KR102438938B1 - Vitalmelon (KCTC14699BP) and anti-obesity composition comprising vitalmelon extract - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or alleviating obesity or metabolic-related diseases by comprising vital melon (KCTC14699BP) and an extract thereof. The vital melon of the present invention is excellent in productivity, as the vitalmelon can bear as much as the number of nodes of main branches and side branches and obtain larger fruits than those of oriental melons. Since an extract thereof can effectively inhibit all of lipid accumulation, weight gain, and fat accumulation in liver and the like, the extract can be used as the composition for preventing, alleviating or treating various obesity or metabolic diseases.

Description

Vitalmelon (KCTC14699BP) and anti-obesity composition comprising extract thereof {Vitalmelon (KCTC14699BP) and anti-obesity composition comprising vitalmelon extract}

The present invention relates to a composition for preventing, treating, and improving obesity or metabolic diseases, comprising vital melon (KCTC14699BP) and an extract thereof.

In modern society, the number of obese people is rapidly increasing due to various causes such as convenient living environment according to rapid automation, excessive nutrition intake and decrease in physical activity due to increase in processed food and eating out. As a result, hypertension, diabetes, arteriosclerosis, hyperlipidemia , metabolic diseases such as insulin resistance and dyslipidemia are on the rise. Among them, in recent years, non-alcoholic fatty liver (NAFLD) is increasing due to an increase in obesity, diabetes, hyperlipidemia, etc., and some of them are known to progress to fatty liver or cirrhosis. Since it is considered an early symptom of a metabolic disease, not a disease, the importance of early diagnosis and treatment of fatty liver is increasing.

On the other hand, metabolic chronic disease is known to be caused by obesity. When excessive energy is supplied beyond the limit of adipose tissue, which is an energy storage tissue, it accumulates in non-adipose tissues such as liver, muscle, pancreas, and heart in the form of triglycerides (TG). As a result, free fatty acids in the blood increase, leading to apoptosis or inflammation. In addition, fatty acid synthesis increases, fatty acid oxidation rate decreases, insulin resistance in muscles and liver increases, insulin production in the pancreas decreases and beta cell death occurs. , heart failure, myocardial infarction, and non-alcoholic fatty liver (NAFLD) in the liver are known to occur. Therefore, tissue fat suppression through fat absorption, fat biosynthesis inhibition, and fatty acid oxidation increase can be an effective strategy for the prevention and treatment of metabolic diseases such as obesity, insulin resistance, diabetes, and fatty liver.

As an anti-obesity drug sold at home and abroad, there is 'Xenical', which uses orlistat as a main ingredient, which has been approved by the US FDA. It is known to have side effects.

Therefore, there is a need for new substances that can effectively prevent, treat, and improve various modern metabolic diseases caused by obesity or obesity without side effects, and there is a high interest in new substances using natural materials.

The present inventors found that by hybridizing melons and cultivated melons during research to obtain new useful crops, new varieties of melons can be produced that can overcome the low productivity of melons while increasing the useful activity of melons. It was confirmed, and further, it was confirmed that the extract of the new variety identified in the present invention exhibits an excellent anti-obesity effect, and the present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical, food, and health functional food composition comprising the vital melon extract and the vital melon having an anti-obesity effect.

In order to achieve the above object, the present invention is i) fruiting as many as the number of nodes on the main and side branches; And ii) provides a vital melon ( Cucumis melo L. var. vitalmelon) with an accession number KCTC14699BP having characteristics such as high content of cucurbitacin A and cucurbitacin B compared to melon varieties.

In addition, the present invention is a parent melon ( Cucumis melo var. makuwa Makino) self-fertilized 5 to 10 times with the target trait for the characteristics of epiphysis and positivity in each node, and the characteristics of leaflets and large fruits as target traits. The step of hybridizing self-fertilized melon 10 times ( Cucumis melo var. reticulatus ); It provides a method for producing vital melon ( Cucumis melo L. var. vitalmelon ) with accession number KCTC14699BP containing a.

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity or metabolic disease comprising an extract of vital melon (accession number KCTC14699BP).

In addition, the present invention provides a health functional food composition for preventing or improving obesity or metabolic disease comprising an extract of vital melon (accession number KCTC14699BP).

In addition, the present invention provides a food composition for preventing or improving obesity or metabolic disease comprising an extract of vital melon (accession number KCTC14699BP).

