WO2023106777A1 - Vital melon (kctc14699bp) and anti-obesity composition comprising extract thereof - Google Patents

Abstract

The present invention relates to a vital melon (KCTC14699BP) and a composition comprising an extract thereof for preventing, treating or alleviating obesity or metabolic-related diseases. A vital melon of the present invention undergoes fruit setting such that the number of joints is the same as the number of main and side branches, and more fruit flesh can be obtained therefrom than from a conventional oriental melon, and thus excellent productivity is provided, and an extract thereof can effectively inhibit all of lipid accumulation, weight gain, fat accumulation in the liver, and the like, and thus can be effectively used as a composition for preventing, alleviating or treating obesity or various metabolic diseases.

Description

Anti-obesity composition containing vital melon (KCTC14699BP) and its extract

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

This application claims priority based on Korean Patent Application No. 10-2021-0176404 filed on December 10, 2021, and all contents disclosed in the specification and drawings of the application are incorporated into this application.

In modern society, the number of obese people is rapidly increasing due to various causes, such as convenient living environment due to rapid automation, excessive nutritional intake due to the increase in processed food and eating out, and decrease in physical activity. Metabolic diseases such as , insulin resistance and dyslipidemia are on the rise. Among them, in recent years, non-alcoholic fatty liver (NAFLD) has been increasing due to an increase in obesity, diabetes, and hyperlipidemia, and some of them are known to progress to fatty liver or cirrhosis, and fatty liver is a simple liver disease. Since it is regarded as an early symptom of a metabolic disease rather than a disease, the importance of early diagnosis and treatment of fatty liver is increasing.

On the other hand, chronic metabolic diseases are known to be caused by obesity. When excessive energy is supplied beyond the limits of adipose tissue, an energy storage tissue, it is accumulated in non-fat tissues such as the liver, muscle, pancreas, and heart in the form of triglycerides (TG). In addition, fatty acid synthesis increases, fatty acid oxidation rate decreases, insulin resistance increases in muscle and liver, insulin production decreases in pancreas, and beta cell death occurs. , heart failure, myocardial infarction, and non-alcoholic fatty liver (NAFLD) in the liver. Therefore, inhibition of tissue fat through fat absorption, inhibition of lipogenesis, and enhancement of fatty acid oxidation can be an effective strategy for preventing and treating metabolic diseases such as obesity, insulin resistance, diabetes, and fatty liver.

There is 'Xenical', which has orlistat as its main ingredient, which has been approved by the US FDA for the treatment of obesity sold domestically and internationally. It is known to have side effects.

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

The inventors of the present invention found that when melons and cultivated melons are hybridized during research to obtain new useful crops, melons of new varieties can be produced that can overcome the low productivity of melons while increasing the useful activity of melons. It was confirmed, and furthermore, 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 vital melon having an anti-obesity effect and the vital melon extract.

In order to achieve the above object, the present invention is a) the number of nodes in the main and side branches; and b) a vital melon with accession number KCTC14699BP having the same characteristics as having high cucurbitacin A and cucurbitacin B content compared to melon varieties ( Cucumis melo L. var. vitalmelon).

In addition, the present invention is a parental melon ( Cucumis ) self-fertilized 5 to 10 times with the characteristics of epiphytic female flowers for each node and the characteristics of benignization as the target trait. melo var. makuwa Makino) and cultivar melons ( Cucumis melo var. reticulatus) self-fertilized 5 to 10 times with target traits for leaflet characteristics and large fruits; Accession number containing a vital melon ( Cucumis melo L. var. vitalmelon ).

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

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

In addition, the present invention provides a method for preventing, improving, or treating obesity or metabolic disease, comprising administering an extract of vital melon (accession number KCTC14699BP) to a subject in need thereof.

In addition, the present invention provides an extract of vital melon (accession number KCTC14699BP) for preventing, improving, or treating obesity or metabolic diseases.

