KR20160059743A

KR20160059743A - Composition for the immunity stimulatory activity comprising orostachys japonicus A. berger water extract

Info

Publication number: KR20160059743A
Application number: KR1020140161651A
Authority: KR
Inventors: 장대자; 양혜정; 김상희; 차환수
Original assignee: 한국식품연구원
Priority date: 2014-11-19
Filing date: 2014-11-19
Publication date: 2016-05-27

Abstract

The present invention relates to an activity composition for enhancing immunity, comprising an Orostachys japonicus A. Berger water extract as an active ingredient. Activity of macrophages is improved by containing extract powder which hot water-extracts the Orostachys japonicus A. Berger as an active ingredient, while activity of anti-cancer and anti-inflammation is weak.

Description

[0001] The present invention relates to an immunostimulatory composition comprising an extract of a plant,

The present invention relates to an immunostimulatory composition containing an extract of Pleurotus ostreatus which is excellent in macrophage activity as an active ingredient.

Recently, life sciences have been prolonged due to the development of life sciences and medical technology, and it is expected to become a full-fledged aging society after 2020 due to the improvement of people's standard of living. Therefore, degenerative diseases such as dementia are increasing, and degenerative immune responses can be classified into congenital immune responses, which are early immune responses, and acquired immune responses, which are late immune responses.

In the initial immune response, macrophages and natural killer cells (NK cells) inhibit the pathogen by inhibiting the pathogen, thereby protecting the host. At this time, macrophages produce TNF-α, which is an active marker, And NK cells produce and secrete perforin, an active marker, to kill pathogenic cells. It then activates cytotoxic T lymphocytes, helper T lymphocytes, and B lymphocytes that are involved in acquired immunity to kill infected cells or protect the host by making antibodies.

Cytotoxic T lymphocytes, like NK cells, produce and release a large amount of perforin to kill pathogen-infected cells, while B lymphocytes protect the host by making antibodies dependent on or dependent on helper T lymphocytes (Choi, In-Sung, -133, 1992; Choi Jaeho et al., J. Ginseng Res., 33 (1): 33-39, 2009).

Except for the United States, immigrants have been actively engaged in research activities in Korea, which are comparable to those of Europe and Japan. A total of 66 patents related to domestic immunological function foods and food materials were filed in 2002 or later, mostly since 1997. It is classified into 7 groups including general vegetable material, seafood material, mushroom material, microbial material such as lactic acid bacteria, chitosan material, amino acid material and other product groups according to the invention contents.

In order to examine the anticancer and immunological activity of polysaccharides derived from natural products and the immunopotentiating activity of glycoproteins extracted from seaweeds, there have been observed blood leukocyte counts, total number of peritoneal cells, weight of immune related organs, and phagocytosis of macrophages (Ryu BH. 1992, J. Korean Soc. Food Nutr., 21, 154-162.). In addition, the immune function-enhancing action of red ginseng polysaccharide has been reported to have lymphocyte proliferative effect and LAK (lymphokine activated killer) cell production ability (Kim KH, 1997, Korean J. Ginseng Sci 21, 78-84).

In the present invention, the present inventors have conducted research to develop a more effective immunostimulating composition and a health functional food for immunity-enhancing activity by extracting an active ingredient from resources that can be used for edible purposes without adverse effects.

Korean Patent Publication No. 2014-0072464 Korea Patent No. 0614222

It is an object of the present invention to provide an immunostimulatory composition containing an extract of Pleurotus ostreatus, which is excellent in macrophage activity, as an active ingredient.

Another object of the present invention is to provide a health functional food for immunoenhancing activity containing the above-mentioned water extract as an active ingredient.

In order to accomplish the above object, the immunostimulatory composition of the present invention may contain an extract of WASONG as an active ingredient.

The above-mentioned water-extracted extract may be prepared by mixing the water and the water at a weight ratio of 1: 5 to 20.

The extraction temperature may be 20 to 100 캜, and the concentration of the washed extract may be 50 to 600 ug / ml.