In addition, the present invention provides a quasi-drug composition for preventing or improving obesity or metabolic disease comprising an extract of vital melon (accession number KCTC14699BP).

The vital melon of the present invention has fruit as many as the number of nodes of the main and side branches, and can obtain larger fruits compared to the existing melon, so it has excellent productivity, and its extract reduces lipid accumulation, weight gain, and fat accumulation in the liver. Since it can be effectively inhibited, it can be utilized as a composition for preventing, improving or treating various obesity or metabolic diseases.

1 is a view showing the appearance of the vital melon (KCTC14699BP) of the present invention obtained by hybridizing the parent melon and the secondary cultivated melon.
2 is a diagram showing the results of confirming the effect of inhibiting adipocyte differentiation and lipid accumulation of vital melon water extract (Hybrid (F1)), melon (mother, M), and cultivated melon (Father, F).
Figure 3 is a diagram showing the results of confirming the effect of inhibiting intracellular glucose uptake (A) and triglyceride accumulation (B) according to the treatment concentration of vital melon water extract (VW) (The values with different letters indicate significant differences as determined by Duncan's multiple range test (p < 0.05)).
Figure 4 is a diagram showing the results of confirming the changes in plasma liver enzymes, lipid levels, total protein and glucose according to the diet of orlistat or vital melon water extract (VW) as a positive control in a high-fat diet mouse (HFD). .
5 is a diagram showing the results of confirming the changes in insulin, leptin, and adiponectin according to the diet of orlistat (OT10) or vital melon water extract (VW) as a positive control in high-fat diet mice (HFD).
6 is a diagram showing the results of confirming the effect of inhibiting liver fat accumulation according to the diet of orlistat (OT10) or vital melon water extract (VW), which is a positive control, in a high-fat diet mouse (HFD).
7 is a diagram showing the results of confirming the effect of suppressing fat cell size according to the diet of orlistat (OT10) or vital melon water extract (VW), which is a positive control, in high-fat diet mice (HFD).
8 is a diagram showing the results of confirming the expression change of PPAR-γ and its target genes LPL, CD35, HMGCR and L-FABP according to the treatment of vital melon water extract (VW) in 3T3-L1 cells.
9 is a diagram showing the results of confirming the expression change of PPAR-γ and its target genes LPL, CD36, HMGCR and L-FABP according to the treatment of vital melon water extract (VW) in the liver of a high-fat diet mouse.

The present invention provides accession number KCTC14699BP vital melon ( Cucumis melo L. var. vitalmelon ).

The vital melon is i) fruiting as many as the number of nodes on the main and side branches; and ii) a high content of cucurbitacin A and cucurbitacin B compared to melon varieties.

The vital melon is obtained by hybridizing a melon ( Cucumis melo var. makuwa Makino ) as a model, and a cultivated melon ( Cucumis melo var. reticulatus ) as a parent.

In order to obtain the vital melon of the present invention, melons that have been self-fertilized 5 to 10 times, preferably 6 to 8 times, can be used for melons that have been self-fertilized 5 to 10 times, preferably 6 to 8 times, with the characteristics of epiphytic and protonation characteristics at each node as the parent melon. Cultivated melons can use self-fertilized melons 5 to 10 times, preferably 6 to 8 times, with the characteristics of leaflets and large fruits as target traits. Through the repetitive self-fertilization process as described above, the traits of the parent and the parent can be fixed.

The hybridization method of the parent and the parent for obtaining the vital melon of the present invention can use methods known in the art without limitation, but in a preferred embodiment of the present invention, 25 to 40 of melon and cultivated melon varieties having the above traits fixed One-year-old seedlings were transplanted into a horticultural medium, and hybridized vital melons were produced by artificial pollination using pollen derived from female flowers of melons and male flowers of cultivated melons.

The first-generation seeds obtained through hybridization showed uniform and stable expression of the target trait and were confirmed to be a new variety that showed different characteristics from the parent and parent. has been granted

Therefore, the present invention is a parent melon ( Cucumis melo var. makuwa Makino) self-fertilized 5 to 10 times with the target trait for the characteristics of epiphysis and positivity at each node, and the characteristics of leaflets and large fruits as target traits. The step of hybridizing self-fertilized melons ( Cucumis melo var. reticulatus ) to 10 times; It provides a method for producing vital melon ( Cucumis melo L. var. vitalmelon ) with accession number KCTC14699BP containing a.

