KR20140029842A - Functional foods comprising the extract of gelidium elegans - Google Patents

Abstract

The present invention relates to a manufacturing method of extract of Gelidium elegans having an anti-obesity activity and an anti-oxidation activity, which comprises a process of drying Gelidium elegans with far infrared rays, pulverizing and extracting the Gelidium elegans with 70% (v/v) ethanol at 25°C for 24 hours; extract of Gelidium elegans manufactured thereby and having an anti-obesity activity and an anti-oxidation activity; a composition having an anti-obesity activity and an anti-oxidation activity including the extract of Gelidium elegans or a fraction thereof; functional food having an anti-obesity activity including the composition; and functional food having an anti-oxidation activity including the composition. The extract of Gelidium elegans of the present invention is able to be usefully used in suppressing obesity caused by oxidative stress as the extract of Gelidium elegans is excellent in a fat accumulation reduction effect and an anti-oxidation activity.

Description

{Functional Foods Comprising the Extract of Gelidium elegans}

The present invention relates to an extract of Opuntia ficus-indica and a functional food containing the same. More specifically, the present invention relates to a method for producing a polyphenol polyol having an anti-obesity and antioxidative activity, comprising a step of drying the pine forest using far infrared rays, followed by pulverization and extraction with 70% (v / v) ethanol at 25 ° C for 24 hours An antiobesity and antioxidant activity composition comprising the extract of Staphylococcus epidermidis extract or the fraction thereof having antioxidant activity and antiobesity activity produced by the method, a functional food having an anti-obesity activity comprising the composition And a functional food having an antioxidative activity comprising the composition.

According to the World Health Organization (WHO), the worldwide obesity population has more than doubled since 1980, and by 2008, 1.5 billion adults over the age of 20 are reported to be overweight. Recently, the obesity population is continuously increasing due to economic growth and westernized diet. Obesity is a major cause of metabolic syndrome and causes various diseases such as type 2 diabetes, cardiovascular disease and hypertension (Eckel RH et al., Lancet, 16-21; 365 (9468): 1414-28, 2005), and in recent years, cancer (see Basen-Engquist et al., Curr Oncol Rep. 13 (1): 71-6, 2011), chronic kidney disease (see Abrass CK JASN 15 (11) 2772, 2011), and asthma (Dixon et al., J. Asthma. 48 (7): 707-13, 2011).

These obesity is caused by excessive accumulation of triglyceride due to excessive nutrition of the fat cells, which is increased by the gene that differentiates adipocytes and lipid droplet accumulates in the cytoplasm. Usually, adipocytes are formed by differentiation of adipose precursor cells into adipocytes, in which a transcription factor called PPAR (peroxisome proliferator activated receptors) -γ C / EBP (CCAAT enhancer binding protein) -α is involved, Progressively induces differentiation and affects lipid accumulation in adipocytes (J.Auwerx et al., J Mol. 74: 347-352, 1996).

PPAR-γ is known to be a key factor in adipocyte differentiation and lipid accumulation. It is expressed by binding to the PPRE (peroxisome proliferator response element) of DNA by forming a complex with RXR (retinoid X receptor) (see Siersbaek R et al., FEBS letters. 584 (15) 3242-3249, 2010). In particular, PPAR-γ interacts with C / EBP-α in adipocyte expression and fat accumulation, and both expresses a lipid-specific gene, FABP4, acting as a transcription factor. FABP4 is a protein that transports fatty acids. As expression increases, the accumulation of fat in the cells increases (Lefterova et al., Trend in Endocrinology and metabolism. 20 (3) 107-114, 2008). Therefore, as the expression of PPAR-γ, C / EBP-α and FABP4 decreases, lipid accumulation in adipocytes can be suppressed.

Recent studies have shown that effective removal of active oxygen as well as genes involved in lipid differentiation and accumulation may contribute to obesity. For example, glucose entering the cell produces active oxygen during the conversion to energy, which is produced more as fat accumulation increases. The resulting active oxygen causes an imbalance in lipid metabolism, resulting in more fat accumulation and active oxygen (Furukawa et al., JCI 114 (12): 1752-1761, 2004). The occurrence of such active oxygen increases the fat accumulation and vicious circulation. Such repetition not only worsens the obesity but also causes secondary diseases caused by obesity, so that the active oxygen generated in the adipocytes is effectively eliminated Is important for inhibiting and treating fat accumulation.

In this situation, there has been a growing interest in the development of functional ingredients which are capable of simultaneously controlling the gene responsible for differentiating adipocytes and accumulating lipids and the active oxygen, while being safe enough to be commonplace.

Accordingly, the inventors of the present invention have made efforts to develop a functional ingredient having excellent safety to such an extent that it exhibits anti-obesity and antioxidative activity, and as a result, it has been found that the ethanol extract of the mugwort can be safe and common And thus the present invention has been completed.

As a result, the first object of the present invention is to provide a process for producing an anti-obesity and antioxidant activity, which comprises a step of drying the bean curd by using far-infrared rays, followed by pulverization and extraction with 70% (v / v) ethanol at 25 ° C for 24 hours And to provide a method for producing an extract of Mugwort.

A second object of the present invention is to provide a mugwort extract having anti-obesity and antioxidant activity, which is prepared by the above-mentioned method.

A third object of the present invention is to provide a composition having anti-obesity and antioxidative activity, comprising the extract or the fraction thereof.

A fourth object of the present invention is to provide a functional food having an anti-obesity activity comprising the composition.

A fifth object of the present invention is to provide a functional food having antioxidative activity comprising the composition.

The present inventors have succeeded in extracting the extracts with 0, 30, 50, or 70% (v / v) ethanol to obtain a dried product by drying natural oriental fungus, which is dominant in Korea, by natural drying, hot air drying or far infrared ray drying method, And then compared them. As a result, it was found that when the far-infrared drying method was used rather than the natural drying method or the hot-air drying method, the extraction yield was relatively high and the ethanol extraction concentration was low, It was analyzed that the polymer polysaccharide component contained in large amount in the waterweed was high solubility in water.

As a result of analyzing the content of total flavonoids and phenol compounds contained in the extract, when the far infrared ray drying method was used instead of natural drying or hot air drying, the content of total phenolic compounds and flavonoids increased, and the concentration of ethanol increased The contents of total phenolic compounds and flavonoids were increased.

As a result of the analysis of the antioxidative activity of the extract, there was no significant difference in antioxidative activity according to the drying method. As the concentration of ethanol increased, the antioxidative activity of the extract increased, And flavonoids. In particular, it was confirmed that the antioxidative activity was highest when the extract was dried using far infrared rays and extracted with 70% ethanol at 25 ° C for 24 hours.

