KR101738753B1 - Healthful composition using Mapleleaf Aisnsliaea extracts, and funtional drinks comprising the same - Google Patents

Healthful composition using Mapleleaf Aisnsliaea extracts, and funtional drinks comprising the same Download PDF

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KR101738753B1
KR101738753B1 KR1020150089861A KR20150089861A KR101738753B1 KR 101738753 B1 KR101738753 B1 KR 101738753B1 KR 1020150089861 A KR1020150089861 A KR 1020150089861A KR 20150089861 A KR20150089861 A KR 20150089861A KR 101738753 B1 KR101738753 B1 KR 101738753B1
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extract
mushroom
sample
enzyme
ethanol
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KR20170000888A (en
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이창호
권정숙
손건호
이정순
임지음
문종태
김강하
박태룡
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경상북도(관련부서:경상북도산림자원개발원)
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/14—Extraction
    • A23Y2220/67—

Abstract

There is provided a health functional composition comprising an effective amount of a plant extract having an antioxidant, anti-inflammatory, anti-obesity and antimutagenic activity and a functional beverage containing the same. This health functional composition contains an extract of a single puff breeze as an active ingredient. The functional beverage also includes such a health functional composition.

Description

The present invention relates to a health functional composition and a functional beverage containing the extract,

The present invention relates to a health functional composition using Mapleae Aisnsliaea extract and a functional beverage containing the same.

As the national standard of living improves as a result of industrial development and economic growth, there is a growing interest in functional health supplements along with a well-being trend around the world, and consumption is increasing rapidly. In order to meet these demands, researches for finding various physiological functional ingredients from plant materials have been actively conducted.

Physiological functions required for health supplements include antioxidant, anti-inflammatory, anti-obesity, and anti-mutation. Antioxidant efficacy refers to inhibiting or preventing active oxygen involved in arteriosclerosis, brain or cardiovascular disorders, aging, or carcinogenesis. The anti-inflammatory effect refers to a function to remove inflammation such as an anti-inflammatory agent, and the anti-obesity effect refers to a function to inhibit the expression of an obesity factor. The antimutagenic effect refers to lowering the mutation rate of a cell or reversing the action of a mutagenic agent.

Ainsliaea acerifolia is a perennial plant of Asteraceae which grows in the mountains of Korea. The growing environment grows in moist, partly cloudy. It is mainly used for edible lentils and distributed in Korea, China and Japan. There are no studies on the physiological functioning of sweetweeds in addition to the use of lice blossoms in folk remedies and edible foods.

Therefore, it is urgently required to research and develop the ventilator as a next-generation health supplement food by proving the physiological function of the sweetpotato.

It is an object of the present invention to provide a health functional composition comprising, as an active ingredient, an extract of P. aeruginosa having antioxidant, anti-inflammatory, anti-obesity and antimutagenic properties.

It is another object of the present invention to provide a functional beverage containing such a single-puff extract as an active ingredient.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the health functional composition according to one embodiment of the present invention includes an extract of Artemisia sp.

The monoptera extract can be extracted with water or ethanol.

In the case of producing a single-blasted water extract, it is preferable to conduct extraction for 1 to 50 hours while adding water of 1 to 20 times (v / w) of the sample air weight at the extraction temperature of 50 to 100 ° C. In the case of producing the single-blasted ethanol extract, it is preferable to conduct the extraction for 1 to 50 hours while adding 50 to 90% ethanol of 1 to 20 times (v / w) of the sample air weight at the extraction temperature of 5 to 50 ° C.

The above-mentioned single-puff extract can be fermented using the mushroom mycelium after the extraction with water or ethanol. Preferably, 1 to 10% (v / v) of the mushroom mycelia is inoculated into the above-mentioned single-bladder water extract, and the mixture is fermented for 1 to 14 days to obtain a biotransfected single-puff extract. The mushroom mycelium may be a mushroom mycelium.

In addition, the monodysteria extract may be fermented using lactic acid bacteria after the extraction with water or ethanol. Preferably, 1 to 10% (v / v) of the above-mentioned lactic acid bacterium is inoculated into the above-mentioned single-puff extract, followed by fermentation for 1 to 5 days to obtain a biotransformed single-puff extract. The lactic acid bacterium may be Lactobacillus plantarum KCCM 11322.

Furthermore, the above-mentioned single-puff extract can be extracted by enzyme treatment. The monoptera extract can be fermented by lactic acid bacteria after the enzyme treatment step. Preferably, the method further comprises an extraction step using water or ethanol between the enzyme treatment step and the lactic acid bacteria fermentation step.

In the case of producing an extract from a pale-green air-polluted enzyme, 50 to 500 ppm of the enzyme is added to a sample of air blast at an extraction temperature of 20 to 60 ° C, and then subjected to an enzyme treatment for 1 to 24 hours to obtain a first extract, 1 to 10% (v / w) of the lactic acid bacterium is added to the second extract to obtain a second extract for 1 to 50 hours while adding 1 to 20 times (v / w) water or ethanol to the weight of the tea extract, v) can be inoculated and fermented for 1 to 5 days to obtain a biodegraded monodyne extract. The enzyme used for the enzyme treatment may be Viscozyme or Promozyme.

According to another aspect of the present invention, there is provided a functional beverage including the health functional composition comprising the extract of the present invention as an active ingredient.

The inventors of the present invention conducted studies on the functional components of the leafhopper, and found that the antioxidative, antiinflammatory, anti-obesity and anti-inflammatory properties of the leaf extract from the biotransformed plant extracts using the hot water / ethanol extract, Mutagenic effect, and the like, and can be usefully used as a functional health drink utilizing the same. The present invention has been completed based on this finding. The functional beverage of the present invention may contain various kinds of supplementary acidulants, acid herbicides, vitamins, minerals, dietary fibers, or other food additives, if necessary, in addition to the extract of A.