The vital melon prepared in the present invention may be characterized as having a medium-sized fruit weight between the mother and the parent, and high content of cucurbitacin A and B, which are not identified in the second version.

'Cucurbitacin' is a kind of biochemical compound produced by some plants, especially Cucurbitaceae, which includes common pumpkin and gourd, and has a protective action against herbivores, and is known as an effective ingredient in Cucurbitaceae. have.

In addition, since the vital melon of the present invention has as many nodes as the number of nodes on the main and side branches, the low productivity of the parent can be overcome.

Accordingly, the vital melon of the present invention is characterized as a new variety that contains more cucurbitacin A and B, which are effective ingredients, than the parent, and has a larger fruit size and higher productivity than the parent.

In addition, since the extract of vital melon of the present invention can exhibit excellent symptoms of obesity and metabolic disease, and therapeutic effect, the present invention provides for prevention, treatment or treatment of obesity or metabolic disease comprising an extract of vital melon (accession number KCTC14699BP) It provides a pharmaceutical composition for improvement, a health functional food composition, a food composition, and a quasi-drug composition.

The vital melon of the present invention is i) fruiting as many as the number of nodes on the main and side branches; and ii) may be a new variety characterized in that the content of cucurbitacin A and cucurbitacin B is higher than that of the melon variety, and various extraction methods known in the art are used to prevent, treat or improve obesity or metabolic disease. Thus, the pulp can be extracted and used.

The extract in the present invention may include all without limitation, extracts and fractions thereof.

In the present invention, the extract of the vital melon may be characterized in that it is extracted with a lower alcohol extract having 1 to 4 carbon atoms or a mixed solvent thereof, and preferably may be extracted using water as an extraction solvent.

In the present invention, when extracting vital melon using water as an extraction solvent, extraction can be performed at room temperature, and 10 to 30 times (w/v), preferably 15 to 25 times (w/v), vital melon samples v) of distilled water is added, and an extract extracted by extracting or sonicating it for 2 to 5 hours in a water bath at 60 to 90° C., preferably at 75 to 85° C. may be used. The ultrasonic treatment may be performed 1 to 5 times.

In the present invention, the vital melon extract exhibits the effects of adipocyte differentiation, lipid accumulation inhibition, intracellular glucose uptake inhibition, triglyceride accumulation inhibitory effects, and lipid accumulation in the liver induced by a high-fat diet, adipocyte size increase, It was confirmed that all of the increase in blood lipids can be effectively suppressed.

Therefore, the vital melon extract of the present invention can exhibit the effect of preventing, treating or improving not only obesity, but also various metabolic diseases.

The metabolic disease may include, without limitation, all of the metabolic diseases widely known in the art as related to obesity, preferably from the group consisting of obesity, hypertension, arteriosclerosis, hepatic steatosis, fatty liver, dyslipidemia, diabetes and hyperglycemia. It may be one or more selected. More specifically, the dyslipidemia may be at least one selected from the group consisting of hyperlipidemia, hyperLDL cholesterolemia, hypertriglyceridemia, and low HDL cholesterolemia.

The 'Vital Melon Extract' of the present invention may be included in any amount (effective amount) depending on the use, formulation, purpose of mixing, etc. as long as the active ingredient can exhibit anti-obesity activity, where the 'effective amount' refers to a mammal to which it is applied. The amount of active ingredient contained in the composition of the present invention that can exhibit intended functional and pharmacological effects, such as anti-obesity effect, when the composition of the present invention is administered to a person during the administration period recommended by a medical professional, etc. say Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In addition to the active ingredient, the composition of the present invention may further include any compound or natural extract whose safety has already been verified for the prevention, treatment or improvement of obesity or metabolic disease.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or the extract or compound of the present invention. It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. Since the compound of the present invention has almost no toxicity and side effects, it is a drug that can be safely used even when taken for a long period of time for prophylactic purposes.

Since the vital melon extract of the present invention is a natural extract, it is not toxic to cells, so it can be used in various ways as health functional food, food, and quasi-drug composition other than pharmaceuticals.

Specifically, the functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions. Foods that are highly effective. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of the drug using food as a raw material, and has excellent portability.

The health functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, a flavoring agent or natural carbohydrate may be included as an additional ingredient in addition to the vital melon extract as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.), and sugar alcohols (eg, xylitol, sorbitol, erythritol, etc.). As flavoring agents, natural flavoring agents (eg, taumarin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) can be used.