Accordingly, the mugwort extract of the present invention can be prepared by drying the mugwort extract using far-infrared rays and then pulverizing it and extracting it with 70% (v / v) ethanol at 25 ° C for 24 hours. In this case, the mugwort used is not particularly limited, but it is preferable to use a fine genus Gelidium elegans.

On the other hand, the inventors of the present invention studied the extracts of the fine Chrysanthemum morifolium obtained by using 70% ethanol, treating each of the first fractions with 50% methanol, 75% methanol, 100% methanol or chloroform, Analysis of the antioxidative activities of the fractions showed that the first fraction did not show cytotoxicity and that the 50% methanol eluate showed the most excellent antioxidative activity among the first fractions.

It was confirmed that the 50% methanol eluate showed the best antioxidative activity. Therefore, the 50% methanol eluate was treated with 5%, 10%, 25%, 50% and 100% methanol to obtain the respective second fractions Analysis of the antioxidative activity of each of the secondary fractions showed that the secondary fraction did not show cytotoxicity and that the 50% methanol eluate showed the best antioxidative activity among the secondary fractions.

In addition, in the cells treated with the second fraction, the 50% methanol eluate, the expression amount of the gene encoding the protein that induces the production of ROS decreases, the expression amount of the gene encoding the protein involved in the ROS cleavage increases, Genetic analysis results were confirmed once again by Western blot analysis.

Accordingly, the antioxidant composition provided in the present invention may comprise the extract or the fraction of the extract, wherein the extract is dried by far infrared rays, powdered and dissolved in 70% (v / v) ethanol At 25 ° C for 24 hours, and the fraction is adsorbed on RP C18 resin and eluted with 50% methanol. The first fraction or the first fraction obtained is dissolved in RP C18 resin A second fraction obtained by adsorption and elution with 50% methanol, and the like, but the present invention is not particularly limited thereto.

In addition, the food exhibiting the antioxidative effect provided by the present invention may contain the antioxidant composition as an active ingredient. That is, the antioxidant composition provided in the present invention is not particularly limited, but may be selected from the group consisting of saccharides (e.g., monosaccharides, disaccharides, polysaccharides, sugar alcohols), flavors (e.g., tau martin, stevia extract, saccharin, aspartame, Food additives such as food additives, such as food additives, edible electrolytes, flavoring agents, coloring agents, aggravating agents (eg cheese, chocolate etc.), alginic acid, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, And can be produced in the form of food which exhibits antioxidative effects. In this case, the form of the food is not particularly limited. However, it may be added to the food during the production of a variety of food products, including a line, a noodle, a confectionery, a beverage, a meat, a fish, a herb, a soup or a rice, The amount of the antioxidant composition of the present invention to be added to the food to be represented is not particularly limited.

On the other hand, the inventors of the present invention have confirmed whether the primary fraction or the secondary fraction of the above-obtained Angelica gigas Nakai extract has anti-obesity activity.

Specifically, it was confirmed that the 50% methanol eluate in the first fraction exhibited the most excellent anti-obesity activity by treating the primary fraction while differentiating the precursor adipocytes into adipocytes. As a result, the same anti-obesity effect was confirmed using the second fraction derived from the 50% methanol eluate. As a result, it was confirmed that the 50% methanol eluate showed the most excellent anti-obesity activity among the second fractions. In particular, it was confirmed that the 50% methanol eluent used as the second fraction improved the anti-obesity activity as the treatment concentration was increased.

In the cells treated with the first fraction, expression levels of PPAR-gamma, C / EBP-alpha and G6PDH genes, which are involved in fat accumulation, decreased, and in the cells treated with the second fraction, The expression levels of PPAR-gamma, C / EBP-alpha and FABP4 genes are decreased. When the 50% methanol eluate of the second fraction is treated at different concentrations, the PPAR-gamma gene And aP2 gene were decreased in proportion to the treatment concentration.

Accordingly, the anti-obesity composition provided by the present invention may comprise the above-mentioned Extract or the fraction of the Extract. The Extract is dried by far-infrared rays, powdered and dissolved in 70% (v / v) ethanol At 25 DEG C for 24 hours, and the fraction is adsorbed on RP C18 resin and eluted with 50% methanol. The first fraction or the first fraction obtained by this step is dissolved in RP C18 resin , Followed by elution with 50% methanol, but the present invention is not limited thereto.

In addition, the food exhibiting the anti-obesity effect provided by the present invention may comprise the anti-obesity composition as an active ingredient. That is, the anti-obesity composition provided by the present invention is not particularly limited, but may be a sugar (eg, Along with food additives such as vitamins, edible electrolytes, flavors, coloring agents, aggravating agents (eg cheese, chocolate etc.), alginic acid, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, Can be produced in the form of food added to food to exhibit an anti-obesity effect. In this case, the form of the food is not particularly limited. However, it may be added to the food during the production of a variety of food products, including a line, a noodle, a confectionery, a beverage, a meat, a fish, a herb, a soup or a rice, The amount of the antioxidant composition of the present invention to be added to the food to be represented is not particularly limited.

The extract of the present invention has excellent fat accumulation-reducing effect and antioxidant activity, and thus can be useful for suppressing obesity caused by oxidative stress. Any of the anti-obesity agents and antioxidants commonly used in the art may be added to the antioxidant for preventing or treating obesity, which comprises the extract of the present invention as an active ingredient.