The details of other embodiments are included in the detailed description.

INDUSTRIAL APPLICABILITY The extract of monodoric breeze according to the present invention exhibits antioxidant, anti-inflammatory, anti-obesity, and antimutagenic activity, and thus can be usefully used as a health functional beverage composition. Particularly, as in the present invention, the effect of these activities is remarkable when biotransformation of a single-puff extract is carried out using water or ethanol extract, mushroom mycelium, lactic acid bacteria or an enzyme.

The present invention will be described more specifically based on examples and experimental examples. However, these examples and experimental examples are provided only for the understanding of the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of an extract

<1-1> Preparation of hot water extract

The dried cutworm (purchased in the market as a wild alive native to Gyeongbuk province) was crushed and weighed to a dry weight of 200 g. (V / w) of distilled water was added to the pulverized short-blasted sample and the mixture was extracted at 85 ° C for 12 hours. After centrifuging at 3500 rpm for 10 minutes, only the supernatant was sampled and the solid content was measured. Lt; / RTI &gt;

<1-2> Preparation of ethanol extract

The dried cutworm (purchased in the market as a wild alive native to Gyeongbuk province) was crushed and weighed to a dry weight of 200 g. 70% ethanol was added to the crushed deodorant sample at a concentration of 10 times (v / w), and the mixture was extracted at room temperature for 10 hours. The supernatant was taken through a centrifuge (3500 rpm, 10 minutes) Was used for the experiment.

Example 2. Preparation of biotransformed odor pans

<2-1> Production of single-pest seaweed extract fermented using mushroom mycelium

Mushroom mycelia were used to convert bioluminescent extracts of leaves into 11 species. The mycelia of each mushroom were cultured on an optimal medium, and 5 mL of the mushroom mycelium (5 v / v) was inoculated to 50 mL of the hot air extract of the A. pneumoniae prepared in Example <1-1>, followed by fermentation for 7 days. The resultant was centrifuged, and only the supernatant was taken to obtain a hot-water extract of monodoxic persimmon fermented with mushroom mycelia. Table 1 below shows the types of mushroom mycelium and the culture conditions used in this Example. The mushroom mycelium shown in Table 1 was sold by the Korean Microorganism Conservation Center (KCCM) or Korea Biotechnology Research Center (KCTC). In the present embodiment, biotransformation was performed using a hot air extract of a single-breeze. However, the present invention is not limited thereto. Even when biotransformation is performed using the single-bladed ethanol extract in Example <1-2> Can be obtained.

Mushroom mycelium Culture conditions badge Temperature (℃) rpm Cloudy mushroom (Coriolus esicola) PDA 24 160 Mushroom (Agaricus bisporus) PDA 24 160 Ganoderma lucidum PDA 24 160 Horsetail mushroom (Fomes fomentarius) PDA 24 160 Leaf mushroom (Grifola umbellata) PDA 26 160 Mushroom (Phellinus linteus) YM 24 160 Agaricus mushroom (Agaricus blazei murii) YM 24 160 Pleurotus eryngii YM 24 180 Mushroom (Gymnopilus spectabilis) Malt 18 180 Mushroom (Leposta nude) Malt 25 160 Schizophyllum commune Malt 24 180 Mushroom (Inonotus obliqus) Malt 25 160

(PDA = Patoato Dextrose Agar, YM = Yeast Malt)

&Lt; 2-2 > Production of monodystrophy extract obtained by fermentation using lactic acid bacteria

Lactobacillus acidophilus and six kinds of lactic acid bacteria and two common bacteria were used for biotransformation of the single blight extract using lactic acid bacteria. Each lactic acid bacterium was cultured on an optimal medium. Then, 5 mL of lactic acid bacteria or Bacillus bacteria (5% v / v) was inoculated to 50 mL of the hot air extract of the dried breeze from Example <1-1> and fermented for 2 days. The resultant was centrifuged and only the supernatant was taken to obtain a hot-air extract of monodoric breeze fermented with lactic acid bacteria or Bacillus bacteria. Table 2 below shows the kinds of lactic acid bacteria and Bacillus bacteria used in this example and the culture conditions. In the present embodiment, biotransformation was performed using a hot air extract of a single-breeze. However, the present invention is not limited thereto. Even when biotransformation is carried out using the ethanol extract of A. barki in Example <1-2> Can be obtained.

Strain Culture conditions badge Temperature (℃) rpm Lactobacillus acidophilus
( Lactobacillus acidophilus ) MRS 37 150 Lactobacillus casei
( Lactobacillus casei ) MRS 37 150 Rucono Stock Lactis Asiatic Lactis
( Leuconostoc lactis subsp. Lactis ) MRS 37 150 Streptococcus thermophilus
( Streptococcus thermophilus ) MRS 37 150 Lactobacillus Florata Room
( Lactobacillus plantarum ) MRS 37 150 Lactobacillus planta room KCCM11322
( Lactobacillus plantarum KCCM 11322) MRS 37 150

(MRS = de Man Rogosa Sharpe, YPD = Yeast Peptone Dextrose, and Lactobacillus plantarum KCCM 11322 were purchased from the Korean Center for Microbiological Conservation (KCCM).)

