KR101493895B1 - Polysaccharide extracted from above ground part of Astragalus membranaceus and uses thereof - Google Patents

Abstract

The present invention relates to: an arabinogalactan composite polysaccharide, wherein a galactose structure isolated from the aerial part of Astragalus membranaceus and a sugar chain structure of 3,6-branched galactose alternately exist, and the galactose is connected with plenty of monosaccharides; an immune adjuvant pharmaceutical composition or health functional food containing the polysaccharide or an Astragalus membranaceus aerial part polysaccharide fragment including the polysaccharide as an active ingredient; an oral or mucous administrated vaccine including the immune adjuvant pharmaceutical composition; a vaccine supplementary agent containing the polysaccharide or the Astragalus membranaceus aerial part polysaccharide fragment including the polysaccharide as an active ingredient; and a method for isolating polysaccharide having an immune adjuvant activity. A galactose structure and a sugar chain structure of 3,6-branched galactose alternately exist in an aerial part of the Astragalus membranaceus, and a polysaccharide fragment including an arabinogalactan composite polysaccharide, in which the galactose is connected with plenty of monosaccharides, is included, so that an activity as a strong immune adjuvant agent is exhibited and a low cythemolysis activity (0.66%) is exhibited. Accordingly, side effects are rare, and a long remaining period of two month is ensured. Moreover, a very stable shape is ensured, so that the same is expected to be utilized as an assisting agent exhibiting an immunity reinforcing effect.

Description

Polysaccharide Extracted from the Above Ground Part and Its Use (Polysaccharide extracted from above ground part of Astragalus membranaceus and uses thereof)

More particularly, the present invention relates to a polysaccharide extracted from a hull and a galactose structure separated from a hull land portion and a sugar chain structure of a 3,6-branched galactose alternately, and a galactose- A pharmaceutical composition for immune adjuvant comprising an arabinogalactan complex polysaccharide to which a saccharide body is connected, a polysaccharide or a polysaccharide fraction containing the above-mentioned hyperglycosylated polysaccharide as an active ingredient, and a health functional food, An oral or mucosal administration vaccine comprising the composition, a vaccine adjuvant containing the polysaccharide or the polysaccharide as the active ingredient, and a polysaccharide separation method having the immune adjuvant activity.

In the latter half of the 20th century, the fact that chronic diseases such as cancer, circulatory system diseases and autoimmune diseases, which are difficult to treat with modern medicines, are on the increase, and in particular the surge of constitutional diseases or psychosomatic diseases, And the serious side effects of the plant have been attracting much attention to the pharmacologically active substances derived from the plant. These studies have concentrated on the immune system activating action such as anticancer action, and in particular, attempts have been made to select biological response modifiers from foods or traditional herbal medicines and use them as medicines or functional foods. Recently, the government has been conducting vaccination since the spread of the foot-and-mouth disease beyond the network of the disinfestation system. However, controversy over the efficacy and safety of the vaccine continues, and in the case of cows and pigs that do not develop antibodies after vaccination, In this case, the cattle and pigs may act as an infection pathway for spreading viruses to other livestock or cubs (such as cattle ruminants, if the virus is latent in the process of antibody formation after vaccination, It may be possible to act as a virus infectious agent for about three years without the vaccine.) In vaccination, it is necessary to develop an adjuvant that can improve the immunity of the individual itself. Infectious diseases such as foot-and-mouth disease are extremely catastrophic to humans and animals, and many infectious diseases can be fatal everywhere. Vaccination is the most effective and valuable technology for infectious diseases and can be applied as a preventive measure against pathogen exposure.

Vaccine adjuvant refers to a substance that, when coadministered with a vaccine, enhances, prolongs, or accelerates the antigen-specific immune response induced by the vaccine antigen It is called. Vaccine adjuvants approved for human use to date include 'Alum (aluminum phosphate and aluminum hydroxide)' and 'squalene emulsion'. Vaccine adjuvants must meet one or more of the following five criteria: 1) to control expression of co-stimulatory molecules on the surface of antigen presenting cells, induce antigen-specific T lymphocyte responses, 2) antigen presentation, 3) CD8 ⁺ T lymphocyte reaction, 4) targeting, and 5) antigen-depot generation. In addition, the ideal vaccine adjuvant should be 1) safe, 2) be biodegradable in the body, 3) exhibit higher defense immunity or therapeutic immunity when administered alone, 4) be chemically or biologically identifiable , 5) it is better to act at a lower concentration than the antigen, and 6) the half-life should be long enough for commercial or clinical application. Currently, vaccine adjuvants are being used or are being considered for use: 1) mineral salts such as aluminum hydroxide gel, 2) surfactants, 3) bacterial-derived substances, 4) cytokines or hormones, 6) a polyanion, 7) a polyacryl, 8) a carrier, 9) a living vector using virus, 10) a vehicle such as mineral oil or liposome, ). Currently, active research is being conducted from Vibrio cholera greatly and are getting a vaccine-derived protein supplements attention (Vibrio cholera toxin (CT) derived from cholerae and heat-labile toxin (LT) derived from E. coli. Such cytokines or hormones are currently commercially available for autologous tumor vaccines or allogeneic tumor vaccines. In particular, products such as GVAX have been shown to enhance the efficacy of cell-based vaccines by expressing granulocyte-macrophage colony-stimulating factor (GM-CSF). However, the use of only granulocyte macrophage colony stimulating factor (GM-CSF) has been shown to emphasize the preventive function, and it has been reported that the ability to fight against micro cancer cells that may remain after cancer removal surgery is reduced. These vaccine adjuvants induce the production of antigen-specific antibodies in the mucosal area and serum and induce B7-2 expression on the surface of antigen presenting cells (APC) It has been reported that CD4 ⁺ cells are stimulated by co-stimulatory signaling. However, since they are exotoxins having a high enterotoxicity, it is strongly desired to develop a safe and effective vaccine adjuvant which reduces toxicity and increases adjuvativity.

Astragalus membranaceus has traditionally been shown to improve not only the various diseases but also to promote health, which is highly utilized as an alternative medicine. Hwanggi is recommended as a medicinal material for general health promotion and its main use root contains various compounds classified as flavonoid, saponin, and polysaccharide, among which astragaloside ), Formononetin, calycosin, etc. have been reported as typical active substances.

In the present cultivation area, the top part of Hwanggi is fed by using a self-feed preparation device through fermentation. However, its utilization is very low and most of it is abandoned as a residue at harvest time. , And the specific beneficial components are higher than those of the underground parts. Therefore, it is supposed that the surface part of the eruptive eruption has the same disease improving effect as the underground part. However, the food and health It is not registered as a functional food raw material and its utilization can not be increased.

