KR20160007762A - Composition for Improving Immunity Containing Complex Polysaccharide from Aloe - Google Patents

Abstract

The present invention relates to a composition for increasing immunity, a health functional food, and a cosmetic composition containing aloe powder, Fucoidan and Phellinus linteus extract. A composition containing Aloe powder, Fucoidan and Phellinus linteus extract according to the present invention has more excellent capacities of activating macrophage and natural killer cells compared to an existing composition using aloe, Fucoidan, or Phellinus linteus alone. A composition for reinforcing immunity contains aloe powder, Fucoidan and Phellinus linteus extract. The aloe powder is Aloe vera gel powder. The composition for reinforcing immunity contains 10 to 60 weight parts of Fucoidan and 10 to 60 weight parts of Phellinus linteus extract with respect to 100 weight parts of Aloe powder. A health functional food contains Aloe powder, Fucoidan and Phellinus linteus extract. A cosmetic composition contains Aloe powder, Fucoidan and Phellinus linteus extract.

Description

TECHNICAL FIELD [0001] The present invention relates to an immunoconjugate composition containing a complex polysaccharide,

The present invention relates to a composition for enhancing immunity containing a complex polysaccharide, and more particularly to a composition for improving immunity, a health functional food and a cosmetic composition containing aloe powder, fucoidan and mushroom extract.

It is already known that immune function defends the body from various pathogens. Such immune function is used as a method to prevent and treat disease, and vaccination and antitoxin use are the most representative methods to be.

Recently, as the mechanism of action of the immune function is known, attempts have been made to directly use an immunoregulatory substance capable of regulating immune function. The immunomodulatory substance nonspecifically stimulates immune cells to enhance the immune function of the living body, thereby enhancing the defense force of the living body from disease factors. Such immunomodulating substances include chemically synthesized substances, microbial compositions or biological preparations. However, most of the above immunomodulating substances are limited to practical living bodies due to side effects or toxicity.

Recently, studies on immunomodulatory substances have been carried out through the use of effective ingredients extracted from food materials or natural substances that do not have toxicity, and the use of existing herbal medicine and verification of efficacy. In fact, It has been proved to be effective for degenerative diseases. Studies on the nutritional physiology and pharmacokinetics of these effects have also been actively conducted.

As the search for physiologically active substances to prevent biological damage and enhance the bio-defense system from these natural materials has been progressing actively, they are now entering into practical use as therapeutic agents or health supplement foods for alternative medicine.

Many immune enhancers have been developed and used to enhance the immune response as described above. (Yu, Z. et al ., Carbohydrate Polymer , 75: 309-320) . In addition, there is a need for an immune-enhancing agent that can be used as an immune enhancer. 307, 2009, Walsh AM et al., J. Anim. Sci., 4: 284, 2012).

However, such a natural extract has a disadvantage in that the immunostimulating effect is not remarkable as compared with the pharmaceutical composition.

Accordingly, the present inventors have made intensive efforts to develop an immunostimulating agent having an excellent immunity enhancing effect while maintaining the stability of the natural extract. Surprisingly, it has been surprisingly found that a complex polysaccharide containing aloe powder, fucoidan and mushroom is used alone The present invention has been completed based on this finding.

It is an object of the present invention to provide a composition for enhancing immunity which contains a complex-derived polysaccharide derived from a natural product having excellent immunity-enhancing effect while maintaining the stability of the natural extract.

It is still another object of the present invention to provide a health functional food containing a complex polysaccharide derived from a natural product.

It is still another object of the present invention to provide a cosmetic composition containing a complex polysaccharide derived from a natural product.

In order to achieve the above object, the present invention provides an immunostimulating composition containing aloe powder, fucoidan and mushroom extract.

The present invention also provides a health functional food containing aloe powder, fucoidan and mushroom extract.

In addition, the present invention provides a cosmetic composition comprising aloe powder, fucoidan and mushroom extract.

The composition containing the aloe powder, fucoidan and mushroom extract according to the present invention has macrophage activation and ability to activate NK cells, which are superior to those of aloe, fucoidan and mushroom, respectively.

