TRPV1 activator

Abstract

To provide a novel TRPV1 activator.SOLUTION: Provided is a TRPV1 activator containing Brazilian green propolis and culifolin as active ingredients.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a TRPV1 (Transient receptor potential cation channel subfamily V member 1) activator. [Background technology]

[0002] In recent years, with the worldwide increase in obesity patients, preventive strategies for obesity through exercise and the intake of dietary supplements have attracted attention. Specifically, obesity prevention strategies that suppress lipid absorption, such as epigallocatechin gallate and indigestible dextrin, are known. However, these strategies only temporarily suppress lipid absorption and do not fundamentally improve physical constitution.

[0003] Adipose tissue is significantly involved in obesity in mammals. Adipose tissue is divided into white adipose tissue and brown adipose tissue. Of these, brown adipose tissue consumes fat to generate body heat and maintain body temperature. Brown adipose tissue also declines with age, resulting in an inability to consume fat and the induction of obesity. Therefore, in order to prevent the decline of brown adipose tissue, attempts have been made to increase brown adipocytes through differentiation into brown adipocytes, which constitute brown adipose tissue. For example, a brown adipocyte differentiation inducer containing artepillin C and curcumin as active ingredients is known (see, for example, Patent Document 1).

[0004] Meanwhile, a new anti-obesity strategy that targets thermosensitive receptor (TRP) channels using food components has attracted attention. TRP channels are known as receptors for cold stimuli, and the cold stimuli they receive induce heat production in brown adipocytes, contributing to maintaining body temperature. The heat production and energy consumption activities of brown adipocytes are expected to not only contribute to the thermoregulation of individual organisms, but also to the prevention of obesity and metabolic diseases by changing the body's constitution to be more energy-efficient.

[0005] For example, a specific target TRP channel is TRPV1 (Transient receptor potential cation channel subfamily V member 1), which belongs to the TRP subfamily. TRPV1 is known as a receptor for capsaicin, the spicy component of chili peppers.

[0006] Activation of TRPV1 induces sympathetic nervous excitation via increased release of catecholamines (norepinephrine; NE) from the adrenal medulla and other areas. Sympathetic nervous excitation activates β3-adrenergic receptors, which are receptors for sympathetic neurotransmitters, and induces the expression of the thermogenic protein UCP1 (uncoupling protein 1) in brown adipocytes. As a result, energy consumption by brown adipocytes is induced, promoting body thermogenesis.

[0007] A TRPV1 activator containing a TRPV1 activator as an active ingredient can activate brown adipocytes, promote body thermogenesis, and exert an anti-obesity effect. A known TRPV1 activator contains menthyl 3-hydroxybutyrate as an active ingredient (see, for example, Patent Document 2). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6488504 [Patent Document 2] Japanese Patent Publication No. 2022-165316 Summary of the Invention [Problem to be solved by the invention]

[0009] The brown adipocyte differentiation inducer described in Patent Document 1 is highly safe because it contains naturally derived artepillin C and curcumin as active ingredients. However, according to research by the present inventors, artepillin C, the main ingredient of these, does not have the effect of activating TRPV1, nor does it have the effect of activating brown adipocytes.

[0010] The TRPV1 activator described in Patent Document 2 contains the active ingredient menthyl 3-hydroxybutyrate, which is a chemically synthesized substance, and therefore has safety concerns for living organisms.

[0011] Therefore, an object of the present invention is to provide a TRPV1 activator that is naturally derived and contains, as an active ingredient, a component that has a TRPV1 activating effect. [Means for solving the problem]

[0012] To achieve the above-mentioned objective, the present inventors conducted extensive research and attempted to identify a natural product with TRPV1 activation activity from among the many available. Among these, they focused on propolis. Propolis is a resinous or waxy substance made by mixing plant buds and resins collected by honeybees with their saliva. Because propolis is derived from plants, its components vary depending on the place of origin, and its physiological effects also differ accordingly.

[0013] For example, in Brazilian green propolis, which is derived from Baccharis dracunculifolia as the main source plant, cinnamic acid derivatives such as artepillin C, drupanin, cliforin, p-coumaric acid, kaempferide, 6-methoxykaempferide, dihydrokaempferide, and baccharin are detected as the main components.On the other hand, in propolis from China, Europe, and Australia, which is derived from poplar as the main source plant, flavonoids such as chrysin and galangin are detected as the main components.

