JP2005213178A - TNF-alpha PRODUCTION INHIBITOR, ESTROGENIC AGENT AND EXTERNAL PREPARATION FOR SKIN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new TNF-α production inhibitor which contains a TNF-α production inhibitory substance as an active ingredient, a new estrogenic agent containing an estrogenic substance as an active ingredient and an external preparation for the skin wherein the TNF-α production inhibitor or the estrogenic agent is compounded. <P>SOLUTION: The TNF-α production inhibitor or the estrogenic agent contains nobiletin, at least one citrus extract chosen from the group consisting of extracts from Citrus hassaku Hort., Citrus unshiu Marcov., grapefruit, Valencia orange, Citrus depressa Hayata, Citrus reticulata Blanco, lemon, marcott and Shiranui or a nobiletin purified product obtained by purifying the citrus extract as an active ingredient. The external preparation for the skin contains the TNF-α production inhibitor or the estrogenic agent. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a TNF-α production inhibitor that can prevent or ameliorate inflammatory diseases, or an estrogenic agent that can prevent or ameliorate skin aging. Moreover, this invention relates to the skin external preparation which mix | blended the TNF- (alpha) production inhibitor or the estrogen-like agent.

  There are various causes and onset mechanisms of inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other skin diseases with rough skin. In addition, a tumor necrosis factor (hereinafter referred to as “TNF-α”) produced from macrophages is known.

  TNF-α was found as a factor that necrotizes tumors, but is recently considered to be a cytokine that plays a mediator role not only for tumors but also for regulating the function of normal cells. TNF-α plays an important role in the process from the onset to the end of inflammation, but its continuous and excessive production causes damage to tissues including the skin, and causes systemic fever and cachexia. And cause worsening of inflammation. As such inflammation, for example, chronic inflammatory diseases such as rheumatoid arthritis and osteoarthritis are representative. Therefore, it is important to suppress excessive production of TNF-α in pathological inflammation.

  Various proposals have been made for TNF-α production inhibitors for that purpose, such as perilla extract (see Non-Patent Document 1), Amaryllidaceae alkaloids ricolin and ricolicidinol (see Non-Patent Document 2).

  On the other hand, the cause of skin aging accompanying aging is that the secretion of estrogen, a type of female hormone, decreases. In other words, estrogen is deeply involved in maintaining the health of adult women, and its lack of secretion leads to various medical illnesses, as well as the cause of unwanted skin changes such as skin irritability, reduced elasticity, reduced moisture, etc. It is known to be.

Therefore, a drug containing a substance having an action similar to that of estrogen is transdermally or orally administered to women in menopause after estrogen secretion declines. Examples of such an estrogen-like agent include an extract from the leaves of pentagon (see Patent Document 1), an extract from a plant belonging to the genus Maple (see Patent Document 2), and a plant belonging to the genus Microcos. Extracts from plants (see Patent Document 3), extracts from plants belonging to the genus Sauropus (see Patent Document 4), and the like have been proposed.
JP 2002-226323 A JP 2003-1113068 A JP 2003-128562 A JP 2003-128569 A "Inflammation" 1993, Vol. 13, No. 4, p.337-340 “Pharmaceutical Journal” 2001, Vol.121, No.2, p.167-171

  However, in the above extract, a specific compound that suppresses the production of TNF-α and a specific compound that exhibits the same action as estrogen have not yet been identified, and a further TNF-α production inhibitory action or estrogen A natural TNF-α production inhibitor or estrogen-like agent having a like action is strongly desired, but has a high TNF-alpha production inhibitory action while being excellent in safety and productivity and inexpensive. The demand of consumers for a TNF-α production inhibitor or an estrogen-like agent having a high estrogen-like action is very strong, and there is not yet a satisfactory one yet.

  Then, this invention aims at providing the skin external preparation which mix | blended the novel TNF- (alpha) production inhibitor which uses a TNF- (alpha) production inhibitory substance as an active ingredient, and the TNF- (alpha) production inhibitor, An object of the invention is to provide a novel estrogen-like agent containing an estrogen-like agent as an active ingredient, and a skin external preparation containing the estrogen-like agent.

  In order to solve the above-mentioned problems, the present inventor has conducted extensive research on a TNF-α production inhibitory action or an estrogen-like action among many safe plants, and as a result, an extract from a specific citrus fruit In particular, nobiletin contained therein has a high TNF-α production inhibitory action and estrogen-like action, and by using them as active ingredients, TNF-α production effective for prevention and improvement of inflammatory diseases and the like The present inventors have found that an inhibitor and an external preparation for skin can be obtained, and that an estrogen-like agent and an external preparation for skin effective for prevention and improvement of skin aging can be obtained.