The health functional food or food of the present invention may be provided in the form of tablets, capsules, pills or liquids, etc., and in the form to which the extract of the present invention can be added, for example, various drinks, meat, sausage, Bread, candy, snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gum, tea and vitamin complexes.

In addition, the present invention comprises the steps of administering an extract of vital melon (Accession No. KCTC14699BP) to the subject; It provides a method of preventing, improving, or treating obesity or metabolic disease, comprising a.

The subject refers to all animals, including humans, that have or are likely to develop obesity or metabolic disease. The animal may be a mammal, such as a cow, a horse, a sheep, a pig, a goat, a camel, an antelope, a dog, a cat, and the like, in need of treatment for symptoms similar thereto as well as humans, but is not limited thereto. Specifically, the method for preventing or treating the metabolic disease may include administering the composition in a pharmaceutically effective amount to an individual who has or is likely to develop a metabolic disease.

The present invention will be described in detail with reference to the following examples. These examples are for illustrative purposes only and do not limit the scope of the present invention.

Example 1. Obtaining Vital Melon Varieties

In order to obtain a new variety, Cucumis melo var, a cultivated melon grown commercially as a supplement. reticulatus (CR) and melon ( Cucumis melo var. makuwa Makino) (CM) as a model were fixed and used. The melon ( Cucumis melo var. makuwa Makino), which is the starting variety, has a small fruit size, a bisexual flower, and has the characteristics that female flowers are epiphytic at every node, and the cultivated melon Cucumis melo var. reticulatus was lobular, and it was confirmed that the fruit size was large. Among the above characteristics, the melons were self-fertilized 7 times using the characteristics of epiphysis and positivity at each node, which are the characteristics of melons, to fix the characteristics of melons. Also, among the characteristics of cultivated melons, the characteristics of leaflets and large fruits were set as the target characteristics, and the characteristics of melons were fixed by self-fertilization of cultivated melons 7 times.

Thirty-day-old seedlings of cultivated melon and melon varieties were transplanted into horticultural media, and fertilized weekly with a nutrient solution mixed with nutrients. Artificial pollination was performed using pollen derived from female flowers of melons and male flowers of cultivated melons, and hybridized first-generation seeds were obtained the following year. After that, the productivity test of the first-generation hybrid seeds was performed over two years, and it was confirmed that the target trait was uniformly and stably expressed. It was deposited with and was given an accession number KCTC 14699BP.

1 shows melon ( Cucumis melo var. makuwa Makino) and cultivated melon ( Cucumis melo var. reticulatus ), and vital melons obtained by hybridization thereof.

The fruits of Vital Melon were harvested at 10, 20, 30, 40 and 50 days of fruit development based on the mating date and used for subsequent experiments.

Example 2. Comparison of the characteristics of vital melon, mother and parent

To compare the fruit weight and cucurbitacin content of the new variety Vital Melon (Accession No. KCTC 14699BP) with the parents, 6 fruits were selected from each group, and the measured values are shown in Table 1 as mean±SD.

[Table 1]

As shown in Table 1 above, the vital melon exhibited a fruit weight larger than the parent and smaller than the parent. In particular, although cucurbitacin A and B were not confirmed at all in the copy, it was confirmed that vital melon contained both cucurbitacin A and B. In addition, it was confirmed that the content of cucurbitacin B was very high for 10 days after fertilization.

It was confirmed that Vital melons, unlike the subcultured melons, which ripen only on the side branches, exhibited the characteristics of melons as the parent melon by fruiting on the main and side branches.

Example 3. Preparation of vital melon extract

In order to confirm the ingredients and usefulness of a new variety, Vital Melon (Accession No. KCTC 14699BP), an extract thereof was prepared. The fruits of vital melon obtained as in Example 1 were immediately freeze-dried to powder, and filtered through 30 mesh. Then, 20 times (w/v) distilled water was added to 100 g of the powder, followed by extraction in a water bath at 80° C. for 4 hours. The obtained extract was filtered through Whatman No.2 filter paper. The mixtures were continuously mixed at 45° C. using a rotary evaporator and concentrated under reduced pressure. The extracts were sealed in glass bottles and stored in a freezer at -20°C.

