KR102238469B1 - Composition for prevention and treatment of obesity comprising 3-hydroxy phloretin as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating obesity containing 3-hydroxy phloretin (3-OH phloretin) as an active ingredient, and 3-hydroxy phloretin or a pharmaceutical thereof according to the present invention The composition for preventing or treating obesity containing an acceptable salt as an active ingredient is PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α), and fatty acid synthase ( Fatty acid synthase), etc., are expected to be widely used for prevention or treatment of obesity by effectively reducing the fat content in cells.

Description

3-Composition for prevention and treatment of obesity comprising 3-hydroxy phloretin as active ingredient}

The present invention relates to a composition for preventing or treating obesity containing 3-hydroxy phloretin (3-OH phloretin) as an active ingredient, and more particularly, 3-hydroxy phloretin or a pharmaceutical thereof It relates to a composition for preventing or treating obesity containing an acceptable salt as an active ingredient.

Obesity refers to a state in which fat is accumulated in the body more than necessary, and the representative cause of obesity is due to an imbalance between energy intake and consumption due to westernized eating habits. Due to this, as visceral fat-type obesity increases, the invention of metabolic syndromes such as type 2 diabetes and hypertension accompanied by insulin resistance is rapidly increasing. These diseases increase the risk of mutual occurrence and accompany complications such as aging, stress, inflammation, and immune function decline.

Drug therapy is mainly used for the treatment of obesity, and the synthetic drug-type drugs mainly reduce food intake by suppressing appetite, or products that play a role in promoting metabolism by inhibiting the absorption of carbohydrates or fats. It is sold in various countries. However, these products have been reported to cause side effects in many cases, mainly due to suppressing the action of lipase or increasing the concentration of serotonin or noradrenaline, gastrointestinal side effects, headache, poor food consumption, constipation, insomnia, heart rate. Increasing, dizziness, etc. are causing. Due to the side effects of such synthetic drugs, there is an urgent need to find a substance capable of obtaining an excellent obesity suppression effect while having few side effects from substances such as natural products.

Phloretin is the IUPAC name, 3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one, and has the same structure as in Formula 1 below.

[Formula 1]

Floretine is dihydrochalcone, a type of natural phenols, and is known to be found in succulent fruits such as apples and pears, or in plants such as apple tree leaves. Phloretin exhibits various biological and pharmacological activities such as anti-inflammatory and anti-cancer, and is also used as a cosmetic ingredient. In addition, in recent years, as it is known that floretin plays a beneficial role in human health, studies are being conducted for the use of the natural ingredients in various fields, but due to the fact that the metabolic process of floretin has not been specifically identified, It is necessary to study metabolic pathways and metabolites, and from this, it is urgent to identify substances that have a significant effect on preventing or treating obesity while having few side effects.

Republic of Korea Patent Publication No. 2010-0114579. Republic of Korea Patent Publication No. 2016-0101492.

Alsanea S et al. AAPS J. (2017.12.14.)

In order to solve the above problem, the present inventors induce and manufacture 3-hydroxy phloretin from phloretin isolated from apple residue, and use this to prevent lipid accumulation in adipocytes. By suppressing, it was intended to provide a composition containing an active ingredient that can effectively prevent or treat obesity.

The present invention provides a composition for preventing or treating obesity containing 3-hydroxy phloretin (3-OH phloretin) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the active ingredient is not limited as long as it does not inhibit the achievement of the object of the present invention, but can suppress obesity through inhibition of a fat differentiation promoting factor.

In the present invention, the fat differentiation promoting factor is not limited as long as it does not inhibit the achievement of the object of the present invention, but PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α) and fatty acid synthase ( fatty acid synthase) and the like.

In addition, the present invention provides a method for preventing or treating obesity for mammals other than humans, comprising the step of treating the composition for preventing or treating obesity on fat progenitor cells.

In the present invention, the composition for preventing or treating obesity is not limited as long as it does not inhibit the achievement of the object of the present invention, but can be treated at a concentration of 1 to 250 uM, preferably 10 to 150 uM, more preferably 10 to 75 uM have.

