WO2023080725A1 - Composition comprising cassia mimosoides var. nomame as active ingredient for promoting or improving energy metabolism - Google Patents

Abstract

The present invention relates to a composition comprising a (Cassia mimosoides var. nomame extract as an active ingredient for promoting or improving energy metabolism. A composition comprising Cassia mimosoides var. nomame as an active ingredient according to an aspect can promote or improve energy metabolism to exhibit the effect of suppressing lipid accumulation or reducing body fat, thus finding advantageous applications in treating obesity and metabolic diseases.

Description

A composition for accelerating or improving energy metabolism containing an extract of green tea plant as an active ingredient

It relates to a composition for accelerating or improving energy metabolism comprising a green tea extract as an active ingredient.

Obesity is a very serious disease that is rapidly increasing in many countries around the world but has no medically effective treatment. The World Health Organization (WHO) defines a body mass index (BMI) of 30 or more as obesity, and the average obesity rate in OECD member countries is 19.5%, and the rate is gradually increasing.

Obesity refers to the state of excessive accumulation of fat in the body due to genetic or lifestyle causes, and causes diseases such as adult diseases and chronic degenerative diseases. are doing Secondary complications such as hyperlipidemia, hypertension, arteriosclerosis, diabetes, and fatty liver caused by excessive fat accumulation and social disorders that can be caused by obesity are problems.

The world's obese population is rapidly increasing despite the fact that the World Health Organization (WHO) declared obesity as the new epidemic of the 21st century in 2013 and the whole world declared war on obesity. A new problem is emerging in the field of anti-obesity and obesity treatment in that inducing individual behavioral change to prevent obesity is not a perfect alternative for individuals leading busy lives in complex industrial informatization. And, despite the development of basic medical and biological research on obesity, the realistic increase in obesity shows a gap between these two factors. Therefore, there is a need for development and research on food and cosmetics that are more accessible and can achieve a long-term anti-obesity effect.

3T3-L1 Adipocytes (pre-adipocytes) activate many transcription factors during the differentiation process to accumulate fat. Representative differentiation factors include C/EBPa (CCAAT/enhancer binding protein alpha) and PPARγ (Peroxisome proliferator) induced by it. activated receptor gamma). In addition, fat accumulation can be reduced through the regulation of energy metabolism, and a representative factor is AMP-activated protein kinase (AMPK).

Zebrafish is a vertebrate animal that is 80% similar to human genes, small in size, easy to breed at the laboratory level, quick to develop, so that development of all organs can be observed in a short period of time, and embryos are transparent to internal organs. is easy to observe. Also like mammals, zebrafish store excess lipids in visceral, intramuscular and subcutaneous fat deposits. For this reason, zebrafish are used as a good model for obesity research.

Cassia mimosoides var. nomame is an annual herb consisting of 30 to 70 small pinnate compound leaves. In oriental medicine, the leaves and stems are called mountain pyeondu (山扁豆) as a medicinal material, and the whole part is specified as an edible part in the food raw materials notified by the Ministry of Food and Drug Safety. Green tea grass has been known to have the effects of clearing the liver and reducing acidity from old times, and in recent years, effects such as antioxidant, anti-aging, and hair loss prevention have been studied.

However, studies have not been conducted on the energy metabolism-related effects of green tea extract.

One aspect is to provide a health functional food composition for promoting or improving energy metabolism comprising a green tea extract as an active ingredient.

Another aspect is to provide a health functional food composition for preventing or improving obesity, comprising a green tea extract as an active ingredient.

Another aspect is to provide a health functional food composition for the prevention or improvement of metabolic diseases comprising a green tea extract as an active ingredient.

Another aspect is to provide a pharmaceutical composition for the prevention or treatment of obesity comprising a green tea extract as an active ingredient.

Another aspect is to provide a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a green tea extract as an active ingredient.

Another aspect is to provide a cosmetic composition for the prevention or improvement of cellulite comprising a green tea extract as an active ingredient.

Another aspect is to provide a method for stimulating or improving energy metabolism comprising administering an effective amount of an effective amount of an extract of S. chinensis to a subject in need thereof.

Another aspect is to provide a method for preventing, ameliorating or treating obesity or metabolic disease comprising administering an effective amount of a green tea extract to a subject in need thereof.

One aspect provides a health functional food composition for promoting or improving energy metabolism, including an extract of Cassia mimosoides var. nomame as an active ingredient.

The green tea extract may be an extract extracted by a solvent from the whole aerial part of the tree, a part thereof, or a material derived therefrom. The part may be a tree stem, root, leaf, flower, petal, seed (seed), fruit flesh, fruit skin, or fruit, and preferably may be a leaf. Whole trees, parts thereof, or materials derived therefrom used for extraction may be ground or minced or suitably dried.

The green tea extract can be extracted by any extraction method such as hot water extraction, solvent extraction, distillation extraction, supercritical extraction, etc., which have been conventionally used to extract natural plants, and in one embodiment, water, organic solvent, or a mixture thereof. It may be extracted with a solvent. The organic solvent may be at least one selected from the group consisting of alcohols having 1 to 4 carbon atoms, such as ethanol, methanol, isopropanol, and butanol. In addition, the alcohol may include alcohol, and may specifically be fermented alcohol. The alcohol concentration of the aqueous alcohol solution is 1 to 99.5 (v / v)%, for example, 10 to 99.5 (v / v)%, 1 to 70 (v / v)%, 1 to 40 (v / v)% , 5 to 50 (v/v)%, 5 to 40 (v/v)%, 10 to 50 (v/v)%, or 10 to 40 (v/v)%.

