JP5770504B2 - Adiponectin production promoter - Google Patents

Description

  The present invention relates to an adiponectin production promoter.

  Adiponectin is a good adipocytokine produced and secreted specifically from adipocytes, and is known to be closely related to lifestyle-related diseases such as cardiovascular diseases, diabetes and obesity. Research and development of components that promote the production of adiponectin in the blood are underway. Many proposals have been made.

Adiponectin is a good adipocytokine having a molecular weight of about 30 KDa and most abundantly expressed in adipocytes. It has structural homology with complement C1q, and consists of a signal peptide, collagen-like domain, and globular domain from the N-terminus. In blood, three types of multimers of 12-18 mer high molecular weight adiponectin, hexamer medium molecular weight adiponectin, and trimer low molecular weight adiponectin are formed.
The blood level of adiponectin decreases with obesity and visceral fat accumulation. Adiponectin is also reduced by genetic factors such as gene polymorphism. Adiponectin gene-deficient mice exhibit various symptoms of metabolic syndrome such as insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. Adiponectin supplementation improves insulin resistance and metabolic syndrome.

Numerous proposals have been made for promoting adiponectin production. Patent Document 1 (Japanese Patent Application Laid-Open No. 2009-209080) discloses that soy protein hydrolyzate is an active ingredient, and in particular, soy protein hydrolyzate is an oligopeptide mixture mainly composed of dipeptide or / and tripeptide. It is disclosed that the composition has an adiponectin secretion promoting action and strongly enhances the blood adiponectin concentration.
Patent Document 2 (Japanese Patent Application Laid-Open No. 2009-249341) discloses an adiponectin production promoter containing a red yeast rice extract and proanthocyanidins as active ingredients.
Patent Document 3 (Japanese Patent Laid-Open No. 2008-255040) discloses an adiponectin production promoter containing resveratrol as an active ingredient.

JP 2009-209080 A JP 2009-249341 A JP 2008-255040 A

  An object of the present invention is to propose a novel adiponectin production / secretion promoter.

As a result of investigating and analyzing 517 kinds of plant extracts, the present inventor has found that the extracts of plants belonging to the genus Vitex have an adiponectin production / secretion promoting action. Furthermore, since the present inventor has confirmed a substance that plays a central role in the adiponectin production / secretion promoting activity of the extract of the Vitex genus plant, an agent comprising this compound as an active ingredient is proposed.
That is, the main configuration of the present invention is as follows.
1. Adiponectin production promoter (excluding food additives) containing castisin as an active ingredient.
2. Insulin resistance improving agent comprising castisin as an active ingredient.
3. A postprandial blood glucose level-lowering agent comprising castisin as an active ingredient.

The present invention includes a Verbenaceae family (Venaceae) Vitex genus plant extract containing custine effective as an adiponectin production promoting action, improving insulin resistance, and reducing blood glucose after ingestion of food and drink. Clarified that there is. In particular, Vitex agnus-castus L. (chest tree) extract is effective.
By ingesting this Verbenaceae family extract (Viperaceae) Vitex genus plant extract in the form of pharmaceuticals, foods and drinks, feeds, etc., production of adiponectin can be promoted or blood glucose level can be lowered. As a result, various insulin resistances involving adiponectin are expected to be improved and useful for the treatment and prevention of various pathological conditions based on metabolic syndrome.

Schematic process drawing of the experimental method. The graph which shows the amount of adiponectin in blood. The graph which shows insulin resistance improvement. Chest tree extract A graph showing the identification of the castisin fraction. The graph which shows the adiponectin production effect | action by a castisin fraction. The graph which shows a weight change. The graph which shows the change of blood glucose by a glucose tolerance test. The graph which shows the adiponectin production and secretion effect | action of the Vitex genus plant. The graph which shows the result which confirmed the adipocyte differentiation promotion effect of castisin. The graph which shows the result of having measured the adiponectin production and secretion promotion effect of castisin.

