KR102470657B1 - Prepariing method of a composition for precenting, improving and treating allergic disease using fermented barley extract - Google Patents

Abstract

The present invention relates to a method for preparing a composition for enhancing immunity using a fermented barley extract. The composition for enhancing immunity may be a food composition, a feed composition, or a pharmaceutical composition. In addition, the present invention relates to a method for preparing a pharmaceutical composition for inhibiting tumor metastasis using fermented barley extract and a pharmaceutical composition for preventing or treating cancer.

Description

Method for preparing a composition for preventing, improving and treating allergic diseases using fermented barley extract

The present invention relates to a method for preparing a composition for preventing, improving and treating allergic diseases using fermented barley extract.

Barley is one of the world's four major crops, and in Korea, it is one of the five grains (rice, barley, millet, beans, and millet), and is the staple food next to rice. Recently, studies have been conducted to utilize the active ingredients of barley. For example, antidiabetic compositions containing barley (Korean Patent No. 10-1859899), cosmetics (Korean Patent No. 10-1749965), food for improving constipation (Korea Patent No. 10-1749965), Registered Patent No. 10-1744478) and the like.

On the other hand, macrophages regulate immune phenomena by secreting various substances in the process of phagocytosing and removing bacteria or foreign substances. It is related to immune function, and shows direct toxic activity against tumor cells. In addition, a substance (LPS or natural product) that reacts to TLR (toll like receptor) activates macrophage to promote T cell and B cell proliferation, macrophage activation for phagocytosis, and secondary immune response such as defense against microbial infection. It is known to produce cytokines such as IL-1, IL-6, IL-12 and TNF-α that can regulate IL-6 and TNF-α are representative cytokines induced by macrophages, and are known to play a pivotal role in the inflammatory response caused by bacterial infection, and their amounts are increased in inflammatory lesions. IL-6 has been reported to act cooperatively with IL-1 to be involved in the differentiation of T cells and B cells and to have anticancer effects, and TNF-α has cytotoxic and antiviral effects on specific cancer cells, acute and It is known to play an important role in various biological reactions that occur in chronic inflammatory diseases. On the other hand, IL-12 is a cytokine that induces NK cell activation and Th1 type immune response, and is known to increase reactivity to cellular foreign substances such as cancer cells.

The inventors of the present invention completed the present invention by confirming that active ingredients derived from fermented barley extract prepared by a specific method, while studying fermented barley, have anti-allergic efficacy, immune function enhancing activity and anti-cancer anti-metastatic activity.

An object of the present invention is to provide a composition for enhancing immune function.

Another object of the present invention is to provide a composition for preventing, improving or treating cancer.

Another object of the present invention is to provide a composition for preventing, improving or treating allergic diseases.

In order to achieve the above object, the present invention provides a composition for enhancing immunity using a fermented barley extract, a composition for preventing, improving or treating cancer using a fermented barley extract, and a composition for preventing, improving or treating allergic diseases using a fermented barley extract. A manufacturing method is provided.

The composition of the present invention has an ability to enhance cytokine secretion, an ability to enhance the activity of natural killer cells, an ability to inhibit tumor metastasis, and an anti-allergic function.

1 shows a scheme for isolation and purification of immunostimulatory polysaccharides from fermented barley.
Figure 2 is the elution profile of BF-CP by gel permeation chromatography packed with a Sephadex G-100 column. BF-CP was applied to a Sephadex G-100 column (3×95 cm) and eluted with 50 mM ammonium formate buffer (pH 5.5) at a flow rate of 1 ml/min. ●, neutral sugar (490 nm); O, uronic acid (520 nm); ▽, pentose (660 nm); ◇, protein (600 nm)
Figure 3 shows the elution patterns of crude polysaccharide (BF-CP) (A) and BF-I (B), BF-II (C), and BF-III (D) isolated from fermented barley. The HPLC was equipped with a Superdex 75TM 10/300 GL column.
4 shows that crude polysaccharide (BF-CP) (A), BF-I (B), BF-II (C) and BF-III (D) isolated from fermented barley were found in peritoneal macrophages of Balb/c mice. show cytotoxic effects. Peritoneal macrophages (2.0×10 5 cells/well) were treated with different concentrations of BF-CP, BF-I, BF-II and BF-III for 24 hours in 96-well plates. NC means negative control.
Figure 5 shows the effect of BF-CP, BF-I, BF-II and BF-III isolated from fermented barley on cytokine production by rat peritoneal macrophages. Peritoneal peritoneal cells (2.0×10 5 cells/well) were treated with different concentrations of BF-I, BF-II and BF-III for 24 hours in 96-well plates. The concentration of cytokines in the medium was determined by ELISA kit. 1 μg/mL lipopolysaccharide (LPS) was used as a positive control (PC, positive control), and the group tested only with medium was used as a negative control (NC, negative control).
Figure 6 shows known immune-modulating active polysaccharides derived from natural products and their related hydrolytic enzymes.
Figure 7 shows the effect of enzymatic hydrolysis on BF-I (A) and BF-III (B) purified from fermented barley.
Figure 8 shows the MW-determination and elution pattern of BF-I purified from barley extract by size exclusion HPLC. The HPLC was equipped with Asahi-Pak GS-520+GS-320+GS-220 linked columns.
9 shows a total ion chromatogram on GC-MS of partially methylated alditol acetates from BF-I.
Figure 10 shows possible structures of BF-I, an immune-stimulating polysaccharide purified from fermented barley.
Figure 11 shows the single radical gel diffusion of purified BF-I from fermented barley identified with β-glucosyl Yariv reagent.
Figure 12 shows the continuous digestion procedure for the structural analysis of BF-I and the purification method of fermented barley-derived crude polysaccharide (BF-CP).
Figure 13 shows the single radical gel diffusion of BF-I and its sub-nucleates obtained by enzymatic hydrolysis.
Figure 14 shows the possible structure and MALDI-TOF spectrum of BF-I-1b purified from BF-1.
15 shows the MALDI-TOF/TOF spectrum of nonasaccharide unit (m/z) 1229 derived from BF-I-1b.
Figure 16 presents the possible structure and MALDI-TOF spectrum of BF-I-2c purified from BF-I.
17 shows the MALDI-TOF/TOF spectrum of hexasaccharide unit (m/z) 1013 derived from BF-I-2c.
Figure 18 shows the possible structure and MALDI-TOF spectrum of BF-I-3b purified from BF-I.
Figure 19 shows the total ion chromatogram on GCMS of BF-I and its subfractions derived from enzymatic hydrolysis.
20 is a possible microstructure of BF-I, a polysaccharide derived from fermented barley.
Figure 21 shows the effect of polysaccharide-subfractions obtained by hydrolysis I to V on cytokines produced by murine peritoneal macrophages.
Figure 22 shows the effect of BF-I on the production of antibodies to OVA. ○ Normal mice, ● OVA-administered mice, □ BF-I 5 μg-administered mice, ■ BF-I 50 μg-administered mice, △ BF-I 500 μg-administered mice.
Figure 23 shows Th1 and Th2 type cytokine production profiles when OVA and BF-I are administered in combination (□ BF-I alone, ■ OVA and BF-I mixed administration).
24 shows the results of subisotype analysis of antibodies against OVA after secondary immunization.
25 shows the effect of BF-I on CTL activity when immunized with a tumor vaccine.
Figure 26 shows the pattern of cytokine production in spleen cells in immunized mice upon combined administration of F/T-vaccine and BF-I.
Figure 27 shows the pattern of cytokine production by stimulation of splenocytes according to the concentration of BF-I.
Figure 28 shows the pattern of cytokine production by macrophage stimulation according to the concentration of BF-I.
29 shows the effect of BF-I on cytokine production in the co-culture of cancer cells and macrophages.
30 shows the synergistic effect of NK-cell cytotoxicity by the administration of BF-I.
31 shows the positive metastasis inhibitory effect of BF-I.

The present invention,

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for preparing a food composition for enhancing immunity.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for preparing a pharmaceutical composition for enhancing immunity.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for preparing a feed composition for enhancing immunity.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for preparing a pharmaceutical composition for inhibiting tumor metastasis.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for preparing a pharmaceutical composition for preventing or treating cancer.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for producing a food composition for preventing or improving allergic diseases.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for preparing a pharmaceutical composition for preventing or treating allergic diseases.

Also, the present invention

Enzymatically hydrolyzing barley to produce a barley hydrolysate;

Fermenting the barley hydrolysate to prepare a fermented barley product;

Extracting the fermented barley to prepare a fermented barley extract; And

Obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract,

It relates to a method for producing a feed composition for preventing or improving allergic diseases.

In addition, the present invention relates to a food composition for enhancing immunity comprising barley-derived polysaccharide.

In addition, the present invention relates to a pharmaceutical composition for enhancing immunity comprising barley-derived polysaccharide.

In addition, the present invention relates to a feed composition for immunity enhancement comprising barley-derived polysaccharide.

In addition, the present invention relates to a pharmaceutical composition for inhibiting tumor metastasis comprising barley-derived polysaccharide.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating cancer containing barley-derived polysaccharide.

In addition, the present invention relates to a food composition for preventing or improving cancer containing barley-derived polysaccharide.

In addition, the present invention relates to a method for enhancing immunity comprising administering a composition containing barley-derived polysaccharide to a subject.

In addition, the present invention relates to a method for inhibiting tumor metastasis comprising administering a composition containing barley-derived polysaccharide to a subject.

In addition, the present invention relates to a method for preventing or treating cancer comprising administering a composition containing barley-derived polysaccharide to a subject.

In addition, the present invention relates to a method for preventing or improving cancer, comprising administering a composition containing barley-derived polysaccharide to a subject.

In addition, the present invention relates to a food composition for preventing or improving allergic diseases containing barley-derived polysaccharide.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating allergic diseases containing barley-derived polysaccharide.

In addition, the present invention relates to a feed composition for preventing or improving allergic diseases containing barley-derived polysaccharide.

In addition, the present invention relates to a method for preventing or treating allergic diseases comprising administering a composition containing barley-derived polysaccharide to a subject.

In addition, the present invention relates to a method for preventing or improving allergic diseases comprising administering a composition containing barley-derived polysaccharide to a subject.

In addition, the present invention relates to a composition containing barley-derived polysaccharide for enhancing immunity, for preventing, treating or improving cancer, and for preventing, improving or treating allergic diseases.

Hereinafter, the present invention will be described in detail.

Method for producing food composition, pharmaceutical composition, and feed composition

The present invention comprises the steps of enzymatically hydrolyzing barley to produce barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Preparing a fermented barley extract by extracting the fermented barley; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract. It's about manufacturing methods.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; fermenting the barley hydrolysate to prepare a fermented barley product; extracting the barley fermented product to prepare a barley fermentation extract; and containing arabino-β-3,6-galactan from the barley fermentation extract. It relates to a method for preparing a pharmaceutical composition for enhancing immunity, comprising obtaining a polysaccharide.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Preparing a fermented barley extract by extracting the fermented barley; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract. It's about manufacturing methods.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Preparing a fermented barley extract by extracting the fermented barley; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract. It relates to a method for preparing the composition.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Preparing a fermented barley extract by extracting the fermented barley; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract. It relates to a method for preparing the composition.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Extracting the fermented barley to prepare a fermented barley extract; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract, preventing allergic diseases or It relates to a method for preparing a food composition for improvement.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Extracting the fermented barley to prepare a fermented barley extract; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract, preventing allergic diseases or It relates to a method for preparing a pharmaceutical composition for treatment.

In another aspect, the present invention includes the steps of enzymatically hydrolyzing barley to produce a barley hydrolyzate; Fermenting the barley hydrolysate to prepare a fermented barley product; Extracting the fermented barley to prepare a fermented barley extract; and obtaining a polysaccharide containing arabino-β-3,6-galactan from the fermented barley extract, preventing allergic diseases or It relates to a method for producing a feed composition for improvement.

In the step of preparing the barley hydrolyzate, first enzyme hydrolyzate is prepared by adding alpha amylase to barley to produce a first enzyme hydrolyzate, and then a mixture of protease, alpha-amylase and glucoamylase is added to the first enzyme hydrolyzate. It can be performed by adding a second enzyme hydrolyzate to prepare a second enzyme hydrolyzate, and adding a saccharification enzyme to the second enzyme hydrolyzate to perform third enzyme hydrolysis to prepare a barley hydrolyzate.

In the step of preparing the fermented barley product, yeast is added to the barley hydrolysate to perform yeast fermentation to prepare a yeast fermentation product, and lactic acid bacteria are added to the yeast fermentation product to perform lactic acid fermentation to prepare a fermented barley product can be done

The step of preparing the fermented barley extract may be performed by centrifuging the fermented barley to obtain a supernatant.

barley-derived polysaccharide

The present invention relates to a composition comprising barley-derived polysaccharide containing arabino-β-3,6-galactan prepared by the production method of the present invention. Preferably, the present invention relates to a composition for enhancing immunity and a composition for preventing, improving, or treating cancer containing barley-derived polysaccharide. The polysaccharide is a polysaccharide derived from fermented barley extract. The polysaccharide includes arabino-β-3,6-galactan, and includes arabinoxylan and/or β-glucan. The β-glucan includes yeast-derived β-glucan and barley-derived β-glucan. The polysaccharide includes arabinoxylan in which arabinose linked to the β-(1→4)-xylan backbone by i) C3 or ii) (1→5) bonds at positions C2 and C3 is branched, and in this case, arabinoxylan The content is 8 to 11% by weight. The polysaccharide includes yeast-derived β-glucan, which is β-(1→3)(1→6)-D-glucan, wherein the yeast-derived β-glucan content is 4 to 7% by weight. The polysaccharide includes barley-derived β-glucan, which is β-(1→3)(1→4)-D-glucan, wherein the content of barley-derived β-glucan is 30 to 39% by weight. The polysaccharide includes arabino-β-3,6-galactan derived from barley pectin containing 3,6-linked Galp, and the content of arabino-β-3,6-galactan is 5 to 9 weight %to be. The polysaccharide increases secretion of interleukin-6, interleukin-12 or TNF-alpha. In addition, the polysaccharide inhibits tumor metastasis and has an effect of preventing, treating and/or improving cancer. In addition, the polysaccharide is selected from the group consisting of immunodeficiency, immunosuppression, disease due to immune system damage, immune function decrease due to anticancer therapy, immune function decrease due to bone marrow transplant, AIDS due to immune system damage, and cancer due to immune function decrease. In a patient with a disease, the immunity is enhanced.

The fermented barley extract of the present invention is prepared as follows. First, prepare raw barley and grind it. At this time, grinding may be performed using a hammer mill under conditions of 875 rpm and 337 kg/ 40 min. Purified water is added to ground barley and soaked for 10 to 14 hours. At this time, ground barley:purified water is used in a weight ratio of 1:3 to 6. After soaking is completed, gelatinization is performed by steaming barley at 90 ° C. for 1 to 3 hours.