The polysaccharide in the undergrowth of Hwanggi has activity of macrophage, activation of T cell, activation of NK cell, increase production of interferon, increase phagocytosis activity, and treatment of Hwanggi saponin as an immunosuppressant for vaccine treatment of Newcastle disease which is a rapid infectious disease of poultry It has been reported that lymphocyte proliferation and antibody production in serum are increased. Specific polysaccharide in the epigallocatech is similar to the polysaccharide in the undergrowth, and can enhance the intestinal immune activity and inhibit immune degradation, thereby acting as a vaccine adjuvant .

Korean Patent No. 10-1173042 discloses a 'recombinant cytokine fusion protein and a vaccine adjuvant for preventing or treating tumors using the same as an active ingredient.' Korean Patent Laid-Open Publication No. 2013-0056273 discloses a trisaccharide derivative and its adjuvant Has been disclosed, but the polysaccharide extracted from the shade-ground portion of the present invention as well as its use has not been disclosed.

The present invention has been made in view of the above-described needs. The present inventors prepared a polysaccharide fraction from the upper part of hwanggi to investigate a utilization method for the upper part of the hwanggi soil, By identifying the structure of the active ingredient in the polyunsaturated fatty acid fraction, the polysaccharide, which is an active ingredient showing immunity enhancement and vaccine assistant effect in the hyaluronic acid, is present alternately in a galactose structure and a sugar chain structure of 3,6-branched galactose, Which is a complex arabinogalactan polysaccharide having a sugar body connected thereto. The aeruginosa polysaccharide fraction containing arabinogalactan complex polysaccharide showed similar intestinal immune activity, suppression of immune decline, and low hemolytic activity and side effects, similar to the polysaccharide of the undergrowth. When the erythrocyte polysaccharide fraction was treated with a typhoid vaccine, proliferation of macrophages and splenocytes following vaccine adjuvant treatment, increase of production of TNF-α and nitric oxide, secreted by macrophages, Gamma increase and immunoglobulin G production were improved. From these results, the present invention has been accomplished by confirming the vaccine-assisting activity of the fragrance-like polysaccharide fragrant part including arabinogalactan complex polysaccharide.

In order to solve the above-described problems, the present invention provides a method for producing a galactose-containing galactose having a galactose structure of Formula 1 and a sugar chain structure of 3,6-branched galactose separated from the hully root portion and having a galactose- Arabinogalactan complex polysaccharide.

The present invention also provides a pharmaceutical composition for immune adjuvant and a health functional food containing the polysaccharide or the polysaccharide-containing polysaccharide fraction as an active ingredient.

The present invention also provides a vaccine comprising the above immunocompromising pharmaceutical composition.

In addition, the present invention provides a vaccine adjuvant comprising the polysaccharide or the polysaccharide fraction comprising the polysaccharide as an active ingredient.

The present invention also provides a method for separating polysaccharides having immune adjuvant activity.

One of the active substances exhibiting immunostimulating activity in Hwanggi is a heteropolysaccharide polysaccharide including glucan, which is a monosaccharide in the underground part, while it is confirmed that an immunoglobulin effective polysaccharide is an arabinogalactan complex polysaccharide did. As a result of assaying the bone marrow cell proliferative capacity mediated by phycoplasma, arabinogalactan complex polysaccharide of Shijiazhuang was 2.5 times higher than that of the active ingredient polysaccharide in the ground part. In addition, it exhibits low hemolytic activity (0.66%), exhibits a low side effect, exhibits a long stay period of 2 months, and exhibits a highly stable form. Thus, it is expected that it can be used safely as a vaccine adjuvant showing immunity enhancement effect.

Figure 1 is an enzymatic hydrolysis and purification process of the intestinal mucosal immunosuppressive polysaccharide of AMA-1-b-PS2, a superficial polysaccharide of Astragalus membranaceus .
Fig. 2 shows the proposed structure of AMA-1b-PS2, wherein the polysaccharide has a galactose structure and a sugar chain structure of 3,6-branched galactose alternately and has a galactose-rich monosaccharide Galactan complex polysaccharide.
Figure 3 shows the results of the improvement of the production of bone marrow cell proliferation cytokines from Peyer's patch cells cultured in the presence of the untreated polysaccharide fraction obtained from the aerial part of Akihabara (AMA, AMMA) and the ground part (AMR, AMMR) . The enhanced activity was measured at a concentration of 100 占 퐂 / ml (black bars) and 50 占 퐂 / ml (white bars) of the polysaccharide fraction in the raw state. The gray bars represent control cultures in the absence of the polysaccharide fraction.
Fig. 4 shows the effect on the proliferation of splenocytes of the hyperkeratotic polysaccharide fraction (AMAO) in ICR mice immunized with OVA. 100 μg of OVA alone or 100 μg of OVA in saline containing Alum (200 μg), QuilA (10 and 20 μg) or AMA (50, 100 or 200 μg) were immunized on ICR mice at day 1 and 15 Respectively. Splenocytes were prepared two weeks after the last immunization and cultured for 72 hours in ConA, LPS, OVA or RPMI 1640. Splenocyte proliferation was measured by the MTT method and expressed as SI (stimulation index) as described in the method. Values for these were expressed as mean ± standard error (number of samples = 5). Significant differences from OVA group were * P <0.05, ** P <0.01 and *** P <0.001.
FIG. 5 shows the effect of IgG, IgG1, and IgG2b antibodies on the expression of the hyperglycosylated polysaccharide fraction (AMAO) in ICR mice immunized with OVA. 100 μg of OVA alone or 100 μg of OVA was dissolved in saline containing Alum (200 μg), QuilA (10 μg and 20 μg) or AMA (50 μg, 100 μg or 200 μg) Immunization was induced in 5 male ICR mice. Serum was collected two weeks after the last immunization. In the serum, OVA specific IgG, IgG1 and IgG2b antibodies were measured by indirect ELISA as described in the method. Values for these were expressed as mean ± standard error (number of samples = 5). Significant differences from OVA group were * P <0.05, ** P <0.01 and *** P <0.001.
FIG. 6 shows the results of observation of intestinal tissue changes in the SD rats (Sprague-Dawley rats) induced by haemorrhagic shock induced by treatment with erythrocyte polysaccharide. (A) Normal group (intestinal tissue of normal animals), B) Model group (intestinal tissue of hemorrhagic shock induction animal), C) Low dose group (inducing hemorrhagic shock, intraperitoneal injection of 10 mg / High dose group (inducing hemorrhagic shock, injecting 20 mg / kg intraperitoneal injection of erbotropic topical polysaccharide)
FIG. 7 shows the results of the effect of the aerobic polysaccharide-assisted treatment on the purified Vi capsular polysaccharide of Salmonella typhi on the RAW 264.7 cell proliferation rate.
FIG. 8 shows the results of the effect of erythrocyte polysaccharide-assisted treatment on the purified Vi capsular polysaccharide of Salmonella typhi on NO production rate in RAW 264.7 cells.
FIG. 9 shows the effect of erythrocyte polysaccharide-assisted treatment on purified Vi capsular polysaccharide of Salmonella typhi on TNF-a secretion in RAW 264.7 cells.
10 is a graph showing the effect of the aeruginosa polysaccharide-assisted treatment on the purified Vi capsular polysaccharide of Salmonella typhi on the splenocyte proliferation rate.
FIG. 11 shows the results of the effect of the aerial polysaccharide-assisted treatment on the purified Vi capsular polysaccharide of Salmonella typhi on body weight change and spleen enlargement in experimental animals.
12 shows the results of the effect of the emetic adjuvant treatment on the purified Vi capsular polysaccharide of Salmonella typhi on interferon gamma production in experimental animals.
FIG. 13 shows the results of the effect of the aerial polysaccharide-assisted treatment on the purified Vi capsular polysaccharide of Salmonella typhi on interleukin-2 production in experimental animals.
FIG. 14 shows the results of the effect on the immunoglobulin G production in laboratory animals of the vaccine-containing polysaccharide-assisted treatment of the purified Vi capsular polysaccharide of Salmonella typhi.