&Lt; 2-3 > Preparation of an extract of an antiseptic enzyme

Seventeen enzymes produced by Merck, Novo Nordisk, and Sigma-Aldrich were used for enzymatic treatment of cutworms to obtain the extract. To select enzymes with excellent activity, 5 μL of the enzyme solution was added to 1 mL of the substrate and allowed to react at a constant reaction temperature for 12 hours. As an experimental condition for the enzyme treatment, a substrate having a pH of 5.0 or 7.0 was prepared, and the reaction temperature was set at 37 ° C or 50 ° C. The reaction was then stopped by heating in boiling water for 10 minutes to inactivate the enzyme, and the supernatant was obtained by centrifugation for 10 minutes in a centrifuge (12,000 rpm). Table 3 below shows the enzymatic activities of the enzymes used in the enzymatic treatment of this example. One unit of enzymatic activity unit means that 1 μmol of substrate per minute is converted to produce reducing sugar.

Cellulose activity [unit = unit] No. Product Name (Manufacturer) pH = 5.0
37 ℃ pH = 5.0
50 ℃ pH = 7.0
37 ℃ pH = 7.0
50 ℃ One Sprizyme plus (M) 9.93 9.61 2.41 2.04 2 Viscozyme (M) 16.50 15.45 5.42 5.58 3 AMG300L (N) 10.21 9.32 3.50 2.89 4 Viscozyme (N) 16.65 16.57 3.91 5.17 5 Pectinex ultra (M) 7.61 7.71 1.12 1.14 6 Fungamyl (M) 3.24 5.38 1.15 1.18 7 Celluclast (N) 11.34 11.82 8.51 1.12 8 Promosy (N) 12.97 9.65 2.15 7.59 9 Cytolase (M) 8.10 7.90 1.18 1.17 10 Pectinex (M) 13.18 26.61 1.16 1.18 11 Termamyl (N) 3.35 5.32 1.13 1.09 12 Shearzyme (N) 11.33 11.23 1.18 1.18 13 Citrozyme (M) 13.29 6.73 1.26 1.09 14 Pectinex (N) 13.05 7.04 1.29 1.20 15 Fungamyl (N) 4.37 5.08 1.21 1.15 16 Cellulase from Aspergillus aculeatus (S) 12.29 6.11 1.28 1.22 17 Pectinase from Aspergillus spices (S) 7.83 8.60 1.26 1.10

(M = Merck, N = Novo Nordisk, S = Sigma-Aldrich)

As shown in Table 3, the enzymatic activity of Viscozyme and Promozyme was relatively good, but the present invention is not limited thereto.

In order to biotransform the cutworm odor using the enzyme treatment, first, the dried cutworm odor at room temperature was pulverized and weighed so that the dry weight was 200 g. 200 ppm of the above-mentioned enzyme was added to the pulverized short-blasted sample, followed by reaction at 37 ° C or 50 ° C for 12 hours to obtain an enzyme-treated first-strand extract of dried pine needles. Next, distilled water (v / w) 10 times its weight was added to the first extract, followed by secondary extraction at 85 ° C for 12 hours. 5 mL of the above-mentioned <2-2> lactic acid bacterium (v / v) was inoculated to 50 mL of the hot-air extract, and fermented for 48 hours. The resultant was centrifuged, and only the supernatant was taken to obtain an extract of treated with the pale-airflow enzyme. In the present embodiment, the secondary extract was obtained using hot water extraction. However, the present invention is not limited to this, and the same operation and effect can be obtained even when the ethanol extraction in the embodiment <1-2> is used instead of the hot water extraction . In addition to the fermentation by the lactic acid bacteria used in the present embodiment, substantially the same action and effect can be obtained even if the fermentation step using the mushroom mycelium in the above Example <2-1> is carried out.

Example 3. Validation of Physiological Functional Effectiveness of Odor Blast

<3-1> Antioxidant Efficacy Measurement

a) DPPH radical scavenging rate

In order to measure the antioxidative effect of the extract of the present invention against the electron donating ability (EDA), the Blois method using DPPH (1,1-diphenyl-2-picrylhydrazyl) (Blois MS, Antioxidant determination by the of a stable free radicals (Nature 1954; 181: 1198-1200). 0.050 mL of 0.2 mM DPPH solution was added to 0.1 mL of each sample solution, and the mixture was left to stand for 10 minutes. Then, the absorbance was measured at 517 nm using a spectrophotometer. As shown in the following formula 1, the DPPH radical scavenging rate was expressed as a percentage (%) in absorbance difference between the addition group and the no addition group of the sample solution.

[Formula 1]

DPPH radical scavenging rate (%) = [1 - (absorbance of sample addition group) / (absorbance of no addition group)] × 100

In addition, the DPPH radical scavenging activity was represented by the concentration value (DPPH RC 50 ) of the sample required to reduce the absorbance of the control group to which the sample was not added, that is, the no-added group, to ½.

b) ABTS radical scavenging ability

In order to measure the antioxidative effect of the extract of the present invention, ABTS radical cation decolorization method (Re R, Pellegrini N, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying and improved ABTS radical cation decolorization 1999, 26: 1231-1237). 7 mM ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) and 2.45 mM potassium persulfate were mixed at a final concentration and left in a dark room for 24 hours ABTS was formed and diluted with ethanol to a absorbance value of 0.70 (± 0.02) at 732 nm using a spectrophotometer. 0.05 mL of the sample was added to 0.1 mL of the diluted solution, and the solution was allowed to stand for 6 minutes. Then, the absorbance was measured at 734 nm using a spectrophotometer. The ABTS radical scavenging activity was measured by the antioxidant activity measurement using ABTS + , which was obtained by the reaction with potassium persulfate, by the antioxidative substance in the sample, and decolorized. In the case of DPPH radical scavenging ability, free radicals were eliminated, while ABTS was a cation radical And the degree of binding between the two substrates and the reactants is different. The ABTS radical scavenging activity was expressed as the concentration of the sample (ABTS RC 50 ) required to reduce the absorbance of the control without addition of the sample to 1/2.

