KR101947276B1 - Composition for immune enhanvement comprising sargassum coreanum enzymatic extract - Google Patents

Abstract

The present invention relates to a composition for enhancing immunization containing an enzymatic degradation product of Sargassum coreanum as an active component. The enzymatic degradation product of Sargassum coreanum manufactured in the present invention: increases the secretion rate of cytokine in spleen cells in vitro; promotes proliferation of spleen cells by oral administration; increases immune globulins, white blood cells and lymphocytes in serum; and increases an activity of NK cells. Therefore, the enzymatic degradation product of Sargassum coreanum can be used for enhancing immunization.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for enhancing immunity,

The present invention relates to a composition for enhancing immunity, which contains an enzymatic degradation product of a big leaf moth as an active ingredient.

Immunity refers to a biological function that protects the normal tissue of a human body from a variety of external harmful substances and internal factors such as tumors. The immune system is a defense system that maintains the life of our bodies. There are many factors that degrade the functions of the immune system, for example, antibiotics, stress, allergen foods, and obesity.

Decreased immunity results in a weakening of the body's resistance to bacteria and viruses and can be direct or indirect causes such as headache, insomnia, indigestion, depression, obesity, hypertension, diabetes, allergies and irritable bowel syndrome. Accordingly, various methods for preventing the decrease of the surface force and for increasing the immunity have been proposed. Immune disorders are diseases in which constituents of the mammalian immune system cause, mediate, or contribute to the pathology of mammals, particularly inflammatory disorders being one of the most important health problems in the world. Inflammation is generally a localized protective response of the body tissue to host infiltration by foreign substances or by harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial formulations; An immune response such as an allergic and autoimmune response, or a condition associated with oxidative stress

On the other hand, researches for the development of medicines, cosmetics, functional foods and the like have been widely carried out in the world, and in particular, in the case of pharmaceuticals, since the number of conventional compounds is limited, And the development of new chemicals that can be used as medicines is declining. Therefore, many attempts have been made to develop physiologically active substances effective in natural resources. In order to isolate the active ingredients from these natural sources and identify their structure and efficacy, it is essential to establish appropriate biological activity targets and ensure sufficient natural resources.

Although marine organisms on the earth have numerical superiority and diversity over land plants, relatively few studies have been conducted due to problems such as collection costs. Among these marine organisms, seaweeds have an infinite possibility of being used as novel bioactive materials since they have biosynthetic pathways that are difficult to find in land plants. Seaweeds can be divided into green algae, brown algae, and red algae among the species that can be collected in Korea. The green algae include hearing, true blue feathers, true feathered horses, leafy blue and leaf vein horses, and brown algae include L, Tart, large leaves, large netted horse, true netted horse, And red algae may be selected from the group consisting of true curls, true black curls, true gambling, chaenginari, true chestnut, coral reef coral, chestnut grass, chestnuts, humpback, baboons, Eggplant grass, chewing gum horses, horseradish, horse chestnut, chestnut tree

As a result of studies on active substances derived from natural products having low toxicity risk, studies for finding functional natural substances for treating immune enhancement effect and diseases through it have been carried out. However, studies on natural substances derived from Sargassum coreanum ) have not been studied.

Prior art related to immunity enhancement, which is a technical field to which the present invention belongs, includes Korean Patent No. 10-1509220, which relates to a method for preparing a composition for enhancing immunity using a coffee grounds fermented product, Korean Patent No. 10-1484021 on the composition.

It is an object of the present invention to enzymatically degrade large leaf mackerel and to elucidate its immunostimulating effect in vitro and in vivo, and to use it as a pharmaceutical composition for immunity enhancement and a food composition for immunity enhancement.

In order to achieve the above object, the present invention provides a pharmaceutical composition for enhancing immunity, which comprises an enzymatic hydrolyzate of Sargassum coreanum as an active ingredient.

In addition, the present invention provides a food composition for enhancing immunity, which contains a large-leaf-leaf enzyme hydrolyzate as an active ingredient.

In addition, the present invention provides a method for producing a fibrinolytic enzyme degradation product.