  That is, the TNF-α production inhibitor of the present invention includes nobiletin; an extract from Yawata, an extract from Wenzhou mandarin orange, an extract from grapefruit, an extract from Valencia orange, an extract from Seaquars, and a ponkan 1 extract or two or more citrus extracts selected from the group consisting of an extract of lemon, an extract from lemon, an extract from marcot, and an extract from Shiranui (hereinafter sometimes referred to as “specific citrus extract”). Or a nobiletin purified product obtained by purifying the specific citrus extract (hereinafter sometimes referred to simply as “nobiletin purified product”) as an active ingredient, and the estrogenic agent of the present invention comprises: , Nobiletin, a specific citrus extract or a purified nobiletin product as an active ingredient, and the skin external preparation of the present invention comprises the above T It is characterized by containing a combination of F-alpha production inhibitor and / or estrogenic agent.

  In addition, nobiletin, a specific citrus extract and a nobiletin purified product, which are active ingredients of the TNF-α production inhibitor and estrogen-like agent of the present invention, have a TNF-α production inhibitory action and inhibit an excessive inflammatory reaction. It is effective for prevention or treatment of inflammatory diseases by suppressing, and prevention of skin aging such as formation of wrinkles on skin and decreased elasticity due to decreased estrogen secretion accompanying aging with estrogen-like action. Alternatively, it is not known at all to be effective for improvement, and these are new findings of the present invention.

  According to the TNF-α production inhibitor of the present invention, the inflammation associated with excessively produced TNF-α is effectively prevented by the TNF-α production inhibitory action of nobiletin, the specific citrus extract or the purified nobiletin product. Or it can be improved.

  Further, according to the estrogen-like agent of the present invention, skin aging phenomena such as skin wrinkle formation and reduced elasticity are effectively prevented by the estrogen-like action possessed by nobiletin, a specific citrus extract or a purified nobiletin product. Or it can be improved.

  Furthermore, according to the skin external preparation of the present invention containing the TNF-α production inhibitor and / or the estrogen-like agent, the skin inflammation is suppressed, or the skin is activated from the inside and kept in a youthful state. be able to.

The present invention will be described in detail below.
[TNF-α production inhibitor / estrogenic agent]
The TNF-α production inhibitor and estrogen-like agent of the present invention include nobiletin; an extract from Yawata, an extract from Satsuma mandarin, an extract from grapefruit, an extract from Valencia orange, and an extract from sequercer One or more citrus extracts selected from the group of: an extract from Ponkan, an extract from lemon, an extract from Marcot and an extract from Shiranui; or the citrus extract is purified The obtained nobiletin purified product is contained as an active ingredient.

Here, the “purified product of nobiletin” includes a crude product of nobiletin.
Nobiletin is a kind of polymethoxyflavonoid and can be produced by synthesis. However, it is preferable to use one obtained by extraction and purification from the following citrus fruits (including a crude product). In addition, the synthesis | combining method of nobiletin is not specifically limited, A conventionally well-known method can be utilized.

  As the citrus fruits used as the raw material for nobiletin, it is preferable to use one or more of Hachiman, Satsuma mandarin, grapefruit, Valencia orange, Siquarcer, Ponkan, lemon, Marcot and Shiranui. In this case, nobiletin may be obtained by mixing two or more kinds of extraction raw materials, extracting the mixed extraction raw materials by an extraction process, and purifying the extract by the extraction process. Each extract extracted from the seed starting material by an extraction process may be mixed and the mixture may be purified.

  Moreover, the specific citrus extract which uses the TNF- (alpha) production inhibitor or estrogen-like agent of this invention as an active ingredient is obtained by using the said citrus as an extraction raw material, and the extraction method is not specifically limited.

  In the present invention, the term “citrus extract” refers to an extract obtained from citrus as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product thereof. Both purified products and purified products are included.

The citrus fruits used in the present invention will be described below.
<Yawata>
Yawata (scientific name: Citrus hassaku Hort.) Is an evergreen tree of the mandarin orange family that is cultivated in Wakayama Prefecture. The pericarp is used as an aromatic stomachic medicine for the treatment or prevention of abdominal pain, bloating, constipation and the like, and is also used to calm cough or sputum.

<Wenzhou mandarin orange>
Citrus unshiu (scientific name: C.unshiu Marcov.) Is an evergreen shrub of the mandarin family that is widely cultivated in the warm areas of central and southern parts of Japan, and its pericarp is frequently used in traditional Chinese medicine. Used for the treatment or prevention of gallstones.

<Grapefruit>
Grapefruit (scientific name: C. paradisii) is an American mandarin orange plant.