In addition, the present invention provides the use of an extract of vital melon (Accession No. KCTC14699BP) for producing a drug for preventing, improving, or treating obesity or metabolic diseases.

The vital melon of the present invention bears fruit as much as the number of nodes of the main branch and side branches, and can obtain larger fruits than conventional melons, so not only is it excellent in productivity, but its extract prevents lipid accumulation, weight gain, and fat accumulation in the liver. Since it can effectively inhibit, it can be used 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 with the parent cultivated melon.

Figure 2 is a view showing the results confirming the effect of inhibiting fat cell differentiation and lipid accumulation of vital melon water extract (Hybrid (F1)), melon (mother, M), and cultivated melon (Father, F). Specifically, A in FIG. 2 is a photograph confirming the effect using a camera and a microscope after staining with Oil Red O, and B in FIG. 2 is a graph in which the effect is quantitatively analyzed using an ELISA reader.

Figure 3 is a diagram showing the results of confirming the effect of inhibiting glucose absorption (A) and triglyceride accumulation (B) in cells according to the 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 changes in plasma liver enzymes, lipid levels, total protein and glucose according to the positive control group orlistat or vital melon water extract (VW) diet in high fat diet mice (HFD). .

5 is a view showing the results of confirming changes in insulin, leptin, and adiponectin according to the positive control group, orlistat (OT10) or vital melon water extract (VW) diet in high-fat diet mice (HFD).

6 is a view showing the results of confirming the effect of inhibiting liver fat accumulation according to the positive control, orlistat (OT10) or vital melon water extract (VW) diet in high-fat diet mice (HFD).

7 is a view showing the results of confirming the fat cell size inhibitory effect according to the diet of orlistat (OT10) or vital melon water extract (VW) as a positive control in high fat diet mice (HFD).

8 is a view showing the results of confirming the expression changes of PPAR-γ and its target genes LPL, CD35, HMGCR and L-FABP according to treatment with vital melon water extract (VW) in 3T3-L1 cells.

Figure 9 is a diagram showing the results of confirming the expression changes of PPAR-γ and its target genes LPL, CD36, HMGCR and L-FABP according to treatment with vital melon water extract (VW) in the liver of mice on a high fat diet.

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

The vital melon is a) as much as the number of nodes in the main and side branches; and b) a high content of cucurbitacin A and cucurbitacin B compared to melon varieties.

The vital melon is melon ( Cucumis melo var. makuwa Makino) as a model, cultivated melon ( Cucumis melo) var. reticulatus ) was obtained by hybridizing with the male.

In order to obtain the vital melon of the present invention, the parental melon can use melons self-fertilized 5 to 10 times, preferably 6 to 8 times, with the characteristics of epiphytic female flowers per node and the characteristics of benignization as the target trait. Cultivated melons can be self-pollinated cultivated melons 5 to 10 times, preferably 6 to 8 times, with the characteristics of lobules and large fruits as the target traits. The traits of the mother and father can be fixed through the repeated self-fertilization process as described above.

Methods known in the art can be used without limitation for the hybridization method of the mother and father to obtain the vital melon of the present invention, but in a preferred embodiment of the present invention, 25 to 40 One-year-old seedlings were transplanted to a horticultural medium, and hybridized vital melons were produced by artificial pollination using pollen derived from female flowers of melon and male flowers of cultivated melon.

The first-generation seeds obtained through hybridization were uniformly and stably expressed in the target trait, and it was confirmed that they were new varieties that exhibited characteristics different from those of the mother and father. It was entrusted to the Korea Research Institute of Bioscience and Biotechnology on 2021.09.13 and was given the KCTC14699BP number.

Therefore, the present invention is a parental melon ( Cucumis ) self-fertilized 5 to 10 times with the characteristics of epiphytic female flowers and benignization characteristics for each node as target traits. melo var. makuwa Makino) and a domestic melon ( Cucumis melo var. reticulatus ) self-fertilized 5 to 10 times with the characteristics of leaflets and large fruits as target traits; Accession number containing a vital melon ( Cucumis melo L. var. vitalmelon ).

