JP2012020963A - Melanin production inhibitor, and drug, food or cosmetic having melanin production inhibiting action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a melanin production inhibitor which has no risk of side effects, is safe, and has excellent melanin production inhibiting action, and to provide a drug, food or a cosmetic having a melanin production inhibiting action containing the melanin production inhibitor.SOLUTION: The melanin production inhibitor contains an extract from Saussurea involucrata (Kar.et Kir.) Sch.Blp., column fractional ingredients from the extract or dihydrodehydrocostuslactone as active ingredients. Further, the drug, food or cosmetic comprises the melanin production inhibitor.

Description

  The present invention relates to a melanin production inhibitor and a pharmaceutical, food or cosmetic having a melanin production inhibitory action.

  Melanin is produced from tyrosine via dopa through enzymatic or non-enzymatic oxidation reaction by the action of tyrosinase present in skin melanocytes. It is considered that pigmentation such as skin spots and buckwheat is caused by excessive production of melanin and deposition on the skin due to abnormal hormone secretion or UV exposure. Conventionally, hydroquinone, L-ascorbic acid and derivatives thereof, kojic acid, arbutin and the like are blended in whitening agents (whitening cosmetics) for the purpose of preventing and treating pigmentation such as stains and buckwheat (for example, (See Patent Document 1).

  However, these substances have poor stability and solubility in the formulation system, and some of them may discolor or generate starch over time, and some may cause safety problems when mixed in a concentration that allows whitening effects. , Was not satisfactory.

  On the other hand, it is known that an extract from a plant belonging to the genus Saussurea is used in skin cosmetics as a collagen production promoter or as an estrogen-like agent (for example, see Patent Document 2). In addition, it is also known that an extract from Saussurea laniceps Hand.-Mazz. Among plants belonging to the genus Saussurea is used as a tyrosinase inhibitor or melanin production inhibitor in whitening cosmetics. (For example, refer to Patent Document 3). In recent years, with the progress of research on extracts from plants belonging to the genus Saussurea, it is also known to use extracts from plants belonging to the genus Saussurea in cosmetics (for example, Patent Document 4). And 5).

  However, among the plants belonging to the genus Saussurea, an extract from Saussurea involucrata (Kar.et Kir.) Sch.Blp., Saussurea involucrata (Kar.et Kir.) Sch. Suppressed melanin production by certain components fractionated from extracts from Blp. Or dihydrodehydrocostuslactone contained in Saussurea involucrata (Kar. Et Kir.) Sch.Blp. It is not known to have an effect.

JP 2009-256326 A JP 2001-316240 A Japanese Patent No. 3658548 JP 2004-331546 A JP 2004-331547 A

  An object of the present invention is to provide a “melanin production inhibitor that is safe and has a significant melanin production-inhibiting action without fear of side effects”. It is another object of the present invention to provide a “medicine, food or cosmetic having a melanin production inhibitory effect” containing the melanin production inhibitor.

  As a result of intensive studies on substances having a melanin production inhibitory effect, the present inventors have heretofore been known not to have a remarkable melanin production inhibitory action "Saussurea involucrata (Kar. Et Kir.) "The extract from Sch.Blp." Is effective as a melanin production inhibitor, and it was fractionated from the extract from "Saussurea involucrata (Kar. Et Kir.) Sch.Blp." The inventors have found that “predetermined components” and “dihydrodehydrocostus lactone” are also effective as melanin production inhibitors, and have completed the present invention. The present invention includes the following items.

[1] A melanin production inhibitor containing an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp as an active ingredient.

[2] A column in which a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp in 30% (V / V) methanol is used as a carrier with porous polystyrene resin The melanin production inhibitor which contains as an active ingredient the component adsorb | sucked to the said porous polystyrene resin when letting it pass.

[3] A column in which a solution obtained by dissolving an extract from Southus involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol is used as a carrier with porous polystyrene resin A melanin production inhibitor containing, as an active ingredient, a component adsorbed on the porous polystyrene resin when 50% (V / V) methanol as an elution solvent is passed through the column.

[4] A column in which a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp in 30% (V / V) methanol is used as a carrier with porous polystyrene resin When 50% (V / V) methanol as an elution solvent is passed through the column, and then methanol as an elution solvent is passed through the column, the methanol is eluted from the column. Melanin production inhibitor containing the active ingredient as an active ingredient.

[5] A melanin production inhibitor containing dihydrodehydrocostus lactone represented by the following formula (1).

[6] A pharmaceutical, food or cosmetic having a melanin production inhibitory effect, comprising the melanin production inhibitor according to [1].

[7] A pharmaceutical, food or cosmetic having a melanin production inhibitory effect, comprising the melanin production inhibitor according to [2].

[8] A pharmaceutical, food or cosmetic having a melanin production inhibitory action, comprising the melanin production inhibitor according to [3] above.

[9] A pharmaceutical, food or cosmetic having a melanin production inhibitory effect, comprising the melanin production inhibitor according to [4].

[10] A pharmaceutical, food or cosmetic having a melanin production inhibitory effect, comprising the melanin production inhibitor according to [5] above.

  According to the present invention, as can be seen from the test examples described later, “a melanin production inhibitor that is safe and has a significant melanin production inhibitory action without fear of side effects” and “a pharmaceutical product that has a melanin production inhibitory action” , "Food or cosmetics".