In order to accomplish the object of the present invention, the present invention provides a galactose structure of formula 1 and a 3,6-branched galactose sugar chain separated from the hully root part alternately and having a galactose-rich monosaccharide To provide an arabinogalactan complex polysaccharide.

Wherein Ara represents arabinose, Gal represents galactose, and f represents forward.

In the above formula, the value of n is not clear in each fraction and the connection structure is also different, but polysaccharide and polysaccharide compound have a common link structure showing regeneration of vital defense function and expression of immunity enhancement. In the case of polysaccharides, there is a connection flow and in the arrangement of one polysaccharide it means that the first one to come is arabinose.

In addition, the present invention relates to an arabinogalactan complex polysaccharide in which a galactose structure and a sugar chain structure of 3,6-branched galactose are alternately present and a galactose-rich monosaccharide is connected, or a polysaccharide fraction containing a polysaccharide- A pharmaceutical composition for an immune adjuvant containing the active ingredient and a health functional food.

The pharmaceutical composition having a bio-defense function or immunostimulating activity of the present invention may comprise 0.02 to 50% by weight of the arabinogalactan complex polysaccharide or the macroporous superfine polysaccharide fraction containing the arabinogalactan complex polysaccharide based on the total weight of the composition, The content of the galactan complex polysaccharide or the emulsifier-containing polysaccharide fraction containing the same is not limited to the above values, and it is more preferable to appropriately adjust the content according to the method of use and the formulation.

The pharmaceutical composition comprising the arabinogalactan complex polysaccharide of the present invention or the hyperglycosylated polysaccharide fraction containing the same may further comprise an appropriate carrier, excipient and diluent commonly used in the production of a pharmaceutical composition.

The pharmaceutical dosage forms of the arabinogalactan complex polysaccharide of the present invention or the herbaceous topical polysaccharide fraction containing the same may be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmacologically active compounds It can be used as an appropriate set.

The pharmaceutical composition comprising the arabinogalactan complex polysaccharide according to the present invention or the pharmaceutical composition comprising the epidermal polysaccharide fraction containing the same may be administered orally or rectally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, The composition can be formulated in the form of a tablet, an external preparation, a suppository, and a sterile injection solution, but is not limited thereto, and can be appropriately adjusted according to the use method and use. Examples of carriers, excipients and diluents that can be contained in the composition comprising arabinogalactan complex polysaccharide or the emulsifiable starch polysaccharide fraction containing the same include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethylcellulose, But is not limited thereto. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include, but are not limited to, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like, but the present invention is not limited thereto. Examples of suppositories include, but are not limited to, witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

 The preferred dosage of the arabinogalactan complex polysaccharide of the present invention or the emeralgia topical polysaccharide fraction comprising the arabinogalactan complex polysaccharide of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period of time, . However, for the desired effect, the arabinogalactan complex polysaccharide of the present invention or the herbaceous topical polysaccharide fraction containing the same is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The arabinogalactan complex polysaccharide of the present invention or the hyperglycosylated polysaccharide fraction containing the same may be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food comprising the arabinogalactan complex polysaccharide exhibiting a bioprotective function or an immunostimulating activity effect or a fragrant topical polysaccharide fraction containing the same and a pharmaceutically acceptable food supplementary additive. Examples of foods to which the arabinogalactan complex polysaccharide or the herbaceous topical polysaccharide fraction containing the same are added include various foods, beverages, Do not.

In addition, it can be added to food or beverage for the purpose of bio-defense function or immune enhancement activity effect. At this time, the amount of arabinogalactan complex polysaccharide in the food or drink or the amount of the emulsifier fraction containing the emulsifier may be 0.01 to 15% by weight of the total food, and the health beverage composition may contain 0.02 to 5 g, Preferably 0.3 to 1 g, and it is preferable to adjust them appropriately according to the type of food and the method of use.

The health functional beverage composition of the present invention is not particularly limited as long as it contains the arabinogalactan complex polysaccharide or an emulsifiable terpolymer polysaccharide fraction containing the arabinogalactan polysaccharide fraction as an essential ingredient in the indicated ratios, Or natural carbohydrate as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those described above, natural flavoring agents (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, arabinogalactan complex polysaccharide of the present invention or a hyperglycosylated polysaccharide fraction containing the same may be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, Chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the arabinogalactan complex polysaccharide of the present invention or an embrasic topical polysaccharide fraction containing the arabinogalactan compound may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of these additives is not so important, it is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the arabinogalactan complex polysaccharide of the present invention or the emulsifier component of the present invention.

The present invention also provides an oral or mucosal administration vaccine comprising the above immunocompromising pharmaceutical composition.

The present invention also relates to an arabinogalactan complex polysaccharide having a galactose structure and a sugar chain structure of 3,6-branched galactose alternately and having a galactose-rich monosaccharide linked thereto, or a polysaccharide fraction containing the polysaccharide As an active ingredient.

Vaccine adjuvant refers to a substance that, when coadministered with a vaccine, enhances, prolongs, or accelerates the antigen-specific immune response induced by the vaccine antigen It is called. Vaccine adjuvants approved for human use to date include 'Alum (aluminum phosphate and aluminum hydroxide)' and 'squalene emulsion'.

