KR101552821B1

KR101552821B1 - Enzymatic hydrolized Formica truncicola extract powder having vasodilatation, sexual function improvement and anti-inflammatory effect

Info

Publication number: KR101552821B1
Application number: KR1020150112211A
Authority: KR
Inventors: 김용수; 백영미; 이수형; 한진택; 정택근; 김완영; 심태경; 장성일; 전용우; 이수정; 채종걸; 김대겸; 김병학; 정영목; 이대웅; 최한나; 고대경; 서지영; 박효진; 박성현
Original assignee: 김용수; 백영미; 이수형; 한진택; 정택근; 김완영; 심태경; 장성일
Priority date: 2015-08-10
Filing date: 2015-08-10
Publication date: 2015-09-16

Abstract

The present invention relates to a method for manufacturing Formica truncicola enzymatic decomposition extract powder having effects of sexual function enhancement and anti-inflammation, Formica truncicola enzymatic decomposition extract powder manufactured by the same method, and processed food containing the same Formica truncicola enzymatic decomposition extract powder. The method for manufacturing Formica truncicola enzymatic decomposition extract powder includes the steps of: (a) immersing dried Formica truncicola in an alkali solution and the taking Formica truncicola out; (b) adding an ethanol solution to the Formica truncicola, taken out in step (a), extracting and filtrating the Formica truncicola to separate an ethanol extract; (c) separating the ethanol extract, obtained in step (b), and adding water and a protein decomposition enzyme into a remaining sold matter of the Formica truncicola to extract the a remaining sold matter of the Formica truncicola; (d) mixing, filtrating, and then drying the extract, obtained in step (c), and the ethanol extract, obtained in step (b); and (e) adding dextrin and water to a dried Formica truncicola extract, obtained in step (d), spraying and drying the dried Formica truncicola extract.

Description

[FIELD OF THE INVENTION] [Technical Field] The present invention relates to an antimicrobial enzyme-degrading extract powder having vasodilatation, enhanced sexual function and anti-inflammatory effect,

(A) immersing and drying the dried firefly in an aqueous alkaline solution; (b) adding an aqueous ethanol solution to the firewood taken out in step (a), extracting it, filtering and separating the ethanol extract; (c) separating the ethanol extract of step (b) and adding water and proteolytic enzyme to the remaining firewood to extract the extract; (d) mixing the extracted extract of step (c) and the ethanol extract of step (b), filtering and drying; And (e) adding dextrin and water to the dry seaweed extract of step (d) and spray drying the seaweed extract. The method of claim 1, And a processed food containing the fragrant enzyme decomposition extract powder and the fragrant enzyme decomposition extract powder produced by the above method.

Fireworks ( Formica truncicola ) is a species of ants belonging to the ants. The body has a dark red color and a light yellow hairs throughout the body. Fire ants live in rotten trees, soil, and stones in Korea, Japan, China, Taiwan, and Russia, and do not live under live trees. Fireworms are widely distributed inland in Korea and fewer in the south. Currently, there are about 60 species of ants in 1, 26, and 60 species including ants and fire ants in Korea.

The anemia is known as a fire ant, and its efficacy is known to be effective for joint pain, cardiovascular disease, respiratory system bronchitis, urinary system, digestive system, nervous system, pulmonary tuberculosis, It is an important medicinal drug that has been used mainly when the wound is not healed. 100 grams of dried ants contain over 70 grams of protein and contain 28 free amino acids and 8 amino acids, including isoxanthopterin, biotin, vitamin B2, 2-amino-6-hydro And cyperteridine.

The ant ingredient contains a venomous solution in the gall bladder, the most important of which is formic acid, and contains 90% of the antialdehyde. The antimutant is known to have the above nutritional elements and efficacy, but the formic acid which shows the toxicity of fire ants is strong enough to be 20 times that of Japanese royal family, and it is difficult to completely neutralize the formic acid in general processing, The development of processed food using

Korean Patent Laid-Open Publication No. 2004-0023459 discloses a known technology for manufacturing health supplement foods by mixing fire antiseptics with various medicines and Korean Patent No. 1136090 discloses an antifungal agent There is known a functional food including a liquid obtained by mixing and then heating a medicinal herb, and Korean Patent No. 1102831 discloses a method of manufacturing a health food which is produced by fire antibiotics and various natural medicines in the form of a bath.