It is a flowchart shown in order to demonstrate Embodiment 1-5. It is a flowchart shown in order to demonstrate the preparation example of sample df and the comparative sample 1. FIG. It is a graph which shows the result of a test example (melanin production suppression effect with respect to B16 mouse | mouth melanoma cell).

  Hereinafter, the “melanin production inhibitor” and the “medicine, food or cosmetic having a melanin production inhibitory action” of the present invention will be described in more detail based on embodiments.

FIG. 1 is a flowchart for explaining the first to fifth embodiments.
The melanin production inhibitor of the present invention includes “an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp.”, “Saussurea involucrata (Kar. Et Kir.) Sch. Blp.), A component obtained by dissolving a 30% (V / V) methanol solution through a column using a porous polystyrene resin as a carrier. A solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol was passed through a column using a porous polystyrene resin as a carrier, Then, when 50% (V / V) methanol is passed through the column, the component is adsorbed on the porous polystyrene resin ”,“ Saussurea involucrata (Kar. Et Kir.) Sch. Blp. ” Extraction A solution in which a product is dissolved in 30% (V / V) methanol is passed through a column using a porous polystyrene resin as a carrier, then 50% (V / V) methanol is passed through the column, and then methanol is added to the column. The component eluted from the column with methanol when it is passed through is contained as an active ingredient "or" dihydrodehydrocostus lactone "as an active ingredient (see FIG. 1 and FIG. 2 described later).
Hereinafter, each above-mentioned active ingredient is demonstrated by Embodiment 1-5.

[Embodiment 1]
The melanin production inhibitor according to Embodiment 1 contains “an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” As an active ingredient (see FIG. 1).

  In the melanin production inhibitor according to Embodiment 1, “extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp.” Includes “Saussurea involucrata (Kar. Et Kir. ) The extract obtained by extraction from Sch. Blp.), A diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product or a purified product thereof. included.

  In the extraction process, Saussurea involucrata (Kar. Et Kir.) Sch.Blp. (Chinese name: Xinjiang Snow Lotus, Tianshan Snow Lotus or Xinjiang Snow Lotus) As an extraction raw material, whole grass can be used, but it is preferable to use the above-ground part.

Saussurea involucrata (Kar.et Kir.) Sch.Blp. Is a herbaceous herbaceous plant distributed mainly in Xinjiang Autonomous Region, Sichuan Province, Ulsan Province and Tibet Province in China. It has been used for rheumatoid arthritis, impotence, irregular menstruation, uterine bleeding, coldness and pain in the lower abdomen of women, colorless belt, kidney collapse and snow blindness, and is known to be used for wound bleeding.
Saussurea involucrata (Kar.et Kir.) Sch.Blp. Is available in Xinjiang Autonomous Region of China.

  As a raw material for extraction, Southrea involucrata (Kar. Et Kir.) Sch.Blp. Is suitably dried and pulverized immediately after collection. Drying may be performed in the sun or using a commonly used dryer.

  In the extraction process, it is preferable to use a polar solvent as the extraction solvent. A component having an inhibitory action on melanin production can be easily extracted from Southus involucrata (Kar. Et Kir.) Sch. Blp. By extraction using a polar solvent.

  Specific examples of the polar solvent include water and water-soluble organic solvents, and these may be used alone or in combination of two or more. Of these polar solvents, water, a water-soluble organic solvent, or a water-containing water-soluble organic solvent is preferably used as the extraction solvent.

Water that can be used as the extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Examples of various treatments include heating, sterilization, sterilization, filtration, and ion exchange. Accordingly, the water that can be used as the extraction solvent in the present invention includes hot water, ion-exchanged water, and the like.
Further, as the extraction solvent, various waters subjected to treatments such as adjustment of osmotic pressure and buffering (physiological saline, phosphate buffer, phosphate buffered saline, etc.) can also be used.

  Examples of water-soluble organic solvents that can be used as the extraction solvent include lower aliphatic alcohols and acetone. Specific examples of the lower aliphatic alcohol include monohydric alcohols such as methanol, ethanol, propanol and butanol, and polyvalents such as 1,3-butylene glycol, propylene glycol, 1,3-propanediol, pentylene glycol and isoprene glycol. Alcohol can be illustrated.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when a water-containing water-soluble organic solvent is used as the extraction solvent, a water-containing water-soluble organic solvent preferably containing 50% by weight or more of a monohydric alcohol, a polyhydric alcohol, acetone or the like can be used.

  An intermediate polar solvent such as ethyl acetate or butyl acetate may be used as the extraction solvent.

  As the extraction solvent, 50% (V / V) ethanol and 80% (V / V) ethanol can be suitably used.

  The extraction treatment is not particularly limited as long as a soluble component contained in Saussurea involucrata (Kar. Et Kir.) Sch. Blp. Can be eluted in the extraction solvent, and can be performed according to a conventional method. In the extraction process, it is not necessary to adopt a special extraction method, and any apparatus can be used at room temperature or under reflux heating.

  Extraction methods include extraction by immersing the extraction raw material in the extraction solvent at room temperature for a long time, extraction with heating to a temperature below the boiling point of the extraction solvent and stirring, or heating to reflux near the boiling point of the extraction solvent. The method of extracting below can be illustrated. At this time, the amount of the extraction solvent is usually 5 to 50 times (weight ratio), preferably 5 to 15 times (weight ratio) of the extraction raw material, and the extraction time is usually 1 to 10 hours, preferably 1 to 1. It is 4 hours, and the extraction temperature is usually 50 to 95 ° C, preferably 80 to 95 ° C.

