WO2019050123A1 - Food composition for improving obesity or hyperlipidemia comprising terminalia chebula fruit extract and phyllanthus emblica extract - Google Patents

Abstract

The present invention relates to a food composition for improving weight loss and blood lipids comprising a Terminalia chebula extract and a Phyllanthus emblica extract. The food composition exhibits an excellent effect of inhibiting body fat formation and promoting body fat degradation and an excellent effect of improving blood lipids. Also, the food composition for improving obesity and hyperlipidemia of the present invention exhibits an excellent fat accumulation inhibitory effect in fat cells, a neutral fat degrading effect, and a fat synthase expression inhibitory effect. Moreover, since the present invention relates to a complex composed of a natural extract having no toxicity, no side effects and no harm to a human body, the present invention has an advantage of being closely accessible to consumers and being able to help consumers lead a healthy life.

Description

A food composition for improving obesity or hyperlipemia, comprising an extract of Ganoderma lucidum and an extract of Amara

The present invention relates to a functional food composition for improving obesity or hyperlipidemia, and more particularly to a food composition for reducing weight and improving blood lipid comprising Gadja fruit extract and Amla extract.

Contemporary society has changed the standards of beauty pursued in contemporary society, unlike the previous days when food shortages and infectious diseases prevailed. As the importance of extrovert beauty was emphasized in the days when only the problem of health was considered, It was worth it. Therefore, the slim and sleek body rather than the bulky body is perceived as a target of envy, so the so-called 'diet' for the purpose of preventing or treating obesity has grown so much that the size of the domestic market can not be ignored.

Obesity is the result of excessive body fat accumulation and an increase in the number and size of fat cells due to the imbalance between energy intake and consumption (Cell. 2001. 104: 531-543). The energy in the body is stored in fat cells in the form of triglycerides. When the energy source is depleted, the stored fat is decomposed into free fatty acids and glycerol and used as an energy source. However, over-consumption of energy promotes differentiation of adipocytes and increases the amount of stored fat in the body, which is a direct cause of obesity (Biochem J. 2011. 435 (3): 723-732).

Obesity is not a direct cause of death but it is associated with metabolic diseases such as hypertension, atherosclerosis, stroke, heart attack, and diabetic and hyperlipidemia, and is a factor causing cancer or sleep apnea, with extensive and fatal complications It is also perceived as related to the average life expectancy of humans. In addition, complaints about his own body, anxiety, personality disorder, depression and other mental illness, such as cause the source of all sickness.

Hyperlipidemia is an abnormally increased concentration of cholesterol, triglyceride, phospholipid and free fatty acid in blood. The most directly influencing factors are blood cholesterol and LDL (low density lipoprotein) - cholesterol, Hypercholesterolemia is known to cause atherosclerosis. Atherosclerosis is a clinically important problem because of the accumulation of lipids along the wall of the blood vessels, resulting in decreased blood flow, resulting in ischemic heart disease, angina pectoris and myocardial infarction (Korean J. Food Sci. Technol. 2003. 35: 720- 725). Therefore, attention has been focused on health functional foods as part of therapeutic and preventive measures to lower these lipid levels in the blood.

Methods for relieving obesity include the use of drug therapies such as appetite suppressants, fat absorption inhibitors, energy metabolism accelerators, hormone preparations, gastrectomy, and liposuction. However, the persistence of the therapeutic effect and the increase in body weight during drug withdrawal are problems (Curr Opin Chem Biol 2004. 4 (4): 452-460). At one time, the market for obesity drugs such as Xenical and Reductil was established, but sales were prohibited because it was known to have serious side effects such as vomiting, constipation, gastrointestinal disorders, and cardiovascular disease (Int J Obes Relat Metab Disord. 25 (7): 1095-1099, Lancet, 2007. 369 (9555): 71-77, Obes Res 2000. 8 (6): 431-437).

Pancreatic lipase acts as an important enzyme that degrades triglyceride into 2-monoacylglycerol and fatty acids. Typical pancreatic lipase inhibitors are tetrahydrolipstatin, a derivative of lipstatin derived from streptomyces toxitricini, which is excellent in efficacy to inhibit the degradation of about 30% of the fat ingested, Currently, it is being marketed as a medicinal product, but side effects such as gastrointestinal disorder, hypersensitivity, bile secretion disorder, etc. are present.

The present invention aims to develop and provide an anti-obesity medicament or a food composition having an effect of preventing obesity or reducing body fat derived from a natural product which has no adverse effect even when taken for a long period of time.

The present invention provides a food composition for improving obesity, which comprises an extract of Ganoderma lucidum and an extract of Amara.

Also, the present invention provides a food composition for improving hyperlipemia, which comprises an extract of Ganoderma lucidum and an extract of Amara.

In the food composition of the present invention, the Gadja fruit extract may preferably contain maltodextrin.

In the food composition of the present invention, the Gadja fruit extract or Amla extract may preferably be prepared by using any one selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixture thereof as an extraction solvent as an extraction solvent It may be extracted.

