JP5947061B2 - SREBP1 inhibitor - Google Patents

Description

  The present invention relates to a SREBP1 inhibitor and an agent for preventing or improving seborrheic alopecia.

  SREBP (sterol regulatory element-binding protein) is known as a transcription factor that regulates the synthesis of cholesterol and fatty acids, and is bound to the endoplasmic reticulum membrane as an inactive precursor type. Under certain conditions such as sterol depletion, SREBP can be cleaved by specific proteases, releasing the active nuclear translocation domain and binding to SRE (Sterol responsive element), which activates the expression of the target gene. (Non-Patent Document 1).

  In SREBP, three isoforms called SREBP1a, SREBP1c and SREBP2 have been identified. SREBP1a and SREBP1c are splicing variants with different transcription initiation sites, and they are composed of the same gene (Srebf1) (Non-patent Document 2). These three isoforms have different biological functions, SREBP1a and SREBP2 activate cholesterol biosynthesis in particular, while SREBP1c mainly enhances transcription of genes necessary for fatty acid biosynthesis. Is known (Non-patent Document 3).

  Recent studies have revealed that SREBP1c is expressed in sebaceous gland cells in the human scalp (Non-Patent Document 4), and it has become clear that SREBP1c plays an important role in scalp sebaceous gland differentiation and lipid synthesis. Yes. Furthermore, the activation of sebaceous gland cells by male hormones increases the expression of SREBP1 and the expression of enzymes involved in SREBP1-dependent fatty acid and cholesterol synthesis, so that excessively secreted sebum causes hair growth. It has also been shown to cause obstruction and inflammation (Non-Patent Document 5). From these facts, suppression of SREBP1 is thought to be effective in preventing or improving scalp troubles such as seborrheic alopecia through suppression of excessive lipid production of sebaceous glands by male hormones.

  In addition, since SREBP1 is deeply related to insulin signal (Non-patent Document 6), it has been pointed out that it is associated with diabetes, and reports that suppression of SREBP1 expression in liver tissue contributes to improvement of insulin resistance (Non-patent document 6). There is a report (Non-patent Document 8) that insulin secretion ability is impaired when SREBP1 expression in pancreatic β cells is enhanced.

  So far, although SREBP1 expression inhibitory action has been reported for fats and oils containing highly unsaturated fatty acids such as fish oil (Non-Patent Document 9), these are easily oxidized, such as generation of off-flavors due to oxidation and deterioration of flavor. Problems are likely to occur.

  On the other hand, rice bran is the part other than endosperm that is removed when white rice is refined. It contains various nutrients such as dietary fiber (cellulose), proteins, lipids, vitamins, minerals, γ-oryzanol, phytic acid, free γ- Functional components such as aminobutyric acid are included. The extract of rice bran has been reported to have anti-obesity action, adiponectin secretion promoting action, anti-cholesterol action, anti-cancer action, alcoholic liver disease improving action, etc. (Patent Documents 1 and 2, Non-Patent Documents 10 to 10) 12) The relationship between rice bran and SREBP1 expression is not known at all.

JP 2006-257064 A JP 2005-68132 A

Hua, X., J Biol Chem., 271; 10379-84 (1996) Shimomura, I., J Clin Invest., 99; 838-45 (1997) Horton, J. D., J Clin Invest., 109; 1125-31 (2002) Harrison, W. J., J Invest Dermal., 127; 1309-17 (2007) Rosignoli, C., Exp Dermatol., 12; 480-9 (2003) Shimomura, I., Mol Cell, 6; 77-86 (2000) Muraoka, T., Metabolism, 60; 617-628 (2011) Iwasaki, Y., BBRC, 378; 545-550 (2009) Sekiya, M., Hepatology, 38; 1529-39 (2003) Chen, CW., J Nutr., 136; 1472-6 (2006) Ullah, A., Carcinogenesis, 11; 2219-22 (1990) Oh, CH., J Med Food, 6; 115-21 (2003)

  The present invention is excellent in safety, and is incorporated into a drug, quasi-drug, food, or the like that can exert an effect of preventing or improving scalp trouble accompanied by sebum lipid abnormality such as suppression of SREBP1 and seborrheic alopecia Related to providing materials.