Figure 1 shows the principle of measurement of macrophage activation.
Figure 2 shows the effect of complex polysaccharides, aloe, fucoidan and mushroom on the phagocytosis of macrophages.
3 is a graph showing the IL-2 production ability of complex polysaccharide, aloe, fucoidan, and mushroom mushroom in peritoneal macrophages.
Fig. 4 is a graph showing IFN-y production ability of complex polysaccharide, aloe, fucoidan and mushroom in bovine granular cells.
FIG. 5 is a graph showing the IL-12 production ability of complex polysaccharide, aloe, fucoidan, and mushroom mushroom in peritoneal macrophages.
FIG. 6 is a graph showing the activity of NK cell of complex polysaccharide, aloe, fucoidan and mushroom on the natural kill effect of YAC-1 cancer cells.

In one aspect, the present invention relates to an immunostimulatory composition comprising aloe powder, fucoidan and mushroom extract.

The "complex polysaccharide composition" used in the present invention refers to a composition containing a polysaccharide derived from a natural product, which comprises a composition containing aloe powder, fucoidan and mushroom extract.

In the present invention, the composition may be contained in an amount of 10 to 60 parts by weight of fucoidan and 10 to 60 parts by weight of mushroom extract, based on 100 parts by weight of aloe powder, 40 parts by weight and mushroom extract 20-40 parts by weight.

In the present invention, the aloe powder may be an aloe vera gel powder.

To In one embodiment of the present invention to ensure that the immune enhancing effect of the complex polysaccharide is superior aloe powder, fucoidan and each immunoactive linteus extract, using an aloe, fucoidan and Phellinus, through the in vitro cell line Immunopotentiating activities were compared. Surprisingly, it was confirmed that the compounds exhibited significantly higher activities in the complex polysaccharides used than when each of the above components alone was treated with macrophage activity, immune-related cytokine activating ability and NK activating ability.

The macrophages select solid substances (exogenous pathogens or dead cells in the body) to be consumed through signal substances, and macrophages reach the selected solid substances to adsorb the cell membranes, It is wrapped and put into the cell in the form of food. In this macrophage, there is lysosome, and solid matter is decomposed by mixing with hydrolytic enzyme in lysosome, and it can be said that immune response is appropriately performed as macrophage macrophage activity of pathogen is activated.

In one embodiment of the present invention, the complex polysaccharide activates the immune function of human normal cells to inhibit proliferation and recurrence of cancer cells, activate macrophages, enter the body of cancer cells, and promote the secretion of various cytokines, And the zymosan, which activates the immune function of T cells and B cells, was used to measure the phagocytosis of macrophages (Fig. 1).

The effect of macrophages on the phagocytosis of macrophages was evaluated by measuring the cell activation (%) value of 100% in the zymosan-treated wells of macrophages, and when the complex polysaccharide, aloe, fucoidan, . In the group treated with no treatment and the group treated with inhibitor, the cell activity was remarkably decreased. In the group treated with 500 μg / ml of the complex polysaccharide, the mouse peritoneal macrophages had the highest activity Respectively.

IL-2 and IFN-γ are type 1 cytokines associated with the T-cell mediated immune system. IL-2 and IL-12 are cytokines that are directly involved in the activation of natural killer cells (NK cells).

IL-2 is a cytokine that enhances immune responses by inducing the production of cytotoxic T-cells with virus resistance by T-cell growth factor and Helper t-cell (antibody production). IFN-γ inhibits inflammation-inducing cytokines and inhibits inflammation-related side effects. IL-12 is an activating cytokine of NK-cell, and NK-cell treats cancer and thus has an effect on innate immune enhancement.

Therefore, in another embodiment of the present invention, in order to evaluate the activity in vitro , the activity of macrophage activation, NK cell activation, IL-2, IL-12 and IFN, known as T-cell mediated immune system and natural killer activation factors, -γ was further evaluated. Further, in order to confirm whether the complexed polysaccharide of the present invention causes inflammation in the cells, it was confirmed that the inflammatory reaction was not caused by comparing LPS, ConA treated and untreated.

It was confirmed that IL-2, which is an inflammatory cytokine, was not produced in cells treated with complex polysaccharide, aloe, fucoidan and mushroom, compared with cells stimulated only with ConA. In the control group, the secretion level was the highest. Followed by IL-2 production by 500 μg / ml of fucoidan. In addition, the cells treated with the complex polysaccharide, aloe, fucoidan, and mushroom showed less generation of IFN-γ, which is an inflammatory cytokine, compared with cells stimulated only with ConA. / ㎖ treatment group showed the highest secretion level. Then, IFN-γ production was shown in the treatment group of 500 μg / ml of fucoidan.