In addition, the present invention provides a diet health functional food containing the composition.

The composition for preventing or treating obesity containing 3-hydroxy fluoretine or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient is PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα, which are major fat differentiation promoting factors. (CCAAT/enhancer-binding protein α) and fatty acid synthase (fatty acid synthase) can be effectively suppressed to reduce the intracellular fat content and thus can be widely used for the prevention or treatment of obesity.

FIG. 1 relates to a) HPLC, and b) NMR measurement results at the time of conversion of 3-hydroxy phloretin from phloretin using P450 enzyme.
Figure 2 relates to the results of measuring the viability of the cells and the concentration of 3-hydroxy phloretin does not show cytotoxicity to 3T3-L1 adipocytes through the MTS assay (MTS assay).
Figure 3 is a comparison of the effect of inhibiting differentiation of adipocytes by Oil-red-O staining, b) fat in the cells differentiated in a) when 3T3-L1 adipocytes are treated with 3-hydroxy fluoretin. Lipid accumulation was measured with a spectrophotometer.
4 relates to the results of gene expression of adipogenic differentiation promoting factors.
Figure 5 is a high-fat diet (High Fat Diet, HD) when the oral administration of 3-hydroxy phloretin of the present invention to mice, a) appearance, b) body weight and c) dietary amount of the mouse as a control group (SD ) And the result of comparison.
6 shows a result of comparing a) the distribution of visceral adipose tissue, b) the size of the isolated adipose tissue, and c) the size of the isolated adipose tissue in the mouse of FIG. 5.
FIG. 7 relates to a result of comparing the size of a) visceral and liver tissues and b) adipocytes in visceral tissues in the mouse of FIG. 5.

Hereinafter, a composition for preventing or treating obesity containing 3-hydroxy fluoretine or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient will be described in detail with reference to the accompanying drawings. The drawings introduced below are provided as examples in order to sufficiently convey the spirit of the present invention to those skilled in the art. Accordingly, the present invention is not limited to the drawings presented below and may be embodied in other forms, and the drawings presented below may be exaggerated to clarify the spirit of the present invention.

Unless otherwise defined, technical terms and scientific terms used in the specification of the present invention have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the present invention in the following description and accompanying drawings Descriptions of known functions and configurations that may unnecessarily obscure the gist of are omitted.

In addition, the singular form used in the specification of the present invention may be intended to include the plural form unless otherwise indicated in the context.

In addition, units used in the specification of the present invention are based on weight, and as an example, the unit of% or ratio means weight% or weight ratio.

Hereinafter, a composition for preventing or treating obesity containing 3-hydroxy fluoretine or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient will be described in detail.

The present invention provides a composition for preventing or treating obesity containing 3-hydroxy phloretin (3-OH phloretin) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the active ingredient is not limited as long as it does not inhibit the achievement of the object of the present invention, but can suppress obesity through inhibition of a fat differentiation promoting factor.

In the present invention, the fat differentiation promoting factor is not limited as long as it does not inhibit the achievement of the object of the present invention, but PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α) and fatty acid synthase ( fatty acid synthase) and the like.

The adipogenic differentiation promoting factor is a key among transcriptional regulatory factors and plays a role of promoting the expression of genes related to adipogenesis and differentiation into adipocytes through the interaction of PPARγ and C/EBPα. The accumulation of fat can be suppressed through inhibition.

3-hydroxy phloretin (3-OH phloretin) according to the present invention can be used in the form of a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is by a conventional method in the art. What can be prepared, for example, salts with inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, nitric acid, carbonic acid, etc., formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, Citric acid, maleic acid, malonic acid, mandelic acid, cinnamic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid tartaric acid, gluconic acid, lactic acid, fumaric acid, lactobionic acid, ascorbic acid, salicylic acid, or acetylsalicylic acid (aspirin) and Salts with organic acids such as glycine, alanine, vanillin, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, and salts with amino acids such as proline, methanesulfonic acid, ethanesulfonic acid, benzene And salts with sulfonic acids such as sulfonic acid and toluenesulfonic acid, metal salts obtained by reaction with alkali metals such as sodium and potassium, or salts with ammonium ions.