Primary enzymatic hydrolysis is performed by adding alpha amylase to gelatinized barley and treating it at 70 to 90° C. for 1 to 3 hours. Thereafter, a mixture of protease, alpha-amylase and glucoamylase is added to the first enzyme hydrolyzate and treated at 50 to 70 ° C. for 1 to 3 hours to perform secondary enzyme hydrolysis . In addition, beta-glucanase, a saccharification enzyme, is added to the secondary enzymatic hydrolyzate and reacted at 50 to 70 ° C. for 12 to 36 hours to perform tertiary enzymatic hydrolysis to prepare a saccharification solution.

Yeast (Saccharomyces cerevisiae) is added to the saccharification solution and yeast fermentation is performed at 20 to 40 ° C. for 12 to 36 hours. Thereafter, a lactic acid bacterium, Weissella cibaria, is added to the yeast fermentation product, and lactic acid bacteria fermentation is performed at 20 to 40 ° C. for 12 to 36 hours to prepare a fermented barley product. The fermented barley product is centrifuged at 5,000 to 8,000 rpm for 20 to 40 minutes to separate the supernatant and sterilized under pressure to prepare a fermented barley extract.

As described above, the fermented barley extract of the present invention is obtained by primary enzymatic hydrolysis using alpha amylase, secondary enzymatic hydrolysis using protease, alpha-amylase and glucoamylase, tertiary enzymatic hydrolysis using beta-glucanase, yeast It is prepared by sequentially performing fermentation and Visella cibaria fermentation. In this way, a fermented barley extract with enhanced immune enhancing components can be prepared. If at least one of the first to third enzymatic hydrolysis steps and yeast fermentation and lactic acid bacteria fermentation are omitted, the processed barley product thus prepared has a significantly lower content of immune enhancing components than the fermented barley extract of the present invention, or the present invention There is a disadvantage that the immune enhancing effect is significantly lower than that of barley fermented extract.

The fermented barley extract of the present invention does not include a step of roasting barley in its manufacturing process, which is the result of roasting barley and hydrolyzing or fermenting it, which is a process product of barley (i.e., hydrolyzed barley or fermented barley extract, etc.) This is because the macrophage stimulating activity was very low. This is considered to be because polysaccharides form carbides during the roasting process and toxic substances are also produced.

composition of the present invention

The present invention relates to a composition comprising barley-derived polysaccharide containing arabino-β-3,6-galactan prepared by the production method of the present invention. Preferably, the present invention relates to a composition for enhancing immunity containing barley-derived polysaccharide, a composition for inhibiting cancer or tumor metastasis, a composition for preventing, improving or treating cancer, and a composition for preventing, improving or treating allergic diseases. . The composition may be a food composition, pharmaceutical composition or feed composition. The cancer is a group consisting of pancreatic cancer, ovarian cancer, breast cancer, colon cancer, liver cancer, lung cancer, brain cancer, laryngeal cancer, thyroid cancer, colon cancer, stomach cancer, kidney cancer, lymphoma, acute myelogenous leukemia, osteosarcoma, bronchial cancer, bladder cancer, Wilms' tumor and prostate. It may be one or more selected from among. The allergic disease consists of bronchial asthma, allergic rhinitis, urticaria, anaphylaxis, food allergy, atopic dermatitis, allergic nonhemorrhagic transfusion reaction, and drug allergy. It may be one or more selected from the group. The allergic disease may be induced by one or more selected from the group consisting of pollen, drugs, vegetable fibers, bacteria, food, dyes, and chemicals.

food composition

The food of the present invention includes health supplements, health functional foods, functional foods, exercise supplements, etc., but is not limited thereto, and includes natural foods, processed foods, and general food materials containing the barley-derived polysaccharide of the present invention.

The food composition containing the barley-derived polysaccharide of the present invention may be added as it is or used together with other foods or food compositions, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined depending on the purpose of its use (prevention or health maintenance purpose). In general, the barley-derived polysaccharide of the present invention can be added in an amount of 0.01 to 70.00% by weight, preferably 0.01 to 30.00% by weight, more preferably 0.01 to 10.00% by weight, based on the raw material during production of food or beverage. % can be added.

There is no particular limitation on the type of food. The food composition containing the barley-derived polysaccharide as an active ingredient may be used in the form of preparations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc. Formulations for oral administration may be prepared using excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives, or bulking agents.

Examples of foods to which the barley-derived polysaccharide can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, powder preparations and vitamin complexes may be mentioned, but are not limited to these types of foods.

feed composition

The feed composition containing barley-derived polysaccharide containing arabino-β-3,6-galactan prepared by the production method of the present invention as an active ingredient can be served together with conventional feed, and the feed composition of the present invention It may be added to a conventional feed composition to prepare and use a functional feed composition. In addition, the feed composition of the present invention may further include functional ingredients other than the barley-derived polysaccharide of the present invention. When preparing a functional feed composition in which the conventional feed composition and the barley-derived polysaccharide of the present invention are mixed, the barley-derived polysaccharide of the present invention is 0.01 to 30.00% by weight, preferably 0.01 to 20.00% by weight, based on the total feed composition can be added as The effective dose of the barley-derived polysaccharide of the feed composition may be used according to the effective dose of the food composition, but may be less than the above range in the case of long-term intake for the purpose of continuous immunity enhancement or health control, Since there is no problem in terms of safety, the active ingredient may be used in an amount greater than or equal to the above range.

The feed composition of the present invention is intended for livestock or poultry. The livestock or poultry is cattle, pigs, chickens, horses, sheep, donkeys, mules, wild boars, rabbits, quails, domestic ducks, roosters, game chickens, pigeons, turkeys, dogs, cats, monkeys, hamsters, mice, rats, cockerels , Parrots, parakeets, canaries, etc., but are not limited thereto, and any non-human mammal or bird that can be reared at home will be the subject of the feed composition of the present invention.

pharmaceutical composition

The pharmaceutical composition containing the barley-derived polysaccharide containing arabino-β-3,6-galactan prepared by the production method of the present invention enhances the immune activity of an administrator. In addition, the pharmaceutical composition containing the barley-derived polysaccharide of the present invention suppresses tumor metastasis of the administering agent. In addition, the pharmaceutical composition containing the barley-derived polysaccharide of the present invention prevents or treats cancer by suppressing the progress, continuation, and deterioration of cancer of the administrator. In addition, the pharmaceutical composition containing the barley-derived polysaccharide of the present invention prevents or treats allergic diseases. The pharmaceutical composition containing the barley-derived polysaccharide of the present invention may be a pharmaceutical composition for preventing and treating diseases caused by immunodeficiency, immunosuppression or immune system damage. In addition, the pharmaceutical composition containing the barley-derived polysaccharide of the present invention is effective in reducing immune function due to anti-cancer therapies such as chemotherapy and radiation therapy, diseases caused by immunosuppression after bone marrow transplantation, and AIDS and immune function decrease due to immune system damage. It may be a pharmaceutical composition for the prevention and treatment of cancer caused by cancer. In addition, the pharmaceutical composition containing the barley-derived polysaccharide of the present invention is a pharmaceutical composition for preventing and treating allergic diseases induced by at least one selected from the group consisting of pollen, drugs, vegetable fibers, bacteria, food, dyes, and chemical substances. may be an enemy composition. In addition, the pharmaceutical composition containing the barley-derived polysaccharide of the present invention is effective against bronchial asthma, allergic rhinitis, urticaria, anaphylaxis, food allergy, atopic dermatitis, allergy It may be a pharmaceutical composition for preventing and treating allergic diseases selected from the group consisting of non-hemolytic transfusion reactions and drug allergy.

The pharmaceutical composition of the present invention may contain 0.01 to 80% by weight of barley-derived polysaccharide containing arabino-β-3,6-galactan prepared by the production method of the present invention, preferably 0.02 to 65% by weight. % by weight. However, this may be increased or decreased according to the needs of the administering person, and may be appropriately increased or decreased according to circumstances such as dietary habits, nutritional status, disease progression, and obesity.

The pharmaceutical composition of the present invention can be administered orally or parenterally and can be used in the form of general pharmaceutical preparations. Preferred pharmaceutical preparations include preparations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, and the like, and these pharmaceutical preparations are pharmaceutically acceptable carriers, for example, excipients in the case of preparations for oral administration, It can be prepared using a binder, disintegrant, lubricant, solubilizer, suspending agent, preservative or bulking agent.

The dosage of the pharmaceutical composition containing the barley-derived polysaccharide of the present invention may be determined by experts according to various factors such as the patient's condition, age, sex and complications, but is generally 0.1 mg to 10 g per 1 kg of adult, preferably Preferably, it may be administered in a dose of 10 mg to 5 g. In addition, the daily dose of the pharmaceutical composition per unit dosage form or a dose of 1/2, 1/3 or 1/4 thereof may be contained, and may be administered 1 to 6 times a day. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and the active ingredient may be used in an amount greater than the above range because there is no problem in terms of safety.

Advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various forms different from each other, only these embodiments make the disclosure of the present invention complete, and common knowledge in the art to which the present invention pertains. It is provided to completely inform the person who has the scope of the invention, and the present invention is only defined by the scope of the claims.

<Materials and methods>

Hulled barley from Honam, Korea, purchased through contract cultivation in the Honam region of Korea was used.

<Experimental Example 1>

<1-1> Preparation of fermented barley extract

<1-1-1> Preparation of fermented barley extract

<Production Example 1>

Using a hammer mill, grind barley at a speed of 875 rpm, 337 kg/ 40 min, add purified water to the ground barley so that the weight ratio of barley: water is 1: 4, soak for 12 hours, then steam at 90 ° C for 2 hours and proceeded with luxury,

Alpha amylase was added to gelatinized barley and treated at 80 ° C. for 2 hours to prepare a primary enzyme hydrolyzate. Thereafter, a complex enzyme containing equal amounts of protease, alpha-amylase and glucoamylase was added to the first enzyme hydrolyzate and treated at 60° C. for 2 hours to prepare a second enzyme hydrolyzate. In addition, beta-glucanase as a saccharification enzyme was added to the secondary enzyme hydrolyzate and reacted at 60 ° C. for 24 hours to prepare a saccharification solution.

Yeast (Saccharomyces cerevisiae) was added to the saccharification solution and yeast fermentation was performed at 30 ° C. for 24 hours to prepare a yeast fermentation product. Barley fermented product was prepared by adding lactic acid bacteria, Weissella cibaria, and performing lactic acid fermentation at 30 ° C. for 24 hours. In addition, the fermented barley was centrifuged at 6,500 rpm for 30 minutes to separate the fermentation supernatant and sterilized under pressure to prepare a fermented barley extract.

<Production Example 2>

A fermented barley extract was prepared in the same manner as in Preparation Example 1, except that the yeast fermentation and lactic acid bacteria fermentation processes were performed immediately without enzymatic hydrolysis of the gelatinized barley.

<Production Example 3>

A fermented barley extract was prepared in the same manner as in Preparation Example 1, except that the saccharified solution was directly fermented with lactic acid bacteria without yeast fermentation.

<Production Example 4>

A fermented barley extract was prepared in the same manner as in Preparation Example 1, except that the lactic acid bacteria fermentation process was omitted after yeast fermentation of the saccharification solution.

<Production Example 5>

A fermented barley extract was prepared in the same manner as in Preparation Example 1, except that the barley was roasted at 90 ° C and the roasted barley was ground to prepare a fermented barley extract.

<1-1-2> Investigation of macrophage stimulating activity of crude polysaccharide of fermented barley extract

The fermented barley extracts of Preparation Examples 1 to 5 were concentrated to 40 brix, respectively, diluted to 5 brix, centrifuged to remove the precipitate, and the supernatant was recovered. Ethanol was added to the recovered supernatant to a final concentration of 80 v/v%, left overnight, and then separated into a precipitate and a supernatant by centrifugation. The precipitate was recovered and re-dissolved in a small amount of distilled water, followed by dialysis and freeze-drying to obtain crude polysaccharide derived from fermented barley extract.

After dissolving the crude polysaccharide derived from the fermented barley extract in a small amount of water, gel permeation chromatography using a Sephadex G-100 column (4 × 120 cm) equilibrated with 50 mM ammonium formate buffer (pH 5.5) (GPC) was performed. The eluate was fractionated into 100 fractions of 5 mL each, and each fraction was analyzed for neutral sugar, acidic sugar, pentose sugar and protein content to obtain fractions with different molecular weights, and the macrophage stimulating activity of the fractions was compared. , This was performed by evaluating IL-6, IL-12, and TNF-α production activities using the fractions as samples (the evaluation method is the same as in <1-12> below). As a result, the immunoactivity of the polysaccharide derived from the fermented barley extract of Preparation Example 1 was significantly superior. In the following experiments, the fermented barley extract of Preparation Example 1 was used.

<1-2> Extraction and separation of immunoactive crude polysaccharide from fermented barley extract

The concentrate (40 brix) of the fermented barley extract of Preparation Example 1 (hereinafter referred to as "fermented barley extract") was diluted to 5 brix, centrifuged to remove the precipitate, and then the supernatant was recovered. Half of the recovered supernatant was lyophilized to obtain BF-EX, a stock solution of fermented barley extract, and the other half was left overnight after adding ethanol to a final concentration of 80 v/v%, and then centrifuged to separate the precipitate and supernatant. . The ethanol supernatant was concentrated and lyophilized to obtain an ethanol supernatant BF-S derived from fermented barley extract, and the generated precipitate was recovered and re-dissolved in a small amount of distilled water, followed by dialysis and freeze-drying to obtain crude polysaccharide BF-CP derived from fermented barley extract.

After dissolving crude polysaccharide BF-CP derived from fermented barley extract in a small amount of water, gel permeation chromatography (gel permeation) was performed using a Sephadex G-100 column (4 × 120 cm) equilibrated with 50 mM ammonium formate buffer (pH 5.5). chromatography) (GPC) was performed. The eluate was fractionated into 100 fractions of 5 mL each, and each fraction was analyzed for neutral sugar, acidic sugar, pentose sugar and protein content to obtain three fractions BF-I, BF-II and BF-III with different molecular weights. , Their yields were confirmed to be 2.37%, 0.94% and 1.71%, respectively (FIGS. 1 and 2).

<1-3> Analysis of general chemical properties of samples isolated from fermented barley extract

General component analysis was performed as follows. The neutral sugar content of polysaccharide samples was determined by the phenol-sulfuric acid method using galactose as the standard material, the content of acidic sugar by the m-hydroxybiphenyl method using galacturonic acid as the standard material, and the content of TBA-positive material using the 2-keto-3- The thiobarbituric acid method using deoxy-D-manno-octulosonic acid (KDO) as the standard substance, the protein content by the Bradford method using bovine serum albumin (BSA) as the standard substance, and the phenolic compounds content using gallic acid as the standard substance were analyzed quantitatively by the Folin-Ciocalteu (F-C) method.

