JP7696201B2 - Compositions for external or internal use - Google Patents

Description

The present invention relates to a composition for external or internal use. More specifically, the present invention relates to a composition for external or internal use having improved functionality due to flavonoids such as nobiletin.

Fruits contain functional ingredients such as carotenoids, flavonoids, limonoids, coumarins, and polyphenols. These functional ingredients are added to health foods such as health functional foods with the aim of adding high added value to foods.

Among these functional components, one example of a flavonoid is nobiletin, which is found in large amounts in mandarins and oranges. Nobiletin has been reported to have the effect of suppressing reactions related to carcinogenesis, arteriosclerosis, and inflammation, and research has also been conducted on the prevention and improvement of dementia (Non-Patent Document 1). Another example of a coumarin is auraptene, which is found in pomelo and trifoliate orange. Auraptene has been researched primarily for its cancer-suppressing effect, and other reported effects include activating lipid metabolism and suppressing blood coagulation reactions (Non-Patent Document 1).

Journal of the Japanese Society of Food Chemical Engineering, Vol. 60, No. 10, 2013

Although nobiletin has been reported to have anti-inflammatory and other functional properties, its action alone is not sufficient. In order to obtain sufficient effects by adding nobiletin to food, a large amount of nobiletin would be required, which would make it less cost-effective.

The object of the present invention is to provide a composition that can improve the functionality of flavonoids such as nobiletin.

After extensive research, the inventors discovered that the functionality of a flavonoid such as nobiletin can be improved by combining it with a coumarin such as auraptene. The present invention was completed based on this finding and through further research.

［式中、Ｒ¹～Ｒ⁷は、各々独立して、水素原子、水酸基、アルキル基又はアルコキシ基を表す］で示される化合物１と、
下記式（２）：

［式中、Ｒ⁸は、水素原子、水酸基、アルキル基、アルコキシ基、又はエステル基含有基を表し；Ｒ⁹は、水素原子、水酸基、アルキル基、アルコキシ基、カルボキシル基、アリール基又はクマリニル基を表し；Ｒ¹⁰は、水素原子、水酸基、アルキル基、アルコキシ基、カルボキシル基又はカルボキシアルキル基を表し；Ｒ¹¹は、水素原子、アルキル基、オレフィン系不飽和二重結合含有基を表す。］で示される化合物２と、を含む、外用又は内用組成物。
項２． 抗炎症剤として用いられる、項１に記載の組成物。
項３． 前記請求項１の化合物が、ノビレチン、タンゲレチン、ルテオリン、及びケルセチンからなる群より選択される、項１又は２に記載の組成物。
項４． 前記請求項２の化合物が、下記（２１）～（２８）からなる群より選択される、項１～３のいずれかに記載の組成物。

項５． 前記化合物１がノビレチンであり、前記化合物２がオーラプテンである、項１～４のいずれかに記載の組成物。
項６． 前記化合物１の１モルに対し、前記化合物２を０．１～１０モル含む、項１～５のいずれかに記載の組成物。
項７． 飲食品である、項１～６のいずれかに記載の組成物。 That is, the present invention provides the following aspects.
Item 1. The following formula (1):

[wherein R ¹ to R ⁷ each independently represent a hydrogen atom, a hydroxyl group, an alkyl group or an alkoxy group];
The following formula (2):

[wherein ^R8 represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or an ester group-containing group; ^R9 represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, a carboxyl group, an aryl group, or a coumarinyl group; ^R10 represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, a carboxyl group, or a carboxyalkyl group; and ^R11 represents a hydrogen atom, an alkyl group, or an olefinically unsaturated double bond-containing group.] and a compound 2 represented by the formula:
Item 2. The composition according to Item 1, which is used as an anti-inflammatory agent.
Item 3. The composition according to item 1 or 2, wherein the compound of claim 1 is selected from the group consisting of nobiletin, tangeretin, luteolin, and quercetin.
Item 4. The composition according to any one of items 1 to 3, wherein the compound according to item 2 is selected from the group consisting of the following (21) to (28):

Item 5. The composition according to any one of Items 1 to 4, wherein compound 1 is nobiletin and compound 2 is auraptene.
Item 6. The composition according to any one of Items 1 to 5, comprising 0.1 to 10 moles of the compound 2 relative to 1 mole of the compound 1.
Item 7. The composition according to any one of Items 1 to 6, which is a food or drink.