The vital melon prepared in the present invention can be characterized by having a medium-sized fruit weight between the mother and the female, and high contents of cucurbitacins A and B, which are not found in the female.

'Cucurbitacin' is a kind of biochemical compound produced in some plants, especially in the Cucurbitaceae family, which includes common pumpkins and gourds, and has a protective action against herbivores, and is known as an active ingredient in cucurbits. there is.

In addition, since the vital melon of the present invention bears the number of nodes on the main and side branches, it can overcome the low productivity of the mother plant.

Therefore, the vital melon of the present invention is characterized as a new variety having the advantage of having a larger fruit size and higher productivity than the mother plant while containing more cucurbitacins A and B, which are effective components, than the parent plant.

In addition, since the extract of vital melon of the present invention can exhibit excellent symptoms of obesity and metabolic disease, the present invention can prevent, treat or treat obesity or metabolic disease, including extracts of vital melon (accession number KCTC14699BP). A pharmaceutical composition for improvement, a health functional food composition, a food composition, and a quasi-drug composition are provided.

The vital melon of the present invention is a) fruiting by the number of nodes on the main and side branches; and b) it may be a new variety characterized by a higher content of cucurbitacin A and cucurbitacin B compared to melon varieties, using various extraction methods known in the art for use in preventing, treating or improving obesity or metabolic diseases. So, the fruit flesh can be extracted and used.

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

In the present invention, the extract of 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, preferably extracted with 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) of the vital melon sample An extract obtained by adding distilled water of v) and extracting or ultrasonicating it in a water bath at 60 to 90° C., preferably 75 to 85° C. for 2 to 5 hours may be used. The ultrasonic treatment may be performed 1 to 5 times.

In the present invention, the vital melon extract not only exhibits effects of adipocyte differentiation, inhibition of lipid accumulation, inhibition of intracellular glucose absorption, and inhibition of triglyceride accumulation, but also lipid accumulation in the liver induced by a high-fat diet, increase in fat cell size, It was confirmed that all lipid increases in the blood can be effectively suppressed.

Therefore, the vital melon extract of the present invention may exhibit effects of preventing, treating, or improving various metabolic diseases as well as obesity.

The metabolic disease is associated with obesity and may include all metabolic diseases widely known in the art without limitation, preferably in the group consisting of obesity, hypertension, arteriosclerosis, hepatic steatosis, fatty liver, dyslipidemia, diabetes and hyperglycemia. It may be one or more selected species. The dyslipidemia may be more specifically one or more selected from the group consisting of hyperlipidemia, hyper-LDL cholesterolemia, hypertriglyceridemia, and hypo-HDL cholesterolemia.

The 'vital melon extract' of the present invention may be included in any amount (effective amount) according to the purpose of use, formulation, blending, etc., as long as the active ingredient can exhibit anti-obesity activity. More specifically, when the composition of the present invention is administered to a person during the administration period according to the advice of medical experts, etc., the amount of the active ingredient included in the composition of the present invention that can exhibit the intended functional and pharmacological effects such as anti-obesity effect says Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

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 to prevent, treat, or improve obesity or metabolic disease, or increase or enhance activity.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. there is. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . 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 suspensions. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may 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. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages 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 dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection. Since the compound of the present invention has little toxicity and side effects, it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

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

Specifically, the health functional (sex) food (functional food) is the same term as food for special health use (FoSHU), medicine that is processed to efficiently display bioregulatory functions in addition to nutritional supply, medical care Means highly effective food. Here, "function (sex)" means to obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the preparation. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The food composition of the present invention can be prepared in various types of dosage forms, and unlike general medicines, it has the advantage of not having side effects that may occur when taking medicines for a long time by using food as a raw material, and has excellent portability.