FIG. 1 is a graph showing the contents of total phenolic compounds and flavonoids in ethanol extracts of fine true mugwort.
FIG. 2 is a graph showing the DPPH radical scavenging activity of the ethanol extract of Prunus mume.
FIG. 3 is a graph showing the nitrite scavenging activity of the ethanol extract of Thunberger's mushroom.
FIG. 4 is a graph showing the effect of reducing the amount of ROS produced by the ethanol extract of Prunus mume.
FIG. 5 is a graph showing the effect of reducing the amount of ROS produced by the ethanol extract of the true oriental japonica from mature adipocytes.
FIG. 6 is a graph showing the NO production inhibitory activity of ethanol extracts of Mugwort mugwort.
FIG. 7A is a graph showing the cytotoxicity of the first fraction derived from a fine Chrysanthemum morifolium extract. FIG.
Fig. 7B is a graph showing the cytotoxicity of the first fraction derived from Mugwort extract.
8 is a photograph and a graph showing the effect of decreasing the amount of ROS produced in each of the first fraction derived from the ethanol extract of Prunus mume.
FIG. 9 is a graph showing the cytotoxicity of the second fraction derived from a fine Chrysanthemum morbus extract.
FIG. 10 is a photograph and a graph showing the effect of reducing the amount of ROS produced in each of the secondary fractions derived from the ethanol extract of a fine Chrysanthemum morbus.
FIG. 11 is a photograph and a graph showing the effect of decreasing the amount of ROS produced according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of a fine Chrysanthemum morbus.
FIG. 12 is a photograph and a graph showing the effect of reducing the amount of ROS produced according to the treatment concentration of the 70% ethanol extract of a fine Chrysanthemum morifolium.
FIG. 13 is a photograph and a graph showing changes in the expression amount of the NOX4 gene encoding a protein that induces the production of ROS according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of a fine Chrysanthemum overanensis.
14 is a photograph showing the change in the expression level of the Cu / Zn-SOD gene encoding a protein involved in the elimination of ROS according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract Graph.
15 is a photograph and a graph showing changes in the protein level of GPx, which is a protein involved in the elimination of ROS, according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of gentle green beans.
16 is a photograph and a graph showing the change in the protein level of Mn-SOD, which is a protein involved in the elimination of ROS, according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of fine Chrysanthemum morbus.
17 is a photograph and a graph showing a change in the protein level of Cu / Zn-SOD, a protein involved in the elimination of ROS, according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract .
FIG. 18 is a photograph and a graph showing the anti-obesity activity of each primary fraction derived from the ethanol extract of Ganoderma lucidum.
FIG. 19 is a photograph and a graph showing the anti-obesity activity of each secondary fraction derived from the ethanol extract of a fine Chrysanthemum morifolium.
FIG. 20 is a photograph showing fat cells treated with respective secondary fractions derived from a low-boiling ethanol extract.
FIG. 21 is a photograph and a graph showing anti-obesity activity according to the treatment concentration of 50% methanol eluate, which is one of the second fractions derived from the ethanol extract of a fine Chrysanthemum morbus.
FIG. 22A is a photograph showing the change in expression amount of a gene encoding a protein involved in fat accumulation according to the treatment of the first fraction derived from an ethanol extract of a fine Chrysanthemum morifolium.
22B is a photograph showing the change in the expression level of a gene encoding a protein involved in ROS cleavage and production according to the treatment of the first fraction derived from the ethanol extract of Prunus mugwort.
FIG. 23A is a photograph showing the change in the expression level of a gene encoding a protein involved in fat accumulation according to the treatment of the second fraction derived from an ethanol extract of a fine Chrysanthemum indicum.
FIG. 23B is a photograph showing the change in expression amount of a gene encoding a protein involved in ROS cleavage and production according to the treatment of a second fraction derived from an ethanol extract of a fine Chrysanthemum overglaze.
FIG. 23C is a photograph showing the change in expression level of a gene encoding a protein involved in ROS cleavage and production according to the treatment concentration of a 50% methanol extract, which is a second fraction derived from a fine Chrysanthemum orientalis ethanol extract.
24 is a photograph and a graph showing the change in the expression level of the PPAR-gamma gene according to the treatment concentration of the 50% methanol eluate, which is a secondary fraction derived from the ethanol extract of Prunus orientalis.
25 is a photograph and a graph showing changes in the expression level of aP2 (FABP4) gene according to the treatment concentration of a 50% methanol eluate, which is a secondary fraction derived from a fine Chrysanthemum morifolium ethanol extract.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Preparation and activity analysis of a fine Cherrywood extract

Example 1-1: Preparation of fine Cherrywood extract

The domestic dominant species, Gelidium elegans, was dried by natural drying, hot air drying or far-infrared drying, and then powdered to obtain the respective dry powders. 0, 30, 50 or 70% (v / v) ethanol was added to each of the above dried powders and extracted at 25 ° C for 24 hours. The extract was filtered with a Watson filter paper No. 2 (Whatman Ltd., UK) to obtain a liquid component. The liquid component was applied to a vacuum evaporator, concentrated, and lyophilized to obtain a powdery extract And the extraction yields were compared by measuring the amount of soluble solids (see Table 1).

Extraction Yield of Fine Cherry Blossom Extraction order number Drying method Ethanol concentration (%) Extraction yield (%) One
2
3
4
5
6
7
8
9
10
11
12 Natural drying
Natural drying
Natural drying
Natural drying
hot air dry
hot air dry
hot air dry
hot air dry
Far-infrared drying
Far-infrared drying
Far-infrared drying
Far-infrared drying 0
30
50
70
0
30
50
70
0
30
50
70 25.8
21.9
19.6
19.5
31.8
25.7
23.6
21.3
30.9
26.2
23.4
22.3

As can be seen from the results of Table 1 above, it was found that the extraction yield was relatively good when the far-infrared drying method was used, and the extraction yield was relatively higher as the concentration of ethanol used as the extraction solvent was lower than that of natural drying or hot air drying However, it was found that the ethanol concentration had a direct effect on the extraction yield rather than the drying method. The lower the concentration of ethanol, the better the extraction yield. The higher the concentration of ethanol, the higher the yield of the extract.

Example 1-2: Content analysis of total flavonoids and phenol compounds contained in the fine Chrysanthemum morifolium extract

The contents of total flavonoids and phenolic compounds contained in each of the extracts obtained in Example 1-1 were compared.

First, total flavonoid contents were mixed with 1 ml of each extract or solvent fraction and 5 ml of 50% methanol solution, and 10 ml of diethylene glycol, and 1 ml of 1 N sodium hydroxide solution was further added thereto and mixed. Then, the mixture was reacted at 37 DEG C for 1 hour, and the absorbance was measured at 420 nm, and the measured absorbance was calculated to calculate the total flavonoid content. At this time, the absorbance value conversion was performed using a standard calibration curve (Naringin, Sigma Co., USA) (see FIG. 1).

Next, the total phenol content was analyzed by the Folin-Dennis method. Specifically, 1 ml of each extract or solvent fraction solution dissolved in DMSO and 0.5 ml of Folin-Denis reagent were mixed and reacted for 3 minutes. 1 ml of saturated sodium carbonate anhydrous solution and 0.5 ml of distilled water were added The reaction was terminated. The absorbance was measured at 725 nm, and the measured absorbance was converted into the total phenol content. At this time, the Pauline-Dennis reagent was prepared by dissolving 10 g of sodium tungstate, 2 g of phosphomolybdic acid and 5 ml of phosphoric acid in distilled water and refluxing. As a standard solution, tannic acid (Sigma Co., USA) was used.