[0014] As mentioned above, Brazilian green propolis contains artepillin C as its main component. However, artepillin C does not have the effect of activating TRPV1, nor does it have the effect of activating brown adipocytes. This led to the assumption that Brazilian green propolis does not have the effect of activating TRPV1. However, surprisingly, the present inventors have found that Brazilian green propolis has the effect of activating TRPV1, and also has the effect of activating brown adipocytes and inducing body thermogenesis.

[0015] Furthermore, the present inventors investigated the component in Brazilian green propolis that exerts the above-mentioned effects and discovered that it is cliffolin.

[0016] Based on these findings, the present inventors have succeeded in creating a TRPV1 activator containing Brazilian green propolis and / or cliffolin as active ingredients, which are naturally derived and have TRPV1 activating activity, in order to solve the problems of the present invention. The present invention has been completed based on the findings and successful examples first discovered by the present inventors.

[0017] Thus, the following aspects of the present invention are provided: [1] A TRPV1 activator containing Brazilian green propolis as its active ingredient. [2] A thermogenic agent containing Brazilian green propolis as an active ingredient. [3] A brown fat cell activator containing Brazilian green propolis as an active ingredient. [4] A TRPV1 activator containing clifforin as an active ingredient. [5] A thermogenic agent containing criforin as an active ingredient. [6] A brown fat cell activator containing criforin as an active ingredient. [7] A food, drink, medicine, or quasi-drug containing the agent described in any one of [1] to [6]. [Effects of the Invention]

[0018] Brazilian green propolis and cliffolin have excellent TRPV1 activation properties and are therefore useful as active ingredients in TRPV1 activators.

[0019] Furthermore, the TRPV1 activator of one embodiment of the present invention is highly safe because its active ingredient is naturally derived Brazilian green propolis, which is commonly consumed, and cliffolin, which is contained in Brazilian green propolis. Thus, the TRPV1 activator of one embodiment of the present invention can be used for activating TRPV1 in various forms, such as foods and beverages, pharmaceuticals, and quasi-drugs. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows temperature changes over time in the rectum and brown adipose tissue when artepillin C and a TRPV1 inhibitor were administered to mice in which beige adipose tissue formation was induced. [Figure 2] Figure 2 shows temperature changes over time in the rectum and brown adipose tissue when Brazilian green propolis and a TRPV1 inhibitor were administered to mice in which beige adipose tissue formation was induced. [Figure 3] FIG. 3 shows the amount of norepinephrine in plasma when artepillin C, Brazilian green propolis, and a TRPV1 inhibitor were administered to mice in which beige adipose tissue formation was induced. [Figure 4] Figure 4 shows the change in calcium signal intensity over time and its AUC when each material was added to HEK293 cells overexpressing TRPV1, and the change in calcium signal intensity over time for cliffolin when a TRPV1 inhibitor was added at various concentrations. [Figure 5] FIG. 5 shows temperature changes over time in the rectum and brown adipose tissue when cliffolin and Brazilian green propolis were administered to mice in which beige adipose tissue formation was induced. [Figure 6]FIG. 6 is a graph showing the amount of norepinephrine in plasma when clifolin and Brazilian green propolis were administered to mice in which beige adipose tissue formation was induced. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, the embodiments for carrying out the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0022] The term "content" is synonymous with concentration and amount added (amount used), and refers to the ratio of the amount of a component to the total amount of the composition. However, the total amount of the components does not exceed 100%. When a commercially available product is used, the content of the component is preferably the amount of the component contained in the commercially available product, but may also be the amount of the commercially available product itself. "Food and beverage" is a generic term meaning food, beverages, or both. "Comprise," "contain," and "include" mean that elements other than those explicitly stated as included may be added (same meaning as "comprise at least"), but also encompass "consist of" and "essentially consist of." That is, "comprise" can mean including the explicitly stated elements and any one or more elements, consisting of the explicitly stated elements, or essentially consisting of the explicitly stated elements. Elements include limitations such as ingredients, steps, conditions, and parameters. The "to" in a numerical range means a range that includes both the preceding and following values, and also includes a range excluding one of the included limits. For example, "0% to 100%" means that the range may be greater than or equal to 0% and less than or equal to 100%.