Example 4. Confirmation of the inhibitory effect on the differentiation of pre-adipocytes of the vital melon extract

In order to confirm the anti-obesity activity of vital melon, an experiment was performed to confirm the effect on the differentiation of 3T3-L1 pre-adipocytes. 3T3-L1 cell line was purchased from KCLB (Korea Cell Line Bank, Seoul, Korea), 10% FBS (Welgene, Gyeongsan-si), 25 mM HEPE (Sigma, St Louis, MO, USA) and 25 mM NaHCO ₃ (Sigma, St Louis) , MO, USA) in a DMEM culture medium containing 37 ℃ 5% CO ₂ and cultured and maintained. For differentiation, cells were transferred to 6-well plates and grown for 48 h until complete confluence, 10 μg mL ⁻¹ insulin (Sigma, St Louis, MO, USA), 1 μM dexamethasone (Sigma, St Louis, MO, USA) and 10 μM rosiglitazone (Sigma, St Louis, MO, USA) were added to the differentiation medium (DM) to induce adipocyte differentiation. Distilled water (IN + DEX + ROS + DW) or 1.0, 5.0 and 10.0 μg/ml of vital melon water extract (VW) was added during the whole process of differentiation. The differentiation medium was changed every 2 days. For comparison, the mother melon ( Cucumis melo var. makuwa Makino ) and the parent melon ( Cucumis melo var. reticulatus ) were also prepared and treated with water extracts in the same manner as in Example 3 as in the vital melon. Adipogenesis was confirmed through lipid accumulation evaluation using Oil Red O staining. Oil Red O staining was performed after washing the cells with PBS on the 8th day, fixed with 4% formaldehyde and maintained at room temperature for 30 minutes, then treated with Oil Red O dye and maintained at 4° C. for 60 minutes. After staining, the excess amount of staining was removed and the cells were washed twice with PBS, and then photographing was performed using a microscope and a camera at 200 magnification. The results of confirming the inhibition of adipocyte differentiation and lipid accumulation of the vital melon water extract are shown in FIG. 2 .

As shown in FIG. 2, lipid accumulation was inhibited from the 50 μg/ml concentration of cultivated melon (Father, F), which is the parent, and the melon (Mother, M) treated group and the vital melon treated group (F1) were 1 to 10 From μg/ml, the accumulation of lipids was effectively inhibited. In addition, the vital melon-treated group exhibited a better lipid accumulation inhibitory effect than the parent at a low concentration of 1 to 5 μg/ml. These results show that the water extract of vital melon exhibits better fat suppression ability.

Example 5. Vital Melon Extract Blocking Glucose Absorption and Inhibiting Triglyceride Accumulation

An experiment was performed to confirm whether the treatment with the Vitalmelon water extract blocks glucose uptake from the medium into the cells and inhibits the accumulation of triglyceride (TG) in 3T3-L1 cells. 3T3-L1 cells were placed in differentiation medium (DM), DMSO-added differentiation medium (DMSO), or a differentiation medium containing water extracts of different concentrations of vital melon, and differentiation into adipocytes was induced until day 8. Then, a medium was obtained from each well, and the glucose concentration on the 8th day was measured. In order to measure the glucose content contained in the medium, the medium from each well was collected on day 8 and centrifuged at 1000 g for 5 minutes. The glucose present in the supernatant was then quantified using a commercial glucose kit. To measure the triglyceride content, cells were aliquoted into 12 well plates and incubated for 48 hours to reach complete confluency. Differentiation was induced and triglycerides were extracted from the cells using 5% Triton X-100 on the 8th day, and these were quantified using a commercial TG kit.

The results of confirming the measured content of glucose in the medium and the content of triglyceride in cells are shown in FIG. 3 .

As shown in FIG. 3 , the vital melon water extract inhibited the absorption of glucose in the medium into the cells, and inhibited the accumulation of triglycerides.

Example 6. Confirmation of anti-obesity effect of vital melon extract in high-fat diet mice

Since it was confirmed through the above examples that the vital melon water extract can exhibit an anti-obesity effect at the cellular level, an experiment was performed to determine whether weight gain could be suppressed using a high-fat diet-induced mouse model.