Constituent sugar analysis was performed as follows. In order to carry out the analysis per component, the experiment was conducted by partially modifying the method of Honda et al. (Honda S, Akao E, Suzuki S, Okuda, M., Kakehi, K., & Nakamura, J. (1989). High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3 -methyl5-pyrazolone derivatives.Analytical biochemistry, 180(2), 351-357) After hydrolysis with 2M TFA, each component was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), and DIW After phase separation using chloroform and choloroform, the aqueous layer is recovered, filtered, and equilibrated with a solvent of 0.1M phosphate buffer (pH 6.7): acetonitrile = 82:18 using an HPLC-UVD equipped with an Acclaim C18 column at 254 nm was measured in The mole% of each component was calculated by calculating the peak area, molecular weight, and molecular response factor for each derivative, respectively.

Molecular weight measurement using HPLC was performed as follows. In order to measure the purity of the barley fermentation extract-derived stock solution BF-EX, ethanol supernatant BF-S, and crude polysaccharide BF-CP samples, each sample was equilibrated with 50 mM ammonium formate buffer (pH 5.5) using a Superdex 75 GL column using HPLC - RID was performed. In addition, HPLC-RID was performed on each sample using a Superdex 75 GL column to measure the degree of purification of purified polysaccharides BF-I, BF-II and BF-III isolated from fermented barley extract.

<1-4> Evaluation of cytotoxicity of samples isolated from fermented barley extract

BALB/c mice were intraperitoneally injected with 1 mL of 5% thioglycollate (TG), and 4 days later, the mice were sacrificed by cervical dislocation. ) was collected. The collected PEC was dispensed in a 96-well culture plate at a concentration of 2.0 × 105 cells/well, cultured for 2 hours to attach macrophages to the plate, and then samples were added at various concentrations to the macrophages and cultured for 24 hours. . For the effect of cytotoxicity according to each sample concentration, after diluting EZ-cytox (Dogen, Seoul, Korea) 5 times and adding 50 μL per well, reacting for 30 to 60 minutes in a 37 ℃, 5% CO2 incubator, and The absorbance was measured and evaluated.

<1-5> Evaluation of cytokine activity of samples isolated from fermented barley extract

Macrophage culture medium was prepared as follows. BALB/c mice were intraperitoneally injected with 1 mL of 5% thioglycollate (TG), and 4 days later, the mice were sacrificed by cervical dislocation. ) was collected. The collected PEC was dispensed in a 96 well culture plate at a concentration of 2.0 × 105 cells/well, cultured for 2 hours to allow macrophages to adhere to the plate, and then washed with the culture medium to remove non-adherent cells. Samples were added to Macrophage at various concentrations and incubated for 24 hours. After the end of the culture, 180 μL of the cell culture supernatant was recovered by centrifugation at 900 rpm for 5 minutes. The content of cytokines was measured.

Cytokine measurement by sandwich ELISA was performed as follows. The content of cytokines produced by macrophages was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA). Capture antibody, a specific monoclonal antibody for each cytokine, was diluted in coating buffer according to the manufacturer's instructions, dispensed 100 μL each into a flat-bottomed 96-well microplate (NuncTM, Roskilde, Denmark), and incubated at 4℃ for 12 hours. proceeded. The coated ELISA plate was washed three times using washing buffer (PBS with 0.05% tween® 20, PBST), and 200 μL of assay diluent (PBS with 10% or 2% skim milk) was added and left for 1 hour to detect antibodies. The non-attached well surface was blocked. After blocking was completed, each well was washed three times using washing buffer, and 100 μL of each of the standard recombinant mouse cytokine or macrophage culture medium diluted serially was dispensed into each well. After reacting at room temperature for 2 hours, washing with washing buffer, adding detection antibody-biotin and enzyme reagent (avidin-horseradish peroxidase conjugate), followed by reaction at room temperature for 1 hour. After the reaction is complete, wash 5 times with washing buffer, add 100 μL of substrate solution (Tetramethylbenzidine, TMB), react for 30 to 60 minutes in the dark, and then treat with 50 μL of stop solution (1M H3PO4 or 2N H2SO4). Absorbance was measured at 450 nm.

<1-6> Enzyme selection for structural analysis of immunoactive polysaccharide isolated from fermented barley extract

Before analyzing the microstructures of purified polysaccharides BF-I and BF-III isolated from fermented barley extract, 6 enzymes were selected and screened to find enzymes that selectively hydrolyze sugar chain bonds of polysaccharides. Purified polysaccharides BF-I and BF-III isolated from fermented barley extract were dissolved, and Lichenase (β-1,3-1,4-glucanase), Lyticase (β-1,3-glucanase), endo-laminarinase (Endo- 1,3-glucanase), exo-laminarinase (exo-1,3-glucanse), amyloglucosidase (α-1,4-1,6-glucanase), and β-xylanase (β-1,4-xylanase) at 30 After processing the units, it was cultured for 3 days under the optimal conditions for each enzyme. Then, the amount of reducing sugar was measured in the enzyme-treated reaction solution of purified polysaccharides BF-I and BF-III using the dinitrosalicylic acid (DNS) method. The DNS method was performed by adding 1 mL of sample and 3 mL of DNS, reacting at 100 ° C for 15 minutes, and measuring at 540 nm.

<1-7> Structural analysis of active polysaccharide isolated from fermented barley extract

<1-7-1> Molecular weight measurement using HPLC

In order to measure the molecular weight of the purified polysaccharide BF-I isolated from the fermented barley extract, HPLC-RID was performed on each sample using a Superdex 75 GL column. Molecular weight measurement was performed by calculating the retention time using the standard pullulan series (P-5, 10, 20, 50, 100, 200, 400 and 800), calculating the Kav value for each molecular weight, and converting it from the standard curve prepared. decided.

<1-7-2> Determination of structure and binding site by methylation analysis

Preparation of methylsulfinyl carbanion

To prepare methylsulfinyl carbanion, 20 mL of anhydrous DMSO (dimethylsulfoxide) was added to 1.26 g of anhydrous NaH, filled with nitrogen, and reacted for about 10 to 15 minutes in a 90 °C oil bath. The reaction was terminated with the point at which the reaction solution turned pale green as the end point, cooled to room temperature, centrifuged at 3,000 rpm and 30°C, and the supernatant containing methylsulfinyl carbanion was substituted with nitrogen so as not to come into contact with air. After dispensing, it was stored frozen at -70 ° C and used.

methylation

Methylation to determine the binding site of the polysaccharide sample was performed using the Hakomori method (Hakomori S. A rapid permethylation of glycolipid, and polysaccharidecatalyzed by methylsulfinyl carbanion in mimethyl sulfoxide. J Biochem 1964;55:205-208.). 1 mL of anhydrous DMSO was added to each polysaccharide sample (0.5 mg) sufficiently dried in a desiccator for 1 to 2 days, stirred to completely dissolve, and then 500 μL of methylsulfinyl carbanion (MSCA) was added and reacted for 4 hours. At this time, MSCA was additionally added if necessary so that the polysaccharide could be completely converted to polyalkoxide, and the remaining unreacted MSCA was checked with triphenylmethane. The sample converted to polyalkoxide was methylated by adding an excess of CH3I, and the remaining CH3I was removed through N2 gas flushing and recovered using a Seppak C18 cartridge.

Hydrolysis and acetylation of methylated polysaccharides

The methylated sample was hydrolyzed by adding 1 mL of 2 M TFA and reacting at 121 ° C for 1.5 hours, and then dried. Ring opening and reduction were performed, and an appropriate amount of acetic acid was added to remove residual NaBH4, and excess acetic acid was removed by repeatedly drying while adding methanol. After that, 1 mL of acetic anhydride was added and reacted at 121 ° C for 3 hours to convert to partially methylated alditol acetate, which was separated and extracted with a two-phase solvent system (chloroform, H2O), dissolved in acetone, and gas chromatograph (GC) and analyzed by GC-mass spectrometer (MS).

Analysis by GC and GC-MS of partially methylated alditol acetate

GC analysis was performed using a YoungLin ACME6100 GC equipped with an SP2380 capillary column (0.25 mm × 30 m, 0.2 mm film thickness, Supelco, Bellefonte, PA, USA) under optimal temperature conditions [60 °C (1 min), 60 °C → 180 ℃ (30 ℃ / min), 180 ℃ → 250 ℃ (1.5 ℃ / min), 250 ℃ (5 min)] was analyzed in splitless injection mode (1/20), at which time the flow rate of carrier gas (N2) was adjusted to 1.5 mL/min. On the other hand, GC-MS is performed in splitless injection mode (He flow rate : 1.5 mL/min). Derivatives of methylated samples were identified by combining fragment ion analysis by GC-MS and relative retention time of GC, and the mole% of each peak was converted from the peak area and molecular response factor.

<1-7-3> Confirmation and quantification of arabino-β-3,6-galactan (type II) using βGlucosyl Yariv reagent

Reactivity with β-glucosyl Yariv reagent (Biosupplies, Parkville, Australia) to confirm the presence of aarabino-β-3,6-galactan was measured by single radical gel diffusion method according to Holst and Clarke's method. (van Holst GJ, Clarke AE. Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion. Anal Biochem 1985;148:446-450.) 0.15 M NaCl agarose plate containing 10 μg/mL of β-Glucosyl Yariv reagent was prepared, a well with a diameter of 3 mm was prepared, and a solution containing 5 μg of sample and gum arabic diluted by concentration was injected into each well. The plate was allowed to stand at 25 ° C for 15 hours in a wet state to react, and the presence or absence of arabino-β-3,6-galactan was observed by observing the red precipitate formed, and the reactivity between the sample and β-glucosyl Yariv reagent The area of the generated precipitation transition was calculated and compared with each other.

<1-7-4> Fragment preparation for structural analysis of purified polysaccharide BF-I isolated from fermented barley extract

In order to estimate the overall structure of BF-I, it must be broken down into fragments (oligos with low molecular weight) that are easy to analyze, purified, and analyzed. For this purpose, a series of enzymatic hydrolysis was performed. The enzymes used at this time were provided by the manufacturer through preliminary experiments to set conditions for easy separation of fragment oligosaccharides (Amyloglucosidase, β-Xylanase, Lyticase, Lichenase, Galactan-1,3-β-galactosidase) suitable for degradation. One treatment condition was applied and used according to laboratory conditions.

Hydrolysis and Fragment Preparation by Amyloglucosidase

300 mg of BF-I sample was dissolved in 50 mM ammonium formate buffer (pH 4.5) and 100 units of Amyloglucosidase (α-1,4-1,6-glucanase, from A. niger, Fluka., St. Louis, MO, USA) was added to perform hydrolysis in a constant-temperature water bath at 50°C for 48 hours, and the remaining enzyme activity was stopped by bathing at 100°C for 20 minutes. The reaction mixture was centrifuged to remove the precipitated inactive enzyme protein, and the supernatant was fractionated on HPLC using a Superdex 75 GL column equilibrated with 50 mM ammonium formate buffer (pH 5.5) and analyzed for neutral sugar. Since it was not hydrolyzed, the entire amount was recovered, concentrated, and lyophilized.

Hydrolysis by β-Xylanase and preparation of fragments

After amyloglucosidase treatment, 50 mM ammonium formate buffer (pH 6.5) was added to the recovered BF-I sample to dissolve it, and then β-Xylanase (β-1,4-xylanase, from Cellvibrio mixtus, Megazyme International Ireland Ltd., Ireland) 150 units were added and treated in a constant temperature water bath at 40 ° C for 48 hours. To stop the enzymatic reaction, the supernatant was obtained by bathing in water at 100 ° C for 20 minutes and centrifuging, which was fractionated on HPLC using a Superdex 75 GL column equilibrated with 50 mM ammonium formate buffer (pH 5.5). analysis was performed. At this time, two fractions BF-I-1a and BF-I-1b having different molecular weights and constituents were obtained, which were desalted (Microacylizer, cut-off 100) and lyophilized to prepare fractional fractions.

Hydrolysis by lyticase and preparation of fragments

After adding 0.1 M potassium phosphate buffer (pH 7.0) to the BF-I-1a sample obtained in the β-Xylanase treatment process to dissolve it, lyticase (β-1,3-glucanase, from Arthrobacter Luteus, Sigma, St. Louis, MO , USA) was added in 100 units and treated in a constant temperature water bath at 30 ° C for 48 hours. To stop the enzymatic reaction, the supernatant was obtained by heating in a water bath at 100 °C for 20 minutes and centrifuging, which was fractionated on HPLC using a Superdex 75 GL column equilibrated with 50 mM ammonium formate buffer (pH 5.5). At this time, three fractions BF-I-2a, BF-I-2b, and BF-2c having different molecular weights and components were obtained, and after desalting (Microacylizer, cut-off 100), lyophilization was performed to prepare fractional fractions.

Hydrolysis by Lichenase and preparation of fragments

After adding 50 mM ammonium formate buffer (pH 7.0) to the BF-I-2a sample obtained in the lyticase treatment process to dissolve it, Lichenase (β-1,3-1,4-glucanase, from Bacillus subtilis, Megazyme International Ireland Ltd. , Ireland) 100 units were added and treated in a constant temperature water bath at 60 ° C for 48 hours. To stop the enzymatic reaction, the supernatant was obtained by bathing in water at 100 ° C for 20 minutes and centrifuging. It was fractionated on HPLC using a Superdex 75 GL column equilibrated with 50 mM ammonium formate buffer (pH 5.5) and analyzed for neutral sugar. However, it was not hydrolyzed, so the entire amount was recovered, concentrated, and lyophilized.

Hydrolysis and Fragment Preparation by Galactan-1,3-β-galactosidase

50 mM phosphate buffer (pH 6.5) was added to the BF-I-2a sample obtained in the process of lyticase and lichenase treatment to dissolve it, and then Galactan-1,3-β-galactosidase (from Clostridium thermocellum, Creative Enzymes, Shirley, NY, USA ) 1 unit was added and treated in a constant temperature water bath at 50 ° C for 48 hours. To stop the enzymatic reaction, the supernatant was obtained by bathing in a water bath at 100 ° C for 20 minutes and centrifuging, which was fractionated on HPLC using a Superdex 75 GL column equilibrated with 50 mM ammonium formate buffer (pH 5.5) and analyzed for neutral sugar and pentose sugar was carried out. At this time, two fractions BF-I-3a and BF-I-3b having different molecular weights and constituents were obtained, which were desalted (Microacylizer, cut-off 100) and lyophilized to prepare fractional fractions.