The present invention also relates to

a) extracting the hulls with water to obtain crude extract;

b) filtering and vacuum-concentrating the crude extract and adding ethanol to induce precipitation; And

c) lyophilizing the supernatant obtained by centrifuging the precipitate to obtain a hypergrociferous polysaccharide fraction; And
d) separating arabinogalactan complex polysaccharide in which the galactose structure and the 3,6-branched galactose sugar chain structure are alternately present in the obtained erythrocyte polysaccharide fraction and the galactose-rich monosaccharide is connected thereto A galactose structure having immune adjuvant activity and an arabinogalactan complex polysaccharide in which a sugar chain structure of 3,6-branched galactose is alternately present and a galactose-rich monosaccharide is connected, Separation method.

In one embodiment of the present invention, the hull is characterized by being a hull land portion.

To obtain the extract of the present invention, methods such as hot water extraction, room temperature extraction, warming extraction, and ultrasonic extraction, which are conventionally used, may be used, but the present invention is not limited thereto.

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

Materials and Methods

1. Structural analysis and activity test of polyunsaturated fatty acid

A-1-b-PS2 (see Example 1 of Korean Patent No. 0994444) showing the activity of intestinal immunity via a phy's plate was subjected to exo-β-D- (1 → 3) -galactanase (AFase, exo-β-D- (1 → 3) -galactannase and exo-α-L-arabinofuranosidase (GNase, exo-α-L-arabinofuranosidase) was stepwise enzymatically treated. AMA-1-b-PS2 (17 mg) was dissolved in 4 ml of 50 mM acetate buffer (pH 4.5) and AFase (0.01 U) was hydrolyzed at 40 ° C for 7 hours. The hydrolyzate was eluted with a column of bio-gel P-30 (2 x 50 cm) which had been equilibrated with 50 mM acetate buffer (pH 5.2) and the fraction eluted from the column (AH-AMA-1 -b-PS2, 10 mg). The AH-AMA-1-b-PS2 fraction was dialyzed again, lyophilized and desalted, and then re-dissolved in 5 ml of acetate buffer (pH 4.5), treated with GNase (0.1 U) and hydrolyzed at 37 ° C for 48 hours Respectively. The fraction eluted with the column of bio-gel P-30 (1.5 x 90 cm), in which the hydrolyzate was equilibrated with 50 mM acetate buffer (pH 5.2), fraction eluted with AH-1 (7.5 mg) AH-2 having a molecular weight and AH-3 having a small molecular weight were obtained. AAH-1 (7.5 mg) was further treated with AFase to obtain 2AH-1. GNAH was treated and fractionated with bio-gel P-6 to obtain 2AH-1-A (5 mg). AH-2 and AH-3 were assumed to be monosaccharides and were excluded from hydrolysis. Methylation of the prepared fractions was performed for the structural analysis of the superficial immunoglobulin (EGP) of the epidermis, and the subcomponent method (J. Biochm. 1964; 55: 205 ~ 208) was modified and the methylated product was analyzed by Waeghe et al. Carbohydrate Research 1983; 123: 281-304) using a Sep-pak C18 cartridge. Each polysaccharide was deesterified by treatment with 0.2 M NaOH at room temperature for 2 hours prior to methylation and methylation was performed only once to avoid β-elimination during the reaction. However, when a methylsufinyl carbanion is required (identified by triphenylmethane) several times so that the polysaccharide is completely converted to the polyalkoxide, it is added.

To confirm the binding structure of the uronic acid moiety in the polysaccharide sample, 1 M lithium tetrahydrodeutride dissolved in THF was used to label the C6 position with d2 to facilitate mass spectrometry analysis. The methylated product was converted to partially methylated alditol acetates by the addition of 2M TFA and hydrolysis at 121 ° C for 1.5 hours. Partially methylated alditol acetates were analyzed using GLC and GLC-EIMS. GLC measurements were performed using Hewlett-Packard model 5890 gas chromatography using SP-2380 capillary columns and analyzed using the method by Zhao et al. (Cabohydrate Reserch. 1991; 219: 149-172) and methylated alditol acetates Were analyzed by using fragment ion data obtained by GLC-EIMS (mass spectrometer, Hewlett-Packard 5970B, USA) and relative retention time of GLC. The molar percentages of these fractions were determined by the peak width and the response coefficient . The methylated monosaccharide was analyzed by FAB-Mass (JEOL JMS 505 HA mass spectrometer) and xenon was used as a bambarding gas. The atomosphere was adjusted to 3 kV, 10 mA. Scanning was performed at 20 s per decade at m / z 0-1500, accelerating voltage was 3 kV, and a 1: 1 mixture of glycerol-mono-thiglycerol was used as the matrix 1 μl of a methylated monosaccharide methanol solution was placed in the probe and 1 μl of the matrix was mixed. The connecting sugar chain of the daughter ion had a scanning speed of 120 / s and helium was used as a collision gas.

2. Differentiating polysaccharide from the ground polysaccharide in the Hwanggi area

2-1. Confirmation of sugar composition (ground active polysaccharide and underground active polysaccharide)

In order to ensure the differentiation between the intestinal immunoglobulin and the active polysaccharide in the lower part of the stem, 20 times volume of distilled water was added to 1 kg of the upper part and the lower part of the stem, and after the pulverization and stirring, And heated. The filtrate was collected by suction filtration, and the filtrate was concentrated. The filtrate was concentrated under reduced pressure, and the precipitate was precipitated by adding 5 times of concentrated ethanol. After centrifugation at 7,000 rpm for 30 minutes at 4 ° C, And separated into supernatant and residue. The supernatant was again lyophilized to obtain a hot-water extract fraction (top portion: AMA-0, 19.42 g, yield: 1.942%, underground portion: 25.32 g, yield: 2.532%).

Using the above-ground Astragalus the case with respect to the sample neutral sugars after hydrolysis of the sample for 1.5 h at 121 ℃ by 2.0M TFA (trifluoroacetic acid) by the method of Jones and Albersheim NaBH ₄ obtained in order to check the configuration of the underground part per polysaccharide The neutral sugar was reduced to alditol and converted to an alditol acetate derivative using (CH ₃ CO) ₂ O. The SP-2380 column (0.2 μm film, 0.25 mm id × 30 m, Supelco, USA) The constituent sugars were analyzed by gas-liquid chromatography (HP-6890 II GLC, Hewlett-Packard, USA). In the case of acidic saccharides, GLC was carried out by conversion to alditol acetate derivatives under similar conditions to neutral sugars using NaBD ₄ after hydrolysis with 0.1 M TFA at 100 ° C. for 1 hour. The temperature condition of the GLC for the constitutional analysis was 250 ° C. for the injector and 250 ° C. for the detector. After 1 minute at 60 ° C., the column was raised to 220 ° C. at a rate of 30 ° C./minute and maintained for 12 minutes And the temperature was raised to 250 ° C. at a rate of 8 ° C./minute to maintain the temperature for 15 minutes. The constituent sugar in the sample was analyzed by comparing with the storage time of the standard saccharide. The molar percentage of the constituent sugar was determined by the area ratio of each peak, Calculated from the molecular weight of the alditol acetate derivative and expressed as molar ratio.