In this specification, the term "fermented alcohol" may refer to ethanol made by fermenting starch (cereals, sweet potatoes, tapioca) or sugar (candy) raw materials by adding a saccharification enzyme and then distilling them in a continuous distillation method.

In one embodiment, the green tea extract may be extracted with hot water or fermented alcohol.

The extraction is 3 to 50 (w / w) times, for example, 3 to 40 (w / w) times, 3 to 30 (w / w) times, 5 to 50 (w / w) times the extraction solvent with respect to green tea grass w) times, 5 to 40 (w/w) times, 5 to 30 (w/w) times, or 4 to 40 (w/w) times. For example, it may include adding 3 to 50 kg of the extraction solvent based on 1 kg of the material derived from the tea plantation.

The extraction may be performed by hot water extraction, immersion extraction, supercritical extraction, subcritical extraction, reflux cooling extraction, steam distillation, high pressure enzymatic digestion, ultrasonic extraction, elution, compression, and the like.

The extraction is performed at 4°C to 90°C, such as 4°C to 80°C, 4°C to 70°C, 5°C to 80°C, 10°C to 70°C, 15°C to 70°C or 20°C to 70°C. it may be The extraction time may vary depending on the selected temperature and may be 1 hour to 2 months, for example, 1 hour to 1 month, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 3 days, 1 hour to 2 days. , 1 hour to 1 day, 1 hour to 10 hours, 2 hours to 1 month, 2 hours to 15 days, 2 hours to 10 days, 2 hours to 5 days, 2 hours to 3 days, 2 hours to 2 days, 2 hours to 1 day or 2 hours to 10 hours. The extraction may include mixing the whole green tea grass, a portion thereof, or a material derived therefrom in the solvent and leaving it for a certain period of time. The standing may include suitable agitation. The extraction may be repeated one or more times, for example 1 to 5 times.

In the extraction, plant residues and extracts may be separated by a known method such as filtration. The extraction may also include removing the solvent from the obtained extract by a known method such as concentration under reduced pressure. The extraction may also include preparing a dried extract by drying the obtained extract, such as lyophilization.

In one embodiment, the green tea extract is 0.0001% to 90.0% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, based on the total weight of the composition, 0.01% to 20% by weight, 0.01% to 10% by weight, 0.01% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% by weight % to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 0.1% by weight 20 wt%, 0.1 wt% to 10 wt%, 0.1 wt% to 5 wt%, 5 to 20 wt%, 5 to 18 wt%, 6 to 15 wt%, 8 to 15 wt%, or 7 to 10 wt%. may be included. However, in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

In one embodiment, the green tea extract may increase the activity or expression of AMPK (AMP-activated protein kinase).

As used herein, the term "AMP-activated protein kinase (AMPK)" is an important factor in regulating energy balance in most cells and systemic levels, and may be involved in promoting energy metabolism by phosphorylating and activating AMPK.

In one embodiment, the green tea extract promotes energy metabolism by activating AMPK, thereby suppressing adipogenesis or reducing body fat to be effective in improving obesity, diabetes, and various metabolic diseases. The promotion of energy metabolism may mean promotion of energy metabolism in fat cells or muscle cells.

The tea plant extract may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

Another aspect provides a health functional food composition for preventing or improving obesity, which includes an extract of Cassia mimosoides var. nomame as an active ingredient.

As used herein, the term "obesity" refers to a state in which body fat is excessive, and generally refers to a case in which the body weight is higher than normal, but even if the body weight is not much, if the proportion of body fat among the body components is high, it is diagnosed as obesity, It is a disease that affects both adults and children.

Another aspect provides a health functional food composition for the prevention or improvement of metabolic diseases comprising a green tea extract as an active ingredient.

As used herein, the term "metabolic disease" may refer to a disease caused by excessive synthesis or accumulation of fat due to abnormal energy metabolism in the body due to various causes such as excessive energy intake or hormonal imbalance. Thus, metabolic diseases can be caused by obesity. The type of the metabolic disease is not particularly limited, but may be at least one selected from the group consisting of fatty liver, diabetes, hypertension, hyperlipidemia, hypercholesterolemia, dyslipidemia, and arteriosclerosis.

The "health functional food" refers to a food manufactured and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient contained in a food raw material for the purpose of supplementing health. It refers to food designed and processed to sufficiently exert biological control functions such as defense, regulation of circadian rhythm, prevention and recovery of disease, etc. The health functional food composition may promote or improve energy metabolism, or perform functions related to prevention and improvement of obesity, prevention and improvement of metabolic diseases, and the like.

There is no particular limitation on the type of food. Examples of foods to which the above substances may be added include powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leached teas, and formulations selected from the group consisting of health drinks, etc. It includes all health foods in the usual sense.

The health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

The health functional food may include food additives acceptable in food science, and may include appropriate carriers commonly used in the manufacture of health functional food.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages; and the like. In addition, the composition of the present invention may include fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01 to 0.1 part by weight per 100 parts by weight of the composition of the present invention.

Another aspect provides a pharmaceutical composition for the prevention or treatment of obesity comprising a green tea extract as an active ingredient.

Another aspect provides a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a green tea extract as an active ingredient.

Another aspect provides a pharmaceutical composition for accelerating or improving energy metabolism comprising a green tea extract as an active ingredient.

Another aspect provides a method for stimulating or improving energy metabolism comprising administering an effective amount of a green tea extract to a subject in need thereof.

Another aspect provides a method for preventing, ameliorating or treating obesity or metabolic disease comprising administering an effective amount of a green tea extract to a subject in need thereof.