<3-2> Measurement of anti-inflammatory effect

a) Cytotoxicity measurement

The cytotoxicity of the extract of the present invention was measured by MTT (3- (4,5-methylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay. RAW264.7 cells (1 × 10 5 cells / mL) were cultured overnight in 0.1-mL DMEM (dulbecco's modified eagle medium) in 96-well plates, and each sample was treated for each concentration and cultured for another 24 hours . A reducing reaction was induced by adding 0.01 mL of 5 mg / mL MTT solution to each well and culturing for 3 hours. The formed purple formazan crystals were completely dissolved by adding 0.1 mL of dimethyl sulfoxide (DMSO) solution. The degree of color development was measured at 570 nm using a microplate reader. Cytotoxicity was expressed using the relative cell viability of the drug-treated group based on the survival rate of 100% of the untreated group cultured only with the cells.

b) Measurement of NO (Nitric oxide) content

Further, in order to verify the NO (nitric oxide) inhibitory activity of the extract of the present invention, NO production amount was measured. Nitrite (nitrite, NO 2-) Griess reagent concentrations Reaction present in the cell culture to measure the amount of NO (Wang S, Chen Y, He D, He L, Yang Y, Chen J, Wang X 2007 Inhibition (3)) 458 - 462). The results are shown in Table 1. The results are shown in Table 1. The results are shown in Table 1. In other words, each well was pretreated with LPS at a concentration of 1 μg / mL for 1 hour, and RAW264.7 cells were treated with various concentrations of the drug and incubated for 24 hours. The cell culture medium was collected, and the same amount of Griess reagent (0.1% NED / 1% sulfanilamide in 5% H 3 PO 4 ) was added to 0.1 mL of the culture solution. The degree of color development was measured at 540 nm using a microplate reader. The NO inhibitory activity of each cell line was determined based on the difference in the relative amount of nitrite generated in the LPS-only control group and the LPS-untreated control group (% of control).

c) Western blot

In addition, westen blot analysis was performed to confirm the effect of the samples on iNOS and COX-2 protein expression. Samples were cultured in a culture dish using each cell line, followed by sample treatment, and then washed with PBS 24 hours later. Cells were then collected by centrifugation and cell lysate was prepared using RIPA buffer. Bradford method was used to measure the amount of protein contained in the cell lysate. 20 μg of the protein was separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (polyvinylidenedifluoride membrane) at 120 V for 1 hour. Then, the PVDF membrane was left in 5% skim milk for 1 hour, and the primary antibody was reacted overnight at 4 ° C. After washing three times with TBST (Tris-buffered saline and tween 20), the secondary antibody was reacted for 1 hour at room temperature. After washing three times with TBST, the ECL kit (Amersham-Pharmacia Biotech, Piscataway, And exposed to an X-ray film. Biorad geldoc was used to quantify the developed band on the x-ray film.

<3-3> Anti-obesity Efficacy Verification

a) Cell viability measurement

Cytotoxicity assays were performed according to the method of Carmichael et al. (Carmichael, J., WG DeGraff, AF Gazdar, JD Minna, and JB Mitchell, 1987. Evaluation of a tetrazolium based semimicrotomic colorimetric assay. Cancer Res. 47: 936- 942.). RAW264.7 cells were dispensed in a 96-well plate at a concentration of 1 × 10 4 cells / well in an amount of 0.18 mL. Each sample was added at a concentration of 0.02 mL and cultured at 37 ° C. in a 5% CO 2 incubator for 24 hours. In the control group, the same amount of distilled water as that of the sample was added and the cells were cultured under the same conditions. After adding 0.02 mL of the MTT solution prepared at the concentration of 5 mg / mL for 4 hours, the culture solution was removed. 0.1 mL of DMSO was added to each well, followed by reaction at room temperature for 30 minutes, and the absorbance was measured at 550 nm using an ELISA reader. As shown in the following Equation 2, the cell viability was expressed as a percentage (%) of the absorbance difference between the addition group and the no addition group of the sample solution.

[Formula 2]

Cell survival rate (%) = [1 - (absorbance of sample addition group) / (absorbance of no-addition group)] × 100

b) 3T3-L1 culture and induction of differentiation

Mouse embryo 3T3-L1 whole fat cell line was purchased from American Type Culture Collection (ATCC). The cells were suspended in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin streptomycin at a concentration of 2.5 × 10 5 cells / ml and incubated for 72 hours at 37 ° C. in a 5% CO 2 incubator The sample was treated while inducing differentiation when confluent. At the onset of differentiation, the cells were replaced with differentiation medium containing 10% FBS, 1% penicillin streptomycin, 5 μg / ml insulin, 1 μM DEX (dexamethasone) and 0.5 mM IBMX (3-isobutyl-1-methylxanthine) Lt; / RTI &gt; After that, the cells were cultured for 48 hours in DMEM supplemented with 10% FBS and 5 μg / ml insulin to promote differentiation into adipocytes. Followed by 2 changes in 10% FBS DMEM medium at 48 hour intervals to induce differentiation into adipocytes.

c) Oil-red staining of cells using oil red O

And stained with Oil red O that specifically reacted to lipids for lipid droplet staining of differentiated 3T3-L1 adipocytes. After removing the medium, 1 mL of 10% formalin was treated once for 5 minutes and then treated with 1 mL of fresh 10% formalin for 1 hour. After washing with 60% isopropanol, each well was thoroughly dried. Oil red O stock was treated for 10 minutes and washed four times with distilled water. Then, the cells differentiated by the microscope were observed to confirm that the differentiation was inhibited. After observation, the wells from which distilled water had been removed were completely dried. For the elution of oil red O combined with the lipid, 100% isopropanol was treated for 10 minutes and absorbance was measured at 520 nm.

