KR102141862B1 - Increasing method of cordycepin of Cordyceps millitaris and bioactive compounds of strawberry and Composition for preventing or treating obesity comprising fermented extracts - Google Patents

Abstract

The present invention relates to a method for increasing the production amount of a functional substance of a cordycepin and a strawberry, and a composition for increasing the production amount of a cordycepin of a strawberry using a strawberry, pupa, and a military cordyceps. The present invention also relates to a pharmaceutical composition for preventing or treating obesity and a food composition for preventing or improving obesity, comprising a fermented extract of a mixture comprising strawberries and pupae. According to the present invention, when fermentation pupae, which is a fermentation source, is added to a strawberry and fermented with Cordyceps sinensis, the content of cordycepin in Cordyceps is increased and the production of major bioactive substances in strawberries is increased, which can be usefully utilized as a functional material. . In addition, according to the present invention, the fermented extract of the mixture of pupae mixed with strawberry suppresses adipocyte differentiation and inhibits triglyceride (TG) accumulation and glucose absorption in adipocytes, which can prevent, improve or treat obesity. As it can be usefully used in the manufacture of pharmaceuticals and functional foods, it can be used in various fields in obesity improvement, where continuous growth is predicted.

Description

Increasing method of cordycepin of Cordyceps millitaris and bioactive compounds of strawberry and Composition for preventing or treating obesity comprising fermented extracts}

The present invention relates to a method for increasing the production amount of a functional substance of a cordycepin and a strawberry, and a composition for increasing the production amount of a cordycepin of a strawberry using a strawberry, pupa, and a military cordyceps. The present invention also relates to a pharmaceutical composition for preventing or treating obesity and a food composition for preventing or improving obesity, comprising a fermented extract of a mixture comprising strawberries and pupae.

Cordyceps genus belongs to Ascomycetes, Clavicipitales, and Clavicipitaceae. About 800 species of Cordyceps sinensis are known to date and 78 are classified in Korea. The types of cordyceps presented in the "Food Ingredients Classification Table" in the Food Code are Paecilomyces japonica/Paecilomyces tenuipes and Cordyceps militaris . Currently, the domestic cordyceps market is about 12 million tons per year, of which the military leash cordyceps occupies about 15%. Cordycepin, cordyceptic acid, ophiocordin, complex polysaccharide, ergosterol, etc. are known as active ingredients of Cordyceps sinensis. In addition, Militaris cordyceps protects against foreign substances such as anti-inflammatory, antibacterial, anti-tumor, and immune-enhancing effects, and enhances basic metabolic activity, such as nourishing tonic, stabilizing, anti-fatigue, improving athletic performance, preventing aging, and prolonging life. It is known to possess various functions such as disease suppression and alleviation, such as suppressing arteriosclerosis, lowering cholesterol and neutral lipids, strengthening blood sugar, and treating hypertension (Park et al., 2002; Lee et al., 2002, 2004; Kim et al., 2001, 2002 , 2004, 2005; Lim et al., 2004; Koh, 2001, 2002; Kwon et al., 2001).

Indigenous Cordyceps did not become a popular medicine because it was difficult to collect a large amount, but many studies have been conducted and popularized as artificial cultivation technology has been developed and mass production is possible, especially cordycepin, a secondary metabolite. Recognition as a health functional food is increasing due to its bioactive substances. When cultivating Cordyceps sinensis, the pupae is used to increase the content of cordycepin, and pupae cordyceps is reported to have various physiological activities such as immuno-enhancing activity, anti-cancer activity, antiviral effect, and anti-inflammatory effect. It is widely used as a tonic food or tonic, and their active ingredients are known as polysaccharides and nucleoside derivatives such as cordycepin (3'-deoxyadenosine). In Korea, pupa cordyceps is recognized as a food raw material, and it has become important to develop codice pin content of pupa cordyceps with the development of various foods and expansion of cultivated farms. Pupa cordyceps has been artificially cultivated using pupae, which is an animal medium, but has recently been cultivated using brown rice, which is a vegetable medium, due to controversy over food consumption due to the aversion of the animal medium. In order to increase the usability of food raw materials of pupa cordyceps, as well as developing cultivation methods using vegetable medium components, it is known that codycepin is the main active component of pupa cordyceps so as to increase the content of codysepin, carbohydrates, sugars, inorganic elements, environmental conditions Various methods, such as, have been tried, but few studies have tested the content change of main ingredients by adding fermentation sources to raw materials.

Meanwhile, obesity is one of the most common medical problems worldwide, and it causes various health problems such as dyslipidemia, fatty liver and hypertension. In addition, adipocytes are the main component of body cell tissue, and the increase in adipocytes leads to an increase in adipocyte tissue.

During the differentiation of adipocyte precursors, many transcription factors affect adipogenesis and lipid accumulation. For example, CCAAT enhancer-binding proteins (C/EBPs) and PPARs (peroxisome proliferator-activated receptors) Promotes differentiation of prisoners. In addition, PPAR-γ among PPARs is fatty-acid binding protein (A-FABP), fatty acid synthase (FAS), lipoprotein lipase (LPL), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and glucose transporter Adjust type 4 (Glut4).

Various methods to suppress obesity by controlling fat cells that cause obesity have been proposed, but there are problems such as causing significant side effects even when there is no obesity suppression effect or an obesity suppression effect. Accordingly, there is a need for a method to safely and effectively prevent or treat obesity.

In the present invention, by adding pupae, which is a fermentation source, to strawberries that are consumed a lot as vegetable crops, a method of increasing the content of cordisipine, a major bioactive component of military cordyceps sinensis, and The present invention was completed during research to develop a functional material by analyzing the content change. In addition, the present invention was completed by confirming that these ferment extracts can effectively inhibit obesity while studying the efficacy of the military-containing Cordyceps sinensis extract of strawberries and pupae of the present invention.

Accordingly, the present invention relates to a method for increasing the production amount of functional substances of cordycepin and strawberries of military cordyceps using strawberries, pupae, and silkworm cordyceps, and compositions for increasing production thereof. In addition, the present invention relates to a pharmaceutical composition for preventing or treating obesity and a food composition for preventing or improving obesity, comprising the extract of Militaris cordyceps sinensis of a mixture comprising strawberries and pupae.

In order to achieve the above object, the present invention (S1) preparing a mixture comprising a strawberry and pupa; And (S2) inoculating the mixture by inoculating Militaris Cordyceps militaris to ferment; and providing a method for increasing the production of functional substances of Cordycepin and Strawberry of Militaryis Cordyceps.

In addition, the present invention provides a composition for increasing the production of functional substances of cordycepin and strawberries of Militaryis Cordyceps, comprising a fermentation product of Cordyceps militaris of strawberry and pupae mixture.

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity, comprising the extract of Militaris cordyceps sinensis of a mixture comprising strawberries and pupae.

In addition, the present invention provides a food composition for preventing or improving obesity, comprising the extract of Militaris cordyceps sinensis of a mixture comprising strawberries and pupae.