[0023] A TRPV1 activator according to one embodiment of the present invention comprises at least one active ingredient selected from the group consisting of Brazilian green propolis and cliffolin. In this specification, Brazilian green propolis, cliffolin, and both are collectively referred to as the "active ingredient." The TRPV1 activator according to one embodiment of the present invention can activate TRPV1 by comprising the active ingredient.

[0024] Activation of TRPV1 leads to increased release of catecholamines (norepinephrine; NE) from the adrenal medulla and other organs, inducing sympathetic nerve excitation. Sympathetic nerve excitation induces the expression of the thermogenic protein UCP1 (uncoupling protein 1) in brown and beige adipocytes via the β3-adrenergic receptor, a sympathetic nerve receptor, thereby inducing energy consumption and promoting body heat production. Therefore, activating TRPV1 with a TRPV1 activator according to one embodiment of the present invention is expected to promote body heat production and activate brown adipocytes. Therefore, another embodiment of the present invention is a body heat production promoter containing an active ingredient. Another embodiment of the present invention is a brown adipocyte activator containing an active ingredient. The TRPV1 activator will be described in detail below, but these descriptions can also be used for the body heat production promoter and brown adipocyte activator.

[0025] [Brazilian Green Propolis] One embodiment of the TRPV1 activator of the present invention contains Brazilian green propolis as an active ingredient. Propolis is a resinous or waxy substance that forms the walls of beehives and is made by mixing plant shoots or resins collected by bees with bee saliva. Propolis can be obtained, for example, as a beekeeping product using conventional methods. Brazilian green propolis is selected from those produced in the state of Minas Gerais in the Federative Republic of Brazil. Propolis from Minas Gerais in the Federative Republic of Brazil (hereinafter referred to as Brazilian green propolis) may also be used in combination with propolis from other regions, such as China, Taiwan, Europe, Russia, Oceania, and the United States. Brazilian green propolis may be of any grade, such as super green or ultra green.

[0026] Brazilian green propolis is not particularly limited as long as it is collected in the state of Minas Gerais in the Federative Republic of Brazil. Among them, Brazilian green propolis whose source plant is alexrin is preferred. Alexrin is Baccharis dracunculifolia, a member of the Baccharis genus in the Asteraceae family.

[0027] Brazilian green propolis may be, for example, raw propolis or a processed propolis product obtained by subjecting raw propolis to some processing. The processed propolis product may be, for example, raw propolis that has been subjected to processing such as pulverization, extraction, concentrating or powdering the extract, or granulating the powder, or may be the extraction residue remaining after extraction. That is, the processed propolis product may be, for example, a pulverized propolis product, an extract, a concentrated extract, an extract powder, an extract granules, or an extraction residue. The extraction may be, for example, water extraction, hydrophilic organic solvent extraction, or supercritical extraction. Examples of hydrophilic organic solvents include ethanol, glycerin, and 1,3-butylene glycol. The propolis extract may be obtained by extraction from raw propolis or by further extraction from the residue after extraction. One processing method may be used, or two or more methods may be combined. A hydrophilic organic solvent extract of propolis is preferred as the processed propolis product, since it allows the active ingredients of propolis to be extracted efficiently and in a balanced manner in a short period of time. The processed propolis is preferably a propolis ethanol extract.

[0028] The propolis extract includes the recovered extract (including further purified extracts as necessary), concentrates obtained by concentrating the extract, and solids obtained by removing the solvent from the extract by freeze-drying, spray-drying, etc. The concentration, freeze-drying, and spray-drying of the extract can be carried out according to conventional methods. The propolis extract of the present invention also includes powders obtained by powdering such extracts and granules obtained by granulating the powders.

[0029] Commercially available propolis or its extract may be used. Specific examples of commercially available products containing Brazilian green propolis include Yamada Bee Farm's Propolis 300, Propolis Liquid 30, Propolis Granules, Propolis Granules APC, Propolis Mild, and Propolis Drink.