However, the prior art discloses only simple addition of an antidote to a food composition, but specific and scientific contents and data such as an antimutagenic sexual function improvement and anti-inflammatory effect are not disclosed and are different as in the present invention.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide an antifungal agent which can effectively neutralize formic acid contained in fire ants, thereby solving the problems of human toxicity during ingestion, The object of the present invention is to establish a method for producing an antimicrobial extract powder having enhanced sexual function and anti-inflammatory effect by extracting and then drying the fire ants.

In order to solve the above-described problems, the present invention provides a method for producing a firefly, comprising the steps of: (a) immersing a dried firefly in an aqueous alkaline solution and then taking it out; (b) adding an aqueous ethanol solution to the firewood taken out in step (a), extracting it, filtering and separating the ethanol extract; (c) separating the ethanol extract of step (b) and adding water and proteolytic enzyme to the remaining firewood to extract the extract; (d) mixing the extracted extract of step (c) and the ethanol extract of step (b), filtering and drying; And (e) adding dextrin and water to the dry antifoulant extract of step (d) and spray-drying the antifouling extract of step (d). to provide.

In addition, the present invention provides a fragrant enzyme decomposition extract powder prepared by the above method.

In addition, the present invention provides a processed food containing the above cracked enzyme decomposition extraction powder.

The present invention effectively cleanses the formic acid contained in the fire ants through an antifouling pretreatment process, efficiently extracts the functional components contained in the fire ants, and improves the sexual function by drying and pulverizing the extracted extract, , There is an advantageous effect that the extracted powder can be used for food, pharmaceutical industry and the like.

FIG. 1 is a graph comparing the amount of NO production in mouse macrophages (RAW 267.4) according to the antiseptic powder treatment.
(A) LPS (2 ㎍ / ㎖) + Fire Fighting Enzymatic Extraction Powder Treatment, (A-1) LPS 4 ㎍ / ㎖ + Fire Fighting Enzymatic Extraction Powder Treatment, (B) LPS 2 ㎍ / B-1) LPS 4 ㎍ / ㎖ + antiseptic ethanol extract powder treatment
Figure 2 compares the NF-κB signaling pathway in mouse macrophages (RAW 267.4) following antimony powder treatment.
DMSO: dimethyl sulfoxide, Cilostazol: Cilostazol, red A: fire-cracking enzyme decomposition powder, red B: antimony ethanol extract powder
FIG. 3 is a graph comparing the NO production amount after incubation for 12 hours in mouse vascular endothelial cells (MOVAS) according to the antiseptic powder treatment.
HA: firefly enzyme decomposition extraction powder, HB: antiseptic ethanol extract powder
Figure 4 compares the NF-κB signal transduction mechanism in mouse vascular endothelial cells (MOVAS) following an antiseptic powder treatment.
Figure 5 compares the NF-κB signaling pathway in human vascular endothelial cells (HUVEC) following an antiseptic powder treatment.
DMSO: dimethyl sulfoxide, Cilo: Cilostazol, HA: firepot enzyme decomposition extraction powder, HB: antiseptic ethanol extract powder in FIGS. 4 to 5.
FIG. 6 is a graph comparing the activities of production of endothelial nitric oxide synthase (eNOS) according to the antifungal treatment in mouse vascular endothelial cells (MOVAS) and human vascular endothelial cells (HUVEC).
Red A: Fire Fighting Enzyme Decomposition Extract Powder, Red B: Antimony Ethanol Extraction Powder

In order to achieve the object of the present invention,

(a) immersing the dried firefly in an aqueous alkaline solution and then taking it out;

(b) adding an aqueous ethanol solution to the firewood taken out in step (a), extracting it, filtering and separating the ethanol extract;

(c) separating the ethanol extract of step (b) and adding water and proteolytic enzyme to the remaining firewood to extract the extract;

(d) mixing the extracted extract of step (c) and the ethanol extract of step (b), filtering and drying; And

(e) adding dextrin and water to the dry antler extract of step (d) and spray-drying the antler extract; and do.

In the method for producing the fragrant enzyme decomposition extraction powder of the present invention, the aqueous alkaline solution of step (a) may be an aqueous 0.1 to 10% by weight alkali solution preferably having a pH of 8 to 9, more preferably 0.1 % Alkali aqueous solution. The alkali used in the aqueous alkali solution is preferably at least one selected from the group consisting of sodium bicarbonate, sodium acetate, potassium acetate, sodium citrate, potassium citrate and potassium carbonate, more preferably sodium bicarbonate . Through the alkali treatment as described above, the formic acid contained in the fire ants could be partially neutralized.

