JP5923381B2 - PPARγ activity inhibitor - Google Patents

Description

  The present invention relates to a material capable of suppressing the activity of a peroxisome proliferator activated receptor γ (referred to as Peroxisome Proliferator Activated Receptor gamma, PPARγ).

  Peroxisome proliferator-activated receptor (PPAR) is a transcription factor that is activated by binding of a ligand to the C-terminal ligand binding region, and is a nuclear receptor having glucocorticoid, estrogen, vitamin D, etc. as ligands. It is one of the body superfamily (Non-patent Document 1). When PPAR is activated by binding to a ligand, it forms a heterodimer with a retinoid X receptor (RXR) and binds to a PPAR response element (peroxisome proliferator response element: PPRE) existing upstream of the target gene. Thus, expression of the target gene is activated (Non-patent Documents 2 and 3).

  Three isoforms called PPARα, PPARδ and PPARγ have been identified in PPAR. It has been reported that these have different expression tissues and have different biological functions (Non-Patent Document 4). For example, PPARγ is a molecule that acts on energy storage in the presence of sufficient nutrition and acts as a so-called thrifty gene. In particular, PPARγ2 is expressed with relatively strong specificity in adipocytes, and has been shown to play a central role in adipocyte differentiation. Thiazolidine derivatives, known as PPARγ ligands, are known to downsize adipocytes and increase insulin sensitivity by strongly inducing adipocyte differentiation, and are widely used as insulin resistance improvers and diabetes therapeutics. (Non-Patent Document 5).

  By inhibiting the activity of PPARγ, adipocyte differentiation and / or maturation is suppressed, so that fat synthesis can be suppressed. As a result, obesity, dyslipidemia, fatty liver can be prevented or improved, and lifestyle It becomes possible to prevent or improve diseases and visceral fat syndrome (metabolic syndrome).

  Furthermore, it is known that PPARγ is expressed in sebaceous cells. In a culture test using rat sebaceous gland cells, it has been shown that the PPARγ agonist BRL-49653 promoted differentiation of sebaceous gland cells into lipid-producing cells (Non-patent Document 6). When PPARγ in epidermoid cancer cells or sebaceous gland cells is activated by ultraviolet radiation (UVB) in the B region or drug-induced oxidative stress stimulation, the expression of cyclooxygenase 2 dependent on PPARγ activity occurs and an inflammatory reaction is triggered. It has also been reported (Non-Patent Document 7).

  Therefore, by suppressing the activity of PPARγ, it is also possible to prevent or ameliorate skin diseases or skin troubles associated with abnormal skin lipids such as acne through suppression of excessive lipid production in sebaceous glands.

  Citral (3,7-dimethyl-2,6-octadienal) is an aroma component contained in citrus fruits and lemongrass, and is mainly used as a perfume and is also known as a blood flow promoting component. (Patent Document 1). Piperine ((2E, 4E) -1-piperidino-5- (1,3-benzodioxol-5-yl) -2,4-pentadien-1-one), a spicy component of pepper, has long been an insecticide. It is a substance that has been used as a component, and is also known as a warming substance (Patent Document 2).

JP 2010-024184 A Special table 2011-516092 gazette

Ann. N.Y. Acad. Sci. (1993) 684: 157-173 Cell (1999) 31 (6): 218-234 J Lipid Res. (1996) 37: 907-925 Endcrinology (1996) 137: 354-366 Diabetes. (2000) 49: 759-767 Dermatol. (1998) 196: 43-46 Dermatol. (2006) 126: 42-48

  The present invention relates to the provision of a material capable of suppressing the activity of PPARγ.

  The present inventors have found that citral or piperine has PPARγ antagonist activity, suppression of adipocyte differentiation and / or maturation, suppression of fat synthesis, suppression of sebum, skin diseases or skin with abnormal skin lipids such as acne It was found useful as an active ingredient for preventing or ameliorating troubles, or preventing or ameliorating diseases or conditions such as obesity, dyslipidemia, fatty liver, lifestyle-related diseases and visceral fat syndrome.

That is, the present invention provides the following.
(1) A PPARγ activity inhibitor containing citral or piperine as an active ingredient.
(2) An adipocyte differentiation and / or maturation inhibitor containing citral or piperine as an active ingredient.
(3) A fat synthesis inhibitor containing citral or piperine as an active ingredient.
(4) A sebum inhibitor containing citral or piperine as an active ingredient.
(5) Acne preventive or ameliorating agent containing citral or piperine as an active ingredient.
(6) An obesity preventing or improving agent comprising citral or piperine as an active ingredient.
(7) A prophylactic or ameliorating agent for dyslipidemia comprising citral or piperine as an active ingredient.

