JP2010083787A - Cinnamic aldehyde and cassia extract as cb1 receptor antagonist - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition which is used for treating, improving, controlling or preventing insulin resistance, hyperlipemia, diabetes and obesity, is obtained from a raw material originated from natural products, has high safety, and has CB1 receptor-inhibiting activity, and to provide a method for producing the same. <P>SOLUTION: There are provided a food and a medicinal composition which each contains a cassia extract and cinnamic aldehyde of raw materials originated from natural products, as a CB1 receptor-specific antagonist/inverse agonist, and is effective for treating, improving, controlling or preventing metabolic syndromes and the like; and the method for producing the same. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a composition having CB1 receptor (cannabinoid receptor) inhibitory activity, comprising cinnamon extract as an active ingredient, and more specifically, insulin resistance, hyperlipidemia, It also relates to foods used for the treatment, amelioration, suppression or prevention of diabetes and obesity and pharmaceutical compositions used for the treatment, amelioration, suppression or prevention of diseases or conditions associated with CB1 receptors.

  The cannabinoid (CB) receptor belongs to a G protein-coupled receptor having a seven-transmembrane domain. Among these, the CB1 receptor is mainly distributed in the central nervous system, but it is reported that it is also expressed in peripheral tissues such as stomach, adipose tissue, liver, skeletal muscle, pancreas (non-patent document). 1). CB2 receptors have been found to be distributed mainly in cells of the immune system (see Non-Patent Document 1). The CB1 and CB2 receptors show 48% homology, and the CB1 receptor retains 97-99% amino acid sequence homology in rats, mice, and humans.

  Today, obesity is recognized as one of the major public health problems because of the prevalence and associated health risks. This is associated with increased mortality, where increased obesity correlates with coronary heart disease, stroke, hypertension, type 2 diabetes, dyslipidemia, osteoarthritis, nonalcoholic steatohepatitis, cancer and respiratory disorders Because.

  Currently available prescription drugs for obesity management generally reduce weight by inducing satiety or reducing absorption of dietary fat. On the other hand, adrenergic weight loss drugs such as amphetamine, methamphetamine and phenmetrazine are no longer recommended due to their risk of abuse. In addition, serotonergic weight loss drugs such as fenfluramine and dexfenfluramine can no longer be used due to severe side effects in the lungs and heart.

  The CB1 receptor and its ligand, an endocannabinoid, are involved in the regulation of energy balance. During obesity, CB1 receptor gene expression and endogenous cannabinoid levels are increased, suggesting that CB1 receptor antagonist / inverse agonist functions as an obesity inhibitor (see Non-Patent Documents 2 and 3). CB1 receptor antagonists act on the hypothalamus to transiently reduce food intake. However, since the effect of suppressing body weight is more persistent than the effect on eating behavior, an effect on peripheral tissues is also suggested. Examples of the mechanism of action on peripheral tissues by the CB1 receptor antagonist include the following. Increases oxygen consumption and increases sugar uptake in skeletal muscle. In white adipose tissue, it promotes the gene expression level of adiponectin, an adipokine that functions to suppress the expression of enzyme genes involved in lipid synthesis. In the liver, it suppresses the expression of adipose synthesis transcription factor. Although it has been suggested through these effects on peripheral tissues to increase energy expenditure and maintain insulin sensitivity, the major mechanisms in the anti-obesity effect of CB1 receptor antagonists are still debated (non- Patent Document 1).

  In addition, since CB2 receptor has been suggested to be involved in the immune system, an antagonist specific for CB1 receptor is desirable.

  CB1 receptor antagonists have also been demonstrated in humans from clinical trials.

  Thus, the CB1 receptor antagonist is expected to improve obesity and prevent and / or improve metabolic syndrome related to hyperlipidemia and diabetes. Known CB1 receptor antagonists include rimonabant, bicyclic pyrazoyl and imidazolyl compounds (see Patent Document 1 and Patent Document 2).

Special table 2007-509113 gazette JP-T-2006-524228 Diabetes Metab Res Rev 2007; 23: 507-517. FASEB J.2005 Sep; 19 (11): 1567-9 Cell Metab. 2008 Jan; 7 (1): 68-78

  Conventionally known CB1 receptor antagonists as described above are expensive and have disadvantages such as side effects. Therefore, a CB1 receptor antagonist agent that is highly safe, highly effective, and easily available is desired.

  An object of the present invention is to provide a composition having a CB1 receptor antagonist activity obtained from a natural product-derived material and having high safety, and a method for producing the same. Furthermore, the present invention relates to a food effective for the treatment, amelioration, suppression or prevention of metabolic syndrome related to obesity, insulin resistance, hyperlipidemia and diabetes, and the treatment or amelioration of a disease or symptom associated with CB1 receptor. It is an object of the present invention to provide a pharmaceutical composition used for suppression, prevention.