In addition, in the method for producing the fragrant enzyme decomposition extraction powder of the present invention, the protease of step (c) may be an alkaline, but is not limited thereto.

In order to further improve the anti-inflammatory effect of the antirheumatic powder in the method for producing ungermant enzymatic decomposition extraction powder of the present invention, (f) a step of adding ethanol to the spray dried antifungal enzyme decomposition extraction powder followed by extraction, More specifically, 60 to 80% (v / v) ethanol is mixed in the ratio of 0.8 to 1.2: 8 to 12 (w: v) to the spray-dried antibiotic decomposition extract powder, More specifically, 70% (v / v) ethanol was mixed at a ratio of 1:10 (w: v) to spray-dried antibiotic-decomposed enzyme-extracted powder, and then the mixture was extracted three times It can be repeatedly extracted.

The method for producing an ungermant enzyme decomposition extraction powder of the present invention is more specifically

(a) immersing the dried firefly in an aqueous alkaline solution having a pH of 8 to 9 for 50 to 70 minutes and then taking out the same;

(b) adding an aqueous 45 to 55% (v / v) ethanol solution to the fire ants taken in step (a), extracting the mixture at 50 to 70 ° C for 8 to 12 hours, filtering and separating the ethanol extract;

(c) extracting the ethanol extract of step (b), adding water and proteolytic enzyme to the remaining anthrax extract, extracting it at 40 to 50 ° C for 65 to 80 hours while maintaining the pH at 8 to 9;

(e) adding dextrin and water to the dry antimycotic extract of step (d) to adjust the brix to 25 to 35 brix, bringing the temperature to 170 to 190 DEG C and the air temperature to 100 to 120 DEG C at a rate of 0.2 to 0.4 L / Spray drying,

More specifically,

(a) immersing the dried firefly in an aqueous alkaline solution of pH 8.5 for 60 minutes and then taking it out;

(b) adding 50% (v / v) ethanol aqueous solution to the fire ants taken in step (a), extracting at 60 ° C for 10 hours, filtering and separating the ethanol extract;

(c) separating the ethanol extract of step (b), adding water and proteolytic enzyme to the remaining anthrax extract, and extracting it at 40 to 50 ° C for 72 hours while maintaining the pH at 9;

(e) spray-drying at a rate of 0.3 L per minute at an inlet temperature of 180 ° C and an air-ventilation temperature of 110 ° C by adjusting dextrin and water to 30 brix by adding the dried antler extract of step (d) have.

The present invention also provides a fire-fighting enzyme decomposition extraction powder having the sexual function and anti-inflammatory effect prepared by the above method. The antifouling enzyme extracted powder of the present invention can extract functional ingredients more effectively than conventional antifouling powders and can provide an antifungal powder having improved sexual function and anti-inflammatory effect.

The present invention also provides a processed food containing the above cracked enzyme decomposition extraction powder. There is no particular limitation on the kind of the processed food. Examples of the food to which the above cracked enzyme decomposition extraction powder can be added include dairy products including pie, tablet, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include processed foods in a conventional sense.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Manufacturing example 1: Preparation of fire-cracking enzymatic decomposition extraction powder

(a) 1 kg of dry fire ants were immersed in a 0.1% aqueous solution of sodium bicarbonate of pH 8.5 for 1 hour and then recovered.

(b) 5 kg of a 50% aqueous ethanol solution was added to the recovered ants of the step (a), stirred at 60 ° C for 10 hours, filtered and the ethanol extract was separated.

(c) 5 kg of purified water was added to the remaining antimony ethanol-extracted leaves, and 5 g of protease (alkalazine, Novozymes) was added while maintaining the pH at 9 to 40 ° C at 50 ° C. The mixture was stirred for 72 hours The enzyme was degraded.

(d) The extracted extract of step (c) and the ethanol extract of step (b) were mixed and filtered with a 200 mesh microfilter, followed by vacuum drying.

(e) 50% (w / w) of dextrin is added to the dried antler extract of step (d), and purified water is further added thereto to adjust the concentration to 30 brix. And spray-dried at a rate of 0.3 L per minute to obtain a fire-extinguishing enzyme-extracted powder.