  The PPARγ activity inhibitor of the present invention has PPARγ antagonist activity and can inhibit PPARγ activation. The PPARγ activity inhibitor of the present invention suppresses differentiation and / or maturation of adipocytes, suppression of fat synthesis, and further diseases such as obesity, dyslipidemia, fatty liver, lifestyle-related diseases and visceral fat syndrome (metabolic syndrome) Or it can be used for prevention or amelioration of the condition. Furthermore, the PPARγ activity inhibitor of the present invention can be used for the suppression of sebum production and the prevention or improvement of skin diseases or skin troubles accompanied by abnormal skin lipids such as acne.

  In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by therapy.

  In the present specification, “improvement” refers to improvement of a disease, symptom or condition, prevention or delay of deterioration of the disease, symptom or condition, or reversal, prevention or delay of progression of the disease, symptom or condition.

  As used herein, “prevention” refers to preventing or delaying the occurrence of a disease, symptom or condition in an individual, or reducing the risk of occurrence of a disease, symptom or condition in an individual.

Citral (3,7-dimethyl-2,6-octadienal) can be chemically synthesized according to the methods described in JP-A-52-148209, JP-A-9-188645, JP-A-2006-124348, and the like. Can be synthesized. Alternatively, it can be purchased from Sigma Aldrich, Wako Pure Chemical Industries, etc.
Piperine ((2E, 4E) -1-piperidino-5- (1,3-benzodioxol-5-yl) -2,4-pentadien-1-one) is an example of piperine such as pepper plant. Herbal medicine extract and dry extract can be used. Alternatively, it can be purchased from Sigma Aldrich, Wako Pure Chemical Industries, etc.

  As will be described later in Examples, citral or piperine acts as an antagonist to PPARγ and has an effect of suppressing the transcriptional activity of genes dependent on PPARγ. As described above, PPARγ is a molecule that acts on energy storage, and PPARγ ligand promotes adipocyte differentiation (non-patent document 5) and sebaceous gland cell differentiation into lipid-producing cells (non-patent document 6). It is known. That is, citral or piperine is a novel PPARγ antagonist and can be used to suppress PPARγ activity. Furthermore, citral or piperine is used for the suppression of adipocyte differentiation and / or maturation, for the suppression of adipocyte fat synthesis, for the suppression of sebum, for skin diseases or skin troubles such as acne and other skin lipid abnormalities. It can be used for prevention, treatment or improvement, or prevention, treatment or improvement of diseases or conditions such as obesity, dyslipidemia, fatty liver, lifestyle-related diseases and visceral fat syndrome (metabolic syndrome). The use can be in humans or non-human animals, or tissues, organs, cells, etc. derived therefrom, and can be therapeutic or non-therapeutic. Citral and piperine may each be used alone or in combination.

Therefore, in one aspect, the present invention provides a PPARγ activity inhibitor containing citral or piperine as an active ingredient. The present invention also provides an adipocyte differentiation and / or maturation inhibitor containing citral or piperine as an active ingredient. The present invention also provides a fat synthesis inhibitor containing citral or piperine as an active ingredient. The present invention also provides a sebum inhibitor containing citral or piperine as an active ingredient. Moreover, this invention provides the acne prevention or improvement agent which uses citral or piperine as an active ingredient. Moreover, this invention provides the obesity prevention or improvement agent which uses citral or piperine as an active ingredient. The present invention also provides a preventive or ameliorating agent for dyslipidemia containing citral or piperine as an active ingredient.
In one embodiment, the agent may consist essentially of citral or piperine.

In another aspect, the present invention provides the use of citral or piperine for the manufacture of a PPARγ activity inhibitor. The present invention also provides the use of citral or piperine for the production of an adipocyte differentiation and / or maturation inhibitor. The present invention also provides the use of citral or piperine for the manufacture of a fat synthesis inhibitor. The present invention also provides the use of citral or piperine for the manufacture of sebum inhibitors. The present invention also provides the use of citral or piperine for the manufacture of an acne prevention or amelioration agent. The present invention also provides use of citral or piperine for the manufacture of an agent for preventing or improving obesity. The present invention also provides use of citral or piperine for the manufacture of an agent for preventing or improving dyslipidemia.
In one embodiment, the above-mentioned agent suppresses adipocyte differentiation and / or maturation, suppresses fat synthesis, suppresses sebum, prevents or improves acne, prevents or improves obesity, or prevents or improves dyslipidemia. Is caused by suppression of PPARγ activity.