  The present inventors searched for CB1 receptor antagonists using various in vitro systems derived from various edible materials. As a result, it has been found that cinnamon extract and cinnamaldehyde, which are materials derived from natural products that can be easily obtained or used daily, have high activity as CB1 receptor-specific antagonists / inverse agonists. It came to.

That is, the present invention includes the following features [1] to [15].
[1] A composition for inhibiting CB1 receptor, comprising cinnamon extract as an active ingredient.
[2] The composition of [1], wherein the cinnamon extract is cinnamaldehyde.
[3] The composition according to [1] or [2], which has an anti-obesity effect.
[4] The composition according to [3], wherein the anti-obesity effect is an effect of improving insulin resistance or lipid metabolism, an effect of suppressing weight gain, or a slimming effect.
[5] A food or drink comprising the composition according to any one of [1] to [4].
[6] The food or drink according to [5], which is labeled to treat, improve, suppress and / or prevent obesity.
[7] The composition according to any one of [1] or [2] for treating, ameliorating, suppressing and / or preventing a disease or symptom associated with the CB1 receptor.
[8] The composition of [7], wherein the disease or symptom associated with the CB1 receptor is insulin resistance, hyperlipidemia, diabetes and obesity.
[9] A pharmaceutical composition comprising the composition of [7] or [8].
[10] A method for producing a composition having CB1 receptor inhibitory activity, comprising adding a cinnamon extract.
[11] The method according to [10], wherein the cinnamon extract is cinnamaldehyde.
[12] The method according to [10] or [11], wherein the composition having CB1 receptor inhibitory activity is a composition having an anti-obesity effect.
[13] The method according to [12], wherein the anti-obesity effect is an effect of improving insulin resistance or lipid metabolism, an effect of suppressing weight gain, or a slimming effect.
[14] The method of [10] or [11], wherein the composition having CB1 receptor inhibitory activity is a composition for treating, ameliorating, suppressing and / or preventing a disease or symptom associated with CB1 receptor.
[15] The method according to [14], wherein the disease or symptom associated with the CB1 receptor is insulin resistance, hyperlipidemia, diabetes and obesity.

  The composition of the present invention can significantly inhibit the activity of the CB1 receptor.

  In addition, since the composition of the present invention is a composition derived from a natural product (food), there is little concern about side effects and the like, and it can be ingested continuously over a long period of time at a low cost.

  The composition of the present invention can be used as a food for the treatment, amelioration, suppression or prevention of lifestyle-related diseases such as insulin resistance, hyperlipidemia, diabetes and obesity due to antagonistic activity against the CB1 receptor. I can do it. Furthermore, the composition of this invention can be utilized for the pharmaceutical composition used for the treatment, improvement, suppression, or prevention of the disease or symptom relevant to CB1 receptor by antagonistic activity with respect to CB1 receptor.

  The composition for inhibiting CB1 receptor in the present invention includes a cinnamon extract obtained by extraction from cinnamon, which is a material derived from a natural product. The cinnamon extract contains cinnamaldehyde as an active ingredient for CB1 receptor inhibition.

  The cinnamon bark is a bark of the plants of the genus Cynonymaceae, Cinnamomum cassia, Annan Nikkei (Cinnamomum obtussifolium), Ceylon nikkei (Cinnamomum verum). As the cinnamon used as the extract extract, those obtained by pulverizing the bark with a mixer or the like, or cinnamon dry powder can be used.

  In the present invention, the bark preservation state, the pulverization method, the variety, or the production area are not particularly limited.

  The cinnamon extract can be obtained by an extraction method known to those skilled in the art, and examples of the extraction method include organic solvent extraction, supercritical fluid extraction method, warming pressure extraction method and the like. Each of the extraction methods may be used alone, or a plurality of extraction methods may be used in combination.

  In the present invention, a particularly preferred extraction method is solvent extraction. Solvent extraction can be performed using general techniques known to those skilled in the art. Specifically, first, water and an organic solvent are added to a pulverized or dried crushed cinnamon bark and extracted at room temperature or with heating.

  Examples of the extraction solvent include, but are not limited to, ethanol, methanol, ethyl acetate, acetone, water, and the like. These solvents may be used alone or in combination of two or more. Preferably, it is desirable to use ethanol in view of safety for use in foods and the like. The amount of the solvent to be added is 1 to 100 times, preferably 5 to 50 times, for example, 10 times the weight of the cinnamon material.