In the food composition of the present invention, the Gadum fruit extract and Amla extract may preferably be prepared by mixing 0.3 to 0.6 parts by weight of Amla extract with 1 part by weight of Gadja fruit extract.

In the food composition of the present invention, it is preferable that the food composition contains 0.01 to 50% by weight of Gamadzu extract and Aramra extract, based on the total weight of the composition.

The complex composed of the Gamadzu fruit extract and the Arama extract of the present invention exerts an effect of inhibiting the production of body fat and accelerating the degradation, and also has an excellent effect of improving blood lipid. More specifically, the food composition of the present invention for improving obesity and improving hyperlipemia exhibits excellent fat cell accumulation inhibitory effect, neutral fat decomposing effect, and inhibitory effect on lipogenesis enzyme expression. In addition, since the present invention is a complex composed of a natural extract having no toxicity, no side effects, and harmless to the human body, it has an advantage of being intimately accessible to consumers and helping a healthy life.

FIG. 1 is a graph showing the results of measurement of the degree of accumulation of intracellular fat in order to examine the effect of the complex of the present invention on fat accumulation in adipocytes.

FIG. 2 is a graph showing an experiment result of measuring glycerol secreted from a cell to confirm the effect of the complex of the present invention on fat decomposition in adipocytes.

FIG. 3 is a graph showing an experiment result of measuring the content of intracellular triglyceride in order to examine the effect of the complex of the present invention on fat decomposition in adipocytes.

FIG. 4 is a graph showing the results of measurement of gene expression levels of intracellular LPL (FIG. 4 a) and FAS (FIG. 4 b) in order to confirm the lipid synthesis inhibitory effect of the complex of the present invention.

FIG. 5 is a graph showing the results of measurement of gene expression levels of intracellular PKA (FIG. 5 a) and HSL (FIG. 5 b) in order to confirm lipolysis promoting effect of the complex of the present invention.

FIG. 6 is a graph showing an experiment result of measuring changes in body weight of an experimental animal in order to examine the effect of the complex of the present invention on body weight of an obese animal model.

FIG. 7 is a graph showing an experiment result in which blood adiponectin was confirmed from an experimental animal in order to confirm the body fat decomposition effect of the composite of the present invention.

FIG. 8 is a graph showing the results of experiments in which the degree of gene expression of LPL (FIG. 8 a) and FAS (FIG. 8 b) was measured from an experimental animal in order to confirm the effect of inhibiting lipid synthesis in the complex of the present invention.

FIG. 9 is a graph showing the results of experiments in which the degree of gene expression of PKA (FIG. 9 a) and HSL (FIG. 9 b) was measured from an experimental animal in order to confirm the lipolysis promoting effect of the complex of the present invention.

FIG. 10 is a graph showing an experiment result of measuring the total fat volume of the abdomen from an experimental animal in order to confirm the effect of reducing the body fat of the complex of the present invention.

FIG. 11 is a graph showing the results of an experiment to determine the neutral lipids in blood from an experimental animal in order to confirm the effect of the composite of the present invention on blood lipid improvement.

FIG. 12 is a graph showing the results of an experiment in which blood total cholesterol (FIG. 12 a) and LDL-cholesterol (FIG. 12 b) were observed from an experimental animal in order to confirm the effect of the complex of the present invention on blood lipid improvement.

The present invention provides a food composition for improving obesity, which has a body fat reducing effect, which comprises a gadu fruit extract and an aroa extract.

Also, the present invention provides a food composition for improving hyperlipemia, which comprises an extract of Ganoderma lucidum and an extract of Amara.

In the food composition for improving hyperlipemia according to the present invention, the hyperlipemia may be a food composition for improving hyperlipemia characterized by an increase in blood LDL (Low Density Lipoprotein) - cholesterol.

As a result of research on new food materials that exhibit anti-obesity effects from natural substances free from toxicity and side effects, the present inventors found that when a combination of Ganoderma lucidum extract and Amaranth extract was used, It was confirmed that the cell differentiation inhibitory effect, fat accumulation inhibitory effect, lipogenic enzyme inhibitory effect, and lipolytic protein regulatory gene expression enhancing effect were exhibited. In addition, it was confirmed that weight loss effect, blood lipids (total cholesterol, triglyceride, LDL cholesterol) were reduced in experimental animals.

In the present invention, the seeds of Ganoderma lucidum are the mature fruit of Ganoderma ( Terminalia chebula Retz.) Belonging to Combfreetaceae . The medicinal terminalia fruit is a medicinal ingredient used in Asia because it is said to be a goose, a giraffe, a scoundrel, a giraffe, and a goose. In oriental medicine, it has a pleasant taste and warm tolerance. It is widely used for the treatment of convergence, hemostasis, chronic laryngitis, laryngeal tuberculosis, intestinal hemorrhage, chronic uterine inflammation and dysentery. It is known that Ganoderma lucidum extract has various physiological activities such as anticancer activity, antidiabetic activity, antimutagenic activity, antimicrobial activity, and prevention of tooth decay (J. Ethnopharmacol. 2002. 81: 327-336, Food Chem. 2002. 40: 527-534; Int. J. Antimicrob. Ag 2001. 18: 85-88).