Finally, it was confirmed that IL-12, which is an inflammatory cytokine, was not produced in cells treated with complex polysaccharide, aloe, fucoidan, and mushroom, compared with cells stimulated only with LPS. / ㎖ showed the highest level of secretion. Then, IL-12 production ability was shown at 300 / / ml of complex polysaccharide and 500 / / ml of fucoidan.

Although NK cells occupy a very small part of lymphocytes, they are very important part of the immune defense mechanism. Unlike T lymphocytes and B lymphocytes, NK cells are immunologically produced by antigen. Protects our body from infectious or proliferating cell death by killing target cells that show spontaneous attacking cytotoxicity. It is also the suppressor cells of the immune system that regulate the primary immune response as well as the immunoglobulin production of B lymphocytes by interfering with the antigens.

In another embodiment of the present invention, activation of NK cells was evaluated by measuring LDH isolated from YAC-1 cells destroyed by NK cells attacking YAC-1 cells as target cells, and 500 μg / ml of complex polysaccharide The highest cytotoxicity (%) was observed in the treated group of 300 ㎍ / ㎖ of complex polysaccharide and 500 ㎍ / ㎖ of fucoidan. The complex polysaccharide increased the activity of NK cells and attacked YAC-1 cells. Thus, it was confirmed that the natural killing effect of NK cells on Yac-1 cells was more apparent.

In another aspect, the present invention relates to a health functional food containing aloe powder, fucoidan and mushroom extract.

The functional food of the present invention can be used in the form of powder, granule, tablet, capsule or beverage, for example, various foods, candy, chocolate, beverage, gum, tea, vitamin complex and health supplement.

The complex polysaccharide of the present invention may be added to food or beverage for the purpose of enhancing immunity. At this time, the amount of the above extract in the food or beverage may generally be 0.01 to 50% by weight, preferably 0.1 to 20% by weight, of the total food weight of the health functional food composition of the present invention, In a proportion of 0.02 to 10 g, preferably 0.3 to 1 g.

The health beverage composition of the present invention has no particular limitation on the liquid ingredient other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aggravating agents (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In another aspect, the present invention relates to a cosmetic composition comprising aloe powder, fucoidan and mushroom extract.

Useful substances which can be further used in the cosmetic composition of the present invention include antioxidants, binders, bulking agents, chelating agents, pigments, softeners, emulsion stabilizers, film formers, fillers, directional ingredients, gelling agents, But are not limited to, hair fixatives, wetting agents, plasticizers, preservatives, skin conditioners, solvents, sunscreens, surfactants, ultraviolet absorbers, viscosity modifiers, waxes and the like.

The cosmetic composition of the present invention may contain a cosmetically acceptable carrier and may be an aqueous solution such as water or a polar organic solvent, alcohol such as ethanol or other polar solvent, natural or synthetic oil; Oil-in-water emulsion; Water-in-oil emulsion; Or wax. The carrier is clearly non-toxic. A preferred carrier for the composition of the present invention is an aqueous solution.

Depending on the function, the cosmetic composition of the present invention can be applied to a variety of cosmetic compositions such as a solution (lotion-like composition), a concentrated solution, a gel, ointment, emulsion (cream, emulsion), vesicle dispersion, powder, dense powder, And the like. More specifically, the cosmetic composition of the present invention can be used as a cosmetic composition for oral use such as ball puff, cream (facial cream, hand cream, moisturizing cream, sunscreen cream), cream powder, eyeliner, eye shade, eyebrow pencil, foundation, lotion, Emulsions, ointments, pomades, lozes, and the like, but are not limited thereto. They may be packed in a squeeze pack containing a filler that can be applied in the form of a foam or spray.

The cosmetic composition may contain natural or artificial wax. Natural waxes include animal and carnauba such as lanolin, beeswax, spermaceti or lanolin alcohol, hardened or acetylated lanolin, lanolin derivatives such as lanolin fatty acids or acetylated lanolin alcohols, carnauba, candelilla, There are vegetable such as kapok, rice, hardened jojoba, alfa or japanese wax or cork fiber, sugar cane wax, cocoa butter and the like. Alternatively, the inorganic wax may include paraffin, montan, lignite, petrolatum, petrolatum wax or microcrystalline wax, ceresin, or ozokerite. Examples of synthetic waxes used include linear esters such as myristyl myristate, a reaction product of a polyethylene wax obtained by Fischer-Tropsch synthesis and a saturated C10-C40 carboxylic acid and a saturated C10-C40 alcohol. Other waxes used include calcium lanolate or stearate or hardened coconut oil and the like.