<3-4> Tyrosinase inhibitory effect (Tyrosinase inhibitory effect)

After centrifuging the enzyme extract, add 0.2 mL of 5 mM L3,4-dihydroxyphenylalanine (L-DOPA) and 0.2 mL of 0.1 M sodium phosphate buffer (pH 6.0) to 0.5 mL of the supernatant and add 1 unit of tyrosinase And the mixture was reacted at 35 DEG C for 2 minutes. The absorbance (B Abs ) obtained by adding 0.1 mL of distilled water instead of the supernatant of the change of the initial absorbance (S Abs ) changed by the unit time at 475 nm and the centrifuged sample, the value obtained by adding 0.5 mL of distilled water instead of the sample solution , And the absorbance was measured by the same method (C Abs ) to calculate tyrosinase inhibitory activity as shown in the following Equation 3.

[Formula 3]

Tyrosinase inhibitory activity (%) = [1- (S Abs- B Abs ) / C Abs )] × 100

In addition, the inhibitory concentration required to reduce the activity of tyrosinase by 50% was expressed as IC 50 (μg / mL).

<3-5> Measurement of ACE (Angiotensin converting enzyme) inhibitory activity

Measurement of ACE inhibitory activity was performed according to the method of Cushman and Cheung (Cushman DW, Cheung HS, 1971. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem P harmacol 20: 1637-1648). 0.05 mL of the substrate (L-hippuryl-histidyl-leucine: HHL, Sigma-Aldrich Co.) was added to 0.05 mL of the supernatant liquid sample and left at 37 ° C for 5 minutes. After adding 0.05 mL of the ACE solution and reacting at 37 ° C for 1 hour, the reaction was stopped by adding 0.25 mL of 1 N HCl solution. Then, 1.5 μL of ethyl acetate was added, homogenized for 15 seconds, centrifuged at 5,000 × g for 10 minutes, and 1 μL of supernatant was taken. The supernatant was heated at 80 ° C for 1 hour to completely dry, and 3 ml of 1 M NaCl solution was added and dissolved. Then, the absorbance at 228 nm was measured to calculate the ACE inhibitory activity as shown in Equation 4 below.

[Formula 4]

ACE inhibitory activity (%) = [(B-A) / (B-C)] 100

(Where A = absorbance at the time of adding the sample, B = absorbance at the time of adding distilled water instead of the sample, and C = absorbance at the time of addition of the sample after stopping the reaction)

<3-6> Measurement of antimutagenic activity

To determine the antimutagenic activity, we used the preincubation test with an improved Ames test. Specifically, 0.5 ml of a sodium phosphate buffer solution or S9 mix (S9 mix), 0.1 ml of S. enterica serovar Typhimurium culture solution (0.1 ml) was added to a sterilized glass cap tube while the test tube was kept in an ice bath. mL, 0.05 mL of each of the sample and mutant were added, mixed, and cultured at 37 ° C for 30 minutes. Then, 3 mL of the top agar at 45 ° C was dispensed into each glass cap tube test tube, mixed for 3 seconds, and then layered on MGA (minimal glucose agar) medium and cultured at 37 ° C for 48 hours. After measuring the number of his + revertant colonies grown on MGA medium, the inhibition ratio of return mutation was calculated according to the following equation (5).

[Formula 5]

Return mutation inhibition rate (%) = [(a-b) / (a-c)] 100

(Where, a = variation induced by the one-His + reverse mutation number of colonies, b = transition source and the sample number of His + reverse mutation colonies derived during processing, c = transition source and sample the His + return induction untreated Number of mutant colonies)

Reference Example 1: Statistical processing

Statistical analysis was performed using the SPSS v12.0 (SPSS Inc., Chicago, IL, USA) program and the results were expressed as mean ± SD. For each variable, One-way ANOVA was performed. Duncan's multiple range test was used for post-test and the significance was verified at P <0.05 level.

EXPERIMENTAL EXAMPLE 1. Antioxidative Effectiveness of the Hot Water Extract of the Single Wind Blast and the Ethanol Extract of the Single Wind Blast

Table 4 below shows the measured DPPH radical scavenging activity of DPPH RC 50 for the hot-water extract and the 70% ethanol extract of the single-blind spot according to an embodiment of the present invention.

sample DPPH RC 50 (μg / mL) Hot water extract 368.34 ± 16.09 70% Ethanol Extract 122.67 + - 3.58

Table 5 below shows the ABTS radical scavenging activity of ABTS RC 50 measured for the hot-water extract and 70% ethanolic extract of the monochoria bark according to one embodiment of the present invention.

sample ABTS RC 50 (μg / mL) Hot water extract 185.72 + 5.06 70% Ethanol Extract 115.10 + - 6.58

As shown in Tables 4 and 5, the DPPH RC 50 was found to be 368.34 μg / mL and the ABTS RC 50 was found to be 185.72 μg / mL in the case of the single-stranded hot-water extract according to an embodiment of the present invention. 50 was 122.67 μg / mL and ABTS RC 50 was 115.10 μg / mL. Thus, the antioxidant activity of both the hot and cold water extracts as well as the ethanol extract of A. Furthermore, the ethanol extract of the present invention exhibited a relatively superior antioxidant activity than the hot water extract of the family P. monodon.