2-2. Comparison of the production ability of bone marrow cell proliferation factor mediated by phyeric plate

In order to differentiate the active polysaccharide from the intestinal immunoglobulin and the active polysaccharide from the underground part, the effect of promoting the proliferation of the intestinal Pai lepan lymphocytes was investigated. C3H / He female mice, 5 weeks of age and in good condition, were purchased from Korea Experimental Animal Center, Chungbuk, Korea. The environment was maintained under constant temperature and humidity conditions (temperature 22 ± 0.5 ℃, relative humidity 55 ± 5%) and artificial light for 1 day and 1 day in artificial light. The animals were fed diets for experimental animals (Samyang Feed, Incheon) And 12 hours (from 9:00 am to 9:00 pm). The proliferation promoting effect on lymphocytes was measured according to the method of Hong et al. (Phytomdecine. 1998; 5 (5): 353-360). That is, carefully remove the phycoplane present on the small barrier of the C3H / He mouse and transfer it to a Petri dish containing HBSS (Hank's balanced salt solution, Sigma, USA), and disrupt the tissue to release the cells from the physis plate. The cell suspension was filtered through a metal sieve (No. 200), washed with RPMI 1640-FBS (RPMI-1640 containing 5% FBS) and adjusted to a concentration of 2 × 10 ⁶ cells / ml RPMI 1640- - Dissolve 180 μl each in the well plate, dilute the sample to measure the activity to an appropriate concentration, add 20 μl each, incubate in a 5% CO ₂ incubator at 37 ° C for 5 days, recover the supernatant, Respectively. Bone marrow cells were recovered using femoral bones of the same species mouse. That is, HBSS was injected into the bone using a syringe, and the bone marrow cells were collected in a test tube. After filtration and washing as described above, 2.5 x 10 ⁵ cells / Adjust the cell concentration with 1640-FBS and dispense 100 μl into a 96-well plate. Next, 50 μl of the supernatant for measuring the proliferation activity of bone marrow cells obtained above and 50 μl of RPMI 1640-FBS are added, and the mixture is cultured in a 5% CO ₂ incubator at 37 ° C. for 6 days. Direct intestinal immune activity is expressed as the degree of proliferation of bone marrow cells by reacting the supernatant recovered after the reaction between the sample and the Peyer's patch cells with the bone marrow cells. The degree of proliferation is measured by the fluorescence reagent of Alamar Blue ^TM , And a measurement method in which light is reduced by emitted electrons is used. Namely, 20 μl of Alamar Blue ^™ was added 12 hours before the culture of the bone marrow cells and the culture supernatant obtained above, and then the fluorescence was emitted at excitation 544 nm using SpectroFluor Plus (Tecan, Durham, USA) 590 nm, and the proliferation degree of bone marrow cells was compared with that of the control using physiological saline instead of the sample.

3. Gastrointestinal Immunity Assay for Vaccine Supplementation of Polyunsaturated Fraction of Above-Ground

3-1. Preparation of Polysaccharide Fraction from Above Ground

To 1 kg of the shiitake (except the root), 20 times volume of distilled water was added, followed by pulverization and stirring, and then heated to half the amount of distilled water added for the first time. The filtrate was collected by suction filtration, and the filtrate was concentrated under reduced pressure. The filtrate was concentrated by adding 5 times of concentrated ethanol and precipitated. The residue was centrifuged at 7,000 rpm for 30 minutes at 4 ° C Centrifuged and separated into supernatant and residue. The supernatant was again lyophilized to obtain a hot-water extract fraction (AMA-0, 19.42 g, yield: 1.942%).

3-2. Measurement of Hemolytic Activity of Polysaccharide Fraction of Abalone

Blood samples were collected from healthy New Zealand rabbits (Korea BioLink, Korea) using BD Vacutainer ^™ (NH 143 IU, Belliver Industrial Estate, UK). The cell suspension pellet was prepared so that 7 ml of blood was washed with saline (0.89% w / v NaCl, pyrogen free) and centrifuged (180 × g, 5 minutes) to obtain a saline solution of 0.5%, and 0.5 ml of cell suspension pellet The reaction was continued for 30 minutes at 37 ° C, followed by centrifugation (70 × g, 10 minutes), and the supernatant And the amount of free hemoglobin in the supernatant was assayed using a spectrophotometer (412 nm). Controlled saline was used as the minimal hemolytic control and sterile water was used as the maximum hemolytic control to calculate the hemolytic rate of each treatment to the saline control.

3-3. Induction of immune response and immunosuppressive effects of polysaccharide fraction

Immunoreactivity was induced by dividing 6 - week - old ICR mice into 8 groups and each group consisted of 5 mice. Each group contained 100 μg / ml of OVA (ovalumin) alone, 100 μg / ml of OVA + Aluminum hydroxide gel, 200 μg of Aluminum hydroxide gel, 100 μg / ml of OVA + QuilA (10, (100, 200, ug / ml) of OVA 100 μg / ml + hyperkeratotic polysaccharide solution to induce an immune response two times on the 1st and 15th days of the experiment. Saline alone was used as a control. Immunoreactivity was induced two weeks after each treatment, and serum and spleen cell proliferation and erythropoietin - specific antibody were measured two weeks later. Spleen cell proliferation was measured in immunized mice in each treatment group by adding HBSS (Hank's balanced salt solution, Sigma, USA) under aseptic condition to the spleen of immunized mice and passing through a steel mesh to obtain homogenized The cell suspension was obtained and treated with ammonium chloride (0.8%, w / v) to decompose the erythrocyte. The resulting cell suspension was centrifuged (380 x g, 4 캜, 10 min), and the pellet formed was washed three times with PBS and washed twice with complete medium [RPMI 1640 medium containing 12 mM HEPES (pH 7.1), 0.05 mM 2-mercaptoethanol, / Ml penicillin, 100 g / ml streptomycin and 10% newborn rats serum (FCS)]. Cell counts were measured in the hemocytometer using the trypan blue dye exclusion method and in the spleen cell proliferation assay, spleen cells were initially cultured in 4-5 wells of a 96-well flat microtiter plate and 100 Lt; ⁶ &gt; cells / ml in the complete medium. LPS (final concentration 10 占 퐂 / ml) and OVA (final concentration 10 占 퐂 / ml) were added to the medium to a final volume of 200 占 퐇 in ConA (final concentration 5 占 퐂 / ml). The plate was further cultured at 37 ° C under 5% CO _2. After 68 hours, 50 μl of MTT solution (2 mg / ml) was added to each well and treated again with 200 μl of DMSO working solution DMSO with 8 μl 1N HCl) was added and the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay reader. The SI was calculated using the absorbance obtained (SI = absorbance of the mitotic material culture / Absorbance of non-stimulated culture. IgG, IgG1, and IgG2b antibody levels were measured using an antibody against IgG, IgG1, and IgG2b, bound with mustard serum peroxidase, to detect IgG, IgG1, and IgG2b antibody levels in the blood. ELISA analysis. Well microtiter plate coated with 100 μl of OVA solution (50, 25 and 50 μg / ml for IgG, IgG1 and IgG2b antibodies in 50 mM carbonate-bicarbonate buffer, pH 9.6) Was stored at 4 ° C for 24 hours. Each well was washed three times with PBS containing 0.05% (v / v) Tween 20 and blocked with 5% FCS / PBS at 37 ° C for 1 hour. After washing, 100 [mu] l of diluted serum sample or control and 0.5% FCS / PBS were added to the wells and the plates were again incubated at 37 [deg.] C for 1 hour and washed again three times. A horseradish peroxidase-conjugated antibody against IgG, IgG1 and IgG2b was added to the wells, incubated at 37 DEG C for 1 hour, and the oxidase activity was measured after washing. Oxidase activity was determined by adding 100 μl of substrate saline (10 mg O-phenylenediamine and 37.5 μl 30% H ₂ O ₂ in 25 ml 0.1 M citrate-phosphate buffer (pH 5.0) Was added to each well and the plate was incubated again at 37 ° C for 10 minutes. After addition of 50 μl of 2N H ₂ SO ₄ per well, the reaction was terminated and the absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay reader Respectively.