<Results and discussion>

<1-8> General chemical properties of crude polysaccharide derived from fermented barley extract

As a result of general analysis of crude polysaccharide BF-CP derived from fermented barley extract and refined polysaccharide BF-I, II and III, crude polysaccharide BF-CP derived from fermented barley extract contained 91.1% neutral sugar, 4.9% acidic sugar, and 4.9% protein. 2.0% and polyphenol 2.0%. As a result of confirming constituent sugars, glucose (70.7%) was mostly occupied, and arabinose and xylose occupied 9.0% and 11.4%, respectively. As a result of analyzing the general components of purified polysaccharides BF-I, BF-II and BF-III derived from fermented barley extract, it was confirmed that all three fractions with different molecular weights contained 90% or more of neutral sugar. However, the high molecular fraction BF-I was composed of glucose (40.9%), xylose (26.7%) and arabinose (19.9%) in high proportions, and BF-II contained glucose (62.0%), arabinose (11.6%) and xylose ( 10.8%), and finally, the low molecular weight fraction BF-III was mostly composed of 94.9% glucose (Table 1).

Summarizing the above results, purified polysaccharide BF-I derived from fermented barley extract suggests the possibility that arabinoxylan and β-glucan, which are a kind of hemicellulose derived from barley, and β-glucan derived from yeast may exist in a mixture. From the increase in the content of BF-III, it was assumed that BF-III would exist in the form of limit dextrin as starch is decomposed by fermentation. As a result of measuring the degree of purification of each purified polysaccharide using HPSEC, crude polysaccharide BF-CP derived from fermented barley extract was found to be in the form of a mixture of high and low molecules. As a result, it was confirmed that BF-I was purified as a single peak with left-right symmetry, BF-II was found to be a mixture of substances with various molecular weights, and BF-III was composed only of low molecular weight substances. was confirmed (Fig. 3).

Chemical properties of BF-CP, BF-I, BF-II and BF-III isolated from fermented barley

<1-9> Evaluation of immune activity of polysaccharide isolated from fermented barley extract

<1-9-1> Cytotoxicity of polysaccharide samples derived from fermented barley extract

A direct toxicity test was performed on cells of crude polysaccharide BF-CP and purified polysaccharide BF-I, II and III derived from fermented barley extract using mouse-derived normal cell macrophages. As a result, all four samples of fermented barley extract-derived crude polysaccharide BF-CP and purified polysaccharide BF-I, II, and III did not show significant toxicity to macrophages, which are normal cells.

<1-9-2> Measurement of cytokine production of polysaccharide samples derived from fermented barley extract

Macrophage regulates immune phenomena by secreting various cytokines in the process of phagocytosing and removing bacteria or foreign substances, and is a cell that plays a central role in immune action against antigens. It is related to antigen presentation and non-specific immune action of lymphocytes. It exhibits direct cytotoxic activity against tumor cells. In addition, substances (LPS or natural products) that respond to TLR (toollike receptor) activate macrophages to regulate secondary immune responses such as proliferation of T cells and B cells, activation of macrophages for phagocytosis, and defense against microbial infection. It is known to produce cytokines such as IL1, IL6, IL-10, IL-12, and TNF-α. IL1, IL6, and TNF-α are representative cytokines induced by macrophages and play a pivotal role in the inflammatory response following bacterial infection, and are known to increase in inflammatory lesions. IL-6 has been reported to act cooperatively with IL-1 to be involved in the differentiation of T cells and B cells and to have anticancer effects, and TNF-α has cytotoxic and antiviral effects on specific cancer cells, acute and It is known to play an important role in various biological reactions that occur in chronic inflammatory diseases. On the other hand, IL-12 is a cytokine that activates NK cells and induces a Th1 type immune response, and is known to increase reactivity to cellular foreign substances such as cancer cells. In addition, IL-10 cross-regulates the production of TNF, IL-1, chemokine and IL-12 by macrophages, thereby inhibiting the secondary function of macrophages (T lymphocyte-mediated specific immune inflammatory response) in the activation of T lymphocytes. It is known to do

As a result of in vitro measurement of macrophage cytokine production by direct stimulation of crude polysaccharide BF-CP derived from fermented barley extract and purified polysaccharide BF-I, BF-II and BF-III samples, all four samples showed IL-6, IL- It was confirmed that the production of 12 and TNF-α was promoted (FIG. 5). However, in the case of BF-II and BF-III, the ability to produce cytokines such as IL-6, IL-12, and TNF-α began to increase at concentrations above 10 μg/mL, and the activity was activity was lower. On the other hand, in the case of BF-I, the activity was the most excellent among the three purified fractions, and showed excellent activity even at a low concentration of 1 μg/mL. This showed almost similar activity to BF-CP at all concentrations, and it was confirmed that the immune enhancing activity of the fermented barley extract was mainly due to BF-I.

In the case of IL-12 (FIG. 5(B)), it was confirmed that BF-I showed excellent activity at various concentrations. On the other hand, it was confirmed that BF-II and BF-III showed low activity even at a concentration of 1000 μg/mL. These results suggest that purified polysaccharide BF-I derived from fermented barley extract is expected to have activity in cell-mediated immunity, such as activation of NK cells and activation of cytotoxic T lymphocytes (CTL) through the induction of Th1-type immune responses.

In addition, there is an activity to significantly produce IL-6 and TNF-α, which are classified as inflammatory cytokines directly related to the homing of immune cells to the inflammatory site, and IL-12, which is directly related to the activation of cellular immunity. Since this was confirmed, it was judged that purified polysaccharide BF-I derived from fermented barley extract has a function of activating (regulating) the immune mechanism acting in biological defense. In particular, IL-12 is recognized as an essential cytokine for inducing anticancer activity because it is directly involved in activating NK cells that kill cancer cells when cancer cells are present. Therefore, purified polysaccharide BF-I derived from fermented barley extract is expected to have NK cell activation function as well. It became. Through this result, it was finally confirmed that the main active body of crude polysaccharide BF-CP derived from fermented barley extract was the BF-I fraction, and then structural analysis of BF-I was conducted to identify immune-active polysaccharide substances derived from fermented barley extract. did

<1-10> Structural analysis of immune active polysaccharide BF-I derived from fermented barley extract

Except for starch, which is a storage polysaccharide, polysaccharides generally present in barley include cellulose, a polysaccharide present in the primary cell wall and middle lamella, hemicellulose recently identified as xyloglucan and arabinoxylan, pectic substances, and β-glucan. It consists of four main types. Among them, cellulose is a polysaccharide that is extremely difficult to dissolve in water because it forms macromolecules due to high intermolecular hydrogen bonds. are classified as However, the presence of large amounts of arabinose and xylose in BF-I, a refined polysaccharide derived from fermented barley extract, suggested the possibility that arabinoxylan contained in hemicellulose was selectively water-solubilized during the fermentation process. In addition, the fermented barley extract provided in this experiment is prepared through three-stage fermentation, wherein yeast such as Saccharomyces cerevisiae is used for the two-stage fermentation. Since the cell wall of yeast is reported to be composed of β-glucan and chitin as main components and surrounded by mannoprotein, the yeast responsible for alcohol fermentation during the fermentation and maturation process is autolyzed to produce β-glucan, chitin and mannan can be selectively eluted. Therefore, the presence of a large amount of glucose in purified polysaccharide BF-I derived from fermented barley extract may be starch present in barley, but the possibility that it is β-glucan derived from barley or β-glucan derived from yeast cannot be ruled out.

<1-10-1> Enzyme screening for structural identification of BF-I

As a result of the above experiments, before identifying the structure of BF-I, which was identified as the active body of fermented barley extract-derived crude polysaccharide BF-CP, the purified polysaccharide BF-I derived from fermented barley extract was analyzed to determine the -glucan and yeast-derived β-glucan were presumed to exist, and barley fermented extract-derived purified polysaccharide BF-III was presumed to be mixed with barley-derived limit dextrin and oligosaccharide-type barley and yeast-derived β-glucan through constituent sugar analysis Therefore, we tried to confirm whether the expected structure exists through various enzyme treatments. To this end, after hydrolysis by treatment with various enzymes, the amount of reducing sugar was measured by the DNS method (FIG. 6).

In general, starch is composed of α-(1→4)-glucan type amylose and α-(1→4)-glucan main chain α-(1→4)-glucan branched amylopectin structure at position C6, and arabinoxylan is widely known as a structure in which the OH group at C2, C3, or C2, C3 is substituted with T-Araf in the β-(1→4)-D-xylan main chain. In addition, barley-derived β-glucan has a linear form consisting of β-(1→3,1→4)-D-glucan repeatedly, and [-4)2~3-β-D-Glc-(1 It exists in the form of ,3)-β-Glc-(1] or [-4)5~28-β-D-Glc-(1,3)-β-Glc-(1] Yeast-derived β-glucan has a structure in which the side chain extends in the form of β-(1→3)-glucan at the C6 position in the main chain of β-(1→3)-D-glucan.

Therefore, in this experiment, in order to predict the structure of fermented barley extract-derived polysaccharides, β-xylanase (β-1,4-xylanase), β hydrolyzing barley-derived arabinoxylan to purified polysaccharides BF-I and BF-III derived from fermented barley extracts. -Lichenase (β-1,3-1,4-glucanase) that hydrolyzes glucan, Amyloglucosidase (α-1,4-1,6-glucanase) that hydrolyzes starch and β-glucan derived from yeast After treating lyticase (β-1,3-glucanase), endo-laminarinase (endo-1,3-glucanase) and exo-laminarinase (exo-1,3-glucanase), reducing sugar The amount of was measured.

In addition, after treating the purified polysaccharide BF-I derived from fermented barley extract with various enzymes, the results of measuring reducing sugar using the DNS method were compared and analyzed based on the absorbance of BF-I at a concentration of 5 mg/mL.

As a result, the purified polysaccharide BF-I derived from fermented barley extract was detected with high reducing sugar content in the group treated with Lichenase, Lyticase, exo-laminarinase, and β-Xylanase enzymes. Therefore, it was confirmed that the purified polysaccharide BF-I derived from fermented barley extract was composed of barley-derived arabinoxylan and β-glucan and yeast-derived β-glucan (Fig. 7(A)).

On the other hand, in purified polysaccharide BF-III derived from fermented barley extract, a high level of reducing sugar was detected in the group treated with Amyloglucosidase enzyme, and a small amount of reducing sugar was detected in the group treated with lyticase and exo-laminarinase enzymes. Therefore, it is presumed that most of the glucose detected in purified polysaccharide BF-III derived from fermented barley extract is starch present in the form of limit dextrin, which is not degraded by amylase during fermentation. It was estimated that this was contained in a small amount (Fig. 7 (B)).

<1-10-2> Molecular weight measurement of purified polysaccharide BF-I derived from fermented barley extract

In order to identify the exact structure of the purified polysaccharide BF-I derived from fermented barley extract, the molecular weight was measured through HPSEC. As a result of standard calculation of the pullulan series, BF-I was confirmed to be about 380 kDa (FIG. 8).

<1-10-3> Analysis of binding pattern by methylation of purified polysaccharide BF-I derived from fermented barley extract

In the structural analysis of polysaccharides, the binding pattern of sugar chains is known to be the most important analysis in that it elucidates the basic structure of polysaccharides. Nucleic acids and proteins are connected by the same bonding pattern of phosphodiester bond or peptide bond (amide bond) between nucleotides or amino acids, which are component molecules, whereas polysaccharides have 1→2, 1→3, 1→4, It is difficult to analyze the structure compared to other biomolecules because it can form 1→6 bonds and can exist in a branched form at each carbon. However, the analysis of the binding mode is the most important step in structural characterization of polysaccharides. Methylation analysis, which is widely used for sugar chain linkage analysis, methylates and hydrolyzes the hydroxyl attached to the carbon not participating in the linkage to open the ring of the partially methylated constituent sugar, then acetylates the remaining hydroxyl and uses GC-MS. It is a complicated process to identify the structure by analyzing the fragment ion.

In this experiment, in order to confirm the binding pattern of purified polysaccharide BF-I, which was identified as the active body of crude polysaccharide BF-CP derived from fermented barley extract, by the Hakomori method (Hakomori S. A rapid permethylation of glycolipid, and polysaccharidecatalyzed by methylsulfinyl Carbanion in mimethyl sulfoxide. J Biochem 1964;55:205-208) As a result of conversion to partially methylated alditol acetate (PMAA) derivative and GC-MS analysis, a total of 29 sugar chain bonds were identified (Fig. 9 and Table 2 )

Purified polysaccharide BF-I derived from fermented barley extract was presumed to be composed of barley-derived arabinoxylan, β-glucan, and yeast-derived β-glucan, as confirmed in the previous enzyme screening, suggesting a high possibility of detecting the binding thereto. Subsequently, as a result of analyzing the binding pattern, it was assumed that several polysaccharides with similar molecular weights were mixed, as the binding of hemicellulose-derived arabinoxylan, barley and yeast-derived β-glucan and some pectin substances were confirmed. First, looking at the binding pattern of the barley-derived arabinoxylan structure, it was confirmed that it was composed of terminal-Araf, 5-linked-Araf, 4-linked-Xylp, 3,4-linked-Xylp and 2,3,4-linked-Xylp. did In particular, terminal-Araf was detected at a high rate (10.7%), which confirmed that arabinose was present in a short form by one molecule mainly at the non-reducing terminal (Table 2). On the other hand, in the case of the xylose residue, xylose at the non-reducing end was hardly detected and 4-linked Xylp was detected as high as 13.9%, confirming that xylose is the main chain linked by (1→4) linkage. Xylp and 2,3,4-branched Xylp were detected. This fact indicates that the main chain of arabinoxylan present in purified polysaccharide BF-I derived from fermented barley extract exists in the form of (1→4) bonded xylan, and some xylose have two strands at the C3 or C2 and C3 positions at the same time. , it was possible to infer that arabinose exists in a short form one molecule at a time. Taken together, the arabinoxylan present in the purified polysaccharide BF-I derived from fermented barley extract is a single strand at the C3 position of xylose or two strands at the C2 and C3 positions of xylose in the main chain in which xylose is linked by a (1→4) bond. It was assumed that the (1→5)-arabinan chain would be connected as a side chain structure while extending into a strand. Therefore, based on this, the predicted structure of BF-1 was modeled (FIG. 10).

Second, refined polysaccharide BF-I derived from fermented barley extract was detected at a high rate (29.2%) of 4-linked Glcp. Could. Also, since 10.8% of 3-linked Glcp and 0.4% of 6-linked Glcp were detected, it was assumed that they were in the form of yeast-derived β-glucan.

Finally, 3,6-linked Galp was detected, which was presumed to have a small amount of arabino-β-3,6-galactan present in the side chain of rhamnogalacturonan (RG)-I derived from pectin, which was not identified in the previous enzyme screening. Therefore, the reactivity with β-glucosyl Yariv reagent was examined afterwards.