In addition, Ganoderma lucidum is an important plant in the ancient Indian medicine Ayurveda Medicine. The taste is worn and tangy, salty and sweet, and the temperament is warm and contains five of the six flavors of ayurveda, with a particularly bitter taste. It is also known as Haritaki, one of the three components of Triphala, a medicinal ingredient widely used in India, and is known to have the highest potency as a laxative. Fruits, leaves, and stems are all used as medicines, but mostly mature fruits are harvested and dried in autumn. Let's go out of the room, and it is called Jangchung, which is used for sore throat and pneumonia. It drops the face and chest, removes the old wall, helps digestion, and stops diarrhea. I take the fruit as it is, or roast it in bran, or dip it in boiling water, and steam it, soak it in a weak fire and put it on fire (Buddhist plants in the scriptures.

Gadza contains tannins, ellagic acid, punicalagin, flavonoids, gallic acid, chebulinic acid, which are known to have antioxidant, antibacterial and wound healing properties. acid, and chebulagic acid (Inter. J. Fres. Pharm. Biomed. Sci., 2012. 3 (2): 679-683).

Amla ( Phyllanthus emblica ) is also called "Indian gooseberry" and is also called " Emblica officinalis" in the tinnitus. It is a medium-sized deciduous arboreous tree that grows throughout India and is mainly grown in the Himalayas of India. It is medium sized and the length of small branches is 10 ~ 20cm. The leaves are lemon scent, and the flowers are small and yellowish green. The fruit is a flat ball with a diameter of 2 cm, ripe when it is ripe, greenish-green with a six-piece stripe. It is also one of the three fruits of the widely used formula Triphala, which is called amalaki and contains vitamin C, minerals, amino acids, tannins, rutin, etc. (J. Nat. Orod. 2000. 63: 1507-1510). Amaranthus vitamin C is 20 times more than orange and is very stable in heat, so even if it is exposed to high temperature for a long time, vitamins are hardly destroyed. The vitamin C heat resistance of amla is believed to be derived from the tannin components contained together, and these components show antioxidant, antitumor and anti-inflammatory activity (J. Ethnopharmacology. 2006. 104: 113-118).

On the other hand, in the food composition of the present invention, the Gamadzu fruit extract or the Arama extract is preferably extracted with water as an extraction solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, or a mixture thereof . In addition, the above-mentioned Gadja fruit extract or Amara extract may be extracted using, for example, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction. The extract of the present invention may be in the form of a powder obtained by lyophilization or hot-air drying, including a concentrate obtained by extraction, filtration and concentration under reduced pressure or vacuum concentration.

In the food composition of the present invention, the Gadja fruit extract may preferably contain maltodextrin. Maltodextrin can be added and used to make an extract from the extract of Gadja fruit through spray drying.

On the other hand, the food composition of the present invention is preferably prepared by mixing 0.3 to 0.6 parts by weight of Amla extract with 1 part by weight of Gadja fruit extract. More preferably, it is mixed with 7 parts by weight of Gramineae extract and 3 parts by weight of Amla extract.

On the other hand, in the food composition of the present invention, it is preferable that the Gamadzu fruit extract and Amla extract are contained in an amount of 0.01 to 50% by weight based on the total weight of the composition.

Meanwhile, the food composition of the present invention is preferably any one selected from meat, cereal, caffeinated beverage, ordinary beverage, jelly, noodle, gourmet, vitamin complex, and other health supplement foods, but is not limited thereto .

In addition, the food composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in food manufacture. The carrier, the negative active material and the diluent are, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinyltolydone, water, honey, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, it is prepared using a diluent or excipient such as a filler, a weighting agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. which is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have.

In addition, the food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloids, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain natural fruit juice and flesh for the production of fruit drinks or vegetable drinks. These components may be used independently or in combination.

Hereinafter, the structure of the present invention will be described in detail with reference to the following examples and experimental examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.

[Preparation Example 1: Preparation of fruit extract of Ganoderma lucidum]

1 kg of dried gherkin was put in a bowl and 5 L of water was added and extracted at 40 ° C for 3 hours. Thereafter, the mixture was filtered through a 10-mesh sieve, and then maltodextrin was added to 10% of the final powder, followed by spray drying to prepare a ghattique fruit extract powder to be used in the present invention.

[Preparation Example 2: Preparation of Amla Extract Powder]

1 kg of Amaranth Fruit was dried and then ground, and 300 g of the powder was placed in a bowl and 3 L of water was added thereto. The mixture was repeatedly extracted three times at 60 ° C for 6 hours to obtain Amara Fruit Extract. Thereafter, the mixture was filtered under reduced pressure through a filter paper (whatman no. 1, England), concentrated under reduced pressure at 60 ° C in a vacuum rotary condenser, and spray dried to prepare an extract powder for use in the present invention.