According to the present invention, the enzymatic hydrolyzate of the folium increased the secretion amount of cytokines in spleen cells in vitro, promoted the proliferation of splenocytes by oral administration, increased the secretion of immuno-related cytokines, Increased globulin, leukocyte and lymphocyte, and increased the activity of NK cells, which can be used for immunity enhancement purposes.

Fig. 1 is a diagram showing the low molecular weight of TLC obtained by the enzymatic hydrolyzate by time.
FIG. 2 is a graph showing the effect of the enzymatic degradation product of the large-leaf moth on the secretion amount of IFN-y, IL-2, IL-6 and IL-10 in splenocytes.
FIG. 3 is a graph showing the effect of enzyme hydrolyzate of each of the large-leaf mothballs on the splenocyte proliferative capacity of the mice not treated with mitogen (A), those with LPS (B), and those with the Con-A activated (C) .
FIG. 4 is a graph showing the activity of IL-2 from splenocytes activated by LPS-activated (B), Con-A (C) It is a graph that confirms the increase of the secretion amount.
FIG. 5 is a graph showing the results of immunohistochemical analysis of the enzyme-degrading products of the extracts of IFN-.gamma. In the splenocytes treated with mitogen-treated (A), LPS-activated (B) and Con- Of the amount of secretion.
FIG. 6 is a graph showing the effect of the enzyme hydrolyzate of each of the large-leaf mothballs on the amount of IgG1 and IgG2a secreted in the serum of mice administered orally.
FIG. 7 is a graph showing the increase in NK cell activity in mice to which the enzymatic hydrolyzate of each time interval of the larvae was orally administered.
8 is a graph showing increase in the numbers of leukocytes (WBC), lymphocytes (LY), mononuclear cells (MO), and neutrophils (NF) in the whole blood of mice administered orally with the enzymatic hydrolysates at different time points.

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention. It should be understood, however, that the invention is not limited thereto and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. .

In one aspect, the present invention relates to a pharmaceutical composition for enhancing immunity, which contains a large-leaf-leaf enzyme hydrolyzate as an active ingredient.

In one embodiment, the foliar enzymatic degradation product may be the result of the reaction of a large leaf mushroom with a culture medium of Schwannella onidensis, wherein the Schwannellaonidensis is S. oneidensis PKA 1008 strain deposited with Deposit No. KCTC 18588P The large-leaf-leaf enzyme hydrolyzate may be one obtained by low-molecular-weighting a large-leaf moth with the above-mentioned culture medium. The culture medium of the strain may be used as a crude enzyme solution, and the crude enzyme solution may have alginate decomposing activity.

The optimal growth conditions of the strain of Shwanaellaonaidensis PKA 1008 (Accession No. KCTC 18588P) of the present invention were incubated at pH 9.2% NaCl and 30 ° C for 24 hours. The crude enzyme solution contained enzyme activity at pH 9 and 30 ° C And can produce 1.001 g / l of reducing sugar at 3.5% working concentration during 1 hour reaction.

In one embodiment, the fibrinolytic enzymes of the foliar enzymes are obtained by mixing the supernatant obtained by centrifuging the S. oneidensis PKA 1008 culture broth with the foliar larvae at a ratio of 1: 1 (v / v) and then reacting for 12 to 60 hours Lt; / RTI >

In one embodiment, the large leaf mackerel may be a pulverized material obtained by drying the large leaf mackerel, a lysine powder obtained by extracting the large leaf mackerel, or a lyophilized powder or a large leaf mash extract. The extraction method of the mash extract is not particularly limited, Extraction, shaking extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction. As the extraction solvent, water, a polar solvent such as a C ₁ -C ₄ lower alcohol, a nonpolar solvent such as hexane, chloroform, dichloromethane or ethyl acetate, or a mixture of two or more thereof may be used.

As used herein, the term " extract " means an active ingredient isolated from a natural product, i.e., a substance exhibiting the desired activity. The extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes all the forms that are formulated using the dry powder of the extract. In addition, the above-mentioned extract also includes fractions obtained by fractionating the extract obtained through the above extraction process.