The metabolic disease may be one or more selected from the group consisting of fatty liver, diabetes, hypertension, hyperlipidemia, hypercholesterolemia, dyslipidemia, and arteriosclerosis.

As used herein, the term "pharmaceutical composition" can refer to a molecule or compound that imparts some beneficial effect upon administration to a subject. Beneficial effects may include enabling diagnostic determination; amelioration of a disease, symptom, disorder or condition; reducing or preventing the occurrence of a disease, condition, disorder or condition; and responding to a disease, symptom, disorder or condition in general. The pharmaceutical composition according to one embodiment may impart beneficial effects on obesity and metabolic diseases by promoting energy metabolism.

As used herein, the term “prevention” refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its attendant symptoms, preventing the acquisition or re-acquisition of a disease or disorder, or preventing the occurrence or recurrence of a disease or disorder. means of reducing the risk of acquisition. For example, the prevention refers to any action that suppresses or delays the occurrence of obesity and metabolic disease symptoms by administration of the composition according to one embodiment.

As used herein, the term "treatment" includes inhibition, alleviation or elimination of the development of diseases, such as obesity and metabolic diseases.

As used herein, the term "improvement" may refer to any activity that at least reduces a parameter associated with alleviation or treatment of a condition, for example, the degree of obesity and metabolic disease symptoms.

The pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may refer to administration through an administration route other than oral, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous administration. When the pharmaceutical composition of the present invention is used as a medicine, it may additionally contain one or more active ingredients exhibiting the same or similar functions.

Types of pharmaceutically active ingredients capable of delivering the active ingredient into the subject include anticancer agents, contrast agents (dye), hormones, anti-hormonal agents, vitamins, calcium agents, mineral preparations, saccharide preparations, organic acid preparations, protein amino acid preparations, detoxifying agents, and enzymes. Preparations, metabolic preparations, diabetes concomitant medicines, tissue regeneration medicines, chlorophyll preparations, pigment preparations, tumor medicines, tumor treatment agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapy agents, antibiotics preparations, antiviral agents, complex antibiotics preparations, chemicals Therapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood products, biological agents, analgesics, immunogenic molecules, antihistamines, allergy medications, non-specific immunogenic agents, anesthetics, stimulants, psychoactive agents, small molecule compounds, nucleic acids, It may include aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs, and micro RNAs.

The pharmaceutical composition may be prepared by further including one or more pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier may be a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and, if necessary, antioxidants and buffers. , bacteriostatic agents and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art.

When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, liurine fat, glycerogeratin and the like may be used.

The pharmaceutical composition may include carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, and small molecules in order to increase stability or absorption. Proteins or other Stabilizers can be used as pharmaceuticals.

Another aspect provides a method of treating obesity and metabolic disease comprising administering the pharmaceutical composition to a subject.

As used herein, the term "administration" means providing a pharmaceutical composition to a subject by any suitable method.

A pharmaceutically effective amount and an effective dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition. In addition, the type and degree of response to be achieved by administration of the pharmaceutical composition, the type of subject to be administered, age, weight, general health condition, symptom or degree of disease, gender, diet, excretion, simultaneous or It may vary according to various factors including drugs and other components of the composition used together in this case, and similar factors well known in the medical field. A person of ordinary skill in the art can readily determine and prescribe an effective dosage for the desired treatment. Administration of the pharmaceutical composition according to the present invention can be administered once a day, divided into several administrations. Therefore, the dosage is not intended to limit the scope of the present invention in any way. The dosage of the pharmaceutical composition may be 1 ug/kg/day to 1,000 mg/kg/day.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual who needs to improve or promote energy metabolism, needs treatment for obesity and metabolic diseases, or has a desire to lose weight.

As used herein, “treatment” or “treating” or “mitigating” or “improving” are used interchangeably. These terms refer to methods that obtain beneficial or desired results, including but not limited to therapeutic benefit and/or prophylactic benefit. By therapeutic benefit is meant any therapeutically significant improvement in or effect on one or more diseases, conditions or symptoms under treatment. For prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, condition or condition or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition or condition has not yet been manifested.

The term “effective amount” or “therapeutically effective amount” refers to an amount of an agent sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the subject and condition being treated, the weight and age of the subject, the severity of the condition, the mode of administration, and the like, and can be readily determined by one skilled in the art. The term also applies to a capacity that will provide an image for detection by any of the imaging methods described herein. The specific dosage may vary depending on one or more of the particular agent selected, the dosage regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the body delivery system that delivers it.

Another aspect provides a cosmetic composition for the prevention or improvement of cellulite comprising a green tea extract as an active ingredient.

As used herein, the term "cellulite" refers to a condition in which fat protrudes in places, particularly in the thigh or buttocks, due to circulation disorders caused by accumulation of excessive fat and waste products. The cause of cellulite is basically the increase or hypertrophy of fat cells.

As used herein, the term "prevention or improvement of cellulite" means a decrease in the volume of cellulite, a decrease in the height of cellulite, removal of cellulite, flattening of cellulite and its surroundings, and the like. it may be

The composition has an effect of suppressing body fat accumulation through promotion of energy metabolism, and thus has an effect of preventing or improving cellulite. In addition, due to this effect, the composition has a body slimming effect. The term "body slimming" refers to promoting even redistribution of fat, reducing the volume of a body or a part of the body, eliminating puffiness as a result, in body lines that appear bumpy due to uneven distribution of fat under the skin. It may mean reducing the volume of the body or a part of the body, improving the state of local obesity, dissolving and excreting fat in fat cells, reducing triglyceride and body fluid accumulation in fat cells, and the like.