  In the extraction process, before performing the extraction with the polar solvent, a degreasing process using a nonpolar solvent such as pentane, hexane, heptane, octane, petroleum ether or the like may be performed. Can be efficiently performed.

  After the soluble component is eluted by the extraction treatment, an extraction liquid can be obtained by performing a treatment such as filtration or centrifugation to remove the extraction residue. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed.

  Since the extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp has a characteristic odor and taste, it may be subjected to purification for decolorization, deodorization and the like. Specifically, purification can be performed by activated carbon treatment adsorption resin treatment, ion exchange resin treatment, etc., and these treatments are extracted from Southus invollucata (Kar. Et Kir.) Sch.Blp. In a range that does not cause a decrease in the melanin production inhibitory effect of the product.

[Embodiment 2]
The melanin production inhibitor according to Embodiment 2 is “a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” In 30% (V / V) methanol. A component that is adsorbed by the porous polystyrene resin when passed through a column using the porous polystyrene resin as a carrier is contained as an active ingredient (see FIG. 1).

  “An extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” Can be obtained by the method described in the first embodiment.

  Examples of the porous polystyrene resin used as the carrier include Diaion (registered trademark of Mitsubishi Chemical Corporation) HP20 and HP21, and Sepabeads (registered trademark of Mitsubishi Chemical Corporation) SP850, SP700 and SP70.

  In Embodiment 2, the component adsorbed on the porous polystyrene resin is an extract from Southus involucata (Kar. Et Kir.) Sch. Blp. Dissolved in 30% (V / V) methanol. It is possible to elute the solution by passing a solution having a relatively low polarity through the column after passing the solution through the column. Examples of the solvent that can be used for elution of the component include intermediate polar solvents such as lower aliphatic alcohols, acetone and ethyl acetate, and nonpolar solvents such as hexane. Of these solvents, methanol can be particularly preferably used.

In this specification, “a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol is used as a porous polystyrene resin. A component that is adsorbed to the porous polystyrene resin when it passes through a column as a carrier is referred to as an “adsorption component”.
In addition, a column in which a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol is used as a column with a porous polystyrene resin as a carrier The component that is eluted from the column with 30% (V / V) methanol when passed through is referred to as “non-adsorbed component”.

[Embodiment 3]
The melanin production inhibitor according to Embodiment 3 is “a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” In 30% (V / V) methanol. A component that is adsorbed to the porous polystyrene resin when 50% (V / V) methanol is passed through the column after passing through the column using the porous polystyrene resin as a carrier is contained as an active ingredient (see FIG. 1). .)

  “An extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” Can be obtained by the method described in the first embodiment.

  Examples of the porous polystyrene resin used as the carrier include Diaion (registered trademark of Mitsubishi Chemical Corporation) HP20 and HP21, and Sepabeads (registered trademark of Mitsubishi Chemical Corporation) SP850, SP700 and SP70.

  In Embodiment 3, the component adsorbed on the porous polystyrene resin can be eluted by passing 50% (V / V) methanol through the column and then passing a lower polarity solvent through the column. Examples of the solvent that can be used for elution of the component include intermediate polar solvents such as lower aliphatic alcohols, acetone and ethyl acetate, and nonpolar solvents such as hexane. Of these solvents, methanol can be particularly preferably used.

[Embodiment 4]
The melanin production inhibitor according to Embodiment 4 is “a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” In 30% (V / V) methanol. The component that is eluted from the column by methanol when the 50% (V / V) methanol is passed through the column and then methanol is passed through the column is effective. Contains as a component (see FIG. 1).

  “An extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.” Can be obtained by the method described in the first embodiment.

  Examples of the porous polystyrene resin used as the carrier include Diaion (registered trademark of Mitsubishi Chemical Corporation) HP20 and HP21, and Sepabeads (registered trademark of Mitsubishi Chemical Corporation) SP850, SP700 and SP70.

  In this specification, “a solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol is used as a porous polystyrene resin. “Methanol-eluted fraction” is a component that is eluted from the column by methanol when 50% (V / V) methanol is passed through the column and then methanol is passed through the column. That's it.

[Embodiment 5]
The melanin production inhibitor which concerns on Embodiment 5 contains dihydrodehydrocostus lactone represented by following formula (1) as an active ingredient.

  Dihydrodehydrocostus lactone is contained in an extract from Southus involucrata (Kar. Et Kir. Sch. Blp.) As shown in the preparation examples described later (see FIG. 1 and FIG. 2 described later). .) The dihydrodehydrocostus lactone used for the melanin production inhibitor may be a product separated from an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp. (Saussurea involucrata (Kar.et Kir.) Sch.Blp.) Which is a natural product separated from a natural product containing dihydrodehydrocostus lactone, or a commercially available product or synthetic product It may be used.

The melanin production inhibitor according to Embodiments 1 to 5 (that is, the melanin production inhibitor of the present invention) can be expected to prevent, treat or improve pigmentation such as spots and buckwheat through a melanin production inhibitory action.
Moreover, since the melanin production inhibitor of this invention contains the component contained in the natural product used for a long time as a tradition medicine as an active ingredient, it becomes a "safe melanin production inhibitor without a fear of a side effect." Moreover, the melanin production inhibitor of this invention turns into "a melanin production inhibitor which has a remarkable melanin production inhibitory effect" so that the test example mentioned later may show. In addition, the melanin production inhibitor of this invention may contain any 1 type in the active ingredient in the said Embodiment 1-5, and contains 2 or more types in the active ingredient in the said Embodiment 1-5. You may contain. In the case where two or more of the active ingredients in Embodiments 1 to 5 are contained, the blending ratio thereof may be appropriately determined according to the degree of their action.