In yet another aspect, the present invention provides the use of citral or piperine for inhibiting PPARγ activity. The present invention also provides the use of citral or piperine for inhibiting adipocyte differentiation and / or maturation. The present invention also provides the use of citral or piperine for inhibiting fat synthesis. The present invention also provides the use of citral or piperine for sebum control. The present invention also provides the use of citral or piperine for the prevention or amelioration of acne. The present invention also provides the use of citral or piperine for the prevention or amelioration of obesity. The present invention also provides the use of citral or piperine for the prevention or amelioration of dyslipidemia.
The present invention further provides citral or piperine for use in the prevention, treatment or amelioration of acne. The present invention further provides citral or piperine for use in the prevention, treatment or amelioration of obesity. The present invention further provides citral or piperine for use in the prevention, treatment or amelioration of dyslipidemia.

  Furthermore, citral or piperine is used to inhibit PPARγ activity, inhibit adipocyte differentiation and / or maturation, inhibit fat synthesis, inhibit sebum, prevent or improve acne, or obesity, dyslipidemia Can be used for the manufacture of compositions, medicines, quasi-drugs, cosmetics, foods and drinks, etc. for the prevention or improvement of diseases or conditions such as liver disease, fatty liver, lifestyle-related diseases and visceral fat syndrome .

  The above composition, medicine, quasi drug, cosmetic, food and drink, etc. can be produced or used for human or non-human animals. Citral or piperine is blended as a raw material in the composition, medicine, quasi-drug, cosmetics, food and drink, etc., to suppress PPARγ activity, to inhibit adipocyte differentiation and / or maturation, It may be an active ingredient for suppression, sebum suppression, prevention or improvement of acne, or prevention or improvement of diseases or conditions such as obesity, dyslipidemia, fatty liver, lifestyle diseases and visceral fat syndrome .

  The medicine, quasi drug or cosmetic contains citral or piperine as an active ingredient. The medicine, quasi drug or cosmetic may contain citral or piperine alone, or may contain a combination of pharmaceutically or cosmetically acceptable carriers. Examples of such carriers include excipients, coating agents, binders, extenders, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, dispersing agents, emulsifiers, preservatives, and stabilizers. , Antioxidants, colorants, ultraviolet absorbers, thickeners, activity enhancers, anti-inflammatory agents, bactericides, moisturizers, fragrances, flavoring agents, flavoring agents, and the like, and in cosmetics, whitening agents , UV protection agents, cell activators, detergents, keratolytic agents, makeup ingredients (for example, makeup bases, foundations, irresistible powders, teak, lipsticks, eye makeup, eyebrow, mascara, etc.). Furthermore, the said medicine, quasi-drug, or cosmetic may contain other active ingredients, pharmacological ingredients, or cosmetic ingredients as long as citral or piperine does not lose the PPARγ activity-suppressing action.

  The pharmaceutical or quasi drug can be administered in any dosage form. Administration may be oral or parenteral. Dosage forms for oral administration include, for example, solid dosage forms such as tablets, coated tablets, granules, powders, capsules, and liquid dosage forms such as elixirs, syrups and suspensions. Examples of the dosage form for oral administration include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppository, bolus, and patch. The cosmetic may be in any form that can be used for cosmetics such as cream, emulsion, lotion, suspension, gel, powder, pack, sheet, patch, stick, cake, and the like.

  The medicine, quasi-drug or cosmetic can be produced from citral or piperine or, if necessary, by combining the carrier and / or other active ingredients, pharmacological ingredients or cosmetic ingredients by a conventional method. . 1-100 mass% is preferable, as for content of the citral or piperine in the said medicine or quasi drug, 10-90 mass% is more preferable, and 20-80 mass% is more preferable. Moreover, 0.001-10 mass% is preferable, as for content of the citral or piperine in the said cosmetics, 0.01-5 mass% is more preferable, 0.1-3 mass% is further more preferable.

  The said food-drinks contain citral or piperine as an active ingredient. The food and drink are PPARγ activity inhibition, adipocyte differentiation and / or maturation inhibition, fat synthesis inhibition, sebum inhibition, acne prevention or improvement, or obesity, dyslipidemia, fatty liver, lifestyle-related diseases and visceral fat syndrome Food and drink, functional food and drink, food and drink for the sick, food and drink for specified health use, livestock for which effects such as prevention or improvement of diseases or conditions such as It may be feed, livestock beverages, pet foods and the like.