In order to accomplish the above-mentioned other objects, the health functional food for immunoenhancing activity of the present invention may contain an extract of Wansong water as an active ingredient.

The concentration of the washed extract may be 50 to 600 ug / mL, and the extraction temperature may be 70 to 110 캜.

The health functional food may be selected from the group consisting of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels, jellies, and the like.

However, the extract of water of the present invention, which is different from the case of using the extract extracted with the above organic solvent, can be used as an anticancer agent and anti-inflammatory agent It has no activity against inflammation and promotes the activity of macrophages, and thus can be preferably used for immunity enhancement. In addition, the immunostimulatory composition of the present invention, which contains an extract of water as an active ingredient, can be ingested in the form of food since it is not toxic.

FIG. 1A is a graph showing the NO production of the goose extract prepared according to Examples and Comparative Examples of the present invention in macrophages (RAW264.7 cells). FIG.
FIG. 1B is a graph showing cell viability of macrophages (RAW 264.7 cells) treated with the Salmonella extract prepared according to Examples and Comparative Examples of the present invention.
FIG. 2 is a graph showing the DPPH radical scavenging activity of the dried water extract prepared according to an embodiment of the present invention. FIG.
FIG. 3A is a photomicroscopic photograph of macrophages (RAW 264.7 cells) treated with the wash extract prepared according to an embodiment of the present invention.
FIG. 3B is a graph of a macrophage (RAW 264.7 cells) treated with a fresh water extract prepared according to an embodiment of the present invention, using a flow cytometer.
FIG. 3C is a graph obtained by quantitatively analyzing the measurement value of FIG. 3B by MFI.
4 is a photograph showing the immunological efficacy of the water extract prepared according to the example of the present invention.
FIG. 5 is a graph showing the cell viability of skin cancer cells (B16F10 melanoma cell line) treated with the Wassong extract prepared according to Examples and Comparative Examples of the present invention.
FIG. 6 is a graph showing NO production of the goose extract prepared according to Examples and Comparative Examples of the present invention in macrophages (RAW 264.7 cells) increased by LPS.

The present invention relates to an immunostimulatory composition comprising as an active ingredient, an extract of Pleurotus ostreatus, which has weak anticancer activity and antiinflammatory activity while enhancing macrophage activity.

Hereinafter, the present invention will be described in detail.

The immunostimulatory composition of the present invention contains an extract of onion as an active ingredient.

Orostachys japonicus A. Berger is a perennial herbaceous plant with a crinoid called Iwamatsu and Oyasho which is used for the fever, swelling, hemostasis, and humidification in Oriental medicine. It is widely used for cancer treatment by therapy. It is a fleshy perennial herb that grows on the side of a rock, but has a characteristic of blossoming and fruiting when it blooms.

In order to prepare the above-mentioned dried extract, firstly, it is washed with water to remove the moisture of the wastes from which the foreign substance is removed, and then dried and pulverized.

Next, the thus prepared wash powder and water are mixed at a weight ratio of 1: 5 to 20, preferably 1: 7 to 15, at a temperature of 20 to 120 ° C, preferably 40 to 90 ° C for 0.1 to 48 hours, Preferably 1 to 20 hours, to prepare an extract.

When the weight ratio between the powder and the water during the extraction is out of the above range, the effective amount of the extract can be extracted in a small amount.

In addition, when the extraction temperature is lower than the lower limit value, the immunity enhancing activity can not be expected, and when the temperature is higher than the upper limit value, the immunity enhancing activity can be lowered.

The water extract of the present invention is used at a concentration of 50 to 600 ug / mL. If the concentration is below the lower limit, the immunity enhancing activity is not expected or weak. If the concentration is above the upper limit, toxicity may be caused.

The thus-prepared dried water extract can be used as an extract itself or as powder for convenient use. In the method of pulverizing the above-mentioned water extract, the extract is concentrated under reduced pressure to reduce its volume and then lyophilized by a freeze dryer to be pulverized.

The immunostimulatory composition containing the extract of the present invention as an active ingredient contains 0.01 to 80% by weight, preferably 0.1 to 50% by weight, based on the total weight of the composition.