[Example 1: Preparation of composite material]

The gherkin extract powder and amla extract powder prepared in Preparation Examples 1 and 2 were blended in a weight ratio as shown in Table 1 to prepare a composite.

Weight ratio of extract powder of Ganoderma lucidum and Amara extract powder

	Garnet extract powder (% by weight)	Amla extract powder (% by weight)
Example 1	70	30

[Experimental Example 1: Confirmation of Effect of Complex of the Present Invention on Fat Accumulation in Adipocyte Differentiation]

In this Experimental Example, the effect of the single extract prepared in Preparation Examples 1 and 2 and the complex prepared in Example 1 on fat accumulation in adipocyte differentiation was examined.

The preadipocytes, 3T3-L1 preadipocytes, were pretreated with 1% penicillin-streptomycin, 1% L-glutamine, 1% sodium pyruvate, and cultured in a high-glucose Dulbeco's modified Eagle's medium (DMEM) containing 1% NEAA mixture at 37 ° C and 5% CO ₂ . When the cells were adhered to the bottom of the flask at a density of 80% or more as a single layer, the cell surface was washed with a PBS solution, and 0.25% trypsin-EDTA was added, followed by incubation in an incubator for 3 minutes Cells were isolated. Cells were then collected by centrifugation at 1,600 rpm for 5 minutes using a centrifuge (GYROZEN 416G). These cells were treated with 10% fetal bovine serum and 1% penicillin-streptomycin, 1% L-glutamine, 1% hepes, 1% NEAA mixture, 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), adipogenic cocktail (MDI solution), and insulin were added to DMEM culture containing gentamycin 10 μg / mL) and dexamethasone (DEX, 1 μM) were mixed to induce differentiation. The differentiation period lasted for a total of 9 days, and the same culture medium was exchanged every day for the first 3 days of differentiation. The medium was exchanged every day for 3 days with medium supplemented with 10% FBS containing insulin (10 / / mL) only for 3 days. During the 3 days after the differentiation, DMEM culture medium containing 10% FBS I exchanged it every day.

Oil red O staining was performed to measure the effect of the complex of the present invention on adipocyte differentiation in the process of differentiation of adipocytes into adipocytes. In other words, the cells were differentiated according to the cell culture and differentiation induction method and treated with 80 占 퐂 / mL of Preparation Examples 1 to 2 and Example 1, respectively, at the beginning of differentiation. DMEM culture medium and extracts containing 10% FBS containing adipogenic cocktail were inoculated daily for 24, 48, and 72 hours, and oil red O staining was performed at the end of differentiation (day 9). The culture medium was removed and washed twice with PBS solution. The PBS solution was completely removed, and 10% formalin was added and fixed at room temperature for 5 minutes. Formalin was then removed and fresh 10% formalin was added and fixed at room temperature for 2 hours or more . After that, formalin was removed and 60% isopropanol was immediately added to remove the flask. The flask was completely dried, and oil syrup was stained for 60 minutes by adding oil red O solution. After dyeing, the plate was washed 4 times with distilled water, and 100% isopropanol was added to elute the oil red o dye. The absorbance was measured at 520 nm using an ELISA reader (Molecular Devices, USA). The percentage of fat globules accumulated in the degree of staining in Production Examples 1 to 2 and Example 1 was determined as 100% in the group (control group) differentiated into adipocytes without any treatment. The results are shown in FIG. 1, and the significant differences between the experimental groups were analyzed using ANOVA and the significance was evaluated as p <0.05.

As a result of the experiment, it was confirmed that the fat accumulated in each of Production Examples 1 to 2 and Example 1 group was smaller than that of the control group. In particular, it was confirmed that the fat accumulation amount in the adipocyte was smaller in the group of Example 1 than that of the single extracts of Preparation Examples 1 and 2.

[Experimental Example 2: Confirmation of effect of complex of the present invention on intracellular lipolysis]

In this experiment, the content of triglyceride in adipocytes and glycerol, which is increased during lipolysis, were measured in order to confirm the effect on fat decomposition in adipocytes.

In order to determine the content of glycerol, glycerol phosphate oxidase-free glycerol reagent (TRINDER) was applied according to the method of McGowan et al. (Clin Chem. 1988: 29: 538-542) ) Was used to measure the glycerol content in the culture. The culture solutions were collected at the latter stage of differentiation (day 9) by treating Production Examples 1 to 2 or Example 1 with 80 占 퐂 / mL for 72 hours. 1 ml of the culture and 800 μl of free glycerol reagent were mixed and reacted on a hot plate at 37 ° C for 10 minutes and then absorbance was measured at a wavelength of 540 nm. The glycerol content was measured by making a standard curve using free glycerol as a standard reagent and the protein content was measured using BSA as a standard reagent.