The present invention provides a composition that can improve the functionality of flavonoids such as nobiletin.

1 shows the results of confirming the cytotoxicity of test samples used in the Examples and Comparative Examples.

The composition for external or internal use of the present invention is characterized by containing a specific flavonoid and coumarin. The composition for external or internal use of the present invention is described in detail below.

Certain flavonoids (compound 1)
The specific flavonoid contained in the composition for external or internal use of the present invention is compound 1 represented by the following formula (1).

In formula (1), R ¹ to R ⁷ each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, or an alkoxy group. The alkyl group includes a C1-6 (carbon number 1 to 6) alkyl group, and the alkoxy group includes a C1-6 alkoxy group. Preferred examples of R ¹ to R ⁷ include a hydrogen atom, a hydroxyl group, and an alkoxy group, and more preferred examples include a hydrogen atom, a hydroxyl group, and a C1-6 alkoxy group, preferably a hydrogen atom, a hydroxyl group, and a C1-4 alkoxy group, and more preferably a hydrogen atom, a hydroxyl group, and a C1-2 alkoxy group.

The above compound 1 may be used alone or in combination of two or more.

A particularly preferred example of the above compound 1 is nobiletin in which R ¹ to R ⁷ are all methoxy groups.

In the present invention, the compound of formula (I) also includes salts. Such salts are not particularly limited as long as they are physiologically acceptable salts, and examples of such salts include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium; magnesium salts; ammonium salts; and organic salts such as ethylenediamine, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine, 2-amino-2-methyl-1-propanol, diethanolamine, ethanolamine, N-methylglucamine, and L-glucamine. Examples of such salts include salts with bases, etc.; salts with basic amino acids such as lysine, δ-hydroxylysine, and arginine; salts with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, and salicylic acid; and salts with acidic amino acids such as aspartic acid and glutamic acid.

Selected Coumarins (Compound 2)
The specific coumarin contained in the topical or internal composition of the present invention is compound 2 represented by the following formula (2).

In formula (2), ^R8 represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or an ester group-containing group. The alkyl group includes a C1-6, preferably a C1-4, more preferably a C1-2 alkyl group. The alkoxy group includes a C1-6, preferably a C1-4, more preferably a C1-2 alkyl group. The ester group-containing group includes an alkyloxycarbonylalkyl group, and the alkyloxycarbonylalkyl group includes a C1-6 (preferably C1-4, more preferably C1-2) alkyloxycarbonylC1-6 (preferably C1-4, more preferably C1-2) alkyl group.

In formula (2), R ⁹ represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, a carboxyl group, an aryl group, or a coumarinyl group. The alkyl group includes a C1-6, preferably a C1-4, and more preferably a C1-2 alkyl group. The alkoxy group includes a C1-6, preferably a C1-4, and more preferably a C1-2 alkyl group. The aryl group includes a phenyl group, a naphthyl group, and the like, and is preferably a phenyl group. The coumarinyl group includes a 3'-coumarinyl group, a 4'-coumarinyl group, and is preferably a 3'-coumarinyl group.

In formula (2), R ¹⁰ represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, a carboxyl group, or a carboxyalkyl group. The alkyl group includes a C1-6, preferably a C1-4, and more preferably a C1-2 alkyl group. The alkoxy group includes a C1-6, preferably a C1-4, and more preferably a C1-2 alkyl group. The carboxyalkyl group includes a carboxyC1-6 (preferably C1-4, and more preferably C1-2) alkyl group.