3-hydroxy phloretin (3-OH phloretin) of the present invention may exist in solvated forms, for example, hydrated and unsolvated forms, and 3-hydroxy phloretin according to the present invention Solvates of 3-hydroxy phloretin (3-OH phloretin) include all solvated forms with pharmaceutical activity.

The 3-hydroxy phloretin (3-OH phloretin) of the present invention may have a chiral center and exist as racemates, racemic mixtures and individual enantiomers or diastereomers. These isomers can be separated or resolved by conventional methods and any given isomer can be obtained by conventional synthetic methods or by stereospecific or asymmetric synthesis. All such isomeric forms and mixtures thereof are included within the scope of the present invention.

The 3-hydroxy phloretin (3-OH phloretin) of the present invention can be administered in the form of a prodrug that is degraded in the human or animal body to provide the compound of the present invention. Prodrugs can be used to alter or improve the physical and/or pharmacokinetic profile of the parent compound and can be formed when the parent compound contains suitable groups or substituents that can be induced to form the prodrug.

In addition, the pharmaceutical composition of the present invention is generally used in the 3-hydroxy phloretin (3-OH phloretin), a prodrug thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. By adding non-toxic pharmaceutically acceptable carriers and excipients, such as conventional formulations in the pharmaceutical field, such as tablets, pills, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. It can be formulated as a formulation for oral administration or a formulation for parenteral administration in a sterile aqueous or oily solution for intravenous, subcutaneous, sublingual, or intramuscular administration.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are commonly used in formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin. , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and/or mineral oil, etc. , But is not limited thereto.

Excipients that can be used in the pharmaceutical composition of the present invention include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, tonicity agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances, and the like. The proportions and properties of excipients can be determined by the solubility and chemical properties of the selected tablet, the route of administration chosen, and standard pharmaceutical practice. Examples of excipients include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, stearic acid, magnesium stearate, magnesium aluminum silicate, starch, gelatin, rubber tragacanth, arginic acid, Sodium alginate, methylcellulose, sodium carboxylmethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, and the like.

In addition, the pharmaceutical composition of the present invention may be formulated in a parenteral dosage form. In this case, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration may be used, but are not limited thereto. At this time, in order to formulate the formulation for parenteral administration, the pharmaceutical composition is an active ingredient, that is, 3-hydroxy phloretin (3-OH phloretin), a prodrug thereof, a solvate thereof, A stereoisomer thereof or a pharmaceutically acceptable salt thereof is mixed in water with a stabilizer or buffer to prepare a solution or suspension, and such a solution or suspension may be prepared in unit dosage form of ampoules or vials.

In addition, the pharmaceutical composition of the present invention may be sterilized or further contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsifying accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances. Alternatively, it may be formulated according to conventional methods of mixing, granulating or coating.

In addition, 3-hydroxy phloretin (3-OH phloretin), an active ingredient in the pharmaceutical composition according to the present invention, a prodrug thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof The dosage for mammals including silver humans may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity. In general, it may be included in the pharmaceutical composition in an effective amount of 0.001 to 500 mg/kg (body weight) per day, preferably 0.01 to 100 mg/kg (body weight), and such pharmaceutical compositions may be used once or twice a day. It can be divided and administered through an oral or parenteral route. However, since it may increase or decrease according to the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.

In addition, the present invention provides a method for preventing or treating obesity for mammals other than humans, comprising the step of treating the composition for preventing or treating obesity on fat progenitor cells. The type of mammal is not particularly limited, and any organism of a system having a disease due to obesity can be treated without limitation through a method such as injection or oral administration.