According to the present invention, when fermentation pupae, which is a fermentation source, is added to a strawberry and fermented with Cordyceps sinensis, the content of cordycepin in Cordyceps is increased and the production of major bioactive substances in strawberries is increased, which can be usefully utilized as a functional material. . In addition, according to the present invention, the fermented extract of the mixture of pupae mixed with strawberry suppresses adipocyte differentiation and inhibits triglyceride (TG) accumulation and inhibits glucose uptake in adipocytes, thereby preventing, improving or treating obesity. As it can be usefully used in the manufacture of pharmaceuticals and functional foods, it can be used in various fields in obesity improvement, where continuous growth is predicted.

1 is a view showing the chemical formula of a bioactive substance present in strawberries.
2 is a diagram showing the process of differentiation of 3T3-L1 adipocytes.
3 is a view showing the appearance analysis results according to the addition of pupae.
FIG. 4 is a diagram showing the results of examining the content of physiologically active components of cordycepin and strawberries produced by cordyceps of fermented extracts fermented by adding pupae without added pupae, 25%, 50% and 100% pupae to be.
5 is a view showing the results of PCA analysis according to the extraction using a variety of solvents ((A) the results of clearly separated physiologically active substances in the extract through PCA analysis (B) three extraction solvents (70% ethanol, 50 % Ethanol, water) according to PCA analysis).
6 is a view showing the results of the principal component analysis (principal component analysis) of the fermentation extract according to the added content of the pupa added during fermentation (SC1: 70% of non-fermented strawberries, ethanol, 50% ethanol and water extract (SC1P70) , SC1P50, SC1W).SCP0: 70% of fermented strawberries, ethanol, 50% ethanol and water extracts (SCP0P70, SCP0P50, SCP0W)), SCP25: 70%, ethanol, 50% ethanol of fermented strawberries containing 25% pupae And water extract (SCP25P70, SCP25P50, SCP25W), SCP50: 70% of fermented strawberries containing 50% pupae, ethanol, 50% ethanol and water extract (SCP50P70, SCP50P50, SCP50W), SCP100: 70% of fermented pupae, ethanol , 50% ethanol and water extract (SCP100P70, SCP100P50, SCP100W).
7 is a view showing the results of confirming the physiologically active substance of the fermented strawberry extract according to the content of the extraction solvent and pupa ((A) common / unique physiologically active substance appearing in different types of extraction solvent. (B) shared distribution Venn diagram (C) PCA score plot of bioactives of fermented strawberry extract with pupae (ellipse and shape indicate clustering of samples).
8 is a diagram showing the results of classification of bioactive substances in fermented strawberry extract by agglomerative hierarchical clustering (ACH) (SC1: 70% of non-fermented strawberries, ethanol, 50% ethanol and water extracts (SC1P70, SC1P50, SC1W) SCP0: 70%, ethanol, 50% ethanol and water extract of fermented strawberries (SCP0P70, SCP0P50, SCP0W), SCP25: 70%, ethanol, 50% ethanol and water extract of fermented strawberries containing 25% pupae (SCP25P70 , SCP25P50, SCP25W), SCP50: 70% of fermented strawberries containing 50% pupae, ethanol, 50% ethanol and water extract (SCP50P70, SCP50P50, SCP50W), SCP100: 70% of fermented pupae, ethanol, 50% ethanol and Water extract (SCP100P70, SCP100P50, SCP100W).
9 is a view showing the results of comparative analysis of the bioactive substance content of the fermented strawberry extract according to the fermentation of strawberry and pupa mixture by Cordyceps sinensis through hierarchical cluster analysis (HCA) ((A) HPLC analysis (B) HCA analysis result (Heatmaps) showing the relative level (log10 scale) of bioactive substances in pupa mixed fermented strawberry extract (70%, ethanol, 50% ethanol and water extract) by Cordyceps millitaris (Beatmaps) )).
10 is a view showing the results confirming the effect of inhibiting the adipocyte differentiation of the fermented strawberry extract of the present invention. Here, the results of oil red O staining are shown in FIG. 10A, and the results of OD measurement are shown in FIG. 10B. *P<0.05, **p<0.01 compared to the differentiation medium to which water was added.
11 is a view showing the results of measuring the amount of triglyceride accumulation and glucose uptake to confirm the effect of inhibiting the triglyceride (TG) accumulation and glucose uptake in adipocytes of the present invention FSP25E50. *P<0.05, **p<0.01 compared to the differentiation medium to which water was added.
12 is a view showing the results of confirming the effect of inhibiting the expression of C/EBP-β and PPAR-γ of the present invention FSP25E50 (S4WCP25-P50) through Western blot. The results at day 1 of differentiation are shown in Fig. 12A, and the results at day 8 are shown in Fig. 12B. In FIG. 12A and 12B, *p<0.05 is compared with the In+Dex+Ros group.
13 is a view showing the results of confirming the effect of inhibiting the expression of A-FABP, FAS, LPL, HMGCR and Glut4 of the present invention FSP25E50 through Western blot. *P<0.05, **p<0.01 compared to the In+Dex+Ros group.

The present invention (S1) preparing a mixture comprising strawberries and pupa; And (S2) inoculating the mixture with Militaris cordyceps militaris to ferment; providing a method for increasing the production of functional materials of cordycepin and strawberries of Militaryis cordyceps. The strawberry of the present invention is not limited to a specific variety, and the pupae may preferably be silkworm pupa, but is not limited thereto.

In the step (S1), the pupa is 0.01 to 60% (w/w), preferably 0.01 to 50% (w/w), and most preferably 0.01, 25 and 50% (w/w) compared to the content of the strawberry. It can be included as By mixing strawberries and pupae as in the step (S1), these mixtures act as effective fermenters of Cordyceps militaris in the next step (S2), and the functional substances of cordycepin and strawberry The increase can be further maximized.

In addition, the fermentation in the step (S2) may be solid fermentation. It is possible to effectively enhance the functional substances of cordycepin and strawberry of cordyceps by inoculating and solid-phase fermentation by inoculating the military leash cordyceps. In step (S2), the military cordyceps can be inoculated to the mixture at a concentration of 3 to 7% (v/w), preferably 5% (v/w), but is not limited thereto. In addition, the solid phase fermentation can be carried out for 6 weeks, preferably 4 weeks, but is not limited thereto.

In addition, the functional material of the strawberry is cyanidin-3-glucoside, Cinchonine, Catechin, Epi-gallocatechin, p-coumarinic acid ( p- Coumaric acid) and ellagic acid (Ellagic acid) may be selected from the group consisting of 1 or more, but is not limited thereto. Through the method of the present invention, by further improving the production amount of the functional material of the strawberry as described above, it is possible to further increase the utilization of the strawberry as a functional material.

The present invention also provides a composition for increasing the production of functional substances of cordycepin and strawberries of Militaryis Cordyceps, comprising a fermentation product of Cordyceps militaris of strawberry and pupa mixture.

