):

Or a salt thereof.   An anti-inflammatory agent comprising the compound according to claim 1 or a salt thereof.   An inflammatory cytokine production inhibitor comprising the compound according to claim 1 or a salt thereof. Loading an extract of peanut germ on adsorption chromatography, removing the fraction passed through the column and the fraction eluted with water, then containing the fraction eluted with 40-100% lower alcohol or purified product thereof , An anti-inflammatory agent comprising a compound represented by formula (I) according to claim 1 as an active ingredient . Loading an extract of peanut germ on adsorption chromatography, removing the fraction passed through the column and the fraction eluted with water, then containing the fraction eluted with 40-100% lower alcohol or purified product thereof , And the inflammatory cytokine production inhibitor which contains the compound shown by the formula (I) of Claim 1 as an active ingredient . The inflammatory cytokine production inhibitor according to claim 5, wherein the inflammatory cytokine is tumor necrosis factor α.   A method for producing a compound according to claim 1, comprising isolating the compound according to claim 1 from an extract of peanut germ.

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