In the present invention, the composition for preventing or treating obesity is not limited as long as it does not inhibit the achievement of the object of the present invention, but can be treated at a concentration of 1 to 250 uM, preferably 10 to 150 uM, more preferably 10 to 75 uM And, more specifically, it can be treated at a concentration of 12.5 uM, 25 uM, or 50 uM, and a synergistic effect that can minimize stress or damage to the cells to be treated in the above range while at the same time maintaining the effect of inhibiting fat accumulation in a high state. Can be obtained, and is more preferable.

In addition, the present invention reacts with phloretin any one or two or more enzymes selected from the group consisting of bacterial cytochrome P450 enzyme, amino acid mutant of P450 enzyme, chimeric P450 enzyme, and amino acid point mutant of chimeric P450 enzyme. It provides a method for preparing 3-hydroxy fluoretine for the prevention or treatment of obesity, including the step of preparing 3-hydroxy fluoretine.

Although 3-hydroxy fluoretine is a metabolite of floretin having a structure similar to that of floretin, it is known that it cannot be chemically synthesized in the general field of organic chemistry. Accordingly, one method for producing 3-hydroxy fluoretine through an enzyme and a mutant thereof, which is a biological catalyst, has recently been identified by the research team.

In the present invention, the P450 enzyme is not limited as long as it does not inhibit the achievement of the object of the present invention, but may be a wild-type CYP102A1 of P450 BM3.

In the present invention, the CYP102A1 mutant may refer to the entire CYP102A1 having a sequence changed by natural or artificial substitution, deletion, addition and/or insertion of amino acids based on wild-type CYP102A1. Preferably, it may include all individuals and the like in which point mutations have occurred based on the amino acid sequence of wild-type CYP102A.

In the present invention, the CYP102A1 mutant can be prepared by a known mutation method. For example, deletion-mutation method, PCT method, Kunkel method, point mutation method, DNA shuffling, StEP (staggered extension process), error-prone PCR method may be used, but is not limited thereto.

In the present invention, the CYP102A1 mutant may be expressed as an amino acid before mutation, a position of an amino acid, and an amino acid that has undergone mutation. For example, R47L refers to a CYP102A1 mutant in which arginine (R), the 47th amino acid of SEQ ID NO: 1, which is the amino acid sequence of wild-type CYP102A1, is naturally or artificially substituted with leucine (L) by mutation induction. When there is more than one amino acid substituted by mutation in the mutant, it may be represented by'/'. For example, in R47L/F87V/L188Q, arginine (R), which is the 47th amino acid of SEQ ID NO: 1, is substituted with leucine (L), and phenylalanine (F), which is the 87th amino acid of SEQ ID NO: 11, is substituted with valine (V), It means a mutant of wild-type CYP102A1 in which leucine (L), which is the 188th amino acid of SEQ ID NO: 1, is substituted with glutamine (Q).

In the present invention, the group consisting of the mutant of CYP102A1 is not limited as long as it does not inhibit the achievement of the object of the present invention, but as an example, wild-type CYP102A1 R47L/F87V/L188Q, R47L/F81I/F87V/E143G/L188Q/E267V, R47L/ F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H102Y/A1008D R47L/K59N/F81I/F87V/E143G/L188Q/G240E/E267V/S383R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/S887NM967V894S Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/E143G/L188Q/E267V/Y334C/A335V/D369G/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K8816H/R825N/R8816H/R825S/ E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/E143G/L188Q/Q206R/E267A/V297A/K336E/A474V/E558D/T664A/P67587A6781A/E687E6781 R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/A474V/E558 A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, I39V/R47 L/F81I/F87V/E143G/L188Q/N213D/I259T/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894V967S/S954R/M967V96 It may be a group consisting of A1008D/H1021Y/Q1022E.

According to an embodiment of the present invention, the mutant of wild type CYP102A1 may include a mutant in which point mutation has occurred in wild type CYP102A1, specifically R47L/F87V/L188Q of wild type CYP102A1.