[0030] [Clifolin] Another embodiment of the TRPV1 activator of the present invention comprises clifforin as an active ingredient. The structural formula of clifforin is shown below. [ka]

[0031] Clifolin can be used in either an unisolated or unpurified state (crude extract) or an isolated or purified state. Criforin can be used whether it is a homemade product or a commercially available product. Here, the method for homemade preparation of clifforin is not particularly limited, and examples thereof include a method of extracting clifforin from plants or natural products containing clifforin, and a method of chemically synthesizing clifforin.

[0032] [Active ingredient] The TRPV1 activator may contain Brazilian green propolis, cliffolin, or both as active ingredients.

[0033] The content of Brazilian green propolis in the TRPV1 activator is not particularly limited as long as it is within a range in which the effects of the present invention can be obtained, and can be appropriately adjusted depending on the final form, etc., and can be, for example, 0.00001% by mass or more, 0.0001% by mass or more, 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, or 30% by mass or more, based on the total solid content of the TRPV1 activator. % by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60 quality The amount may be % or less, 55% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less.

[0034] Furthermore, the content of clifforin in the TRPV1 activator is not particularly limited as long as it is within a range in which the effects of the present invention can be obtained, and can be appropriately adjusted depending on the final form, etc., and can be, for example, 0.00000001 mass% or more, 0.0000001 mass% or more, 0.000001 mass% or more, 0.00001 mass% or more, 0.0001 mass% or more, 0.0005 mass% or more, 0.001 mass% or more, 0.003 mass% or more, 0.005 mass% or more, 0.007 mass% or more, 0.01 mass% or more, 0.015 mass% or more, or ...15 mass% or more, based on the total solid content of the TRPV1 activator. 0.02 mass% or more, 0.03 mass% or more, 0.04 mass% or more, 0.05 mass% or more, 0.06 mass% or more, 0.07 mass% or more, 0.08 mass% or more, or 0.09 mass% or more, and 100 mass% or less, 90 mass% or less, 80 mass% or less , 70% by mass or less, 60% by mass or less, 55% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less.

[0035] The TRPV1 activator can be administered to humans, for example, for use in activating TRPV1 in the body. The TRPV1 activator can be administered orally or parenterally. Oral administration includes enteral administration, and parenteral administration includes topical administration and transdermal administration.

[0036] The dosage of a TRPV1 activator when administered orally varies depending on the active ingredient, composition form, and application method / amount. For example, if the active ingredient is Brazilian green propolis, it can be used at a daily dose of 1 μg to 10,000 mg, preferably 2 μg to 9,000 mg, more preferably 3 μg to 8,000 mg, and even more preferably 4 μg to 7,000 mg, calculated on dry solids for a 60 kg adult. For example, if the active ingredient is cliffolin, it can be used at a daily dose of 1 ng to 10,000 μg, preferably 2 ng to 9,000 μg, more preferably 3 ng to 8,000 μg, and even more preferably 4 ng to 7,000 μg, calculated on dry solids for a 60 kg adult. The dosage can be increased or decreased as appropriate depending on factors such as the health condition of the person taking the drug, the method of administration, the type of active ingredient, and the combination with other agents.

[0037] As long as the effective daily dose is within the above-mentioned range, the TRPV1 activator may be administered once a day or in divided doses such as twice or three times a day. The TRPV1 activator exhibits its effects immediately after administration, but continuous administration for 1 to 4 weeks, or for 1 month or more, 6 months or more, or 1 year or more is preferred because the effect can be sustained longer.

[0038] TRPV1 activators can be used as foods and beverages (particularly foods and beverages intended for health, health maintenance, or health promotion (e.g., health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. The term "TRPV1 activator" also encompasses the meaning of additives that impart TRPV1 activation activity.

[0039] A food or drink, quasi-drug, or pharmaceutical product containing a TRPV1 activator as one of its ingredients can be produced, for example, by adding the TRPV1 activator to an intermediate product in the manufacturing process of these products.

[0040] The above-mentioned foods and beverages contain, in addition to the active ingredient, other ingredients, such as minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, fragrances, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solubilizers, humectants, etc.

[0041] Food and beverage products include any food and beverage product that can be consumed by animals (including humans). The types of food and beverage products are not particularly limited, and examples include dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and alcoholic beverages such as fruit wine); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery products (chocolate, donuts, pies, cream puffs, gum, gummy candies, jellies, candies, cookies, cakes, puddings, etc.); Japanese confectionery products (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, popsicles, sorbets, etc.); foods (curry, beef bowls, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0042] The method for producing the food or drink is not particularly limited, and any known method can be used as appropriate.