The cosmetic composition includes lotion (skin lotion), toner, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, hand sanitizer, foundation, essence, Nutritional essence, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, suspensions, gels, powders, pastes, mask packs, and can be prepared into formulations including sheets. Compositions of such formulations may be prepared according to conventional methods in the art. The blending amount of the additional ingredients such as the moisturizing agent can be easily selected by those skilled in the art within a range that does not impair the objects and effects of the present invention.

The cosmetic composition may further include functional additives and ingredients included in general cosmetic compositions in addition to the active ingredients disclosed herein, and commonly used purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, A flavoring agent or a combination thereof may be further included. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts. Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, coloring agents, perfumes, and the like may be exemplified as the carrier. Compounds/compositions that can be used as the alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, coloring agent, fragrance, etc. are already known in the art. Since there is, those skilled in the art can select and use an appropriate corresponding material / composition. In addition, the cosmetic composition, if necessary, sunscreen, antioxidants (butylhydroxyanisole, gallic acid propyl, ellisorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben , propylparaben, phenoxyethanol, imidazolidinylurea, chlorphenesin, etc.), colorant, pH adjuster (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid , sodium succinate, sodium hydroxide, sodium hydrogen phosphate, etc.), humectants (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methylglue Seth-20, etc.) and lubricants may be further added.

In addition, in the cosmetic composition of each formulation, appropriate components may be selected and blended according to the cosmetic formulation or purpose of use. Since the formulation components and methods may follow conventional techniques, detailed descriptions thereof are omitted herein.

The terms and methods described for the above invention are equally applied between each invention.

According to the composition containing the extract of green tea plant according to one aspect as an active ingredient, it has the effect of improving or accelerating energy metabolism and reducing body fat, so it can be usefully used for improving or promoting energy metabolism, obesity and metabolic diseases. there is.

1 is a graph showing the results of analyzing a hot water extract of green tea extract and a 30% alcohol extract by high performance liquid chromatography (HPLC).

Figure 2 is a graph confirming the toxicity of green tea extract ( Cassia mimosoides water Ex) and 30% alcohol extract ( Cassia mimosoides 30% EtOH Ex) to adipocytes. (Above: HPLC of hot water extract of green tea grass, bottom: HPLC of 30% alcohol extract of green tea grass)

Figure 3 is a photograph confirming the change in neutral fat accumulation in adipocytes when the hot water extract of green tea plant and 30% alcohol extract were treated. (GM: general medium treatment; DM: differentiation medium treatment)

Figure 4 is a graph of the results of quantitative analysis of the change in neutral fat accumulation in adipocytes when the hot water extract and 30% alcohol extract were treated. (MDI: treatment with differentiation medium)

Figure 5 is a graph showing the change in body length when zebrafish are administered with hot water extract and 30% alcohol extract after induction of obesity. Hot water extract; CM30E: 30% alcohol extract of green tea grass)

Figure 6 is a photograph showing the change in body length when zebrafish are administered with hot water extract and 30% alcohol extract after induction of obesity. (NOR: normal control; CON: obesity induced control; PC: positive control; CMW: green tea Hot water extract; CM30E: 30% alcohol extract of green tea grass)

Figure 7 is a graph showing the change in the circumference of the belly when the hot water extract of zebrafish and 30% alcohol extract were administered after induction of obesity. (NOR: normal control group; CON: obesity induced control group; PC: positive control group; CMW: Green tea plant hot water extract; CM30E: tea plant 30% alcohol extract)

Figure 8 is a graph showing the change in body weight when a hot water extract and 30% alcohol extract of zebrafish were administered after induction of obesity. Hot water extract; CM30E: 30% alcohol extract of green tea grass)

Figure 9 is a photograph confirming the amount of fat when zebrafish were administered with hot water extract and 30% alcohol extract after induction of obesity. (NOR: normal control group; CON: obesity induced control group; PC: positive control group; CMW: Green tea plant hot water extract; CM30E: tea plant 30% alcohol extract)

Figure 10 is a graph confirming the accumulation of fat in the liver when a hot water extract and 30% alcohol extract were administered to zebrafish after induction of obesity. (NOR: normal control group; CON: obesity induced control group; PC: positive control group; CMW: hot water extract of green tea plant; CM30E: 30% alcohol extract of green tea grass)

11 is a graph showing the change in body weight when 30% alcohol extract of green tea grass was simultaneously administered with the induction of obesity in mice.

12 is a dual-energy X-ray absorptiometry of mice when 30% alcohol extract of green tea grass was simultaneously administered with induction of obesity in mice to see the effect of reducing body fat of 30% alcohol extract of green tea grass. It is a picture of fat distribution image and body fat content measured using this method.

Figure 13a is a graph showing the weight of body fat in the case of simultaneous administration of 30% alcohol extract of green tea plant along with induction of obesity in mice; And Figure 13b is a graph showing the fat content in the body when 30% alcohol extract of green tea plant was simultaneously administered with the induction of obesity in mice.

Figure 14a is a graph showing the weight of subcutaneous fat in the case of simultaneous administration of 30% alcohol extract of green tea plant along with induction of obesity in mice; 14B is a graph showing liver weight; 14C is a graph showing mesenteric fat weight; 14D is a graph showing epididymal fat weight; 14E is a graph showing renal fat weight; and Figure 14f is a graph showing brown fat weight.

Figure 15a is a photograph of adipocytes observed under a microscope to see the effect of reducing the size of fat globules of 30% alcohol extract of green tea grass; and FIG. 15B is a graph showing the size of adipocytes.