<Valencia Orange>
Valencia orange (scientific name: C. sinensis cv. Valencia) is a plant of the mandarin orange family and has the highest production in orange and is cultivated all over the world.

<Seek Arthur>
Shiku Arthur (scientific name: C.depressa Hayata) is also known as Hiramemon, a plant of the native Okinawan mandarin.

<Ponkan>
Ponkan (scientific name: Citrus reticulata Blanco) is an evergreen small Takagi of the mandarin orange family and is cultivated in South China, Taiwan, India, Sri Lanka, Philippines, etc. In Japan, it is also cultivated in Kagoshima, Miyazaki, Kochi, etc. Has been.

<Lemon>
Lemon (scientific name: C. limon (Linn.) Burm.) Is a plant of the mandarin family and is known to have a bactericidal and disinfecting action.

<Marcot>
A marcot is a plant of the mandarin family that is a kind of a hybrid of oranges and oranges. Florida in the United States is the birthplace, and in Japan it is cultivated in Nagasaki, Kumamoto, Saga, etc.

<Shiranui>
Shiranui is a plant of the mandarin family that is a hybrid between Kiyomi Orange and Ponkan, and is cultivated in Kumamoto, Aichi, Kagoshima, Ehime, and others. Of the Shiranui, those with a sugar content of 13 degrees or more and a sourness of 1.0% or less are referred to as Decopon (registered trademark).

  Hereinafter, the preferable purification method of nobiletin which uses the said citrus fruits as a raw material is demonstrated.

  Examples of the citrus part that can be used as an extraction raw material include fruits, pericarps, leaves, and the like, and one or more of these can be used as the extraction raw material. It is preferable to use as. As the fruit, it is preferable to use the whole fruit including the skin. As the fruit or pericarp, any of immature fruit (plucked fruit) or pericarp and mature fruit or pericarp may be used. The extraction raw material can be subjected to pretreatment such as cutting, crushing, and drying.

  Examples of the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, and glycerin. A mixed solvent of a hydrophilic organic solvent such as a polyhydric alcohol having 2 to 5 carbon atoms such as water and the like can be used, and it is particularly preferable to use an ethanol aqueous solution having an ethanol concentration of 0 to 40% by volume. In the ethanol aqueous solution, the ethanol concentration can be appropriately changed within the range of 0 to 40% by volume.

  The ethanol aqueous solution having an ethanol concentration of 0% by volume means water. The water 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 the treatment applied to water include purification, heating, sterilization, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like.

  The extraction treatment is not particularly limited as long as nobiletin contained in the citrus can be extracted, and can be performed according to a conventional method. For example, by immersing the extraction raw material in an extraction solvent 1 to 20 times the mass of the extraction raw material (mass ratio), extracting for 0.1 to 3 hours at room temperature or under reflux heating, and then filtering to remove the residue, An extract containing nobiletin can be obtained. The obtained extract may be concentrated according to a conventional method. The obtained extract may be treated with activated carbon for the purpose of decolorization and the like. The extract obtained here can be a “citrus extract” in the present invention.

  When the extraction raw material is extracted with an ethanol aqueous solution having an ethanol concentration of 0 to 40% by volume, the extraction raw material is preferably extracted with hot water before or after the treatment. Thereby, the nobiletin yield from an extraction raw material can be improved.

  The temperature of the hot water is usually 40 to 100 ° C, preferably 60 to 90 ° C. When extracting with hot water before extraction with an aqueous ethanol solution with an ethanol concentration of 0 to 40% by volume, the extraction raw material is extracted with hot water and filtered to obtain an extract containing nobiletin, and then the residue Is extracted with an aqueous ethanol solution having an ethanol concentration of 0 to 40% by volume and filtered to obtain an extract containing nobiletin, and both extracts are mixed. In the case of extracting with hot water after extracting with an ethanol aqueous solution having an ethanol concentration of 0 to 40% by volume, the raw material for extraction is extracted with an aqueous ethanol solution having an ethanol concentration of 0 to 40% by volume and filtered to contain nobiletin. After obtaining the extract, the residue is extracted with hot water and filtered to obtain an extract containing nobiletin, and the two extracts are mixed.

  An extract obtained by the above method, particularly an extract obtained using an aqueous ethanol solution having an ethanol concentration of 0 to 40% by volume or a mixture of the extract and an extract obtained using hot water, It is preferable that the nobiletin adsorbed on the porous adsorption resin is eluted after passing through the porous adsorption resin to adsorb nobiletin onto the porous adsorption resin.

  The porous adsorption resin is not particularly limited as long as it can adsorb nobiletin. Examples of the porous adsorption resin include synthetic adsorption resin, activated carbon, and the like. Among these, one type or two or more types can be used, and it is preferable to use a synthetic adsorption resin.