As a result of studying various natural substances, the present inventors have demonstrated that SREBP1 expression is excellent in triterpene alcohol typified by cycloartenol, and the rice bran hydrolyzate rich in the triterpene alcohol or the triterpene alcohol Was found to be useful as a SREBP1 inhibitor, a preventive or ameliorating agent for seborrheic alopecia.
That is, the present invention relates to the following 1) to 10).
1) SREBP1 inhibitor containing rice bran hydrolyzate as an active ingredient.
2) The SREBP1 inhibitor according to 1) above, wherein the rice bran hydrolyzate contains at least a cycloartane type triterpene or a derivative thereof.
3) The SREBP1 inhibitor according to 2) above, wherein the rice bran hydrolyzate further contains β-sitosterol and / or campesterol.
4) SREBP1 inhibitor of said 1) -3) whose rice bran hydrolyzate contains 1-100 mass% of cycloartenol or its derivative (s).
5) SREBP1 inhibitor containing cycloartane type triterpene or a derivative thereof as an active ingredient.
6) The SREBP1 inhibitor according to 5) above, wherein the cycloartane-type triterpene is cycloartenol and / or 24-methylenecycloartanol.
7) A preventive or ameliorating agent for seborrheic alopecia comprising a rice bran hydrolyzate as an active ingredient.
8) The agent for preventing or improving seborrheic alopecia according to 7) above, wherein the rice bran hydrolyzate contains cycloartane type triterpene or a derivative thereof.
9) The agent for preventing or improving seborrheic alopecia according to 8) above, wherein the rice bran hydrolyzate further contains β-sitosterol and / or campesterol.
10) The agent for preventing or improving seborrheic alopecia according to 7) to 9) above, wherein the rice bran hydrolyzate contains 1 to 100% by mass of cycloartenol or a derivative thereof.
11) A preventive or ameliorating agent for seborrheic alopecia comprising cycloartane type triterpene or a derivative thereof as an active ingredient.
12) The agent for preventing or improving seborrheic alopecia according to 11) above, wherein the cycloartane-type triterpene is cycloartenol and / or 24-methylenecycloartanol.

  According to the SREBP1 inhibitor and the agent for preventing or improving seborrheic alopecia of the present invention, it is possible to safely prevent or improve scalp troubles associated with sebum lipid abnormalities such as seborrheic alopecia.

The graph which shows the expression change of SREBP1 gene by rice bran hydrolyzate intake. The graph which shows the expression change of SREBP1 gene by the sterol group addition contained in a rice bran hydrolyzate.

Brown rice consists of seed coat, pericarp, endosperm and germ, and there is a paste layer on the outer layer of the endosperm tissue. When brown rice is refined, depending on the degree, even the paste layer is removed as rice bran (ginger).
As the rice bran in the present invention, a mixture of parts other than endosperm (seed coat, pericarp, starch layer and germ) removed when white rice is refined may be used, or a separated one may be used. There are no particular limitations on the classification and type of rice used as a raw material for rice bran, and for example, any of japonica, indica, and jabanica rice, sticky rice, red rice, purple black rice, and the like can be used.

  As the rice bran hydrolyzate of the present invention, the rice bran as it is or after being dried and freeze-dried and then appropriately pulverized, or these extracts (rice bran extract) hydrolyzed with enzymes, acids, alkalis, etc. Is mentioned. Here, the rice bran extract includes rice bran oil and the like.

  Extraction to obtain a rice bran extract is performed by using a distillation method such as steam distillation in addition to solvent extraction performed by impregnation in a solvent at room temperature or in a heated state, or using extraction equipment such as a Soxhlet extractor. Or a supercritical extraction method in which carbon dioxide gas is put into a supercritical state, or a pressing method for obtaining an extract by pressing. For extraction, for example, 1 to 50 parts by mass of a solvent is used per 1 part by mass of rice bran, and it is preferably immersed or heated to reflux at room temperature (25 ° C.) to 100 ° C. for several hours to several weeks.