The immunostimulatory composition may be formulated into various oral or parenteral dosage forms.

Examples of formulations for oral administration include tablets, pills, hard, soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, (For example, silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycol), in addition to the active ingredient (s). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid Or a disintegrating or boiling mixture such as its sodium salt and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.

Representative examples of formulations for parenteral administration include injectable preparations, and water, Ringer's solution, isotonic saline or suspensions may be mentioned as a solvent for the injectable preparation. The sterile, fixed oils of the injectable preparations may be used as a solvent or suspending medium, and any non-irritating fixed oils, including mono-, di-glycerides, may be used for this purpose. The injectable preparation may be a fatty acid such as oleic acid.

The dosage of the immunostimulatory composition containing the extract of the present invention as an active ingredient varies depending on the age, sex, weight, and severity of the disease of the patient. However, the composition of the present invention is not more than 300 mg per kg of the patient's weight , Preferably 100 to 200 mg is administered 1 to 4 times a day, but is not limited thereto.

The immunosuppressive activity-containing health functional food containing the above extract of the present invention as an active ingredient contains 0.01 to 80% by weight of a dried water extract based on the total weight of the health functional food.

The health functional food may be in the form of a capsule, tablet, powder, granule, liquid, ring, flaky, paste, syrup, gel, jelly and jute for the purpose of supplementing nutrients that may be lacking in daily eating or supplementing functional raw materials useful in the human body And is easily manufactured and operated once.

In addition, the health functional food may contain at least one selected from the group consisting of glucose, citric acid, liquid oligosaccharide, corn syrup, soybean lecithin, butter, vegetable hardening oil, skim milk, sugar, margarine, salt, starch, And can be prepared by using commonly used components such as sodium bicarbonate and sugar ester.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Example 1:

The wastes were washed with water to remove water and then dried by hot air blowing. The thus prepared salted powder and water were mixed at a weight ratio of 1:10, and the mixture was extracted at 40 ° C for 15 hours. The obtained extract was filtered and concentrated under reduced pressure to reduce the volume to 1/5. The reduced-pressure concentrated water extract was pulverized with a freeze-dryer to prepare an extract powder of dried matter.

Comparative Example 1. Aqueous solution of aqueous solution of < RTI ID = 0.0 >

The procedure of Example 1 was repeated except that 70% alcohol was used instead of water as an extraction solvent.

Comparative Example 2. Wassin ethanol extract

The same procedure as in Example 1 was carried out except that 99.9% ethanol was used instead of water as an extraction solvent to prepare a wastepan ethanol extract powder.

Comparative Example 3: Waste methanol extract

The procedure of Example 1 was repeated except that methanol was used instead of water as an extraction solvent to prepare an aqueous methanol extract powder.

Comparative Example 4. Wasson's ethyl acetate extract

The procedure of Example 1 was repeated except that ethyl acetate in place of water was used as an extraction solvent to prepare an extract of Wassong ethylacetate.

Comparative Example 5: Wassong hexane extract

The same procedure as in Example 1 was carried out except that hexane as the extraction solvent was used instead of water to prepare an aqueous wastes hexane extract powder.

< Test Example >

- Cell culture

Human fetal bovine serum (FBS; Hyclone, Logan, Utah, USA) and 100 U / mL of mouse macrophage RAW264.7 or skin cancer cell line B16F10 melanoma were purchased from the American Type Culture Collection (ATCC, Manassas, were cultured in DMEM (Dulbecco's Modified Eagles Media (Gibco Invitrogen, Carlsbad, Calif., USA) or MEM (Minimum Essential Medium) supplemented with penicillin (Gibco Invitrogen) at 37.1 캜 and 5% CO ₂ .

- Statistical analysis

The results are mean ± standard error (SE). One-way ANOVA / Dunnetts t-test was used to verify the significance of the control and test groups. Statistical analysis was performed using SPSS, version 12 (SPSS Inc., Chicago, IL, USA).