In formula (2), R ¹¹ represents a hydrogen atom, an alkyl group, or an olefinically unsaturated double bond-containing group. Examples of the alkyl group include C1-15 alkyl groups. Examples of the olefinically unsaturated double bond-containing group include alkenyl groups [e.g., C2-30, preferably C2-16 alkenyl groups], alkadienyl groups [e.g., C4-30, preferably C4-16 alkadienyl groups such as 2,4? alkadienyl groups and 2,6? alkadienyl groups], alkatrienyl groups [e.g., C6-30, preferably C6-16 alkatrienyi groups], alkatetraenyl groups [e.g., C8-30, preferably C8-16 alkatetraenyl groups], and alkpentaenyl groups [e.g., C10-30, preferably C10-16 alkpentaenyl groups].

In the present invention, suitable examples of compound 2 include the compounds shown in (21) to (28) below.

The above compound 2 may be used alone or in combination of two or more.

A particularly preferred example of the above compound 2 is auraptene shown in formula (21).

In the internal or external use composition of the present invention, the blending ratio of compound 1 to compound 2 is not particularly limited, and for example, the blending amount of compound 2 per mole of compound 1 is 0.1 to 10 moles. From the viewpoint of more efficiently improving the functionality of compound 1, such as anti-inflammatory properties, the blending amount of compound 2 per mole of compound 1 is preferably 0.15 to 5 moles, more preferably 0.2 to 4 moles, even more preferably 0.25 to 2.5 moles, even more preferably 0.25 to 2 moles, even more preferably 0.25 to 1.5 moles, and particularly preferably 0.25 to 1 mole.

Other components The internal or external use composition of the present invention may be composed of only the above-mentioned compound 1 and compound 2, which are the active ingredients, or may further contain additives and/or bases according to the formulation form and use. Such additives and bases are not particularly limited as long as they are pharmacologic acceptable, and include, for example, excipients, binders, disintegrants, lubricants, isotonicity agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, gelling agents, coating agents, glossing agents, water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, water-soluble polymers, surfactants, metal soaps, lower alcohols, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV protection agents, preservatives, flavorings, fragrances, powders, thickeners, colorants, chelating agents, sweeteners, etc. These additives may be used alone or in combination of two or more. The content of these additives and bases is appropriately set depending on the types of additives and bases used, the formulation form and use of the internal or external composition of the present invention, etc.

In addition, the internal or external use composition of the present invention may contain other nutritional components or pharmacological components as necessary, in addition to the above-mentioned compound 1 and compound 2, which are the active ingredients. Such nutritional components and pharmacological components are not particularly limited as long as they are pharmacologic acceptable, and examples thereof include antacids, stomachic agents, digestive agents, intestinal regulators, antispasmodics, mucosal repair agents, anti-inflammatory agents, astringents, antiemetics, antitussives, expectorants, anti-inflammatory enzymes, sedatives, hypnotics, antihistamines, caffeine, cardiac diuretics, antibacterial agents, vasoconstrictors, vasodilators, local anesthetics, herbal extracts, vitamins, and menthols. These nutritional components and pharmacological components may be used alone or in combination of two or more. The content of these components is appropriately set depending on the type of component used, the formulation form of the internal or external use composition of the present invention, and the purpose.

Formulations The formulations of the internal or external use compositions of the present invention are not particularly limited, as long as they are capable of oral administration in the case of internal compositions, and transdermal or transmucosal administration in the case of external compositions.

For example, formulations of compositions for internal use include solid preparations such as powders, fine granules, granules (including dry syrups), tablets, pills, and capsules (soft capsules and hard capsules); semi-solid preparations such as jellies; and liquid preparations such as solutions, suspensions, and syrups. Formulations of compositions for external use include creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, and packs.

To prepare the internal or external composition of the present invention in these formulation forms, the active ingredients, Compound 1 and Compound 2, as well as additives, bases, and/or pharmacological ingredients, which are added as necessary, may be formulated according to conventional formulation methods.

Uses The uses of the internal or external use composition of the present invention are not particularly limited so long as they are for internal or external use.

In the case of compositions for internal use, examples include pharmaceuticals and food and beverage products, preferably food and beverage products. Specific examples of food and beverage products include health foods, functional foods, nutritional supplements, and foods for specified health uses. Specific forms of food and beverage products include supplements (tablets, hard capsules, soft capsules, etc.), beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, milk drinks, etc.), as well as concentrated concentrates of the beverages and powders for preparation when used.