  For example, rice bran oil is a vegetable oil extracted from rice bran and rice germ as raw materials. As an extraction method, for example, rice bran is compressed into a pellet and then extracted. Specifically, it is obtained by adding 10 parts by mass of lyophilized rice bran with respect to 100 parts by mass of the solvent, stirring and extracting for several hours, removing solids by filtration, and then removing the solvent by distillation. . Moreover, you may use the deoxidation foots byproduced in the deoxidation process which is a rice bran oil manufacturing process, the refined product and coarse refined product of gamma-oryzanol extracted from the rice bran.

  Examples of the extraction solvent used in the solvent extraction include alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, and butylene glycol; ketones such as acetone and methyl ethyl ketone; Esters such as ethyl acetate; Chain and cyclic ethers such as tetrahydrofuran and diethyl ether; Polyethers such as polyethylene glycol; Halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; Hexane, cyclohexane and petroleum ether Hydrocarbons; aromatic hydrocarbons such as benzene and toluene; pyridines; supercritical carbon dioxide; oils and fats, waxes, and other oils. These can be used alone or in combination of two or more. It is also possible to carry out repeatedly changed. Among these, it is preferable to use fat-soluble solvents such as hydrocarbons, and it is particularly preferable to use hexane.

  Examples of the means for separating and purifying the extract include degumming treatment, activated carbon treatment, liquid-liquid distribution, column chromatography, liquid chromatography, gel filtration, and precision distillation.

  The hydrolysis treatment can be performed using an acid, an alkali, or an enzyme, and among these, an alkali decomposition treatment is preferable from the viewpoint of the uniformity of the product.

Examples of the hydrolysis treatment with an acid include a method in which hydrochloric acid, sulfuric acid, or the like is used, the pH of the treatment liquid is usually adjusted to pH 0 to 1, and the reaction is performed at 25 to 200 ° C. for 0.5 to 50 hours.
As the hydrolysis treatment with alkali, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate or the like is used, and the pH of the treatment liquid is adjusted to normal pH 12 to 14 at 25 to 200 ° C. for 0.5 to 50 hours. The method of making it react is mentioned.

Examples of the enzyme used in the hydrolysis with an enzyme include lipase, cholesterol esterase, oryzanol esterase and the like.
Examples of the hydrolysis treatment with an enzyme include a method of reacting rice bran or rice bran extract with an appropriate amount of the enzyme for 0.5 to 50 hours under conditions near the optimum pH and temperature of the enzyme. .

The rice bran hydrolyzate of the present invention can be produced by the method described above. More specifically, for example, it is described in JP-B-55-2440, JP-A-2006-27364, JP-A-2006-257064. Method. Among these, rice bran described in JP-A-2006-257064 is dissolved by heating with ethanol (80 ° C., 1 hour), concentrated and dissolved by adding hexane, and further, sulfuric acid is added to adjust the pH to 3, followed by filtration. The filtrate is preferably subjected to reflux extraction with ethanol and sodium hydroxide (80 ° C., 1 hour), neutralized by adding hydrochloric acid to the standing supernatant, and concentrated to dryness.
Commercial products such as “Olisa Triterpenoid-P” (manufactured by Oriza Oil Co., Ltd.) can also be used.

  The rice bran hydrolyzate of the present invention includes plant sterols such as β-sitosterol and campesterol in addition to triterpene alcohol or derivatives thereof. Here, as the triterpene alcohol or a derivative thereof, a lupine type triterpene or a derivative thereof, a cucurbitan type triterpene or a derivative thereof, or a cycloartane type triterpene or a derivative thereof can be given. Of these, cycloartane type triterpenes such as cycloartenol, 24-methylenecycloartanol, cyclobranol, cycloartanol, 9,19-cyclolanostane-23,25-dien-3-ol, and derivatives thereof are included. The thing containing a cycloartenol or its derivative (s) is more preferable. Examples of these derivatives include ester forms such as fatty acid esters, ferulic acid esters and cinnamic acid esters, and glycosides such as saponins.