As used herein, the term " immunological enhancement " means any action that increases the immunity by administration of the foliar enzymatic degradation product of the present invention or a composition comprising the same. In other words. Enhancing the immune response by increasing the secretion of cytokines in the immune cells, or enhancing the immunity by increasing the activity of the cells of the immune system.

The composition of the present invention can exhibit an immunopotentiating effect by increasing the production of cytokines. According to one embodiment of the present invention, the composition of the present invention increases the production of cytokines selected from the group consisting of IL-2, IL-6, IFN-y and IL-10. IL-2, IL-6 and IFN-y are Th1 cytokines that regulate cellular immunity and IL-10 is a Th2 cytokine that regulates humoral immunity. The composition of the present invention inhibits the production of Th1 and Th2 cytokines Can be increased, so that the immune enhancement effect can be exerted on both cellular immunity and humoral immunity.

The composition of the present invention increases the activation of NK cells, and NK cells are natural killer cells that attack virus-infected cells and cancer cells and belong to lymphocytes, but the main histocompatibility complex (MHC) It is a congenital immune system because it has a characteristic that it attacks only nonspecific cells.

The pharmaceutical composition for immunoenhancing of the present invention can be used for prevention or treatment of immune diseases through its immunoenhancing effect. The immunological disease may be selected from the group consisting of cold, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergy, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, But is not limited thereto.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, external preparations, suppositories, sterile injectable solutions and sprays.

The therapeutically effective amount of the composition of the present invention may vary depending on a variety of factors, such as the method of administration, the site of administration, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington ' s Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Inc. A buffered saline solution, a buffer solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and if necessary, an antioxidant, a buffer, Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the protein, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Wherein the starch is selected from the group consisting of lactose, mannitol, sugar, arabic gum, pregelatinized starch, cornstarch, powdered cellulose, hydroxypropyl cellulose, opaques, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, Calcium, white sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 part by weight to 90 parts by weight, but is not limited thereto.

The composition of the present invention may be administered orally or non-orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered in accordance with a desired method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, more preferably administered once to several times a day.

Examples of the liquid preparation for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Etc. may be included together. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention relates to a food composition for enhancing immunity, which contains a large-leaf-leaf enzyme hydrolyzate as an active ingredient.

The term " food " as used in the present invention means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, It is intended to include food, food additives, functional foods and beverages as a meaning.

The food composition of the present invention can be manufactured in the form of, but not limited to, various drinks, gums, tea, confectionery, vitamin complexes, health supplements and the like.

The preferred intake amount of the food composition of the present invention varies depending on the condition and the weight of the recipient, the degree of symptoms, the type of food, the period of consumption, and can be appropriately selected. It is preferable for the food composition of the present invention that the active ingredient is ingested at 0.2 mg / kg to 200 mg / kg per day for optimal effect.

In one aspect, the present invention is directed to a method for preparing a foliar enzymatic degradation product.

In one embodiment, the method for producing the foliar enzymatic degradation product comprises: culturing S. oneidensis PKA 1008 strain at pH 9 and 25 to 35 ° C for 20 to 30 hours; Obtaining a crude enzyme solution by centrifuging the S. oneidensis strain PKA 1008; Preparing a large leaf mash powder; And mixing the crude enzyme solution of step 2) with the large leaf mash powder of step 3), and then allowing to react at pH 9 for 12 to 60 hours.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

<Example> Production of Enzymatic Hydrolyzate of Small-leaf mushroom

It was separated from U. pertusa , which is undergoing decomposition, at the off-shore of Songjeong, Pusan coast, and was deposited at the Korea Research Institute of Bioscience and Biotechnology ( Shewanella oneidensis, S. oneidensis ) PKA 1008 strain (Accession No. KCTC 18588P) was cultivated for 24 hours at pH 9, 2% NaCl and 30 ° C, which are optimal conditions. After culturing, the supernatant was centrifuged at 10,000 xg for 30 minutes at 4 ° C using a centrifuge (UNION 32R, Hanil Co., Kimpo, Korea). The activity of the crude enzyme was 517 U / mL.