Methylation analysis of BF-I purified from fermented barley

<1-10-4> Review of reactivity between BF-I and β-glucosyl Yariv reagent

In <1-10-3>, 3,6-linked Galp was detected in methylation, which was expected to be arabino-β-3,6-galactan present in pectin-derived RG-I structure. Therefore, the presence or absence of arabino-β-3,6-galactan was tested using β-glucosyl Yariv reagent.

The specific method is as follows. βGlucosyl Yariv reagent [1,3,5-tri-(4-β-glucopyranosyl-oxyphenylazo)-2,4,6-trihydroxybenzene] reacts specifically with type II arabino-β-3.6-galactan among arabinogalactan, resulting in a red color. It is known to form precipitates. Since this is known as the only color developing reagent that specifically reacts with polysaccharide, it was judged that the existence of the arabino-β-3.6-galactan structure in the purified polysaccharide BF-I fraction derived from fermented barley extract could be clarified. Therefore, a 0.15 M NaCl agarose plate containing 10 μg/mL of β-Glucosyl Yariv reagent was prepared, a well of 3 mm in diameter was prepared, and a solution containing 1,000 μg/mL of BF-I and standard gum arabic diluted by concentration was prepared in the well. After each injection, the reaction was allowed to stand at room temperature for 15 hours, and the presence or absence of arabino-β-3,6-galactan was observed by observing the resulting red precipitate.

As a result, the standard arabino-β-3,6-galactan, gum arabic, showed an increase in precipitation in a concentration-dependent manner. In addition, in the case of BF-I, it was observed that about 3.39% of precipitation conversion was formed compared to the concentration of 1000 μg/mL of the standard material (FIG. 11). Therefore, it was confirmed that in addition to barley-derived arabinoxylan and yeast-derived β-glucan, pectin-derived arabino-β-3,6-galactan was also partially present in the purified polysaccharide BF-I derived from fermented barley extract.

<1-11> Microstructure identification and activity evaluation of purified polysaccharide BF-I derived from fermented barley extract of β-glucosyl Yariv

Purified polysaccharide BF-I derived from fermented barley extract, which had excellent immune activity through the previous experiments, was a polysaccharide with a molecular weight of about 380 kDa and relatively excellent degree of purification. It was confirmed that arabinoxylan, β-glucan, and yeast-derived β-glucan were present in a mixed form.

Since polysaccharides have not been developed to analyze the entire structure at once due to the characteristics of polymers, fragments are prepared by chemical or enzyme treatment, the microstructure of each fragment is identified, and the overall structure is finally estimated. It is common to compare the activity of each fragment to evaluate the main active site that affects immune activity. Therefore, in this experiment, in order to investigate the microstructure of the purified polysaccharide BF-I derived from fermented barley extract, it was fragmented and purified by treatment with various enzymes.

<1-11-1> Preparation of fractional fractions of purified polysaccharide BF-I derived from fermented barley extract

In order to analyze the exact structure of purified polysaccharide BF-I derived from fermented barley extract, BF-I was dissolved in 50 mM ammonium formate buffer under pH 4.5 conditions for the purpose of removing a small amount of remaining starch before treatment with a specific enzyme, and Amyloglucosidase 100 Unit was added and incubated at 50 ° C for 48 hours to hydrolyze. Subsequently, as a result of fractionation using a Superdex 75 GL column, neutral sugars were detected only in the polymer fraction. Therefore, it was confirmed again that BF-I did not contain starch, and enzyme treatment experiments were conducted later.

The polymer fraction from the Hydrolysis I process was dissolved in 50 mM ammonium formate buffer at pH 6.5, and 150 units of β-Xylanase, which selectively hydrolyzes (1→4)-Xyl, was added, followed by incubation at 40 ° C for 48 hours for hydrolysis (Hydrolysis II).

Then, as a result of fractionation using a Superdex 75 GL column, two fractions, a high molecular fraction BF-I-1a and a low molecular fraction BF-I-1b, could be obtained.

After the low molecular weight oligosaccharide (BF-I-1b) is removed through the Hydrolysis II process, the remaining BF-I-1a derived from fermented barley extract is dissolved in 0.1 M potassium phosphate buffer at pH 7.0, and (1→3)-Glc is selectively 100 units of lyticase hydrolyzed were added and hydrolyzed by incubation at 30 ° C for 48 hours (Hydrolysis III).

Then, as a result of fractionation using a Superdex 75 GL column, it was possible to obtain three fractions: high molecular fraction BF-I-2a, low molecular fraction BF-I-2b and BF-I-2c.

Low-molecular-weight oligosaccharides (BF-I-2b, BF-I-2c) were removed through Hydrolysis III, and the remaining BF-I-2a derived from fermented barley extract was dissolved in pH 7.0 50 mM ammonium formate buffer (1→3). (1→4)-glucan was hydrolyzed by adding 100 units of Lichenase, which selectively hydrolyzes, and incubated at 60°C for 48 hours (Hydrolysis IV).

However, as a result of fractionation using a Superdex 75 GL column and measurement of neutral sugars and pentoses, neutral sugars and pentoses were detected only in the polymer fraction, so that the enzymatic action of Lichenase did not occur and all of them were recovered.

During Hydrolysis IV, Lichenase enzyme action did not occur, so the entire amount of BF-I-2a recovered was dissolved in pH 6.5, 50 mM ammonium formate buffer, and arabino-β-(3,6)-galactan structured (1→3)-galactan was obtained. 1 unit of galactan-β-(1,3)-galactosidase that selectively hydrolyzes was added and incubated at 50°C for 48 hours to hydrolyze (Hydrolysis V).

Then, as a result of fractionation using a Superdex 75 GL column, two fractions, a high molecular fraction BF-I-3a and a low molecular fraction BF-I-3b, could be obtained (FIG. 12).

All the fractions recovered through each hydrolysis process described above were desalted and lyophilized and used for structural analysis later.

<1-11-2> Constituent sugar analysis of purified polysaccharide BF-I fraction derived from fermented barley extract

Constituent sugar analysis was performed on the fragment fractions (BF-I-1a to BF-I-3b) recovered through each hydrolysis process in <1-11-1>.

As a result of analyzing the constituent sugars of BF-I-1b recovered in the Hydrolysis II process, it was confirmed that arabinose (34.6%) and xylose (53.7%) were detected, suggesting that arabinoxylan was decomposed by β-Xylanase. (Table 3).

As a result of analyzing the constituent sugars of the polymeric fraction BF-I-2a recovered in the hydrolysis III process, the glucose content was detected as very low as 5.7%, suggesting that most of the glucose was fractionated in the hydrolysis III process. On the other hand, since it is composed of large amounts of arabinose (31.3%), galactose (30.1%), and xylose (10.3%), it is presumed that highly branched arabinoxylan that has not yet been degraded in the hydrolysis II process remains, and galactose was previously detected. Arabino-β-3,6,-galactan was judged to be prominent as molecules such as arabinoxylan and β-glucan were removed (Table 3).

In addition, as a result of analyzing the constituent sugars of BF-I-2b and BF-I-2c recovered in the hydrolysis III process, the BF-I-2c fraction was found to have a glucose content of 96.9%, and was converted by lyticase (1→3 ) It was judged that the glucose of the bond was degraded and appeared as a low molecular weight. However, in BF-I-2b, despite treatment with lyticase, the content of glucose (9.6%) was low and large amounts of arabinose (38.3%) and xylose (25.3%) were detected. It was determined that the form of arabinoxylan was fractionated in the hydrolysis III process due to non-specific hydrolysis by enzyme treatment (Table 3).

As a result of analyzing the constituent sugars of BF-I-3a and BF-I-3b recovered from the Hydrolysis V process, in the case of BF-I-3b, it was found to be mainly composed of galactose (53.7%) and arabinose (32.4%), indicating that galactan It was confirmed that the branch part of type II arabino-β-3,6,-galactan was partially hydrolyzed by -β-(1,3)-galactosidase enzyme.

On the other hand, since BF-I-3a was composed of arabinose (28.5%), galactose (26.0%) and mannose (20.6%), it was presumed that some arabinogalactan still remained.

Subsequently, the fractions BF-I-2a and BF-I-3a of purified polysaccharide BF-I derived from fermented barley extract were tested for the presence or absence of type II arabino-β-3,6-galactan using β-glucosyl Yariv reagent. experiment was conducted.

Monosaccharide composition of BF-I and its derived fractions obtained from enzymatic hydrolysate

<1-11-3> Examination of reactivity of fractions derived from fermented barley extract with β-glucosyl Yariv reagent

As a result of component sugar analysis of the fractions BF-I-2a and BF-I-3a recovered in the Hydrolysis III process, galactose was detected in large amounts. Therefore, the presence or absence of arabino-β-3,6-galactan was confirmed using β-glucosyl Yariv reagent.

The specific method is as follows. Prepare a 0.15 M NaCl agarose plate containing 10 μg/mL of β-Glucosyl Yariv reagent, make wells with a diameter of 2.5 mm, and inject solutions containing diluted standard gum arabic and sample 1,000 μg/mL into each well. After reaction was allowed to stand at room temperature for 15 hours, the presence or absence of arabino-β-3,6-galactan was observed by observing the resulting red precipitate.

Gum arabic, a standard arabino-β-3,6-galactan, showed a concentration-dependent increase in precipitate, and in the case of BF-I-2a, formation of a precipitate equivalent to about 22.04% of the standard material was observed ( Figure 13). This was a result of about 7 times higher precipitate formation than 3.39% of the purified polysaccharide BF-I derived from fermented barley extract. Therefore, it was confirmed again that pectin-derived arabino-β-3,6-galactan was present in BF-I-2a.

On the other hand, in the case of BF-I-3a, the formation of a precipitate equivalent to about 7.24% compared to 1000 μg / mL of the standard material was observed, showing less precipitation than BF-I-2a. This was judged to be a result of hydrolysis by galactan-β-(1,3)-galactosidase enzyme in Hydrolysis IV and only remaining arabino-β-3,6-galactan was formed by precipitation (FIG. 13).

<1-11-4> MALDI-TOF analysis of purified polysaccharide BF-I fragments derived from fermented barley extract

MALDI-TOF (positive ion mode) analysis was performed to accurately confirm the molecular weight and predict the structure of the fragments per small molecular weight oligosaccharide recovered from the hydrolysis process.

As a result of molecular weight measurement through MALDI-TOF of the fraction BF-I-1b recovered in the Hydrolysis II process, several kinds of molecular weight peaks were observed. Most of the various molecular weight peaks identified in the measured spectrum were confirmed to show a difference in molecular weight 132 of the pentose residue, and the molecular weight of m / z 437 detected as the smallest molecular weight was three pentose (P) [P = ( 132*3)+18] was judged to have added Na+ ions (MW: 23). In addition, m/z 569, 701, 833 . 2945 peaks were observed, which is the molecular weight corresponding to [P4+Na]+, [P5+Na]+, [P6+Na]+ to the maximum [P22+Na]+. At this time, [P3+Na]+ is shown in Fig. As shown in Fig. 14, the structure could be predicted by presuming that arabinoxylan was present in several cases, such as when only the xylose main chain was linked and when one arabinose was linked to the xylose main chain as a side chain (FIG. 14).

As a result of MALDI-TOF measurement, it was estimated that all molecular weights detected in the spectrum were in the form of [P+Na]+. To confirm this, daughter fragment ions analysis was performed by measuring MALDI-TOF/TOF.

Daughter fragment ions analysis was performed for [P+Na]+ nonasaccharide m/z 1229 ions. As a result, m/z 1229, 1097, 965, 833, 701 and 569 ions were found, and they were released from nonasaccharide[P9+Na]+ to tetrasaccharide[P4+Na]+ form with -132 (pentose) difference. It was confirmed again that it was composed only of pentose. As a result, it was confirmed that barley-derived arabinoxylan was hydrolyzed and existed in the form of an oligosaccharide, as estimated in the previous results (FIG. 15).

As a result of molecular weight measurement through MALDI-TOF (positive ion mode) of the fraction BF-I-2c recovered in the Hydrolysis III process, several kinds of molecular weight peaks were observed. Most of the various molecular weight peaks identified in the measured spectrum were confirmed to show a difference in the molecular weight of 162 of the hexose residue, and the molecular weight of m / z 527 detected as the smallest molecular weight was three hexose (H) [H = ( 162*3)+18] was judged to have added Na+ ions (MW: 23). In addition, m/z 689, 851, 1013 . 2309 peak was observed, which is the molecular weight corresponding to [H4+Na]+, [H5+Na]+, [H6+Na]+ to the maximum [H14+Na]+ (FIG. 16). Barley-derived β-glucan has a linear structure, and yeast-derived β-glucan has a linear β-(1→3)-D-glucan main chain with β-(1→3)-D-glucan at the C6 position. Since the side chain extends in the form of -(1→3)-glucan, the structure of [H3+Na]+ is expected to exist in a linear form or when one molecule of glucose is linked to the glucose main chain through a side chain. estimated, from which the structure could be predicted.

As a result of MALDI-TOF measurement, it was estimated that all molecular weights detected in the spectrum were in the form of [H+Na]+. To confirm this, daughter fragment ions analysis was performed by measuring MALDI-TOF/TOF. Specifically, daughter fragment ions analysis was performed for [H+Na]+ type hexasaccharide m/z 1013 ion.

As a result, m/z 851, 688, 527, and 365 ions were found, and they were released from hexasaccharide[P6+Na]+ to trisaccharide[P3+Na]+ forms with -162 (hexose) differences, indicating that they are hexose It was confirmed again that it was composed of only (FIG. 17). As estimated from the previous results, it was a result that confirmed that barley or yeast-derived β-glucan was hydrolyzed and existed in the form of an oligosaccharide.

As a result of molecular weight measurement through MALDI-TOF (positive ion mode) of the fraction BF-I-3b recovered in the Hydrolysis V process, several molecular weight peaks were observed. Several types of molecular weight peaks identified in the measured spectrum were confirmed to show a difference in molecular weight between pentose (-132) and hexose (-162) residues, and the molecular weight of m/z 497 detected as the smallest molecular weight was 1 pentose (-162). P) and two hexoses (H), it was determined that Na+ ions (MW: 23) were added to [ 132(P)+(162(H)*2)+18(H2O)].

In addition, m/z 791, 953, 1085 . 3599 peaks were observed, which is the molecular weight corresponding to [2P+3H+Na]+, [2P+4H+Na]+, [3P+4H+Na]+ to the maximum [11P+13H+Na]+ (FIG. 18 ).

Therefore, from the above results, it was finally confirmed that type II arabino-β-3,6-galactan was hydrolyzed to low molecular weight in BF-I-3b. It was judged that the phosphorus arabinose appeared alternately.