16 is a graph confirming the expression of p-AMPK protein in mouse liver tissue when 30% extract of green tea grass was administered.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Tea pool ( Cassia mimosoides var. nomame) Preparation of extract

The 30% alcohol extract contained in the composition was prepared by the following process. First, domestically grown tea grass was purchased, washed thoroughly with distilled water, and then completely dried in a cool place without sunlight until there was no change in weight. Then, after preparing 30 kg of 30% fermented alcohol (30 times, w/w) to the completely dried dried product, 1 kg of the dried product was added. At this time, the mixing ratio of the dry matter and 30% fermented alcohol was 30 times the weight ratio. Next, 30% fermented alcohol mixed with the dried product was put in a constant temperature water bath and stirred and extracted at 80 ° C. for 5 hours. The extracted sample was filtered and concentrated under reduced pressure to obtain a yellow-brown water-soluble powder.

The hot water extract contained in the composition was prepared by the following process. First, domestically grown tea grass was purchased, washed thoroughly with distilled water, and then completely dried in a cool place without sunlight until there was no change in weight. Then, after preparing 30 kg of purified water (30 times, w/w) to the completely dried dried product, 1 kg of the dried product was added. At this time, the mixing ratio of the dried material and the purified water was 30 times the weight ratio. Next, purified water mixed with the dried material was put in a constant temperature water bath and stirred and extracted at 100 ° C. for 5 hours. The extracted sample was filtered and concentrated under reduced pressure to obtain a yellow-brown water-soluble powder.

Experimental Example 1: Component Analysis of Green Tea

The extract powder obtained in Example 1 was analyzed by high performance liquid chromatography (HPLC) and a UV/Vis detector. HPLC instruments were Waters e2695 Series system, Waters 24489 UV/Vis detector (Worcester, MA, USA), Xselect (5 μm, 250 Х 4.6 mm, Phenomenex, Torrance, CA, USA) column, and all solvents used in the analysis were used. is J.T. HPLC grade solvents purchased from Baker (Phillipsburg, NJ, USA) were used. At the time of analysis, the temperature of the column was set to 30°C, the injection volume was 10 μl, and the measurement wavelength was set to 285 nm. As the mobile phase, acetonitrile (ACN) with 0.1% formic acid and tertiary distilled water (D.W) with 0.1% formic acid were used, and 0.1% formic acid ACN was added at a rate of 1 ml/min. - 0.1% formic acid D.W (1:9 - 10:0, v/v) mixed solution was analyzed for 65 minutes. For the analysis sample, 200 mg of hot water extraction powder and alcohol extraction powder obtained in Example 1 were precisely weighed, 10 ml of methanol was added, dissolved in an ultrasonic shaker for 20 minutes, cooled at room temperature, and the supernatant was filtered through a 0.45 μm membrane filter. used For each analysis sample, the chromatogram was extracted at 285 nm, and the hot water extract of Chapule and the 30% alcohol extract were compared and analyzed (FIG. 1).

Experimental Example 2: Confirmation of cytotoxicity

The cytotoxicity of the extract of Example 1 on adipocytes was measured.

To confirm cytotoxicity, first, 3T3-L1 was attached to a 96-well plate at a concentration of 1.0 X 10 ⁴ cells/well for 24 hours to stabilize. Thereafter, the hot-water extract and 30% alcohol extract of green tea plant were exchanged with the medium treated with the sample for each concentration and cultured for 24 hours. Then, the degree of cell viability was measured by MTT assay.

Figure 2 is a graph confirming the toxicity of green tea extract ( Cassia mimosoides water Ex) and 30% alcohol extract ( Cassia mimosoides 30% EtOH Ex) to adipocytes.

As shown in Figure 2, it was confirmed that the green tea extract was not cytotoxic.

Experimental Example 3: Assessment of inhibition of fat accumulation through Oil Red O staining

In this experiment, in order to induce adipocyte differentiation, 3T3-L1 cells were seeded on a plate and cultured in 10% BS medium until the cell density reached 100%. In the stage of cell differentiation, 10% FBS differentiation medium (Insulin 5μg/ml, Dexametasone 1μM, 3-Isobutyl-1-methylxanthine 0.5mM) at concentrations of 10 ppm, 25 ppm, 50 ppm, and 100 ppm, respectively, of Example 1 was applied. Extract and 30% alcohol extract were treated, respectively. After 8 days, how much fat accumulation can be inhibited was measured through Oil Red-O staining and quantitative analysis. For visual evaluation, images were taken after staining. Then, the stained cells were completely dried, dissolved in DMSO (Dimethyl Sulfoxide), transferred to a 96-well plate, and absorbance was measured at 450 nm.

Figure 3 is a photograph confirming the change in neutral fat accumulation in adipocytes when the hot water extract of green tea plant and 30% alcohol extract were treated. (GM: general medium treatment; DM: differentiation medium treatment)

Figure 4 is a graph of the results of quantitative analysis of the change in neutral fat accumulation in adipocytes when the hot water extract and 30% alcohol extract were treated. (MDI: differentiation medium treatment)

As shown in Figures 3 and 4, it was confirmed that the green tea extract inhibits fat accumulation. These results indicate that the green tea extract promotes energy consumption, furthermore effectively inhibits or improves fat accumulation, and also effectively prevents or treats metabolic diseases caused by obesity.