Immunological activity test

Test Example One. NO Generation and cell Survival Measure

1-1. NO production (uM): RAW 264.7 cells (3 × 10 ⁴ cells / well) cultured on 96-well plates were incubated for 20 h with 0, 100, 200 and 400 mg / Lt; / RTI > 100 mL of the culture was then reacted with 100 mL of a grease reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) at room temperature for 10 minutes. Absorbance was measured at 560 nm using a microplate reader (Tecan, Mnnedorf, Switzerland) and the concentration of NO in each culture was calculated using a standard concentration curve using sodium nitrite (NaNO ₂ ).

1-2. Cell viability (%) was measured using the CCK-8 (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) assay. Cells (3 × 10 ⁴ cells / well or 1 × 10 ⁴ cells / well) in 96-well plates were incubated with the Wassin extract. Cell viability was calculated using CCK-8 kit 24 hours or 72 hours after sample treatment. The cell survival rate was expressed as the survival rate (%) of the group treated with the sample for the absorbance of the control group without any treatment.

FIG. 1A is a graph showing the NO production of the saliva extract prepared according to the examples and comparative examples of the present invention in macrophages (RAW264.7 cells), FIG. 1B is a graph showing the NO production of the saliva extract prepared according to the examples and the comparative examples of the present invention, (RAW264.7 cells) treated with the extract of the present invention.

Nitric oxide (NO) is a kind of ROS (reactive oxygen species) secreted in cells for the phagocytosis of macrophages. Since nitrite or nitrate is the end product of NO, measurement of nitrite or nitrate is the same as indirectly measuring the production of NO. Therefore, nitrite or nitrate in the macrophage culture solution Was measured.

As shown in FIG. 1A, it was confirmed that the amount of NO production of macrophages was increased only by the extracts of freshwater produced according to Example 1, compared with the fresh extracts prepared according to Comparative Examples 1 to 5. This means that the extract of Example 1 can enhance the activity of macrophages.

In addition, as shown in FIG. 1B, all of the Wassong extracts prepared in Example 1 and Comparative Examples 1 to 5 were found to have excellent cell viability.

Test Example 2. DPPH Radical Scatters Measure

Antioxidant activity was measured by the DPPH method using an antioxidant such as vitamin C as a comparative sample.

The DPPH radical scavenging activity (%) was determined by adding 2 mL of 0.1 mM DPPH to 200 mL of the Salmon extract diluted to the indicated concentration, reacting at room temperature for 30 minutes, measuring the absorbance at 517 nm, Respectively.

[Equation 1]

DPPH radical scavenging ability (%) = (1 - absorbance of control group / absorbance of sample addition group) * 100

FIG. 2 is a graph showing the DPPH radical scavenging activity of the dried water extract prepared according to Example 1 of the present invention. FIG.

As shown in FIG. 2, it was confirmed that the DPPH radical scavenging ability was improved as the concentration of the water extract of Example 1 was increased.

Test Example 3. Microscopic observation and Flow cell analyzer analysis

Macrophage activity was measured and measured by optical microscopy and flow cytometry using the Phagocytosis assay kit (Cayman Chemical, Ann Arbor, MI).

Macrophages (RAW 264.7 cells, 5 × 10 ⁵ cells / well) cultured on a 12-well plate were incubated with the freshwater extract of Example 1 and Latex Beads-Rabbit IgG-FITC solution for 24 hours. Then, morphological changes of the cells were observed under an optical microscope, and the macrophage activity was measured using a flow cytometer (FACS Calibur, Becton Dickinson) after cell collection. Fluorescence intensity of RAW264.7 cells (fluorescence IgG-opsonized particles entering into RAW264.7 cells by macrophage) was measured by flow cytometry and MFI mean fluorescence intensity).

FIG. 3A is a photograph of a macrophage (RAW 264.7 cell) treated with a fresh water extract prepared according to Example 1 of the present invention by an optical microscope, FIG. 3B is a photograph FIG. 3C is a graph showing the quantitative analysis of the measurement value of FIG. 3B by MFI (* p <0.05, ** p <0.01). FIG. *** p < 0.001 vs. control group).