<1-11-5> Analysis of binding patterns by methylation of purified polysaccharide BF-I fragments derived from fermented barley extract

As a result of structural analysis of purified polysaccharide BF-I derived from fermented barley extract, it was assumed that barley-derived arabinoxylan and β-glucan, yeast-derived β-glucan, and pection-derived acabinogalactan were present. Therefore, in order to identify the structure thereof, a fractional fraction was prepared through continuous enzymatic hydrolysis to make the polysaccharide BF-I into a microstructure.

As a result of methylation analysis of purified polysaccharide BF-I derived from fermented barley extract, a total of 29 sugar chain bonds were identified.

As a result, 28 and 11 sugar chain bonds were identified in BF-I-1a and BF-I-1b fractionated in Hydrolysis II, respectively, and BF-I-2a and BF-I-1b fractionated in Hydrolysis III 2b and BF-I-2c were found to have 28, 24 and 12 sugar chain bonds, respectively. BF-I-3a and BF-I-3b fractionated in the Hydrolysis V process identified 27 and 19 sugar chain bonds, respectively. Although the number of analyzed sugar chain bonds did not show a significant difference, it was confirmed that a significant difference was observed in the area of the peak (FIG. 19, Table 4).

Subsequently, as a result of analyzing the binding mode, the purified polysaccharide BF-I fragment fraction derived from fermented barley extract showed a difference in binding mode and content according to enzyme treatment.

First, as a result of analyzing the binding form of BF-I-1a and BF-I-1b separated in the hydrolysis II process, most of BF-I-1b hydrolyzed in oligosaccharide form by β-Xylanase treatment showed arabinose and xylose It was composed of β-Xylanase, and arabinoxylan was enzymatically hydrolyzed to low molecular weight by β-Xylanase treatment, as the content of arabinose and xylose binding forms was rapidly reduced in the fractional fraction BF-I-1a in the form of a polymer. It was judged to remain in BF-I-1a without being hydrolyzed.

Therefore, as estimated from the methylation results of purified polysaccharide BF-I derived from fermented barley extracts, it was finally confirmed that arabinoxylan derived from barley has an arabinose branch at the C3, C2, or C3 position of the β-(1→4)-xylan backbone.

Subsequently, as a result of analyzing the binding patterns of BF-I-2a, BF-I-2b and BF-I-2c separated in the hydrolysis III process, BF-I-2c hydrolyzed into oilgo sugar form by lyticase treatment was It was mostly composed of glucose. In particular, as the binding patterns of terminal-Glcp (30.3%), 3-linked-Glcp (25.1%) and 4-linked-Glcp (39.5%) were mainly detected, β-(1→3)(1→6) -D-glucan form of yeast-derived β-glucan and β-(1→3)(1→4)-D-glucan form of barley-derived β-glucan were assumed to have a linear structure (Table 4).

From these results, the lyticase used in the hydrolysis III process can hydrolyze not only the β-1,3 linkage of yeast-derived β-glucan but also the β-1,3 linkage of barley-derived β-glucan. It was judged that 4-linked Glcp was detected in large amount.

As a result of analyzing the binding pattern of the fraction BF-I-2b, similarly to BF-I-1b hydrolyzed in the form of an oligosaccharide during hydrolysis II, high contents of arabinose and xylose were detected. It was judged that the highly branched arabinoxylan remained in BF-I-1a due to non-specific hydrolysis by enzyme treatment and was fractionated during hydrolysis III.

As a result of analyzing the binding pattern of the fragment fraction BF-I-2a, the contents of galactose and arabinose were detected as high as in the result of the constituent sugars, and compared to the purified polysaccharide BF-I derived from fermented barley extract or the fraction fraction BF-I-1a. The content of galactose was markedly elevated. In particular, the content of 3,6-linked Galp, which is a characteristic of the pectin-derived arabino-β-3,6-galactan structure, was increased, which was consistent with the previous β-Glucosyl Yariv reagent results. It was judged to be prominent as the glucan was removed.

In the case of the hydrolyzed fraction BF-I-3b in the form of an oligosaccharide separated in the process of Hydrolysis V, arabinose and galactose were mainly composed, especially terminal-Araf (25.3%), 3-linked Galp (8.1%), 6- Binding patterns of linked Galp (13.8%) and 3,6-linked Galp (29.1%) were observed (Table 4).

In summary, the purified polysaccharide BF-I derived from fermented barley extract contained highly branched arabinoxylan (9.4%) and β-glucan (35.0%) derived from barley and arabino-β-3,6-galactan (7.0%) derived from pectin. It was finally determined that yeast-derived linear β-glucan (6.0%) would exist in a mixed form (Table 4).

Therefore, the analysis results of the purified polysaccharide BF-I derived from fermented barley extract to identify the microstructure, that is, the constituent sugar analysis of the prepared fragments, methylation analysis, and MALDI-TOF analysis results, are all combined to reveal BF-I was finally confirmed as a structure in which the structures modeled in FIG. 20 were mixed.

Analysis of glycosylation of BF-I and its fragments obtained from enzymatic hydrolysates

<1-12> Comparison of macrophage stimulating activity for evaluation of active moiety of purified polysaccharide BF-I fragment derived from fermented barley extract

In previous experiments, it was confirmed that purified polysaccharide BF-I derived from fermented barley extract enhances immune function by stimulating macrophages to induce the production of various cytokines. Therefore, barley-derived arabinoxylan, β-glucan, yeast-derived β-glucan, and pectin-derived arabino-β-3,6-galactan structural parts of purified polysaccharide BF-I derived from barley fermented extract were identified to contribute to the immune activity of barley. Production activities of IL-6, IL-12, and TNF-α were evaluated for fractions obtained by continuous enzymatic treatment of purified polysaccharide BF-I derived from fermentation extract.

The evaluation method is as follows. Murine peritoneal macrophages (2.0×10 5 cells/well) were treated with various concentrations of BF-I and its subfractoins in 96-well plates for 24 hours. The concentration of cytokines in the medium was determined by ELISA kit. 1 μg/ml lipopolysaccharide (LPS) was used as a positive control (PC, positive control) and the group used only with the medium was used as a negative control (NC, negative control).

As a result, in the case of high molecular fraction BF-I-1a and low molecular weight fraction BF-I-1b obtained by treating purified polysaccharide BF-I derived from fermented barley extract with β-Xylanase, BF-I-1a from which some arabinoxylan was removed was BF-I-1a. Compared to -I, it showed slightly lower IL-6, IL-12 and TNF-α production stimulating activity, and oligosaccharide type low molecular weight fraction BF-I-1b showed cytokine production stimulating activity only at a high concentration of 1000 μg/mL. showed (Fig. 21).

If arabinoxylan present in BF-I did not contribute to the activity, the cytokine production-stimulating activity of the BF-I-1a fraction, in which the content of β-glucan was increased due to partial removal of arabinoxylan, would increase. However, when compared to BF-I, the activity of BF-I-1a after removing arabinoxylan through β-Xylanase treatment showed that the activity was partially reduced, suggesting that arabinoxylan contributes to the activity and β-glucan also contributes to the activity. It was judged to be

As a result of treating BF-I-2a, BF-I-2b, and BF-I-2c obtained by re-processing the fraction BF-I-1a with lyticase for the purpose of removing β-glucan, It was confirmed that the production stimulating activity of IL-6, IL-12 and TNF-α was significantly reduced at the concentration (FIG. 21).

Therefore, compared to BF-I-1a, the cytokine production stimulation activity was reduced in BF-I-2a from which β-glucan was removed. It was confirmed again, and the remaining activity of BF-I-2a was determined to be due to highly branched and unhydrolyzed arabinoxylan, as confirmed in the constituent sugar analysis (FIG. 21).

In addition, in the case of fraction BF-I-2b and BF-I-2c, at a concentration of 100 μg/mL, the production of IL-6, IL-12 and TNF-α was similar to or slightly lower than that of the native polysaccharide BF-I. Although stimulatory activity was shown, it was confirmed that the activity was almost absent at concentrations of 100 μg/mL or less (FIG. 21).

Therefore, as confirmed in the constituent sugar analysis, in BF-I-2b, arabinoxylan remaining in a low-molecular form is judged to show some activity, and in BF-I-2c, although the oligosaccharide form of β-glucan is relatively low, it is not active. was judged to be contributing.

In the case of the fractional fractions BF-I-3a and BF-I-3b, even at a concentration of 100 μg/mL, little IL-6, IL-12, and TNF-α production ability was observed. It is judged that the activity is lowered as the (1→3)-galactan part of arabino-β-(3,6)-galactan is hydrolyzed by β-(1,3)-galactosidase. It was confirmed that ,6)-galactan also contributes to the immune activity of purified polysaccharide BF-I derived from fermented barley extract (FIG. 21).

Through the above results, it was finally confirmed that the immunostimulatory activity of purified polysaccharide BF-I derived from fermented barley extract was due to the immune activity shared by arabinoxylan and β-glucan derived from barley, β-glucan derived from yeast and arabinogalactan derived from pectin.

<Experimental Example 2>

<2-1> Evaluation of activity of humoral immunity

<2-1-1> Evaluation of antibody production enhancing ability

Ovalbumin (OVA) was used as an antigen for antibody production. For the production of antibodies for each antigen, 6-week-old BALB/c mice were used. In order to examine the effect of BF-I, an active ingredient of fermented barley extract, on antibody production, immunization was performed with antigen OVA (10 μg/mouse) alone or in combination with BF-I. The dose of BF-I was 500, 50, or 5 µg/mouse. Both groups were immunized with a mixture of aluminum hydroxide (1 mg/mouse) as an adjuvant for antigen immunization. Each immunogen was subcutaneously immunized twice at 2-week intervals, and antisera were collected one week after the final immunization. Antiserum collection was prepared by separating serum from the blood collected from each mouse after 5 days of boosting immunization, and stored at -20 ° C until the measurement of the antibody titer. The total antibody titer against the antigen (OVA) present in serum was measured by ELISA method. 100 μl of each antigen at 50 μg/ml was dispensed per well in each well of a flat-bottomed microtiter plate and attached for 16 hours at 4° C. for antigen coating. After washing each well three times with PBS-Tween 20 (0.05%; PBST), blocking using 3% skim milk and washed again with PBST. Serum for each prepared antigen was diluted from 100-fold to 2-fold dilution, added to each well, and reacted at room temperature for 2 hours. After washing the ELISA plate, a secondary antibody (HRP-conjugated goat anti mouse IgAM) conjugated with peroxidase to mouse immunoglobulin (mouse Ig) was diluted and added to each well, followed by reaction at room temperature for 1 hour. A TMB solution was used as a substrate for color development, and after stopping the reaction by adding 2N sulfuric acid, absorbance was measured at 450 nm.

After the secondary immunization, the total antibody titer was examined from serum. When a foreign substance enters the host, the host induces an antigen-specific immune response to counter the antigen, and this immune response is a cellular immune response involving humoral and cytotoxic T-cells by antibody production depending on the type of antigen. will induce a reaction. The antigen OVA used in this experiment is a protein antigen widely used in antibody production experiments.

As a result, as a result of simultaneous administration of 5, 50, and 500 μg as in the case of OVA monoimmunization, simultaneous administration of 50 μg showed the highest antibody production effect, and it was confirmed that 50 μg is the appropriate concentration to increase the antibody titer in mice (Fig. 22). Therefore, the polysaccharide component obtained from fermented barley was judged to have an adjuvant activity that increases the antibody titer upon simultaneous immunization with a protein antigen.

Adjuvant is a substance that induces immunostimulatory activity against a specific antigen by regulating the function of immune cells, ultimately inducing antigen-specific humoral and cellular immune enhancing activity. Since its introduction, studies on various adjuvant formulations have been conducted for more than 100 years, but the only clinically applicable substance is an aluminium-containing compound (alum), in which immune enhancing activity is induced by antigen storage. However, alum exhibits many differences in induction of activity depending on the antigen, making it difficult to apply widely, and has the disadvantage of overproducing IgE antibodies that induce allergy for some antigens and inducing mainly only humoral immunity enhancing activity. In order to apply to vaccines, the development of new substances to replace alum is required. In particular, polysaccharide substances such as β-glucan induce mitogen activity on B and T cells and show high activity even when administered orally, and are attracting attention as a next-generation adjuvant as clinical trials for application to animals as well as humans are in progress. . Therefore, the polysaccharide obtained from the fermented barley extract is expected to be applicable as an immune enhancer for various antigens.

<2-1-2> Evaluation of Th1/Th2 type immune response activation ability

The effect of BF-I on T cells as an adjuvant in the production of OVA-specific antibodies was investigated by measuring the amounts of antigen-specific Th1-type and Th2-type cytokines produced in the T cell culture supernatant.

Spleens were taken from mice immunized with 10 μg of OVA alone or BF-I or normal mice, and the cell concentration was adjusted to 3 × 10 6 /well, and then seeded in a 24-well culture plate. OVA with an antigen final concentration of 10 μg/mL was simultaneously added to each well in which splenocytes were dispensed and incubated for 72 hours in a 37°C, 5% CO2 incubator. After completion of the culture, the culture supernatant was prepared by centrifugation (900 rpm/10 min) and stored at -80°C until cytokine measurement. Cytokines such as IL-2, IFN-γ, IL-4, GM-CSF, and IL-10 induced in the spleen cell culture supernatant were measured using an ELISA kit for each cytokine.

The regulatory ability of cytokines related to antibody production is mainly determined by cytokines produced by T-cells. Th1-type cytokine is reported to be involved in the production of IgG2 type antibodies by B cells, and Th2-type cytokines promote differentiation into B cells that mainly produce IgG1 type antibodies.

As a result, mice immunized with a mixture of antigens OVA and BF-I had an activity that significantly increased IFN-γ, a Th1-type applied to the experiment, compared to mice in the antigen-only immunization group, and a typical Th2-type, IL- 4, it was confirmed that the production activity of IL-5 and IL-10 has a function of lowering (FIG. 23). Since all of these tendencies were concentration-dependent of BF-I, the adjuvant effect related to antibody production by BF-1 was mainly due to the enhancement of Th1 type cytokine and the function of lowering the production of Th2 type cytokine.

In conclusion, as a result of this experiment, it was investigated that BF-I has an adjuvant activity against the production of antibodies against protein antigens and some cellular immune systems. It was judged to act by activating the immune response of

<2-1-3> Determination of antibody isotype

When a foreign substance enters the host, the host induces an antigen-specific immune response to counter the antigen, and this immune response can be largely divided into two types, that is, the humoral and cellular immune systems. In this experiment, the humoral immunity enhancing effect of BF-I against the protein antigen OVA was investigated by its ability to produce antibodies against BSA. Analysis of subisotypes of immunoglobulin IgG was investigated by ELISA using secondary antibodies (peroxidase-conjugated rabbit anti-mouse IgG1, G2a and G2b, Zymed, USA) specific for each subisotype of mouse immunoglobulin.

