KR20210089345A - Manufacturing method of the Poria cocos Wolf extract - Google Patents

Abstract

The present invention relates to a method for producing a Wolfiporia extensa extract and the like. The method for producing a Wolfiporia extensa extract of the present invention can produce an extract with a very high flavonoid content by greatly improving an extraction yield. In addition, the Wolfiporia extensa extract produced by the method of the present invention has almost no cytotoxicity, and exhibits remarkably excellent activity in anti-obesity and antioxidant effects, and thus can be used in food for preventing or alleviating obesity and oxidative stress. Moreover, the Wolfiporia extensa extract produced by the method of the present invention maintains a beta-glucan content at a higher level, and thus can strengthen immune enhancing effects due to the intake of Wolfiporia extensa.

Description

Manufacturing method of the Poria cocos Wolf extract

The present invention relates to a method for preparing a bokryeong extract and the like.

As human lifespan is extended along with advances in science and the development of medical technology, interest in people's health is increasing, as well as their daily eating habits, including drinking water.

Bokryeong (Wolfiporia cocos, syn. Poria cocos Wolf.) is a kind of fungus belonging to the fungus family. It is known as a parasite that forms amorphous sclerotia around it. The sclerotia of Bokryeong are divided into white bokryeong with firm flesh and soft and tender jeokbokryeong according to the color of the inside. It can be used as medicine.

Bokryeong contains various kinds of triterpenes and polysaccharides as its main ingredients, and it is known for its anticancer action, antioxidant action, nematicide, antihyperlipidemic action, antibacterial action, anti-inflammatory action, antihypertensive action, immune enhancement and brain cell activity. In particular, physiological activities such as immune enhancement and brain cell activity are known. Among the components of bokryeong, polysaccharides such as U-pachyman, pachymaran, carboxymethyl pachymarna, and (1,3)-(1,6)-β-D-glucan have excellent antioxidant effects, and triterpenes components have effects on oxidation, inflammation and pancreatic cancer. is known to have The main pharmacological components of bokryeong include β-glucan, triterpene, and pachymaran. Among the pharmacological components, β-glucan and triterpene have been reported to have biological tissue regeneration, healing function, antioxidant function, antibacterial, anti-inflammatory, and antiviral effects. As a bokryeong polysaccharide without 6 branches, pachyman has no antitumor activity, but it has been reported to exhibit strong anticancer activity when the structure is changed to pachymaran. Bokryeong reduces blood triglycerides, total cholesterol, LDL- cholesterol, and lowers HDL+ cholesterol. It has been reported that it can improve blood fat distribution by increasing it, and research results have been reported that 4 types of anticancer compounds isolated from bokryeong sclerotia have an anticancer effect that induces cancer cell suicide by inhibiting cancer cell proliferation in lung adenocarcinoma. In addition, bokryeong bark and endothelial extracts inhibit elastase and collagenase activity and are effective in wrinkle improvement and inflammation, so they are used as natural cosmetic materials.

Due to the various effects of bokryeong, its use in various fields is increasing every year, and the amount of imports from China is increasing. In particular, according to the Food and Drug Statistical Yearbook of the Ministry of Food and Drug Safety, as of 2015, it ranked 10th in domestic drug production, 7th in import volume, and 2nd in import volume, and is the second most used herbal medicine after licorice. Because there are few domestic bokryeong farms, more than 90% of it is natural, and the supply is limited, so the price is set higher than that of imported bokryeong.

As a herbal medicine, bokryeong has been used to boil or brew soup. However, there is still a lack of an extraction method that can maximize the pharmacological efficacy of bokryeong. Accordingly, in order to expand the usability of bokryeong as functional food, medicine, and quasi-drugs, the present inventors have completed the present invention by intensively researching a method for preparing bokryeong extract that is harmless to the human body and can improve its efficacy.

The technical problem to be achieved by the present invention is to provide a method for preparing a bokryeong extract using a vacuum low temperature extraction method, and to provide a food composition for anti-obesity, anti-aging, and/or antioxidant comprising the bokryeong extract prepared by the method will be.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a method for preparing a bokryeong extract using a vacuum low temperature extraction method.