  The melanin production inhibitor of this invention can be used as a pharmaceutical, a foodstuff, or cosmetics containing the said melanin production inhibitor and having a melanin production inhibitory effect.

  The route of administration of the “internal medicine having melanin production inhibitory activity” of the present invention is not particularly limited, but for example, enteral administration such as oral administration and rectal administration, mucosal administration such as nasal administration, intravenous administration・ Injection administration such as subcutaneous administration can be given. The dosage form of the internal medicine of the present invention can be in the form of a formulation suitable for the administration method, for example, tablet, powder, fine granule, granule, capsule, powder, pill, troche And liquids such as solid preparations, solutions, suspensions, emulsions, syrups, injections, and gel preparations. The pure product, purified product, crude product, etc. of the melanin production inhibitor of the present invention may be administered as it is, or may be administered with a pharmacologically acceptable excipient. As the excipient, any monosaccharides, second class, polysaccharides, inorganic salts, fats and oils, distilled water, etc. that can be generally used as preparations can be used. In formulating, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used.

  The content of the melanin production inhibitor in the internal medicine of the present invention can be set to an optimal amount for each dosage form based on the data of the dosage form as the dosage form, the effective dosage and the dosage form.

  The form of the “food having melanin production inhibitory action” of the present invention includes a pure product of the melanin production inhibitor of the present invention, a partially purified product thereof, a paste containing the melanin production inhibitor, and the melanin production inhibitor. Examples include blended foods. In other words, drinks, jelly, biscuits, tea, tablets, pills, soft capsules, hard capsules, powders, fine granules, granules, etc. are provided as normal foods in the form of “foods that suppress melanin production” Any form can be used as long as it is possible. Additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can also be used as auxiliary materials.

  Examples of the form of the “external medicine or cosmetic having a melanin production inhibitory action” of the present invention are not particularly limited.

  Examples of the form of external medicine include ointments, creams, cloths, tapes, and external preparations. The pharmaceutical preparation for external use of the present invention can contain other pharmaceutical ingredients as necessary in the melanin production inhibitor of the present invention, and also includes a binder, a dispersant, a suspension, an emulsifier, a diluent, and a buffer. Additives such as antioxidants and bacteria inhibitors may be used.

  As the form of the cosmetic, any form that can be used as an external preparation / cosmetic preparation such as lotion, cosmetic liquid, milky lotion, cream, gel, pack, cosmetic powder, facial cleansing foam, bath preparation, etc. can be used. In addition to the melanin production inhibitor of this invention, you may contain the component mix | blended with a cosmetic formulation as needed in the said cosmetic formulation. Examples of the ingredients include solid oil, semi-solid oil, liquid oil, low molecular moisturizer, polymer moisturizer, fat-soluble moisturizer, emollient, surfactant, preservative, antioxidant, pH adjuster, Ethanol, water, etc. can be raised.

The pharmaceuticals, foods and cosmetics of the present invention can be expected to prevent, treat or improve pigmentation such as spots and freckles through the melanin production inhibitory action of the melanin production inhibitor of the present invention. .
Moreover, since the pharmaceutical, food and cosmetics of the present invention contain "a melanin production inhibitor that is safe and has a remarkable melanin production inhibitory action without fear of side effects," It is safe and has a remarkable melanin production-suppressing action, “pharmaceutical, food or cosmetic”.

  Hereinafter, the present invention will be described in more detail with reference to “1. Sample preparation examples” and “2. Test examples of melanin production inhibitory action”, but the present invention is not limited thereto.

1. Sample Preparation Example (Preparation Example 1) Sample Preparation Example Preparation Example 1 is an extract of purified water from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. (Sample a (water extract) )) Is a preparation example.
Heat extraction with 450 mL (milliliter) of purified water for 30.3 g of ground product of Saussurea involucrata (Kar. Et Kir.) Sch.Blp., A plant belonging to the genus Saussurea (After 80 degreeC-90 degreeC, 2 hours), the obtained solution was filtered (2 types of filter paper for qualitative analysis (purchased from Advantech Toyo Co., Ltd.) was used.) An extract was obtained. The obtained extract was concentrated under reduced pressure and lyophilized in order to obtain 8.3 g of sample a (water extract). The solid yield was 27.3%.

(Preparation Example 2) Preparation Example of Sample b Preparation Example 2 is an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp. With 50% (V / V) ethanol (sample b (50 % Ethanol extract))).
450 mL of 50% (V / V) ethanol for 30.5 g of ground pulverized product of Saussurea involucrata (Kar. Et Kir. Sch.Blp.), A plant belonging to the genus Saussurea After heating extraction (80 ° C. to 90 ° C., 2 hours) by filtration, the resulting solution was filtered (uses two types of filter paper for qualitative analysis (purchased from Advantech Toyo Co., Ltd.)) to obtain an extract. . The obtained extract was concentrated under reduced pressure and freeze-dried sequentially to obtain 7.4 g of sample b (50% ethanol extract). The solid yield was 24.2%.

