UCP1 production promoter

Abstract

To provide a novel UCP1 production promoter and the like for administration to humans and the like.SOLUTION: (1) A UCP1 production promoter comprising neem (Melia azadirachta L.) extract. (2) A UCP1 production promoter comprising rutin. (3) A UCP1 production promoter comprising Eisenia arborea extract. (4) A UCP1 production promoter comprising Amacha (Hydrangea serrata (Thunb.) Ser. var. thunbergii (Siebold) H. Ohba (Saxifragaceae)) extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel UCP1 production promoter for use in humans and the like. [Background technology]

[0002] Various effects of neem extract have been discovered (Patent Document 1). However, the effects of neem extract have not yet been fully studied. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-091645 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a novel UCP1 production promoter for use in humans, etc. [Means for solving the problem]

[0005] The present invention has been made to solve the above problems and was invented after discovering new effects of neem extract and the like.

[0006] The present invention includes, for example, the following embodiments. -UCP1 production promoter containing neem extract, rutin, eisenia extract and / or hydrangea extract. [Effects of the Invention]

[0007] The agent of the present invention can promote UCP1 production. DETAILED DESCRIPTION OF THE INVENTION

[0008] (Neem extract) Neem is a plant of the Meliaceae family: Melia azadirachta L. (= Azadirachta indica Juss.) (also known as Indian neem or margosa). The neem extract used in the following examples is a leaf extract. The neem extract used in the examples is neem leaf extract powder (Ichimaru Falcos). Neem leaf extract powder is produced by extracting the leaves of Melia azadirachta L. (Meliaceae) by adding water, adding an excipient to the extract, and drying it as required to produce a powder.

[0009] (Rutin) Rutin is one of the flavone glycosides widely found in nature. It was discovered in the whole plant of the Rutaceae plant, common rue, but was later isolated from the buds of the Sophora japonica tree, the whole plant of buckwheat, tobacco leaves, the flowers of the Viola trifoliata, fig trees, and the leaves of the Chinese parasol tree. It is a quercetin glycoside composed of quercetin and rutinose. The neem leaf extract powder also contains a predetermined amount of rutin as one of its active ingredients. The present invention also includes, for example, enzyme-treated rutin (the above-mentioned rutin treated with a glycosyltransferase in the presence of dextrin).

[0010] (Eisenia extract) Eisenia is the brown algae Eisenia arborea. The Eisenia extract used in the following examples is an extract of the whole algae. The Eisenia extract used in the examples is Eisenia Veil B (Ichimaru Falcos). Eisenia Veil B is produced by extracting the whole algae of the brown algae Phaeophyceae with an ethanol solution, filtering the filtrate, adding 1,3-butylene glycol to the filtrate and concentrating it, adding 1,3-butylene glycol solution, treating in a cold place, adjusting, and filtering.

[0011] (Hydrangea extract) The hydrangea used in the examples is Hydrangea serrata (Thunb.) Ser. var. thunbergii (Siebold) H. Ohba (Saxifragaceae). The hydrangea extract used in the examples below is an extract of the leaves (including the branch tips). The hydrangea extract used in the examples is Falcorex hydrangea B (Ichimaru Falcos). Falcorex hydrangea B is produced by extracting the leaves and branch tips of Hydrangea serrata (Thunb.) Ser. var. thunbergii (Siebold) H. Ohba (Saxifragaceae) with water, filtering the filtrate, adding 1,3-butylene glycol, and subjecting the filtrate to cold treatment, purification, adjustment, and filtration.