In order to prepare the enzymatic degradation products of the leaves, 80 mg / mL of the powder was prepared by pulverizing and drying 10 mg of phosphate buffer (pH 9), and the optimal growth conditions of S. oneidensis PKA 1008 enzyme 1 N NaOH was used to adjust to pH 9. To this, 0.02% sodium azide of the total volume of the reaction was added to the above-prepared S. oneidensis PKA 1008 enzyme solution in a ratio of 1: 1 (v / v) to prevent microbial contamination. The reaction between the crude enzyme solution and the leaves was carried out at 30 ° C for 0, 3, 6, 12, 24, 48 and 60 h, and the decomposed solution was treated with boiling water for 10 minutes to inactivate the enzyme. The 10,000 × g, 10 minutes, followed by freeze the supernatant obtained by centrifugation at 4 ℃ dried and stored at -20 ℃.

<Experimental Example 1> Characterization of enzymatic degradation products

Each of the lyophilized (0, 24, 48 and 60 h) enzymatic degradation products was dissolved at 20 mg / mL in each of the lyophilized reaction samples, and then ethanol was added to 80% of the final solution. Respectively. The supernatant was then removed by centrifugation at 7,500 rpm for 30 minutes at 4 ° C, and the precipitate was dried to remove ethanol and used in the experiment. Low molecular weight degradation products were identified by thin-layer chromatography (TLC). Five microliters of low molecular weight samples were spotted on Silica gel F254 plates and developed using 1: butanol: methanol: water (4: 1: 2 v / v / v). After development, 10% sulfuric acid-added ethanol solution was sprayed and heated at 110 DEG C for 30 minutes. Cellooligosaccharide was used as a standard mixture and cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose were used as a standard mixture. (Megazyme Co., Wicklow, Ireland), and glucose was purchased from Sigma Chemical Co. (St Louis, MO, USA).

The enzymatic reaction was decomposed into tetramer and trimer by decomposing into dimmer and trimer in reaction 48h, and then decomposed into tetramer and trimer by enzymatic reaction after 24h. It was confirmed that the final 60h reaction product was decomposed into dimers (Fig. 1).

Experimental Example 2: Promoting cytokine secretion in spleen cells

In order to confirm the immune enhancing activity of the lipolytic enzyme hydrolyzate prepared in the above example in vitro, it was isolated from mouse. The cytokine (IFN-γ, IL-2, IL-6 And IL-10) secretion levels were measured.

Specifically, a 6-week-old male Balb / c mouse (Orient Bio, Seongnam, Korea) was preliminarily raised for 1 week in an animal breeding room maintained at a temperature of 20 ± 2 ° C and a humidity of 50 ± 10% , Sacrificed by cervical dislocation, and aseptically excised. This experiment was approved by the Animal Experiment Ethics Committee of Pukyong National University (Approval No. 2015-04). The cell suspension homogenized using a tissue grinder was centrifuged at 1,800 rpm for 5 minutes at 4 ° C to liberate the cells from the extracted spleen. Then, red blood cells were removed by standing in an RBC lysis buffer (Tris-buffered ammonium chloride, 0.87% NH ₄ Cl, pH 7.2) for 10 minutes, and 10% FBS (Hyclone, Victoria, Austria) And diluted to a concentration of 2 × 10 ⁶ cells / mL. To the diluted splenocyte suspension, 50% and 100 μg / mL of the proteolytic enzymes were added, respectively. To the control, 0.01 M PBS (Phospate buffered saline, pH 7.2) was added and incubated at 37 ° C in a 5% CO ₂ incubator (MCO- 15AC, Sanyo, Tokyo, Japan) for 72 h. Anti-mouse IFN-y, IL-2, IL-6 and IL-10 mAbs were placed in wells (Nunc, Roskilde, Denmark) and coated overnight at 4 ° C. After washing with PBST (0.01 M PBS, pH 7.2, 0.05% Tween 20) and blocking with 10% FBS solution for 1 h at room temperature, the cell culture supernatant was added and reacted at room temperature for 2 h. Biotinylated anti-mouse IFN-γ, IL-2, IL-6, IL-10 mAb and HRP (streptavidin-horseradish peroxidase) conjugate were added and reacted for 1 h at room temperature. After washing with PBST, citrate buffer (pH 5.0) supplemented with OPD (o-phenylenediamine) and H ₂ O ₂ was added and developed in a dark place for 30 minutes. The enzyme-linked immunosorbent assay (ELISA), which measures the absorbance at 490 nm with a microplate reader (model 550, Bio-Rad, California, USA) after addition of 2 NH ₂ SO ₄ to stop the reaction, The amounts of IFN-y, IL-2, IL-6 and IL-10 cytokines secreted from the cell culture supernatants were measured.