FIG. 1 is a graph showing the contents of total phenolic compounds and flavonoids in ethanol extracts of fine true mugwort. As shown in FIG. 1, when the far-infrared drying method is used instead of natural drying or hot air drying according to the drying method, the content of total phenolic compound and flavonoid tends to increase relatively. It was also found that the content of total phenolic compounds and flavonoids increased with increasing ethanol concentration. The results of FIG. 1 are in contrast to those of Table 1, which indicates that the phenolic compounds and flavonoids are different from the polymer polysaccharides because they are less soluble in water.

Example 1-3: Antioxidant activity analysis of the extract from the finely chestnut tree

Example 1-3-1: DPPH radical scavenging activity

The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of each extract obtained in Example 1-1 was compared with the electron donating ability (EDA) calculated by the Blosis method (See Blosis. Nature. 26: 1199-1200, 1958).

Specifically, 0.4 ml of each extract and 4 ml of 0.15 M DPPH solution were mixed, reacted at room temperature for 30 minutes, and absorbance was measured at 520 nm. At this time, as the control group, the reaction product obtained by using distilled water instead of the sample was used. Each measured absorbance was substituted into the following equation to calculate DPPH radical scavenging activity.

EDA (%) = [1- (A / B)] x 100

       A: Absorbance of the sample

B: Absorbance of the control group

FIG. 2 is a graph showing the DPPH radical scavenging activity of the ethanol extract of Prunus mume. As shown in FIG. 2, the DPPH radical scavenging activity tends to be relatively increased when the hot-air drying method is used, rather than natural drying or far-infrared drying depending on the drying method. It was also found that the DPPH radical scavenging activity increased with increasing ethanol concentration.

Comparing the results of FIG. 2 with those of Table 1 and FIG. 1, the antioxidant activity was analyzed by the total phenol compounds and flavonoids, not by the polymer polysaccharide contained in the fine Cherrywood extract.

Example 1-3-2: Nitrite scavenging activity

The nitrite scavenging activity was measured by a known method. Specifically, 1 ml of each extract obtained in Example 1-1 and 1 ml of 1 mM NaNO ₂ solution having the same concentration were mixed and titrated with 0.1 N HCl (pH 1.2) to adjust the pH of the reaction solution to 1.2 , And the volume of the reaction solution was titrated to 10 ml. The reaction solution was reacted at 37 ° C for 1 hour. 5 ml of a 2% acetic acid solution and 1% of a Griess reagent (1% sulfanilic acid prepared with 30% acetic acid) (TU-1800, USA) at 520 nm was prepared by adding 0.4 ml of naphthylamine at a ratio of 1: 1 and adjusting the mixture immediately before use. The mixture was allowed to stand at room temperature for 15 minutes and then applied to a UV / Vis spectrophotometer The absorbance was measured and the absorbance was substituted into the following equation to calculate the nitrite scavenging activity of each extract. At this time, as a control group, a reaction product using distilled water instead of a grease reagent was used.

N (%) = [1- (A-C) / B] 100

       N: nitrite scavenging activity

A: Absorbance of 1 mM NaNO ₂ after 1 hour of sample treatment

B: Absorbance of 1 mM NaNO ₂

C: Absorbance of the control group

FIG. 3 is a graph showing the nitrite scavenging activity of the ethanol extract of Thunberger's mushroom. As shown in FIG. 3, there was no difference in nitrite scavenging activity according to the drying method. In addition, nitrite scavenging activity was increased with increasing ethanol concentration, and nitrite scavenging activity was highest when 50% ethanol was used.

Comparing the results of FIG. 3 with those of Table 1 and FIG. 1, it can be confirmed that the antioxidant activity is exhibited by the total phenol compounds and flavonoids, rather than the polymer polysaccharides contained in the fine Cherrywood extract.

Example 2: Further analysis of antioxidant activity of Thin Cherrywood extract

Example 2-1: NBT analysis

From the results of Examples 1-3, it was confirmed that the antioxidative activity of the extract of Chrysanthemum morifolium was shown by the total phenolic compounds and flavonoids contained in the fine Chrysanthemum morifolium extract. Thus, it was confirmed whether or not these extracts could lower intracellular ROS NBT assay.

First, differentiation of adipocytes was induced from precursor adipocytes. Specifically, 3T3-L1 precursor adipocytes obtained from the American Type Culture Collection (ATCC, CL-173) were cultured by a known method. Specifically, the precursor adipocytes were inoculated into a DMEM medium containing 3.7 g / L sodium bicarbonate, 1% P / S and 10% calf serum for 2 days, and the medium was supplemented with a hormone additive (0% FBS , 0.5 mM IBMX, 0.25 [mu] M DEX and 10 [mu] g / ml insulin) for 2 days to differentiate the precursor adipocytes into adipocytes. After the differentiation, the medium was replaced with DMEM medium containing 10% FBS and 10 μg / ml insulin, and the culture medium was changed every two days in the same medium. At this time, each of the extracts obtained in Example 1-1 was treated at a concentration of 100 / / ml while inducing differentiation into the adipocytes, and finally adipocytes treated with each extract were obtained.

Each of the obtained adipocytes was washed twice with PBS, and 0.2% NBT (nitroblue tetrazolium) diluted in PBS was added thereto, followed by reaction at 37 캜 for 90 minutes. After the reaction was completed, the reaction solution was washed with distilled water, dried, treated with 99% acetic acid, and then absorbed at 570 nm. At this time, cells not treated with the extract were used as a negative control, and cells treated with 1 mM NAC (N-acetylcysteine) instead of the extract were used as a positive control.

FIG. 4 is a graph showing the effect of reducing the amount of ROS produced by the ethanol extract of Prunus mume. As shown in FIG. 4, there was no difference in the effect of decreasing ROS production depending on the drying method. In addition, it was confirmed that the activity of decreasing ROS production was increased as the ethanol concentration was increased, and that the activity of decreasing ROS production was the highest when 70% ethanol was used.

Example 2-2: DCFH-DA analysis

The effect of the mugwort extract, which cleaves excess ROS from mature fat cells, was analyzed using the DCFH-DA (2 ', 7'-dichlorodihydro fluroescin diacetate) method. DCFH-DA is converted into DCFH by intracellular estrase through the cell membrane of the cell, and the converted DCFH is converted into fluorescent DCF by reacting with the accumulated ROS in the cell, and the converted DCF is detected by fluorescence microscope and And can be measured using a fluorescence detector.

FIG. 5 is a graph showing the effect of reducing the amount of ROS produced by the ethanol extract of the true oriental japonica from mature adipocytes. As shown in FIG. 5, when the natural drying method or the far-infrared drying method was used, the effect of reducing the ROS production was exhibited, and the ethanol extract produced using the same drying method exhibited an excellent effect of reducing the ROS production , Especially 70% ethanol, showed the highest activity of reducing ROS production.