The cosmetic composition may include modified or unmodified oils of vegetable or animal oils. For example, sweet almond oil, avocado oil, castor oil olive oil, jojoba oil, sunflower oil, turmeric oil, sesame oil, peanut oil, grape seed oil, soy milk, safflower oil, coconut oil, maze oil, hazelnut oil, Butter, palm oil, apricot seed oil, calf pill oil or perhydro-squalene. Furthermore, the oil phase may be an inorganic oil such as liquid paraffin, liquid petrolatum, and the like. The oil may be selected from the group consisting of fatty acid esters such as isopropyl myristate, isopropyl palmitate, 2-ethylhexyl palmitate, penicillin oil (stearyloctonate), unsaturated fatty acids such as oleate, palmitate, stearate, behen, linoleic, Volatile or non-volatile isoparaffins such as fatty acids or C8-C16 isoparaffins.

Moreover, the oil may be C12-C18 fatty alcohols such as oleyl alcohol, cetyl alcohol and stearyl alcohol.

If provided as an emulsion, the emulsified composition of the present invention comprises an oil phase and an aqueous phase. The oil phase is preferably provided in a range of about 1 to about 75 wt%, more preferably about 5 to about 60 wt%, and most preferably about 40 to about 60 wt%, based on the total weight of the composition desirable.

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

Example 1: Preparation of a complex polysaccharide composition

2.5 g of aloe vera gel powder (IMPROVE USA, INC., Mexico), 0.75 g of Fucoidan (MSC, Korea) and 0.75 g of Sasa mushroom extract powder (World EL, North Korea) were mixed to prepare a complex polysaccharide.

Example 2: Cytotoxicity test of complex polysaccharide, aloe, fucoidan and mushroom

The complex polysaccharides, aloe, fucoidan and mushroom were confirmed to be cytotoxic.

The cytotoxicity of the complex polysaccharide prepared in Example 1 and aloe (IMPROVE USA, INC., Mexico), fucoidan (MSC, Korea) and mushroom (WFP, North Korea) Cell viability was measured. The toxicity of the sample was measured, and the samples showing the maximum activity, efficacy and non-toxicity in the toxicity test were selected as final samples and the remaining in vitro tests were conducted.

Macrophages were isolated from the abdominal cavity of mice (7 weeks old, male, Balb / C) using thioglycollate, and the cells were treated with 10% Fetal Bovine Serum, 1% Penicillin, 0.1% Gentamicin, 1% L- sodium pyruvate and 1% Hepes 1M. After seeding to a cell number of 5 × 10 ⁴ cells / well in a 96-well plate, the complex polysaccharide (100 μg / ㎖ to 500 μg / ml) (100 μg / ml to 500 μg / ml), fucoidan (100 μg / ml to 500 μg / ml) and mushroom (100 μg / ml to 500 μg / ml), respectively.

Twenty-four hours after the sample treatment, the WST-1 assay solution was treated in each well and incubated at 37 ° C and 5% CO ₂ for 1 hour. OD was measured at 450 nm using a microplate rederer for 30 minutes. Cell viability Respectively.

As a result, cell viability up to 1000 μg / ml of the complex polysaccharide, aloe, fucoidan and mushroom was not statistically significantly decreased. Therefore, the experiment was carried out by selecting 500 μg / ml of the complex polysaccharide, aloe, fucoidan and mushroom.

Example 3: Measuring the phagocytosis of peritoneal macrophages of complex polysaccharides, aloe, fucoidan and mushroom

Macrophages were isolated from the peritoneal cavity of mice and the phagocytosis of macrophages was measured against the complex polysaccharide, aloe, fucoidan and mushroom to the maximum concentration.

 By activating the immune function of normal human cells, it inhibits the proliferation and recurrence of cancer cells, activates macrophages, enters the body of cancer cells, and promotes the secretion of various cytokines, thereby activating immune functions of immune T cells and B cells (Cytoselect 96-well phagocytosis assay kit, CELL BIOLABS, INC. USA, Sandiego) was used to measure the phagocytosis of macrophages using zymosan.

Seven-week-old Balb / c mice were intraperitoneally injected 3 days before the autopsy of 2 ml of thioglycollate medium (which stimulates the peritoneal cavity to stimulate the peritoneal cavity to secrete macrophages in the peritoneal cavity as a primary immune response) 8 mL of DMEM medium was added to the abdominal cavity and the cells were thoroughly massaged and recovered. The cells were seeded at 10 ⁵ cells per well in a 96-well plate and cultured overnight at 37 ° C in 5% CO ₂ .