Experimental Example 4: Tea pool ( Cassia mimosoides var. nomame) extract in vivo ( in vivo ) effect analysis

4-1. Zebrafish and experimental design

In order to analyze the in vivo anti-obesity efficacy of the green tea extract obtained in Example 1, zebrafish fry were prepared by inviotec. First, females and males were placed in a 1:1 ratio in a tank equipped with a net to separate eggs and adult fish, and zebrafish embryos were collected after 12 hours. After collection, it was washed three times using 0.03% sea salt solution to remove foreign substances and reared in an incubator with a 28.5 ° C photoperiod (14 light / 10 dark). Zebrafish fry (3 dpf) after 3 days of fertilization were placed in a petri dish, and then treated for 2 days of rotifer, 6 days of artemia, and 4 days of feed, for a total of 12 days, to prepare an overfeed-induced obese zebrafish model. The overfeed-induced obese zebrafish model was treated with rotifer, artemia, and feed 4 times a day, and the normal control group was treated with rotifer, artemia, and feed twice a day. From the 11th day, the overfeed-induced obese zebrafish model was treated with 100 μg/ml of the hot-water extract or 30% alcohol extract of Example 1 and 50 μg/ml of Cissus extract as a positive control group and exposed for 24 hours.

4-2. Analysis of weight and fat reduction effect by green tea extract

In order to confirm that the body weight and fat of zebrafish were reduced when the green tea extract obtained in Example 1 was administered, an experiment was performed as follows. After the experiment was completed, the zebrafish fry were anesthetized with 0.02% tricaine, and the body length and belly length of the zebrafish were observed using an optical microscope (Olympus 1Х70, Olympus, Japan). Afterwards, body length and belly length were measured using Focus Lite software, and the weight was checked with a precise electronic scale for analysis and graphed. Statistical analysis was performed using Sigma Plot software (Systat software Inc., USA) and paired t-teat and repeated ANOVA to confirm whether there was a difference between groups in each experimental condition.

Figure 5 is a graph showing the change in body length when zebrafish were administered with hot water extract and 30% alcohol extract after induction of obesity.

Figure 6 is a photograph showing the change in body length when zebrafish were administered with hot water extract and 30% alcohol extract after induction of obesity.

7 is a graph showing changes in the circumference of the stomach when zebrafish are administered with hot water extract and 30% alcohol extract after induction of obesity.

8 is a graph showing changes in body weight when zebrafish are administered with hot water extract and 30% alcohol extract after induction of obesity.

9 is a photograph confirming the amount of fat when zebrafish were administered with hot water extract and 30% alcohol extract after induction of obesity.

NOR in the figure: normal control; CON: obesity induced control; PC: positive control; CMW: Chapul hot water extract; CM30E: It means 30% alcohol extract of green tea plant.

As shown in Figures 5 and 6, Tables 1 and 2, when compared to the zebrafish (CON) in which obesity was induced, the positive control (PC), green tea extract (CMW) and 30% alcohol extract (CM30E) Although there was little change in body length in the group administered with, it was confirmed that weight, abdominal circumference and fat were reduced as shown in FIGS. 7 to 8 and Table 3. Specifically, compared to the normal control group (NOR), obesity induced zebrafish (CON) increased in weight and showed a large amount of fat in the abdomen, but the positive control (PC), green tea extract (CMW) and 30% alcohol extract In the group administered with (CM30E), it was confirmed that body weight decreased and abdominal fat decreased.

These results indicate that the green tea extract effectively prevents or improves obesity by inhibiting fat accumulation, and also effectively prevents or treats metabolic diseases caused by obesity.

	NOR	CON	Positive	CMW	CM30E
Body length (mm)	7.134 ± 0.1571	7.204 ± 0.1202	7.118 ± 0.2969	7.173 ± 0.2629	7.186 ± 0.1771

	NOR	CON	Positive	CMW	CM30E
Belly length (μm)	1530 ± 73.3	1804 ± 151.0	1643 ± 64.2	1702 ± 109.7	1615 ± 102.7

	NOR	CON	Positive	CMW	CM30E
body weight (mg)	0.05 ± 0.006	0.09 ± 0.006	0.04 ± 0.006	0.07 ± 0.006	0.05 ± 0.006

4-3. Confirmation of inhibition of fatty liver by green tea extract

In order to confirm the reduction of fatty liver in the liver of obese zebrafish when the extract of Example 1 was administered, an experiment was performed as follows. First, the sacrificial zebrafish fry at the end of the experiment were fixed in 4° C. paraformaldehyde (PFA) overnight, and then washed twice with 1X PBS. Thereafter, 80% and 100% propylene glycol were sequentially treated for 20 minutes at room temperature, and zebrafish fry were dyed by exposing overnight to 0.5% Oil Red O in 100% propylene glycol at room temperature to block light. The dyed fry was washed with 1 X PBS, treated with 100% and 80% propylene glycol at room temperature for 30 minutes, respectively, to wash the dye, stored in 80% propylene glycol at 4℃, and then examined under an optical microscope (Olympus 1Х70, Olympus , Japan) was used to observe liver fat, and fat size was measured using Focus Lite software.

9 is a graph confirming the accumulation of fat in the liver when the hot water extract and 30% alcohol extract were administered after induction of obesity in zebrafish.

As shown in FIG. 10 and Table 4, compared to obesity-induced zebrafish (CON), the positive control (PC), green tea extract (CMW) and 30% alcohol extract (CM30E) were administered to the liver in the group. It was confirmed that the accumulated fat was reduced. These results indicate that the green tea extract effectively prevents or improves obesity by inhibiting fat accumulation, and also effectively prevents or treats metabolic diseases caused by obesity.