4. Analysis of biochemical markers in the intestinal tract by oral administration of the polyunsaturated fatty acid fraction

In order to evaluate the biochemical markers and intestinal protective effects of oral polysaccharide fractions of Hwanggi-gyeonggi, we used hematopoietic shock by reperfusion in SD rats (Sprague-Dawley rats) (MDA), nitric oxide (NO), endothelin (ET), and endothelin (ET) in the intestinal tract by administration of the intraperitoneal injection of the erythrocyte polysaccharide fraction into the hemorrhagic shock- Of the biochemical markers. Twenty-four SD rats (200-300 g) were divided into four groups. Each group was divided into three groups: sham-treated group (hemorrhagic shock-treated group by perfusion injury), control group, low- The control group was treated with saline alone before the induction of hemorrhagic shock and the treated group was treated with low dose (10 mg / kg) and high dose (20 mg / kg) group before the induction of hemorrhagic shock, / Kg). Male SD rats (Sprague-Dawley rats) were used and the temperature was adjusted to 25 ~ 27 ℃. Two weeks before the start of the experiment, the animals were fed general diet and water and adjusted to the environment. Fasted. 3 mL of erythrocyte polysaccharide was prepared by saline solution and concentration, and administered intraperitoneally 3 min before induction of perfusion injury. To induce hemorrhagic shock, an ischemia-reperfusion was performed. The rats were anesthetized by inserting a white glass for 20 seconds into a glass container containing ether, and then ketamine hydrochloride (75 mg / kg) and xylazine ) (25 mg / kg) was intramuscularly anesthetized and the abdomen was shaved. The laparotomy was performed by wiping the laparotomy area with a potas- tine solution. Tweezers were used to carefully dissect the superior hepatic artery without damage to the organs and blood vessels. Then, a small blood vessel clamp (Bulldog clamp) was used to block the hepatic arterial origin and to minimize water and heat loss The abdominal cavity was closed with a 3-0 suture. The intervertebral artery occlusion was performed for 30 minutes. In order to replenish the fluid, all groups were subcutaneously injected with 50 ml / kg of physiological saline. After 45 minutes of interruption, the abdominal cavity was rewarmed to remove the forceps, and the flow of the hepatic artery was resumed to allow reperfusion. Each animal was sacrificed 2 hours after reperfusion and specimens were collected. In order to observe the intestinal tract, 5 cm of the small intestine was fixed with formaldehyde polymerisatum, dehydrated with alcohol, and embedded in paraffin. The embedded specimens were cut into 4-5 μm slices using a sliding microtome, stained with hematoxylin-eosin staining method, treated with a microscope, , And antrum damage were divided into 10 grades (Chiu's score).

4-1. Quantification of malondialdehyde (MDA)

MDA measurements for lipid peroxidation were quantified using the TBA method. 1 g of the intestinal tissue obtained in the above manner was mixed with 9 ml of 1.15% KCl, cut into small pieces with scissors, and homogenized 10 times at 7 ° C for 10 seconds with a homogenizer. Homogenized tissue fluid 0.1 ml of 8.1% SDS, 0.8 ml of acetic acid buffer, 0.8 ml of 0.8% TBA and 0.2 ml of distilled water was added, and the mixture was heated at 100 ° C for 1 hour, cooled at -20 ° C for 5 minutes, Of n-butyl alcohol was added and centrifuged (1000 rpm, 10 minutes), and the absorbance of the supernatant was measured at 533 nm. The degree of lipid peroxidation was expressed in nmol (nmol / mg) of MDA per gram of tissue.

4-2. Determination of NO, Nitric oxide content

The NO contents in the mucosal tissues of the intestinal tract were obtained by homogenizing 100 ml of intestinal mucosal tissues with 0.9 ml of saline, centrifuging (1000 rpm, 10 minutes), heating the supernatant again at 100 ° C for 3 minutes, , 10 minutes). To 0.1 ml of the obtained supernatant, 0.2 ml of 35% sulfosalicylic acid was added, and the mixture was homogenized and centrifuged, and the supernatant was stored at -20 ° C. 100 μl of the supernatant was measured by absorbance at 530 nm using an indirect nitric acid deoxidized enzyme method and the nitrogen monoxide content was determined and used as a standard curve using KNO ₂ .

4-3. Test for content of endothelin-1 (ET-1, endotelin-1)

Samples with 0.9 ml of saline solution in the mucosal tissue fluid (10%) were analyzed by radioimmunoassay.