The health functional food of the present invention includes components commonly added during food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared 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, dextrins, cyclodextrins, etc.), and and sugar alcohols (eg, xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumarin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

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

In addition, the present invention provides a method for preventing, improving, or treating obesity or metabolic diseases, comprising administering an extract of vital melon (accession number KCTC14699BP) to a subject in need thereof.

In addition, the present invention provides an extract of vital melon (accession number KCTC14699BP) for preventing, improving, or treating obesity or metabolic diseases.

In addition, the present invention provides the use of extracts of vital melon (Accession No. KCTC14699BP) for producing drugs for preventing, improving, or treating obesity or metabolic diseases.

The subject refers to all animals, including humans, who have or are likely to develop obesity or metabolic diseases. The animal may be not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but are not limited thereto. The method for preventing or treating the metabolic disease may include, in detail, administering the composition in a pharmaceutically effective amount to a subject who has or is likely to have a metabolic disease.

The present invention will be described in detail through examples below. These examples are for illustrative purposes only and do not limit the scope of the present invention.

Example One. vital melon breeding

In order to obtain a new variety, Cucumis melo var. reticulatus (CR) and melon ( Cucumis melo var. makuwa Makino) (CM) were fixed and used as a model. The melon ( Cucumis melo var. makuwa Makino), which is the starting variety, has the characteristics of small fruit size, bisexuality, and epiphytic female flowers on each node, and Cucumis , a cultivated melon melo var. reticulatus is a leaflet, and it was confirmed that the fruit size is large. Among the above characteristics, melons were self-fertilized 7 times with the characteristics of melon epiphytic female flowers per node and benignization characteristics as target traits, and through this, the melon traits were fixed. In addition, among the characteristics of cultivated melons, leaflet characteristics and large-fruit characteristics were set as target traits, and the cultivated melons were self-fertilized 7 times to fix the melon traits.

30-day-old seedlings of cultivated melon and melon varieties were transplanted into a horticultural medium, and fertilized weekly with a nutrient solution mixed with nutrients. Artificial pollination was performed using pollen derived from female flowers of melon and male flowers of cultivated melon, and first-generation hybrid seeds were obtained the following year. Over the next two years, the productivity test of the first-generation hybrid seeds was performed, and it was confirmed that the target trait was expressed uniformly and stably, and it was named as a new variety 'Vital Melon', and as of 2021.09. and received the accession number KCTC 14699BP.

Melon with fixed traits ( Cucumis melo var. makuwa Makino) and cultivated melon ( Cucumis melo) var. reticulatus ), the vital melons obtained by their hybridization are shown in FIG.

Fruits of vital melon were harvested at 10, 20, 30, 40, and 50 days of fruit development based on the mating date and used in subsequent experiments.

Example 2. with vital melon example , Comparison of traits of copies

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

As shown in Table 1, the vital melon showed a fruit weight larger than that of the parent and smaller than that of the female. In particular, it was confirmed that the vital melon contained both cucurbitacins A and B, although no cucurbitacins A and B were found in the part. In addition, it was confirmed that the content of cucurbitacin B was very high for 10 days after fertilization.

Unlike the main grown melons, which bear fruit only on the side branches, the vital melon showed the characteristics of the parent melon by settling on the main and side branches, and it was confirmed that it was possible to harvest continuously after fruit set after settling on the main and side branches as much as the number of nodes.

Example 3. vital melon Preparation of extract

In order to confirm the components and usefulness of vital melon (accession number KCTC 14699BP), a new variety, an extract thereof was prepared. The fruit of the vital melon obtained in Example 1 was immediately lyophilized to powder and filtered through 30 mesh. Thereafter, 20 times (w/v) distilled water was added to 100 g of the powder and extracted 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 still and concentrated under reduced pressure. Extracts were sealed in glass bottles and stored in a freezer at -20°C.