  The Verbenaceae family extract (Viperaceae) Vitex genus plant extract used in the present invention is one of the 517 plant extracts that have been investigated for adiponectin production activity. As a result of testing with respect to changes in sex fat and changes in body weight, it was possible to deepen until good results were obtained. Furthermore, as a component exhibiting this action, a compound called castisin contained in the genus Vitex was examined. As a result, it was confirmed that castisin alone exhibits the same effect, and therefore an agent containing castisin as an active ingredient is also proposed.

The present invention can be used as a medicine, food additive, food and pet food, and animal medicine. The dosage form can be formulated into an arbitrary form together with an auxiliary agent by a known method and administered orally. It can be administered as a capsule or tablet, granule, fine granule, powder, or liquid.
The dose may vary depending on the administration method and the patient's age, medical condition, general condition, etc., but 0.4 to 600 mg is usually appropriate as an active ingredient per day for 1 kg of body weight in adults based on the results of animal tests. .

The adiponectin production promoter of the present invention can be added to general foods and health foods, and can also be used as a component of food additives. The food to be blended is not particularly limited, for example, bakery foods such as bread, confectionery bread, pies, Danish, donuts, cakes, noodles such as udon, soba, Chinese noodles, yakisoba, pasta, fries such as tempura, croquettes, curry, Sources such as stew and dressing, sprinkles, kneaded products such as kamaboko, beverages such as juice, snacks, rice confectionery, rice cake, confectionery such as gum.
Suitable as feed for mammals such as dogs, cats, hamsters and squirrels. The form of the pet food of the present invention is not particularly limited, and examples thereof include dry type, wet type, semi-moist type, biscuit type, sausage type, jerky type, powder, granule, capsule and the like.

<Verbenaceae (Viperaceae) Vitex genus plant extract screening test>
The test method will be described according to the process outline shown in FIG. Mouse-derived preadipocytes (3T3-L1) were subcultured and cultured in Dulbeccos modified Eagle medium (DMEM) containing 10% FBS according to a conventional method. As an evaluation for the search, a multi-well plate coated with rat tail collagen I (48 well, BD BioCoat, manufactured by BD Bioscience) was seeded with 5 × 10 ⁴ 3T3-L1 cells per well, and confluent. Cultured until complete (6-8 days). Thereafter, the cells were cultured in an induction medium containing dexamethasone, isobutylmethylxanthine and insulin for 3 to 4 days to induce differentiation from preadipocytes to immature adipocytes, and then cultured in a maintenance medium containing insulin and a test sample for 1 week. . After culturing for one week, the bottom is measured by the oil red O staining method for the amount of lipid droplets in mature adipocytes (adipocyte differentiation promoting action), and the culture supernatant is a commercially available ELISA kit (mouse / rat). Evaluation was performed by measuring the amount of adiponectin produced and secreted using an adiponectin ELISA kit (Otsuka Pharmaceutical).

  From a number of test agents, test agent no. Adiponectin producing action was observed for E10004. As shown in FIG. 2, the effect was 5.9 times that of the control, and an effect of more than 50% was confirmed in Pioglitazone marketed as an adiponectin producing agent.

  Test agent No. E10004 is an extract of Vitex agnus-castus L. (chest tree) belonging to the family of the Verbenaceae family (Viraceae) Vitex. 200 ng / ml or more is shown. It is a deciduous shrub belonging to the plant of the Verbenaceae family (Viraceae) Vitex genus (Villus genus). In addition, Pioglitazone used as a medicine for adiponectin production was used as a positive control. Vitex agnus-castus L. (chest tree) extract showed more than 50% of the activity of the positive control and about 6 times the activity of the control.