As shown in FIG. 3A, the PBS-treated RAW 264.7 cells (control group) showed almost no change compared with before treatment with Latex Beads-Rabbit IgG-FITC, while the freshwater extract of Example 1 or RAW 264.7 cells treated with LPS It was confirmed that the cell size was larger than that before and the surface of the cell was changed into a rough and sharp shape. These morphological changes are the result of proving that the water extract or LPS can stimulate RAW264.7 cells to activate macrophages.

As shown in FIGS. 3B and 3C, the MFI values of RAW264.7 cells treated with the wash extract of Example 1 were found to be about 1.35 times higher than those of the control group. This is a result of proving that the extract of Example 1 can stimulate RAW264.7 cells to act more aggressively.

Test Example 4. Flavonoid Analysis

4-1. Total flavonoid measurement: Take 0.5 mL of the sample solution into a test tube, add 5 mL of diethylene glycol, mix with 0.5 mL of 1N NaOH, and allow to stand in a 37 ° C water bath for 1 hour. The absorbance was then measured by UV spectrophotometer at 420 nm. The calibration curves for quantification were prepared by dissolving 1 mg of Rutin (Sigma) in 1 mL of 70% methanol to 100, 200, 300, and 400 ug / mL, Respectively.

4-2. Measurement of flavonoid content: Samples prepared in Examples and Comparative Examples were analyzed for flavonoids by UPLC-MS / MS. Twenty standard products were diluted with methanol and the standard stock solution was diluted to a concentration of 0.05, 0.1, 0.5, 1, 2, 3, 4 and 5 ug / mL using Ultra Performance Liquid Chromatography / Tandem Mass Spectrometer (UPLC-MS / MS) To prepare a mixed standard solution for calibration curve preparation. Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters Milford, Mass., USA) was used for the UPLC-MS / MS and Waters Xevo-TQ A: An aqueous solution containing 0.05% formic acid and 0.05% ammonium acetate, B: an ACN solution containing 0.05% formic acid. When the flow rate of the mobile phase was 0.65 mL / min, the column temperature was 40 ° C., 5 uL. MRM (Multiple Reaction Monitoring) conditions were optimized by optimizing each parameter of MS in negative ion mode. Source temperature was 150 ℃, cone gas flow was 50 L / h and desolvation gas temperature was 400 ℃.

division Example 1 Comparative Example 1 Total flavonoid content (mg / g) 10.47 + 0.02 64.19 + - 0.88 Flavonoid type Gallic acid 963.96 + 110.21 335.41 + - 96.51 Apigenin ND ND Naringenin ND 0.84 + 0.07 Luteolin 0.06 ± 0.01 2.14 ± 0.25 Kaempferol 18.65 ± 1.13 168.47 ± 37.90 Epicatechin 405.41 ± 17.20 120.72 ± 22.50 Catechin 1.38 + - 0.34 4.44 ± 0.03 Quercetin 15.14 + - 0.51 114.41 + - 20.89 Chlorogenic acid 0.12 + 0.08 0.15 + 0.07 Naringin 0.39 + 0.07 0.40 ± 0.05 Routine 16.76 ± 1.27 28.11 ± 2.87 Hesperidin 0.44 ± 0.09 0.61 + 0.03 Protocatechuic acid 38.02 + - 4.91 34.77 + - 8.15 Caffeic acid 15.32 ± 5.29 25.86 ± 12.93 Methyl caffeate 0.03 ± 0.00 0.02 ± 0.00 Hesperin ND 0.02 ± 0.00 ECG 71.71 + - 3.66 635.14 ± 133.15 Kaemperol-3-glucoside 16.40 ± 0.63 330.63 ± 89.50 EGCG 16.04 + - 0.62 1441.44 + 561.14 Quercetin-3-glucoside 46.76 ± 0.38 1162.16 + 346.40

As shown in Table 1, the amount of total flavonoid was significantly smaller than that of the aqueous solution of the aqueous solution of salty water of Comparative Example 1 prepared according to Example 1 of the present invention, and the content of measured flavonoid was also measured Example 1 was generally lower than Comparative Example 1, but it was confirmed that only two kinds of materials, Gallic acid and Epicatechin, were contained in a larger amount in Example 1 than in Comparative Example 1.