The preparation method of the present invention specifically comprises the following steps:

(1) washing and drying bokryeong; (2) crushing the dried bokryeong; (3) drying the crushed bokryeong with hot air at 35 to 45° C. for 10 to 14 hours; (4) mixing 8 to 12 parts by weight of distilled water based on 1 part by weight of the hot air dried bokryeong; and (5) extracting bokryeong mixed with the distilled water at 75-85° C. for 10 to 14 hours under vacuum.

As an embodiment of the present invention, the hot air drying in step (3) may be performed at 40° C. for 12 hours.

As another embodiment of the present invention, in step (4), 10 parts by weight of distilled water may be mixed based on 1 part by weight of hot-air dried bokryeong.

As another embodiment of the present invention, the vacuum low temperature extraction of step (5) may be performed at 80 °C for 12 hours.

In addition, the present invention provides a bokryeong extract prepared by the above method without cytotoxicity.

In addition, the present invention provides a health functional food composition for preventing or improving obesity comprising the extract of bokryeong.

In addition, the present invention provides a food composition for anti-aging or antioxidant comprising the extract of bokryeong.

As an embodiment of the present invention, the anti-aging means damage to cells or extracellular matrix caused by oxidative stress.

The bokryeong extract preparation method of the present invention can greatly improve the extraction yield to produce an extract having a very high flavonoid content. Also, the bokryeong extract prepared by the method of the present invention has almost no cytotoxicity, and has anti-obesity and antioxidant effects. It also exhibits a remarkably excellent activity in the bar, it can be used in food for preventing or improving obesity and oxidative stress. In addition, the bokryeong extract prepared by the method of the present invention can further strengthen the immune enhancing effect due to the intake of bokryeong bar content of beta-glucan is maintained higher.

1 is a schematic diagram of a method for preparing a bokryeong extract of the present invention.
Figure 2 is a graph showing the cytotoxicity of the extract prepared by the production method of the present invention.
Figure 3 is a graph showing the total flavonoid content of the extract prepared by the method of the present invention.
Figure 4 is a graph confirming the antioxidant activity of the extract prepared by the method of the present invention.
5 is a graph showing changes in blood sugar of zebrafish according to administration of a bokryeong extract prepared by the method of the present invention.
6 is a graph showing the change in BMI of zebrafish according to the administration of the extract prepared by the method of the present invention.
7 is a result of analyzing the sugar component of the extract prepared by the method of the present invention.
8 is a UPLC component analysis result of a bokryeong extract prepared by the method of the present invention.
9 is a graph showing the beta-glucan content of the extract prepared by the method of the present invention.

The present invention relates to a vacuum low temperature concentrated hot water extract of bokryeong, and by extracting bokryeong containing bokryeong blood by vacuum low temperature concentration method, bokryeong extract containing a large amount of physiologically useful substances of bokryeong and harmless to the human body can be provided.

The bokryeong extract prepared by the method of the present invention has no or very low cytotoxicity, is harmless to the human body, and has excellent effects on antioxidant, anti-obesity, and immune enhancement. Food includes functional food and health functional food.

In the manufacturing method of the present invention, the extraction yield of bokryeong is 70% or more, and more specifically, the extraction yield is 70-85%.

On the other hand, the bokryeong used in the preparation method of the bokryeong extract of the present invention preferably includes bokryeongpi, and may be non-reclaimed artificially cultivated bokryeong more than half a year old.

The bokryeong extract manufacturing method of the present invention is to wash the non-reclaimed artificially cultivated bokryeong for 30 minutes, remove the surface moisture, and then crush it finely. Then, the crushed bokryeong was dried with hot air at 40° C. for 12 hours, and sterilized purified water corresponding to 10 times the weight of the dried bokryeong was added, cooled at 80° C. for 12 hours, and vacuum low temperature concentration extraction was performed.

The results of performing HPLC-ELSD to confirm the sugar components of the prepared bokryeong extract are shown in Table 1 below.