Example 2-3: NO production inhibitory activity

To investigate the inhibitory effect of the extracts on the production of nitric oxide (NO) by the macrophage-treated extracts, RAW 264.7 cells were treated with LPS (10 μg / ml) to produce large amounts of NO, To analyze the NO production inhibitory activity.

FIG. 6 is a graph showing the NO production inhibitory activity of ethanol extracts of Mugwort mugwort. As shown in FIG. 6, it was confirmed that the NO production amount was significantly decreased as the concentration of the alcohol in all the drying methods increased.

Example 3: Preparation of fractions derived from a fine Chrysanthemum morbus extract and its antioxidant activity

It was confirmed through the above Example 2 that the best extract of Angelica japonica obtained by using 70% ethanol showed the best antioxidative effect. Therefore, the extract of Angelica japonica, obtained by far-infrared ray drying and 70% ethanol, The antioxidative activity of each fraction was analyzed.

Example 3-1: Preparation of the first fraction derived from a fine Cherrywood extract

100 g of RP C18 resin was added to the mixture, and the mixture was applied to a rotary evaporator and concentrated under reduced pressure to give the extract, which was then dissolved in 100 ml of the above resin Absorbed.

On the other hand, 400 g of dried RP C18 resin was added to the column, filled with 3% methanol, and the resin adsorbed on the above-obtained extract was further filled in the top of the filled resin. Subsequently, 50% methanol, 75% methanol, 100% methanol and chloroform were sequentially added as an eluent to sequentially obtain each eluate and a primary fraction which is a component remaining in the resin.

Example 3-2: Cytotoxicity analysis of the first fraction of a thin Cherrywood extract

To confirm the cytotoxicity of each of the primary fractions obtained in Example 3-1 above.

The cytotoxicity of each primary fraction was analyzed using a cell viability assay kit (WELGENE, Korea). Specifically, cells inoculated at a concentration of 10 ⁴ cells / cm 2 were cultured for 12 hours, and the respective primary fractions (50% methanol eluate, 75% methanol eluate, 100% methanol eluate, chloroform eluate and residue) (25, 50, 100, 200 or 400 占 퐂 / ml) and cultured again for 24 hours. After the culture was completed, the cells were added with XTT (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide inner salt and the absorbance was measured at 2 and 4 hours Respectively. At this point, cells not treated with the primary fraction were used as a negative control, and cells treated with 10 mM NAC were used as a positive control instead of the first fraction.

FIG. 7A is a graph showing the cytotoxicity of the first fraction derived from a fine Chrysanthemum morifolium extract. FIG. As shown in Figure 7a, not all primary fractions showed any cytotoxicity.

On the other hand, in order to confirm whether or not the same cytotoxicity was not exhibited also in the case of Gelidium amansii, instead of the fine genus Gelidium elegans, it was used as a first fraction and a 50% methanol eluate Was treated at a concentration of 1, 10, 20, 40, 80 or 100 占 퐂 / ml, the same procedure as in Examples 3-1 and 3-2 was carried out. Fig. 7B is a graph showing the cytotoxicity of the first fraction derived from Mugwort extract. As shown in FIG. 7 (b), when treated at a concentration of 100 μg / ml, it was found to be toxic, so it was preferable to treat it at a concentration lower than 100 μg / ml.

Example 3-3: NBT analysis of the first fraction of a thin Cherrywood extract

Except that each of the first fraction (50% methanol eluate, 75% methanol eluate, 100% methanol eluate, chloroform eluate and residue) was treated with 50 占 퐂 / 1, the antioxidative activity of the first fraction was analyzed.

8 is a photograph and a graph showing the effect of decreasing the amount of ROS produced in each of the first fraction derived from the ethanol extract of Prunus mume. As shown in FIG. 8, the 50% methanol eluate exhibited the best ROS production reduction activity.

Example 3-4: Preparation of a second fraction derived from a fine Cherrywood extract

From the results of Example 3-3, it was confirmed that the 50% methanol eluate in the first fraction showed the most excellent antioxidative activity. Thus, a second fraction was prepared from the 50% methanol eluate.

That is, the eluate obtained by using 50% methanol in the obtained first eluate was adsorbed on RP C18 resin, and various eluents (2 L of 5% methanol, 2 L of 10% methanol, 2 L of 25% Methanol, 2 L of 50% methanol and 3 L of 100% methanol) were sequentially added to obtain each secondary fraction.

Example 3-5: Cytotoxicity analysis of the second fraction of a thin Cherrywood extract

The cytotoxicity of each of the secondary fractions obtained in Example 3-4 was examined. That is, except for treating the secondary fraction (5%, 10%, 25%, 50% and 100% methanol eluate) with various concentrations (10, 25, 50, 100 or 200 μg / , The cytotoxicity of the second fraction was analyzed by the same method as in Example 3-2.

FIG. 9 is a graph showing the cytotoxicity of the second fraction derived from a fine Chrysanthemum morbus extract. As shown in Figure 9, not all secondary fractions showed any cytotoxicity.

Example 3-6: NBT analysis of the second fraction of a thin Cherrywood extract

The same procedure as in Example 2-1 was repeated except that the second fraction (5%, 10%, 25%, 50% and 100% methanol eluate) of each of the second fraction To analyze the antioxidant activity of the second fraction.

FIG. 10 is a photograph and a graph showing the effect of reducing the amount of ROS produced in each of the secondary fractions derived from the ethanol extract of a fine Chrysanthemum morbus. As shown in FIG. 10, the 50% methanol eluate exhibited the best ROS production reduction activity.

On the other hand, it was tried to determine whether the treatment concentration of the eluate exhibited a significant effect on the decrease of the ROS production amount, while changing the treatment concentration of the 50% methanol eluate showing the best ROS production reduction activity. Thus, the same procedure as in Example 2-1 was carried out, except that the 50% methanol eluate as the second fraction was treated at various concentrations (1, 5, 10 and 20 / / ml) The antioxidant activity of 50% methanol extract was analyzed.

FIG. 11 is a photograph and a graph showing the effect of decreasing the amount of ROS produced according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of a fine Chrysanthemum morbus. As shown in FIG. 11, the treatment concentration of 50% methanol eluate did not significantly affect the activity of reducing ROS production.

On the contrary, it was tried to determine whether the treatment concentration of the extract had a significant effect on the decrease of the ROS production while changing the treatment concentration of the extract. Thus, the same method as in Example 2-1 was carried out, except that the 70% ethanol extract was treated at various concentrations (1, 10, 20, and 40 / / ml) Respectively.