Comparative Example 1: Manufacture of antimony powder

50% (w / w) of dextrin was added to powder obtained by pulverizing 1 kg of dried fire ants, and further purified water was added thereto to adjust to 30 brix. The powder was adjusted to have a draw temperature of 180 ° C and an air temperature of 110 ° C, L to produce antifungal powders.

Experimental Method

1. Sexual Enhancement Experiment

1) Sexual function experiment

Male sexually active male New Zealand Hight Rabbit (Samutobi Bio Korea), which has a similar structure and physiological erectile mechanism to the penile corpus cavernosum of the human body, was tested with the test animals by the following method Were used for the experiment.

That is, the animals were anesthetized by inhalation of ether, and the penis was excised to separate the cavernosal smooth muscle from the low-temperature Krebs-Henseleit's solution, which was supplied with a mixture of 95% oxygen and 5% carbon dioxide Respectively. 2 x 2 x 2 mm sections were prepared from the separated cavernosal smooth muscle and fixed in an organ bath containing Krebs hexellate solution.

One end of the section was fixed to the lower part of the tissue container and the other end was connected to a power shrinkage recorder to record the movement state of the cavernosal smooth muscle. The Krebs hexenylate solution in the tissue container was maintained at 37 占 폚 and the oxygen mixed gas was continuously supplied. The smooth muscle slices produced by the above method induced contraction by phenylephrine (1 × 10 ^-5 M) and the existence of endothelial cells was confirmed by the presence or absence of relaxation by acetylcholine (Acetycholine). After removal of endothelial cells, only a sample with relaxation of less than 10% of acetylcholine relaxation was used as a smooth muscle slice with endothelial cells removed. The tip of the clamped rabbit corpus cavernosum was connected to an isometric force-displacement tranducer (FT03, Grass, AD instrumuent, Colorado springs, Co. USA) PowerLab 4/30, AD instrument) and analyzed with Labchart software (AD instrument).

In order to confirm the effect of the antimicrobial powder on the basic tension of the cavernosal sections, the stability of the corpus cavernosum tissue was stabilized and the concentrations of 0.1 mg / ㎖, 0.5 mg / ㎖, 1 mg / Ml, and the change of the tension and the change of the amplitude were measured with the passage of time.

2) Maternal behavior change

In order to examine the effective doses of the fragrant enzyme decomposition extract powder according to the present invention, 50 mice of SD rats and 20 females were sprayed with 1 mg / kg, 10 mg / kg, 100 mg / kg was taken for 1 day, and the number of mating, number of inserting, mating behavior regression time were measured for 4 weeks.

2. Anti-inflammatory effect

(1) Production of ethanol extract powder

100 g of the fragrant enzyme-decomposing extracted powder of Preparation Example 1 was added to 1,000 ml of a 70% ethanol solution, and the mixture was heated under reflux for 2 hours. This process was repeated two more times to obtain an extract. The extracted extract was filtered, and the filtrate was centrifuged at 5,000 × g for 30 minutes to obtain supernatant. The supernatant was taken in a rotary vacuum concentrator (EYELA, Japan), concentrated under reduced pressure, and lyophilized in a freeze dryer to obtain 7.86 g of the extract.

(2) In vitro experiments

1) Preparation of vascular endothelial cells

For in vitro experiments to confirm the anti-inflammatory effect of antimicrobial powder, mouse vascular endothelial cells (MOVAS, mouse vascular endothelial cells) and human vascular endothelial cells (HUVEC, human umbilical vein endothelial cells) Respectively.

2) Measurement of NO (Nitric Oxide) production

Antiserum extract was dissolved in dimethyl sulfoxide (DMSO, Sigma Chem, CO.) To a final concentration of 0.05% and used by filtration sterilization with a filter (0.02 ㎛ pore size, Millipore). The concentrations of the samples were 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 ㎍ / ㎖, which were determined through preliminary experiments. The grease reagent, which is a reagent for measuring NO production, is prepared by adding Solution A (0.2% Naphthylethylene diamine dihydrochloride in DW) and Solution B (2% Sulfonylamide in 5% H ₃ PO ₄ ) and stored in a cool dark place. Solution was mixed at a ratio of 1: 1 and a mixed solution was used. 100 μl of the culture supernatant was dispensed into a 96-well plate, and 100 μl of the mixed solution was dispensed. The absorbance at 540 nm was measured using an ELISA reader. The standard calibration curve was 0 to 80 μM Lt; / RTI >