(Preparation Example 3) Preparation Example of Sample c Preparation Example 3 is an extract from Saussurea involucrata (Kar. Et Kir. Sch. Blp.) With 80% (V / V) ethanol (sample c (80 % Ethanol extract))).
450 mL of 80% (V / V) ethanol with respect to 30.7 g of ground pulverized product of Saussurea involucrata (Kar. Et Kir. Sch.Blp.), A plant belonging to the genus Saussurea After heating extraction (80 ° C. to 90 ° C., 2 hours) by filtration, the resulting solution was filtered (uses two types of filter paper for qualitative analysis (purchased from Advantech Toyo Co., Ltd.)) to obtain an extract. . The obtained extract was concentrated under reduced pressure and freeze-dried sequentially to obtain 5.4 g of sample c (80% ethanol extract). The solid yield was 17.5%.

(Preparation Example 4) Preparation Examples of Samples df and Comparative Sample 1 FIG. 2 is a flowchart shown for explaining preparation examples of Samples df and Comparative Sample 1.
Preparation Example 4 is a sample prepared from Saussurea involucrata (Kar. Et Kir.) Sch.Blp., Comparative sample 1 (non-adsorbed component), sample d (adsorbed component), sample e (methanol elution fraction) and It is an example of preparation for obtaining the sample f (dihydrodehydrocostus lactone). The preparation example was performed according to the flow shown in FIG.

  Saussurea involucrata (Kar. Et Kir.) Sch.Blp. 4 kg ground ground was digested with 80% (V / V) ethanol (80 ° C, 2 hours). After performing twice, the resulting solution was filtered (uses two types of filter paper for qualitative analysis (purchased from Advantech Toyo Co., Ltd.)) to obtain an extract. Further, the extract was concentrated under reduced pressure at 60 ° C. and dried (normal temperature, 10 hours) to obtain 680 g of an extract. Next, the extract was dissolved in 30% (V / V) methanol (3 L, 40 ° C.) to form a solution, and the solution was preliminarily equilibrated with 30% (V / V) methanol, Diaion HP20 (1500 g, (Purchased from Mitsubishi Chemical Corporation) was passed through a column packed as a carrier. Thereafter, elution was performed with 6 L of 30% (V / V) methanol, and the 30% (V / V) methanol elution fraction was recovered as a non-adsorbed component (Comparative Sample 1). Subsequently, elution was performed successively with 7 L of 50% (V / V) methanol, 6 L of methanol, 5 L of ethanol, 5 L of ethyl acetate and 5 L of hexane, and the elution fractions were collected. Each elution fraction was made into a dried product by removing the solvent, 399 g of non-adsorbed component (Comparative Sample 1), 38 g of 50% (V / V) methanol elution fraction, 100 g of methanol elution fraction (Sample e), 21 g of ethanol elution fraction, 18 g of ethyl acetate elution fraction and 0.11 g of hexane elution fraction were obtained. The total of the elution fractions other than the non-adsorbed component was defined as the adsorbed component (sample d), and the total weight of each elution fraction other than the non-adsorbed component (ie, adsorbed component) was 177.11 g. .

Among these, the methanol elution fraction (sample e) (100 g) was subjected to silica gel column chromatography (80 mmφ × 350 mm, purchased from Fuji Silysia Chemical Ltd.) using chloroform / methanol / water as an elution solvent. That is, elution was performed while sequentially changing the ratio of the elution solvent from 90: 10: 0, 40: 10: 1, and 30: 10: 1 (both chloroform: methanol: water), and the eluate was 150 mL per fraction. The fractions were collected one by one, and these fractions were combined to obtain 8 fractions A to H. Among these, the solvent was removed from the eluate containing fraction A (elution amount: 450 mL to 1200 mL) to obtain a dry solid, and 40.9 g of fraction A was obtained.
Next, fraction A (40.9 g) was subjected to silica gel column chromatography (60 mmφ × 250 mm, purchased from Fuji Silysia Chemical Ltd.) using hexane / acetone as an elution solvent. That is, elution was carried out while sequentially changing the elution solvent ratio to 9: 1, 6: 1, 4: 1, 2: 1 (both hexane: acetone), and 60 mL of the eluate was fractionated. These fractions were combined to obtain 8 fractions AA to AH. Among these, the solvent was removed from the eluate containing fraction AB to obtain a dry solid, and 13.1 g of fraction AB (elution amount: 180 mL to 600 mL) was obtained.
Further, fraction AB (13.1 g) was subjected to ODS silica gel column chromatography (60 mmφ × 150 mm, purchased from Nacalai Tesque, Inc.) using methanol / water as an elution solvent. That is, elution was carried out while sequentially changing the elution solvent ratios to 7: 3, 8: 2, 9: 1 (both methanol: water), and the eluate was fractionated 40 mL per fraction, and these fractions were combined. Ten fractions of AB-1 to AB-10 were obtained. Among these, the solvent was removed from the eluate containing fraction AB-3 (elution amount: 1200 mL to 1440 mL) to obtain a dry solid to obtain 2.1 g of fraction AB-3.
Further, fraction AB-3 (2.1 g) was subjected to silica gel column chromatography (60 mmφ × 150 mm, purchased from Fuji Silysia Chemical Ltd.) using hexane / ethyl acetate as an elution solvent. That is, elution was carried out while sequentially changing the elution solvent ratio to 19: 1 and 9: 1 (both hexane: ethyl acetate), and the eluate was fractionated 40 mL per fraction, containing dihydrodehydrocostus lactone. Eluate (elution amount: 1240 mL to 1520 mL) was obtained. The solvent was removed from the eluate to obtain a dry solid, and 161 mg of dihydrodehydrocostus lactone (sample f) was obtained.