Test Example 5. Immunological analysis

All the proteins (25 mg) in the RAW 264.7 cells cultured for 10 minutes and the wash extract of Example 1 were separated by molecular weight by electrophoresis on a 12% SDS-PAGE polyacrylamide gel, and then polyvinylidene fluoride membranes (PVDF, Bio-Rad Laboratories, Berkeley, Calif., USA). The membranes were then blocked with 5% skim milk and placed in a 5% BSA solution diluted in primary antibody for 12 hours at 4 ° C. After that time, the membranes were washed with TBS-T buffer, Secondary antibodies were placed in diluted skim milk (Santa Cruz, CA, USA, 1: 5,000) for 1 hour. Immunoreactivity was confirmed using a chemiluminescence western blotting detection system (Biosesang, Seoul, Korea).

4 is a photograph showing the immunological efficacy of the water extract prepared according to Example 1 of the present invention.

Changes in protein expression in signal transduction pathways known to be altered by Fc gamma receptor-activated tyrosine kinase to examine the mechanism of activation of RAW264.7 cells treated with the extract of Example 1 Respectively.

As shown in FIG. 4, the expression of p-Lyn, a Src family, and p-Syk, a non-receptor tyrosine kinase, which play a major role in RAW264.7 intracellular immunoreceptor signals, And it was confirmed that it was higher in the group treated with the extract. In addition, it was confirmed that the phosphorylation of PLC-γ was also high in the group treated with the extract of T. trachomatis in Example 1.

However, it was confirmed that the water extract of Example 1 did not significantly affect the expression of p-PKC alpha / beta II protein.

Anti-cancer and anti-inflammatory test

Test Example 6. Skin cancer cell line (B16F10 melanoma cell line ) Proliferation Low performance Measure

The proliferation inhibition test of the cancer cells (B16F10 melanoma cell line) was performed using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Cells (3 × 10 ⁴ cells / well or 1 × 10 ⁴ cells / well) dispensed into 96-well plates were incubated with the washout extract of Example 1. After 24 hours or 72 hours of sample treatment, cancer cell proliferation inhibition was calculated using CCK-8 kit. The inhibition of cancer cell proliferation was expressed as the inhibitory effect (%) of the cancer cell proliferation in the sample treated with the absorbance of the control group without any treatment.

FIG. 5 is a graph showing the cell viability of skin cancer cells (B16F10 melanoma cell line) treated with the Wassong extract prepared according to Examples and Comparative Examples of the present invention.

As shown in FIG. 5, it was confirmed that the wakis extract prepared according to Comparative Examples 1 to 5 inhibited skin cancer cells, but it was confirmed that the wash extract prepared according to Example 1 does not inhibit skin cancer cells.

Test Example 7. Anti-inflammatory efficacy

NO production measurement (uM) RAW 264.7 cells (3 × 10 ⁴ cells / well) cultured on a 96-well plate were treated 30 minutes after the treatment of the extracts (0, 100, 200 and 400 mg / LPS was stimulated with 1 mg / mL and incubated for 20 hours. 100 mL of the culture was then reacted with 100 mL of Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylene diamine dihydrochloride in 5% phosphoric acid) for 10 minutes at room temperature. The absorbance was measured at 560 nm using a microplate reader (Tecan, Mnnedorf, Switzerland). The concentration of NO in each culture was calculated using a standard concentration curve using sodium nitrite (NaNO ₂ ).

FIG. 6 is a graph showing NO production of the goose extract prepared according to Examples and Comparative Examples of the present invention in macrophages (RAW 264.7 cells) increased by LPS.

As shown in FIG. 6, mouse-derived macrophages (RAW 264.7) treated with the saliva extract of Example 1 and Comparative Examples 1 to 5 were stimulated with LPS, and the amount of secretion of each nitrite was measured using a Griess reaction assay . As a result, it was confirmed that the Salmonella extract of Comparative Examples 1 to 5, except for the onion extract of Example 1, inhibited the secretion amount of nitrite (Nitrite) increased by LPS in a concentration-dependent manner.