To measure the neutral lipid content, 'Triglyceride Quantification colorimetric kit (BioVision Inc. Milpitas Blvd., Milpitas, CA USA)' was used. Adipocytes were treated with 80 μg / mL of Preparation Example 1 or 2 or Example 1 and adipocytes were harvested 72 hours later and the cells were lysed with distilled water containing 5% NP-40. The mixture was boiled at 80 to 100 ° C for 2 to 5 minutes, rapidly cooled, centrifuged, and the supernatant was separated and diluted 10 times with distilled water to prepare a test solution. The test solution and the standard reagent were dispensed into each well of a 96-well plate, and the total volume was adjusted to 50 μL with assay buffer. Then, a lipase reagent was added to each well, And the mixture was mixed well. Then, the mixture was allowed to stand at room temperature for 20 minutes. After 20 minutes, 50 μL of the reaction mix was dispensed every well, and the mixture was allowed to stand at room temperature for 30 to 60 minutes and absorbance was measured at a wavelength of 570 nm.

The results are shown in FIG. 2 and FIG. 3, and the significant difference between the experimental groups was analyzed by ANOVA and the significance was evaluated as p <0.05.

As a result of the measurement of glycerol shown in FIG. 2, the glycerol secretion amount was higher in the preparation examples 1 to 2 and the example 1 than in the control, and it was found that the compound was more effective in lipolysis due to the higher glycerol secretion amount at the same concentration I could.

In the case of the intracellular neutrophils identified in FIG. 3, low neutral lipid contents were observed in both Preparation Examples 1 and 2 and Example 1, especially in the case of complexes, at the same concentration The low neutral lipid content was effectively confirmed.

[Experimental Example 3: Measurement of Gene Expression of Lipolytic Enzyme and Lipolytic Enzyme in Adipocyte Complex of the Present Invention]

In the present experimental example, the complex of the present invention contains lipid peroxidase (LPS), fat-degrading enzyme PKA (protein kinase A), HSL (hormone sensitive lipase), fatty acid synthetase (FAS) Were analyzed by RT-PCR (Real-time PCR).

Adipocytes were treated with 80 μg / mL of each of Production Examples 1 to 2 or Example 1, and adipocytes were collected after 72 hours. Extraction of total RNA from adipocytes was performed using the RNeasy lipid tissue mini kit (QIAGEN sciences, Maryland, USA) and the manufacturer's method. cDNA synthesis was performed with iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA, USA) using 1 μL of RNA per sample. SUBR Green (iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc., CA, USA)) was used to measure the expression of the genes and 'one step real-time PCR (Applied biosystems)' was used. The PCR product size of all genes was set at about 100 bp and the melting temperature was designed around 54 ℃. The nucleotide sequences of the PCR primers for each gene are shown in Table 2. In real-time PCR, 1 μL of cDNA, 10 μL of 2X SYBR mix, and 1 μL of 100 pmol / μL of primer were added to the total 20 μL, respectively, and the rest were filled with distilled water. For all genes, the PCR amplification steps were as follows, and the amplification cycle was performed for 40 cycles. Denaturation at 95 ° C for 2 seconds, annealing at 60 ° C for 6 seconds and extension at 72 ° C for 10 seconds for a hot start at 95 ° C for 10 minutes, denaturation of the amplification step at 95 ° C for 2 seconds, , And fluorescence values were recorded after each cycle extension. Melting curve analysis was performed to confirm the specificity of the primer after all cycles were completed. The analysis of the results was analyzed by 'one step system software v2.1' provided by 'applied biosystems'.

The results are shown in FIG. 4, and the significant difference between the experimental groups was analyzed by ANOVA and the significance was evaluated as p <0.05.

The nucleotide sequence of the PCR primer for each gene

Genes (Gene)	Primer sequences
LPL	F 5'-CAA GAT TCA CTT TTC TGG GAC TGA-3 '
	R 5'-GCC ACT GTG CCG TAC AG AGA-3 '
FAS	F 5'-GAA GTG TCT GGA CTG TGT CAT TTT TAC-3 '
	R 5'-TTA ATT GTG GGA TCA GGA GAG CAT-3 '
PKA	F 5'-TTC ACT CAG AGC CGC TTA AGG-3 '
	R 5'-CTC TAT CCG AAG TAT TGC TGC TAC CT-3 '
HSL	F 5'-CAC TAG TCC CTC CCC CAG TTT-3 '
	R 5'-AGC TGG CAC AGC AGG TCT GT-3 '
GAPDH	F 5'-CAT GGC CTT CCG TGT TCC TA-3 '
	R 5'-GCG GCA CGT CAG ATC CA-3 '

As a result of measurement of LPL and FAS expression levels shown in Fig. 4, the expression level of the gene measured in the general group was set to 1, with general preadipocytes not inducing differentiation as a general group. The expression level of LPL and FAS in the adipocytes of the control group after induction of differentiation was increased, and the expression levels of the preparation examples 1 to 2 or the example 1 group were lower than those of the control group. From these results, it was confirmed that the complex showed lipid synthesis and lipid accumulation inhibitory effect in adipocytes, and the effect of each complex was higher than that of each single extract.