Six-week-old C57BL/6 male mice were purchased from Samtako Bio-Korea Inc. (Osan, Korea) with water and a standard normal diet (10 kcal% fat, Research Diets, New Brunswick, NJ, USA) and a high-fat diet (60 kcal% fat). , Research Diets, New Brunswick, NJ, USA). During the experiment, mice were maintained in a sterile environment, 23±2° C., 50±10% relative humidity, and a strict lighting cycle (lighting at 08:00 and lights off at 20:00). All mouse experiments were conducted at the Pusan National University-Laboratory Animal Resource Center accredited by the Korean FDA (Accredited Unit no. 000231) and AAALAC International (Accredited Unit no. 001525), and the Animal Ethics Committee of Pusan National University (PNU-2020-2709) was approved for animal studies. All animals were acclimatized to a normal diet (D12450K; Research Diets, New Brunswick, NJ, USA) for 1 week.

A total of 50 C57BL/6 mice were randomly divided into 5 groups (10 mice/group), and the experiment was conducted for 8 weeks by setting up the experimental groups as follows: 1) a control group fed a normal diet, 2) a high-fat control group HFD group fed the diet, 3) OT group fed 10 mg/kg Orlistat on a high-fat diet (positive control group), 4) VW1 group fed 1 mg/kg Vitalmelon water extract on a high-fat diet, 5) 25 high-fat diet VW25 group provided mg/kg vital melon water extract.

The composition of the diet used in the present invention is shown in Table 2.

[Table 2]

Mice body weight and food intake were measured every 3 days for 10 weeks according to the KFDA guidelines using an electronic scale (cat. no. AD-2.5; Mettler Toledo, Greifensee, Switzerland) at 9 am. At the end of the experiment, mice were sacrificed by CO ₂ asphyxiation, and blood samples were collected for biochemical analysis. Liver, kidney and adipose tissue from sacrificed C57BL/6 mice were collected, each tissue was weighed, photographed and immediately stored at -70 °C until analysis.

Table 3 shows the effect of weight change according to the treatment of the vital melon water extract.

[Table 3]

- All data presented mean ± SEM of 10 mice per group. Food efficiency ratio (%) = [total weight gain / total food intake] Х 100

As shown in Table 3, there was no difference in the initial body weight (BW) for each group, but in the high-fat diet group, the weight gain (52.1%) was distinctly higher than in the normal diet group, and the weight of adipose tissue increased significantly. . On the other hand, it was confirmed that in the experimental group fed together with the vital melon water extract of the present invention, the tendency of weight gain and tissue weight increase induced by a high-fat diet could be alleviated.

Example 7. Serum Biochemical Analysis

As in Example 6, after feeding the experimental diet for 8 weeks, and after fasting for 12 hours, blood was collected from abdominal veins of all C57BL/6 mice, and blood samples were collected in serum separation tubes (BD Biosciences, Franklin Lakes, NJ). , USA) for 30 min at room temperature. Serum was obtained by centrifugation at 1,500 x g for 15 min. Blood sugar, total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol levels were measured with an automated chemical analyzer (7180 Automatic Analyzer, Hitachi High-Technology Cor., Tokyo, Japan) was analyzed with In addition, serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), BUN (blood urea nitrogen), and creatinine (Crea) were analyzed using an automatic biochemical analyzer (7180 Automatic Analyzer). ; Hitachi High-Technology Cor., Tokyo, Japan) was used for analysis. All assays were repeated twice using fresh serum, and plasma insulin, creatine, leptin, and adiponectin measurements were also performed using an ELISA kit. The results of serum biochemical analysis are shown in FIGS. 4 and 5 .

As shown in FIG. 4, plasma liver enzymes AST and ALT of the high-fat diet group (HFD) were significantly increased compared to the normal diet group (Nor), and the group fed with the vital melon water extract (VW) compared with HFD. Thus, it was confirmed that this increase was significantly decreased. In addition, it was confirmed that the increase in lipid levels (total cholesterol, HDL, cholesterol, LDL cholesterol, triglycerides), total protein and glucose induced by a high-fat diet was improved in the Vitalmelon water extract feeding group.

As shown in FIG. 5 , the concentration of insulin and leptin increased in the high-fat diet group, which was determined to be related to the increase in abdominal fat in the high-fat diet group. The increase in insulin concentration decreased in the OT group, but did not decrease to a significant extent. On the other hand, it was confirmed that insulin and leptin were significantly decreased in the VW25 group of soldiers fed Vital Melon for 70 days compared to the HFD group (P<0.05). On the other hand, serum adiponectin was all increased in the Vital Melon-treated group compared to the HDF group.