5. Vaccine activity control of polysaccharide fraction vaccine

5-1. Effect of vaccine (Vac) combination treatment of macrophage-like polysaccharide fractions on macrophages

(AMA-0), LPS, Aluminum hydroxide gel (Invitrogen), a commercial vaccine adjuvant, were added to the purified Vi capsular polysaccharide of Salmonella typhi against the salivary macrophages (Raw 264.7) USA), and MTT assay was performed to compare the degree of cell activation. Macrophages were cultured on RAW 264.7 cells, which were cultured on a suture type macrophage. They were obtained from American Type Culture Collection (ATCC) on April 3, 2013. The cells were subcultured every 2-3 days and the cells diluted in 5% dimethylsulfoxide (DMSO) were placed in a freezing vial and stored in liquid nitrogen. RAW 264.7 cells were cultured in the presence of DMEM containing 10% fetal bovine serum (Gibco BRL, USA), 100 U / ml penicillin, 100 μg / ml streptomycin (P / S, Gibco BRL, USA) modified Eagle's medium, Gibco BRL, USA) with 5% CO ₂ at 37 ° C. This test substance was dissolved in PBS, and the test substance was dissolved in the solvent at the highest concentration, and then diluted at the highest concentration in accordance with the concentration. The dose of the test substance was 5% of the volume of the medium. Cell proliferation was measured by dividing RAW 264.7 cells into 24-well plates at 2 × 10 ⁵ cells / ml and 2 × 10 ⁶ cells / ml, and AMA-0 was added at various concentrations (100, 200, 500, 1000, 2000 mg / ) And added with Vac. Lipopolysaccharide (LPS; 0.1 mu g / ml), a mitogen of macrophages, and concanavalin A (ConA; 1 mu g / ml), a mitogen of lymphocytes, were used as a positive control for cell activation efficacy. In addition, aluminum hydroxide gel {Alum; 10 [mu] g / ml (Raw 264.7), 2 [mu] g / ml (splenocytes)} were used to compare efficacy as a vaccine adjuvant. After 24 or 72 hours of treatment of the test substance, the absorbance was measured at 570 nm by MTT measurement. The absorbance ratio of the pharmacologically treated group to the absorbance of the untreated control group was calculated as%.

RAW 264.7 cells were seeded at 2 × 10 ⁵ cells / ml in a 24-well plate, and each test substance was treated in a concentration-free range. After culturing for 24 hours, the supernatant was collected, and the amount of nitrite was measured with a grease reagent. RAW 264.7 cells were divided into 2 × 10 ⁵ cells / ml in a 24-well plate, . After 24 hours, the culture supernatant was collected and the amount of TNF-α was measured using a TNF-α enzyme-linked immunosorbent assay kit.

5-2. Effect of Polysaccharide Fraction of Shijiazhuang Area on Splenocytes in Combination with Vac

Isolation and culture of splenocytes were performed by sacrificing female ICR mice at 6 weeks of age and then harvesting the spleen. Splenocytes isolated from the harvested spleen were removed with erythrocyte disruption buffer (Biolend, Canada). And cultured in DMEM medium containing 10% FBS and 1% P / S at 5% CO ₂ and 37 ° C. Proliferation assays of splenocytes were performed as in macrophages.

6. Verification of application of shiitake polysaccharide fraction vaccine adjuvant

Other adjuvant efficacy evaluations were performed in combination with the vaccine combination treatment with the Shigella topoisomeric polysaccharide fraction using animal models. A 6-week-old female ICR mouse (Korea BioLink, Korea) was purchased on June 4, 2013, and maintained at 23 ± 3 ° C and 50 ± 20% relative humidity at room temperature 1 The system was reared and used for experiments. 6 weeks old female ICR mice were immunized for 1 week and then treated with PBS (Con), a vaccine-only control (Vac), a vaccine-treated group (Vac + AMA-0) And a control group (Vac + Alum) in which a positive control substance was administered together. Animal experiments were conducted in accordance with the regulations for animal control and approved by the Animal Experiment Ethics Committee of Catholic University of Daegu. The vaccine was prepared by intraperitoneally administering PBS containing 5 μg / mouse of AMA-0, 100 and 200 mg / kg of Alum, and 200 μg of Alum on days 1 and 15, and the same amount of PBS was administered Respectively. The weight change (g) was determined by measuring the body weight at the start and the day of sacrifice, and the weight of the spleen was measured and the weight of the spleen was calculated as a percentage of the body weight of the final sacrifice day. In order to measure the amount of cytokine and antibody produced in blood, whole blood collected is put into a tube for serum separation and centrifuged to obtain serum. Separated serum was assayed for IFN-y, IL-2 and total IgG using an enzyme-linked immunosorbent assay kit.

Example  One. Hwanggi  Structural analysis and activity assay of surface polysaccharides

AH-AMA-1-b-PS2 obtained by treating AMA-1-b-PS2 with AFase showed about 61% destruction of arabinose. In AH-AMA-1-b-PS2, Linked 3,4-or 3,5-branched arabinose, 3,6-branched galactose decreased, 4- or 5-linked arabinose and 6-linked galactose increased . The sugar component of fraction AH-1 obtained by treating GNase with AH-AMA-1-b-PS2 showed that the proportion of galactose degraded in AH-AMA-1-b-PS2 by GNase (3.6% 3-linked and 3,6-branched forms that can be degraded by GNase in AH-AMA-1-b-PS2 are very small. This means that the GNase treatment for AH-AMA-1-b-PS2 is not perfect and therefore the fraction obtained by further treating GNase with AH-AMA-1-b- 2AH-1 decreased the galactose content to about 31% with respect to AH-1, indicating that AMA-1-b-PS2 could be hydrolyzed by stepwise GNase treatment (Table 1).

The sugar structure of the product decomposed from AMA-1-b-PS2 by exo-beta-D- (1 → 3) -galactanase (AFase) and exo-alpha-L-arabinofuranosidez Component sugar
(molar ratio) AMA-1-a-PS2 AH-AMA-1-b-PS2 AH-1 2AH-1-A AH-2 AH-3 Arabinose 12.8 7.6 2.4 2.6 24.6 3.8 Fucose 4.5 3.8 - - - - Galactose 25.6 30.6 26.7 8.3 74.2 92.6 Glucose 23.6 25.6 32.1 49.6 0.8 1.8 Mannose 24.8 24.5 29.6 31.9 - - Rhamnose 5.1 7.0 8.2 7.6 - - Xylose 0.7 0.9 1.0 - - - Galacturonic acid 1.5 - - - 0.4 1.2 Glucuronic acid 1.4 - - - - 0.6

Methylation analysis of 2AH-1-A revealed that about 66% of the 3-linked form was destroyed compared to AH-AMA-1-b-PS2 and 3,6-branched galactose was degraded to AH-AMA-1 -b-PS2 (Table 2).