  As an extraction method, for example, cinnamon and the extraction solvent are mixed and stirred for 1 to 5 hours at room temperature or refluxed at the boiling temperature of the extraction solvent for 1 to 5 hours, followed by extraction or filtration or centrifugation. A method of removing the sample residue from the extract and concentrating the extract by reduced pressure or ultrafiltration can be mentioned. Further, the extract can be dried by a general method such as drying, freeze drying, spray drying and the like.

  From the cinnamon extract obtained as described above, cinnamaldehyde can be purified and used in combination with an appropriate separation method and purification method. Examples of the purification method include liquid-liquid partition, organic solvent precipitation, various column chromatography (for example, HPLC, silica gel chromatography, molecular sieve chromatography, ion exchange chromatography, reverse phase chromatography, etc.), crystallization and the like. Can be used.

  The obtained cinnamon extract and / or cinnamaldehyde can be directly ingested as a CB1 receptor antagonist as it is, or can be blended in known carriers and various base materials.

  Cinnamic aldehyde (also known as cinnamaldehyde, (E) -3-phenylpropenal) is known to have agonistic effects on TRP calcium channel proteins, and TRP channel agonists generally induce adipose differentiation. It is thought that aldehydes can also regulate the differentiation-inducing action of adipocytes (Japanese Patent Laid-Open No. 2006-199647). However, the activity of cinnamaldehyde as a CB1 receptor antagonist described below has not been reported so far. Furthermore, none of the cinnamic aldehyde is disclosed as being capable of treating, ameliorating, suppressing or preventing diseases or symptoms such as insulin resistance, hyperlipidemia, diabetes and obesity via the CB1 receptor. The present inventors have found that cinnamaldehyde has CB1 receptor antagonist activity for the first time.

  The composition of the present invention has CB1 receptor inhibitory activity. The activity can be evaluated by a known method, but in vitro, it can be evaluated by an intracellular cAMP accumulation assay using CB1 receptor stably expressing cells. The assay is an experimental system that directly measures CB1 receptor inhibitory activity. Furthermore, it is known that a compound having agonist activity at the CB1 receptor exhibits an effect of lowering body temperature in vivo (J. Pharmacol. Exp. Ther., 1994, 270, 219-227; The Journal of Pharmacology and Experimental Therapeutics, Vol. 284, No. 3, 1209-1217, 1998). Therefore, the CB1 receptor inhibitory activity is determined by pre-administering the composition of the present invention, then administering a CB1 receptor agonist, for example, HU210, and then determining whether the composition of the present invention inhibits the subsequent decrease in body temperature. It can be evaluated by doing.

  The composition of the present invention may also have an anti-obesity effect. “Anti-obesity effect” refers to an effect of suppressing appetite, suppressing body weight gain, reducing body weight, suppressing body fat accumulation, reducing body fat, etc., and is effective in preventing, suppressing, improving and treating obesity. Means effect. In particular, the composition of the present invention may have an anti-obesity effect by inhibiting CB1 receptor, particularly an effect of improving insulin resistance or lipid metabolism, an effect of suppressing weight gain, or a slimming effect.

  Rimonabant, which has been shown to have anti-obesity effects in vivo, is an in vitro intracellular cAMP accumulation assay using human CB1 receptor stably expressing CHO cells (The Journal of Neuroscience, July 15, 1997, 17 (14): From the results of 5327-53333), it has become clear that it has CB1 receptor inhibitory activity. In addition, rimonabant has been shown to have an effect of suppressing body weight gain by oral administration in vivo (Am J Physiol Regul Integr Comp Physiol 284: R345-R353, 2003). From these results, it is clear that there is a correlation between the in vitro CB1 receptor inhibitory activity and the in vivo weight gain inhibitory effect.

  The composition of the present invention has CB1 receptor inhibitory activity, can have an effect of suppressing weight gain due to the inhibitory activity, produces an anti-obesity effect, and can be effective in the prevention, suppression, improvement and treatment of obesity.

  Furthermore, the compositions of the present invention can be used to treat, ameliorate, suppress or prevent diseases or conditions associated with the CB1 receptor. “Disease or condition associated with CB1 receptor” refers to a disease or condition that can be treated, ameliorated, suppressed, or prevented by inhibiting the activity of CB1 receptor. Examples of the disease or symptom include those caused by factors such as increased expression of CB1 receptor gene, activation of CB1 receptor, and increased amount of endogenous cannabinoid as a ligand, such as insulin resistance, hyperlipidemia Disease, diabetes, obesity and the like.