Example 8. Histopathological analysis

Liver and adipose tissue were isolated from all sub-mouse groups fed by the method of Example 6 and fixed overnight in 10% neutral buffered formaldehyde (pH 6.8). Dehydrated liver tissue was fixed in paraffin wax. Liver and fat sections (4 μm) were cut from paraffin-embedded tissues using a Leica microtome (cat. no. DM500; Leica Microsystems, Bannockburn, IL, USA). The sections were then deparaffinized with xylene, rehydrated with grade ethanol (100-70% reduced concentration) and finally washed with distilled water. Slides with liver sections were stained with hematoxylin and eosin, washed with dH2O, and pathological changes were evaluated and fat size was measured using Leica Application Suite (Leica Microsystems). The results of confirming the size of fat and lipid droplets in the liver are shown in FIG. 6 .

As shown in FIG. 6 , in the high-fat diet group, it was confirmed that lipid droplets were significantly increased in the liver, lipids were accumulated in the hepatocytes, and differentiated into giant vesicular adipocytes, thereby accumulating fat in the liver. On the other hand, it was confirmed that the lipid accumulation in the liver tissue was remarkably inhibited in the group administered with the vital melon water extract of the present invention. Through the above results, it was confirmed that the vital melon extract of the present invention can significantly improve macrovesicular steatosis.

Also, as shown in FIG. 7 , the size of adipocytes in the HFD group was confirmed to be about 100 μm, which is about a 2.7-fold increase compared to about 37 μm in the normal control group. In the positive control group, the OT group, the adipocyte size was about 50 μm, which is about twice the size of the HFD group. Compared with the HFD group, it was confirmed that a significant concentration-dependent effect on the size of adipocytes was reduced in all VW-treated groups. The adipocyte size in the VW treatment group was about 65 μm and 60 μm, respectively, depending on the concentration (P<0.05).

Example 9. Results of protein expression analysis for in vitro and in vivo samples

The PPAR-γ pathway is known to play an essential role in the differentiation of adipocytes by regulating the transcription of various genes responsible for lipid transport and accumulation. Therefore, an experiment was performed to determine whether the treatment of the vital melon water extract inhibits the protein expression of these PPAR-γ and its target genes.

For in vitro sample preparation, culture cells in culture medium, differentiation medium (DM), differentiation medium (DM) + DMSO or differentiation medium containing various concentrations of vital melon water extract VW (1, 5 and 10 µg/ml). Then, total protein was extracted with RIPA buffer containing PMSF for 30 min on ice. The lysate was centrifuged at 12,000 g at 4°C for 15 minutes, and the total protein concentration of the supernatant was quantified using BioRad protein assay reagent (BioRad, Hercules, CA, USA).

For in vivo sample preparation, the liver tissue (50 mg) collected from each group was homogenized using PRO-PREPTM Solution (cat. no. 170841; iNtRON Biotechnology Inc., Sungnam, Korea) and centrifuged at 13,000 rpm for 5 minutes. Separated to obtain a total protein extract. The protein was then mixed with 4X loading buffer (BioRad, Hercules, CA, USA) and 2-mercaptoethanol (Bioshop, Burlington, Ontario, Canada) and heated in boiled water for 7 minutes. Next, 12% SDS-PAGE with a protein marker (BioRad, Hercules, CA, USA) was continuously applied to the prepared protein. The protein was then transferred from the gel to the PVDF membrane using a semi-dry transfer system (BioRad, Hercules, CA, USA) followed by membrane transfer for 1 h at room temperature using 1% BSA in PBST buffer (0.1% Tween-20/PBS). blocked. Thereafter, the parallel primary antibody was immediately probed overnight at 4°C. The membranes were then washed 3 times for 5 min each time with PBST solution and then incubated with parallel HRP-conjugated secondary antibody for an additional 1 h at room temperature. Then, the membrane was washed 3 times with PBST solution for 5 minutes each. Finally, immunoreactive bands were visualized using an ECL detection kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Finally, the bands were analyzed using ImageJ software (Wayne Rasband, NIH, USA). The results of confirming the protein expression in each dietary group are shown in FIGS. 8 and 9 .