In the present invention, the method for producing 3-hydroxy fluoretine may further include a NADPH-producing system. In the present invention, the NADPH-producing system may use a system known in the field to which the present invention belongs, for example, glucose 6-phosphate, NADP+, and yeast glucose 6-phosphate dehydrogenase. ) Can be used, but is not limited thereto.

In addition, the present invention provides a diet health functional food containing the composition.

In the present invention, the term'health functional food' refers to a natural product or processed product containing one or more nutrients, and preferably uses physical, biochemical, and biotechnological methods in the food to determine the function of the food for a specific purpose. It refers to foods that have been designed and processed to sufficiently express the function of exercise to the living body, such as regulation of biological defense rhythm, disease prevention and recovery, etc. of a food group or food composition that has added value to act and express The health functional food may include food additives that are acceptable food additives, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of health functional foods.

Health functional foods to which the active ingredient of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and the like. In addition, special nutritional foods (e.g., formula, infant, baby food, etc.), health supplements, confectionery (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, beverages (e.g. fruits, vegetables, etc.) Beverages, soy milk, fermented beverages, etc.) The food, beverage or food additive described above can be prepared by a conventional manufacturing method.

The health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and heavy ingredients (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, water, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination.

As described above, the health functional food of the present invention may have various 　 formulations as described above, and in particular, may have any one 　 formulation in powders, granules, tablets, capsules and beverages, but is not limited thereto.

Hereinafter, the content of the present invention will be described in more detail through examples. The examples are only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.

[Reagents and Materials]

P450 BM3 (wild type CYP102A1): Produced and used in Room 303, Chonnam National University Joint Laboratory, where inventors Chul-ho Yun and Nguyen Ngok-an conduct their research.

(References: Kang JY, Kim SY, Kim D, Kim DH, Shin SM, Park SH, Kim KH, Jung HC, Pan JG, Joung YH, Chi YT, Chae HZ, Ahn T, Yun CH.Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium.AMB Express.2011 Mar 28;1(1):1.doi: 10.1186/2191-0855-1-1.)

Adipocyte progenitor cells: 3T3-L1 was sold and used from the American Type Culture Collection (ATCC, USA).

CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS): G5430, purchased from Promega and used.

All other reagents were purchased from Sigma-Aldrich and used.

[Mouse, feed and diet]

As a mouse, a 10-week-old female C57BL/6 was purchased from Damul Science Co., Ltd. (Korea) and used.

As for the feed for mice, a standard diet (SD) or a high fat diet (HD) diet was performed in the form of free feeding for 12 weeks.

The high fat diet was used as a product of catalog number HFD D12451 (Research Diets Co.; New Brunswick, NJ). The composition of the high fat feed is shown in Table 1 below.

Nutrients Kcal (%) Protein 20 Carbohydrate 35 Fat 45 Total (kcal/gram) 100

Mice were subjected to a 12-hour light-dark cycle, individually housed in a cage dedicated to mice, and then subjected to a diet for 12 weeks using the feed.

[Example 1] Preparation of 3-hydroxy phloretin

3-hydroxy phloretin was prepared using a mutant of the enzyme CYP102A1, which is a P450 enzyme, in phloretin. The preparation of the 3-hydroxy fluoretine was based on the method of Korean domestic application No. 10-2017-0124724 (filed on September 27, 2017) of the present inventor.

The mutant of CYP102A1, a P450 enzyme, was used in the following preparations by making R47L/F87V/L188Q point mutations in the CYP102A1 protein.

50 uM phloretin, 60 pM CYP102A1 mutant, 100 mM potassium phosphate (pH 8.0) and NADPH-producing system (final concentration 0.5 mM NADP ⁺ , 10 mM glucose-6-phosphate ( Glucose-6-phosphate; pH 8.0) and 1.0 IU glucose-6-phosphate dehydrogenase per 1 mL were mixed, followed by reaction for 40 minutes to prepare 3-hydroxy fluoretine.