Item unit Way detection limit result Ribose % HPLC-ELSD 0.01 none Xylose % HPLC-ELSD 0.0025 none Fructose % HPLC-ELSD 0.001 none Glucose % HPLC-ELSD 0.001 0.116 Sucrose % HPLC-ELSD 0.001 none Lactose % HPLC-ELSD 0.0025 none Maltose % HPLC-ELSD 0.0025 none Arabinose % HPLC-ELSD 0.005 none

On the other hand, the prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed. For example, the filtration may use a filter paper or a reduced pressure filter, but the filter paper is preferably a Grade 1 filter paper. In addition, the concentration may be performed by a vacuum concentrator, and drying may be performed by a spray drying method, a freeze drying method, and the like, but is not limited thereto.

Bokryeong extract prepared by the method of the present invention exhibited superior activity than extracts using other organic solvents in antioxidant and anti-obesity effects, and showed low cytotoxicity in MTT assay, anti-obesity, anti-aging, and/or anti-oxidation. It can be included in health functional food for The anti-aging means aging by free radicals, and the bokryeong extract of the present invention can prevent or improve cellular aging caused by oxidative stress.

Therefore, the present invention can provide a health functional food composition for preventing or improving obesity or cellular oxidative stress comprising the extract of bokryeong as an active ingredient, and more specifically, the composition of the present invention is anti-obesity, anti-aging, And/or it may be added to health functional food for the purpose of antioxidant, and when the bokryeong extract having an anti-obesity, anti-aging, and/or antioxidant effect of the present invention is used as a food additive, the extract may be added as it is or other food or It can be used together with food ingredients, and can be used appropriately according to a conventional method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the compound of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene purposes or for health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substance can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in the ordinary sense.

The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.20 g, preferably about 0.04 to 0.10 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain a carbonation agent used in carbonated beverages, and the like. In addition, the composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Example]

Example 1. Confirmation of cytotoxicity

To prepare MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent, it was diluted with PBS to a concentration of 2 mg/mL.

Since the reagent does not dissolve well in PBS, it was sonicated for 30 minutes. When completely dissolved, it was used by filtering with 0.2 μM. Since MTT reagent is weak to light, it was stored by covering it with foil to block the light.

The bokryeong extract extracted by low temperature vacuum concentration hot water extraction method was equally aliquoted to 2.0 × 10 cells/well of RAW264.7 cells in a 96 well plate and pre-cultured for 18 hours.

The old medium was removed, and the sample according to the new medium and concentration was treated and incubated for 24 hours again. MTT reagent was diluted in fresh medium and wrapped in foil.

After removing the medium in the well, 100 μL of MTT reagent was treated. After reagent treatment, incubation was performed at 37°C for 3-4 hours.

After removing the MTT reagent, 100 μL of DMSO was added per well. Formazan was uniformly dissolved in DMSO to develop purple color. Absorbance was measured at 540 nm.

The MTT measurement test result of the sample is shown in FIG. 2 . When the cell viability of the control group not treated with the sample was 100%, the group treated with the hot water extract showed no toxicity to the cells. On the other hand, the ethanol extract-treated stock solution group showed 40% survival rate inhibition, and it was confirmed that the 1/2 dilution group, 1/4 dilution group, and 1/8 dilution group also showed inhibition of cell viability.

In order to be harmless to the human body in the cytotoxicity test, it is considered that the extraction method using organic solvents, including the alcohol extraction method, is not safe to use as a composition of food processing products, beverages, tea, and drugs.

Example 2. Determination of total flavonoid content

Add 200 µL of standard or test substance and 800 µL of ultrapure water to the test tube.

Add 60 μL of 5% NaNO2 (w/v) solution here.

After adding 60 μL of 10% AICI3 (w/v) solution, react for 5 minutes.

Add 400 μL of 1M NaOH solution and 480 μL of deionized water, and react for 6 minutes.

Absorbance is measured at 510 nm using a UV-Vis spectrophotometer (Libra S70, Biochrom, UK).

A calibration curve of the standard material was prepared using the standard material quercetin, and the absorbance of the sample was applied to the calibration curve and expressed as QE mg/100 mL extract.

Example 3. Measurement of DPPH radical scavenging ability

After dissolving 24 mg of DPPH reagent in 100 mL of methanol, it was stored at -20°C.

Stored DPPH solution should be O.D. It was used by adjusting it to 0.98±0.02.

1.5 mL of DPPH solution was added to 50 μL of sample and reacted in the dark for 30 minutes.