FIG. 12 is a photograph and a graph showing the effect of reducing the amount of ROS produced according to the treatment concentration of the 70% ethanol extract of a fine Chrysanthemum morifolium. As shown in FIG. 12, it was confirmed that the activity of decreasing ROS production was improved as the treatment concentration of 70% ethanol extract increased.

Example 3-7: Genetic analysis

Cells treated with various concentrations (1, 10, 20 and 40 / / ml) of 50% methanol eluate as one of the above secondary fractions were treated with TRIzol reagent (Invitrogen, USA) to obtain total RNA and its absorbance As a result of the measurement, it was confirmed that the value of OD260 / 280 was 1.8 to 2.0.

CDNA was synthesized by performing RT-PCT using the obtained total RNA using Taq DNA polymerase kit (Solgent, Korea) and PCR DNA thermocycler. At this time, cDNA was synthesized at 45 ° C for 60 minutes, and RTase was inactivated at 95 ° C for 5 minutes.

Using the synthesized cDNA as a template, PCR was carried out using the following primers capable of amplifying a gene encoding a protein involved in the production or elimination of ROS (95 DEG C for 5 minutes; 30 cycles (95 DEG C for 30 seconds, annealing of each primer annealing temperature of 30 seconds and 72 ° C for 1 minute); 72 ° C for 5 minutes) to amplify the cDNA. At this time, beta-actin and GAPDH were used as control groups.

NOX4 F: 5'-ccagaatgaggatcccagaa-3 '(SEQ ID NO: 1)

NOX4 R: 5'-accacctgaaacatgcaaca-3 '(SEQ ID NO: 2)

SOD1 F: 5'-cagcatgggttccacgtcca-3 '(SEQ ID NO: 3)

SOD1 R: 5'-cacattggccacaccgtcct-3 '(SEQ ID NO: 4)

SOD2 F: 5'-gggttggcttggtttcaata-3 '(SEQ ID NO: 5)

SOD2 R: 5'-aggtagtaagcgtgctccca-3 '(SEQ ID NO: 6)

Gpx F: 5'-gggcaaggtgctgctcattg-3 '(SEQ ID NO: 7)

Gpx R: 5'-agagcgggtgagccttctca-3 '(SEQ ID NO: 8)

beta-actin F: 5'-agccatgtacgtagccatcc-3 '(SEQ ID NO: 9)

beta-actin R: 5'-ctctcagctgtggtggtgaa-3 '(SEQ ID NO: 10)

GAPDH F: 5'-acacattgggggtaggaaca-3 '(SEQ ID NO: 11)

GAPDH R: 5'-aactttggcattgtggaagg-3 '(SEQ ID NO: 12)

FIG. 13 is a photograph and a graph showing changes in the expression level of the NOX4 gene encoding a protein that induces the production of ROS according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of a fine Chrysanthemum morbus. 14 is a photograph showing the change in the expression level of the Cu / Zn-SOD gene encoding a protein involved in the elimination of ROS according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract Graph.

As shown in FIGS. 13 and 14, as the treatment concentration of the 50% methanol eluate increases, the expression level of the gene (NOX4) encoding the protein that induces the production of ROS decreases and the gene encoding the protein involved in the ROS cleavage Cu / Zn-SOD) was increased.

Example 3-8: Western blot analysis

From the results of the above Examples 3-7, it was confirmed that the 50% methanol eluate, which is one of the above-mentioned second fractions, increased the expression amount of the gene encoding the protein involved in the ROS cleavage, Western blot analysis was performed to confirm whether or not it was reflected. Antibodies against Gpx, Mn-SOD and Cu / Zn-SOD, which are related to the elimination of ROS, were used as primary antibodies.

15 is a photograph and a graph showing changes in the protein level of GPx, which is a protein involved in the elimination of ROS, according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the ethanol extract of fine Chrysanthemum morifolium, FIG. 17 is a photograph and a graph showing the change in the protein level of Mn-SOD, which is a protein involved in the elimination of ROS, according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the low- Fig. 2 is a photograph and a graph showing changes in the protein level of Cu / Zn-SOD, which is a protein involved in the elimination of ROS, according to the treatment concentration of 50% methanol eluate, which is one of the secondary fractions derived from the true mugwort ethanol extract.

As shown in FIGS. 15 to 17, it was found that as the treatment concentration of the 50% methanol eluate was increased, the level of the protein involved in erasing ROS was increased.

Example 4: Analysis of anti-obesity activity of fractions derived from a fine Chrysanthemum morbus extract

Example 4-1: Analysis of anti-obesity activity of the first fraction derived from a fine Chrysanthemum morbus extract

Using the method of Example 2-1, the primary fraction prepared in Example 3-1 was treated at a concentration of 50 / / ml while inducing differentiation of adipocytes from precursor adipocytes, Each of the fat cells treated with the tea fraction was obtained. Each of the obtained adipocytes was washed twice with PBS, treated with 10% formalin, and allowed to stand at room temperature for 5 minutes. The formalin was then removed, treated with 10% formalin again, then left at 37 ° C for 1 hour, washed with 60% isopropanol and dried. Oil Red O (Sigma Aldrich) was added to the dried fat cells to stain the triglyceride and lipid. The reaction was allowed to proceed at room temperature for 1 hour. After completion of the reaction, the cells were washed with distilled water four times and dried. 100% isopropanol was added to the dried sample for 10 minutes, and the absorbance was measured at 490 nm.

FIG. 18 is a photograph and a graph showing the anti-obesity activity of each primary fraction derived from the ethanol extract of Ganoderma lucidum. As shown in FIG. 18, the 50% methanol eluate showed the most excellent anti-obesity activity.

Example 4-2: Analysis of anti-obesity activity of the second fraction derived from the finely cherries extract

From the results of Example 4-1, it was confirmed that the 50% methanol eluate showed the most excellent anti-obesity activity among the first fractions. Therefore, the anti-obesity activity of the second fraction derived from the 50% methanol eluate was compared.

Namely, the anti-obesity activity of the second fraction was measured in the same manner as in Example 4-1, except that the second fraction prepared in Example 3-4 was used instead of the first fraction.

FIG. 19 is a photograph and a graph showing the anti-obesity activity of each secondary fraction derived from the ethanol extract of a fine Chrysanthemum morifolium. As shown in FIG. 19, the 50% methanol eluate exhibited the most excellent anti-obesity activity.