[ Example 2] Measurement of cytotoxicity of 3-hydroxy fluoretin

The 3T3-L1 adipocytes were divided into 7 experimental groups, treated with 200, 100, 50, 25, 12.5, 6.25 uM of 3-hydroxy fluoretine prepared in Example 1, respectively, and the other experimental group was an untreated control group. After setting as (control), cytotoxicity was measured using the MTS Assay Kit.

As a comparative experimental group, after the treatment at the same concentration using floretin instead of 3-hydroxy floretin, cytotoxicity was measured by the same method.

In addition, DMSO (dimethyl sulfoxide) was treated with 0.2 and 0.1%, respectively, and cytotoxicity was compared with that of the untreated control group.

The results are shown in FIG. 2.

As can be seen from this, it was confirmed that the 3-hydroxy fluoretine of the present invention did not significantly affect cell survival even when the concentration was increased to a certain level as well as at a low concentration. However, it was confirmed that the cell viability slightly decreased when treated with a concentration of 200 uM.

[ Example 3] Effect of 3-hydroxy Fluoretin on Adipocyte Differentiation and Fat Accumulation Inhibition

After incubating the 3T3-L1 adipocytes until they become confluent in a 12-well plate, 3-hydroxy phloretin was treated, and MDI culture solution (10% fetal bovine serum (FBS)), which is an adipose differentiation medium, was cultured. ) Containing high-concentration glucose, DMEM (Dulbecco's Modified Eagle's Medium), 500 uM methylisobutylxanthine, 1 uM dexamethasone, 10 ug/mL insulin mixture).

It was replaced with a medium containing 10% FBS DMEM, 500 uM methylisobutylxanthine, 1 uM dexamethasone and 10 ug/mL insulin, and after 3 days, it was replaced with a medium containing 10 ug/mL insulin. After 6 days, differentiation was induced for 10 days by replacing the 10% FBS DMEM medium every two days.

After 10 days, the differentiated cells were fixed in a phosphate buffered-saline (PBS) solution mixed with 4% paraformaldehyde for 1 hour, washed twice with PBS containing 60% isopropanol, and then oil- After dyeing for 15 minutes with Red-O dyeing solution, it was washed with running water.

After completion of staining, a photograph of the cells was taken, and the stained portion was quantified using the'Image J'program, and then shown as a graph.

In the above experiment, the optical density was measured by performing the same procedure for each of phloretin and untreated control group instead of 3-hydroxy phloretin.

Figure 3 shows the fat accumulation photograph and the fat accumulation amount of the experimental group.

From this, it was confirmed that the differentiation of adipocytes was significantly suppressed in the group treated with 3-hydroxy phloretin, which was significantly superior to the group treated with phloretin (FIG. 3A).

In addition, as a result of measuring the amount of fat accumulation in the cell, it was confirmed that it was significantly suppressed at about 30% compared to the control, and showed an excellent fat accumulation inhibitory effect more than twice as compared to the case of treatment with phloretin (FIG. 3b).

[ Example 4] Measurement of expression of obesity-related adipogenic differentiation promoting factor genes in adipocytes

After the adipocytes were differentiated in the same manner as in Example 3, the differentiation-completed adipocytes were harvested for the three experimental groups of 3-hydroxy phloretin, phloretin, and untreated control, respectively.

Thereafter, primers for amplifying the obesity-related genes PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α), and fatty acid synthase (FAS) genes as shown in Table 2 below. It was prepared, and RT-PCR was performed. PCR was performed 35 times of denaturation at 95° C. for 1 minute, annealing at 54° C. for 1 minute, and extension at 72° C. for 1 minute.

primer Base sequence GAPDH-F 5'-CCC TTA TTG ACC TCA ACT ACA TGG T-3 GAPDH-R 5'-GAG GGG CCA TCC ACA GTC TTC TG-3' PPARγ2-F 5'-GTT TTA TGC TGT TAT GGG TG-3' PPARγ2-R 5'-GTA ATT TCT TGT GAA GTG CT-3' C/EBPα-F 5-TTA CAA CAG GCC AGG TTT CC-3' C/EBPα-R 5'-GGC TGG CGA CAT ACA GTA CA-3' FAS-F 5'-TTG CTG GCA CTA CAG AAT GC-3 FAS-R 5-AAC AGC CTC AGA GCG ACA AT-3'

The results after performing RT-PCR are shown in FIG. 4.