3) Western blot analysis

Cells were lysed with lysis buffer (20% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM iodoacetoamide, 1 mM leupeptin, 1 mM antipain, 0.1 mM sodium orthovanadate, 5 mM sodium fluoride) Proteins were quantified using a DC protein assay kit from Hercules, CA, USA. 20 to 60 占 퐂 of proteins were separated by size by SDS-PAGE, transferred to nitrocellulose membrane, and then incubated with blocking solution at room temperature for one hour. Then, the cells were incubated with primary antibody at 4 ° C for one day, and cultured at room temperature for 2 hours using a secondary antibody conjugated with HRP (horseradish peroxidase). Antibody-bound proteins were developed by exposure to X-ray film using an ECL kit (Perkin Elmer Life and Analytical Science, Boston, Mass., USA).

4) NO (Nitric Oxide) analysis

LNCaP prostate cancer cell line cells were divided into 24 well plates at a density of 1 x 10 ⁴ cells / ml, stabilized for 24 hours, treated with cilostazol and antler extract, and cultured for 12 hours and 24 hours, respectively. The cell culture was separated from each well, placed in a 1.5 ml tube, refrigerated, and analyzed for NO. 50 μl of the cell culture was taken and mixed with 50 μl of a grease reagent (1% sulfanilamide / 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% H ₃ PO ₄ ) and dispensed into a 96-well plate. After 5 to 10 minutes of incubation in the dark room, the NO content was measured at 530 nm in an ELISA reader after 30 minutes of change to purple / magenta, and the NO concentration was calculated based on the standard curve of sodium nitrite (NaNO ₂ ) .

(3) Effect on anti-inflammation in RAW264.74 cell line

1) Cell line and cell culture

The mouse macrophage cell line (RAW264.7) was purchased from Seoul National University, Seoul, Korea. Mouse macrophage cell line (RAW264.7) supplemented with 10% FBS and 1% antibiotics added to the temperature of 37 ℃ in MEM medium with 5% CO ₂ condition CO ₂ that is maintained And cultured in an incubator (Forma Sci, USA).

2) RAW264.7 Analysis of macrophage culture and NO production

The culture medium (37 ° C, humidified, 5% CO ₂ /95% air environment) was placed in a 24-well culture plate with 2 ml of DMEM medium containing 10% FBS and 2 × 10 ⁵ cells / well of RAW 264.7 cells ), And then the supernatant was removed. Then, cilostazol (10 μg / ml) treated with LPS at a concentration of 2 μg / ml and 4 μg / ml and the antiserum extract (10, 50, 100 μg / Ml) and then cultured again. The supernatant was separated and stored in a freezer. ELISA was performed by using Mouse NO (Intron, Korea) ELISA kit. 100 쨉 l of cilostazol and supernatant were added to each well according to the manufacturer's instructions. The wells were incubated for 2 hours and then incubated at room temperature. The grease reagent, which is a reagent for measuring NO production, is prepared by adding Solution A (0.2% Naphthylethylene diamine dihydrochloride in DW) and Solution B (2% Sulfonylamide in 5% H ₃ PO ₄ ) and stored in a cool dark place. Solution was mixed at a ratio of 1: 1 and a mixed solution was used. 100 μl of the culture supernatant was dispensed into a 96-well plate, and 100 μl of the mixed solution was dispensed. The absorbance at 540 nm was measured using an ELISA reader. The standard calibration curve was 0 to 80 μM Lt; / RTI >