First, 100 μl of each antigen at 50 μg/ml was dispensed per well in each well of a flat-bottomed microtiter plate and attached for 16 hours at 4° C. for antigen coating. After washing each well three times with PBS-Tween 20, blocking using 3% BSA and washed again with PBST. Serum for each prepared antigen was appropriately diluted (IgG1: 400 times, IgG2a/b: 100 times), added to each well, and reacted at room temperature for 2 hours. Immunoglobulin subisotype analysis was performed by ELISA using specific secondary antibodies (peroxidase-conjugated rabbit anti-mouse IgG1, G2a and G2b, Zymed, USA) for each subisotype of mouse immunoglobulin. In the case of IgE antibody, it was investigated using an ELISA kit having specificity for IgE antibody. TMB solution was used as a substrate for color development, and after stopping the reaction by adding 2N H2SO4, absorbance was measured at 450 nm.

As a result of examining the antibody titer against OVA, the antibody titer of IgG1 type showed some higher antibody production as a result of simultaneous administration of 50 ug of BF-I compared to antigen-only immunization. There was no difference between groups for IgG2a, but in the case of IgG2b Compared to the single group, the group simultaneously administered with BF-I showed a higher antibody production effect. In general, it has been reported that IgG2 type antibodies are mainly produced by Th1-type cytokines, and IgG1 and IgE type antibody titers are produced by Th2-type cytokines. Therefore, the BF-I component has the function of stimulating the Th1 immune response, such as IFN production, when the immune response to the antigen is induced, and the function of suppressing the production of Th2 type cytokines, such as IL-4 and IL-5. It was judged to have an activity to inhibit the production of IgE antibody, which is an antibody that induces a reaction (FIG. 24).

<2-2> Evaluation of cellular immune activity

<2-2-1> Evaluation of antitumor activity by immunization of tumor vaccines

The nature of an antigen that induces an effective immune response for the removal of cancer has not yet been identified. However, when the cancer is transplanted into the same host again after induction of cancer by carcinogen, the experimental results in which the cancer is removed suggest the possibility of inducing an immune response in the host that can remove the cancer. In addition, as a result of research for overcoming malignant tumors, it is recognized that cancer cells induce an immune response by themselves. In other words, in several studies for the development of immunotherapy by immunization of tumor vaccines for the induction of antitumor activity, some effective activities have been recognized for the prevention and treatment of tumors due to the induction of effective antitumor immunity. A method of practically effective application has not been developed. It is known that the most important reason why tumor immunotherapy fails is due to the immune evasion mechanism of tumor cells themselves, including their low immunogenicity. That is, since cancer cells themselves have suppressed class I- and II-MHC expression, antigen presentation using MHC is not performed, and even if tumor antigens are present, CTLs and helper T cells against tumors are activated by the action of CD4+CD25+ regulatory T cells. It is known to disable the killing mechanism of cancer effector cells by inhibiting the immune response to tumors of the body's immune system by interfering with cell functions and producing immunosuppressive cytokines such as TGF-β produced from tumor cells or T cells. have. In the induction mechanism of tumor vaccines for the treatment or prevention of malignant tumors, the antigen presenting ability of denditic cells (DC), which are proffesional APCs, and the role of macrophages are regarded as the most important factors in the induction stage of tumor immunity. Currently, several researchers co-cultivate DCs or macrophages and cancer cells (apoptotic or nectotoc tumor cells) killed as tumor vaccines in vitro, and then induce significant inhibitory activity on tumor proliferation and metastasis by immunizing them to cancer-bearing hosts. have. Ultimately, this implies that the above-mentioned immune evasion mechanism of tumors can be overcome by using strong antigen-presenting cells to present tumor antigens. Efforts to use adjuvants are also underway. These efforts are ultimately efforts to increase the immunogenicity of low-expressed cancer antigens, and several research results have become important basic studies for the study of the activity and mechanism of action of tumor vaccines for clinical application. Therefore, in order to induce an effective immune response of tumor antigens, the grafting of the concept of an adjuvant related to the enhancement of antigen presentation ability is the most important factor in inducing antitumor immunity along with the identification of malignant tumor antigens inherent in tumor cells. judged by

In <2-1>, the adjuvant activity related to antibody production against protein antigens was demonstrated using BF-I, and a subsequent experiment was conducted to confirm whether or not the cellular immunity enhancement activity was induced for the tumor vaccine. The cellular immunity enhancing effect of BF-I was evaluated by examining the tumor metastasis suppression ability by applying it to a tumor metastasis model in which living cancer cells are inoculated after coimmunization with the prepared tumor vaccine. First, in order to collect colon26-M3.1 carcinoma grown in EMEM medium, cultured cells are separated using trypsin-EDTA, then culture medium is added and washed once, then PBS is added and washed three times to remove the FBS component. Removed. To prepare the tumor vaccine, the cells (1×10 7 /ml) were suspended in PBS, followed by rapid freezing and thawing using liquid nitrogen three times. The prepared tumor vaccine was stored at -70°C and was referred to as F/T vaccine. In order to investigate the cellular immunity enhancing function of BF-I, each vaccine was mixed with BF-I and immunized, and the tumor metastasis inhibitory effect was examined using an experimental animal tumor metastasis model using colon26-M3.1 carcinoma. For immunization with the prepared F/T tumor vaccine, subcutaneous immunization was performed at 5 × 10 5 cells/mouse. Since the appropriate concentration of BF-I in the humoral immune response was 50 to 500 μg, the dose of BF-I was set at an intermediate concentration of 200 μg and immunized by mixing with each vaccine. The immunogen was immunized twice at 2-week intervals, and cancer cells were intravascularly injected 2 weeks after the final immunization. 14 days after the tumor inoculation, the mice were sacrificed, the lungs, the target organs of the tumors, were removed, and the metastasized tumors were fixed in Bouin's solution, and then the number of tumor clusters was measured.

As a result of the experiment, when immunized with the F/T vaccine, the vaccine alone showed a significant tumor metastasis inhibitory activity of about 48.9% compared to the tumor control group. I showed the result of an immune enhancing effect upon immunization with a tumor vaccine (Table 5). In animal studies by immunization of tumor vaccines for the induction of antitumor immunity, necrotic cells such as F/T cell lysates activate macrophages to produce cytokines such as TNF-α, IL-1 and IL-6, , reported that active immunity of macrophages exposed to these vaccines not only prevented tumors in vivo, but also significantly inhibited tumor proliferation therapeutically. That is, it is known that the most important factor for inducing tumor immunity is the acquisition of antigen presenting ability (maturation) of antigen presenting cells to induce the activation of cytotoxic T cells, which are cellular immune systems as effector cells for tumor cells. As a result of tests on the effect of BF-I on the innate immune system, BF-I inhibits cytokines such as TNF-α and IL-12, which are recognized as important cytokines in inducing antigen-specific immune enhancing activity from macrophages in vitro. It was confirmed that by induction, there is an effect of increasing the antigen presenting ability for the induction of antigen-specific immune-enhancing activity. Therefore, it was confirmed that BF-I has adjuvant activity to increase tumor immunity induced by F/T vaccine by the inhibitory effect of BF-I on tumor metastasis in F/T vaccine immunity in Table 5.

Effects of BF-I on tumor metastasis in immunization of tumor vaccines

<2-2-2> Evaluation of CTL activity

In order to obtain successful immunity against tumors, tumor cell phagocytosis by dendritic cell (DC) or macrophage, which is a professional antigen presenting cell (APC), processing and antigen presentation to T cells ( Antigen presenting) is an essential element in the induction phase of tumor immunity. In other words, the production of CTL with tumor-killing activity by enhancing the antigen-presenting ability of antigen-presenting cells is the most important factor in inducing tumor immunity. Therefore, the introduction of an adjuvant with an APC activating function is the intrinsic malignancy of tumor cells. It is considered the most important factor in inducing anti-tumor immunity along with the identification of tumor antigens.

As an interpretation of the results of animal experiments, the cytotoxic T lymphocyte (CTL) activity of spleen cells of immune mice was investigated when F/T vaccine and BF-I were administered in combination. F/T vaccine alone or F/T+BF-I (200 μg) was immunized twice at 1-week intervals. One week after the final immunization, live colon26-M3.1 carcinoma cells were injected, and spleens were prepared from mice or normal mice 14 days later. Splenocytes and colon26-M3.1 carcinoma cells of each group were added to a round bottom 96 well plate and cultured for 6 hours. After the completion of the culture, 10 μl of the supernatant was collected and the LDH kit solution was added according to the manufacturer's instructions to measure the degree of tumor cells killed by the splenocytes, and the absorbance was measured at 450 nm.

As a result, in the case of immunization with the F/T vaccine, the syngienic tumor (colon26-M3.1 carcinoma) was effectively killed compared to the splenocytes of normal mice and cancer control mice, and F/T vaccine and BF-I As a result of simultaneous immunization, it was confirmed that higher killing activity was induced compared to the case of F/T alone (FIG. 25). In Experimental Example <1-9-2>, BF-I showed a BRM activity to produce IL-12 from innate immune cells. IL-12 acts as an important factor in activating natural and acquired immune systems in antitumor immunity by acting mainly on the activation of NK-cells or Th1 type cells. The superiority of BF-I in inducing production of IL-12 seemed to be involved in the production of T-cells against tumors at the initiation stage of the immune response. It is a well-known fact that IL-12 is required for the development of Th1 cells or IFN-g secreting CD8+ cells, and CD8+ cells (cytotoxic T cells; CTL) that have acquired cell-killing ability produce IFN, resulting in self-toxicity and activation of the innate immune system. known to contribute to In vivo, BF-I immunized with F/T vaccine acquires the ability to kill cancer cells, including maturation of APCs induced upon immunization with vaccine immunogens and activation of Th1 cells that induce tumor-specific cellular immune responses. It was expected to be directly related to the induction of one CTL. In fact, the production of granzyme enzyme and the production of IFN, which are one of the components that cause cytotoxicity, showed the same tendency as CTL activity, so it was confirmed that BF-I has the function of enhancing CTL activity against tumor cells (FIG. 25 )..

<2-2-3> Yang-specific cytokine production pattern

In order to interpret the antitumor immune response enhancement effect (Table 5) by BF-I confirmed as a result of animal experiments, the effect of BF-I on the cellular immune response to tumor cells was investigated as a tumor-specific cytokine production function. After immunization with each vaccine, splenocytes of mice injected with live cancer cells were added to the culture medium or F/T vaccine prepared as a syngeneic tumor, cultured for 3 days, and then Th-1 (IFN-γ and Th-1 produced in the culture supernatant) The cytokine production patterns of GM-CSF) were investigated.

The forms of these antigen-specific (vaccine-specific) cytokines are classified into Th1 cytokines and Th2 cytokines according to the T-cells that produce them. The cytokines of the Th1 type are mainly IFN-γ, GM-CSF, and TNF-α, and the Th2 type includes IL-4 and IL-10. It is known that Th1 cytokines induce enhancement of cellular immunity, which mainly includes activation of CD8+ CTL cells, and Th2 type cytokines enhance humoral immunity, which is mainly involved in antibody production.

After re-stimulation with living cancer cells in each immune group, the production pattern of GM-CSF was investigated. As a result, the tumor control, F/T-vaccine, and BF-I+F/T vaccine immune groups were 76.0, 127.8, and 168.9 pg/g/T, respectively. As the production pattern of GM-CSF in ml was shown, an increase in the production amount of GM-CSF by BF-I was observed in F/T immunization (FIG. 26). It is known that GM-CSF induces maturation of DC and directly enhances antigen presenting ability, thereby activating T cells against tumors. The production activity of Il-2, which is involved in T-cell proliferation, also showed a similar trend. On the other hand, as a result of the IFN-γ production ability test, the F/T vaccine immune group produced about 1.5 times higher IFN-γ (1082.2 pg/ml) than the control group (729.9 pg/ml) in which only tumors were transplanted. seemed IFN-γ is a cytokine mainly produced by cytotoxic T cells (CTL), which have the ability to kill Th1 type T cells or tumor cells. It was strongly suggested that the F/T vaccine could directly activate CTL or induce CTL activation by cross-priming via helper T cells. In addition, the F/T+BF-I immune group showed a statistically higher IFN-γ production (1565.3 pg/ml) activity than the F/T vaccine, thereby showing the same anticancer activity as each immune group in Table 5. got the result of However, it did not have any effect on the production of representative cytokines of the Th2 type.

The result of inducing tumor immunity only by immunization with the F/T vaccine, which is an inactivated tumor cell, is that the antigen-presenting ability is improved by transforming the antigen recognition pattern of cancer cells into a form that can be easily recognized by antigen-presenting cells during the F/T vaccine manufacturing process. High (IL-12) and induces an increase in cellular immunity by the mechanism of cross primig via Th-cell for cancer cells in which MHC class-II is lost in producing CTLs against tumors (GM-CSF) In addition, it can overcome (partially) the immunosuppressive ability by cancer cells (production of IL-2), and eventually, effective tumor-specific antitumor immunity is achieved by inducing T cells (production of IFN-g) with killing activity. thought to have been induced. In conclusion, the enhancement of antitumor activity by the combined administration of BF-I during immunization with the F/T vaccine is due to two characteristics, such as the production of GM-CSF in T-cells and the induction of CTL by the F/T vaccine. It was judged to be the result due to the activation of cellular immune-competent cells.

<2-3> Evaluation of innate immune stimulation activity

<2-3-1> Cytokine production pattern by stimulation of splenocytes and macrophages

The ability of BF-I to directly stimulate spleen cells, which are immune cells, was investigated. The experimental method is as follows. The spleens were removed from BALB/c mice, dispensed in a 96 well culture plate at a concentration of 2.5×10 6 /ml, and various concentrations of BF-I were added and cultured for 72 hours. After the culture was completed, the amount of various cytokines induced and secreted in the culture supernatant was measured according to the manufacturer's instructions by purchasing an ELISA kit (Pharmingen, San Jose, CA, USA) for each cytokine. At the same time, the effect on the proliferation of splenocytes was measured using a cell counting kit (CCK-8; Dojindo Laboratories, Kumamoto, Japan).

As a result, BF-I weakly induced splenocyte proliferation in a dose-dependent manner (FIG. 27). These results indicate that BF-I induces the differentiation and proliferation of immune-related cells. At the same time, stimulation with BF-I for 3 days induced production of cytokines such as IFN-g and GM-CSF in splenocytes, but did not affect IL-4 production. These results suggest that GF-1 is involved in the differentiation and proliferation of immune-related cells.

Activated macrophages produce various cytokines, such as TNF-α and IL-12, and exert a host defense function against foreign substances including cancer by regulating the immune response. The amount of cytokines produced in the macrophage culture supernatant was investigated to see if BF-I had an activity to directly stimulate macrophages.