On the other hand, fat cells can visually confirm the accumulation of fat through an optical microscope. Each of the adipocytes treated with the second fraction was photographed by an optical microscope on the eighth day after the differentiation. FIG. 20 is a photograph showing fat cells treated with respective secondary fractions derived from an ethanol extract of a fine Chrysanthemum morifolium. As shown in FIG. 20, it was confirmed that almost no fat of the 50% methanol fraction was visually observed.

On the other hand, it was tried to determine whether the treatment concentration of the eluant exhibited a significant effect on the anti-obesity activity while changing the treatment concentration of the 50% methanol eluate exhibiting the best anti-obesity activity. Thus, the same method as in Example 4-1 was carried out except that the 50% methanol eluate as the second fraction was treated at various concentrations (1, 10, 20 and 40 占 퐂 / ml) instead of the first fraction And the anti-obesity activity of the second fraction, 50% methanol eluate, was analyzed.

FIG. 21 is a photograph and a graph showing anti-obesity activity according to the treatment concentration of 50% methanol eluate, which is one of the second fractions derived from the ethanol extract of a fine Chrysanthemum morbus. As shown in FIG. 21, it was confirmed that the anti-obesity activity was improved as the concentration of 50% methanol eluate was increased.

Example 4-3: Genetic analysis

Example 4-3-1: Gene analysis using the first fraction

Cells treated with 50 ㎍ / ml of the first fraction (50% methanol eluate, 75% methanol eluate, 100% methanol eluate, chloroform eluate and residue) were used as primers. As a primer, a representative gene involved in fat accumulation, PPAR- The same method as in Example 3-7 was used, except that the following primers capable of amplifying?, C / EBP- ?, and G6PDH were used, and a PPAR encoding a protein involved in fat accumulation -γ, C / EBP-α, and G6PDH genes, catalase and NOX4 genes encoding proteins involved in ROS clearing and production, and their expression patterns were compared.

C / EBP-alpha F: 5'-tgaaggaacttgaagcacaa-3 '(SEQ ID NO: 13)

C / EBP-alpha R: 5'-tcagagcaaaaccaaaacaa-3 '(SEQ ID NO: 14)

PPAR-y F: 5'-ctgtcagaccaacagcctga-3 '(SEQ ID NO: 15)

PPAR-y R: 5'-aatgcgagtggtcttccatc-3 '(SEQ ID NO: 16)

G6PDH F: 5'-cgatggcagagcaggt-3 '(SEQ ID NO: 17)

G6PDH R: 5'-gatctggtcctcacg-3 '(SEQ ID NO: 18)

FIG. 22A is a photograph showing the change in the expression level of a gene encoding a protein involved in fat accumulation according to the treatment of a primary fraction derived from an ethanol extract of a fine Chrysanthemum morbus. As shown in FIG. 22 (a), expression levels of PPAR-γ, C / EBP-α and G6PDH genes involved in fat accumulation were significantly lowered when 50% methanol eluate was used.

FIG. 22B is a photograph showing the change in expression amount of a gene encoding a protein involved in ROS cleavage and production according to the treatment of the first fraction derived from the ethanol extract of a fine Chrysanthemum morbus. As shown in FIG. 22B, catalase, which is a gene that decomposes hydrogen peroxide into water and oxygen, is most abundantly expressed in 50% methanol extract, and expression of NOX4 gene, which is a protein that produces ROS in the cell membrane, 50% methanol extract. Therefore, it was confirmed that the 50% methanol extract had an effect on the ROS cleavage by regulating the genes involved in ROS cleavage and production.

Example 4-3-2: Genetic analysis using the second fraction

Cells treated with 50 μg / ml of the second fraction (5%, 10%, 25%, 50% and 100% methanol eluate) were used as primers and PPAR-γ, C / The following procedures were carried out in the same manner as in Example 3-7, except that the following primers capable of amplifying EBP-alpha and FABP4 were used, and PPAR-gamma, C / EBP-alpha and FABP4 genes, and the NOX4 gene, which is a gene encoding a protein involved in ROS elimination and production, and their expression patterns were compared.

C / EBP-alpha F: 5'-tgaaggaacttgaagcacaa-3 '(SEQ ID NO: 13)

C / EBP-alpha R: 5'-tcagagcaaaaccaaaacaa-3 '(SEQ ID NO: 14)

PPAR-y F: 5'-ctgtcagaccaacagcctga-3 '(SEQ ID NO: 15)

PPAR-y R: 5'-aatgcgagtggtcttccatc-3 '(SEQ ID NO: 16)

FABP4 F: 5'-tcacctggaagacagctct-3 '(SEQ ID NO: 19)

FABP4 R: 5'-aatccccatttacgctgatg-3 '(SEQ ID NO: 20)

FIG. 23A is a photograph showing the change in the expression level of a gene encoding a protein involved in fat accumulation according to the treatment of the second fraction derived from an ethanol extract of a fine Chrysanthemum indicum. As shown in FIG. 23 (a), the expression level of PPAR-γ, C / EBP-α and FABP4 genes involved in fat accumulation was significantly lowered when 50% methanol eluate was used.

FIG. 23B is a photograph showing the change in expression amount of a gene encoding a protein involved in ROS cleavage and production according to the treatment of a second fraction derived from an ethanol extract of a fine Chrysanthemum overglaze. As shown in FIG. 23B, gene expression of NOX4 gene involved in ROS elimination and production was significantly reduced only when treated with 50% methanol extract, and the level of decrease was similar to that of positive control NAC. Therefore, it was analyzed that 50% methanol extract inhibited the intracellular ROS production by inhibiting the expression of NOX4, a protein that induces ROS production.

As described above, it has been confirmed that the 50% methanol extract inhibits the expression of NOX4, which is a protein that induces ROS production. Therefore, when the 50% methanol extract is treated at various concentrations, it is related to ROS elimination and production We examined how the expression of Gpx (glutathione peroxidase), catalase, SOD1, SOD2 and NOX4 genes, which encode proteins, is affected. 23C is a photograph showing the change in the expression level of a gene encoding a protein involved in ROS cleavage and production according to the treatment concentration of a 50% methanol extract, which is a secondary fraction derived from a fine Chrysanthemum orientalis ethanol extract. As shown in FIG. 23C, among the genes encoding proteins related to ROS cleavage and production, Gpx, catalase, SOD1, and SOD2 genes exhibited increased expression levels as the treatment concentration of 50% methanol extract increased, while the NOX4 gene As the concentration of 50% methanol extract increased, the expression level of the extract decreased.

Since the Gpx, catalase, SOD1 and SOD2 genes encode proteins involved in ROS cleavage and the NOX4 gene is known to encode a protein involved in ROS production, the 50% methanol extract inhibits ROS production, It is clear that it plays a role of erasing.