3) Western blot analysis

Western blot analysis was performed using the anti-inflammatory factor NF-kB antibody in order to determine whether the antiserum extract was involved in the anti-inflammatory mechanism in the RAW264.7 cell line. The RAW264.7 cell line was divided into 6-well plates at 5 × 10 ⁶ cells / ml and cultured for 24 hours. Antifungal extracts were treated at 10, 50 and 100 μg / ml, / Ml and then cultured. After incubation, the cells were washed with cold PBS, and cells were obtained with a scraper and centrifuged to discard the supernatant. Whole cell lysate was reacted with 100 μl of Lysis buffer (980 μl of RIPA buffer + 100 μl of protease inhibitor cocktail + 10 μl of PMSF 100 mM) for 15-20 minutes on ice, and the supernatant was obtained. Cytoplasmic fractions were collected by centrifugation using ice / cytoplasmic extraction kit (Active motif, USA) for 15 min on ice. Lysis buffer was added to the nuclei remaining in the tube, reacted on ice for 30 seconds, and centrifuged to obtain a nuclear fraction. The obtained protein was quantified by BCA protein determination method. A 10% SDS-PAGE gel was prepared, filled with running buffer, pre-run for 20 minutes, the protein was diluted with a loading buffer and boiled for 5 minutes in boiling water, I released this twist. The first blank of the gel was floated, the protein marker was inserted in the second column, and the sample was added from the next column to 120V. The densified gel was cut to size and immersed in a transfer buffer for 20 minutes, during which time the membrane was cut to the size of the gel and pre-soaked in transfer buffer. The gel and the membrane were closely adhered, then filled with transfer buffer, ice was not added, and the protein was transferred to the membrane at 150V for 1 hour and 30 minutes. Protein-transferred membranes were blocked with 5% skim milk (in TBS / T buffer) for 1 hour. As a primary antibody, goat anti-iNOS was reacted at 4 ° C for one day, and all primary antibodies used were those from Santa Cruz Biotechnology (Santa Cruz, Calif.). The next day, the secondary antibody was reacted for 1 hour at room temperature using anti-rabbit IgG (Amersham, Buckinghamshire, UK) and anti-goat IgG (Dako, Glostrup, Denmark). The membrane was reacted with a detection solution (Amersham, USA), and the membrane was lightly dried and transferred to a film to emit light. The film was immersed in the developing solution and developed, fixed on a fixing solution and dried. The band densities of the results were compared with those of YY-1 using Image-Rab densitometer (Bio-Rad, CA, USA).

4) Statistical analysis

Statistical analysis of all experimental results was performed using SAS statistical program. The results were expressed as mean value and standard error, and statistical significance ( p <0.05) was verified with ANOVA.

Example 1: Measurement of sexual function improvement by antiseptic powder treatment

To confirm the effect of the antifungal powder on the basic tension of the penile corpus cavernosum, the motility of penile cavernosal tissues was stabilized. When the tension was maintained constant, Table 1 shows the results of confirming the degree of enhancement of erection according to the concentration of the powder using the difference in the lowest point at which the smooth muscle of the penile cavernosal smooth muscle was relaxed against the peak at which the penile cavernosum smooth muscle was contracted by phenylephrine. As a result, it was confirmed that the antifungal enzyme extract of Preparation Example 1 according to the present invention can enhance the erectile power of the penis compared with the antifungal powder of Comparative Example 1. [

Measurement of Erectile Enhancement by Fire Fighting with Enzymatic Hydrolyzed Powder (%) Extracted powder concentration 0.1 mg / ml 0.5 mg / ml 1 mg / ml Antimony powder 9.2 34 65 Firefly enzyme decomposition extraction powder 13.5 53 87

As a result of observing mating behavior change, the mating frequency, mating behavior regression time, and number of insemination were significantly higher in the 10 mg / kg fed group and 100 mg / kg fed group than in the control group Significant results were observed (Table 2).

Maternal behavioral changes in SD rats at 4 weeks division Mating behavior regression time (sec) Number of matings (times) Number of insertions (times) Control group 40.6 11.2 0 1 mg / kg intake group 38.3 10.9 0 10 mg / kg intake group 26.7 14.8 Episode 2 100 mg / kg intake group 21.2 16.2 4 times

Example 2: Mouse In the macrophage line (RAW 264.7) Anti-inflammatory effect of antler extract

(1) Measurement of NO production in mouse macrophages (RAW264.7 cell line)

NO reacts very quickly with lipid alkoxyl, a peroxyl radical intermediate, with or without an enzyme, to produce nitrification intermediates. NO is converted from a high concentration to a reactive oxidant causing pathological damage to the cell. LPS is a bacterial endotoxin that when administered to macrophages produces an inflammatory response mediator, such as inflammatory cytokines and NO, leading to pathological responses.

To investigate the effect of antimycotic extract and Cilostazol on NO production in RAW264.7 macrophages, RAW264.7 macrophages were treated with various concentrations of antiseptic extract and cilostazol (10 ㎍ / ㎖) as a positive control ) And stimulated with LPS. After 24 hours, the production of inflammatory cytokine NO was measured by ELISA.

FIG. 1 (A) shows the anti-inflammatory effect of the extract of ungulate enzymatic decomposition. As a result, the NO production amount measured by stimulation with LPS 2 μg / ml in RAW264.7 macrophages was higher than that of the normal group (RAW264.7, Normal) The control group (LPS) treated with LPS significantly increased. The positive control group, cilostazol, showed a statistically significant decrease in NO production compared to the control (LPS) ( p <0.01). The NO production of the experimental group treated with antiseptic powder was significantly lower than that of control (LPS) at 100 ㎍ / ㎖ ( p <0.05).