The fermentation product of Cordyceps militaris of the strawberry and pupa mixture of the present invention is 0.01 to 60% (w/w), preferably 0.01 to 50% (w/w) of pupae in strawberries , Most preferably 0.01, 25 and 50% (w/w) to prepare a mixture, and then 3 to 7% (v/w), preferably 5% (v/w) ) Refers to the fermentation product obtained by inoculating at a concentration and solidifying.

When using the composition for increasing the production amount of functional substances of cordycepin and strawberry of the militaryis cordyceps of the present invention, cordycepin and various kinds of strawberries can effectively increase the production of functional substances, and cordycepin as a functional material And the utilization of strawberries.

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity, comprising a mixture of strawberries and pupae, Militaris cordyceps ferment extract.

The fermentation extract, (S1) preparing a mixture comprising strawberries and pupa; (S2) step of obtaining a fermentation product by inoculating the mixture with Cordyceps militaris and fermenting it; And (S3) extracting the fermentation product with lower alcohols of C ₁ to C ₄ .

In the step (S1), the mixing ratio of strawberry and pupae may be 0.5:1 to 3.5:1, preferably 0.7:1 to 3.3:1, more preferably 1:1 to 3:1 (w/w). . By mixing the strawberry and pupae as in the mixing ratio, the mixture can be fermented and the effect of inhibiting fat production of the extracted extract can be further increased.

In step (S2), the military cordyceps can be inoculated into the mixture at a concentration of 3 to 7% (v/w), preferably 5% (v/w), but is not limited thereto.

In the step (S3), the lower alcohol may be 40 to 80%, preferably 45 to 75%, more preferably 50 to 70% (v/v) ethanol. By using ethanol having the above ratio for extraction, it is possible to further increase the effect of inhibiting fat production of the extract.

According to the present invention, when the fermented product obtained by inoculating the military and cordyceps by inoculating Military Lee Cordyceps with a mixture of strawberries and pupae 3: 1 (w/w) is extracted with 50% (v/v) ethanol, the prepared fermentation Extracts can most effectively prevent or treat obesity.

In addition, the fermentation extract inhibits adipocyte differentiation, inhibits triglyceride accumulation, inhibits intracellular glucose absorption, inhibits C/EBP-β (CCAAT-Enhancer-Binding Protein-beta) expression, and peroxisome proliferator-activated receptor-gamma (PPAR-γ). ) Expression inhibition, A-FABP (Adipocyte fatty acid-binding protein) expression suppression, FAS (fatty acid synthase) expression suppression, LPL (lipoprotein lipase) expression suppression, HMGCR (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase ) Inhibition of expression and inhibition of expression of Glut4 (glucose transporter type 4) may have one or more inhibitory effects. The ferment extract of the present invention can effectively suppress or treat obesity by having the above-described effects.

The pharmaceutical composition for preventing or treating obesity, comprising the extract of Militaris cordyceps sinensis of the mixture comprising the strawberry and pupae of the present invention, further comprises suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Can. In addition, additives for the preparation of a solid or liquid may be used in the preparation of the pharmaceutical composition. The additive for formulation may be either organic or inorganic.

Excipients include, for example, lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, silicon dioxide, and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and cured vegetable oil. Any colorant can be used as long as it is normally permitted to be added to pharmaceuticals. These tablets and granules may be appropriately coated according to sugar content, gelatin coating, or other needs. In addition, preservatives, antioxidants, and the like can be added as necessary.

In addition, the pharmaceutical composition of the present invention may be prepared in any formulation conventionally prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. However, it can be formulated and used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods. .

The pharmaceutically effective amount of the present invention may vary depending on the type of wound, the application site, the number of treatments, the treatment time, the formulation, the patient's condition, and the type of adjuvant. The amount of use is not particularly limited, but when the daily effective amount of the pharmaceutical composition of the present invention is applied to a patient, it may be 0.00001 to 10000 μg. The daily amount may be administered once a day or divided into 2-3 times a day at appropriate intervals, or may be administered intermittently at intervals of several days.

However, the amount of the pharmaceutical composition of the present invention is the administration route, the patient's age, gender, weight, the severity of the patient, the type of wound, the application site, the number of treatments, the treatment time, the formulation, the patient's condition, the type of adjuvant, etc. As determined in light of a number of related factors, the effective amount should not be construed as limiting the scope of the invention in any aspect.

The present invention also provides a food composition for preventing or improving obesity, comprising the extract of Militaris cordyceps sinensis of a mixture comprising strawberries and pupae.

The "food" of the present invention can be prepared in all forms, such as functional food, nutritional supplement, health food and food additives. For example, as a health food, sialyl lactose of the present invention may be prepared in the form of tea, juice, and drink for drinking or granulated, encapsulated, and powdered. In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (e.g. canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (e.g. ham, sausages, corn beef, etc.) , Breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheats, etc.), edible vegetable oils, margarine, vegetable protein, retort foods , Frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the bee venom of the present invention.

When using a pharmaceutical composition or a food composition for preventing or treating obesity, which includes a fermented extract of a mixture comprising strawberries and pupae of the present invention, it is possible to effectively suppress obesity, thereby preventing, improving or treating obesity, and As it can be usefully used for the production of functional foods, it can be used in various fields in the field of obesity improvement where continuous growth is predicted.

Hereinafter, the present invention will be described in more detail based on examples. The examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the present invention.

Materials and methods

(1) Experimental materials

Seolhyang varieties grown in Miryang, Gyeongnam were purchased directly from farms in 2016, and raw crushed raw fruits were used as ingredients. Pupa was made in China and used for purchase at a mart. Strains used for fermentation of strawberries were Paecilomyces tenuipes and Militaris cordyceps militaris , which were used for fermentation after being sold from Prof. Sang-Mong Lee's laboratory at Pusan National University.

(2) Reagents and equipment

As an organic solvent used for HPLC analysis and sample extraction, an HPLC grade solvent from Fisher scientific (Fisher scientific Korea, Seoul, Korea) was used. All others used express reagents. The HPLC analyzer used a system controller of Agilent 1100 Series LC system (Agilent Technologies, Santa Clara, CA, USA), photodiode array detector (PDA), auto sample injector, and degasser.The HPLC column was Phenomenex's Luna C18 column (150× 4.6 mm, 5 μm, Phenomenx, USA) was used.

Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Paso Robles, CA, USA), paraformaldehyde from TCI (TCI, Tokyo, Japan), and isopropanol from GENEray (GENEray, Shanghai, China). In addition, Western loading buffer, Bio-Rad protein assay reagent and protein marker were purchased from BioRad (BioRad, Hercules, CA, USA). The triglyceride detection kit and glucose detection kit were purchased from Asan Pharmaceutical (Seoul, Korea). NaHCO ₃ , HEPEs, insulin, rosiglitazone, dexamethasone, oil red O and other drugs were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA).