As shown in FIG. 5, the amount of expression of PKA and HSL was measured. As a result, the amount of gene expression measured in the general group was set to 1, with general preadipocytes that did not induce differentiation. In the case of PKA, the amount of expression in the control group after induction of differentiation was remarkably lower than that in the general group, but it was confirmed that the expression levels were increased by Production Examples 1 to 2 or Example 1. In the case of HSL, the expression level in the control group was comparable to that in the general group, but it was confirmed that the expression level was increased by Production Examples 1 to 2 or Example 1. From these results, it was confirmed that the complex showed the lipolytic effect by increasing the amount of fat lipase in the adipocyte, and it was found that the complex is more effective at the same concentration than the effect of each single extract .

[Experimental Example 4: Confirmation of effect of the complex of the present invention on weight change of obese-induced mice]

In this experiment, we investigated the effect of compound on weight change of high fat diet induced obese mice.

Experimental animals were fed with a normal diet (AIN 93G; D10012G) in the rodent's breeding room for one week by purchasing 6 weeks old male C57BL / 6J mice (n = 56) mice from Charles River laboratory animal company After adaptation, normal subjects were observed during adaptation period and healthy individuals were selected and grouped by randomization. Seven animals were divided into 5 groups and breeding was carried out for 12 weeks. The classification of experimental group and the composition of empirical formula are shown in Table 3.

Classification of experimental group and empirical formula

	(n = 7 / group)
Experimental group	Empirical formula
General group	The general diet (AIN93G)
Control group	High fat diet (60% fat)
Preparation Example 1 150 mg / kg	High fat diet (60% fat) + Garnet fruit extract 0.12%
Preparation Example 2 150 mg / kg	High fat diet (60% fat) + Amla extract 0.12%
Example 1 150 mg / kg	High fat diet (60% fat) + complex 0.12%

The incubation environment was such that the light cycle was maintained at 12 hours at a temperature of 23 ° C and a humidity of 50%. Diet and drinking water were freely consumed during the experiment, and body weight and dietary intake were measured at regular intervals twice a week. The food efficiency ratio (FER) was calculated by dividing the weight gain during the entire experimental period from the feeding day to the sacrifice day by the dietary intake during the experimental period. The measured body weight results are shown in Table 4, and the weight increase amount is shown in FIG. ANOVA analysis was used for the significant difference between the experimental groups, and significance was evaluated as p <0.05.

Weight measurement results

	Initial bodyweight (g)	Final bodyweight (g)	Weight gain (g / 12weeks)	Food intake (g / day)	FER
General group	22.33 ± 1.021 a	33.51 ± 3.165 c	11.19 ± 2.417 d	3.28 ± 0.461 a	2.89 ± 0.625 d
Control group	22.47 ± 1.267 a	50.10 + - 0.316 a	27.03 + - 1.417 a	3.04 ± 0.313 bc	7.32 ± 0.528 a
Production Example 1	22.07 ± 1.370 a	42.33 ± 6.333 b	23.40 ± 2.825 b	3.10 ± 0.460 ab	5.66 ± 2.126 b
Production Example 2	22.77 ± 1.083 a	42.70 + - 4.008 b	20.23 ± 3.354 bc	2.79 ± 0.289 d	5.52 + 1.790 bc
Example 1	22.93 + 0.709 a	42.30 ± 1.200 b	19.13 ± 2.003 c	2.88 ± 0.287 cd	7.03 ± 1.001 ab

As a result, obesity - induced control group showed higher body weight than normal group. In comparison with the control group, the weight was lower in the preparation examples 1 to 2 and the example 1 group, and the effect was more effective when the composition was used as compared with the effect of each single extract. From the above results, it can be confirmed that the complex of the present invention shows excellent weight loss effect.

[Experimental Example 5: Confirmation of Effect of Complex of the Present Invention on Blood Adiponectin Changes in Obese-Induced Mice]

After the feeding of experimental animals was completed, the animals were fasted for 12 hours before sacrifice and then anesthetized with isofluorane and blood was collected through the hepatic vein. Blood was separated by centrifugation (14,000 rpm, 20 minutes, 4 ℃) into serum and plasma and analyzed.

Adiponectin was separated and analyzed using the R & D system Quantikine ELISA kit (Bio-Techne co., Minneapolis, MN, USA). Serum samples were diluted 2000 fold with 'calibrator diluent RD5-26' reagent and used in the experiment. 50 μL of assay diluent RD1W was dispensed into each well of a 96-well plate. 50 μL of each sample and standard reagent was dispensed into each well, followed by gentle shaking for 1 minute. After coating on a 96-well plate, the plate was allowed to stand at room temperature for 3 hours, and 400 μL / well was washed with wash buffer for 5 times in total. Then, 100 μL of adiponectin conjugate reagent was dispensed into each well and allowed to stand at room temperature for 1 hour. Then, the plate was washed 5 times with wash buffer, and the substrate solution (100 μL) of the substrate solution was added to each well and left at room temperature for 30 minutes while blocking light. Each 100 μL stop solution was dispensed into each well and absorbance was measured at a wavelength of 450 nm. The measurement results are shown in FIG. 7, and the significant differences between the experimental groups were analyzed using ANOVA and the significance was evaluated as p <0.05.