As shown in Figure 8, it was confirmed that the expression of PPAR-γ and its target genes LPL, CD36, HMGCR and L-FABP in 3T3-L1 cells was significantly increased, but this increase was treated with the vital melon water extract for 8 days. It decreased in a concentration-dependent manner in one experimental group. These results show that vital melon water extract can suppress the protein expression of PPAR-γ and also suppress the increase in their gene expression, which is upregulated during adipocyte differentiation.

As shown in FIG. 9 , as a result of confirming the PPAR-γ pathway signal in the liver by western blot, PPAR-γ and its target genes LPL, CD35, HMGCR and L-FABP were all reduced compared to HDF in the VW25 treatment group. Confirmed.

All experiments described in the present invention were expressed as mean ± SD over three replicates.

Hereinafter, formulation examples of the pharmaceutical composition for preventing or treating obesity including the composition for anti-obesity of the present invention will be described, but it is not intended to limit the present invention, but merely to describe it in detail.

Formulation Example 1. Preparation of powder

100 mg of vital melon extract of the present invention

1 g lactose

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2. Preparation of tablets

100 mg of vital melon extract of the present invention

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional method for manufacturing tablets.

Formulation Example 3. Preparation of capsules

100 mg of vital melon extract of the present invention

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, the capsules were prepared by filling in gelatin capsules according to a conventional manufacturing method of capsules.

Formulation Example 4. Preparation of pills

100 mg of vital melon extract of the present invention

1.5 g lactose

1 g of glycerin

0.5 g of xylitol

After mixing the above components, it was prepared so as to be 4 g per ring according to a conventional method.

Formulation Example 5. Preparation of granules

100 mg of vital melon extract of the present invention

Soybean extract 50 mg

glucose 200 mg

Starch 600 mg

After mixing the above ingredients, 100 mg of 30% ethanol was added and dried at 60° C. to form granules, and then filled in a bag.

Formulation Example 6. Tablet-type health functional food

Octacosanol powder 15% by weight, lactose hydrolyzate powder 15% by weight, soy protein isolate 15% by weight, chitooligosaccharide 15% by weight yeast extract powder 10% by weight, vitamin and mineral mixture 10% by weight, magnesium stearate 4.6% by weight, titanium dioxide 0.2% by weight, and 0.2% by weight of glycerin fatty acid ester, and 20% by weight of the vital melon extract of the present invention are blended to prepare a tablet-type health functional food by a conventional method.

Formulation Example 7. Health Drink

Honey 5% by weight, fructose 3% by weight, sodium riboflavin hydrochloride 0.0001% by weight, pyridoxine hydrochloride 0.0001% by weight, water 86.9998% by weight and 5% by weight of the vital melon extract of the present invention are blended to prepare a health drink by a conventional method.

Korea Institute of Bioscience and Biotechnology KCTC14699BP 20210913

Claims (13)

Vital melon with accession number KCTC14699BP having the characteristic of fruiting as much as the number of nodes on the main and side branches ( Cucumis melo L. var. vitalmelon ).
According to claim 1, wherein the vital melon is melon ( Cucumis melo var. makuwa Makino ) as a model, cultivated melon ( Cucumis melo var. reticulatus ) It is a hybrid of a parent, vital melon.
The vital melon according to claim 2, wherein the mother melon is self-fertilized 7 times with the characteristics of epiphysis and positivity characteristics of each node.
The vital melon according to claim 2, wherein the subcultured melon is self-fertilized 7 times with the characteristics of leaflets and large fruits as the target characteristics.
The parent melon ( Cucumis melo var. makuwa Makino) self-fertilized 7 times for the characteristics of epiphysis and bisexuality at each node as the target trait, and the double-cultivated melon self-fertilized 7 times with the characteristics of leaflets and large fruits as the target trait ( Cucumis melo var. reticulatus ) hybridizing; The accession number KCTC14699BP containing the vital melon ( Cucumis melo L. var. vitalmelon ) Manufacturing method.
A pharmaceutical composition for preventing or treating obesity comprising an extract of vital melon, which is accession number KCTC14699BP.
[Claim 7] The pharmaceutical composition for preventing or treating obesity according to claim 6, wherein the vital melon has a characteristic of fruiting as many as the number of nodes on the main and side branches.
The pharmaceutical composition for preventing or treating obesity according to claim 6, wherein the extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
delete delete A health functional food composition for preventing or improving obesity comprising an extract of vital melon with accession number KCTC14699BP.
A food composition for preventing or improving obesity comprising an extract of vital melon, which is accession number KCTC14699BP. delete

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