  Examples of the form of the food include various foods such as breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, soups, edible oils, seasonings, etc. Examples of forms similar to those for oral administration (tablets, capsules, granules, powders, syrups, etc.), livestock feeds, pet foods, etc., include beverages such as fruit juices, carbonated drinks, teas Beverages, near water, sports drinks, milk drinks, alcoholic drinks, soft drinks and the like can be mentioned.

  The above-mentioned various forms of food and drink are citral or piperine alone, or other food and drink materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, moisturizing agents. It can be produced by a known method for producing foods and drinks by appropriately combining agents, thickeners and the like. The content of citral or piperine in the food or drink is preferably 0.0005 to 1.5% by mass, more preferably 0.0025 to 0.3% by mass, and still more preferably 0.005 to 0.1% by mass. .

  Alternatively, the dose (effective intake) of the above-mentioned pharmaceutical, quasi-drug or food or drink is preferably 2 to 5000 mg / 60 kg body weight per day in terms of the mass of citral or piperine, and 10 to 1000 mg / 60 kg body weight. Is more preferable, and 20 to 500 mg / 60 kg body weight is more preferable.

In yet another embodiment of the present invention, citral or piperine is used to inhibit PPARγ activity, to inhibit adipocyte differentiation and / or maturation, to inhibit fat synthesis, to inhibit sebum, and to prevent or improve acne. Therefore, for prevention, treatment or improvement of diseases or conditions such as obesity, dyslipidemia, fatty liver, lifestyle-related diseases and visceral fat syndrome, it can be administered in an effective amount to a subject in need thereof.
In one embodiment, the administration may be performed for the purpose of promoting health or beauty, preventing or ameliorating skin diseases or skin troubles associated with abnormal skin lipids such as acne, preventing or improving obesity, preventing weight loss or increase, The treatment is performed non-therapeutically for the purpose of preventing a decrease or increase in fat mass, or preventing a decrease or increase in blood lipid content.

  The subjects of administration include animals that need to suppress PPARγ activation, animals that need to suppress adipocyte differentiation and / or maturation, animals that need to suppress fat synthesis, and animals that need to suppress sebum. And animals that need prevention or improvement of acne, or animals that need prevention or improvement of obesity and dyslipidemia. Alternatively, the subject of administration includes animals suffering from skin diseases such as acne and skin diseases with abnormal skin lipids, or diseases or conditions such as obesity, dyslipidemia, fatty liver, lifestyle-related diseases and visceral fat syndrome, and the like Or animals with high risk. Alternatively, the subject of administration has not been diagnosed as suffering from or at risk of the above-mentioned diseases or conditions, but for the purpose of promoting health or cosmetics, skin diseases with abnormal skin lipids such as acne or Examples include animals that want to prevent or ameliorate skin problems, prevent a decrease or increase in body weight or body fat mass, or prevent a decrease or increase in blood lipid levels. The animal is preferably a human or non-human mammal, more preferably a human.

  The preferred dosage may vary according to the subject's species, weight, sex, age, condition or other factors. The dose, route, interval, and amount and interval of intake can be determined appropriately by those skilled in the art. For example, when orally administered to humans, the dose is preferably 2 to 5000 mg / day, more preferably 10 to 1000 mg / day, more preferably 20 to 500 mg per adult in terms of the mass of citral or piperine. / Day is more preferable.

  Alternatively, the administration subject can be an animal-derived tissue, organ, cell, or a culture or fraction thereof. The tissue, organ, cell, or fraction thereof is a naturally-derived or biologically or bioengineered tissue, organ, cell, or fraction thereof that expresses or has PPARγ It is a thing. Therefore, the present invention also provides a method for suppressing the activity of PPARγ in the tissue, organ, cell, or culture or fraction thereof. The method includes, for example, a step of culturing a cell having PPARγ and suppressing PPARγ activity in the presence of citral or piperine. When the cell is a cell culture, the concentration of citral or piperine added is 1 to 100 μM, preferably 5 to 50 μM, more preferably 10 to 30 μM.