[0012] (UCP1) UCO1 is a molecular uncoupling protein 1 (UCP1) that is specifically expressed in the mitochondria of brown fat cells, for example. UCP1 has the activity of uncoupling oxidative phosphorylation in mitochondria. When this activity is activated, the energy generated by the oxidative decomposition of fatty acids and glucose is directly converted into heat, dissipated, and consumed, rather than used for ATP synthesis. Brown fat thermogenesis by UCP1 is directly controlled by the sympathetic nervous system and contributes to body temperature increases during awakening from hibernation or anesthetic hypothermia, and during cold exposure. For example, when physiological stimuli such as cold exposure are applied, the activity of the sympathetic nervous system, which is densely distributed in brown fat, is increased, and fatty acids are released through a series of reactions: norepinephrine → beta-adrenergic receptor → hormone-sensitive lipase. These fatty acids are oxidatively decomposed and become a substrate for thermogenesis, while at the same time activating UCP1, causing fever. When norepinephrine acts on white fat, Similarly, lipolysis occurs, but the fatty acids produced here are released into the blood and consumed by brown fat and muscles. Thermogenesis by the sympathetic nervous system - brown fat beta receptor - UCP1 system is not merely a heat-generating mechanism for regulating body temperature, but can also be considered as an energy-consuming mechanism that dissipates fat energy as heat. In experiments using many hypothalamic or UCP genetic obesity model animals, not only excessive eating but also energy depletion was observed. It has been shown that UCP1 reduces energy consumption and brown fat function, and when diet-induced obesity is induced in normal animals through excessive eating, an inverse correlation between obesity level and UCP1 has been observed. It is believed that activating the sympathetic nervous system-brown fat system can prevent and alleviate obesity (Chemistry and Biology Vol. 50, No. 1, 2012, p. 23 to p. 29) (Journal of Japanese Biochemical Society 94(1):97-101(2022)).

[0013] (Oral Composition) The UCP1 production promoter according to the present invention is contained in, for example, an oral composition, such as a food or drink (including functional foods, foods for specified health uses, and supplements), or a pharmaceutical.

[0014] For example, when the oral composition is a food or beverage, the food or beverage may take the form of various foods and beverages such as breads, cakes, noodles, confectioneries, jellies, frozen foods, ice cream, dairy products, beverages, etc., as well as forms similar to those of the oral administration preparations described above (tablets, capsules, syrups, etc.) Foods in various forms can be prepared by using the active ingredient of the present invention alone or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc.

[0015] For example, when the oral composition is a pharmaceutical, the pharmaceutical is generally easy to formulate a convenient daily dosage regimen that can be adjusted according to the degree of pain, and the pharmaceutical form can be, for example, a solid form or a liquid form.The solid form can include, for example, powder, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules.For example, in powders, the carrier is generally a finely divided solid that is mixed with a finely divided active ingredient.For example, in tablets, the active ingredient is generally mixed with a carrier having the necessary binding capacity in an appropriate ratio and formed into the desired shape and size.Suitable carriers can include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting wax, cocoa butter, etc. The pharmaceutical may also contain ingredients such as excipients, stabilizers, preservatives, binders, disintegrants, hydrocarbons, fatty acids, alcohols, esters, pH adjusters, and preservatives as needed, within the range that does not impair the desired effects.

[0016] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the content of components contained in the agent of the present invention means % by mass. [Example]

[0017] Examples of the present invention will be described below.

[0018] (Regarding the samples used in the experiment below) The following was used: Mouse fibroblast cells: 3T3-L1 (CL-173, ATCC). Using these cells, we confirmed the amount of glycerol produced, UCP1 gene expression, and lipid droplet reduction after adding samples (neem extract, eisenia extract, and hydrangea extract).

[0019] Culture medium: Prepared with the following additions: DMEM (high glucose, phenol red): Fujifilm Wako Pure Chemical Industries, Ltd. (product number: 045-30385) 10% FBS 1 mM pyruvate: 100 mM pyruvate (Life Technologies, 11360070) was added at a 100-fold dilution. 2 mM L-glutamic acid: 200 mmol / L L-glutamic acid (Fujifilm Wako Pure Chemical Industries, 073-05391) was added at a 100-fold dilution. Antibiotic-Antimycotic: Gibco's Antibiotic-Antimycotic (100X, product number: 15240096) was diluted 100 times and added.

[0020] Differentiation medium 1: A culture medium supplemented with the following factors was prepared. 5 μg / ml insulin (SIGMA, 16634-100MG) 0.25 μM dexamethasone (Fujifilm Wako Pure Chemical Industries, 041-18861) 0.5 mM isobutylmethylxanthine (Fujifilm Wako Pure Chemical Industries, 095-03413)

[0021] Differentiation medium 2: A culture medium supplemented with the following factors was prepared. 5 μg / ml insulin (SIGMA, 16634-100MG)

[0022] Neem extract: Neem leaf extract powder (Ichimaru Falcos) Eisenia extract: Eisenia Veil B (Ichimaru Falcos) Hydrangea extract: Falcorex Hydrangea B (Ichimaru Falcos)

[0023] The sequence used for UCP1 gene expression is described below. The UCP1 sequence was engineered to correspond to the sequence encoding mouse ucp1 (NM_009463.3). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for relative abundance analysis. The RPS18 sequence used was engineered to correspond to the sequence encoding mouse RPS18 (NM_011296.3).