Example 4. vital melon of the extract preadipocytes Confirmation of differentiation inhibitory effect

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. The 3T3-L1 cell line was purchased from KCLB (Korea Cell Line Bank, Seoul, Korea), 10% FBS (Welgene, Gyeongsan), 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° C., 5% CO ₂ and maintained. For differentiation, cells were transferred to 6-well plates and grown for 48 hours until full confluency was reached, 10 μg/mL insulin (Sigma, St Louis, MO, USA), 1 μM dexamethasone (Sigma, St Louis, MO, USA). St. Louis, MO, USA) and 10 μM rosiglitazone (Sigma, St Louis, MO, USA) were added to induce differentiation of adipocytes in differentiation medium (DM). Distilled water (IN + DEX + ROS + DW) or vital melon water extract (VW) at 1.0, 5.0 and 10.0 μg/ml was added throughout the differentiation process. Differentiation medium was changed every 2 days. For comparison, the model melon ( Cucumis melo var. makuwa Makino) and its counterpart, cultivated melon ( Cucumis) melo var. reticulatus ), as in Example 3, a water extract was prepared and treated in the same manner as with vital melon. Adipogenesis was confirmed by evaluation of lipid accumulation using Oil Red O staining. Oil Red O staining was performed on day 8 after washing the cells with PBS, fixing with 4% formaldehyde, maintaining at room temperature for 30 minutes, and then treating with Oil Red O dye and maintaining 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 images were taken using a 200 magnification microscope and camera. 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 Figure 2, lipid accumulation was suppressed from the concentration of 50 μg/ml of cultivated melon (Father, F), the parent melon (Mother, M) treatment group and vital melon treatment group (F1) were 1 to 10 μg/ml. The accumulation of lipids was effectively inhibited from ml. In addition, the vital melon treatment group showed a better lipid accumulation inhibitory effect than the mother plant at a low concentration of 1 to 5 μg/ml. These results show that the water extract of vital melon exhibits a more excellent ability to suppress fat.

Example 5. vital melon Blocking glucose uptake of the extract and triglyceride Confirm accumulation inhibitory effect

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

3 shows the results of confirming the measured glucose content in the medium and intracellular triglyceride content.

As shown in Figure 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. In mice on a high-fat diet vital melon of the extract anti-obesity Check the effect

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

Six-week-old C57BL/6 male mice were purchased from Samtako Bio-Korea Inc. (Osan, Korea) and supplied 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, the mice were maintained in a sterile environment, 23±2%, 50±10% relative humidity, and strict lighting cycle conditions (08:00 on and 20:00 off). All mouse experiments were handled by 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 Pusan National University Animal Ethics Committee (PNU-2020-2709) approved for animal studies. All animals were acclimated to a normal diet (D12450K; Research Diets, New Brunswick, NJ, USA) for one 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 the experimental groups as follows: 1) a control group fed a normal diet, 2) a high-fat group HFD group fed diet, 3) OT group fed 10mg/kg Orlistat to high-fat diet (positive control), 4) VW1 group fed 1mg/kg vital melon water extract to high-fat diet, 5) 25mg/kg high-fat diet fed VW25 group provided kg of vital melon water extract.

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

The body weight and food intake of the mice were measured every 3 days for 10 weeks at 9 am using an electronic balance (cat. no. AD-2.5; Mettler Toledo, Greifensee, Switzerland) according to KFDA guidelines. 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 were collected from sacrificed C57BL/6 mice, each tissue was weighed, photographed and immediately stored at -70°C until analysis.

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

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

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

Example 7. Serum biochemical analysis

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

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

As shown in Figure 5, the concentrations 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 fed group. The increase in insulin concentration was reduced in the OT group, but not to a significant extent. On the other hand, in the VW25 group, which was fed vital melon for 70 days, it was confirmed that insulin and leptin decreased significantly compared to the HFD group (P<0.05). On the other hand, serum adiponectin was increased in the Vitalmelon treated group compared to the HDF group.