Example 1: PPARγ Activity Inhibition Test Pipeline (Sigma Aldrich) and citral (Sigma Aldrich) were used as test substances, and PGWγ antagonist GW9662 (Wako Pure Chemical Industries) was used as a positive control.
African green monkey kidney cell line CV-1 was plated on a 24-well plate and cultured in DMEM (5% charcoal-treated fetal bovine serum) for 1 day. A reporter plasmid (pG5-Luc; invitrogen) containing a GAL4 binding sequence upstream of the firefly luciferase gene and a human PPARγ2 ligand binding site (NCBI Ref Seq NM — 015869, nt703-1606, SEQ ID NO: 1) were inserted into a pBIND vector (invitrogen) pBIND-PPARγ-LBD was introduced into the cells at the same time using a transfection reagent (Superfect Transfection Reagent; QIAGEN) at 0.25 μg / well. When introduced into a cell, the pBIND-PPARγ-LBD vector expresses a fusion protein of a PPARγ2 ligand binding site and a site binding to a GAL4 binding sequence. The fusion protein also binds to the GAL4 binding sequence by binding to the PPARγ2 ligand and activates the transcription of the downstream firefly luciferase gene. Therefore, the amount of PPARγ2 ligand bound can be determined by measuring the firefly luciferase activity. In addition, since the Renilla luciferase gene is incorporated into the vector, the introduction efficiency of the vector can be determined by measuring the Renilla luciferase activity.

Three hours after the introduction of the vector, the culture medium was replaced with DMEM (5% charcoal-treated fetal bovine serum), and further three hours later, the culture liquid was placed in a DMEM (-fetal bovine serum) medium containing the test substances shown in Table 1. Exchanged. After 1 hour, pioglitazone, a PPARγ agonist, was added and cultured overnight. After washing with PBS, cells were lysed using a dual luciferase assay system (Promega), and a substrate solution containing luciferin was added to the lysate, Firefly and Renilla luciferase activities were each measured with a luminometer. The PPARγ antagonist activity of the test substance was evaluated by measuring the PPARγ-dependent gene transcription activity (luciferase activity: Luc activity) induced by the PPARγ agonist in the presence of the test substance. PPARγ-dependent gene transcriptional activity (Luc activity) was defined as follows.

PPARγ-dependent gene transcriptional activity (Luc activity)
= (Firefly Luc activity by pG5-Luc) / (Renilla Luc activity by pBIND-PPARγ-LBD)

Using the Luc activity in the control (no test substance added) as 100, the relative value (%) of the Luc activity in the presence of each test substance was determined. The significant difference test for the control was performed using student's t-test. The results are shown in Table 1.

Example 2: Effect on adipocyte differentiation Mouse preadipocyte-derived cell line 3T3-L1 was plated on a 12-well plate and cultured for one day in DMEM containing 10% fetal bovine serum. After adding each test substance and further culturing for one day, the medium was replaced with a differentiation induction medium (5 μg / mL insulin, 1 μM rosiglitazone, 10% fetal bovine serum-containing DMEM) to which the test substance was added. Thereafter, the medium was changed every 2 days in the differentiation induction medium to which the test substance was added, and the cells were collected after 6 days. As a negative control, cells cultured for the same period without changing to the differentiation induction medium (undifferentiated group) were prepared.
Total RNA was prepared from the obtained cells, and cDNA was obtained by reverse transcription reaction. Using the obtained cDNA as a template, the expression levels of Fabp4, SREBP1c, and FAS genes were measured by quantitative PCR using 7500 Fast Real-Time PCR System (Applied Biosystems). Fabp4 is known as an adipocyte differentiation marker. SREBP1c and FAS are major lipid synthesis-related genes in adipocytes (The Journal of Nutritional Biochemistry., 2009, 20 (2): 140-148).
The measured expression level of each gene was corrected by an internal standard (36B4 gene expression level). The obtained value was determined as a relative value when the expression level in the control (no test substance added) was 1. The obtained values were graphed as an average value ± standard error (n = 3). The significant difference test for the control was performed using student's t-test. The results are shown in Table 2.

Claims (5)

PPARγ activity inhibitor as an active ingredient Shitora Le (where sebum suppression, acne prevention or amelioration or obesity due to excessive intake of fructose, except when used for the prevention or amelioration of hyperlipidemia or fatty liver) . Adipocyte differentiation and / or maturation inhibitor for the Shitora Le an active ingredient (except obesity due to excessive intake of fructose, a case of use in the prophylaxis or amelioration of hyperlipidemia or fatty liver). Fat synthesis inhibitors for the Shitora Le an active ingredient (except obesity due to excessive intake of fructose, a case of use in the prophylaxis or amelioration of hyperlipidemia or fatty liver). Preventing or improving agent for obesity (except for obesity due to excessive intake of fructose) that the Shitora Le as an active ingredient. Preventing or ameliorating agent for dyslipidemia (excluding hyperlipidemia caused by excessive intake of fructose) that the Shitora Le as an active ingredient.