[0043] The dosage unit form when used as a supplement is not particularly limited and can be selected appropriately, and examples include chewable tablets, troches, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

[0044] The above pharmaceuticals contain, in addition to the active ingredient, other ingredients, such as vitamins, herbal medicines, etc. The active ingredient and other ingredients can also be mixed with other pharmaceutical ingredients listed in the Japanese Pharmacopoeia before use.

[0045] When preparing a TRPV1 activator as a pharmaceutical, the active ingredient can be prepared together with pharmaceutical acceptable ingredients into a form such as a tablet (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, and lozenges), capsule, pill, powder (powder), fine granules, granules, liquid, suspension, emulsion, syrup, paste, or injection (including when mixed with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion to prepare a liquid at the time of use), to form a pharmaceutical preparation.

[0046] Pharmaceuticals may be administered locally or systemically. There are no particular limitations on the administration method, and they may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intraarterial administration, and transdermal administration.

[0047] In addition to the active ingredient, pharmaceuticals may contain, as appropriate, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, antiseptics, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances.

[0048] The pharmaceuticals of the present invention also include quasi-drugs.

[0049] The TRPV1 activator according to one embodiment of the present invention described above is applicable to mammals including humans (preferably humans).

[0050] Furthermore, the TRPV1 activator of one embodiment of the present invention is highly safe because it uses Brazilian green propolis, which has traditionally been used as a food ingredient, and its component, cliffolin. [Example]

[0051] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0052] [Thermogenesis Assessment (1)] The thermogenic potential of Brazilian green propolis (BGP, Yamada Bee Farm) and artepillin C (ArtC, Yamada Bee Farm) was evaluated by measuring tissue temperature using a temperature probe.

[0053] Four-week-old male C57BL / 6J mice (Japan SLC) were purchased and used in the experiment. Mice were housed individually, one per cage, at a room temperature of 22±3°C with a 12-hour day / night cycle (light period: 8:00-20:00). CE-2 feed (CLEA Japan) was used. A 7-day acclimation period was set aside to allow the mice to become accustomed to oral administration, during which time all mice were orally administered 100 μl of saline per day.

[0054] After the acclimation period, mice were intraperitoneally administered 1 mg / kg of β3-adrenergic receptor agonist CL316,243 (Sigma-Aldrich, C5976-5MG) for 7 days to induce beige fat formation. After administration, mice were used as beige fat-induced mice for the study. To eliminate the effects of CL316,243 remaining in the body, saline was then intraperitoneally administered for 3 days.

[0055] The beige adipose tissue-induced mice were divided into six test groups as shown in Table 1. The AMG517-administered group received 40 mg / kg of AMG517 (Selleck, S7115), a TRPV1 inhibitor, orally for three days. According to Table 1 below, the test groups were designated as the control group (Control group), Control + AMG517 group, ArtC group, ArtC + AMG517 group, BGP group, and BGP + AMG517 group, depending on the sample administered at the time of tissue temperature measurement.

[0056] [Table 1]

[0057] Ten minutes before the start of tissue temperature measurement, AMG517 (40 mg / kg) was orally administered to the AMG517-treated group, and vehicle (5% DMSO-4% Tween 80; 5 ml / kg) was orally administered to the AMG517-free group. Subsequently, as shown in Table 1, vehicle (5 ml / kg) was orally administered to the Control and Control + AMG517 groups, ArtC (10 mg / kg) was orally administered to the ArtC and ArtC + AMG517 groups, and BGP (115 mg / kg) was orally administered to the BGP and BGP + AMG517 groups.

[0058] For mice receiving each sample and undergoing beige adipose tissue induction, rectal and interscapular brown adipose tissue (iBAT) tissue temperatures were measured every 5 minutes using a digital thermometer (Physitemp, BAT-12R) and a needle-type temperature probe (Physitemp, MT-29, RET-3) under inhalation anesthesia with gaseous isoflurane (0.8 L / min). Rectal tissue temperature was measured as core body temperature, which is less affected by the external environment, and was used as an index of changes in body heat production.