Example 8. Histopathological analysis

Liver and adipose tissue were isolated from all subgroups of mice 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 graded ethanol (100-70% decreasing 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 the Leica Application Suite (Leica Microsystems). The results of confirming fat size 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, and lipids were accumulated in hepatocytes, differentiated into macrovesicular adipocytes, and fat was accumulated in the liver. On the other hand, in the group administered with the water extract of vital melon of the present invention, it was confirmed that lipid accumulation in liver tissue was significantly inhibited. Through the above results, it was confirmed that the vital melon extract of the present invention can significantly improve macrovesicular steatosis.

In addition, as shown in FIG. 7, the size of adipocytes in the HFD group was confirmed to be about 100 μm, which is about 2.7 times higher than about 37 μm in the normal control group. In the OT group, which is a positive control group, the fat cell size was about 50 μm, which is about twice as small as that of the HFD group. Compared to the HFD group, it was confirmed that all VW-treated groups showed a concentration-dependently significant reduction in fat cell size. The size of adipocytes in the VW-treated group was approximately 65 μm and 60 μm, respectively, depending on the concentration (P<0.05).

Example 9. In vitro, in vitro vivo Protein expression analysis results for the sample

It is known that the PPAR-γ pathway plays 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 confirm whether the treatment of the vital melon water extract inhibits the protein expression of PPAR-γ and its target genes.

For in vitro sample preparation, cells were cultured 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). After that, total protein was extracted with RIPA buffer containing PMSF for 30 minutes on ice. After the lysate was centrifuged at 12,000 g at 4° C. for 15 minutes, the total protein concentration of the supernatant was quantified using BioRad protein assay reagent (BioRad, Hercules, CA, USA).

For in vivo sample preparation, liver tissues (50 mg) collected from each group were homogenized using PRO-PREPTM Solution (cat. no. 170841; iNtRON Biotechnology Inc., Sungnam, Korea) and centrifuged at 13,000 rpm for 5 minutes. Total protein extract was collected by separation. The protein was then mixed with 4X loading buffer (BioRad, Hercules, CA, USA) and 2-mercaptoethanol (Bioshop, Burlington, Ontario, Canada) and then heated in boiled water for 7 minutes. Next, 12% SDS-PAGE accompanied by a protein marker (BioRad, Hercules, CA, USA) was continuously applied to the prepared protein. Then, transfer the proteins from the gel to the PVDF membrane using a semi-dry transfer system (BioRad, Hercules, CA, USA) followed by 1% BSA in PBST buffer (0.1% Tween-20/PBS) for 1 hour at room temperature. Blocked. They were then immediately probed overnight at 4°C with parallel primary antibodies. The membrane was then washed three times for 5 minutes each time with PBST solution and then incubated with parallel HRP-conjugated secondary antibodies for an additional hour at room temperature. The membrane was then washed three times for 5 minutes each with PBST solution. Finally, immunoreactive bands were visualized using an ECL detection kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Finally, bands were analyzed using ImageJ software (Wayne Rasband, NIH, USA). The results of confirming protein expression in each diet 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 increased markedly, but this increase was not observed after treatment with the vital melon water extract for 8 days. It decreased in a concentration dependent manner in one experimental group. These results show that the water extract of vital melon can suppress the protein expression of PPAR-γ and also suppress the increase in their gene expression, which is up-regulated during adipocyte differentiation.

As shown in Figure 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 in the VW25-treated group compared to HDF. Confirmed.

All experiments described in the present invention are expressed as the mean ± SD through three replicates.