As can be seen from this, PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-), which play a pivotal role in promoting adipocyte differentiation compared to the control group, in the 3-hydroxy fluoretin-treated experimental group. The expression of binding protein α) and fatty acid synthase (FAS) genes was remarkably suppressed, and in particular, it was confirmed that there was a remarkably excellent effect compared to phloretin.

[Example 5] Induction and measurement of obesity in mice receiving oral administration of 3-hydroxy phloretin to mice fed high fat diet

Using a 10-week-old 21-22 g C57BL/6 female mouse, the standard diet and high-fat diet described above for 12 weeks at room temperature at 24±2° C., while supplying sufficient water, HFD D12451 (Research Diets Co.; New Brunswick, NJ) diet was applied to freely diet.

High fat diet Experimental group

After dissolving 3-hydroxy phloretin and phloretin in 0.5% DMSO, respectively, mice of the experimental group were orally administered at 20 mg/kg every other day for 14 weeks. DMSO was administered to the other experimental group of mice in the same manner.

Body weight was measured in units of 0.01 g at the same time every day, and the dietary amount of feed was measured once a week at the time of 7 days.

Thereafter, 12 weeks after the high-fat feed diet, the adipose tissue of each mouse was separated, and its size and weight were checked.

Standard diet Experimental group (Negative control)

In the other experimental group of mice, a standard diet (SD) was applied under the same temperature and environmental conditions as the high-fat diet, and DMSO was administered in the same manner, and then the body weight was measured and the adipose tissue of each mouse was measured 12 weeks after the diet. Separated, its size and weight were checked.

Adipose tissue biopsy

Adipose tissue isolated from the high fat diet experimental group and the standard diet experimental group was subjected to histological examination using a conventional hematoxylin and eosin (H&E) staining method.

Specifically, the adipose tissue was frozen by embedding it with paraffin, cut to a thickness of 8 um using a cryo-cut microtome, and then attached to a slide glass. The slide to which the tissue section was attached was immersed in xylene for 5 minutes to remove paraffin, and the ethanol concentration was gradually lowered to 100%-95%-85%-70% for 2 minutes each to hydrate.

Thereafter, the remaining ethanol was removed by washing with water, stained with hematoxylin for 6 minutes, and then immersed in a 1% hydrochloric acid-ethanol (HCl-EtOH) mixed solution 3 times and then removed. The dye was fixed by allowing it to be absorbed, soaking it in 0.5% ammonia water 10 times, and then taking it out.

The tissue sections stained with hematoxylin were stained again with eosin for 1 minute, and the concentration of ethanol was increased to 70%-85%-95%-100% for 2 minutes each to be dehydrated.

The dehydrated tissue slide was immersed in xylene for 5 minutes, washed, and completely dried at room temperature, and then the tissue section was observed under a microscope and photographed.

Measurement of fat accumulation in liver tissue

The liver tissues isolated from the high fat diet experimental group and the standard diet experimental group were measured for fat accumulation using a conventional Oil-Red-O staining method.

Specifically, the liver tissue was fixed in a 10% para-formaldehyde solution dissolved in PBS (phosphate buffered-saline) for 1 hour, and washed twice with PBS containing 60% isopropanol. Thereafter, dyeing was performed at room temperature for 1 hour with an Oil-Red-O staining solution (0.5% Oil Red-O staining reagent dissolved in isopropanol and water, diluted in a ratio of 6 to 4).

After staining, the remaining solution was washed with running water, dried completely, and the tissue section was photographed under a microscope.

The results of dietary application to mice are shown in FIG. 5.