  In one embodiment, the composition of the present invention can be provided as a pharmaceutical composition. The pharmaceutical composition of the present invention has CB1 receptor inhibitory activity. The pharmaceutical composition of the present invention may be effective in treating, ameliorating, suppressing or preventing a disease or condition associated with the CB1 receptor.

  When the composition according to the present invention is provided as a pharmaceutical composition, it can be produced by mixing the cinnamon extract and / or cinnamaldehyde with a pharmaceutically acceptable additive.

  The pharmaceutical composition according to the present invention can be administered orally or parenterally. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. Examples of parenteral preparations include injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, and external preparations (for example, nasal preparations, transdermal preparations, ointments) ), Suppositories (for example, rectal suppositories, vaginal suppositories). These preparations can be formulated using a pharmaceutically acceptable carrier by a technique usually performed in this field. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. For example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter can be used as a carrier.

  Oral preparations contain active ingredients such as excipients (eg lactose, sucrose, starch, mannitol), disintegrants (eg calcium carbonate, carboxymethyl cellulose calcium), binders (eg pregelatinized starch, gum arabic, carboxy Methylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose) or lubricant (eg talc, magnesium stearate, polyethylene glycol 6000) and compression molded, then, if necessary, taste masking, enteric or sustained purposes Therefore, it can be produced by coating by a method known per se. As the coating agent, for example, ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate and Eudragit (Rohm, Germany, methacrylic acid / acrylic acid copolymer) can be used.

  An injection comprises an active ingredient containing a dispersant (for example, Tween 80 (manufactured by Atlas Powder, USA), HCO 60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), a preservative (for example, methylparaben). , Propylparaben, benzyl alcohol, chlorobutanol, phenol), isotonic agents (eg, sodium chloride, glycerin, sorbitol, dextrose, invert sugar) and aqueous solvents (eg, distilled water, physiological saline, Ringer's solution, etc.) Alternatively, it can be produced by dissolving, suspending or emulsifying in an oily solvent (for example, vegetable oil such as olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol). At this time, if desired, additives such as a solubilizing agent (for example, sodium salicylate, sodium acetate), a stabilizer (for example, human serum albumin), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride) are added. Also good.

  The external preparation can be produced by making the active ingredient into a solid, semi-solid or liquid composition. For example, the solid composition is prepared by using the active ingredient as it is, or an excipient (eg, lactose, mannitol, starch, microcrystalline cellulose, sucrose), a thickener (eg, natural gums, cellulose derivatives, acrylic acid). Polymer) and the like can be added and mixed to form a powder. The liquid composition can be produced in almost the same manner as in the case of an injection. The semi-solid composition is preferably an aqueous or oily gel or a cartilage. In addition, these compositions are all pH adjusters (for example, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide), preservatives (for example, p-hydroxybenzoates, chlorobutanol, benzalkonium chloride). Etc. may be included. Suppositories can be produced by making the active ingredient into an oily or aqueous solid, semi-solid or liquid composition. Examples of the oily base used in the composition include higher fatty acid glycerides [for example, cacao butter, witepsols (manufactured by Dynamite Nobel)], intermediate fatty acids [for example, miglyols (manufactured by Dynamite Nobel)], or vegetable oils (for example, , Sesame oil, soybean oil, cottonseed oil). Examples of the aqueous base include polyethylene glycols and propylene glycol. Examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers.

  By administering the pharmaceutical composition of the present invention to a patient suffering from or at risk of suffering from a disease or symptom associated with the CB1 receptor, the disease or symptom can be treated, ameliorated, suppressed or prevented.

  The dosage or intake of cinnamon extract and / or cinnamaldehyde, which are the active ingredients in the pharmaceutical composition of the present invention, is an amount that can give a therapeutic effect for a given symptom or usage, and the amount is used for animals. However, the age, sex, body weight, applicable disease and symptoms, dosage form, administration method, etc. of the subject should be considered. For example, when the pharmaceutical composition of the present invention is an oral preparation, the dose or intake is 0.01 to 20 mg / Kg body weight / day, preferably 0.01 to 8 mg / Kg body weight / day. be able to.

  In another embodiment, the composition of the present invention can be provided as a food or drink. The food / beverage products of this invention have CB1 receptor inhibitory activity. Moreover, the food / beverage products of this invention have an anti-obesity effect by CB1 receptor inhibitory activity, and may be effective for improvement of insulin resistance or lipid metabolism, suppression of weight gain, or slimming.

  The food of the present invention comprises an effective amount of the cinnamon extract and / or cinnamaldehyde. The food can be blended with the cinnamon extract and / or cinnamaldehyde as it is or in the form of a composition containing them.