  The extract is preferably passed through a porous adsorption resin after being concentrated. Thereby, the liquid passing time to porous adsorption resin can be shortened. Concentration can be carried out according to a conventional method such as vacuum concentration, and is usually concentrated 3 to 10 times, preferably 4 to 6 times.

  The space velocity of the extract is usually 0.1 to 4, preferably 0.8 to 1.5. In addition, the space velocity (Space Velocity; SV) = 1 sets the speed | rate which passes 1 time of resin amount per hour.

  The extraction liquid is passed through the porous adsorption resin, and the liquid flowing out of the porous adsorption resin may contain nobiletin that has not been adsorbed on the porous adsorption resin. It is preferable to collect it.

  Examples of the solvent capable of eluting nobiletin adsorbed on the porous adsorption resin include an ethanol aqueous solution, a methanol aqueous solution, and an isopropanol aqueous solution. Among these, it is preferable to use an ethanol aqueous solution. The ethanol concentration of the aqueous ethanol solution used for elution of nobiletin is usually 55 to 95% by volume, preferably 60 to 90% by volume.

  Before eluting nobiletin adsorbed on the porous adsorption resin, it is preferable to wash the porous adsorption resin and elute components other than nobiletin adsorbed on the porous adsorption resin. Examples of the cleaning liquid for the porous adsorption resin include water, an aqueous ethanol solution, an aqueous methanol solution, an aqueous isopropanol solution, and an aqueous sodium hydroxide solution. Of these, an aqueous ethanol solution is preferably used. The ethanol concentration of the aqueous ethanol solution used for washing the porous adsorption resin is usually 10 to 50% by volume, preferably 30 to 50% by volume.

  A crude nobiletin product can be obtained by drying the eluate from the porous adsorption resin according to a conventional method such as drying under reduced pressure. A nobiletin crude product can also be obtained by mixing the porous adsorbent resin washing solution with the eluate from the porous adsorbent resin and drying the mixed solution.

  The nobiletin crude purified product usually contains polymethoxyflavonoids other than nobiletin contained in the extraction raw material (eg, tangeretin, pentamethoxyflavone, tetramethoxyflavone, heptamethoxyflavone, etc.).

  The extract from the citrus fruits has a high TNF-α production inhibitory action and an estrogen-like action, and in particular, the nobiletin crude product has an extremely high TNF-α production inhibitory action and an estrogen-like action. It can be formulated as it is or with other active substances and molding aids according to a conventional method and provided as an arbitrary dosage form such as powder, granule, tablet, etc. It can be used as an injection, a suppository or the like.

  When formulating the obtained citrus extract or crudely purified nobiletin product, a pharmaceutically acceptable carrier such as dextrin or cyclodextrin may be added in order to facilitate storage and handling. it can.

  In addition, the TNF-α production inhibitor and / or estrogen-like agent of the present invention is an active ingredient by blending with other natural extracts having a TNF-α production inhibitory effect and / or an estrogen-like action, if necessary. Can be used as As an example of the blending ratio in this case, nobiletin: natural extract = 1: 0.01-1: 5 (mass ratio) can be mentioned.

  The TNF-α production inhibitor of the present invention can suppress the production of TNF-α through the TNF-α production inhibitory action possessed by the active ingredient nobiletin, the specific citrus extract or the nobiletin purified product. Therefore, the TNF-α production inhibitor of the present invention is useful for the prevention or improvement of inflammatory diseases involving excessively produced TNF-α, such as contact dermatitis (rash), psoriasis, pemphigus vulgaris It is.

  Further, the estrogenic agent in the present invention is an aging phenomenon of skin typified by the formation of wrinkles on the skin and reduced elasticity through the estrogenic action of nobiletin, a specific citrus extract or purified nobiletin, which is an active ingredient. It is useful for prevention or treatment.

  Further, in addition to these uses, the TNF-α production inhibitor of the present invention can be used for all uses meaningful in suppressing the production of TNF-α, and the estrogenic agent of the present invention. Can be used for all purposes that are meaningful for exerting the same action as estrogen.

[Skin external preparation]
The external preparation for skin of the present invention contains the above-described TNF-α production inhibitor and / or estrogen-like agent (including nobiletin, a specific citrus extract or a purified nobiletin product) as an active ingredient.

  Nobiletin, a specific citrus extract or a nobiletin purified product has a TNF-α production inhibitory action and / or an estrogen-like action, and is effective in treating inflammatory diseases such as contact dermatitis (rash), psoriasis and pemphigus vulgaris. It is possible to prevent and / or improve, prevent and / or improve skin aging, and is excellent in usability and safety when applied to the outer skin. Is preferred. In this case, nobiletin, a specific citrus extract or a purified nobiletin product may be blended as it is, or a TNF-α production inhibitor or an estrogenic agent formulated from nobiletin, a specific citrus extract or a purified nobiletin product. May be.