[0024] (Preparation of cells used in the following experiments) The cells were prepared as follows. First, 3T3-L1 cells were cultured in culture medium at 37°C and 5% CO2 until the cell density reached 80% confluency. After reaching this cell density, the cells were detached from the culture plate using TrypLE Select (Gibco, 12563011) and subcultured. The cells obtained by this procedure were seeded into a 24-well cell culture plate at 20,000 cells / well. The seeded 3T3-L1 cells were cultured in culture medium at 37°C, 5% CO2 until the cell density reached 100% confluency. After reaching this cell density, the medium was replaced with culture medium, and the cells were cultured for an additional 2 days at 37°C, 5% CO2. After this culture, the medium was replaced with differentiation medium 1, and the cells were cultured for 2 days at 37°C, 5% CO2. After this culture, the medium was replaced with differentiation medium 2, and the cells were cultured for 2 days at 37°C, 5% CO2. After this culture, the medium was replaced with culture medium, and the cells were cultured for 3 days at 37°C, 5% CO2. After this culture, the medium was replaced with culture medium, and the cells were cultured for an additional 3 days at 37°C, 5% CO2. After this culture, the lipid droplets in the cells were confirmed by bright field photography, and the cells were used in various tests as differentiated adipocytes.

[0025] [Experiment 1: Confirmation of glycerol production, UCP1 gene expression, and lipid droplet reduction after the addition of neem extract] (Experiment 1-1: Glycerol production promotion test (measurement of cellular lipase activity)) Lipid breakdown was measured by utilizing the fact that triglycerides are broken down into fatty acids and glycerol in the cells and released into the culture medium (Folia Pharmacol. Jan. 146, 93-97, 2015). The following samples were added to differentiated adipocytes. Sample 1-1: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and neem extract were added to a final concentration of 8 μg / ml. Sample 1-2: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and neem extract were added to a final concentration of 40 μg / ml. Samples 1-3: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and neem extract were added to a final concentration of 100 μg / ml. Samples 1-4: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and rutin were added to a final concentration of 0.7 μg / ml. For comparison, a test group (control group) to which the sample was not added was also prepared. A test group to which only norepinephrine was added was designated the NC group (negative control group). N=3 of each test group was prepared.

[0026] The cells prepared as described above were either added or not added to the sample. After addition, the cells were cultured at 37°C under 5% CO2 for 24 hours. After the culture, the glycerol released into the culture supernatant was measured using the EnzyChrom Adipolysis Assay Kit (BAS, EAPL-200). The cell count was measured using the WST8 method, and the glycerol production in each group was corrected using the cell count in the control group as the relative value. After this correction, the average value for each test group (N = 3) was calculated, and the glycerol production (%) in the sample-added group was calculated when the glycerol production in the control group was set at 100%.

[0027] The measurement results are as follows: the control group was 100.0, the NC group was 112.7, the sample 1-1 addition group was 168.2, the sample 1-2 addition group was 351.8, the sample 1-3 addition group was 419.3, and the sample 1-4 addition group was 157.9. A significance test (Turkey test, using JMP8) was performed and a significant difference (p<0.05) was confirmed in the neem extract and rutin addition groups (samples 1-1 to 1-4 addition groups) compared to both the control group and the NC addition group, confirming that glycerol production (lipase activity) is being exerted in conjunction with norepinephrine.

[0028] (Experiment 1-2: Confirmation of lipid droplet shrinkage, etc.) Samples 1-2 and 1-3 listed in Experiment 1-1 were added to the cells prepared above. The comparison group (control group) and NC group were the same as in Experiment 1-1. As in Experiment 1-1, N=3 for each test group was prepared. After adding the sample to the cells prepared as described above, the cells were cultured at 37°C and 5% CO2 for 96 hours. After the culture, the culture supernatant was removed, and the cultured cells were washed with PBS. After the wash, the cells were fixed by leaving them to stand at 25°C for 20 minutes using 10% neutral buffered formalin (Fujifilm, 062-01661). After the fixation, the fixed cells were washed with ultrapure water. After the wash, the fixed cells were treated with 60% diluted 2-propanol at 25°C for 1 minute to dehydrate them. After the wash, the cells were stained with Oil Red O (Chroma-Gesellschaft, 1320-06-5) at 25°C for 20 minutes. After the staining, the cells were washed with ultrapure water. After the wash, the cells were stored in ultrapure water, and lipid droplets were confirmed by bright-field photography (10x objective) using an Olympus CKX-53 culture microscope. The ultrapure water used for storage of the confirmed lipid droplets was removed, and the plates were dried overnight at 25°C. After drying, the remaining oil red on the plate was washed with 60% diluted 2-propanol. After washing, extraction was performed using 4% Triton X-100 (Kishida Chemical, 020-81155) / 2-propanol. The extract was measured at 492 nm using an absorbance system, and the amount of oil red was converted into the amount of fat using a calibration curve created with the oil red solution. After this conversion, the average value for each test group (N = 3) was calculated.