  More specifically, the food according to the present invention is prepared by pulverizing cinnamon or the above cinnamon extract and / or cinnamaldehyde as food, various proteins, sugars, fats, trace elements, vitamins, etc. Further, it may be prepared by blending, liquid, semi-liquid or solid, paste, or added to general food or drink.

  In the present invention, “food” includes foods classified as health foods, functional foods, foods for specified health use, dietary supplements, foods with a disease risk reduction label, or foods for the sick. The Examples of the disease risk reduction display include a display indicating that it is for treating, improving, suppressing and / or preventing obesity. Therefore, the composition of the present invention is a food or drink with a label indicating that it is for treating, ameliorating, suppressing and / or preventing obesity containing, for example, cinnamon extract and / or cinnamaldehyde.

  Not only the shape of a normal food but also the shape of a dietary supplement such as a supplement may be used.

  According to the present invention, a food comprising an effective amount of an extract having CB1 receptor antagonist activity, the treatment of diseases or conditions such as lifestyle-related diseases such as insulin resistance, hyperlipidemia, diabetes and obesity, Foods used for prevention, control or amelioration are provided.

  Here, the function indications attached to these foods can be any of the main body of the product, the container, the packaging, the instructions, the package insert, or the promotional material.

  Specific examples of the food according to the present invention include carbohydrate-containing foods and drinks such as rice, rice cakes, noodles, breads and pasta; Western confectionery such as cookies and cakes; Japanese confectionery such as buns and sheep candy; candy and gum , Various confectioneries such as frozen confectionery such as yogurt and pudding; ice confectionery; juices, soft drinks, milk beverages, tea beverages, functional beverages, nutritional supplement beverages, non-alcohol beer, etc .; Alcoholic beverages; Food and drink such as soup, miso soup and soup; processed products using eggs, processed products of seafood (squid, octopus, shellfish, eel, etc.) and livestock meat (including organs such as liver) Dashi, soy sauce, mirin and other seasonings;

  The foods to be added and blended are not limited to the following, but as examples of preferred foods, the composition of the present invention can be used for beverages such as cappuccino and Western confectionery such as apple pie.

  For example, the composition of the present invention can be drunk as tea or liquor, or can be added to and blended with confectionery such as jelly or gum.

  Moreover, the food / beverage products of this invention can contain other components, such as a nutritional supplement component, in addition to the composition of this invention, as long as CB1 receptor antagonist activity is not suppressed. Such ingredients include vitamins (eg, vitamin A, vitamin B group, vitamin E, vitamin C, vitamin D, vitamin K, niacin, pantothenic acid, folic acid, etc.), carotenoids (eg, β-carotene, lycopene, fucoxanthin, etc.), Minerals (eg seaweed components, CCM, heme iron, iron salt system, whey calcium, fermented calcium lactate, beef bone calcium, salmon calcium, eggshell calcium, etc.), various plants and their extracts, purified products and fractions (E.g. psyllium, chlorella, spirulina, garlic, ginkgo biloba, gymnema, tochu leaf, shiso leaves, pearl barley, soy globulin, rutin, green tea extract, theanine, polyphenols, licorice, yucca, soy saponin, caffeine, huahua Torberry extract, Champignon extract, Garcinia cambodia ), Microorganisms and their growth factors and microbial products (eg, lactic acid bacteria, yeast, lactic acid bacteria growth factors, etc.), dietary fiber and its enzymatic degradation products (eg, apple fiber, corn fiber, starch-derived dietary fiber, indigestible dextrin) , Guam gum enzyme degradation product, sweet potato fiber, soybean fiber, seaweed fiber, mushroom fiber, tea fiber, acidic polysaccharide, plant mucilage, wheat bran, etc.), animal body and its extract, purified product, decomposed product and product ( For example, royal jelly, propolis, oyster extract, chitin, chitosan, taurine, collagen, gelatin, etc.), various oligosaccharides (eg, galactooligosaccharide, xylo-oligosaccharide, soybean oligosaccharide, fructooligosaccharide, isomaltoligosaccharide, whey oligosaccharide, etc.), lipid (For example, unsaturated fatty acids (DHA, EPA, etc.), phospholipids, salatrim Etc.), various proteins and protein degradation products (for example, corn protein, soy protein, TMP (total milk protein), lactalbumin, casein, whey, glutathione, soy peptide, egg white peptide, glutamine peptide, etc.), wheat germ such as defatted germ Etc.

  Ingestion of the food or drink product of the present invention can produce an anti-obesity effect, and can be effective for improving insulin resistance or lipid metabolism, suppressing weight gain, or slimming.