(3) Culture of Cordyceps strains

The strains were passaged at 30-day intervals while preserving at 4°C in a PDA or MEA plate medium. PDA or MEA plate medium was used for mycelial growth and incubated for 7 days at 25°C in an incubator. PDB or MEB medium was added to each 100 ml in a 300 ml Erlenmeyer flask, and sterilized at 121°C for 15 minutes, followed by cooling. After picking up 3 pieces of each of the edges of the hyphae cultured for 7 days in a PDA plate medium using a 5 mm diameter cork borer, inoculating them in a PDB and MEB liquid medium, shaking and incubating them at 25°C for 7 days (150 rpm), and seeding the liquid. Produced.

(4) Preparation of solid-phase fermentation and sample solution from Cordyceps sinensis

For the solid-phase fermentation of the cordyceps of the strawberry and pupa mixture, fresh strawberries were first washed and then mixed with a mixer. Then mixed strawberries (300 g) and 100% pupa (FP) containing 0%, 25% and 50% pupa (FS, FSP25 and FSP50) were placed in autoclavable vinyl. That is, the experimental groups used for fermentation are FS (0% pupa, i.e. strawberry), FSP25 (strawberry with 25% pupa), FSP50 (strawberry with 50% pupa), FP (100% pupa), and Cordyceps (C. militaris) Non-fermented strawberries (NS) without inoculation were used as a control. Vinyl was autoclaved at 121°C for 30 minutes using Hyundai Autoclave (Hyundai Co., Ltd., Seoul, Korea), and then cooled at room temperature for 30 minutes. After that, the Cordyceps sinensis suspension was inoculated to sterilized strawberries (FS, FSP25 and FSP50) and pupae (FP) at a level of 5% (v/w) and solidified for 4 weeks in an incubator at 25±1°C. After fully fermenting the fermented product with a grinder, 10 ml of 95% (v/v) and 50% (v/v) ethanol and distilled water were added to 3.0 g of each sample, followed by ultrasonic extraction for 1 hour and then centrifuged at 3,000 rpm. The supernatant was taken, 9 ml was added again, and repeated twice to combine the ultrasonically extracted solutions to adjust the total amount to 30 ml. 5 ml of the extract was taken and the filtrate filtered with a 0.45 μm syringe filter was used as the sample solution. The remaining 25 ml was filtered with Whatman filter paper No 2 (No. 2 filter paper, Whatman, Clifton, NJ, USA) and concentrated under reduced pressure at 45°C with a rotary vacuum evaporator (rotavator R-200, Bchi, Flawil, Switzerland). Used as NO or MTT analysis data.

(5) Fermentation strawberry extract manufacturing method

In order to confirm the effect according to the extraction solvent, NS, FS, FSP25, FSP50 and FP were extracted with 70% ethanol, 50% ethanol and water, and according to the 70% ethanol, 50% ethanol and water extraction solvent, E70, E50 and DW. The extraction was used by extracting the experimental samples with a 10-fold solvent 3 times for 1 hour through an ultrasonic sonicator at room temperature. The extract is Whatman No. 2 After filtering with filter paper, the residue was re-extracted twice under the same conditions. The extract was concentrated using a rotary evaporator (Heidolph Instruments Co., Ltd, Schwabach, Germany) under reduced pressure at 45°C. It was then weighed for calculation of extraction yield and sealed to prevent moisture absorption and then stored at -18 °C for later analysis. The lyophilized extract was dissolved in DMSO at a concentration of 20 mg/ml, before being used for analysis, and then diluted for further analysis.

(6) Quantification and simultaneous analysis method establishment through HPLC analysis

The main compounds of fermented strawberry extract including non-fermented strawberry and pupa were analyzed by Ko et al. (Ko, Jayaramaiah, Gupta, Kim, An, Wang, et al., 2017). Also, in order to simultaneously analyze the major bioactive substances of cordycepin and strawberry, the major components of Cordyceps sinensis, 14 kinds of major components reported so far were purchased from reagent companies to establish an analysis method. Cyanidin-3-glucoside (Cyanidin-3-), which is known as a major component of strawberries, to cultivate highly functional strawberry varieties based on the analysis of the content of the main varieties cultivated in Korea and the bioactive substances in the growing system. glucoside), callistephin (pelargonidin 3-O-glucoside chloride), pelarrgonin, cinchonine, epi-gallocatechin, chlorogenic acid, (+)-catechin, epi-catechin, p-coumaric acid, elllagic acid, quercetin-3-glucuronide, fisetin , 15 kinds of regression linear, detection limit (LOD) and quantitative limit (LOQ) values such as quercetin, cinnamic acid and kaempferol were obtained to establish a simultaneous analysis method.

Specifically, the quantitative analysis of the standard product was used by filtering the extract by solvent with a 0.45 μm syringe filter (Millipore, Billerica, MA, USA). The high performance liquid chromatography (HPLC) used in this experiment is the Agilent 1100 series (Agilent Technologies Inc., CA, USA), and the column is a Luna C18 (5 μm, 4.6 mm×150 mm), a reverse phase column. UV detector (254 nm) was used. For the mobile phase, 0.025% formic acid (A solution) and acetonitrile (B solution) were used. The gradient was set through a pump program and the flow rate was 0.8 ml/min. Each prepared solution was injected with 10 μl of an autosampler to conduct an analysis experiment. The composition and gradient conditions of the mobile phase solvent were set to various conditions as shown in Table 1 to perform the analysis experiment. The area of chromatogram obtained by analysis by HPLC was substituted into the regression linear equation to obtain the content of each standard, and the content of the index component was calculated by repeating 3 times for each sample.

In order to verify the reliability and reproducibility of the method using HPLC, analysis according to ICH guidelines was performed. For linearity, the peak area ratio was calculated for each of the five concentrations (6.25, 12.5, 25, 50, and 100 μg/ml) to prepare a calibration curve for the standard concentration (X-axis) and the peak area ratio (Y-axis). The correlation coefficient of linearity was obtained and confirmed.

(7) 3T3-L1 cell culture

3T3-L1 cells were purchased from KCLB (Korea Cell Line Bank, Korea) and DMEM (Gibco, Gland Island, NY, USA) containing 10% FBS (Welgene, Gyeongsina-si, Korea), 25 mM NaHCO ₃ (Sigma , St. Louis, MO, USA) and 25 mM HEPEs (Sigma, St. Louis, MO, USA) (culture medium). Differentiation of adipocytes after growth of cells until complete fusion in 48-well plates is performed with 10 μM rosiglitazone (ROS, Sigma, St. Louis, MO, USA), 10 μg/ml insulin (In , Sigma, St. Louis, MO, USA) and 1 μM dexamethasone (DEX, Sigma, St. Louis, MO, USA) (differentiation medium). After 48 hours, differentiation medium was replenished for the next 48 hours of culture, and differentiation medium was replenished every 2 days. During the differentiation process, the experimental group of the present invention (non-fermented strawberry (NS), FS (0% pupa, ie strawberry), FSP25 (strawberry with 25% pupa), FSP50 (strawberry with 50% pupa), FP (100% Pupa)) and extracting the extraction solvent (70% ethanol, 50% ethanol and water) was dissolved in distilled water to observe the effect on 3T3-L1 adipocyte differentiation by adding it to the differentiation medium during the induction process of 3T3-L1 cells. . The differentiation process is shown in FIG. 2.