1 (A-1) shows the anti-inflammatory effect of the extract of the fire-fighting enzyme. As a result, the amount of NO produced by stimulation with LPS 4 μg / ml in RAW 264.7 macrophages was normal (RAW 264.7, Normal) Compared to control (LPS) treated with LPS. The positive control group, cilostazol, showed a statistically significant decrease in NO production compared to the control (LPS) ( p <0.01). The NO production of the experimental group treated with antiseptic powder was significantly decreased in the concentration - dependent manner at 50, 100 ㎍ / ㎖ ( p <0.01) compared to the control (LPS).

FIG. 1B shows the results of observation of the anti-inflammatory effect of the ethanol extract of ungulate ethanol. As a result, the NO production amount measured by stimulation with 2 μg / ml of LPS in RAW264.7 macrophages was higher than that of the normal group (RAW264.7, Normal) Treated group (LPS) significantly increased. The positive control group, cilostazol, showed a statistically significant decrease in NO production compared to the control (LPS) ( p <0.001). The amount of NO produced in the experimental group treated with antimutagenic ethanol extract was decreased in a concentration - dependent manner at 50, 100 ㎍ / ㎖ ( p <0.01) compared to the control (LPS).

Also, B-1 of FIG. 1 shows the anti-inflammatory effect of the ethanol extract of the antiseptic ethanol. As a result, the NO production measured by stimulation with LPS 4 ㎍ / ㎖ in RAW264.7 macrophages was normal (RAW264.7, Normal) In contrast, the LPS-treated control (LPS) significantly increased. The positive control group, cilostazol, showed a statistically significant decrease in NO production compared to the control (LPS) ( p <0.001). The NO production in the experimental group treated with antiseptic ethanol extract powder was decreased in a concentration - dependent manner at 10 ( p <0.01), 50 ( p <0.001) and 100 ㎍ / ㎖ ( p <0.001) compared to the control (LPS).

From the above results, it is considered that the antimutagenic extract powder inhibits the NO production directly involved in the inflammatory reaction.

(2) NF-κB signal transduction mechanism in mouse macrophage (RAW264.7) by firefly extract

NF-κB consists of five constitutive proteins (NF-κB1 [p105 / p50], NF-κB2 [p100 / p52], RelA / p65, RelB and c-Rel) And RHD (Rel-homology domain) composed of amino acids. This domain plays an important role in the NF-κB signaling pathway involved in homotypic or heterodimer formation of the NF-κB constituent protein, interaction with the IκB (Inhibitor of κB) protein and binding to the target gene promoter. It also contains a Nuclear Localization Signal (NLS) to move into the nucleus and act as a transcription factor.

As shown in FIG. 2 A of FIG. 2, when the incubation time was adjusted to 6 hours with RAW264.7 cells, the expression of NF-κB gene was measured by treating 2 μg / ml of LPS with NF-κB expression, KB protein levels (Fig. 2 (A)). On the other hand, it was found that the antifungal ethanol extract decreased the protein level of NF-κB in a dose-dependent manner when LPS was treated (FIG. 2B). These results suggest that NF-κB is involved in the signaling pathway of NO production, and NF-κB activation is reduced by the antimutagenic ethanol extract powder, and IκB, which is a transcription factor, is decreased and binding to the nuclear promoter is inhibited. Which means that NO production is reduced by inactivation.

Example 3: Antimutagenic extract from vascular endothelial cells NO Production activity and mechanism research

(1) Measurement of NO production in mouse vascular endothelial cells (MOVAS, Mouse vascular endothelial cell)

Nitric Oxide promotes vascular endothelial protection, strong antioxidant production, circulatory system, immune system, nervous system, etc., and has a function of signaling to all the organs of the human body. As a result, NO production was significantly higher in 100 ㎍ / ㎖ HA (100 ㎍ / ㎖) and 100 ㎍ / ㎖ (100 ㎍ / ㎖) treated with MOVAS than in the control group (Fig. 3).