  Furthermore, the rice bran hydrolyzate of the present invention preferably contains 0.01% by mass or more, more preferably 0.1 to 100% by mass, and more preferably 1 to 90% by mass of triterpene alcohol or a derivative thereof. It is more preferable that 10 to 70% by mass is contained. The cycloartane type triterpene or derivative thereof is preferably contained in an amount of 0.01% by mass or more, more preferably 0.1 to 100% by mass, more preferably 1 to 90% by mass, More preferably, the content is 70% by mass. Further, cycloartenol or a derivative thereof is preferably contained in an amount of 0.01% by mass or more, more preferably 0.1 to 100% by mass, more preferably 1 to 90% by mass, It is preferably contained in an amount of 10 to 70% by mass, more preferably 20 to 50% by mass.

  More specifically, as the rice bran hydrolyzate of the present invention, cycloartenol is 10% by mass to 30% by mass, preferably 15% by mass to 25% by mass, more preferably 18% by mass to 22% by mass, 24- Methylenecycloartanol is 25% by mass to 45% by mass, preferably 30% by mass to 40% by mass, more preferably 32% by mass to 36% by mass, and β-sitosterol is 3% by mass to 20% by mass, preferably 7% by mass. % To 16% by weight, more preferably 10% to 14% by weight, campesterol 5% to 25% by weight, preferably 10% to 20% by weight, more preferably 13% to 17% by weight. Including.

  For the rice bran hydrolyzate of the present invention, the hydrolyzed product thus obtained may be used as it is, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or dry powder, It may be prepared as follows. It can also be lyophilized and diluted with a solvent usually used for extraction at the time of use. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  In the present invention, cycloartane type triterpenes such as cycloartenol and 24-methylenecycloartanol or derivatives thereof can be used singly or in combination. Such cycloartane type triterpenes or derivatives thereof are described above. It can be obtained by separating / purifying it from wheat, corn, coffee beans, millet, millet, aloe and the like. Furthermore, it can also be prepared by a synthesis method described in J. Chem. Soc., Chem. Commun., 1968, 643, J. Chem. Soc., Perkin. Trans. 1, 1979, 563, etc. Moreover, it can also use a commercial item.

As shown in Examples below, rice bran hydrolyzate or cycloartane triterpene has an action of suppressing the expression of SREBP1 gene. As described above, SREBP1 is expressed in sebaceous gland cells in the human scalp and plays an important role in scalp sebaceous gland differentiation and lipid synthesis (Non-Patent Document 4). In addition, the activation of sebaceous gland cells by male hormones causes an increase in the expression of SREBP1 and an increase in the expression of fatty acids and cholesterol-dependent enzymes dependent on SREBP1, thereby preventing excessively secreted sebum from inhibiting hair growth. And non-patent document 5). Therefore, suppression of SREBP1 suppresses excessive lipid production due to the activation of sebaceous gland cells, and can be said to be effective in preventing or improving seborrheic alopecia.
Therefore, a rice bran hydrolyzate or cycloartane type triterpene or a derivative thereof is ingested or administered to animals including humans to suppress SREBP1, and further, symptoms associated with abnormal sebum lipids resulting from the activation of SREBP It can be used to prevent or ameliorate diseases such as seborrheic alopecia and seborrheic dermatitis (eczema). That is, the rice bran hydrolyzate or cycloartane type triterpene or a derivative thereof can be a SREBP1 inhibitor, a seborrheic alopecia and / or a seborrheic dermatitis preventive or ameliorating agent, and a SREBP1 inhibitor, a seborrheic agent It can be used to produce a preventive or ameliorating agent for alopecia and / or seborrheic dermatitis.
Here, “suppression of SREBP1” means that the expression of SREBP1 gene is suppressed, and further, the production of SREBP1 is suppressed, and preferably, the expression of SREBP1c gene is suppressed, It means that generation is suppressed.