The experimental method is as follows. BALB/c mice were intraperitoneally injected with 1 ml of 3% thioglycollate, and 3 days later, the mice were sacrificed by cervical dislocation, and 10 ml of RPMI-1640 medium was injected into the peritoneal cavity to collect peritoneal exudative cells (PEC). did The harvested PEC was dispensed in a 24 well culture plate at a concentration of 1.5×10 6 /ml. After culturing for 2 hours to attach macrophages to the plate, washing with culture medium to remove non-adhered cells, adding various concentrations of BF-I, and culturing for 24 hours. After completion of the culture, the induced secretion of TNF-α, IL-6 and IL-12 in the culture supernatant was measured according to the manufacturer's instructions by purchasing an ELISA kit (Pharmingen, San Jose, CA, USA) for each cytokine. .

As a result, as a result of the co-culture of BF-I and macrophages, it was confirmed that cytokine production was induced in a concentration-dependent manner of added BF-I (FIG. 28). In the innate immune system, macrophages and NK cells are typical effector cells capable of killing tumor cells, and cytokines produced by macrophages contribute to the activation of effector cells with cancer cell killing functions. TNF-α is a pro-inflammatory cytokine produced by macrophages and is a typical multifunctional cytokine. It is known to play an important role in suppressing cancer by inducing immune-related cells in the inflammatory region and participating in the differentiation and proliferation of T cells. IL-6 has the function of inducing inflammation, promoting phagocytosis and complement production. IL-12 is a cytokine produced in the early stage of the immune response. It is directly involved in the production of IFN-γ and has the function of directly activating NK cells and cytotoxic T lymphocytes, so it is the most important cytokine in terms of cellular immunostimulation activation. It is recognized as one of Cain. Therefore, the cytokine production function of BF-I by stimulating macrophages is expected to directly affect the killing activity of foreign substances, including cancer cells, macrophage polarization and NK cell activation of macrophages in the innate immune system. It was judged.

The effect of BF-I on cytokine production in the co-culture of macrophages and cancer cells was investigated. Colon26-M3.1 tumor cells alone stimulated macrophages to induce the production of various cytokines such as TNF. The concentration of BF-I applied to the experiment was selected at a concentration of 0.08 mg / ml, which does not produce significant cytokines by itself. As a result, BF-I at a concentration of 0.08 ug/ml, which alone could not produce cytokines, increased the production of IL-12 and TNF compared to the culture of cancer cells alone, but had no effect on the production of IL-6 and IL-10. No (FIG. 29).

Macrophages are polarized through an activation process that differs in characteristics according to specific activation signals, and it has been reported that macrophages differentiate into tumor associated macrophages (TAMs) under the influence of cancer cells. In the classical concept, activated macrophages (M1) are characterized by increased expression of MHC class II, expression of IL-12 and TNF-α, production of reactive oxygen species and NO, and bactericidal activity. In contrast, macrophages (M2), which are activated by cytokines such as IL-4 and IL-13, have potent tumor-promoting activity. Most macrophages in the tumor microenvironment have a phenotype similar to M2, and these TAMs express vascular endothelial growth factor, arginase-1 and IL-10, and low levels of MHC class II, IL-12 and TNF. do. Therefore, it has been reported that the immune evasion mechanism of tumors is related to the conversion of macrophages to M2 phenotype instead of activation to M1 type during tumor progression. In conclusion, clinical studies report that high M2 macrophage density indicates poor prognosis in cancer patients. Therefore, in the present invention, we investigated whether BF-I treatment induces changes in the cytokine production pattern produced by TG-induced macrophages co-cultured with tumor cells. As a result, BF-1 treatment of macrophages in the presence of tumor cells increased the production of IL-12 and TNF-α but did not increase the production of IL-6 and IL-10. The reason why BF-I treatment did not inhibit the production of IL-10, a typical M2 macrophage marker, and did not affect IL-6 production by M1 macrophages was not identified. However, since at least BF-1 promotes the production of TNF-α and IL-12 in the presence of tumor cells, it was judged to have a function of partially suppressing polarization to TAM-like cells.

<2-3-2> Effect on NK-cell activation

Activated NK cells are one of the most important effector cells of the innate immune system that increase cytotoxicity to cancer cells and suppress cancer metastasis or growth. In fact, many animal studies have shown that activation of NK cells by immune stimulants inhibits cancer metastasis. Therefore, experiments were conducted on whether administration of BF-I induces NK-cell activation and enhances the killing effect on cancer cells in animal experiments.

The experimental method is as follows. Two 6-week-old BALB/c mice per group were intravenously injected with 200 μg of BF-I, and 2 days later, the spleens of the mice were sterilely harvested to prepare effector cells (E). Splenocytes obtained from mice and YAC-1 cells (target cells; T), known as NK-sensitive cells, were placed in a U-bottomed 96-well plate and adjusted so that the E/T ratios were 10:1, 5:1, and 2.5:1. and incubated for 6 hours. After the end of the culture, the culture supernatant was taken through centrifugation, and the amount of LDH that was beneficial to the killed cancer cells was measured using a kit according to the manufacturer's instructions (Promega, Fitchburg, WI. USA).

NK cells and cytotoxic T cells contain granules, which are secretory lysosomes, and contain proteins such as granzyme and perforin, which are related to the induction of cytotoxic activity. When NK-cells recognize the target cell, these toxic substances are released and produce a cytotoxic effect. NK cells activated in this process produce IFN-g to activate macrophages to fight against tumor cells. As a result of this experiment, the splenocytes (200 ug) of rats administered with BF-I significantly improved the killing effect of YAC-1 cells compared to the splenocytes of normal rats, and at the same time increased the todtkseh of INF and grazyme. showed (FIG. 30). Therefore, it was confirmed that BF-I administration of NK cells directly induces NK cell activation.

<2-3-3> Inhibitory effect of BF-I on tumor metastasis

In order to confirm the function of BF-I to activate the innate immune system against tumors, it was applied to an experimental metastasis model using colon26-M3.1 carcinonma and B16-BL6 melanoma cells in mice to test the induction function of suppressing tumor metastasis. proceeded. Female BALB/c and C57BL/6 mice aged 6 to 8 weeks were distributed from Orient Co., Ltd. and put 5 to 10 mice each in a breeding tank and were provided with purified water and pellet feed for laboratory animals (Samyang Co Ltd, Incheon, Korea) freely. It was supplied, and the temperature was 22 ℃, humidity 50%, in a state of automatic lighting at 12-hour intervals, care was taken not to be stressed. All animal experiments in this study were conducted with the approval (2018-002) of the Animal Experimentation Ethics Committee of Kyonggi University. BF-I was administered by intravascular injection (5, 50, 500 μg/mouse) once 2 days before cancer cell administration. To measure the antitumor effect, each cancer cell was inoculated, and after 14 days, the lung, the target organ of the tumor, was removed, the metastasized tumor was fixed in Bouin's solution, and the number of tumor clusters was measured.

Administration of BF-I (5 to 500 ug/mouse) before tumor inoculation suppressed metastasis to the lung by two types of tumor cells in a dose-dependent manner. When 500 μg of BF-I was administered, lung metastasis was inhibited by more than 76% in colon26-M3.1 carcinoma and by more than 77% in B16-BL6 melanoma, and tumor metastasis was significantly suppressed up to 50 μg dose. Administration of BF-I in this range did not show obvious side effects such as weight loss or piloelection (FIG. 31).

It has been well reported that the tumor metastasis inhibition effect by administration of herbal medicines in a tumor metastasis model is an effect attributable to the activation of the innate immune system such as cytotoxicity or macrophage or NK-cell. was judged to be an effect by activation of macrophages and NK-cells.

Claims (10)

Water is added to ground barley at a weight ratio of 1:3 to 6, soaked for 10 to 14 hours, and gelatinized by steaming for 1 to 3 hours, then alpha amylase is added thereto at 70 to 90 ° C for 1 to 3 hours. After preparing a first enzyme hydrolyzate by first enzyme hydrolysis, a mixture of protease, alpha-amylase, and glucoamylase is added to the first enzyme hydrolyzate, followed by second enzyme hydrolysis at 50 to 70 ° C. for 1 to 3 hours. Digesting to prepare a secondary enzyme hydrolyzate, adding beta-glucanase to the secondary enzyme hydrolyzate and performing tertiary enzyme hydrolysis at 50 to 70 ° C. for 12 to 36 hours to prepare a barley hydrolyzate;
Yeast was added to the barley hydrolysate to perform yeast fermentation at 20 to 40 ° C. for 12 to 36 hours to prepare a yeast fermentation product, and Weissella cibaria was added to the yeast fermentation product to 20 to 40 Performing lactic acid fermentation for 12 to 36 hours at ℃ to prepare fermented barley;
Preparing a fermented barley extract by centrifuging the fermented barley at 5,000 to 8,000 rpm for 20 to 40 minutes to obtain a supernatant; and
After diluting the concentrate of the fermented barley extract, removing the precipitate by centrifugation, adding ethanol to the supernatant and leaving it to stand, obtaining the precipitate by centrifugation, dissolving the precipitate in water, dialysis and drying to obtain crude polysaccharide fractionating the crude polysaccharide to obtain a polysaccharide;
The polysaccharide is barley-derived pectin containing 3,6-linked Galp, arabino-β-3,6-galactan, barley-derived arabinose branched arabinoxylan, barley-derived β-glucan, and yeast-derived Contains β-glucan, the content of arabino-β-3,6-galactan is 5 to 9% by weight, the content of arabinoxylan is 8 to 11% by weight, and the barley-derived β-glucan The content of is 30 to 39% by weight, and the content of the yeast-derived β-glucan is 4 to 7% by weight,
bronchial asthma (asthma), allergic rhinitis (allergic rhinitis), hives (urticaria), anaphylaxis (anaphylaxis), food allergy (food allergy), allergic non-hemorrhagic transfusion reaction and drug allergy selected from the group consisting of allergic diseases Method for producing a food composition for prevention or improvement.
delete delete delete delete According to claim 1,
The allergic disease is a method for producing a food composition for preventing or improving allergic diseases, characterized in that induced by one or more selected from the group consisting of pollen, drugs, vegetable fibers, bacteria, food, dyes and chemicals.
delete According to claim 1,
The method for producing a food composition for preventing or improving an allergic disease, characterized in that the food composition for preventing or improving the allergic disease increases the secretion of interleukin-6, interleukin-12 or TNF-alpha.
Water is added to ground barley at a weight ratio of 1:3 to 6, soaked for 10 to 14 hours, and gelatinized by steaming for 1 to 3 hours, then alpha amylase is added thereto at 70 to 90 ° C for 1 to 3 hours. After preparing a first enzyme hydrolyzate by first enzyme hydrolysis, a mixture of protease, alpha-amylase, and glucoamylase is added to the first enzyme hydrolyzate, followed by second enzyme hydrolysis at 50 to 70 ° C. for 1 to 3 hours. Digesting to prepare a secondary enzyme hydrolyzate, adding beta-glucanase to the secondary enzyme hydrolyzate and performing tertiary enzyme hydrolysis at 50 to 70 ° C. for 12 to 36 hours to prepare a barley hydrolyzate;
Yeast was added to the barley hydrolysate to perform yeast fermentation at 20 to 40 ° C. for 12 to 36 hours to prepare a yeast fermentation product, and Weissella cibaria was added to the yeast fermentation product to 20 to 40 Performing lactic acid fermentation for 12 to 36 hours at ℃ to prepare fermented barley;
Preparing a fermented barley extract by centrifuging the fermented barley at 5,000 to 8,000 rpm for 20 to 40 minutes to obtain a supernatant; and
After diluting the concentrate of the fermented barley extract, removing the precipitate by centrifugation, adding ethanol to the supernatant and leaving it to stand, obtaining the precipitate by centrifugation, dissolving the precipitate in water, dialysis and drying to obtain crude polysaccharide fractionating the crude polysaccharide to obtain a polysaccharide;
The polysaccharide is barley-derived pectin containing 3,6-linked Galp, arabino-β-3,6-galactan, barley-derived arabinose branched arabinoxylan, barley-derived β-glucan, and yeast-derived Contains β-glucan, the content of arabino-β-3,6-galactan is 5 to 9% by weight, the content of arabinoxylan is 8 to 11% by weight, and the barley-derived β-glucan The content of is 30 to 39% by weight, and the content of the yeast-derived β-glucan is 4 to 7% by weight,
bronchial asthma (asthma), allergic rhinitis (allergic rhinitis), hives (urticaria), anaphylaxis (anaphylaxis), food allergy (food allergy), allergic non-hemorrhagic transfusion reaction and drug allergy selected from the group consisting of allergic diseases Method for producing a pharmaceutical composition for prevention or treatment.
Water is added to ground barley at a weight ratio of 1:3 to 6, soaked for 10 to 14 hours, and gelatinized by steaming for 1 to 3 hours, then alpha amylase is added thereto at 70 to 90 ° C for 1 to 3 hours. After preparing a first enzyme hydrolyzate by first enzyme hydrolysis, a mixture of protease, alpha-amylase, and glucoamylase is added to the first enzyme hydrolyzate, followed by second enzyme hydrolysis at 50 to 70 ° C. for 1 to 3 hours. Digesting to prepare a secondary enzyme hydrolyzate, adding beta-glucanase to the secondary enzyme hydrolyzate and performing tertiary enzyme hydrolysis at 50 to 70 ° C. for 12 to 36 hours to prepare a barley hydrolyzate;
Yeast was added to the barley hydrolysate to perform yeast fermentation at 20 to 40 ° C. for 12 to 36 hours to prepare a yeast fermentation product, and Weissella cibaria was added to the yeast fermentation product to 20 to 40 Performing lactic acid fermentation for 12 to 36 hours at ℃ to prepare fermented barley;
Preparing a fermented barley extract by centrifuging the fermented barley at 5,000 to 8,000 rpm for 20 to 40 minutes to obtain a supernatant; and
After diluting the concentrate of the fermented barley extract, removing the precipitate by centrifugation, adding ethanol to the supernatant and leaving it to stand, obtaining the precipitate by centrifugation, dissolving the precipitate in water, dialysis and drying to obtain crude polysaccharide fractionating the crude polysaccharide to obtain a polysaccharide;
The polysaccharide is barley-derived pectin containing 3,6-linked Galp, arabino-β-3,6-galactan, barley-derived arabinose branched arabinoxylan, barley-derived β-glucan, and yeast-derived Contains β-glucan, the content of arabino-β-3,6-galactan is 5 to 9% by weight, the content of arabinoxylan is 8 to 11% by weight, and the barley-derived β-glucan The content of is 30 to 39% by weight, and the content of the yeast-derived β-glucan is 4 to 7% by weight,
bronchial asthma (asthma), allergic rhinitis (allergic rhinitis), hives (urticaria), anaphylaxis (anaphylaxis), food allergy (food allergy), allergic non-hemorrhagic transfusion reaction and drug allergy selected from the group consisting of allergic diseases Method for producing a feed composition for prevention or improvement.

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