When ¹ H-NMR spectrum data and ¹³ C-NMR spectrum data were obtained by nuclear magnetic resonance spectroscopy for dihydrodehydrocostus lactone (sample f) fractionated and isolated by the above method, the following peaks were observed. It almost agreed with the value in the literature (Journal of Natural Products, Vol. 62, page 27, 1999). The molecular formula of dihydrodehydrocostus lactone (sample f) is C ₁₅ H ₂₀ O ₂ .

¹ H-NMR (CDCl ₃ , 400 MHz) δ: 1.23 (3H, d, J = 7.0 Hz, Me-12), 1.30 (1H, m, H-8), 1.85 (1H, m, H-2), 1.94 (2H, m, H-2, H-7), 2.04 (1H, m, H-9), 2.10 (1H, m, H-3), 2.21 (1H, m, H-11), 2.50 (3H, m, H-3, H-8, H-9), 2.79 (1H, dd, J = 8.9, 8. 9 Hz, H-5), 2.87 (1H, m, H-1), 3.91 (1H, dd, J = 9.3, 9.3 Hz, H-6), 4.77 (1H, br) s, H-15a), 4.87 (1H, br s, H-15b), 5.04 (1H, d, J = 2.0 Hz, H-14a) 5.19 (1H, d, J = 2) .0Hz, H-14b).

¹³ C-NMR (CDCl ₃ , 100 MHz) δ: 13.22 (C-12), 30.17 (C-2), 32.48 (C-3, 8), 37.65 (C-9), 42.04 (C-11), 47.04 (C-1), 49.85 (C-7), 51.93 (C-5), 85.28 (C-6), 109.15 (C -14), 111.81 (C-15), 149.94 (C-10), 151.70 (C-4), 178.70 (C-13).

  Further, when mass spectrometry was performed on the dihydrodehydrocostus lactone (sample f) fractionated and isolated by the above method, the following results were obtained, and the measured mass was approximately the same as the mass estimated from the molecular formula did.

HR-ESI-TOF-MS m / z: 233.1550 [M + H] ⁺ (Calcd for C ₁₅ H ₂₀ O ₂ : 233.1542).

  In the above, general-purpose laboratory instruments and experimental apparatuses were used for column chromatography with Diaion HP20, silica gel column chromatography, and ODS silica gel column chromatography. As a nuclear magnetic resonance spectrum apparatus, a Bruker DRX-400 type nuclear magnetic resonance spectrum apparatus (manufactured by Bruker BioSpin Corporation) was used. As a mass spectrometer, a Waters-Micromass LCT mass spectrometer (manufactured by Waters) was used.

2. Test example of melanin production inhibitory action The test example of melanin production inhibitory action was performed by measuring the melanin production inhibitory action on B16 mouse melanoma cells.

(A) Test method A medium supplemented with Dulbecco's modified Eagle medium (DMEM, purchased from GIBCO-BRL) with 10% (V / V) fetal calf serum (FBS, purchased from GIBCO-BRL) (DMEM supplemented with 10% FBS) Medium) and seeded B16 mouse melanoma cells (B16 melanoma 4A5, purchased from RIKEN BioResource Center) in a 6-well plate (purchased from NUNC) at 3.5 × 10 ⁵ cells / well, and CO ₂ incubator The cells were cultured at 37 ° C., 5% carbon dioxide, 95% air. After 24 hours, the medium was exchanged with 10% FBS-added DMEM medium containing 10 nmol / L α-MSH (melanocyte stimulating hormone, purchased from SIGMA) adjusted to contain the sample at a predetermined concentration (20 μg / mL or 10 μg / mL). The culture was further performed for 48 hours.
As samples, sample a (water extract, see Preparation Example 1 above, concentration 200 μg / mL), sample b (50% ethanol extract, see Preparation Example 2 above, concentration 200 μg / mL), sample c (80 % Ethanol extract, see Preparation Example 3. Concentration 200 μg / mL), sample d (Adsorption component. See Preparation Example 4 above, concentration 20 μg / mL), sample e (Methanol elution fraction. Preparation Example 4 above) Reference: concentration 20 μg / mL) and sample f (dihydrodehydrocostus lactone, see Preparation Example 4 above, concentration 20 μg / mL) were tested for each. For comparison, comparative sample 1 (non-adsorbed component, see Preparation Example 4 above, concentration 200 μg / mL) and comparative sample 2 (arbutin (4-hydroxyphenyl-β-D-glucopyranoside), purchased from SIGMA. The test was also conducted for a concentration of 20 μg / mL.

  Next, trypsin treatment was performed by a conventional method, and cells were collected from each well. Thereafter, the collected cells were transferred to a 1.5 mL microtube, and a pellet was obtained by centrifugation (800 G, 10 minutes). 200 μL of 1 mol / L NaOH aqueous solution was added to the pellet, dissolved by heating at 80 ° C. for 15 minutes, and absorbance at a wavelength of 475 nm (blank: 1 mol / L NaOH aqueous solution) was measured. Simultaneously with the measurement, the number of viable cells without trypan blue was counted using a hemocytometer and evaluated as the amount of melanin per live cell, and compared with the amount of melanin per live cell when using a medium without sample Thus, the relative amount of melanin (an index when the amount of melanin per living cell when no sample was added was taken as 100%) was calculated.