As can be seen from this, the appearance of the mice to which the standard diet was applied is the smallest, compared to the case of administering DMSO and phloretin among the mice to which the high fat diet was applied, the 3-hydroxy phloretin component of the present invention It can be seen that the appearance of the mice administered with the experimental group was smaller (FIG. 5A), and the weight gain was significantly lower compared to the case of administering DMSO and floretin among the mouse experimental groups to which the high-fat diet was applied (Figure 5a). 5b).

From this, it can be seen that there is an effect of suppressing weight gain by the administration of 3-hydroxy phloretin, and this weight gain inhibiting effect is due to the difference in diet amount from the point that there is no change in the amount of feed between the experimental groups. It was confirmed that it was not an effect (Fig. 5c).

In addition, the results of separating and analyzing adipose tissue are shown in FIGS. 6 and 7.

As can be seen in Figure 6, the gonadal white adipose tissue (Gonadal White Adipose Tissue, GWAT) and the retroperitoneal white adipose tissue (Retroperitoneal White Adipose Tissue, RWAT) of the mouse administered with 3-hydroxy phloretin It was confirmed that the visceral adipose tissue distribution was significantly reduced compared to the mice administered with phloretin (FIG. 6a), and the size of these visceral adipose tissues (FIG. 6b) and the weight of visceral adipose tissue (FIG. 6c) were also significantly reduced.

As a result of histologic examination of the white adipose tissue of the gonads using the H&E staining, as can be seen in FIG. 7A, the size of adipocytes isolated from the mice administered with 3-hydroxy phloretin of the present invention is It was significantly reduced compared to the adipocytes of the mice administered with the liver, and it was confirmed that the fat accumulation was significantly reduced when 3-hydroxy fluoretine was administered in the results of measuring the degree of fat accumulation in the liver tissue.

The size of the adipocytes in FIG. 7A was measured and shown as a graph (FIG. 7B).

From this, it was confirmed that the size of adipocytes was significantly reduced through the treatment of 3-hydroxy fluoretine of the present invention.

The 3-hydroxy phloretin of the present invention is significantly superior in weight loss and improvement of fatty liver compared to the experimental groups treated with phloretin and DMSO on a high fat diet, and the above effect is not a phenomenon due to suppression of appetite. Was confirmed.

Accordingly, the present invention was completed by confirming that the 3-hydroxy proletin of the present invention has a remarkably excellent inhibitory effect on obesity and can be usefully used for prevention or treatment.

<110> University Industry Liaison Office Of Chonnam National University <120> Composition for prevention and treatment of obesity comprising 3-hydroxy phloretin as active ingredient <130> P18080740893 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> Wild Type CYP102A1 <400> 1 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 2 cccttattga cctcaactac atggt 25 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 3 gaggggccat ccacagtctt ctg 23 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma2-F <400> 4 gttttatgct gttatgggtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma2-R <400> 5 gtaatttctt gtgaagtgct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C EBPalpha-F <400> 6 ttacaacagg ccaggtttcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C EBPalpha-R <400> 7 ggctggcgac atacagtaca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS-F <400> 8 ttgctggcac tacagaatgc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS-R <400> 9 aacagcctca gagcgacaat 20

Claims (6)

A pharmaceutical composition for preventing or treating obesity containing 3-hydroxy phloretin (3-OH phloretin) or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 1,
The active ingredient is a pharmaceutical composition for preventing or treating obesity to suppress obesity through inhibition of a fat differentiation promoting factor. The method of claim 2,
The fat differentiation promoting factor is a pharmaceutical composition for preventing or treating obesity, including PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α), and fatty acid synthase. A method for preventing or treating obesity in mammals other than humans, comprising the step of treating fat progenitor cells with 3-hydroxy phloretin (3-OH phloretin) or a pharmaceutically acceptable salt thereof. The method of claim 4,
The 3-hydroxy phloretin (3-OH phloretin) or a pharmaceutically acceptable salt thereof is a method for preventing or treating obesity in mammals other than humans treated at a concentration of 10 to 75 uM. Dietary health functional food containing 3-hydroxy phloretin (3-OH phloretin) or a pharmaceutically acceptable salt thereof as an active ingredient.

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