Experimental Example 2. Measurement of anti-obesity efficacy of hot water extract of leafy odor and ethanol extract

Table 6 below shows anti-obesity efficacy using Oil red O staining of the single-blasted hot-water extract and the single-bladed 70% ethanol extract according to one embodiment of the present invention. Here, the MDI referred to as a sample represents a control group for adipocytes not treated with the monocotyledonous extract as a control group cultured using only MDI (0.5 μM IBMX, 1 μM DEX, 10 μg / ml insulin) as a differentiation inducing substance.

sample Oil red O staining (% of MDI) MDI 100 Hot water extract 0.05 (mg / mL) 88.52 + 2.09 0.2 (mg / mL) 63.92 + - 3.41 70% Ethanol Extract 0.05 (mg / mL) 83.49 + 4.18 0.2 (mg / mL) 29.22 + 2.06

As shown in Table 6, both the hot-water extract of the single-bladder and the ethanol extract of the single-blind airflow according to an embodiment of the present invention showed excellent adipocyte differentiation inhibitory activity. In other words, compared with the control group cultured with MDI alone, the amount of triglyceride was decreased in a concentration-dependent manner in the treatment group treated with 0.05 and 0.2 mg / mL for each of the hot airwax extract and the deodorized ethanol extract. Furthermore, triglyceride was reduced to about 70% at 0.2 mg / mL of ethanol extract of the cutworm.

Experimental Example 3. Measurement of anti-inflammatory activity of the hot-water extract and the ethanol extract

Table 7 below shows the cell survival rate and the amount of NO production measured to examine anti-inflammatory effects on the hot-water extract of monoculture and the ethanol extract of monocotyledonous fungus according to an embodiment of the present invention.

First, MTT assay was used to determine the cytotoxicity of Raw 264.7 cells from both the hot and cold water extracts of leaves and the ethanol extract of the leafhopper. The cell survival rate was expressed as the relative survival rate of the treatment group treated with the solar wind plexus extract on the basis of the survival rate of 100% of the untreated group cultured only with the cells. The results showed that both the hot and cold water extracts and the ethanol extracts of the sweet - breeze were similar to those of the untreated group and showed no toxicity at all.

In addition, the amount of NO produced when Raw 264.7 cells were treated with LPS was increased by four times as compared with the untreated group. When treated with 100 μg / mL of monohydrate extract, the NO production was reduced to less than 20% compared with the LPS-treated group. Furthermore, when treated with 100 μg / mL of ethanol extracts from leaves, the production of NO decreased to less than 30% compared to the LPS-treated group. Therefore, it can be seen that the ethanol extract of the present invention has an anti-inflammatory effect by inhibiting the production of NO, an inflammatory factor.

sample Cell survival rate
(cell viability,%) NO production (%) Untreated group 100 23.97 ± 1.85 LPS - 100 Hot water extract 103.34 + 3.94 79.42 ± 1.90 70% Ethanol Extract 104.13 ± 3.99 66.76 + - 4.22

EXPERIMENTAL EXAMPLE 4. Physiological Functional Measurement of Enzymatically Treated Deep-blight Extract

<4-1> Antioxidative Effect of Enzymatically Treated Leaf Blight Extract

Table 8 below shows the ABTS radical scavenging activity (ABTS RC 50 ) and total phenol content measured for biotransformed odor pests using an enzyme according to one embodiment of the present invention. In addition, in general, the radical scavenging activity of ABTS and DPPH is known to increase as the phenolic substance content increases (Kang YH, Park YK, Oh SR and Mood KD: Studies on the Physiological Function of Pine Needle and Mugwort Extracts. J. Food Sci. Technol., 27, 978-984 (1995)).

sample Total phenol content (mg / g) ABTS RC 50 (μg / mL) Enzymatic deodorant extract 94.31 + - 4.58 121.11 ± 46.21

As shown in Table 8, the antioxidant efficacy of ABTS RC 50 of 121. 1 μg / mL was confirmed by the enzyme treated monodoric breeze extract according to an embodiment of the present invention.

<4-2> Determination of tyrosinase inhibitory activity of the enzyme treated monodyne leaf extract

Table 9 below shows the tyrosinase inhibitory activity measured for biotransformed odor pests using an enzyme according to an embodiment of the present invention.

sample Tyrosinase inhibitory activity (%) Enzymatic deodorant extract 71.38

The biosynthetic pathway of the main production process of melanin pigment is formed by polymerization reaction with amino acids and proteins via derivatives such as dopaquinone produced by tyrosinase enzymatic action using tyrosine as a starting material (Lerner and Fitzpatrick, 1950; Pawelek and Korner, 1982). Therefore, a whitening effect can be expected by inhibiting tyrosinase enzyme itself, which is an enzyme of melanin production. As shown in Table 9, the tyrosinase inhibitory activity of the enzyme treated monodysteria rumen extract according to an embodiment of the present invention was measured to be 71.38%, and it was confirmed that there was an excellent whitening effect.

<4-3> Measurement of ACE inhibitory activity of enzyme treated monodyne leaf extract

Table 10 below shows the ACE (Angiotensin converting enzyme) inhibitory activity measured for biotransduced odor pests using an enzyme according to an embodiment of the present invention. In this Example, a viscozyme having excellent activity was used for enzyme treatment, but the present invention is not limited thereto, and various enzymes listed in Table 3 may be used.

sample ACE inhibitory activity (%) Enzymatic deodorant extract 59.21

Hypertension is one of the major risk factors for atherosclerosis, stroke, or myocardial infarction. ACE is known to contract arteries, causing direct vasoconstriction and increasing blood volume, thereby increasing blood pressure (Soffer, 1976; Ganong, 1997). As shown in Table 10, the enzyme treated monodoric breeze extract according to an embodiment of the present invention has an excellent ACE inhibitory activity of 59.21%.