In the case of a composition for external use, preferred examples include medicines, quasi-drugs, and cosmetics. Specific examples of these medicines, quasi-drugs, and cosmetics include creams, lotions, gels, milky lotions, poultices, sprays, ointments, packs, cleansers, etc., and examples of application sites include the skin, mucous membranes, hair, etc.

The internal or external composition of the present invention can improve the functionality of compound 1, and can therefore be used for the purpose of obtaining an effect based on the functionality of compound 1. Preferably, the internal or external composition of the present invention can be used as an anti-inflammatory agent. The internal or external composition of the present invention can also be used as an IL-6 production inhibitor, a TNF-α production inhibitor, and/or an NO production inhibitor.

Dosage and Usage The dosage of the internal or external composition of the present invention is appropriately set according to the type of active ingredient, the age, sex, constitution, etc. of the subject of administration, but for example, the dosage (dosage or intake) of the internal composition is, for example, about 0.1 to 500 mg of the above-mentioned compound 1 per day (in the case of a body weight of 60 kg) for humans. Since the contents or external composition of the present invention can improve the functionality of compound 1, even if the amount of compound 1 is small enough that it alone cannot fully exert its functionality, it can effectively obtain an action effect based on the functionality. From this viewpoint, preferred examples of the dosage of the internal composition include, for humans, preferably about 0.1 to 100 mg, more preferably about 0.2 to 50 mg, and even more preferably about 0.5 to 20 mg of the above-mentioned compound 1 per day (in the case of a body weight of 60 kg). The internal or external composition of the present invention may be used internally or externally 1 to 3 times a day, preferably 2 or 3 times a day.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

(1) Test Method (1-1) Cell Culture Mouse macrophage cell line RAW264.7 cells were used and cultured in 10% FBS-DMEM medium (containing 100 U/mL penicillin, 100 μg/mL streptomycin) in a carbon dioxide incubator under humidified conditions at 37° C. and 5% CO ₂ . Before the test, the RAW264.7 cells attached to the culture dish were detached with a cell scraper, and the cells were prepared in 10% FBS-DMEM medium using a cell counter to obtain the desired cell number.

(1-2) Test Method RAW264.7 cells were seeded in a 96-well culture plate at 2 x ¹⁰⁴ cells/well. After pre-culture, a dimethyl sulfoxide (DMSO) solution of nobiletin and/or auraptene was added as a test sample. Nobiletin and/or auraptene were added to the final concentrations shown in Tables 1 to 5, and dimethyl sulfoxide was added to a final concentration of 0.1%. Then, culture was carried out for 24 hours.

(1-3) Method for evaluating anti-inflammatory effect After the end of the culture, the amount of interleukin (IL)-6 produced and the amount of tumor necrosis factor (TNF-α) produced in the collected culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) to evaluate the anti-inflammatory effect on macrophages in a hyperinflammatory state. In addition, the amount of nitric oxide (NO) produced was quantified by the Griess method. The amount of IL-6 produced, the amount of TNF-α produced, and the amount of NO produced in the control (Comparative Example 1) were each set to 100%, and the relative amount (%) in each test sample was derived. The smaller these relative amounts are, the better the effect can be evaluated.

The ratio of the amount of IL-6 production inhibition obtained in the examples to the amount of IL-6 production inhibition equivalent to the additive effect (hereinafter referred to as the "effect of synergistically improving the inhibitory effect on IL-6 production") was calculated based on the following formula.

Similarly, based on the following formula, the ratio of the amount of TNF-α production inhibition obtained in the examples to the amount of TNF-α production inhibition equivalent to the additive effect (hereinafter referred to as the "effect of synergistically improving the inhibitory effect of TNF-α production") and the ratio of the amount of NO production inhibition obtained in the examples to the amount of NO production inhibition equivalent to the additive effect (hereinafter referred to as the "effect of synergistically improving the inhibitory effect of NO production") were calculated.