Absorbance was measured at 517 nm using a UV-Vis spectrophotometer (Libra S70, Biochrom, UK).

Example 4. ABTs assay measurement

ABTS radical cation (ABTS·+) was generated by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate (final concentration) and reacting for 12-16 hours in a dark place at room temperature.

The ABTS·+ solution was used by adjusting the absorbance to 0.70 (±0.02) at 734 nm by using ethanol before use.

1.0 mL of ABTS solution was added to 10 μL of the sample and reacted at 30 °C in a dark place for 15 minutes.

Absorbance was measured at 734 nm using a UV-Vis spectrophotometer (Libra S70, Biochrom, UK).

Example 5. Anti-obesity effect BMI and blood glucose measurement

Individuals to be subjected to the experiment were bred in a water tank (water temperature 26-28.5℃, pH 6.8-7.5), one each (total of 40), and fed the same amount of general feed and bokryeong mixed feed.

Individuals to be fed general feed and bokryeong mixed feed were separated and bred in each water tank (water temperature 26-28.5℃, pH 6.8-7.5), and two types of feed were fed each for about 2 months.

General feed or bokryeong mixed feed was fed to each tank 3 times a day.

Total length and weight were measured daily.

The BMI index was calculated using the measured length and weight, and observation was performed for 2 months.

During the measurement, 0.2% stock of Ethyl 3-aminobenzoate methanesulfonate (98%, Sigma, E10521-10G) was prepared and used.

Example 5. Beta glucan content measurement

The beta-glucan content of bokryeong was measured using a megazyme kit (mushroom and yeast β-glucan assay procedure kit).

To 100 mg of the freeze-dried sample, 1.5 mL of 37% HCl was added, stirred in a water bath at 30°C for 45 min, and then 10 mL of tertiary distilled water was added, followed by stirring in a water bath at 100°C for another 2 hours.

At room temperature, 10 mL of 2N-KOH was added and mixed to the reaction solution, and 0.2 M sodium acetate buffer (pH 5.0) was added thereto to make 100 mL.

The refined sample was centrifuged (2,000 g, 5 min) to obtain a supernatant, and in 0.1 mL of the obtained supernatant, 20 U/mL of external 1,3-beta-glucanase (exo-1,3-β-glucanase) After adding 0.1 mL of a 4 U/mL mixed solution of glucosidase (β-glucosidase), it was reacted for 1 h in a water bath at 40°C.

3 mL of glucose oxidase/peroxidase (GOPOD) reagent was added to the reaction solution, and after 20 minutes of reaction, absorbance was measured at a wavelength of 510 nm to determine the total glucan content.

Alpha glucan (α-glucan) content was obtained.

The absorbance of total glucan and alpha glucan was calculated as mg/100g value using the absorbance of a reaction solution in which a standard material, a glucose solution, was reacted with a GOPOD reagent.

The beta-glucan content was calculated by subtracting the alpha-glucan content from the total glucan content.

The result is as follows.

Total glucan 7.2±0.91 - a glucan 2.7±1.18 = b glucan 4.5±0.77 (mg/g)

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

(1) washing and drying bokryeong;
(2) crushing the dried bokryeong;
(3) drying the crushed bokryeong with hot air at 35 to 45° C. for 10 to 14 hours;
(4) mixing 8 to 12 parts by weight of distilled water based on 1 part by weight of the hot air dried bokryeong; and
(5) extracting bokryeong mixed with the distilled water at 75-85 ℃ for 10 to 14 hours at a vacuum low temperature; a method for preparing bokryeong extract comprising.
According to claim 1,
The hot air drying of step (3) is characterized in that it is carried out at 40 ℃ for 12 hours, Bokryeong extract preparation method.
According to claim 1,
In step (4), 10 parts by weight of distilled water based on 1 part by weight of hot-air dried bokryeong is mixed, the bokryeong extract manufacturing method.
According to claim 1,
The vacuum low temperature extraction of step (5) is characterized in that performed at 80 ℃ for 12 hours, Bokryeong extract preparation method.
An antioxidant composition comprising the extract of bokryeong prepared by any one of claims 1 to 4 as an active ingredient.
An anti-obesity composition comprising the extract of bokryeong prepared by any one of claims 1 to 4 as an active ingredient.

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