7, the content of adiponectin in blood was lowered in the obesity-induced control group as compared with that in the general group, and the content of the adiponectin in the group of Example 1, which was a single extract, Production Examples 1 to 2, Which was significantly increased.

[Experimental Example 6: Measurement of gene expression of lipogenic enzymes and lipolytic enzymes in adipose tissue of obese-induced mice of the present invention]

After the experiment was completed, the blood was collected on the day of sacrifice and immediately opened. The organ and fatty tissue were removed, washed with physiological saline, the water was removed with a filter paper, and the weight was measured. Respectively.

RT-PCR was performed to confirm the expression of lipid metabolism enzymes in adipose tissue. For each group, 100 mg of visceral fat tissue stored in 1 mL of QIAzol lysis reagent was homogenized and 200 μL of chloroform reagent was dispensed and vortexed for 15 seconds. After centrifugation at 12,000 g at 4 ° C for 15 minutes, the supernatant was transferred to a new tube and RNA was extracted using the RNasy lipid tissue mini kit and manufacturer's instructions. cDNA synthesis was performed with iScript cDNA synthesis kit using 1 μL of RNA per sample. SYBR Green was used to measure the expression of genes and 'one step real-time PCR' was used. The PCR product size of all genes was 100 bp and Tm was designed around 54 ℃. The nucleotide sequences of the PCR primers for each gene are shown in Table 2. &lt; tb &gt; &lt; TABLE &gt; In a real-time PCR reaction, 1 μL of cDNA, 10 μL of 2X SYBR mix, and 1 μL of 100 pmol / μL of primer were added to a total of 20 μL, and the remainder was filled with distilled water. For all genes, the PCR amplification steps were as follows, and the amplification cycle was performed for 40 cycles. Denaturation at 95 ° C for 2 seconds, annealing at 60 ° C for 6 seconds and extension at 72 ° C for 10 seconds for a hot start at 95 ° C for 10 minutes, denaturation of the amplification step at 95 ° C for 2 seconds, , And fluorescence values were recorded after each cycle extension. Melting curve analysis was performed to confirm the specificity of the primer after all cycles were completed. Analysis of the results was done by 'one step system software v2.1' provided by 'applied Biosystems'.

The results are shown in FIG. 8 and FIG. 9, and the significant differences between the experimental groups were analyzed using ANOVA and the significance was evaluated as p <0.05.

As a result of measurement of LPL and FAS expression levels shown in FIG. 8, the amount of gene expression measured in the general group was set to 1, using general mice not inducing obesity as a general group. The expression levels of LPL and FAS in adipose tissues of the obesity-induced control group were increased, and the expression levels of Preparation Examples 1 to 2 or Example 1 were lower than those of the control group. From these results, it was found that the complex showed lipid synthesis and lipid accumulation inhibition in adipose tissue, and the effect of each complex was higher than that of each single extract.

As a result of confirming the expression levels of PKA and HSL shown in Fig. 9, the gene expression level measured in the general group was set to 1, using general mouse as a general group. In the case of PKA, the amount of expression in the control group was significantly lower than that in the general group, but it was confirmed that the expression level was increased by Production Examples 1 to 2 or Example 1. In the case of HSL, the expression level in the control group was higher than that in the general group, and the expression levels of the extracts of Preparation Examples 1 and 2 were similar to those of the control group. However, the amount of expression in the complex of Example 1 was significantly increased. From these results, it was confirmed that the complex showed the lipolytic effect by increasing the amount of fat lipid degradation enzyme in fat tissues of obese-induced mice. The effect of each extract was higher at the same concentration than the effect of each single extract .

[Experimental Example 7: Abdominal total fat volume CT measurement of a subject's obesity-induced mice]

The abdominal micro-CT of the animal was photographed by a specialist at a nortus laboratory, and the entire fat volume was photographed with Direct-TV. Direct-BV (direct- BV) were used to quantify visceral fat volume. The measurement results are shown in FIG. 10, and the significant differences between the experimental groups were analyzed using ANOVA, and the significance was evaluated as p <0.05.

As a result, the abdominal total fat volume of the obesity-induced control group was significantly increased compared to the abdominal total fat volume of the general group, and the abdominal total fat volume of the group of the recipients of Production Example 1 to 2 or Example 1 was significantly lower than that of the control group I could confirm. In addition, it was confirmed that the amount of decrease in the amount of the compound when ingested was significantly increased than that of the single extract.