(8) Oil red O dyeing

After washing the adipocytes with PBS on the 8th day of differentiation, they were fixed using 4% paraformaldehyde (TCI, Tokyo, Japan) at room temperature for 20 minutes and oil red O (Sigma, St. Louis, MO, USA) It was dyed at 4°C for 30 minutes. After dyeing, excess dye was removed, and the plate was washed twice with PBS, and then photographed using a Motic microscope (Moitc, Xiamen, China).

(9) OD value measurement

After photographing, PBS was removed and 150 μl isopropanol (GENEray, Shanghai, China) was added to each well to dissolve oil red O. Then 100 μl from each well was transferred to a 96-well plate and OD values were measured at 500 nm using a BioTek elisa reader (BioTek, Winooski, VT, USA).

(10) Triglyceride (TG) content determination

Cells were grown to fully fuse in a 12-well plate and differentiated according to the differentiation method described in the'(7) 3T3-L1 cell culture' method above. On day 8, triglyceride was extracted using 5% triton X-100 (Bioshop, Burlington, Ontario, Canada) and the TG content was quantified using a commercially available TG kit (Asan Pharmaceutical, Seoul, Korea) according to the protocol. .

(11) Determination of glucose content in the medium

Cells were grown to fully fuse in a 12-well plate and differentiated according to the differentiation method as described in the'(7) 3T3-L1 cell culture' method above. On the 8th day, the medium of each well was collected and the glucose was quantified using a commercial glucose kit (Asan Pharmaceutical, Seoul, Korea) according to the protocol.

(12) Western Blot

3T3 cells were cultured (control), In+Dex, differentiation medium (In+Dex+Ros) and 50% ethanol extract (FSP25E50) of fermented strawberries containing 25% pupae at different concentrations (2.5 μg/ml, 5.0 μg /ml, 10.0 μg/ml) and cultured for 24 hours or 8 days in differentiation medium. Cultured cells were obtained and lysed with ice for 30 min in RIPA buffer (150 mM Nacl, 1% Trition X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl and 1 mM phenylmethylsulfonyl flouride). The lysate was centrifuged at 12,000 g, 4 °C for 15 minutes. After centrifugation, protein concentration was measured using a Bio-Rad protein analysis reagent (BioRad, Hercules, CA, USA). Then, the protein mixed with 4-loading buffer (BioRad, Hercules, CA, USA) and 2-mercaptoethanol (Bioshop, Burlington, Ontario, Canada) was heated at 95°C for 7 minutes, and protein markers (BioRad, Hercules, CA, USA) by 12% SDS-PAGE. The protein was then electro-transfered from the gel to the PVDF membrane and placed for about 20 minutes at 20 V using a Semi Dry Transfer system (BioRad, Hercules, CA, USA). The membrane was blocked with 1% BSA in PBST buffer (0.1% Tween-20/PBS) for 1 hour at room temperature, and then detected as a primary antibody diluted overnight at 4°C with 3% BSA-PBST solution. In addition, the membrane was washed with PBST solution three times for 5 minutes each time, and then left at room temperature for 1 hour to react with the corresponding HRP-binding secondary antibody. After washing the membrane three times with PBST solution for 5 minutes, the immuno-reactive band was visualized using the ECL detection kit (Thermo Scientific, Rocford, IL, USA) according to the manufacturer's instructions. The band was analyzed by Image J software (Wayne Rasband, NIH, USA).

(13) Statistical analysis

Dancan's multiple range test was used to compare the content of bio-activity substances. Student's t-test was used to analyze the differences between the two groups. All data are expressed as means±SD from at least 3 independent experiments. P<0.05 was judged to have a significant statistical difference.

Example 1. Confirmation of enhancement effect of major bioactive substances in strawberries

In order to increase the added value and utilization of strawberries through the enhancement of the main physiologically active substances of strawberries, according to the method described in the previous materials and methods, the pupa weight of strawberries is added at 0%, 25%, 50% and 100% (NS, FS, FSP25, FSP50, and FP) were inoculated for 4 weeks after inoculation with Cordyceps militaris . The result of photographing the appearance of the fermentation is shown in FIG. 3.

As shown in FIG. 3, it was confirmed that the fermented product fermented by the military leash Cordyceps by adding pupae to the strawberry had a substantial change in appearance.

In addition, in order to confirm the effect according to the extraction solvent, the NS, FS, FSP25, FSP50 and FP were extracted with 70% ethanol, 50% ethanol and water, and E70 according to the 70% ethanol, 50% ethanol and water extraction solvent, E50 and DW. In extracts with different extraction solvents (70% ethanol, 50% ethanol, and water), changes in the content of the major bioactive substances of strawberries and cordycepin, the main component of cordyceps, were investigated through HPLC quantitative analysis. The results of radar plotting the results are shown in FIG. 4 and the specific values are shown in Table 2.

As shown in FIG. 4 and Table 2, changes in the main bioactive substances of cordisipine and strawberry were different according to the pupa content and the extraction solvent.

First of all, in the case of cordisipine, the content was highest in the fermentation extract with 25% pupae, followed by the fermentation extract with 50% pupae, fermentation extract without pupa (100% strawberry), and fermentation extract with 100% pupae in that order. Confirmed. In particular, it was confirmed that the content of cordisipine increased more than 15 times in the fermented extract with 25% pupae compared to the fermented extract without pupae. In the change according to the extraction solvent, the content was the highest in the case of extraction with 50% ethanol in the case of cordisipine, and then the content in the extraction in 70% ethanol. On the other hand, in water extract, it was confirmed that the content of cordispin was significantly reduced.

In the case of the main physiologically active substance of the strawberry, the content of cyanidin-3-glucoside was not produced at all when fermented with only strawberries (without pupa), but the higher the addition rate of pupa added to strawberries It increased, but this trend was similar regardless of the extraction solvent. Cinchonine was increased by about 30 times by fermentation, and tended to decrease as the amount of pupa increased, and there was little difference depending on the extraction solvent (70% ethanol, 50% ethanol and water). Callistephin was not detected during fermentation of strawberries, but the content was increased in the fermented extract of mixed strawberries mixed with pupae regardless of the extraction solvent. Catechin (Catechin) was produced in a large amount by fermentation, the more the amount of pupa was decreased, the more. Epi-gallocatechin was slightly increased during fermentation, most of which was added with 50% pupae, and was not detected at all when fermented with 100% pupae. p- Coumaric acid is about 40 μg/g in 25% and 50% pupa added fermentation extract when 70% ethanol is extracted, about 30 μg in 50% pupa fermentation extract when extracted with 50% ethanol /g It was confirmed that the body weight was contained. Ellagic acid contained about 29 μg/g fresh weight when extracted with 70% and 50% ethanol from unfermented strawberries, and 152.1 μg/g live weight and 133.2 μg/g respectively in pupa-free fermented extracts, respectively. It was confirmed that the content increased rapidly by fermentation by increasing the content by 5 times and 4 times or more as the live weight, and the content decreased as the amount of pupa added increased.