As a result, the amount of IFN-γ secretion was increased in the treated group compared to the PBS treated group (35.15 ± 2.91 pg / ml) as compared with the control, and when the enzyme hydrolyzate was treated at 50 μg / And 50.30 ± 6.79 pg / mL, respectively, depending on the reaction time of 0 h, 24 h, and 48 h, respectively. The results showed that IFN-γ secretion was increased in a concentration-dependent manner as the reaction time of the big leaf mallow and coenzyme increased, indicating 61.88 ± 3.88 pg / mL, 80.39 ± 8.72 pg / mL and 93.41 ± 5.82 pg / mL. In addition, the amount of IL-2 secreted at each concentration was higher than that of the control (74.90 ± 2.91 pg / ml), and the response time of the crude enzyme solution and the leaves was 24 h at a concentration of 50 μg / ml The highest value was 87.24 ± 0.97 pg / mL in the group and 85.18 ± 5.82 pg / mL in the 48 h group. In addition, the amount of IL-6 secretion was significantly increased at 50 and 100 μg / mL in the treated group compared to the control (8.42 ± 5.55 pg / mL). In particular, The highest values were obtained at the concentrations of 50 and 100 μg / mL, 118.82 ± 12.68 pg / mL and 268.45 ± 3.96 pg / mL, respectively. In addition, the IL-10 secretion level was significantly increased at a concentration of 50 μg / mL as compared with the control amount of 121.29 ± 0.00 pg / mL, which was 424.03 ± 13.38 pg / mL at 48 h treatment, The levels of IL-2, IL-6, and IFN-γ increased in the treated group at 687.36 ± 2.23 pg / mL at the concentration of 100 μg / mL and increased about 1.5-fold in the secretion of cytokines from the 0 h treatment (438.23 ± 11.15 pg / mL) (Fig. 2). The results showed that the enzyme hydrolyzate of S. oneidensis PKA 1008 - derived enzyme hydrolyzate was able to control the immunoreactivity of the enzyme.

<Experimental Example 3> In vivo immune-enhancing activity was confirmed by administration of enzyme hydrolyzate

3-1. Spleen and Thymus Weighing

The mice were anesthetized and sacrificed using diethyl ether for 2 weeks at 50, 100, and 200 mg / kg body weight concentrations, respectively. The spleen and thymus, the immune organs, were removed from the sacrificed mice and the weight of the spleen and thymus was measured to calculate the percentage of body weight to derive the relative organ weights for each organ.

Group Dose
(mg / kg BW) Spleen indexa
(mg / g) Rate (group / NC) Thymus indexb (mg / g) Rate
(group / NC) NC 0 3.8 ± 0.1 1.00 1.6 ± 0.2 1.00 0 h 50 3.6 ± 0.3 0.95 1.5 ± 0.1 1.00 100 3.5 ± 0.2 0.93 1.4 ± 0.3 0.92 200 3.6 ± 0.2 0.99 1.5 ± 0.1 1.01 24 h 50 3.5 ± 0.1 0.92 1.5 ± 0.2 0.94 100 3.4 ± 0.2 0.91 1.7 ± 0.2 1.10 200 3.7 ± 0.3 0.98 1.7 ± 0.2 1.02 48 h 50 3.5 ± 0.1 0.93 1.6 ± 0.2 1.00 100 3.5 ± 0.3 0.88 1.5 ± 0.1 0.91 200 3.6 ± 0.3 0.89 1.5 ± 0.2 0.91

Spleen index = spleen weight / body weight.

Thymus index = thymus weight / body weight.

As a result, the spleen and thymus weights were not significantly changed by the reaction time of the large leaf mallow and the coenzyme compared to the control group (Table 1).