[Experimental Example 8: Confirmation of Effect of Complex of the Present Invention on Blood Lipid Changes in Obese-Induced Mice]

In this experiment, blood neutrality, total cholesterol, and LDL-cholesterol ratio were measured in order to examine the effect of the complex ingestion of the present invention on blood lipid.

Neutral lipid, total cholesterol and LDL-cholesterol were analyzed using an enzyme assay kit (BioVision Inc. Milpitas Blvd., Milpitas, CA, USA) using the serum isolated from the experimental animals. In the case of neutrophil, 5 μL of each serum sample and standard reagent is dispensed into a 96-well plate, the total volume is adjusted to 50 μL with assay buffer, and then the lipase reagent is added to each well well), mixed well and left at room temperature for 20 minutes. After 20 minutes, 50 μL of the reaction mix was dispensed every well, left at room temperature for 30 to 60 minutes, and absorbance was measured at a wavelength of 570 nm.

For total cholesterol, the entire serum sample was used. For LDL-cholesterol, the serum was diluted 1: 1 with 2X precipitation buffer, left at room temperature for 10 minutes, and then centrifuged to separate the remaining sample from the supernatant. Respectively. Separate samples and standard reagents were dispensed into each well of a 96-well plate in 5 μL increments, and the total volume was adjusted to 50 μL with assay buffer. The reaction mix was then dispensed every 50 μL per well and incubated at 37 ° C for approximately 60 minutes before absorbance at 570 nm. The measurement results are shown in FIGS. 11 and 12, and the significant differences between the experimental groups were analyzed using ANOVA and the significance was evaluated as p <0.05.

In the case of the blood neutrophil quality confirmed in FIG. 11, a much higher content was observed in the obesity-induced control group than in the general group, and the content was similar to that in the general group in the groups in which Preparation Example 1 to 2 or Example 1 was consumed. The results of this experiment showed that the neutral lipids in the blood could be reduced to a similar level to that of the normal group when the complex was ingested.

In the case of total cholesterol and LDL-cholesterol identified in Fig. 12, the content was much higher in the obesity-induced control group than in the general group, and it was confirmed that the groups were significantly reduced in comparison with the control group in the groups in which Production Example 1 to 2 or Example 1 was consumed , And in the case of Production Example 2 or Example 1, it was reduced to a level similar to that of the general group. The results showed that both total cholesterol and LDL-cholesterol were reduced when the complex was consumed.

[Example 2: Production of a food composition having an effect of inhibiting the production and accumulation of body fat and improving blood lipid]

In this Example, a food composition having the effect of inhibiting the production and accumulation of body fat and inhibiting blood lipid was prepared as follows.

(1) Manufacturing of wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then the mixture was prepared as a powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame seeds and perilla seeds were each steamed and dried by known methods, and were then distributed and pulverized to prepare powder having a particle size of 60 mesh. Thereafter, 30% by weight of brown rice, 15% by weight of yulmu, 20% by weight of barley, 9% by weight of glutinous rice, 7% by weight of perilla seeds, 8% by weight of black soybeans, 7% by weight of black sesame seeds, By weight and 0.5% by weight of sulfuric acid were mixed to prepare an electric wire.

(2) Health drinks manufacturing

0.0001 wt.% Of niacinamide, 0.0001 wt.% Of sodium riboflavin hydrochloride, 0.0001 wt.% Of pyridoxine hydrochloride, 0.001 wt.% Of inositol, 0.002 wt.% Of orthoacetic acid, 98.7362 wt. 1% by weight of Example 1) was blended to prepare a health drink.

(3) health supplement manufacturing

, 50% by weight of the complex (Example 1), 16% by weight of guar gum enzyme hydrolyzate, 0.01% by weight of vitamin B1 hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, And 1.85% by weight of corn starch were blended to prepare a refillable health supplement.

Claims (5)

1. 70% by weight of Ganoderma extract and 30% by weight of Amla extract.
2. A mixture of 70% by weight of Ganoderma extract and 30% by weight of Amla extract.
3. 3. The method according to claim 1 or 2,

   The Gramineous fruit extract or Amara extract may be obtained by, for example,

   Wherein the extractant is extracted using water or any one selected from the group consisting of lower alcohols having 1 to 4 carbon atoms or a mixture thereof as an extraction solvent.
4. Extracting the fruit of Ganoderma lucidum by hot water and spray drying it to obtain an extract of Ganoderma lucidum;

   Extracting amaranth fruits with hot water and spray-drying to obtain an extract of Amara; And

   And a mixture of the gherkin extract powder and the amaranth extract powder at a weight ratio of 7: 3 to prepare a composite.
5. Extracting the fruit of Ganoderma lucidum by hot water and spray drying it to obtain an extract of Ganoderma lucidum;

   Extracting amaranth fruits with hot water and spray-drying to obtain an extract of Amara; And

   And a mixture of the gherkin extract powder and the amaranth extract powder at a weight ratio of 7: 3 to prepare a composite material.

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