In addition, among the physiologically active substances of strawberries, seven major substances whose contents change due to fermentation of strawberries, cyanidin-3-glucoside, cinchonine, callistephin, catechin ( Catechin), ellagic acid, and quercetin and C. militaris are listed in different tabular forms in Table 3.

As shown in Table 3, it was more clearly confirmed that changes in major physiologically active substances appeared due to fermentation of strawberries. Specifically, when looking at the differences in NS, FS, FSP50, FSP25, and FP, cyanidin-3-glucoside was not detected in FS fermented without addition of pupae, and was significantly increased by 10 to 248 times compared to NS in FSP25 and FSP50 It was confirmed that the increase. Also, fermentation increased 2.4 to 31.7 times of NS in case of callistin, 2.0 to 5.1 times of NS in ellagic acid, 1.0 to 2.9 times of NS in quercetin, and 2.0 to 31.8 times in NS in case of cinchonine Was confirmed.

In the case of cordycepsine of Cordyceps sinensis, changes due to fermentation were also apparent. Specifically, it was not detected in the non-fermented strawberry, but it was confirmed that the content was significantly increased in the fermented strawberry regardless of the presence or absence of pupae. Comparing the fermentation experiment groups FS, FSP50, FSP25 and FP, it was confirmed that in the case of cordisipine, the content was further increased when a low concentration pupae was added. In the case of the content change according to the extraction solvent, it was confirmed that the cordisipine content showed a high content in the order of 50% ethanol, 70% ethanol, and water in all experimental groups.

Example 2. Correlation analysis of main physiological activity of strawberry fermentation extract

Among the fermented extracts fermented with Cordyceps militaris cordyceps strains, chrysanthemin (cyanidin-3-glucocy) and callistine, catechin and cinconin, catechin and callistine, epigallocatechin and ellagic acid, epi Gallocatechin and quercetin are components of high significance with a correlation value of 0.7 or more, and as the content of chrysanthemin increases, callistin increases, and when catechin increases, cinkonin, catechin and callistin, and epigal Eloagsan and quercetin tended to increase with increasing catechins. Components with a high correlation value of 0.5 or more were quercetin for cortisepine, p-coumarinic acid, ellagic acid and isoquercetin, and ellagic acid for catechin. On the other hand, a component that shows a high degree of significance with a negative correlation value of 0.5 or less is a callipine by showing a negative correlation value between ellagic acid and quercetin in case of callistine, and between cinchonin and catechin in case of cinnamic acid. Ellagic acid and quercetin decreased with increasing content of cinnamon, while cinnakinin and catechin decreased with increasing cinnamic acid.

Example 3. PCA analysis and PCA score plot of fermented strawberry extract

The results of principal component analysis of the fermentation extract according to the content of pupa added during fermentation are shown in FIG. 5.

As shown in FIG. 5, the first principal component (F1) and the second principal component (F2) showed 96.6% and 1.6% explanatory power of the total data, and the total explanatory power was 98.2%. The description of each code in FIG. 5 is as follows: SC1: 70% of non-fermented strawberries, EtOH, 50% EtOH and water extracts (SC1P70, SC1P50, SC1W), SCP0: 70% of fermented strawberries, EtOH, 50% EtOH and water extract (SCP0P70, SCP0P50, SCP0W), SCP25: 70% of fermented strawberries containing 25% pupae, EtOH, 50% EtOH and water extract (SCP25P70, SCP25P50, SCP25W), SCP50: 50% pupa Contains 70%, EtOH, 50% EtOH and water extract of fermented strawberries (SCP50P70, SCP50P50, SCP50W), SCP100: 70% of fermented pupae, EtOH, 50% EtOH and water extract (SCP100P70, SCP100P50, SCP100W).

Looking at each characteristic distribution, SCP25P70 and SCP25P50, SCP50P70 and SCP50P50, SCP25W and SCP50W were distributed in the right or positive direction of F1, and SCP0P70, SCP0P50, SCP0W, SC1P70, SC1P50, SC1W, SCP100P70, SCP100P5 and SCP100W on the left It was distributed. On F2, SCP25P70, SCP25P50, SCP50P70, SCP50P50, SCP0P70, SCP0P50, and SCP0W were distributed in the positive direction, and SCP100P70, SCP100P50, SCP100W, SC1P70, SC1P50, and SC1W were distributed in the negative direction. In the positive direction of the main ingredients 1 and 2, 25% and 50% of pupae were added to the strawberry, fermented, followed by extracts extracted with 50% and 70% of ethanol, whereas in the negative direction, 25% and 50% of pupae were added to the strawberry. SCP25W and SCP50W extracted by adding fermented water with water were distributed, and the characteristics of the extraction solvent tended to be revealed between the water extract and the ethanol extract.

Strawberry fermentation extracts SCP0P70, SCP0P50, and SCP0W were distributed in the negative direction of main component 1 and positive direction of main component 2, and in the negative direction of main components 1 and 2, pupa fermented products SCP100P70, SCP100P50, SCP100W, and strawberry non-fermentation extracts Phosphorus SC1P70, SC1P50 and SC1W were distributed. Therefore, as a result of PCA analysis of the fermentation extract fermented by adding pupae to strawberries, the extract extracted with ethanol by adding pupae to strawberries was in the positive direction of the first main component and the second main component, and the water extract was negative of the second main component. Distributed in the direction. In the negative direction of the first main component and in the positive direction of the second main component, ethanol and water extracts of fermented strawberries were distributed, and in the negative direction, unfermented strawberries and pupa fermented products were distributed. It was confirmed that there was a clear difference in the presence or absence of pupae, fermentation of pupae and extraction solvent of fermented pupae.

In addition, a mixture of strawberries and fermentation pupae was used to classify substances extracted by solvent (70% ethanol, 50% ethanol, and water) from fermented products fermented with a Cordyceps millitaris strain, and analyzed with a PCA scores plot. Shown.

As shown in FIG. 6, in the case of 70% ethanol extract, the first main component (F1) and the second main component (F2) showed 96.3% and 2.5% explanatory power of the total data, and the total explanatory power was 98.8%. In the case of 70% ethanol extract, fermented extracts fermented by adding 25% and 50% pupa to strawberries were distributed in the positive direction of the main component 1 and the negative direction of the 2, and the negative direction of the main component 1 and the positive amount of the main component 2 The pupa fermented extract was distributed in the aroma, and the unfermented extract and the strawberry fermented extract were distributed in the negative direction of the main component 1 and the negative direction of the main component 2. In the case of 50% ethanol extract, 50% pupa was added to the strawberry in the positive direction of the first and second main ingredients, and the pupa fermentation extract was distributed.In the negative direction of the main ingredient 1 and the positive direction of the main ingredient 2, the strawberry fermentation extract and strawberry In the pupa 25% extract, in the negative direction of the main components 1 and 2, unfermented extract and strawberry fermented extract were distributed. In the case of water extract, fermented extract fermented by adding 25% and 50% of pupae to strawberries in the negative direction of main ingredients 1 and 2, the negative direction of the first main ingredient, and the fermented extract of pupa and the first part in the positive direction of the second main ingredient In the negative direction of the second main component, unfermented strawberry extract and strawberry fermented extract were distributed.