  The content of cinnamon extract and / or cinnamaldehyde, which is an active ingredient in the food and drink of the present invention, is usually 0.01 to 50% by weight, preferably about 1 to 30% by weight. The daily intake may be 1 to 500 mg, preferably 1 to 200 mg.

  Since the pharmaceutical composition and food and drink of the present invention contain ingredients derived from natural products as active ingredients as described above, there are few worries about side effects and the like, and they can be taken continuously at a low cost for a long period of time.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples.

(Example 1) Preparation of cinnamon low-polarity extract 50 times 80% acetone-containing aqueous solution was added to 300 mg of cinnamon dry powder and stirred at room temperature for 1 hour. After centrifugation, the supernatant was collected and dried under reduced pressure to obtain 29.4 mg. This was dissolved in a 100-fold amount of an 80% methanol-containing aqueous solution, and distributed three times with an equal amount of hexane. The obtained 80% methanol-containing aqueous solution layer was dried under reduced pressure, dissolved in 200 times the amount of water, and then partitioned three times with ethyl acetate. The ethyl acetate layer was dried under reduced pressure to obtain 4.6 mg of an extract.

(Example 2) Establishment of stably expressing CB1 cells A pcDNA3.2 plasmid containing human CB1 receptor cDNA was introduced into Chinese hamster ovary cells (CHO cells) using FuGENE 6 (Roche). After gene transfer, the cells were selected with 1.5 mg / ml GENETICIN (GIBCO), and a GENETICIN resistant clone was grown to establish a cell line. Similarly, human CB2 receptor stably expressing cells were established.

(Example 3) Examination of CB1 receptor antagonist activity and concentration dependency of extract The cinnamon low polarity extract prepared in Example 1 was dissolved in ethanol so as to be 100 mg / ml, and CB1 receptor antagonist activity was obtained. evaluated.

CHO cells stably expressing the human CB1 receptor prepared in Example 2 were seeded in a 96-well culture plate so as to be 1 × 10 ⁵ cells / well, and the condition was about 18 hours under conditions of 37 ° C. and 5% CO _2. Cultured. As the medium, α-MEM, 100 μl / well containing 10% FBS, 500 U / ml penicillin / streptomycin solution, 1.5 mg / ml GENETICIN was used. 18 hours after sowing, the medium was replaced with a medium containing the cinnamon low polarity extract prepared in Example 1, forskolin, and CB1 receptor agonist (HU210) as shown in Table 1 below, and cultured for 30 minutes.

Thereafter, using a commercially available measurement kit (cAMP-Screen ^™ System, Applied Biosystems), the cell lysate was collected and the amount of intracellular cAMP was quantified. Inhibition of forskolin-dependent cAMP accumulation upon addition of the extract and HU210 to 100% as the degree of inhibition of forskolin-dependent cAMP accumulation upon addition of HU210 (cAMP amount ((2)-(3)), see Table 1) The degree (cAMP amount ((4)-(3)), see Table 1) was relatively expressed as CB1 receptor antagonist activity (%). Experiments were performed on 3 cases per group. As a result of setting and measuring the addition concentration of the cinnamon low polarity extract in several steps, an increase in CB1 receptor antagonist activity dependent on the addition concentration was observed (see FIG. 1).

(Example 4) Evaluation of CB1 receptor specificity In the same manner as in Example 3, the CB2 receptor antagonist activity of the cinnamon low polarity extract prepared in Example 1 was evaluated and compared with the CB1 receptor antagonist activity. The CB1 receptor specificity was evaluated. The Ki value of HU210 is 0.061 nM and 0.52 nM for the CB1 receptor and CB2 receptor, respectively. Therefore, the concentration of HU210 was 1 nM when measuring CB1 receptor antagonist activity, and 10 nM when measuring CB2 receptor antagonist activity. Experiments were performed on 3 cases per group. As a result of measuring the CB1 receptor and CB2 receptor antagonist activity of the cinnamon low polarity extract (added at a final concentration of 0.06 mg / ml), the CB2 receptor antagonist activity was 50% or less of the CB1 receptor antagonist activity. CB1 receptor specificity of cinnamon low polarity extract containing aldehyde was observed (see FIG. 2).

(Example 5) Preparation of ethanol extract of cinnamon 20-fold amount of 100% ethanol was added to 6.35 g of cinnamon dry powder and stirred at room temperature for 1 hour. The supernatant was collected after centrifugation, the same amount of 100% ethanol was added to the residue, and the mixture was stirred at room temperature for 1 hour. The supernatant was collected after centrifugation, combined with the previous supernatant, and dried under reduced pressure to obtain 589.7 mg of extract. This was dissolved in ethanol so as to be 100 mg / ml, and CB1 receptor antagonist activity was evaluated.