To the cultured cell wells were added 100 μg / ml to 500 μg / ml of complex polysaccharide, 100 μg / ml to 500 μg / ml of aloe, 100 μg / ml to 500 μg / ml of fucoidan, / ㎖ ~ handle 500㎍ / ㎖), allowed to react for a Zymosan, Inhibitor 10㎕ as Table 1, was reacted for 2 hours oi 2 hours after treatment of each well, serum-free medium (DMEM, RPMI) , And then immobilized solution 100 μl was added, reacted at room temperature for 5 minutes, and then washed twice with PBS. blocking reagent (component material) was added, and the mixture was stirred at room temperature for 60 minutes, washed three times with PBS, diluted with 1 x permeabilization solution 100 μl of the reaction mixture was added thereto, followed by reaction at room temperature for 5 minutes, followed by washing. After addition of 100 μl of the detection reagent, the mixture was stirred at room temperature for 60 minutes and then washed three times with PBS. 50 μl of detection buffer was added to each well. After stirring for 10 minutes at room temperature, 100 μl of substrate was incubated for 15 minutes, and the absorbance of each well was measured at 405 nm.

Group cell only Zymosan treatment Inhibitor treatment Inhibitor + Zymosan treatment Complex polysaccharide, Aloe, Fucoidan, Situation mushroom + Zymosan treatment

The cell activation of the complex polysaccharide, aloe, fucoidan, zymosan and zymosan was investigated in the cells treated with zymosan at 100% cell activation (%). As a result, as shown in FIG. 2, it was confirmed that the macrophage activity was significantly lowered in the control group and the inhibitor treated group without any treatment. In the 500 μg / ml complex polysaccharide, , Followed by aloe and fucoidan at 500 ㎍ / ㎖. This implies that the complex polysaccharide enhances the ability of the peritoneal cells to innate immunity and increases the immune response to the external antibody.

Example 4: Measurement of IL-2, IL-12 and IFN-y production in peritoneal macrophages of a complex polysaccharide, aloe, fucoidan and mushroom

IL-2 and IFN-γ are type 1 cytokines associated with the T-cell mediated immune system. IL-2 and IL-12 are cytokines that are directly involved in the activation of natural killer cells (NK cells).

The cells were treated with 10 ⁵ cells / well of each well of a 96-well plate and cultured at 37 ° C and 5% CO ₂ for 24 hours. The maximum concentration of complex polysaccharides, aloe, fucoidan and mushroom extract IL-12 and IFN-γ produced by culturing the cells for 24 hours after adding LPS (IL-12), ConA (IL-2, IFN- DuoSet sandwich ELISA Mouse TNF-α kit (R & D systems).

Group cell only LPS or < RTI ID = 0.0 > ConA < Complex polysaccharide, aloe, fucoidan, situation mushroom

  The coated primary 96-well plate was diluted with PBS and diluted with PBS to 100 μl / well for 1 day. The next day, the primary antibody was washed with washing buffer (PBST, 0.05% Tween 20 in PBS), and assay buffer (1% BSA in PBS) was added for 2 hours After washing, it was washed with washing buffer. The standard curve and the RAW 264.7 cell culture solution prepared above were added to each well for 2 hours. After completion of the reaction, the cells were washed with washing buffer, and the secondary antibody was diluted in the assay buffer. Was added to each well and reacted for 2 hours. After the reaction was completed, the plate was washed with a washing buffer, reacted with a reagent for color development, and absorbance was measured at 570 nm. The amount of cytokine produced in the cells was calculated using a standard curve.

As a result, as shown in Fig. 3, the cells treated with the complex polysaccharide, aloe, fucoidan and mushroom showed less generation of IL-2, which is an inflammatory cytokine, compared to cells stimulated only with ConA. And the highest secretion level was found at 500 ㎍ / ㎖ of complex polysaccharide. Followed by IL-2 production at 500 μg / ml of fucoidan.

In addition, as shown in Fig. 4, in the cells treated with the complex polysaccharide, aloe, fucoidan, and mushroom, overall production of IFN-y, an inflammatory cytokine, was not more than that of cells stimulated only with ConA , And the secretion was the highest at 500 ㎍ / ㎖ of complex polysaccharide. Then, IFN-? Production was observed at 500 占 퐂 / ml of fucoidan.