Vitex agnus-castus is a plant from the southern family, Central Europe and Central Asia. The height is 2 to 3 meters, and the whole has a faint fragrance. The lanceolate leaves are palm-shaped compound leaves and 5 to 7 small leaves. From July to September, conical inflorescences are found at the tips of branches. To make a light purple small flower bloom. In Europe, fruit has been used as a fragrance, and has been used as a remedy for malaise and menopause. It is said that the fruit extract, which mainly uses the fruit portion, promotes the secretion of luteinizing hormone, which is a type of female hormone, and has the effect of improving diseases specific to women, including irregular menstruation.
As the plant of the Verbenaceae family (Annelidae), Vitex genus (Annelida), Vitex agnus-castus (scientific name) (English name Chaste berry, Japanese name carrot), Vitex cannabifolla Sieb. Et. Zucc. (Carrot), Vitex negundo L (Taiwan carrot), Vitex rofundifolia L. fill. (Hamago) extract can be used.
Vitex cannabifolla Sieb. Et. Zucc. (Carrot) is a deciduous shrub belonging to the family Pinaceae and is widely distributed in China. The height is 3-5 meters, the branches are square and the leaves are opposite to each other, and the leaves are palm-like compound. In July-August, light purple small lip-shaped flowers form a conical inflorescence about 20 centimeters in length and bloom in steps. In the Chinese name, carrots are called oysters, fruits are oysters, and leaves are oyster leaves. Oysters are used to treat asthma, cold, stomach disease, indigestion, enteritis, etc. as an antitussive, analgesic, healthy stomach, antipruritic agent. In China, leaves, stems, and roots are used almost like fruits.
Vitex negundo L. (V. negundo L.) is a close relative of carrots (Vitex cannabifolla Sieb. Et. Distributed in China, Southeast Asia, India, Madagascar and Africa. In the Chinese name, it is called “Okekei” and is used in the same way as the previous carrot.
Vitex rofundifolia L. fill. (Hamago) is a vine shrub that grows naturally in the sandy beaches of warm coasts such as Japan, Korea, China and Australia. Flowers and leaves have a scent, and the Japanese name “Hamago” means “hamaka”. The mature and dried fruit of this plant is called Viticis Fructus and is used for antipyretic, analgesic, tonic and anti-inflammatory agents for headache, cold, eye disease, stomach pain, joint pain, etc. In addition, it is prescribed as a Chinese medicine for ryukakosan and kikuhanasan.
Other plants belonging to the genus Vitex include honey beetle (Vitex trifolia L.) and yamaha maggot (Vitex trifolia L. var. Bicolor (Willd.) Moldenke). The former is a three-leaved evergreen shrub widely distributed in the Ryukyus, south of Amami Oshima, and the old world. The latter is widely distributed in Southeast Asia, Micronesia, Australia, and other tropics. In Japan, it lives only on Iriomote Island and Ishigaki Island.

<Insulin resistance improvement>
A graph showing the insulin resistance improving action is shown in FIG. An improvement of 3.2 to 6.5 times that of the control is exhibited in a concentration-dependent manner. It shows 40-80% of behavior relative to the positive control Pioglitazone.

<Casticine detection test>
When the chest tree extract was subjected to liquid-liquid partition using ethyl acetate and water, the action of promoting adipocyte differentiation was observed in the ethyl acetate layer.
When the chest tree extract was subjected to G3000S column chromatography and eluted with ethanol solvents of different concentrations, an adipocyte differentiation promoting action was observed in the 70% aqueous ethanol and 100% ethanol elution fractions.
When the ethyl acetate layer of the chest tree extract was subjected to silica gel column chromatography and eluted with an elution solvent with different mixing ratios of chloroform and methanol, Fraction 2, 3 and 4 (fractions containing castisin as shown in FIG. 4) were obtained. A), an adipocyte differentiation promoting action was observed.
Casticin (common name) has the molecular formula: C19H18O8, molecular weight: 374.345, 3 ', 5-Dihydroxy-3,4', 6,7-tetramethoxyflavone 3,4 ', 6,7-Tetramethoxy- 3 ', 5-dihydroxyflavone.
The chest tree fractions Fraction 2, 3 and 4 were subjected to a reproducibility test of adiponectin production using the commercially available Pioglitazone as an adiponectin production agent as an index. The result is shown in FIG. It was confirmed that the production-enhancing effect was about 150% for the control and 75% for Pioglitazone.
This indicates that the chest tree extract can be expected to have a slow effect as an adiponectin producing agent, and the contents indicate that the chest tree extract can be safely taken on a daily basis.