(A) Test results The test results are shown in Table 1 and FIG. Table 1 is a table | surface which shows the result of a test example (melanin production suppression effect with respect to B16 mouse | mouth melanoma cell). FIG. 3 is a graph showing the results of a test example (melanin production inhibitory effect on B16 mouse melanoma cells).
[Table 1]

Relative melanin content in B16 mouse melanoma cells
Sample concentration Relative melanin content
Sample a (water extract) 40 μg / mL 79.5%
Sample b (50% ethanol extract) 40 μg / mL 82.2%
Sample c (80% ethanol extract) 40 μg / mL 84.7%
Sample d (adsorption component) 20 μg / mL 76.6%
Sample e (methanol elution fraction) 20 μg / mL 67.6%
Sample f (dihydrodehydrocostus lactone) 10 μg / mL 73.5%
Comparative sample 1 (non-adsorbing component) 20 μg / mL 101.0%
Comparative sample 2 (arbutin) 20 μg / mL 80.3%
No sample added (control)-100.0%

(A) About Samples a to c As shown in Table 1 and FIG. 3, any extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp. It was confirmed that B16 mouse melanoma cells have an inhibitory effect on melanin production.

(B) Sample d As shown in Table 1 and FIG. 3, sample d (adsorbed component) has a clear melanin production inhibitory effect, as can be seen from comparison with no sample added and comparative sample 1 (non-adsorbed component). It was confirmed that Moreover, it has also confirmed that the sample d (adsorption component) showed the melanin production suppression effect higher than the comparative sample 2 (arbutin).

(B) Sample e As shown in Table 1 and FIG. 3, it was confirmed that sample e (methanol elution fraction) exhibited a higher melanin production inhibitory effect than sample d (adsorbed component).

(C) About sample f As shown in Table 1 and FIG. 3, sample f (dihydrodehydrocostus lactone) was at a concentration (10 μg / mL) lower than that of comparative sample 2 (arbutin) (20 μg / mL). Even if it exists, it has confirmed that the higher melanin production suppression effect was shown.

Example 1. Preparation of tablets Using the water extract prepared according to the method described in "Preparation example 1) Preparation example of sample a", tablets (500 mg per tablet) are prepared according to the following formulation.

Water extract (Preparation Example 1) 10mg
Lactose 470mg
10mg dried corn starch
Talc 9mg
Calcium stearate 1mg

(Preparation method)
Lactose (94 g) is mixed with water extract (2 g), dried corn starch (2 g), talc (1.8 g) and calcium stearate (0.2 g). Subsequently, a tablet is produced by a conventional method using a single-shot tablet press.

Example 2 Production of hard capsules Using the 50% ethanol extract prepared according to the method described in “Preparation Example 2) Preparation Example of Sample b” above, hard capsules (360 mg per capsule) are produced with the following formulation. .

50% ethanol extract (Preparation Example 2) 5mg
Lactose 220mg
Corn starch 110mg
Hydroxypropylcellulose 25mg

(Preparation method)
Lactose (220 g) and corn starch (110 g) are added to and mixed with 50% ethanol extract (5 g), and an aqueous solution of hydroxypropylcellulose (25 g) is added thereto and kneaded. Next, granules are produced by an ordinary method using an extrusion granulator. A hard capsule is prepared by filling the granule into a gelatin hard capsule.

Example 3 Production of soft capsules Using the 80% ethanol extract prepared according to the method described in “Preparation Example 3) Preparation Example of Sample c” above, soft capsules (170 mg per capsule) are produced according to the following formulation. .

80% ethanol extract (Preparation Example 3) 5mg
Soybean oil 165mg

(Preparation method)
Add 80% ethanol extract (5 g) to soybean oil (165 g) and mix. Next, soft capsules are prepared by filling the soft capsules using a rotary die type automatic molding machine according to a conventional method.

Example 4 Preparation aforementioned pill with adsorbate prepared according to the method described in "(Preparation Example 4) Sample d~f and Preparation of Comparative Sample 1", pills in the following formulation (per one grain 100mg) Is made.

Adsorption component (Preparation Example 4) 0.5mg
Morohaya powder 20.0mg
Starch 30.0mg
Molasses 20.0mg
Tea extract 15.0mg
Soy fiber 14.0mg
Shellac 0.5mg

(Preparation method)
After mixing the raw materials with the above blending, adding a suitable amount of water, producing a homogeneous kneaded product with a kneader, rolling the kneaded product, making a round shape using a round machine and drying it to produce a pill To do.

Example 5 FIG. Preparation of Jelly Using the water extract prepared according to the method described in “(Preparation Example 1) Preparation Example of Sample a”, a jelly (100 g) is prepared by a conventional method with the following formulation.

Water extract (Preparation Example 1) 0.02g
2.00 g of gelatin
Orange juice 20.00g
77.98 g of water

(Preparation method)
The above ingredients are mixed and heated to 90 ° C. After confirming the dissolution of gelatin, fill the container and cool. A jelly is prepared by solidifying gelatin.

Example 6 Preparation of ointment Using the dihydrodehydrocostus lactone prepared according to the method described in "Preparation Example 4) Preparation Examples of Samples df and Comparative Sample 1" above, the ointment ( 100 g).