<4-4> Antimutagenic Activity of Enzymatically Treated Leaf Blight Extract

Table 11 below shows the result of measuring the antimutagenic activity by carrying out the Ames test for the biotransduced odor of leaves by using the enzyme according to an embodiment of the present invention. Specifically, S. mutans serovar Typhimurium TA100 and TA98 were used to investigate the mutagenic effect of biotransduced monocultures against three direct mutagenic agents (MNNG, NPD, and NQD) with different concentrations of cutoffs. In the case of S. enterica serovar Typhimurium TA100, a high reversion mutation inhibition rate was measured when 75-150 μL of the cutworm was added. Preferably, 100 to 125 μL of the odor blotting was added to obtain a more excellent mutation inhibiting effect. More preferably, the antimutagenic activity against mutagenic MNNG and NPD was 61.34% and 51.85%, respectively, when 100 μL of monoculture was added.

Likewise, in the case of S. enterica serovar Typhimurium TA98, a high reversion mutation inhibition rate was also measured when 75-150 μL of biotransduced monocots were added. Preferably, 100 to 125 μL of the odor blotting was added to obtain a more excellent mutation inhibiting effect. More preferably, the antimutagenic activity against the mutagen NQO and NPD was 50.47% and 40.12%, respectively, when 100 μL of the ventilator was added.

sample Addition amount
(μL) TA100 TA98 MNNG
(5 μg / plate) NPD
(15 μg / plate) NQO
(2.5 μg / plate) NPD
(0.25 μg / plate) Biotransformed odor blast using enzymes 25 10.38 7.75 6.11 4.33 50 25.64 20.42 18.47 15.29 75 58.39 48.55 42.22 33.67 100 61.34 55.85 50.47 40.12 125 61.05 55.13 49.23 39.68 150 58.79 52.17 47.26 37.46

EXPERIMENTAL EXAMPLE 5. Measurement of physiological function of a single-pest extract obtained by fermentation using mycelium of mushroom

<5-1> Antioxidative Effect of Leaf Blight Extracts Fermented by Mycelial Mycelia

Tables 12 and 13 below show DPPH and ABTS radical scavenging activities measured for biotransformed odor pans using DPPH RC 50 and ABTS RC 50 , respectively, using mushroom mycelia according to one embodiment of the present invention.

sample Mushroom strain name DPPH RC 50 (μg / mL) Control group - 173.91 ± 36.11 Biotransformed odor of broodstock using mushroom mycelium Cloudy mushroom (Coriols esicola) 208.64 ± 20.88 Mushroom (Agaricus bisporus) 193.21 + - 19.52 Ganoderma lucidum 259.83 + - 22.48 Horsetail mushroom (Fomes fomentarius) 211.14 ± 25.47 Leaf mushroom (Grifola umbellata) 159.38 ± 16.81 Mushroom (Phellinus linteus) 189.09 ± 15.87 Agaricus mushroom (Agaricus blazei murii) 196.17 ± 19.11 Pleurotus eryngii 218.34 + 24.85 Mushroom (Gymnopilus spectabilis) 253.39 ± 25.66 Mushroom (Leposta nude) 261.24 + - 22.14 Schizophyllum commune 201.29 ± 23.57 Mushroom (Inonotus obliqus) 213.47 ± 21.12

As shown in Table 12, DPPH RC 50 was measured to be 159.38 μg / mL in the case of biotransformation of the hot-water extract from the leaf mushroom mycelium, and compared with DPPH RC 50 (= 173.91 μg / mL) And it was found to have efficacy.

sample Mushroom strain name ABTS RC 50 (μg / mL) Control group - 166.47 ± 31.85 Biotransformed odor of broodstock using mushroom mycelium Cloudy mushroom (Coriols esicola) 180.55 + - 33.66 Mushroom (Agaricus bisporus) 189.44 + - 38.23 Ganoderma lucidum 195.68 ± 33.22 Horsetail mushroom (Fomes fomentarius) 189.11 + - 24.66 Leaf mushroom (Grifola umbellata) 161.34 ± 29.24 Mushroom (Phellinus linteus) 177.36 ± 25.78 Agaricus mushroom (Agaricus blazei murii) 206.33 ± 31.91 Pleurotus eryngii 186.27 + - 34.58 Mushroom (Gymnopilus spectabilis) 198.07 ± 38.11 Mushroom (Leposta nude) 209.22 + - 42.11 Schizophyllum commune 193.81 + - 33.75 Mushroom (Inonotus obliqus) 196.78 ± 35.21

As shown in Table 13, the ABTS RC 50 was 161.34 μg / mL when biotransformation of the hot-water extract from the leafy mushroom mycelia was measured and compared with the control ABTS RC 50 (= 166.47 μg / mL) And it was found to have efficacy.