As can be seen from the above, since the component content of strawberry fermentation tends to be significantly different between the main components 1 and 2 depending on the extraction solvent, it is important to create and secure new substances through fermentation, but the content of the functional components according to the extraction solvent Showed a significant difference. Of all 11 types of bioactive components investigated, 11 substances were detected in 70% and 50% extracts, but epigallocatechin, ellagic acid, and isoquercetin were not detected in water extracts, so the extraction solvent had a content of bioactive components. Of course, it was confirmed that it was highly related to detection.

Example 4. Hierarchical cluster analysis

Hierarchical cluster analysis is an analysis method (Huang 2014) that helps to understand the structure of the entire data by grasping the characteristics of a group by grouping similar ones into groups among given observations (Huang 2014). 7 shows the results of the analysis.

As shown in Fig. 7, the results of the clusters were expressed by dendrogram. As a result of colony analysis of the fermentation extract of Cordyceps sinensis of Militaris, it was largely classified into three groups, and as a result of verification based on the distance between the clusters, the group that did not ferment (SC1), the group that fermented with pupa only (SCP100), and the strawberry fermentation group (SCP) Classified into groups. The strawberry fermentation group was further divided into pupa-free fermentation (SCP0) and pupa 25% and 50% addition groups. Based on the distance between clusters, the addition of pupae (SCP0) and the addition of pupae (SCP25, SCP50) were located at close range. These results were largely classified as unfermented strawberries and fermented strawberries, and the fermentation was further classified as fermentation including strawberries (SCP0, SCP25, SCP50) and pupa fermentation without strawberries (SCP100). Fermentation including strawberries was again classified into strawberry fermentation (SCP0) and fermentation with pupae added to strawberry (SCP25, SCP50), and it was possible to clearly classify according to the fermentation, non-fermentation, and pupa added during fermentation.

In addition, agglomerative hierarchical clustering (AHC) results for extracts of fermented strawberries are shown in FIG. 8, and hierarchical cluster analysis (HCA) results are shown in FIG. 9.

8 and 9, it was confirmed that the content of metabolites was significantly different depending on the presence or absence of a pupa as a fermentation source during strawberry fermentation. Cordisipine, quercetin, isoquercetin, and camperol (kaempferol) were classified into one subgroup as a result of cluster analysis.The higher the amount of the pupa, a fermentation source added to strawberries, tended to increase in content, and the pupae alone When fermented, the content was similar to the unfermented strawberry extract. Cinchonine, epigallocatechin, catechin, and ellagic acid were the most sub-groups of fermented extracts without pupae, but tended to decrease as the pupae content increased. The content of cinnamic acid decreased by fermentation, and cyanidin-3-glucoside and callipine were the same subgroup, but there was no difference in fermented extract fermented by adding pupae to strawberries, but the content in pupa fermentation extract increased. Tended to. Therefore, through the hierarchical cluster analysis, the relevant metabolic pathway was derived according to the amount of pupa added during fermentation of strawberries and the presence or absence of fermentation, which was judged to be a good analysis method for comparative analysis of the content of metabolites.

Example 5. Confirmation of the effect of inhibiting adipocyte differentiation of fermented strawberry extract

In this example, the NS, FS, FSP25, FSP50 and FP extracts of the present invention were intended to confirm the effect of inhibiting fat production. To this end, each extract was dissolved in distilled water, and then treated with a 3T3-L1 cell line cultured according to'(7) 3T3-L1 cell culture' to confirm that the effect of suppressing fat production was observed. Rosiglitazone, insulin, and dexamethasone were used to induce fat production in the 3T3-L1 cell line, and it was confirmed that differentiation of adipocytes was induced 8 days after induction. The results of oil red O staining are shown in FIG. 10A, and the results of OD measurement are shown in FIG. 10B. In FIG. 10, *p<0.05 and **p<0.01 were compared with the differentiation medium to which water was added.

As shown in FIG. 10B, the dose-dependent effect was confirmed to 200 μg/ml for FS, 20 μg/ml for FSP50, and 10 μg/ml for FSP25, regardless of the extraction solvent, and all The effect of inhibiting the fat production of the fermented strawberry extract was confirmed. On the other hand, in the case of FP without strawberry, it did not show an inhibitory effect on adipogenesis.

In addition, when looking at the results according to the extraction solvent, in the case of FSP25E70, FSP25E50 and FSP25DW, lipid production was significantly suppressed at dose concentrations of 5.0 μg/ml and 10 μg/ml. FSP50E70, FSP50E50 and FSP50DW extracts significantly inhibited fat production at a concentration of 20 μg/ml. These results are the results of confirming the effect of inhibiting the fat production of the extract of fermented fermented strawberries containing pupae, the results of which can be seen that the extract of FSP25 more effectively inhibits fat production than the extract of FSP50. In addition, it was confirmed that the ethanol extract (70% ethanol and 50% ethanol extract) of the pupa mixed fermented strawberry showed superior fat generation inhibitory effect than the water extract.

Example 6. Confirmation of the inhibitory effect of FSP25E50 on triglyceride (TG) accumulation and glucose uptake in adipocytes

PPAR-γ regulates fatty acid storage and glucose uptake. In this example, differentiation of 3T3-L1 adipocyte precursors (pre-adipocytes) was induced using rosiglitazone, a PPAR-γ agonist. In addition, since it was confirmed that the experimental group showing the most effective inhibitory effect on fat production in the above Example was FSP25E50, the amount of triglyceride accumulation and glucose uptake when FSP25E50 was treated was measured and shown in FIG. 11. In FIG. 11, *p<0.05 and **p<0.01 were compared with the differentiation medium to which water was added.

As shown in FIG. 11, it was confirmed that triglyceride accumulation and intracellular glucose uptake were significantly inhibited in the process of adipogenesis of 3T3-L1 cells depending on the treatment capacity of FSP25E50.

Example 7. Confirmation of the effect of suppressing the expression of C/EBP-β and PPAR-γ of FSP25E50

C/EBP-α, C/EBP-β and PPAR-γ proteins play an important role in the differentiation of adipocyte precursors into mature adipocytes. In this example, 3T3-L1 cells were induced in differentiation medium and cultured with FSP25E50 for 1 and 8 days to reveal the mechanism of inhibition of adipogenesis in the 3T3-L1 cell line. Thereafter, Western blot was used to measure C/EBP-α, C/EBP-β and PPAR-γ in 3T3-L1 cells, which are shown in FIG. 12. The results at day 1 of differentiation are shown in Fig. 12A, and the results at day 8 are shown in Fig. 12B. In FIG. 12A and 12B, *p<0.05 was compared with the In+Dex+Ros group.