(Oil phase component)
Dihydrodehydrocostus lactone (Preparation Example 4) 0.01 g
White petrolatum 20.00g
Mineral oil 20.00g
Stearyl alcohol 5.00g
Steareth-2 3.00g
Propylparaben 0.10g
Natural vitamin E 0.10g
(Aqueous phase component)
1,3-butylene glycol 5.00g
Phenoxyethanol 0.40g
Polysorbate 60 4.50g
Purified water
Total amount 100g

(Preparation method)
The oil phase component and the water phase component were each heated to 80 ° C. to be uniform, and the water phase was added to the oil phase with stirring. After emulsification, the mixture was cooled to prepare an ointment.

Example 7 Preparation of tape agent Using the methanol elution fraction prepared according to the method described in the above-mentioned "(Preparation Example 4) Preparation Examples of Samples df and Comparative Sample 1", the tape agent was prepared in the following manner using the conventional method. (100 g) is prepared.

(Adhesive solvent)
Styrene-isopropylene-styrene block copolymer 7.00 g
Picolite 25.00g
Isopropylene rubber 5.00g
Toluene 15.00g
14.20 g of ethyl acetate
Hexane 25.00g
(Medicinal ingredients)
Methanol elution fraction (Preparation Example 4) 0.01 g
Ethanol 7.9g
(Transdermal absorption enhancer)
Oleyl alcohol 0.80g
Total amount 100g

(Preparation method)
The adhesive solvent and the medicinal component were made uniform, the medicinal component and the transdermal absorption accelerator were added to the adhesive solvent, and the mixture was stirred at room temperature to prepare a composition. This composition is spread on a silicone-treated polyester film, dried at 120 ° C. and cooled, and then the pressure-sensitive adhesive layer is transferred to a polyethylene film to produce a tape agent.

Example 8 FIG. Production of Lotion A lotion (100 g) is produced by a conventional method using the water extract prepared according to the method described in the above-mentioned “(Preparation Example 1) Preparation Example of Sample a”.

(Oil phase component)
Polyoxyethylene (60 mol) hydrogenated castor oil 2.0 g
1,3-butylene glycol 5.0 g
(Aqueous phase component)
Water extract (Preparation Example 1) 0.1g
Glycerin 5.0g
Phenoxyethanol 0.3g
Citric acid 0.1g
Sodium citrate 0.2g
Ethanol 8.0g
Purified water
Total amount 100g

(Preparation method)
An oil phase component and an aqueous phase component are uniformly dissolved, and the oil phase is added to the aqueous phase while stirring to prepare a skin lotion.

Example 9 Preparation of emulsion Using the 50% ethanol extract prepared according to the method described in the above-mentioned "(Preparation Example 2) Preparation Example of Sample b", an emulsion (100 g) is prepared in the usual manner with the following formulation.

Palm oil 8.00g
Ethanol 5.00g
Behenyl alcohol 0.50g
Citric acid 0.10g
Sodium citrate 0.20g
50% ethanol extract (Preparation Example 2) 1.50 g
1,3-butylene glycol 4.00 g
Paraoxybenzoic acid ester 0.10g
Edetic acid disodium 0.01g
Purified water
Total amount 100g

(Preparation method)
An emulsion is prepared by stirring and mixing the above materials.

Example 10 Preparation of cosmetic liquid Using the 80% ethanol extract prepared according to the method described in “Preparation Example 3) Preparation Example of Sample c” above, a cosmetic liquid (100 g) was prepared by the usual method with the following prescription. To do.

80% ethanol extract (Preparation Example 3) 1.50 g
Carboxy vinyl polymer 0.50g
Glycerin 6.50g
1,3-butylene glycol 7.50g
Phenoxyethanol 0.30g
Potassium hydroxide 0.20g
Sodium hyaluronate 0.25g
Squalane 0.50g
Vitamin E acetate 0.05g
Stearic acid 0.20g
Purified water
Total amount 100g

(Preparation method)
A cosmetic solution is prepared by stirring, mixing, and dissolving the above materials.

Claims (10)

  A melanin production inhibitor containing, as an active ingredient, an extract from Saussurea involucrata (Kar. Et Kir.) Sch. Blp.   A solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol was passed through a column using a porous polystyrene resin as a carrier. Sometimes, a melanin production inhibitor containing, as an active ingredient, a component adsorbed on the porous polystyrene resin.   A solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol was passed through a column using a porous polystyrene resin as a carrier, Thereafter, a melanin production inhibitor containing, as an active ingredient, a component adsorbed on the porous polystyrene resin when 50% (V / V) methanol is passed through the column.   A solution obtained by dissolving an extract from Saussurea involucrata (Kar. Et Kir.) Sch.Blp. In 30% (V / V) methanol was passed through a column using a porous polystyrene resin as a carrier, Thereafter, a melanin production inhibitor containing, as an active ingredient, a component that is eluted from the column by methanol when 50% (V / V) methanol is passed through the column and then methanol is passed through the column. The melanin production inhibitor containing the dihydrodehydrocostus lactone represented by following formula (1).

  A pharmaceutical, food or cosmetic comprising the melanin production inhibitor according to claim 1 and having a melanin production inhibitory action.   A pharmaceutical, food or cosmetic comprising the melanin production inhibitor according to claim 2 and having a melanin production inhibitory action.   A pharmaceutical, food or cosmetic comprising the melanin production inhibitor according to claim 3 and having a melanin production inhibitory action.   A pharmaceutical, food or cosmetic comprising the melanin production inhibitor according to claim 4 and having a melanin production inhibitory action.   A pharmaceutical, food or cosmetic comprising the melanin production inhibitor according to claim 5 and having a melanin production inhibitory action.

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