  The SREBP1 inhibitor, seborrheic alopecia and / or seborrheic dermatitis preventive or ameliorating agent itself is for preventing or improving SREBP1 suppression, seborrheic alopecia and / or seborrheic dermatitis It may be a food, a quasi-drug, a pharmaceutical, or the like, or a material or a preparation used by blending in the food, quasi-drug, pharmaceutical or the like. In addition, the food has the concept of SREBP1 suppression, seborrheic alopecia and / or prevention or improvement of seborrheic dermatitis, and if necessary, beauty food, food for the sick or specified health Functional foods such as industrial foods are included.

  The administration form of the above pharmaceutical preparation containing the rice bran hydrolyzate or cycloartane type triterpene or derivative thereof of the present invention is arbitrary, and includes oral, enteral, transmucosal, injection and the like, and oral administration is preferred. Examples of the dosage form of the preparation for oral administration include tablets, coated tablets, capsules, soft capsules, granules, powders, powders, sustained-release preparations, suspensions, emulsions, troches, liquids for internal use, Dragee tablets, pills, fine granules, syrups, elixirs, emulsions and the like can be mentioned. Parenteral administration includes intravenous injection, intramuscular injection, inhalation, infusion, suppository, inhalation, transdermal absorption agent, eye drops, nasal drops, cream, gel, lotion, patch, gel, paste, etc. Can be mentioned.

  Such pharmaceutical preparations of various dosage forms can be prepared by using the rice bran hydrolyzate or cycloartane type triterpene or a derivative thereof of the present invention alone or in combination with other pharmaceutically acceptable carriers. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, fluidity promoters, absorption aids, pH adjusters, emulsifiers, preservatives, stabilization. Agent, antioxidant, colorant, UV absorber, moisturizer, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, flavoring agent, flavoring agent, extender, surfactant, dispersant, Buffering agents, preservatives, fixing agents, fragrances, coating agents and the like can be mentioned. Specific examples of such carriers include lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride and other solid carriers, glycerin, peanut oil And liquid carriers such as polyvinylpyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, and water.

Although the content of the rice bran hydrolyzate in the preparation varies depending on the kind of preparation, it can generally be 0.001 to 100% by mass, preferably 0.01 to 90% by mass, More preferably, it is made -70 weight%.
Further, the content of the cycloartane type triterpene or derivative thereof in the preparation varies depending on the kind of the preparation, but can generally be 0.1 to 100% by mass, preferably 1 to 90% by mass, It is still more preferable to set it as 5-70 mass%.

  Examples of the form of the food containing the rice bran hydrolyzate or cycloartane type triterpene or derivative thereof according to the present invention include confectionery such as breads, noodles and cookies, snacks, jelly products, dairy products, frozen foods, and powdered coffee. Instant foods such as starch processed products, processed meat products, other processed foods, beverages such as coffee beverages, canned beverages such as coffee beverages, soft drinks, fruit juice beverages, soups, seasonings, edible oils, nutritional supplements In addition to various foods such as foods, forms similar to the above-mentioned oral administration preparations (tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form, etc.) can be mentioned.

  Various forms of food include rice bran hydrolyzate, other food and drink materials, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, thickeners, sticking agents , A dispersant, a wetting agent, and the like can be mixed and prepared as appropriate.

Although the content of the rice bran hydrolyzate in the food varies depending on the use form, it can generally be 0.001 to 100% by mass and 0.01 to 90% by mass in terms of dry matter. Is more preferable, and it is still more preferable to set it as 0.05 to 70 weight%.
In addition, the content of the cycloartane type triterpene or derivative thereof in the food varies depending on the type of the preparation, but can generally be 0.1 to 100% by mass, preferably 1 to 90% by mass. 5 to 70% by mass is more preferable.

  The dose or intake of the above-mentioned pharmaceuticals and functional foods may vary according to the subject's condition, weight, sex, age or other factors, but per adult per day for oral administration, per day as cycloartenol It is preferably 50 μg to 500 mg, more preferably 75 μg to 100 mg, further preferably 100 μg to 50 mg, and even more preferably 150 μg to 25 mg. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times a day.