[0059] After 60 minutes of tissue temperature measurement, blood was sampled. Body weight was measured, and blood was collected from the heart under isoflurane anesthesia (0.8 L / min) into a tube containing 5 μL of heparin sodium (10,000 units / 10 mL). Plasma was collected from the collected blood, and the norepinephrine (NE) concentration in the plasma was measured by standard methods. The collected blood was centrifuged at 5,000 rpm at 4°C for 5 minutes to obtain plasma. The amount of norepinephrine (NE) in the plasma was measured using an NE-specific ELISA kit (Abnova, KA1891) according to the kit's protocol.

[0060] Temperature changes were analyzed at each time point, and significant differences between the administration of inhibitors and the non-administration were determined by t-test (*: p<0.05). Plasma NE levels were analyzed for normality, and significant differences between groups were determined by ANOVA analysis of variance and Tukey's test (significant differences between different letters (ab, ac, bc) were detected, p<0.05).

[0061] [Evaluation of BGP-related components] The TRPV1-activating components in BGP were explored and evaluated based on their calcium signal activation ability using TRPV1-overexpressing cells.

[0062] 2 × 10 HEK293 cells, a human embryonic kidney-derived cell line 4 The cells were seeded into a 96-well plate (Perkin) at 100 cells / well and cultured overnight. TRPV1-overexpressing cells were then transfected with the TRPV1 plasmid (Vector Builder) using FuGene IIHD (Promega, E2311) and cultured overnight.

[0063] Calcium signals were analyzed using the Fluo 4-AM kit (Dojin). TRPV1-overexpressing cells were loaded with the Fluo 4-AM calcium indicator according to the Fluo 4-AM kit protocol. The medium was removed and the cells were pretreated with the Fluo 4-AM kit's loading buffer for 30 minutes. The loading buffer was then removed, and assay medium was added. The plate was then placed on a fluorescent plate reader (Envision, Perkin). The fluorescence intensity (λex = 340 nm / 380 nm, λem = 510 nm) was read for 10 seconds to obtain a baseline.

[0064] Next, seven components of BGP that are relatively abundant in BGP—artepillin C (ArtC), drupanin, culirolin, p-cumaric acid, kaemperide, 6-methoxykaemperide, and dihydrokaemperide—were added to TRPV1-overexpressing cells at 10 μM, and the fluorescence intensity was monitored for another 90 seconds. In the AMG517 and culirolin combination experiment, AMG517 was added to TRPV1-overexpressing cells at 0 μM, 1 μM, 2 μM, or 10 μM, and a baseline was obtained 5 minutes later using the same method. Next, culirolin was added to TRPV1-overexpressing cells at 10 μM, and the fluorescence intensity was monitored for another 90 seconds. Vehicle (DMSO) was used as a control.

[0065] The obtained signal was corrected by the average baseline value, and the waveform and signal intensity were evaluated by calculating the difference between the maximum signal value and the minimum signal value, which was then used to evaluate the signal value. The test was performed five times, and the signal value was expressed as the mean ± standard deviation. The signal value was analyzed for normality, and significant differences from the vehicle were determined using ANOVA analysis of variance and Dunnett's test (**: p<0.01).

[0066] [Body heat production evaluation (2)] The thermogenic potential of Brazilian green propolis (BGP, Yamada Bee Farm), artepillin C (ArtC, Yamada Bee Farm), and culifolin (Yamada Bee Farm) was evaluated by measuring tissue temperature using a temperature probe.

[0067] Beige adipose tissue-induced mice were prepared in the same manner as described above in [Evaluation of body heat production (1)].

[0068] Beige adipose tissue-induced mice were divided into three test groups as shown in Table 2. According to Table 2 below, each test group was designated a control group, a BGP group, or a culifolin group, depending on the sample administered at the time of tissue temperature measurement. 0.165 mg / kg body weight of culifolin corresponds to the amount of culifolin contained in 115 mg / kg body weight of BGP.

[0069] [Table 2]

[0070] Immediately before the start of tissue temperature measurement, the control group was orally administered vehicle (5 ml / kg), the BGP group was orally administered BGP (115 mg / kg), and the culifolin group was orally administered culifolin (0.165 mg / kg).