	NOR	CON	Positive	CMW	CM30E
Lipid accumulation In liver (mm 2 )	0.042 ± 0.0155	0.071 ± 0.0140	0.052 ± 0.0136	0.058 ± 0.0174	0.057 ± 0.0148

Experimental Example 5: Analysis of in vivo effect of 30% alcohol extract of green tea grass

5-1. Mice and Experimental Design

In order to analyze the in vivo anti-obesity efficacy of 30% alcohol extract of tea grass, an obese animal model was first prepared. 8-week-old male C57BL/6N mice (specific-pathogen-free (SPF) grade, 20 ± 2 g, Orient Bio) were set into 5 groups as follows, and 8 mice per group were tested: As a normal control, high-fat diet Orlistat, an anti-obesity treatment, was placed in a normal mouse (con), which was not administered with anything, and an obese mouse (HFD), which was induced with a 30% high-fat diet and not administered anything, respectively. was orally administered. As an experimental group, obese mice were orally administered with 100 mg/kg and 300 mg/kg of 30% alcohol extract of green tea plant, respectively. 30% ethyl alcohol extract was orally administered 5 days per week during the administration period. The dark: light cycle was maintained at 12-hour: 12-hour intervals, and water was freely consumed.

5-2. Analysis of weight and fat reduction effect by 30% alcohol extract of green tea plant

An experiment was performed to analyze whether body weight and fat were reduced in mice when 30% alcohol extract was administered. As a result, when looking at the change in body weight of the mice according to each experimental group and time, the positive control group and the group administered with 30% alcohol extract of Japanese tea plant showed a weight loss effect (FIG. 11). In particular, it was confirmed that the group administered with 300 mg/kg of 30% alcohol extract of green tea plant inhibited weight gain to a similar extent to that of the positive control group.

In addition, in the last week of the animal experiment, fat distribution images and body fat content were measured using dual-energy X-ray absorptiometry for mice in each experimental group, and the animals were sacrificed to separate adipose tissue including epididymal fat. fat weight was measured. The experimental results showed significant differences between groups using Dunnett's post hoc test in the GraphPad Prism statistical program (p ^# <0.05 vs normal control group, p ^* <0.05, p ^** <0.01, P ^*** <0.001 vs obese group) . As a result, it was confirmed that the body fat mass and body fat percentage were reduced in the positive control group and the group administered with 30% alcohol extract of green tea plant (Figs. 12 and 13). The weights of various adipose tissue and liver tissue analyzed after animal sacrifice were all increased when a high-fat diet was administered, but it was confirmed that the weight was significantly decreased in the group administered with 30% alcohol extract of Japanese tea plant (FIG. 14).

5-3. Analysis of fat cell size reduction effect by 30% alcohol extract of green tea plant

For histological analysis, mouse epididymal adipose tissue was fixed in 4% paraformalin. After dehydration through continuous alcohol concentration gradient (Graded alcohol series) and washing several times, the tissues were embedded in paraffin. Tissue sections were cut to a thickness of 4 μm, and stained with hematoxylin and eosin. In order to confirm the size of white adipocytes, the area of each adipocyte was measured in each section using CellSence software (Olympus Co., USA). As a result, it was confirmed that the size of fat cells in the group administered with 30% alcohol extract of green tea grass along with induction of obesity was reduced to a degree similar to that of the normal control group (FIG. 15).

5-4. Analysis of protein expression related to energy metabolism by 30% alcohol extract of green tea grass

AMPK (AMP-activated protein kinase) is activated to maintain energy homeostasis, inhibits fat and cholesterol synthesis, and promotes fatty acid oxidation. In addition, during adipocyte differentiation, initial differentiation is initiated by C/EBPs, which are proteins involved in adipogenesis including C/EBPα, peroxisome proliferator-activated receptor (PPAR)γ, and sterol controlled element-binding protein (SREBP)-1. It promotes terminal differentiation by increasing the expression of transcription factors that regulate genes. Therefore, in order to confirm whether the administration of 30% alcohol extract of green tea grass regulates the expression of AMPK, the protein expression level of the group administered with the sample along with induction of obesity was analyzed.

Specifically, after mixing the adipose tissue with the protein extraction solution and crushing it using a tissue grinder, the supernatant was obtained by centrifugation at 4 ° C. and 15,000 rpm for 30 minutes, and then a standard curve was prepared using the Bradford method to quantify the protein. After adding 6-fold sample buffer to 30 μg of protein, incubating in a water bath for 5 minutes, electrophoresis was performed using a 10% SDS-PAGE gel, followed by immunoblotting on a PVDF membrane for 1 hour and 20 minutes. After blocking for 1 hour with TBST buffer solution (Tris-buffered saline-Tween 20) containing 5% (w/v) skim milk, anti-p-AMPK, PPARγ, C/EBPα, and SREBP-1 antibodies were added at 1:1000. and reacted at 4°C for 18 hours. After washing three times for 10 minutes with TBST buffer, the cells were reacted with peroxidase-conjugated secondary antibodies at room temperature for 2 hours. After washing three times for 10 minutes with TBST buffer solution, color was developed and developed as a hyper film using an enhanced chemiluminescence kit (Amersham Life Sciences, Amersham, U.K.), and protein changes in each control and experimental group were confirmed by western blot.

As a result, it was confirmed that energy metabolism was activated while the amount of phosphorylated AMPK protein expression increased when the 30% alcohol extract of green tea grass was administered (FIG. 16).