  Here, “skin external preparation” means various drugs applied to the skin, and includes, for example, cosmetics, quasi drugs, pharmaceuticals and the like.

  Although the form of the skin external preparation of this invention is not specifically limited, As an example, arbitrary forms, such as ointment, cream, emulsion, lotion, pack, jelly, lip balm, lipstick, bathing agent, are possible.

  The compounding amount of the active ingredient in the external preparation for skin can be appropriately adjusted in consideration of the purpose of use, sex, symptoms, etc., but is about 0.0001 to 10% by mass in terms of nobiletin.

  The external preparation for skin includes the above-described TNF-α production inhibitor and / or estrogen-like agent, and any physiologically active substance or auxiliary agent according to the application, for example, astringent, bactericidal / antibacterial agent, whitening agent, ultraviolet absorber If a moisturizer, cell activator, anti-inflammatory / antiallergic agent, antioxidant / active oxygen scavenger, etc. are used in combination, the product becomes more general. In addition, this may lead to a synergistic effect with other active ingredients used in combination, which is more excellent than normally expected.

  The TNF-α production inhibitor and estrogen-like agent of the present invention are preferably applied to humans, but may be applied to animals other than humans as long as the desired action and effect are exhibited. You can also For example, the TNF-α production inhibitor or estrogen-like agent of the present invention can be added to pet foods such as dog foods and cat foods and livestock feed to prevent / treat inflammatory diseases of pet animals and livestock.

  Hereinafter, although a manufacture example, a test example, and a compounding example are shown and this invention is demonstrated concretely, this invention is not limited to each following example at all.

[Production Example 1] Production of nobiletin crude product 50 L of 30% by volume ethanol (mass ratio of water and ethanol 7: 3) as an extraction solvent is added to 2500 kg of Ponkan fruit (whole fruit), and a reflux extractor. And heated at 80 ° C. for 2 hours and filtered while hot. The obtained extract was concentrated under reduced pressure and further dried to obtain an extract (hereinafter referred to as “nobiletin crude product A”). The nobiletin content in the obtained crude nobiletin product A was 0.08% by mass. In addition, nobiletin was identified by measuring by liquid chromatography (HPLC) (carrier: 50% acetonitrile (0.1% phosphoric acid), detection wavelength: 334 nm, column: WAKOSIL-II 5C18HG (Wako Pure Chemical Industries, Ltd.) Manufactured by Kogyo Co., Ltd.)).

  The crude nobiletin product A was treated with column chromatography (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation) and then eluted with 70% by mass aqueous methanol to obtain a fraction. The obtained fraction was treated with activated carbon, further concentrated under reduced pressure, and dried to obtain 1.3 kg of a crude nobiletin product (hereinafter referred to as “nobiletin crude product B”). In addition, the nobiletin content in the crude nobiletin purified product B was 45.6% by mass.

[Test Example 1] TNF-α production inhibitory action test The crude nobiletin product obtained in Production Example 1 was tested for TNF-α production inhibitory action by the following test method.

Mouse macrophage cells (RAW264.7 cells; manufactured by Dainippon Pharmaceutical Co., Ltd.) were pre-cultured in Dulbecco's MEM (Nissui Pharmaceutical Co., Ltd.) medium supplemented with 10% fetal bovine serum (FBS), and then cells were collected from a cell scraper. The cells were seeded in a 96-well microplate at a density of 1 × 10 ⁵ / cells / 100 μL per well and pre-cultured at 37 ° C. under 5% CO ₂ for 4 hours.

Next, the medium in the 96-well plate was discarded, and 100 μL of a test sample previously dissolved in a culture solution containing 2% DMSO was added, and then lipopolysaccharide (LPS, final concentration 1 μg / mL, E. coli 0111; B4, DIFCO 100 μL of Laboratories) was added to stimulate the cells. Thereafter, the amount of TNF-α produced by culturing for 24 hours under the conditions of 37 ° C. and 5% CO ₂ was measured using the following sandwich ELISA method.

  Rat anti-mouse TNF-α monoclonal antibody (Endogen Inc.), which is the primary antibody, was dissolved in 50 mM sodium carbonate buffer (pH 9.6) to 2.5 μg / mL, and 100 μL of the solution was added to a 96-well microplate. And coated overnight at 4 ° C. Next, each well was washed with a washing solution (phosphate buffer containing 0.05% Tween 20) and then blocked with a phosphate buffer containing 1% BSA.