<5-2> Tyrosinase Inhibitory Activity of Leaf Blight Extracts Fermented with Mycelia of Mycelia

Table 14 below shows the IC 50 for tyrosinase inhibitory activity measured against biotransformed odor pests using mushroom mycelium according to an embodiment of the present invention.

sample Mushroom strain name IC 50 (μg / mL) Control group - 1027.33 + - 65.58 Biotransformed odor of broodstock using mushroom mycelium Cloudy mushroom (Coriols esicola) - Mushroom (Agaricus bisporus) - Ganoderma lucidum 1135.99 ± 53.66 Horsetail mushroom (Fomes fomentarius) 1237.58 + - 66.42 Leaf mushroom (Grifola umbellata) 989.63 + - 59.64 Mushroom (Phellinus linteus) 995.36 + - 57.82 Agaricus mushroom (Agaricus blazei murii) - Pleurotus eryngii 1033.87 + - 54.83 Mushroom (Gymnopilus spectabilis) - Mushroom (Leposta nude) - Schizophyllum commune - Mushroom (Inonotus obliqus) -

As shown in Table 14, the IC 50 values were 989.63 μg / mL and 995.36 μg / mL, respectively, when biotransformation of the hot-water extract from the leafy mushroom and mushroom mycelia was carried out, and the IC 50 (= 1027.33 μg / lt; / RTI &gt; mL), it was found to have an effect of effectively inhibiting tyrosinase activity.

Experimental Example 6. Measurement of Physiological Functionality of the Extract of Monascus Bleed with Fermented Lactobacillus

Table 15 below shows the ABTS-scavenging activity of the extract of the single-puff extract fermented using lactic acid bacteria according to an embodiment of the present invention. In other words, ABTS RC 50 (μg / mL) was measured after fermentation of the hot water extract of the single-blasted wastewater using six kinds of lactic acid bacteria for 48 hours.

sample Strain name ABTS RC 50 (μg / mL) Control group 163.33 + - 33.27 A single blow Lactobacillus acidophillus 157.10 + - 43.11 Lactobacillus casei 156.57 ± 40.27 Leuconostoc lactis subsp. lactis 150.11 ± 36.51 Streptococcus thermophillus 145.41 ± 35.56 Lactobacillus plantarum 135.51 + - 23.44 Lactobacillus plantarum KCCM11322 123.25 + 20.57

As shown in Table 15, when compared with the control group ABTS RC 50 , excellent antioxidant efficacy was confirmed by using lactic acid bacteria. Especially, when Lactobacillus plantarum KCCM 11322 was used as a lactic acid bacterium, it was confirmed that ABTS cleavage ability was excellent.

Experimental Example  7. A single blow  Sensory properties of beverages including extracts

Table 16 below shows the sensory characteristics of a beverage to which a single-puff extract is added according to an embodiment of the present invention.

sample Addition amount (v / v%) Incense (odor) sweetness Sour taste Overall appeal Single blight extract 5 3.68 3.57 3.36 3.46 10 4.07 4.53 4.34 4.63 15 3.96 4.38 4.46 4.31

As shown in Table 16, when the beverage was prepared by adding 5 to 15 v / v% of the single-puff extract according to an embodiment of the present invention, the overall acceptability was relatively high in the sensory test. The highest favorability was obtained when 10% v / v%

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, You will understand. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (10)

delete delete delete Water or ethanol is added to the ground single-blasted sample to obtain a single puff extract; Obtained by inoculating mycelia of leaf mushroom with the above-mentioned single-puff extract; A health functional composition for skin whitening comprising fermented monodyne extract as an active ingredient. delete delete delete Adding enzymes to the ground swine samples to effect enzyme treatment; Adding water or ethanol to the resulting enzyme-treated product to obtain a single-bladder extract; Obtained by inoculating lactic acid bacteria into the above-mentioned single-puff extract; A health-functional composition for skin whitening comprising fermented soybean extract as an active ingredient. 9. The method of claim 8,
Wherein the enzyme is Viscozyme or Promozyme, and the lactic acid bacterium is Lactobacillus plantarum KCCM 11322. A functional whitening beverage for skin whitening comprising the health functional composition according to any one of claims 4, 8 and 9.

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KR20180013797A (en) * 2016-07-28 2018-02-07 안동대학교 산학협력단 Vinegar with enhanced anti-obesity, anti-inflammatory and skin whitening effect using Mapleleaf ainsliaea and manufacturing method thereof
KR20210087734A (en) * 2020-01-03 2021-07-13 안동대학교 산학협력단 Method for producing stick type beverage using Ainsliaea acerifolia extract powder
KR20220003893A (en) * 2020-07-02 2022-01-11 충남대학교산학협력단 Composition for preventing, improving or treating prostate disease comprising extract of Ainsliaea acerifolia as effective component

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KR102313648B1 (en) * 2018-12-24 2021-10-18 참자연마을 영농조합법인 Method for producing Chinese cabbage Kimchi with enhanced anti-obesity using Mapleleaf ainsliaea

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KR20180013797A (en) * 2016-07-28 2018-02-07 안동대학교 산학협력단 Vinegar with enhanced anti-obesity, anti-inflammatory and skin whitening effect using Mapleleaf ainsliaea and manufacturing method thereof
KR101937331B1 (en) 2016-07-28 2019-01-11 안동대학교 산학협력단 Vinegar with enhanced anti-obesity, anti-inflammatory and skin whitening effect using Mapleleaf ainsliaea and manufacturing method thereof
KR20210087734A (en) * 2020-01-03 2021-07-13 안동대학교 산학협력단 Method for producing stick type beverage using Ainsliaea acerifolia extract powder
KR102395458B1 (en) * 2020-01-03 2022-05-10 안동대학교 산학협력단 Method for producing stick type beverage using Ainsliaea acerifolia extract powder
KR20220003893A (en) * 2020-07-02 2022-01-11 충남대학교산학협력단 Composition for preventing, improving or treating prostate disease comprising extract of Ainsliaea acerifolia as effective component
KR102376521B1 (en) 2020-07-02 2022-03-21 충남대학교산학협력단 Composition for preventing, improving or treating prostate disease comprising extract of Ainsliaea acerifolia as effective component

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