Formulation Example 1: Preparation of tablets

Tablets were prepared by mixing the ingredients in Table 5 below with respect to the extract of Example 1 according to a conventional tablet manufacturing method and tableting.

raw material name	unit weight (mg)
Example 1	300.0006
silicon dioxide	15.3000
Magnesium Stearate	10.8000
crystalline cellulose	509.4945
Hydroxypropyl Methyl Cellulose	29.0700
Carboxymethyl Cellulose Calcium	27.0000
Glycerin fatty acid ester	0.6930
titanium dioxide	1.4697
red national pigment	4.4082
powdered caramel color	1.7640

Formulation Example 2: Preparation of capsules

Soft capsules were prepared by mixing the ingredients in Table 6 below with respect to the green tea extract of Example 1 according to the usual method for preparing capsules and filling them into gelatin capsules.

raw material name	unit weight (mg)
Example 1	50
vitamin E	2.25
vitamin C	2.25
palm oil	0.5
hydrogenated vegetable oil	2
Yellow lead	One
lecithin	2.25
Soft capsule filling solution	387.75

Formulation Example 3: Preparation of liquid formulation

With respect to the tea plant extract of Example 1, the ingredients in Table 7 below were mixed according to a beverage manufacturing method suitable for taste and filled into a bottle or pouch to prepare a liquid formulation.

raw material name	unit weight (g)
Example 1	2.5050
xanthan gum	0.0075
fructooligosaccharide solution	0.7500
Coconut Flower Extract Powder	1.0500
Ssanghwa Concentrate	1.5000
Red Ginseng Fragrance	0.0450
Purified water	9.1425
raw material name	unit weight (g)
Example 1	2.5050
xanthan gum	0.0075
fructooligosaccharide solution	0.7500
Coconut Flower Extract Powder	1.0500
Ssanghwa Concentrate	1.5000
Red Ginseng Fragrance	0.0450
Purified water	9.1425

Formulation Example 4: Preparation of Jelly

With respect to the tea plant extract of Example 1, the ingredients in Table 8 were mixed according to a jelly manufacturing method suitable for taste and filled in three cotton cloths to prepare jelly.

raw material name	unit weight (g)
Example 1	2.0000
food gel	0.3600
carrageenan	0.0600
calcium lactate	0.1000
sodium citrate	0.0600
complex gold extract	0.0200
Enzymatically Treated Stevia	0.0440
fructooligosaccharide solution	5.0000
Red Grape Concentrate	2.4000
Purified water	13.9560

Formulation Example 5: Preparation of nutrient cream

For the extract of the green tea extract of Example 1, a nutritious cream was prepared according to the composition shown in Table 9 below according to a conventional method.

raw material name	content (%)
Example 1	1.0
sitosterol	4.0
Polyglyceryl 2-oleate 3.0	3.0
Ceteares-4	2.0
cholesterol	3.0
dicetyl phosphate	0.4
concentrated glycerin	5.0
sunflower oil	22.0
carboxyvinyl polymer	0.5
triethanolamine	0.5
antiseptic	a very small amount
Spices	a very small amount
Purified water	balance

Although the above composition ratio is generally prepared as a formulation example by mixing suitable ingredients, the mixing ratio and raw materials may be arbitrarily changed as necessary.

Since the extract of the present invention and components derived therefrom are stable in all formulation test conditions, there was no problem with the stability of the formulation.

Claims (18)

1. A health functional food composition for promoting or improving energy metabolism, comprising an extract of Cassia mimosoides var. nomame as an active ingredient.
2. The health functional food composition for promoting or improving energy metabolism according to claim 1, wherein the green tea extract is extracted with water, C1 to C4 alcohol, or a mixed solvent thereof.
3. The health functional food composition for promoting or improving energy metabolism according to claim 1, wherein the green tea extract is extracted with hot water or fermented alcohol.
4. The method according to claim 1, wherein the green tea extract is contained in 0.0001 to 90% by weight based on the total weight of the composition, the health functional food composition for promoting or improving energy metabolism.
5. The health functional food composition for promoting or improving energy metabolism according to claim 1, wherein the green tea extract increases the activity or expression of AMPK (AMP-activated protein kinase).
6. The health functional food composition for promoting or improving energy metabolism according to claim 1, wherein the green tea extract promotes or improves energy metabolism to inhibit lipogenesis or reduce body fat.
7. The method according to claim 1, wherein the health functional food has a formulation selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leaching teas and health drinks, Health functional food composition for promoting or improving energy metabolism.
8. A health functional food composition for preventing or improving obesity, comprising an extract of Cassia mimosoides var. nomame as an active ingredient.
9. A health functional food composition for preventing or improving metabolic diseases comprising an extract of Cassia mimosoides var. nomame as an active ingredient.
10. The method according to claim 9, wherein the metabolic disease is one or more selected from the group consisting of fatty liver, diabetes, hypertension, hyperlipidemia, hypercholesterolemia, dyslipidemia, and arteriosclerosis, the functional food composition for preventing or improving metabolic diseases .
11. A pharmaceutical composition for preventing or treating obesity comprising an extract of Cassia mimosoides var. nomame as an active ingredient.
12. A pharmaceutical composition for preventing or treating metabolic diseases comprising an extract of Cassia mimosoides var. nomame as an active ingredient.
13. The method according to claim 12, wherein the metabolic disease is at least one selected from the group consisting of fatty liver, diabetes, hypertension, hyperlipidemia, hypercholesterolemia, dyslipidemia, and arteriosclerosis, a pharmaceutical composition for preventing or treating metabolic diseases.
14. A cosmetic composition for preventing or improving cellulite comprising an extract of Cassia mimosoides var. nomame as an active ingredient.
15. A method of promoting or improving energy metabolism comprising administering an effective amount of an extract of Cassia mimosoides var. nomame to a subject in need thereof.
16. A method for preventing, improving or treating obesity or metabolic disease comprising administering an effective amount of Cassia mimosoides var. nomame extract to a subject in need thereof.
17. Use of Cassia mimosoides var. nomame extract for the preparation of a composition for promoting or improving energy metabolism.
18. Use of extract of Cassia mimosoides var. nomame for the preparation of a composition for preventing, improving or treating obesity or metabolic diseases.

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