  After washing each well with a washing solution, the culture supernatant was diluted with a test medium, and 100 μL thereof was added to each well and incubated at 37 ° C. for 60 minutes. After washing each well, 100 μL of rabbit anti-mouse TNF-α polyclonal antibody (manufactured by Endogen Inc.) dissolved in a phosphate buffer containing 0.3% BSA at a concentration of 2.5 μg / mL as a secondary antibody Was added, incubated at 37 ° C. for 60 minutes, and then washed.

  Next, 100 μL of alkaline phosphatase-labeled anti-rabbit IgG antibody (CHEMICON Inc.) diluted 500-fold was added and incubated at 37 ° C. for 60 minutes. After washing each well, 150 μL of a substrate solution prepared by dissolving 50 mg of p-nitrophenyl phosphate in 100 mL of color developing buffer (20 mM magnesium sulfate-containing Tris-HCl buffer, pH 8.0) was added to the well, and the mixture was incubated at 37 ° C. The color was developed by carrying out an enzyme reaction for 20 to 30 minutes, and the absorbance at 405 nm was measured. From the standard curve prepared from the standard solution of recombinant mouse TNF-α (manufactured by Endogen Inc.), TNF-α in the culture supernatant was used. The amount (pg / mL) was determined. The TNF-α production inhibition rate of the test sample was calculated based on the following formula.

TNF-α production inhibition rate (%) = {(B−A) / B} × 100
In the above formula, “A” represents “a TNF-α amount when a sample is added” and “B” represents a “a TNF-α amount when a sample is not added”.

  The TNF-α production inhibition rate is a value calculated when a sample of 50 ppm (μg / mL) is added. The results are shown in Table 1.

[Table 1]
Sample TNF-α production inhibition rate (%)
Nobiletin crude product A (0.08%) 8.3
Nobiletin crude product B (45.6%) 98.5

  From the results shown in Table 1, an excellent TNF-α production inhibitory action was observed in the nobiletin crude product, particularly the nobiletin crude product B. Thus, since the inhibition rate of TNF-α production increases with an increase in the content of nobiletin in the crude purified nobiletin product, it is considered that nobiletin has an excellent TNF-α production inhibitory action.

[Test Example 2] Estrogen-like action test The method of Thomas et al. (In Vitro Cell. Dev. Biol. 28A, 595-602, 1992) for examining the effect of the crude nobiletin product of Preparation Example 1 on the proliferation of estrogen-dependent cells. In accordance with the above, the estrogenic action was tested as follows.

MCF-7 cells derived from human breast cancer are cultured in a 80 cm ² flask until confluent, and the MCF-7 cells are collected by trypsin treatment. 10% FBS (activated charcoal), 1% NEAA and 1 mM sodium pyruvate are added. It was prepared to 3 × 10 ⁴ cells / mL using a MEM medium containing no phenol red (hereinafter referred to as “MEM medium”).

The prepared MCF-7 cells were seeded at 0.9 mL each in a 24-well plate, and cultured at 37 ° C. under 5% CO ₂ -95% air to establish this. Six hours later (Day 0), 100 μL of a sample solution prepared in MEM medium to a concentration 10 times the final concentration (31.25 ppm; μg / mL) was added to the plate, and the culture was continued.

  On the sixth day from the start of the culture, the medium was replaced with a MEM medium containing 0.97 mmol / L MTT, and cultured for 2 hours. Then, the medium was replaced with isopropanol, and blue formazan produced in the cells was extracted. About isopropanol containing the extracted blue formazan, the light absorbency of 570 nm with the absorption maximum of blue formazan was measured.

  In order to correct the influence of adherent cells, the absorbance at 650 nm was also measured at the same time, and the difference between the two absorbances was taken as a value proportional to the amount of blue formazan produced (the absorbance in the formula below is this corrected absorbance). . As a positive control, 0.02 ppm ethinylestradiol was used. The strength of the estrogen-like action (estrogen-dependent growth action) was calculated by the following equation, assuming that the absorbance when no sample was added was 100%.

Estrogen-like action (%) = A / B × 100
In the above formula, “A” represents “absorbance when the sample is added” and “B” represents “absorbance when the sample is not added”.
The results of this test are shown in Table 2.

[Table 2]
Sample Estrogen-like action rate (%)
Nobiletin crude product A (0.08%) 105
Nobiletin crude product B (45.6%) 136

  From the results of Table 2, an excellent estrogen-like action was observed in the nobiletin crude product, particularly nobiletin crude product B. Thus, since the estrogen-like action rate is increased with an increase in the content of nobiletin in the crude nobiletin product, it is considered that nobiletin has an excellent estrogen-like action.