(Example 6) Measurement of CB1 receptor antagonist activity of cinnamon ethanol extract As in Example 3, cinnamon ethanol extract containing cinnamaldehyde prepared in Example 5 (final concentration is 0.2 mg / ml) Were evaluated for CB1 receptor antagonist activity. Experiments were performed on 3 cases per group. As a result, 97.48 ± 9.56% of CB1 receptor antagonist activity was observed.

(Example 7) Identification of active ingredient In order to grasp the component profile contained in the cinnamon low polarity extract prepared in Example 1, a high performance liquid chromatography-photodiode array-mass spectrometer (LC-PDA-MS) was used. We examined using. The conditions are as follows.

[conditions]
Equipment: LC; Waters 2695 separation module, PDA; Waters 2996 photodiode array detector, MS; Waters micromass ZQ (all from Japan Waters)
Column: Atlantis T3 4.6 x 250 mm (Nihon Waters)
Column temperature: 40 ° C
Flow rate: 1.0 ml / min (1/4 volume introduced using splitter for mass spectrometer)
Solvent A: Water (including 0/1% formic acid)
Solvent B: Methanol (including 0/1% formic acid)
Gradient time program: 30% B → 100% B (0min → 25min)
Capillary voltage: 3.0kV
Cone voltage: 30V

  As a result, a plurality of peaks were observed at a detection wavelength of 280 nm (see FIG. 3A). Since the main component of cinnamon essential oil is cinnamon aldehyde, cinnamaldehyde was also examined under the same conditions, and the retention time completely coincided with peak (1), which is the main peak observed in the cinnamon low polarity extract ( (See FIG. 3B). Moreover, the mass spectrum of peak (1) and cinnamaldehyde also coincided (see FIGS. 4A and B, respectively). From the above, it was found that the peak (1) observed in the cinnamon low polarity extract was cinnamaldehyde. A calibration curve was prepared using the sample and the amount of cinnamaldehyde in the cinnamon low polarity extract was determined. The cinnamaldehyde content in the extract was 15% (w / w).

  The CB1 receptor antagonist activity of cinnamic aldehyde was measured by the same method as in Example 3 (only the concentration of HU210 (10 nM) was different), and compared with cinnamon low polarity extract. Cinnamic aldehyde was dissolved in ethanol. Experiments were performed on 3 cases per group. As a result, CB1 receptor antagonist activity dependent on cinnamaldehyde was observed, and 0.2 mg / ml cinnamon low polarity extract and 0.03 mg / ml cinnamaldehyde showed similar activities. From the results of the component analysis described above, it is known that 0.03 mg / ml of cinnamaldehyde is contained in 0.2 mg / ml of cinnamon low polarity extract. From these facts, cinnamaldehyde was identified as the active body of the cinnamon low polarity extract (see FIG. 5).

(Example 8) Evaluation of CB1 receptor antagonist activity intensity of cinnamic aldehyde In order to evaluate the intensity of CB1 receptor antagonist activity of cinnamic aldehyde, the activity of an existing CB1 receptor antagonist agent (AM251) was compared. . The method is the same as in Example 7. Experiments were performed on 3 cases per group. As a result, the IC ₅₀ of cinnamic aldehyde was 172.18 μM, the IC ₅₀ of AM251 was 0.13 μM, and the CB1 receptor antagonist activity of cinnamic aldehyde was about 1/1318 of AM251 (FIG. 6). See).

(Example 9) In vivo evaluation of CB1 receptor antagonist activity of cinnamon low polarity extract In vivo CB1 receptor antagonist activity of cinnamon low polarity extract prepared in Example 1 was pre-administered with the extract. Thereafter, a CB1 receptor agonist (HU210) was administered, and the subsequent evaluation was based on whether the extract inhibited the decrease in body temperature caused by HU210. For the experiment, JcI: ICR male mice (5 weeks old) were used. The cinnamon low polarity extract was suspended in a water-soluble polymer (PUREBRIGHT) at a concentration of 7.5% and administered intraperitoneally at 10 ml / kg body weight (final concentration; 75 mg / kg body weight). Only the solvent was administered to the control group and the vehicle group. Thirty minutes later, rectal temperature was measured with a thermometer (Physitemp BAT-12) connected to a body temperature measurement probe (this value was used as a reference value). Further, 15 minutes later, physiological saline containing HU210 (10 μg / ml) dissolved in 0.15% DMSO and 1% Tween 80 was intraperitoneally administered at 10 ml / kg body weight (final concentration; 100 μg / kg body weight). Only the solvent was administered to the control group. Thereafter, rectal temperature was measured every 30 minutes up to 90 minutes, and after 90 minutes every 90 minutes until 270 minutes. Experiments were conducted with 5 cases per group. The horizontal axis represents the time after administration of HU210, and the vertical axis represents the difference between the rectal temperature at each time and the reference value.