Test Example 8. Sterol Content Measurement

10 mL of Chloroform: Methanol (2: 1) was added to 1 g of each of the samples of Example 1 and Comparative Example 1, and the mixture was stirred at 4 ° C for 10 hours. The supernatant was filtered (Whatman 41) Add 10 mL of EtOH. Then, after stirring at 90 ° C for 1 hour, 9 mL of diethyl ether is added, and the supernatant is separated and dried under reduced pressure. 2 mL of chloroform was added and the mixture was filtered through a 0.45 μm filter paper (PVDF, Whatman) and analyzed by HPLC. For HPLC analysis, the column was measured at drift tube temperature of 90 ° C and spray chamber temperature of 30 ° C using Luna C ₁₈ column (5 μm, 4.6 × 250 mm, Grace) and Evaporative Light Scattering Detector (ELSD) Was separated under isocratic conditions with Acetonitrile: Methanol = 85: 15, column temperature was 30 ° C, injection volume was 20 L and flow rate was 1.0 mL / min. Brassicasterol, Campfesterol, Stigmasterol, B- Sitosterol, and Stigmastanol (Sigma) were used as standards.

Table 2 below shows the content of? -Sitosterol in sterols.

division Example 1 Comparative Example 1 ? -citostererol (mg / g) 0.00 ± 0.00 5.99 ± 0.33

As shown in Table 2 above, unlike Comparative Example 1, no β-sitosterol was detected in the washed extract powder prepared according to Example 1 of the present invention.

The β-sitosterol is a known substance having a function such as preventing cancer, and it was confirmed that the β-sitosterol showed an anticancer activity in Comparative Example 1.

Based on the results of Test Examples 1 to 8, it was confirmed that the extract of the present invention improved macrophage activity and had excellent immunological activity, but had little anticancer activity and anti-inflammatory activity.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example One. Sanje Produce

The washed extract powder obtained in Example 1 500 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example 2. Preparation of tablets

300 mg of the washout extract powder obtained in Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example 3. Preparation of capsules

200 mg of the supernatant extract powder obtained in Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example 4. Preparation of injections

600 mg of the supernatant extract powder obtained in Example 1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

It is prepared by the above-mentioned component content per ampoule according to the usual injection preparation method.

Formulation example 5. Liquid Produce

4 g of the washed extract powder obtained in Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100g by adding purified water, To prepare a liquid agent.

Formulation example 6. Preparation of granules

1,000 mg of the washout extract powder obtained in Example 1

Vitamin mixture quantity

Vitamin A Acetate 70

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2

Vitamin C 10 mg

Biotin 10

Nicotinic acid amide 1.7 mg

Folic Acid 50

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above-mentioned vitamins and minerals is comparatively comparatively mixed with the granules according to the preferred embodiment. However, the blending ratio may be arbitrarily changed, and the above components are mixed according to the ordinary granule preparation method, Can be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Formulation example 7. Manufacture of functional beverages

1,000 mg of the washout extract powder obtained in Example 1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to the flask to obtain a total of 900 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims

Wherein the water-extractable extract is an effective ingredient.

[Claim 2] The immunomodulating composition according to claim 1, wherein the transdermal extract is prepared by mixing the water and the water at a weight ratio of 1: 5 to 20.

The immunomodulating composition according to claim 1, wherein the extraction temperature is 20 to 120 ° C.

2. The immunomodulating composition according to claim 1, wherein the concentration of the dried extract is 50 to 600 ug / mL.

A health functional food for immune enhancing activity containing an extract of water as an active ingredient.

[Claim 6] The health functional food for immunomodulating activity according to claim 5, wherein the concentration of the dried extract is 50 to 600 ug / mL.

[Claim 6] The health functional food for immunomodulating activity according to claim 5, wherein the extraction temperature of the dried extract is 40 to 70 < 0 > C.

[Claim 7] The health functional food according to claim 5, wherein the health functional food is selected from the group consisting of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels, jellies and tablets.