<Mouse intake test>
[Animal testing / feed]
For the test, 5-week-old KKAy / TaJcl mice (Claire Japan), males were used. High-fat, high-sucrose diet (Oriental Yeast Industry, F2HFHSD powdered feed, hereinafter referred to as HFS) as test food, Chest Tree Extract (made by Ask Chemicals, 50% aqueous ethanol extract, 40% excipient) , Hereinafter referred to as VA) was added so as to be 0.5%, 1.0%, and 2.0% as the equivalent amount of the extract. There were 6 animals in each group.
After one week of habituation, each of the above test meals was fed for 3 weeks. During the period, food intake and body weight were measured (twice / week). After feeding for 3 weeks, blood glucose levels were measured under non-fasting conditions, and then an oral glucose tolerance glucose tolerance test (OGTT) was performed under fasting conditions.

  The weight gain is shown in FIG. It has been shown that weight gain is suppressed in the test group to which the chestnut extract was added, compared to a high fat / high sucrose diet (HFS). And the weight suppression effect appears in a concentration-dependent manner.

[Results of glucose tolerance test (OGTT)]
The test results are shown in FIG.
It has been shown that the decrease in blood glucose level is promoted in the test group to which the chest tree extract was added, compared with a high fat / high sucrose diet (HFS). And the concentration-dependent dependence is not recognized by the test addition amount. Therefore, it is possible to reduce the blood sugar concentration after a meal even with a small amount.

  According to "Metabolic syndrome up to date" (edited by the Japan Medical Association), it is reported that the cause of diabetes and abnormal lipid metabolism is due to insulin resistance. That is, metabolic syndrome is improved by improving insulin resistance. The metabolic syndrome has blood glucose level and body weight as typical parameters. Since the present invention has been demonstrated in weight gain and glucose tolerance glucose tolerance test (OGTT), it can be said to have an insulin resistance improving action.

<Adiponectin production / secretion promoting test>
By the method for measuring the amount of adiponectin described in the above [0013] and FIG. 1, the following five Verbenaceae (Viperaceae) Vitex genus plants were further evaluated.
A graph showing the adiponectin production / secretion promoting action is shown in FIG.
Vitex agnus-castus, Vitex rofundifolia L. fill. (Octopus), Vitex cannabifolla Sieb. Et. Zucc., Vitex negundo L., Vitex negundo L. ( Adiponectin production / secretion promoting action tests were conducted on five types of extracts extracted from the leaves of the Thai carrot.
A high value is recognized in all cases compared to the control, and it is higher depending on the concentration. In contrast to the positive control Pioglitazone, it shows 50-90% behavior at high concentrations. In the seed extract of yellow carrots (yellow coconut) and leaf extract (yellow coconut leaves), an excellent effect on the seed extract was observed at a high concentration.

<Castisin Adipocyte Differentiation Promotion Test>
An adipocyte differentiation promotion test was performed using commercially available purified castisin (manufactured by Funakoshi EXTRASYNTHESE). The differentiation promoting activity into adipocytes was measured using the screening test technique of paragraph [0013]. The measurement results are shown in FIG. The castisin-administered group clearly showed the adipocyte differentiation promoting effect as compared with the case where the castisin treatment was not performed.
Castisin induced the differentiation of immature adipocytes into mature adipocytes in a concentration-dependent manner.

<Measurement of adiponectin production and secretion promoting effect of castisin>
The adiponectin production / secretion promoting action of commercially available purified castisin (manufactured by Funakoshi EXTRASYNTHESE) was measured. The measurement results are shown in FIG. The castisin administration group clearly showed an adiponectin production and secretion promoting effect as compared with the case where the castisin treatment was not performed.
It has been clarified that castisin promotes production and secretion of adiponectin at a low concentration of 0.1 μg / ml. Moreover, no cytotoxicity was observed.
Therefore, it was confirmed that castisin can be used as a safe adiponectin production / secretion promoter, insulin resistance improving agent, and postprandial blood glucose lowering agent.

Claims (3)

Adiponectin production promoter (excluding food additives) containing castisin as an active ingredient.   Insulin resistance improving agent comprising castisin as an active ingredient. A postprandial blood glucose level- lowering agent comprising castisin as an active ingredient.