[Test Example 3] Razor loss prevention effect test A lotion-like coating liquid a containing the nobiletin crude purified product B obtained in Production Example 1 and a coating solution having the same composition as the coating liquid a except that it contains no nobiletin crude purified product b (control) were prepared and tested for their anti-razor losing effect. Losing a razor is a symptom in which the skin becomes red, tingling, swelled, feverish, or itchy after shaving the hair such as the beard. The skin after shaving the hair such as the beard with a razor This is caused by the infection of bacteria from the cut. The composition of each coating solution is as shown in Table 3 below.

[Table 3]
Coating liquid a Coating liquid b
Nobiletin crude product B (Production Example 1) 1.0 g None 1,3-butylene glycol 5.0 g 5.0 g
Glycerin 1.0g 1.0g
Ethanol 6.0g 6.0g
Nonionic surfactant 0.5g 0.5g
Purified water balance remainder
(The total amount of both coating liquid a and coating liquid b is 100 g)

  Twenty male subjects losing razors are divided into two groups of 10 each. The application liquid a is applied to one group and the application liquid b is applied to the skin immediately after shaving, and the following criteria are used. The loss prevention (improvement) effect was evaluated.

  If the razor loss disappears, it is “effective”, if the razor loss is weak, “effective”, if the razor loss is slightly weak, “somewhat effective”, no change in the razor loss In this case, it is determined as “invalid”, “A” when less than 20% of the subjects are determined as “invalid”, “B” when 20% or more and less than 50%, and 50% or more and 80% When it was less than 80%, it was evaluated as “C”, and when it was 80% or more, it was evaluated as “D”.

  As a result, the razor loss prevention effect of the coating liquid a was evaluated as “A”, and the razor loss prevention effect of the coating liquid b was evaluated as “D”. In addition, while performing the said test and asking the test subject about the degree of irritation (burning feeling) to the skin, all the test subjects answered that they did not feel any irritation.

  From this result, it was shown that the nobiletin crude purified product has an action to prevent razor loss, that is, an anti-inflammatory action.

[Test Example 4] Skin Roughness Improvement Action Test A milky lotion (hereinafter referred to as “Example milky lotion”) containing the nobiletin crude purified product B obtained in Production Example 1 was prepared according to a conventional method. The composition of the example emulsion is shown below.

Nobiletin crude product B (Production Example 1) 1.0 g
Cetyl alcohol 0.5g
Beeswax 2.0g
Oleic acid polyoxyethylene sorbitan (10E.0.) 1.0g
1.0g glyceryl monostearate
Hyaluronic acid 0.1g
Propylene glycol 5.0g
Ethanol 3.0g
Methyl paraoxybenzoate 0.3g
Perfume 0.03g
Purified water remainder (total amount is 100 g)

  The following evaluation test was performed on the comparative example emulsion having the same composition as the example emulsion except that the example emulsion and the crude purified product of nobiletin were not included.

  Of many women aged 23 to 42, the crevice / cutaneous skin has disappeared, and a wide range of horny skin is turned over (the score shown in Table 4 is 1), or the cutaneous / cutaneous skin is unclear and the keratin is partially Twenty persons judged to be turned up (the score shown in Table 4 is 2) were selected as subjects, and each subject was given the example latex on the right half of the face and the comparative example latex on the left half. Each morning and evening was applied for 30 days.

Judgment 1: After finishing the rough skin application test, a replica of the face was taken by a replica method using Silfro (manufactured by FLEXICL DEVELOPMENTS LTD). The skin condition was determined according to the evaluation criteria shown. Table 5 shows the determination results.

[Table 4]
Score evaluation
1 The skin groove and hill disappear, and a wide range of skin is turned over (rough skin condition)
2 The skin groove and barn are not clear. The keratin is partially turned over (rough skin)
3 Skin grooves and skin hills are observed, but flat (normal skin)
4 Clear skin grooves and hills (relatively beautiful skin)
5 The skin groove and hill are very clear and well-equipped (beautiful skin)

[Table 5]
Example before starting point test Comparative example of emulsion application part Latex application part
1 9 people 0 people 8 people 2 10 people 0 people 8 people 3 1 people 7 people 3 people 4 0 people 11 people 1 people 5 0 people 2 people 0 people

  As shown in Table 5, in the region where the example emulsion was applied, rough skin (skin aging) was remarkably improved as compared with the region where the comparative example emulsion was applied.

Judgment 2: All subjects were asked about superiority or inferiority when the Example emulsion and the Comparative Example emulsion were compared with each other regarding the sensory evaluation usability and the effect on the skin. Table 6 shows the results of the responses.