Example 1 SREBP1 gene expression inhibitory action (1) Rice bran hydrolyzate As the rice bran hydrolyzate, “Oryza triterpenoid-P” (manufactured by Oriza Yuka Co., Ltd.) was used. This product is a powdered triterpene alcohol obtained by hydrolysis from rice bran and rice germ produced from the seeds of Oryza sativa Linne. Table 1 shows the results of component analysis by capillary gas chromatography of this product.

(Analysis conditions)
Column: Capillary GC column DB-1 (manufactured by Agilent Technologies), 30 m × 0.25 mm, film thickness 0.25 μm
Carrier gas: helium, 2.30 mL / min
Split ratio: 40: 1
Injector: T = 300 ° C
Detector: FID, T = 300 ° C
Oven temperature: held at 150 ° C for 1.5 minutes, raised to 250 ° C at 15 ° C / minute, raised to 320 ° C at 5 ° C / minute, held for 20 minutes

(2) Animals and rearing methods C57BL / 6J mice (male, 8 weeks old, Claire Japan) were used for the experiment, and each group was divided so that the body weights were equivalent (N = 12 / group), 1 cage 4 animals were bred per animal.
The diet composition is shown below (Table 2). The content in the test meal was shown as a percentage (W / W%). Low-fat diet containing 5% lipid, high-fat diet containing 30% lipid, rice bran hydrolyzate of (1) above (0.2% or 0.5% orizatriterpenoid-P (manufactured by Oriza Yuka) ) Was fed to each group for 7 weeks. During the breeding period, body weight was measured once a week and food intake was measured twice a week. During the breeding period, free feeding and free drinking were used.

(3) Blood collection and sampling Whole blood was collected from the abdominal vena cava under anesthesia on the last day of feeding, and the blood glucose level was measured using a simple blood glucose measurement device (Accuche Aviva, manufactured by Roche). Saved inside. In addition, liver and visceral fat (peri-testicular peritoneal fat, peri-adrenal fat, retroperitoneal fat, and mesenteric fat) were removed and weighed.

(4) Analysis of blood components Plasma was prepared after the collected blood was centrifuged. Plasma glucose, neutral lipids (triglycerides), free fatty acids, total cholesterol, GOT, and GPT are automatically serum component analyzers (Accute, manufactured by Toshiba) using reagents manufactured by Nitteau Medical or Wako Pure Chemical Industries, Ltd. ) In accordance with a conventional method.

(5) Analysis of genes in mouse liver (RNA extraction, cDNA preparation, and quantitative PCR)
RNA was extracted from the extracted liver using RNeasy Mini Kits (QIAGEN) and RNeasy Lipid Tissue Mini Kits (QIAGEN). CDNA was prepared from 1 μg of each extracted RNA sample using reverse transcriptase. The prepared cDNA sample was diluted 10-fold and quantitative PCR was performed on the SREBP1 (SREBP1c) gene using Taqman Gene Expression Assays (Applied Biosystems). The expression level of SREBP1 gene was corrected with 36B4 gene (Rplp0; ribosomal protein, large, P0).

(6) Statistical analysis All statistics are shown as mean ± standard error. In addition, by using Fisher's PLSD, a test with P <0.05 as the significance level was performed to determine the significant difference between the groups.

(7) Results (i) Body weight, visceral fat weight, liver weight, blood component analysis
Body fat, visceral fat weight, liver weight, and blood components (glucose, triglyceride, free fatty acid, total cholesterol, GOT, and GPT) analysis values after 7 weeks of breeding were compared between the high fat diet group and the orizatriterpenoid-P addition group. There was no significant difference (abnormal value) between them. There was no significant difference in food intake between the control group (high fat diet group) and the orizatriterpenoid-P addition group.