3-2. Identifying the proliferative capacity of splenocytes

Splenocytes obtained from the spleen isolated in Experimental Example 3-1 were dispensed into a well plate at a concentration of 2 × 10 ⁶ cells / mL and pre-cultured for 20 hours. After that, 1 ㎍ / mL LPS and 3 ㎍ / mL ConA were added as mitogen and cultured for 46 h in a 5% CO ₂ incubator (MCO-15AC, Sanyo, Tokyo, Japan) at 37 ° C. Then, 5 mg / mL MTT (thiazol blue tetrazolium bromide, Sigma-Aldrich Co.) solution was added and re-cultured for 2 h. After centrifugation at 879 x g for 10 min at 4 ° C to remove supernatant, DMSO was added to each well and incubated at 540 nm using a microplate reader (Model 550, Bio-rad, Hercules, CA, USA) The optical density (OD) was measured. The cell proliferative capacity using the measured absorbance was calculated by the following formula (1).

As a result, it was shown that the enzyme hydrolyzate obtained by reacting the large-leaf mackerel and crude enzyme solution for 24 h increased the proliferative capacity of spleen cells depending on the oral administration concentration. In the case of splenocytes treated with LPS and ConA, (Fig. 3). &Lt; tb &gt; &lt; TABLE &gt;

3-3. Cytokine secretion measurement

In order to confirm the immuno-promoting activity by oral administration of the enzymatic degradation product of the big leaf mushroom, IFN-? And? In the splenocytes isolated from the mice orally administered with the hydrolyzate of the petiole leaf enzyme by the above Experimental Examples 3-1 and 3-2 The amount of IL-2 secretion was measured in the same manner as in Experimental Example 2.

The amount of IL-2 secretion was 62.36 ± 1.94 pg / mL in the control group without treatment with mitogens. The enzyme-degraded enzyme was reacted at 50, 100 and 200 mg / kg for 24 h In the treatment group, respectively, and it was found to be 75.59 ± 7.75, 89.98 ± 2.91, and 107.80 ± 2.91 pg / mL, respectively. In the case of splenocytes treated with LPS and ConA, it was confirmed that the enzyme hydrolyzate reacted with 24 h was increased in a concentration-dependent manner as compared with the control (Fig. 4). In addition, IFN-γ secretion was 63.97 ± 8.72 pg / mL in the control group, but not in the mitogen treated group. 9.69 pg / mL, and when treated with 200 mg / kg, the secretion amount was increased to 82.44 ± 13.57 pg / mL by treatment with enzyme digest. In the case of splenocytes treated with LPS, the amount of secretion increased by about 1.6 times to 197.59 ± 11.63 pg / mL when treated with 200 mg / kg of enzyme hydrolyzate reacted with 24 h enzyme compared to 118.08 ± 3.87 pg / mL in control Respectively. ConA-treated splenocytes increased in a concentration-dependent manner in a 24 h enzyme-reacted enzyme digest at a higher level than the control (Fig. 5).

3-4. Measurement of IgG1 and IgG2a in serum

ELISA was used to measure the amount of IgG1 and IgG2a in serum. Specifically, anti-mouse IgG1 and IgG2a mAb were diluted at a ratio of 1: 250, put into a 48-well, and coated overnight at 4 ° C. Then, the cells were washed with PBST (0.01 M PBS, pH 7.2, 0.05% Tween 20) and blocked with 10% FBS solution for 1 h at room temperature. Then, the serum isolated from mice orally administered with the fibrinolytic enzymes was added thereto, The reaction was allowed to proceed for 2 h. Biotinylated anti-mouse IgG1, IgG2a mAb and HRP conjugate were diluted in a ratio of 1: 250 to each well and reacted at room temperature for 1 h. Washed with PBST and incubated in a dark place for 30 min with citrate buffer (pH 5.0) supplemented with OPD and H ₂ O ₂ . To stop the reaction, 2 NH ₂ SO ₄ was added and the absorbance was measured at 490 nm with a microplate reader.