[0029] The measurement results are as follows: the control group was 19.25, the NC group was 17.60, the sample 1-2 addition group was 15.33, and the sample 1-3 addition group was 15.64. A significance test (Turkey test, using JMP8) was performed and a significant difference (p<0.05) was confirmed in the neem extract (sample 1-2 and 1-3 addition groups) compared to the control group, confirming that fat reduction was achieved in conjunction with norepinephrine.

[0030] (Experiment 1-3: Confirmation of UCP1 gene expression) Sample 1-3 from Experiment 1-1 was added to the prepared cells. The comparison group (control group) and NC group were the same as those in Experiment 1-1. As in Experiment 1-1, N=3 for each test group was prepared. After adding the sample to the cells prepared as described above, the cells were cultured at 37°C and 5% CO for 24 hours. After adding the sample to the cells prepared as described above, the cells were cultured at 37°C and 5% CO for 96 hours. The purified mRNA was reverse-transcribed using PrimeScript RT Master Mix (TaKaRa, RR036A) to prepare a DNA library. The reverse transcription reaction was carried out using a MiniAmp Thermal Cycler (Thermo Fischer Scientific). The DNA library obtained from this reaction was subjected to qRT-PCR using TB Green Premix ExTaqII (TaKaRa, RR820A). This qRT-PCR was performed using a LightCycler96 (Roche). Among the primers used in qRT-PCR, RPS18 was used as the housekeeping gene and UCP1 as the target gene. The ucp1 gene expression level in each test group was corrected by the RPS18 gene expression level. Furthermore, the ucp1 gene expression level in the control group was set as reference value 1, and the ucp1 gene expression levels in sample groups 1-3, etc. were calculated as relative values. After calculating these relative values, the average value for each test group (N=3) was calculated.

[0031] The measurement results are shown below: the control group was 1, the NC group was 1.25, and the sample 1-3 addition group was 2.91. A significance test (Turkey test, using JMP8) was performed, and a significant difference (p<0.05) was confirmed in the sample 1-3 addition group compared to both the control group and the NC group, confirming the promotion of UCP1 production.

[0032] [Experiment 2: Confirmation of glycerol production, UCP1 gene expression, and lipid droplet reduction after the addition of Eisenia extract] (Experiment 2-1: Glycerol production promotion test (measurement of cellular lipase activity)) Lipid breakdown was measured by utilizing the fact that triglycerides are broken down into fatty acids and glycerol in the cells and released into the culture medium (Folia Pharmacol. Jan. 146, 93-97, 2015). The following samples were added to differentiated adipocytes. Sample 2-1: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and Eisenia extract were added to a final concentration of 0.04%. Sample 2-2: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and Eisenia extract were added to a final concentration of 0.2%. Sample 2-3: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and Eisenia extract were added to a final concentration of 1%. For comparison, a test group (control group) to which the sample was not added was also prepared. A test group to which only norepinephrine was added was designated the NC group (negative control group). N=3 of each test group was prepared.

[0033] The cells prepared as described above were either added or not added to the sample. After addition, the cells were cultured at 37°C under 5% CO2 for 24 hours. After the culture, the glycerol released into the culture supernatant was measured using the EnzyChrom Adipolysis Assay Kit (BAS, EAPL-200). The cell count was measured using the WST8 method, and the glycerol production in each group was corrected using the cell count in the control group as the relative value. After this correction, the average value for each test group (N = 3) was calculated, and the glycerol production (%) in the sample-added group was calculated when the glycerol production in the control group was set at 100%.

[0034] The measurement results are as follows: the control group was 100.0, the NC group was 183.83, the sample 2-1 addition group was 198.13, the sample 2-2 addition group was 289.37, and the sample 2-3 addition group was 685.74. A significance test (Turkey test, using JMP8) was performed, and a significant difference (p<0.01) was confirmed in the Eisenia extract addition group (sample 2-2 or sample 2-3 addition group) compared to both the control group and the NC addition group, confirming that glycerol production (lipase activity) is being exerted in conjunction with norepinephrine.