  As a result, in the above model, it was revealed that the cinnamon low polarity extract can inhibit the decrease in body temperature after administration of HU210 and has an excellent CB1 receptor antagonist activity even in vivo (FIG. 7). See).

(Example 10) In vivo evaluation of CB1 receptor antagonist activity of cinnamic aldehyde In the same manner as in Example 9, CB1 receptor antagonist activity in vivo of cinnamic aldehyde was measured. Cinnamic aldehyde dissolved in ethanol was suspended in physiological saline and administered intraperitoneally at 10 ml / kg body weight (final concentration; 11.25 mg / kg body weight). Experiments were conducted with 5 to 8 cases per group. As a result, it was revealed that cinnamic aldehyde can inhibit a decrease in body temperature after administration of HU210 and has an excellent CB1 receptor antagonist activity even in vivo (see FIG. 8).

  The composition of the present invention has CB1 receptor inhibitory activity and can be used for the treatment, amelioration, suppression and / or prevention of anti-obesity and diseases and / or symptoms associated with CB1 receptor.

FIG. 1 shows the analysis results of CB1 receptor antagonist activity of cinnamon low polarity extract. FIG. 2 shows the analysis results on the specificity of the cinnamon low polarity extract for the CB1 receptor. FIG. 3 shows the analysis result of the component profile contained in the cinnamon low polarity extract using LC-PDA-MS. A shows a chromatogram of a cinnamon low polarity extract, and B shows a chromatogram of cinnamaldehyde. FIG. 4 shows the results of mass spectrometry of peak (1) in FIG. 3 and cinnamic aldehyde. A shows the mass spectrum of peak (1), and B shows the mass spectrum of cinnamic aldehyde. FIG. 5 shows the results of comparative analysis of the CB1 receptor antagonist activity of cinnamon low polarity extract and cinnamaldehyde. FIG. 6 shows the analysis results of the CB1 receptor antagonist activity intensity of cinnamic aldehyde. FIG. 7 shows the analysis result of CB1 receptor antagonist activity in vivo of cinnamon low polarity extract. The horizontal axis represents the time after administration of HU210, and the vertical axis represents the difference between the rectal temperature at each time and the reference value. FIG. 8 shows the results of analysis of CB1 receptor antagonist activity of cinnamic aldehyde in vivo. The horizontal axis represents the time after administration of HU210, and the vertical axis represents the difference between the rectal temperature at each time and the reference value.

Claims (15)

  A composition for inhibiting CB1 receptor, comprising cinnamon extract as an active ingredient.   The composition according to claim 1, wherein the cinnamon extract is cinnamon aldehyde.   The composition according to claim 1 or 2, which has an anti-obesity effect.   The composition according to claim 3, wherein the anti-obesity effect is an effect of improving insulin resistance or lipid metabolism, an effect of suppressing weight gain, or a slimming effect.   Food-drinks containing the composition of any one of Claims 1-4.   The food / beverage product of Claim 5 which attached | subjected the display to the effect of treating, improving, suppressing, and / or preventing obesity.   The composition according to claim 1 or 2, for treating, ameliorating, suppressing and / or preventing a disease or condition associated with the CB1 receptor.   8. The composition of claim 7, wherein the disease or condition associated with the CB1 receptor is insulin resistance, hyperlipidemia, diabetes and obesity.   A pharmaceutical composition comprising the composition according to claim 7 or 8.   A method for producing a composition having CB1 receptor inhibitory activity, comprising adding a cinnamon extract.   The method according to claim 10, wherein the cinnamon extract is cinnamon aldehyde.   The method according to claim 10 or 11, wherein the composition having CB1 receptor inhibitory activity is a composition having an anti-obesity effect.   The method according to claim 12, wherein the anti-obesity effect is an effect of improving insulin resistance or lipid metabolism, an effect of suppressing weight gain, or a slimming effect.   The method according to claim 10 or 11, wherein the composition having CB1 receptor inhibitory activity is a composition for treating, ameliorating, suppressing and / or preventing a disease or condition associated with CB1 receptor.   15. The method of claim 14, wherein the disease or condition associated with the CB1 receptor is insulin resistance, hyperlipidemia, diabetes and obesity.

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