As a result, IgG1 and IgG2a, which are serum IgG1 and IgG2a, were increased by oral administration of enzyme hydrolyzate compared with the control group. Especially, IgG2a showed 7269.84 ± 573.26 ㎍ / mL, The results showed that the degradation products treated with 100 mg / kg of the enzyme were 8616.07 ± 510.15 ㎍ / mL and the enzymes treated with the 48 h enzyme were 9656.97 ± 578.52 ㎍ / mL at the 200 mg / kg treatment (FIG. 6).

3-5. NK cell activity

In Experimental Examples 3-1 and 3-2, YAC-1 cells (KCLB No. 40160, Korean Cell) were used as effect cells and target cells, Line Bank, Korea) (spleen cells: YAC-1 at 200: 1, 100: 1 and 50: 1). The splenocytes isolated from the mouse of each group treated with the enzymatic degradation of the enzymatic reaction time were diluted to 2 × 10 ⁷ cells / mL and 1 × 10 ⁷ cells / mL by adding RPMI 1640 medium. 100 [mu] l of the diluted spleen cell culture was injected into a 96-well plate. Then, 100 μL of YAC-1 cell culture was added to the culture solution at a concentration of 1 × 10 ⁵ cells / mL and cultured in a 5% CO ₂ incubator (MCO-15AC) at 37 ° C. for 44 hours. Then, 5 mg / mL MTT was added, incubated for 4 h, and the absorbance was measured at 540 nm.

As a result, the NK cell activity (% cytotoxicity) of the mice treated with the enzyme-degraded product for 48 h before and after the enzymatic degradation was 200: 1, 100: 1 and 50: 1 in the ratio of splenocytes: YAC- Dependent decrease in the concentration. In contrast, mice treated with the enzyme-degraded product for 24 h showed 27.39 ± 5.02, 33.64 ± 3.99 and 35.45% at 50, 100 and 200 mg / kg, respectively, compared with 24.37 ± 5.00% ± 6.02%, respectively. 100: 1 and 50: 1 were also increased in a concentration-dependent manner compared to the control group (Fig. 7).

3-6. Check blood cells in blood

(CBC) using a hematometer (Hemavet 950 analyzer, Drew Scientific Inc., Waterbury, USA) using whole blood collected from a mouse in order to identify hemocytes in the blood of a mouse administered orally to the hemicellulase , And lymphocytes, monocytes, and neutrophils were measured.

As a result, the values of leukocyte (WBC), lymphocyte (LY), monocyte (MO) and neutrophil count (NE) in blood were increased depending on the oral administration concentration of the enzyme hydrolyzate, and 24 h and 48 h The enzyme degradation products were higher than the control. In particular, the leukocyte and lymphocyte showed the highest increase rate in the group treated with the enzyme treated with the enzymatic reaction for 24 h, showing the best immunity enhancing effect (FIG. 8).

3-7. Statistical processing

Statistical analysis of all the results of the present invention, after the analysis of variance to the mean value using the SAS program (Statistical analytical system V8.2, SAS Institute Inc., Cary, NC, USA), P <0.05 level of a multi-assay of Duncan And the difference between the items was tested.

Korea Biotechnology Research Institute KCTC18588P 20170728

Claims (5)

A pharmaceutical composition for enhancing immunity, which comprises an enzymatic hydrolyzate of Sargassum coreanum mixed with a culture medium of Shewanella oneidensis as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the large-leaf mallow enzyme degradation product is a low-molecular-weight mulberry leaf mushroom cultured in a culture medium of Shewanella oneidensis . The method according to claim 1, wherein the enzymatic hydrolyzate of the leaves is obtained by mixing the supernatant obtained by centrifuging the culture solution of Schwannella oidensis with 1: 1 ratio (v / v) for 12 to 60 hours &Lt; / RTI &gt; A food composition for enhancing immunity, which contains as an active ingredient, a foliar amylolytic enzyme hydrolyzate obtained by mixing a culture medium of Schwannella onidensis. 1) culturing Shwaneella onidensis under conditions of pH 9 and 25 to 35 占 폚 for 20 to 30 hours;
2) centrifuging the culture of Schwannella onidensis to obtain crude enzyme solution;
3) preparing a large leaf mash powder; And
4) mixing the crude enzyme solution of step 2) with the large leaf mash powder of step 3), and then allowing the mixture to react at pH 9 for 12 to 60 hours.

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