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

formulation example One. sanjae manufacturing

100 mg of the vital melon extract of the present invention

lactose 1 g

A powder was prepared by mixing the above ingredients and filling in airtight bags.

formulation example 2. Preparation of tablets

100 mg of the vital melon extract of the present invention

Corn Starch 100 mg

100mg milk sugar

Magnesium stearate 2 mg

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

formulation example 3. Manufacture of capsules

100 mg of the vital melon extract of the present invention

Corn Starch 100 mg

100mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.

formulation example 4. Preparation of pills

100 mg of the vital melon extract of the present invention

lactose 1.5 g

1 g of glycerin

0.5 g xylitol

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

formulation example 5. Preparation of granules

100 mg of the vital melon extract of the present invention

Soybean Extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60° C. to form granules, which were then filled into bags.

formulation example 6. Tablet type health functional food

15% by weight of octacosanol powder, 15% by weight of lactose hydrolysate powder, 15% by weight of soybean protein powder isolated, 15% by weight of chitooligosaccharide, 10% by weight of yeast extract powder, 10% by weight of vitamin and mineral mixture, 4.6% by weight of magnesium stearate, 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 in a conventional manner.

formulation example 7. Healthy drinks

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

[Accession number]

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14699BP

Entrusted date: 20210913

Claims (14)

1. Vital melon ( Cucumis melo L. var. vitalmelon ):

   a) Planting as many nodes as the number of nodes on the main and side branches; and

   b) Higher cucurbitacin A and cucurbitacin B content compared to melon varieties.
2. According to claim 1, wherein the vital melon melon ( Cucumis melo var. makuwa Makino) as a model, cultivated melon ( Cucumis melo var. reticulatus ) as a hybrid, a vital melon.
3. The vital melon according to claim 2, wherein the mother melon is self-fertilized 5 to 10 times with the characteristics of epiphytic female flowers for each node and the characteristics of benignization as target traits.
4. The vital melon according to claim 2, wherein the main grown melon is self-fertilized 5 to 10 times with the characteristics of leaflets and large fruits as target traits.
5. The parent melon ( Cucumis) self-fertilized 5 to 10 times with the characteristics of epiphytic female flowers on each node and the characteristics of benignization as the target trait. melo var. makuwa Makino) and a melon cultivated by melon ( Cucumis melo var. reticulatus ); Accession number containing a vital melon ( Cucumis melo with KCTC14699BP) L. var. Vitalmelon ) Manufacturing method.
6. A pharmaceutical composition for preventing or treating obesity or metabolic disease, comprising an extract of vital melon (accession number KCTC14699BP).
7. The pharmaceutical composition for preventing or treating obesity or metabolic disease according to claim 6, wherein the vital melon has the following characteristics:

   a) Planting as many nodes as the number of nodes on the main and side branches; and

   b) Higher cucurbitacin A and cucurbitacin B content compared to melon varieties.
8. According to claim 6, wherein the extract is water, a lower alcohol extract having 1 to 4 carbon atoms, or a mixture of them extracted with a solvent, obesity or metabolic disease preventing or treating pharmaceutical composition.
9. According to claim 6, wherein the metabolic disease is at least one selected from the group consisting of obesity, hypertension, arteriosclerosis, hepatic steatosis, fatty liver, dyslipidemia, diabetes and hyperglycemia, obesity or metabolic disease prevention or treatment for pharmaceutical composition.
10. 10. The method of claim 9, wherein the dyslipidemia is at least one selected from the group consisting of hyperlipidemia, high LDL cholesterolemia, hypertriglyceridemia and low HDL cholesterolemia, a pharmaceutical composition for preventing or treating obesity or metabolic disease.
11. Health functional food composition for preventing or improving obesity or metabolic disease, comprising an extract of vital melon (accession number KCTC14699BP).
12. A food composition for preventing or improving obesity or metabolic diseases comprising an extract of vital melon (accession number KCTC14699BP).
13. A quasi-drug composition for preventing or improving obesity or metabolic diseases comprising an extract of vital melon (Accession No. KCTC14699BP).
14. A method for preventing, improving, or treating obesity or metabolic diseases, comprising administering an extract of vital melon (accession number KCTC14699BP) to a subject in need thereof.

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