[0035] (Experiment 2-2: Confirmation of UCP1 gene expression) Sample 2-3 from Experiment 1-1 was added to the prepared cells. The comparison group (control group) and NC group were the same as those in Experiment 1-1. As in Experiment 1-1, N=3 was prepared for each test group. After adding the sample to the cells prepared as described above, the cells were cultured at 37°C and 5% CO for 24 hours. After adding the sample to the cells prepared as described above, the cells were cultured at 37°C and 5% CO for 96 hours. The purified mRNA was reverse-transcribed using PrimeScript RT Master Mix (TaKaRa, RR036A) to prepare a DNA library. The reverse transcription reaction was carried out using a MiniAmp Thermal Cycler (Thermo Fischer Scientific). The DNA library obtained from this reaction was subjected to qRT-PCR using TB Green Premix ExTaqII (TaKaRa, RR820A). This qRT-PCR was performed using a LightCycler96 (Roche). Among the primers used in qRT-PCR, RPS18 was used as the housekeeping gene and UCP1 as the target gene. The ucp1 gene expression level in each test group was corrected by the RPS18 gene expression level. Furthermore, the ucp1 gene expression level in the control group was set as reference value 1, and the ucp1 gene expression levels in sample groups 2 and 3, etc. were calculated as relative values. After calculating these relative values, the average value for each test group (N = 3) was calculated.

[0036] The measurement results are as follows: the control group was 1, the NC group was 1.25, and the group with sample 2-3 added was 2.39. It was confirmed that the value of sample 2-3 was higher compared to both the control group and the NC group, confirming the promotion of UCP1 production.

[0037] [Experiment 3: Confirmation of glycerol production and lipid droplet reduction after addition of hydrangea extract] (Experiment 3-1: Glycerol production promotion test (measurement of cellular lipase activity)) Lipid breakdown was measured by utilizing the fact that triglycerides are broken down into fatty acids and glycerol in the cells and released into the culture medium (Folia Pharmacol. Jan. 146, 93-97, 2015). The following samples were added to differentiated adipocytes. Sample 3-1: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and Hydrangea tea extract were added to a final concentration of 0.04%. Sample 3-2: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and Hydrangea tea extract were added to a final concentration of 0.2%. Sample 3-3: 0.1 μM (-)-Norepinephrine (SIGMA, A7257-500MG) and Hydrangea tea extract were added to a final concentration of 1%. For comparison, a test group (control group) to which the sample was not added was also prepared. A test group to which only norepinephrine was added was designated the NC group (negative control group). N=3 of each test group was prepared.

[0038] The cells prepared as described above were either added or not added to the sample. After addition, the cells were cultured at 37°C under 5% CO2 for 24 hours. After the culture, the glycerol released into the culture supernatant was measured using the EnzyChrom Adipolysis Assay Kit (BAS, EAPL-200). The cell count was measured using the WST8 method, and the glycerol production in each group was corrected using the cell count in the control group as the relative value. After this correction, the average value for each test group (N = 3) was calculated, and the glycerol production (%) in the sample-added group was calculated when the glycerol production in the control group was set at 100%.

[0039] The measurement results are as follows: the control group was 100.0, the NC group was 127.8, the sample 3-1 addition group was 114.5, the sample 3-2 addition group was 245.3, and the sample 3-3 addition group was 678.2. A significance test (Turkey test, using JMP8) was performed, and a significant difference (p<0.01) was confirmed in the hydrangea extract addition groups (sample 3-2 or sample 3-3 addition groups) compared to both the control group and the NC addition group, confirming that glycerol production (lipase activity) is being exerted in conjunction with norepinephrine.

[0040] The above has described the embodiments (including examples) of the present invention with reference to the drawings, but the specific configuration of the present invention is not limited to this, and even if there are design changes, etc., within the scope that does not deviate from the gist of the present invention, they are included in the present invention. [Industrial Applicability]

[0041] The present invention can provide a novel UCP1 production promoter for use in humans, etc.

Claims

1. A UCP1 production promoter containing neem extract.

2. A UCP1 production promoter containing rutin.

3. A UCP1 production promoter containing Eisenia extract.

4. A UCP1 production promoter containing amacha extract.