[Table 6]
Evaluation item Example Comparative emulsion is good Comparative emulsion is good Superiority and familiarity to skin 16 2 2 2 Moist feeling 17 1 1 2 Spread to skin 14 2 2 4 Satisfaction of rough skin improvement 17 1 Name 2 Satisfaction with skin tone improvement 13 2 5 Improvement in number and depth of wrinkles 18 1 1

  From the results shown in Table 6, the same effects as in the above-described determination 1 and excellent usability were confirmed by sensory evaluation.

  Based on the results of Judgment 1 and Judgment 2, the topical skin preparation formulated with a nobiletin crude product has an anti-aging / improving effect on skin aging (roughness-improving action), and is excellent in usability and safety when applied to skin. It was confirmed that

[Formulation Example 1]
An emulsion having the following composition was produced by a conventional method.
Nobiletin crude product B (Production Example 1) 1 g
Jojoba oil 4g
2g olive oil
Squalane 2g
Cetanol 2g
2g glyceryl monostearate
Polyoxyethylene cetyl ether (20E.0.) 2.5g
Oleic acid polyoxyethylene sorbitan (20E.0.) 2g
1,3-butylene glycol 3g
Methyl paraoxybenzoate 0.15g
Fragrance 0.05g
Purified water remainder (total amount is 100 g)

[Formulation Example 2]
A lotion having the following composition was produced by a conventional method.
Nobiletin crude product B (Production Example 1) 2 g
Glycerin 3g
1,3-butylene glycol 3g
Oleic acid polyoxyethylene sorbitan (20E.0.) 0.5g
Methyl paraoxybenzoate 0.15g
Citric acid 0.1g
Sodium citrate 0.1g
Fragrance 0.05g
Purified water remainder (total amount is 100 g)

[Composition Example 3]
A cream having the following composition was produced by a conventional method.
Nobiletin crude product B (Production Example 1) 1 g
Liquid paraffin 5g
Salami beeswax 4g
Setanol 3g
Squalane 10g
Lanolin 2g
Stearic acid 1g
Oleic acid polyoxyethylene sorbitan (20E.0.) 1.5g
3g glyceryl monostearate
1,3-butylene glycol 6g
1.5 g of methyl paraoxybenzoate
Fragrance 0.1g
Purified water remainder (total amount is 100 g)

[Composition Example 4]
A pack having the following composition was produced by a conventional method.
Nobiletin crude product B (Production Example 1) 5 g
Polyvinyl alcohol 15g
Polyethylene glycol 3g
7g of propylene glycol
Ethanol 10g
0.05 g ethyl paraoxybenzoate
Fragrance 0.05g
Purified water remainder (total amount is 100 g)

  The external preparations for skin according to Formulation Examples 1 to 4 all showed good storage stability.

The TNF-α production inhibitor according to the present invention is used for the prevention or treatment of inflammatory diseases involving excessive TNF-α production, and the estrogenic agent according to the present invention is used for the formation of skin wrinkles and elasticity. It can greatly contribute to the prevention or treatment of the skin aging phenomenon represented by a decrease.

Claims (8)

  A TNF-α production inhibitor comprising nobiletin as an active ingredient.   Extract from Yawata, Extract from Satsuma mandarin, Extract from grapefruit, Extract from Valencia orange, Extract from Sequarcer, Extract from Ponkan, Extract from Lemon, Extract from Marcot And a TNF-α production inhibitor comprising one or more citrus extracts selected from the group of extracts from Shiranui as an active ingredient.   Extract from Yawata, Extract from Satsuma mandarin, Extract from grapefruit, Extract from Valencia orange, Extract from Sequarcer, Extract from Ponkan, Extract from Lemon, Extract from Marcot And a nobiletin purified product obtained by purifying one or more citrus extracts selected from the group of extracts from Shiranui as an active ingredient, a TNF-α production inhibitor.   An estrogenic agent characterized by containing nobiletin as an active ingredient.   Extract from Yawata, Extract from Satsuma mandarin, Extract from grapefruit, Extract from Valencia orange, Extract from Sequarcer, Extract from Ponkan, Extract from Lemon, Extract from Marcot And an estrogen-like agent comprising one or more citrus extracts selected from the group of extracts from Shiranui as an active ingredient.   Extract from Yawata, Extract from Satsuma mandarin, Extract from grapefruit, Extract from Valencia orange, Extract from Sequarcer, Extract from Ponkan, Extract from Lemon, Extract from Marcot And a nobiletin purified product obtained by purifying one or more citrus extracts selected from the group of extracts from Shiranui as an active ingredient.   A skin external preparation comprising the TNF-α production inhibitor according to any one of claims 1 to 3. An external preparation for skin, comprising the estrogen-like agent according to any one of claims 4 to 6.