Example 4-3-3: Analysis of PPAR-gamma gene using 50% methanol eluent as the second fraction

Cells treated with various concentrations (1, 10, 20 and 40 占 퐂 / ml) of a 50% methanol eluate as a secondary fraction were used, and as a primer, SEQ ID NO: 15 And 16 primers, the PPAR-gamma gene was amplified and the expression patterns thereof were compared by performing the same method as in Example 3-7.

24 is a photograph and a graph showing the change in the expression level of the PPAR-gamma gene according to the treatment concentration of the 50% methanol eluate, which is a secondary fraction derived from the ethanol extract of Prunus orientalis. As shown in FIG. 24, the expression level of the PPAR-γ gene involved in fat accumulation decreased with increasing treatment concentration of 50% methanol eluate.

Example 4-3-4: Analysis of aP2 gene using a 50% methanol eluent as the second fraction

Cells treated with various concentrations (1, 10, 20 and 40 占 퐂 / ml) of 50% methanol eluate as a second fraction were used and as a primer, SEQ ID NO: 19 and 20 were used, amplification of the aP2 (FABP4) gene was carried out in the same manner as in Example 3-7, and the expression patterns thereof were compared

25 is a photograph and a graph showing changes in the expression level of aP2 (FABP4) gene according to the treatment concentration of a 50% methanol eluate, which is a secondary fraction derived from a fine Chrysanthemum morifolium ethanol extract. As shown in FIG. 25, the expression level of the aP2 gene involved in fat accumulation decreased sharply as the treatment concentration of 50% methanol eluate was increased.

Example 5 Preparation of Functional Foods Containing Extract of Mugwort

A composition containing an extract of Mugwort is added at the time of preparing a normal food, and a functional food showing anti-obesity and antioxidative effect was prepared as follows.

Example 5-1: Preparation of functional noodles

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of tea fraction, 700 mg of wheat flour, 80 g of egg, and 200 g of water were mixed to prepare a dough, and a cotton pad was produced by a conventional noodle manufacturing method to produce functional noodles exhibiting anti-obesity and antioxidative effects.

Example 5-2: Preparation of functional biscuit

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of tea fraction, 700 mg of wheat flour, 200 g of milk and 80 g of egg were mixed to prepare a dough, and a functional biscuit exhibiting anti-obesity and antioxidative effect was prepared by a conventional biscuit manufacturing method.

Example 5-3: Preparation of functional beverage

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of tea fraction, 10 mg of fruit flavor, 100 g of fruit juice, 10 mg of sodium benzoate and 900 ml of water were mixed to prepare a functional beverage having anti-obesity and antioxidative effects.

Example 5-4: Preparation of functional seasoning for meat

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of tea fraction, 100 g of juice, 100 g of jujube, 300 g of red pepper paste, and a small amount of salt, garlic, pepper, onion and ginger were mixed to prepare a functional seasoning for meat.

Example 5-5: Preparation of functional bean sprouts

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of tea fractions, 150 g of red pepper powder, a small amount of salt, garlic, pepper, onion and ginger were mixed to prepare spice for bean sprouts, 200 g of the prepared spice and 500 g of bean sprouts were mixed, The functional bean sprouts without antioxidant effect were prepared.

Example 5-6: Preparation of functional miso pot

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of carrot fractions, 600 g of miso, a small amount of salt, garlic, red pepper, onion and ginger were mixed and placed in 500 ml of water and cooked by a conventional method to produce functional soybean soup showing anti-obesity and antioxidative effect.

Example 5-7: Preparation of functional soup

The extracts of fine brown bean curd refuse prepared by the method of Example 1-1, the first fractions of the fine brown bean curd extract prepared by the method of Example 3-1 or the fine bean curd extract prepared by the method of Example 3-4 20 g of tea fraction, 50 g of black soybean, 50 g of sorghum, 50 g of millet, and 300 g of white rice were mixed and cooked by a conventional method to produce functional granulated rice showing anti-obesity and antioxidative effect.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Functional Foods Comprising the Extract of Gelidium elegans <130> P <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NOX4 F primer <400> 1 ccagaatgag gatcccagaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NOX4 R primer <400> 2 accacctgaa acatgcaaca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD1 F primer <400> 3 cagcatgggt tccacgtcca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD1 R primer <400> 4 cacattggcc acaccgtcct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD2 F primer <400> 5 gggttggctt ggtttcaata 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD2 R primer <400> 6 aggtagtaag cgtgctccca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gpx F primer <400> 7 gggcaaggtg ctgctcattg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gpx R primer <400> 8 agagcgggtg agccttctca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin F primer <400> 9 agccatgtac gtagccatcc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin R primer <400> 10 ctctcagctg tggtggtgaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F primer <400> 11 acacattggg ggtaggaaca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R primer <400> 12 aactttggca ttgtggaagg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C / EBP-alpha F primer <400> 13 tgaaggaact tgaagcacaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C / EBP-alpha R primer <400> 14 tcagagcaaa accaaaacaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> PPAR-gamma F primer <400> 15 ctgtcagacc aacagcctga 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma R primer <400> 16 aatgcgagtg gtcttccatc 20 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> G6PDH F primer <400> 17 cgatggcaga gcaggt 16 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> G6PDH R primer <400> 18 gatctggtcc tcacg 15 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FABP4 F primer <400> 19 tcacctggaa gacagctcct 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FABP4R primer <400> 20 aatccccatt tacgctgatg 20

Claims (8)

(B) extracting the bean curds with distilled water at a temperature of 25 캜 for 24 hours with 70% (v / v) ethanol after drying the beans using far infrared rays.
The method according to claim 1,
The mugwort is either Gelidium amansii or Gelidium elegans.
A method for preparing an extract of Euglena japonica having antiobesity and antioxidant activity.
The method according to claim 1,
The far-infrared ray is far-infrared rays at 80 DEG C
A method for preparing an extract of Euglena japonica having antiobesity and antioxidant activity.
An extract of Staphylococcus aureus having antiobesity and antioxidant activity produced by the method of claim 1.
A composition having anti-obesity and antioxidative activity, comprising the extract of Angelica keiskei, or the fraction thereof, of claim 4.
6. The method of claim 5,
The fraction was a second fraction obtained by adsorbing the above extract on RP C18 resin and eluting it with 50% methanol and adsorbing the first fraction or the first fraction on RP C18 resin and eluting it with 50% methanol Featured
Gt; antioxidant &lt; / RTI &gt; activity.
A functional food having an anti-obesity activity comprising the composition of claim 5.
A functional food having antioxidative activity comprising the composition of claim 5.

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