(2) NF-κB signal transduction mechanism in murine vascular endothelial cells (MOVAS)

As shown in Fig. 4A, the NF-κB expression was observed in MOVAS cells for 12 hours. The expression of NF-κB was detected in 10, 50 ㎍ / ㎖ of firefly luciferase (HA) 10, and 50 ㎍ / ㎖, respectively, compared to the control group. IκB expression was increased in 10, 50, 100 ㎍ / ㎖ HA treated group and 10, 50 ㎍ / ㎖ HB treated group compared to the control group.

As shown in Fig. 4B, NF-κB expression was observed in 10, 50 ㎍ / ㎖ HA treated group and 10, 50 ㎍ / ㎖ HB treated group in the control group And 10 ㎍ / ㎖ of HA treatment group, 10, 50 ㎍ / ㎖ of HB treatment group and 10, 50, 100 ㎍ / ㎖ HB treatment group were significantly increased compared to the control group.

These results indicate that NF-κB is involved in the signaling pathway of NO production and NF-κB activation is decreased by the antimycotic extract, and IκB, which is a transcription factor, increases and binds to the nuclear promoter, The output is interpreted as indicating an increase.

(3) NF-κB signal transduction mechanism in human vascular endothelial cells (HUVEC) by antler extract

As shown in FIG. 5, when NF-κB expression was observed in HUVEC cells for 12 hours, 10, 50 μg / ml of HA treatment group and 10, 50 and 100 μg / ml of HB treatment group were decreased compared to the control group IκB expression was increased in 10, 50, 100 ㎍ / ㎖ HA treated group and 10, 100 ㎍ / ㎖ HB treated group compared to the control group.

These results suggest that NF-κB is involved in NO production by HUVEC cells. NF-κB activation is reduced by the antimycotic extract, and IκB, which is a transcription factor, binds to the nuclear promoter. .

(4) Production activity of endothelial nitric oxide synthase (eNOS) in vascular endothelial cells by firefly extract

In the mouse vascular endothelial cell MOVAS and human vascular endothelial cell HUVEC, the production of NO was increased by the antimycotic extract. There were two types of vascular endothelial cell eNOS, activated and inactivated, and eNOS (NO) can be produced only in the activated form, demonstrating a mechanism of increasing NO production and NF-κB phosphorylation in an antimycotic extract.

FIG. 6 shows the expression of eNOS (endothelial NOS) gene in the vascular endothelial cells MOVAS and HUVEC by the antiserum extract. As a result, eNOS mRNA gene expression in human HUVEC cells was increased in a concentration - dependent manner in the ethanol extract of Fusarium oxysporum, although there was no significant difference in MOVAS cells.

Claims

(a) immersing the dried firefly in an aqueous alkaline solution and then taking it out;
(b) adding an aqueous ethanol solution to the firewood taken out in step (a), extracting it, filtering and separating the ethanol extract;
(c) separating the ethanol extract of step (b) and adding water and proteolytic enzyme to the remaining firewood to extract the extract;
(d) mixing the extracted extract of step (c) and the ethanol extract of step (b), filtering and drying; And
(e) adding dextrin and water to the dry antler extract of step (d) and spray-drying the antler extract; and (e) treating the antler extract of step (d).

The method according to claim 1, further comprising the step of (f) adding ethanol to the spray-dried fragrance enzyme decomposition extract powder and then extracting the fragrance. Way.

The method according to claim 1,
(a) immersing the dried firefly in an aqueous alkaline solution having a pH of 8 to 9 for 50 to 70 minutes and then taking out the same;
(b) adding an aqueous 45 to 55% (v / v) ethanol solution to the fire ants taken in step (a), extracting the mixture at 50 to 70 ° C for 8 to 12 hours, filtering and separating the ethanol extract;
(c) extracting the ethanol extract of step (b), adding water and proteolytic enzyme to the remaining anthrax extract, extracting it at 40 to 50 ° C for 65 to 80 hours while maintaining the pH at 8 to 9;
(d) mixing the extracted extract of step (c) and the ethanol extract of step (b), filtering and drying; And
(e) adding dextrin and water to the dry antimycotic extract of step (d) to adjust the brix to 25 to 35 brix, bringing the temperature to 170 to 190 DEG C and the air temperature to 100 to 120 DEG C at a rate of 0.2 to 0.4 L / Wherein the method comprises the step of spray-drying and the step of spray-drying.

4. An anti-inflammatory enzyme decomposition extract powder having improved sexual function and anti-inflammatory effect, prepared by the method of any one of claims 1 to 3.

A processed food containing the unheated enzyme decomposition extraction powder of claim 4.