In addition, as shown in Fig. 5, in the cells treated with the complex polysaccharide, aloe, fucoidan, and mushroom, IL-12, which is an inflammatory cytokine, was less produced than cells stimulated only by LPS, , And the secretion was the highest at 500 ㎍ / ㎖ of complex polysaccharide. Then, IL-12 production ability was shown at 300 / / ㎖ of complex polysaccharide and 500 ㎍ / ㎖ of fucoidan.

Example 5: Activation of natural polysaccharides, aloe, fucoidan and natural mushroom cells (NK cells)

NK cell activation was assessed by measuring YAC-1 cells as target cells and measuring free LDH from YAC-1 cells that were destroyed by NK cells attack. For determination of liberated LDH, a cytotoxicity assay kit (Cytotox 96 Non-radioactivity Cytotoxicity assay, Promega, USA) was used.

For isolation and culture of NK cells (immune cells directly destroying cancer cells), mice (7 weeks old, male, Balb / C) were sacrificed by cervical disassembly and then the abdomen was disinfected with 70% ethanol and the spleen was aseptically extracted Respectively. The spleen was homogenized with a sterilized homogenizer, and 40mesh was soaked in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin in a 50 ml tube. The homogenized spleen was passed through the wells and washed twice with RPMI 1640 Respectively. The red blood cells were ruptured in the same solution of red cell solution and PBS, washed with PBS solution, and incubated after trypan blue dye exclusion test. The tissue culture was cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin at 37 ° C in a 5% CO ₂ incubator.

To examine the cytotoxic ability of NK cells, NK cells were cultured in YAC-1 cells (NK sensitive cell line, (Cytotox 96 Non-radioactive Cytotoxicity assay) was used to measure free LDH from YAC-1 cells that were destroyed by attacking NK-92 (ATCC CRL-2407 ™). The number of YAC-1 cells was adjusted to be 1 × ¹⁰ 3/100 μl in a 96-well plate and cultured in the same manner as in the case of NK cells. The cells were cultured at 37 ° C. in 5% CO ₂ Lt; / RTI > for 4 hours. Target cell maximum 45 minutes before the supernatant was collected. 10 μl of Lysis solution (10 ×) was added to the well with LDH Realease control.

After 4 hours of cell culture, the cells were centrifuged for 4 minutes to collect 50 μl of the supernatant from which the LDH was liberated, and transferred to a 96-well plate. 50 μl of the reconstituted substrate mix was added to each well, followed by incubation at room temperature for 30 minutes The absorbance was then measured at 490 nm. For the spontaneous LDH measurement, only the culture medium was added to the wells. To obtain the maximum value of the LDH released from the YAC-1 cells, the maximum LDH well was added with lysis solution to completely dissolve the cells.

Percentage of toxicity (% of cytotoxicity) was calculated as LDH liberated from each culture using the following equation.

[Formula 1]

Experimental: 1: 5 cell ratio (avg) -culture medium background (avg)

Effector spontaneous: effector spontaneous (avg) -culuture medium backgroun (avg)

Target spontaneous: target spontaneous (avg) -culture medium background (avg)

Target maximum: target maximum (avg) -volume correction control (avg)

As a result, as shown in FIG. 6, the highest cytotoxicity value was measured at a concentration of 500 μg / ml of the complex polysaccharide, and a higher cytotoxicity (%) at a concentration of 300 μg / ml of the complex polysaccharide and 500 μg / Respectively. The complex polysaccharide and fucoidan increased the activity of NK cells and attacked YAC-1 cells. Thus, the natural killing effect of NK cells on Yac-1 cells was more pronounced.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

Aloe powder, fucoidan and mushroom extract.
The immunomodulating composition according to claim 1, wherein the aloe powder is an aloe vera gel powder.
The immunomodulating composition according to claim 1, wherein 10 to 60 parts by weight of fucoidan and 10 to 60 parts by weight of mushroom extract are contained relative to 100 parts by weight of the aloe powder.
Aloe powder, fucoidan and mushroom extract.
The health functional food according to claim 4, wherein the aloe powder is an aloe vera gel powder.
The health functional food according to claim 4, wherein 10 to 60 parts by weight of fucoidan and 10 to 60 parts by weight of mushroom extract are contained relative to 100 parts by weight of the aloe powder.
Aloe powder, fucoidan and mushroom extract.
The cosmetic composition according to claim 7, wherein the aloe powder is an aloe vera gel powder.
The cosmetic composition according to claim 7, wherein 10 to 60 parts by weight of fucoidan and 10 to 60 parts by weight of mushroom extract are contained relative to 100 parts by weight of aloe powder.

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