(Ii) Analysis of SREBP1 gene in mouse liver The expression level of SREBP1 gene, a fatty acid synthase in the liver, is a control by ingesting a high-fat diet supplemented with rice bran hydrolyzate (0.5% oryza triterpenoid-P) It was significantly reduced compared with the high fat diet group (FIG. 1). From this, the hydrolyzate in rice bran is effective in suppressing the expression of SREBP1.

Example 2 Analysis of SREBP1 gene in cultured cells derived from human liver cancer (1) Cultured cells and culture conditions HepG2 cells derived from human liver cancer were 10% (w / v) FBS (Fetal bovine serum) and penicillin (100 unit / ml). ) In a DMEM medium containing streptomycin (100 μg / ml), the cells were cultured under conditions of 37 ° C. and 5% CO ₂ .

(2) Conditions for adding rice bran component To HepG2 cells subjected to overnight serum starvation, triterpene alcohol (0.0003% cycloartenol (manufactured by Sigma-Aldrich, or purified from oryza triterpenoid-P), 24-methylenecycloartanol ( Or a sterol component (β-sitosterol (manufactured by Wako Pure Chemical Industries) or campesterol (manufactured by Wako Pure Chemical Industries))) is added to the DMEM medium of (1) above. And cultured for 24 hours.
In addition, cycloartenol and 24-methylenecycloartanol were obtained by using silica gel column chromatography (column temperature: room temperature, flow rate: 15 mL / min, eluent: hexane / EtOAc, for orizatriterpenoid-P (manufactured by Oriza Yuka). = 9/1 (isocratic)) to obtain a triterpene alcohol fraction, and this triterpene alcohol fraction was further purified by RP-HPLC (column: Inertsil ODS-3 (GL Science) 20 x 250 mm, column temperature: room temperature, Flow rate: 18.9 mL / min, detection: UV = 210 nm, eluent: MeOH / MeCN / THF / H ₂ O = 15/2/2/1 (isocratic)).

(3) Gene analysis of human liver cancer-derived HepG2 cells (RNA extraction, cDNA preparation, and quantitative PCR)
Cells were lysed using RNeasy Mini Kits (QIAGEN) and RNeasy Lipid Tissue Mini Kits (QIAGEN), and RNA was extracted. CDNA was prepared from 1 μg of each extracted RNA sample using reverse transcriptase. The prepared cDNA sample was diluted 10-fold and quantitative PCR was performed on the SREBP1 (SREBP1c) gene using Taqman Gene Expression Assays (Applied Biosystems). The expression level of SREBP1 gene was corrected with 36B4 gene (Rplp0; ribosomal protein, large, P0).
(4) Statistical analysis All statistics are shown as mean ± standard error. In addition, by using Fisher's PLSD, a test with P <0.05 as the significance level was performed to determine the significant difference between the groups.
(5) Results By adding a medium containing triterpene or plant sterol component (0.0003% cycloartenol, 24-methylenecycloartanol, β-sitosterol, or campesterol) in rice bran, the expression level of SREBP1 gene is (Fig. 2). Therefore, triterpene or its derivatives in rice bran is effective for suppressing the expression of SREBP1. Moreover, since the reduction effect of cycloartenol is significantly stronger than 24-methylenecycloartanol and β-sitosterol, increasing the content of cycloartenol in rice bran hydrolyzate is due to the suppression of SREBP1 expression. It is valid.

Claims (6)

  SREBP1 inhibitor comprising rice bran hydrolyzate as an active ingredient.   The SREBP1 inhibitor according to claim 1, wherein the rice bran hydrolyzate contains at least a cycloartane type triterpene or a derivative thereof.   The SREBP1 inhibitor according to claim 2, wherein the rice bran hydrolyzate further contains β-sitosterol and / or campesterol.   The SREBP1 inhibitor according to any one of claims 1 to 3, wherein the rice bran hydrolyzate contains 1 to 100 mass% of cycloartenol or a derivative thereof. A SREBP1 inhibitor comprising cycloartane type triterpene or an ester or glycoside thereof as an active ingredient.   The SREBP1 inhibitor according to claim 5, wherein the cycloartane type triterpene is cycloartenol and / or 24-methylenecycloartanol.