[0071] For the beige adipose tissue-induced mice administered each sample, the tissue temperatures of the rectal and interscapular brown adipose tissue (iBAT) were measured, and then the plasma norepinephrine (NE) concentration was measured, as described above in [Evaluation of body thermogenesis (1)].

[0072] [Evaluation results] The results of thermogenesis evaluation (1) are shown in Figures 1 to 3. Figure 1 shows the temperature changes over time in the rectum (Fig. 1A) and interscapular brown adipose tissue (iBAT; Fig. 1B) of the Control group, Control + AMG517 group, ArtC group, and ArtC + AMG517 group as an evaluation result of the thermogenesis effect of artepillin C (ArtC). As shown in Figure 1, ArtC did not have a thermogenic effect on the rectum or brown adipose tissue. Therefore, as shown in Patent Document 1, ArtC may have a differentiation-inducing effect on brown adipose cells, but it was found that it does not activate brown adipose tissue or its cells, and does not have a thermogenic effect on brown adipose tissue or a thermogenic effect on brown adipose tissue.

[0073] Figure 2 shows the temperature changes over time in the rectum (Fig. 2A) and interscapular brown adipose tissue (Fig. 2B) of the control, control + AMG517, BGP, and BGP + AMG517 groups as an evaluation result of the thermogenic effect of Brazilian green propolis (BGP). As shown in Figure 2, BGP increased the temperature of the rectum and brown adipose tissue over time. Furthermore, this increase in tissue temperature in the rectum and interscapular brown adipose tissue due to BGP was significantly suppressed by coadministration with AMG517.

[0074] The results in Figure 2 indicate that BGP, unlike ArtC, activates brown adipose tissue and its cells, and also has thermogenesis and body heat production effects in brown adipose tissue. Furthermore, the inhibitory effect of AMG517 indicated that the body heat production and brown adipose tissue activation effects of BGP are mediated by TRPV1.

[0075] Figure 3 shows the plasma norepinephrine (NE) levels in each test group. As shown in Figure 3, the plasma norepinephrine levels were significantly increased in the BGP group compared to the control group, but this was suppressed by coadministration with AMG517. This indicates that BGP increases the blood concentration of NE via TRPV1.

[0076] Figure 4 shows the calcium signal intensity when TRPV1-overexpressing cells were treated with the main components of BGP (Figures 4A and 4B) and with 0 μM to 10 μM AMG517 (Figure 4C). As shown in Figure 4A, calcium signals were evaluated when TRPV1-overexpressing cells were treated with each component of BGP. The calcium signal increased immediately after the addition of clifolin alone, and this increase continued over time. Furthermore, as shown in Figure 4B, signal intensity analysis revealed significant signal enhancement with clifolin alone. Furthermore, as shown in Figure 4C, the addition of AMG517 together with clifolin inhibited the calcium signal induction by clifolin in a concentration-dependent manner. These results suggest that the BGP component with TRPV1 activation is clifolin.

[0077] The results of thermogenesis evaluation (2) are shown in Figures 5 and 6. Figure 5 shows the temperature changes over time in the rectum (Fig. 5A) and interscapular brown adipose tissue (iBAT; Fig. 5B) of the control group, BGP group, and Culifolin group as an evaluation result of the thermogenesis effect of Culifolin. As shown in Figure 5, Culifolin increased the temperatures of the rectum and interscapular brown adipose tissue over time.

[0078] Figure 6 shows the plasma norepinephrine (NE) levels in each test group. As shown in Figure 6, the plasma norepinephrine levels were significantly increased in the BGP group and the culifolin group compared to the control group.

[0079] These results indicate that Brazilian green propolis has TRPV1 activation activity, and that it has thermogenesis and brown adipocyte activation effects via TRPV1 activation. Furthermore, it was found that the TRPV1-activating component in Brazilian green propolis is cliffolin. [Industrial Applicability]

[0080] The TRPV1 activator of one embodiment of the present invention is naturally derived, contains active ingredients that are recognized as safe, and can activate TRPV1 in the body, and can be used in various forms such as foods and beverages, pharmaceuticals, and quasi-drugs.

Claims

1. A TRPV1 activator containing clifforin as an active ingredient.

2. A body heat production promoter containing Clifolin as an active ingredient.

3. A brown fat cell activator containing clifforin as an active ingredient.

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