The results regarding the inhibitory effect on IL-6 production are shown in Tables 1 to 3, the results regarding the inhibitory effect on TNF-α production are shown in Table 4, and the results regarding the inhibitory effect on NO production are shown in Table 5.

In addition, to confirm the toxicity of the test sample to RAW264.7 cells, the supernatant was collected, WST-8 solution was added to the cells at 10%, and the cells were incubated at 37°C for 40 minutes. The cell viability was then measured by measuring the absorbance at 40 nm, and the relative viability (%) was calculated with the average value in Comparative Example 1 taken as 100%. The results are shown in Figure 1.

Regarding the IL-6 production inhibitory effect (Tables 1 to 3), nobiletin alone was insufficient (Comparative Examples 2, 6, and 7), and auraptene showed no or almost no effect depending on the concentration (Comparative Examples 3 and 4), and even when it showed an effect, it was insufficient (Comparative Example 5). On the other hand, when nobiletin was combined with auraptene, the IL-6 production inhibitory effect was improved (Examples 1 to 9).

The confirmed IL-6 production inhibitory effect differed depending on the concentration of each component added (Tables 1, 2, 3). In particular, when the ratio of auraptene to nobiletin was as shown in Examples 1, 2, 4, to 9, a particularly remarkable and excellent effect was obtained that exceeded the additive effect of the effects of nobiletin and auraptene alone.

Furthermore, comparing Example 1 and Example 2 in terms of the IL-6 production suppression effect, Example 2 had a higher IL-6 production suppression amount, but Example 1 had a significantly higher effect of synergistically improving the IL-6 production suppression action. Comparing Examples 4 to 6, the IL-6 production suppression amount was higher in Examples 4, 5, and 6 in that order, but the effect of synergistically improving the IL-6 production suppression action was higher in Examples 6, 5, and 4 in that order, with Example 4 being significantly higher. Comparing Examples 7 to 9, the IL-6 production suppression amount was higher in Examples 7, 8, and 9 in that order, but the effect of synergistically improving the IL-6 production suppression action was higher in Examples 9, 8, and 7 in that order (Examples 7 and 8 were equivalent).

Regarding the TNF-α production inhibitory effect (Table 4), there was room for improvement when using nobiletin alone (Comparative Example 2), and auraptene showed no effect at all depending on the concentration (Comparative Examples 3 and 4), and even when it showed an effect, it was insufficient (Comparative Example 5). On the other hand, when nobiletin was combined with auraptene, the TNF-α production inhibitory effect was improved (Examples 1 to 3).

Furthermore, comparing Examples 1 to 3 with respect to the TNF-α production inhibitory effect, when nobiletin and auraptene were combined in the ratios of Examples 1 and 2, a particularly remarkable effect was obtained that exceeded the additive effect of the effects of nobiletin and auraptene alone. Also, comparing Example 1 and Example 2, the amount of TNF-α production inhibition was higher in Example 2, but the ratio of the amount of TNF-α production inhibition (the effect of synergistically improving the TNF-α production inhibitory action) was higher in Example 1.

Regarding the NO production inhibitory effect (Table 5), nobiletin alone had almost no effect (Comparative Example 8), and auraptene showed no effect at all depending on the concentration (Comparative Example 3), and even when it showed an effect, it was insufficient (Comparative Example 4). On the other hand, when nobiletin was combined with auraptene, the NO production inhibitory effect was improved (Examples 10 and 11).

Furthermore, when comparing the NO production inhibitory effect between Examples 10 and 11, not only was the amount of NO production inhibitory effect greater in Example 10, but the effect of synergistically improving the NO production inhibitory effect was also significantly greater in Example 10.

In addition, as shown in Figure 1, it was also confirmed that the test samples had no cytotoxicity.

Claims (2)

A composition for external or internal use , comprising nobiletin and auraptene as active ingredients, containing 0.2 to 2.5 moles of auraptene per mole of nobiletin , and used as an IL6 production inhibitor and/or an NO production inhibitor . The composition according to claim 1 , which is a food or beverage.