As shown in Fig. 12A, FSP25E50 at the treatment concentration standard of 10.0 μg/ml on Day 1 significantly compared the expression of C/EBP-β and PPAR-γ with In+Dex+Ros by 70.2% and 60.1%, respectively. It was confirmed to decrease.

In addition, as shown in FIG. 12B, it was confirmed that C/EBP-β and PPAR-γ were decreased by 47.2% and 68.7%, respectively, compared to In+Dex+Ros when FSP25E50 was treated at a concentration of 10.0 μg/ml on the 8th day. Did.

In addition, it was confirmed through the results in FIGS. 12A and 12B that the protein levels of C/EBP-β and PPAR-γ were reduced in all FSP25E50 treated groups of the present invention.

Example 8. Confirmation of the inhibitory effect of FSP25E50 on the expression of PPAR-γ target genes

A-FABP is a fatty acid carrier protein that is expressed primarily in fat cells and regulates the storage and fat breakdown of fatty acids. It is known that suppressing this protein expression can cure obesity. FAS is a protein that plays an important role in fat production. In addition, LPL is a protein that plays an important role in adipocyte differentiation, and the expression of LPL increases with TG accumulation in cells. HMGCR is an important enzyme in the mevalonate pathway that produces cholesterol and other isoprenoids. Glut4 is a protein essential for the transfer of glucose from the medium to the cells, and is a major glucose transporter for adipocyte precursors to differentiate into adipocytes. Thus, in this example, the effect of inhibiting the expression of A-FABP, FAS, LPL, HMGCR and Glut4 of FSP25E50 was confirmed using Western blot on the 8th day of 3T3-L1 cell differentiation, and the results are shown in FIG. 13. In FIG. 13, *p<0.05 and **p<0.01 were compared with the In+Dex+Ros group.

As shown in Figure 13, it was confirmed that the expression of A-FABP, FAS, LPL, HMGCR and Glut4 was significantly inhibited by FSP25E50. Therefore, through these results, it was confirmed that the FSP25E50 of the present invention can inhibit fat production, inhibit TG accumulation, and inhibit glucose uptake in differentiated 3T3-L1 cells.

Formulation Example 1. Preparation of pharmaceutical products

1.1 Preparation of powder

200mg of Militaris cordyceps fermented extract of a mixture containing strawberries and pupae

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in an airtight fabric to prepare a powder.

1.2 Preparation of tablets

100mg of Militaris cordyceps fermented extract of mixture containing strawberry and pupae

Corn starch 100mg

Lactose 100mg

Magnesium stearate 2mg

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

1.3 Preparation of capsules

100mg of Militaris cordyceps fermented extract of mixture containing strawberry and pupae

Corn starch 100mg

Lactose 100mg

Magnesium stearate 2mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare tablets.

1.4 Preparation of injections

Militaris Cordyceps Fermented Extract 10μg/ml of mixture containing strawberry and pupae

Suitable amount of sterile distilled water for injection

pH Adjuster

It is prepared with the above-mentioned ingredient content per ampoule (2 ml) according to the preparation method of a conventional injection.

1.5 Preparation of liquid

100mg of Militaris cordyceps fermented extract of mixture containing strawberry and pupae

20 g sugar

Iseonghwadang 20g

Lemon flavor

Purified water was added to make a total of 1,00 ml. After mixing the above components according to the manufacturing method of a conventional liquid formulation, a brown bottle is filled and sterilized to prepare a liquid formulation.

Formulation Example 2. Preparation of food

100mg of Militaris cordyceps fermented extract of mixture containing strawberry and pupae

Vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ folic acid

Calcium Pantothenate 0.5 mg

Suitable amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Potassium monophosphate potassium 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The compositional ratio of the vitamin and mineral mixture is a composition suitable for a relatively healthy functional food in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the above components are mixed according to a conventional method for producing a healthy functional food. Then, it can be used in the preparation of a health functional food composition (eg, nutritional candy, etc.) according to a conventional method.

Formulation Example 3. Preparation of beverage

100mg of Militaris cordyceps fermented extract of mixture containing strawberry and pupae

Citric acid 1000 mg

Oligosaccharide 100 g

2 g plum juice

Taurine 1 g

900 ml total by adding purified water

After mixing the above ingredients according to a general health functional beverage manufacturing method, and stirring and heating at 85° C. for about 1 hour, the resulting solution is filtered, obtained by sterilization in a 2 liter container, sterilized and sealed, then refrigerated and stored. It is used for preparing the health functional beverage composition of the present invention.

Although the above composition ratio is a mixture of components suitable for a preference drink in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, country of demand, and usage.

Claims (10)

(S1) preparing a mixture comprising strawberries and pupae; And
(S2) step of obtaining a fermentation product by inoculating the mixture with Cordyceps militaris and fermenting it; And
(S3) extracting the fermentation product with lower alcohols of C ₁ to C ₄ ; Containing, by the method of increasing the production of functional substances of cordycepin and strawberry of Cordyceps of Militaris, the functional substances of the strawberry are cyanidin-3-glucoside, Cinchonine , At least one selected from the group consisting of catechin, epi-gallocatechin, p-Coumaric acid and ellagic acid,
The pupa is contained in 25 to 50% (w/w) per strawberry body weight, a method for increasing the production of functional substances of cordycepin and strawberries of Militaris cordyceps.
According to claim 1,
The fermentation of the step (S2) is characterized in that the solid phase fermentation, the method of increasing the production of functional substances of cordycepin and strawberries of military cordyceps.
delete Cordyceps militaris fermentation product of strawberry and pupa mixture, and the pupae containing 25 to 50% (w/w) of strawberry per live weight of the cordycepin of Cordyceps (Militaris cordyceps) ) And a composition for increasing the production of functional substances of strawberries,
The functional material of the strawberry is cyanidin-3-glucoside, Cinchonine, Catechin, Epi-gallocatechin, p-Coumaric acid ) And 1 or more selected from the group consisting of Ellagic acid.
delete Contains an extract from the extract of the military and cordyceps of the strawberry and pupae, and the pupa contains 25 to 50% (w/w) of strawberries per body weight, and the extract is a lower alcohol extract of C ₁ to C ₄ , Pharmaceutical composition for preventing or treating obesity. The method of claim 6,
The extract,
(S1) preparing a mixture comprising strawberries and pupae;
(S2) step of obtaining a fermentation product by inoculating the mixture with Cordyceps militaris and fermenting it; And
(S3) extracting the fermentation product with lower alcohols of C ₁ to C ₄ ; characterized in that it is prepared, including, pharmaceutical composition for preventing or treating obesity.
delete The method of claim 7,
The lower alcohol in the step (S3), characterized in that 40 to 80% (v / v) ethanol, pharmaceutical composition for preventing or treating obesity.
Contains an extract from the extract of the military and cordyceps of the strawberry and pupae, and the pupa contains 25 to 50% (w/w) of strawberries per body weight, and the extract is a lower alcohol extract of C ₁ to C ₄ , Food composition for preventing or improving obesity.

Images