Methylation analysis of products derived from AMA-1-b-PS2 by AFase and GNase Glucosyl residue Deduced linkage AMA-1-a-PS2 AH-AMA-1-b-PS2 AH-1 2AH-1-A Search terminal ( f ) 32.8 8.2 5.2 5.5 terminal ( p ) 1.4 1.0 - - 4 or 5 5.2 20.0 2.6 2.4 3,4 or 3,5 3.5 - - - Fuc 3 0.6 0.7 - 1.3 Gal terminal 1.3 4.6 4.9 4.8 3 6.2 5.8 5.9 2.1 4 5.9 12.6 12.9 13.8 6 ( f ) 1.9 2.1 3.6 3.6 6 ( p ) 0.9 9.4 11.3 9.4 2,6 0.4 0.5 1.1 - 3.6 20.6 4.5 4.8 5.7 4.6 2.1 2.6 3.1 4.4 Glc terminal ( f ) 8.6 9.6 15.6 15.3 terminal 3.4 4.1 5.6 5.2 3 0.2 1.7 3.4 3.8 4 1.0 1.9 3.6 3.9 6 - 3.3 6.3 7.1 3,4,6 0.5 1.0 1.8 2.0 Man terminal 0.3 0.5 2.1 2.4 2 0.4 0.6 1.8 2.2 4 - 1.6 - - Rha terminal 1.0 0.9 0.9 1.1 2 0.5 0.6 0.9 1.0 3 0.2 0.3 - 0.6 Xyl terminal ( p ) 0.6 0.7 1.0 0.8 2 0.2 0.3 0.4 0.5 4 or 5 0.3 0.9 1.2 1.1

The monosaccharide fractions (AH-2, AH-3) formed by continuous enzymatic treatment of AMA-1-b-PS2 are believed to be present at the non-reducing end of AMA-1-b-PS2. AH-2 was composed mainly of arabinose (25%) and galactose (74%), and the content of glucose and galacturonic acid was extremely low. Terminal galactose, 6-linked, 3,6-branched galactose, and 4,5-linked arabinose were the major sugar chain structures of AH-2. The AH-3 monosaccharide was found to contain about 93% of galactose and the sugar chain was a monosaccharide rich in galactose, composed of 6-linked galactose and 6-linked galactose (Table 3).

Methylation analysis of monosaccharide fractions derived from AMA-1-b-PS2 by AFase and GNase Glucosyl residue Deduced linkage AH-2 AH-3 Search



terminal ( f ) 6.1 3.4 terminal ( p ) 1.5 - 2 ( f ) - 0.6 3 ( p ) 0.9 - 4 or 5 5.3 - Gal





terminal 19.4 32.7 4 - 12.6 6 ( f ) 3.6 21.6 6 ( p ) 49.4 26.8 3.6 6.3 0.5 4.6 1.4 0.5 3,4,6 2.5 - Glc
terminal 0.5 1.1 3 0.2 0.2 GlcA 4 2.9 - Man 2 0.2 -

The AH-2 fraction was isolated from the methylated tri-, tetra-, penta-, hexa- and heptasaccharides of the hexose by the single sugar FAB-mass assay of galactose-containing fractions AH-2 and AH-3. ([M + Na] ⁺ ), and the AH-3 fraction represents a pseudomolecule ion ([M + Na] ⁺ ) due to the methylated tri-, tetra- and pentasaccharide of the hexose, Na] ^&lt; ^{+ &} gt ^; ) (Table 4).

Monosaccharides detected by FAB-mass in the methylated portion of the product degraded from AMA-1-b-PS2 by AFase and GNase Faction m / z ([M + Na] &lt; ^{+ &} gt ^; ) Deduced oligosaccharide AH-2 681 Hex ₃ Na ⁺ 886 Hex ₄ Na ⁺ 1091 Hex ₅ Na ⁺ 1293 Hex ₆ Na ⁺ 1497 Hex ₇ Na ⁺ AH-3 477 Hex ₂ Na ⁺ 681 Hex ₃ Na ⁺ 886 Hex ₄ Na ⁺ 1090 Hex ₅ Na ⁺

Thus, AMA-1-b-PS2 has a 3-linked galactose structure and a 3,6-branched galactose glycan structure interchangeably, and has a galactose- Lactic acid complex polysaccharide type. The structure of AMA-1-b-PS2 was schematized (Fig. 2).

Example  2. Hwanggi  Immune active polysaccharides from the top polysaccharide ) Differentiated from the underground active polysaccharide

1. Identification of the configuration Active polysaccharide  And ground Active polysaccharide )

In the case of the hwanggi region, it is active in the intestinal immunity through the Pael plate. The structures of the 3-linked galactose structure and 3,6-branched galactose are alternately present in the 3-linked galactose structure and galactose-rich (Fig. 6). It was confirmed that the arabinogalactan complex was a polysaccharide-type polysaccharide conjugated with monosaccharides.

When boiling water separation makhyeop Astragalus (Astragalus membranaceus Bunge) and the Mongolian Emperor ( Astragalus There is membranaceus . Physicochemical Properties of Surface Polysaccharides Sample ¹⁾ Component sugar (molar ratio) Astragalus membraanaceus Bunge Astragalus mongholics AMA-0 AMR-0 AMMA AMMR Arabinose 15.6 7.4 13.4 6.9 Fucose 1.1 0.1 1.2 0.1 Galactose 13.5 2.9 14.6 2.4 Glucose 14.8 78.5 14.5 82.3 Mannose 15.2 trace 11.3 trace Rhamnose 3.8 0.9 4.1 1.0 Xylose 3.5 1.0 3.6 0.9 Galacturonic acid 28.5 8.4 32.3 2.4 Glucuronic acid 4.0 0.8 5.0 4.0

2. Pai El Pan  Mediated bone marrow cell proliferation factor Generation  compare

The phagemid cell - mediated phagocyte proliferative activity of the polysaccharide fractions in the aerial part and the lower part of the shrimp was 2.5 times higher than that of the basal part. This study was conducted to investigate the effects of two kinds of Hwanggi ( A. membranaceus, A. mongholics ) which can be used as medicinal herbs in the top and bottom of the polysaccharide. (Fig. 3).

Example 3. Gastrointestinal Immunostaining Assay for Vaccine-Assisted Shaping of the Above-Ground Polysaccharide Fraction

3-1. Measurement of Hemolytic Activity of Polysaccharide Fraction of Abalone

(AMA-0) showed about 0.66% activity at the treatment with AMA-0 concentration of 500 ㎍ / ㎖, and the concentration of 2.5 ~ 250 ㎍ / Ml) showed no hemolytic activity. On the other hand, in the case of QuilA, which is generally used as a vaccine and an immunosuppressant under the same conditions, the HD ₅₀ value showed a hemolytic activity of about 18.56 ± 0.26 μg / ㎖ (Table 7).

Measurement of Hemolytic Activity of Polysaccharide Fraction (AMA-0) Group Absorbance value Haemolytic percent (%) Distilled water 1.731 + 0.040 100.00 0.45 Saline 0.089 ± 0.002 0.00 ± 0.13 AMA-0
(占 퐂 / ml) 500 0.099 ± 0.006 0.66 + - 0.35 250 0.087 ± 0.002 -0.06 ± 0.20 125 0.086 ± 0.001 -0.13 + - 0.10 50 0.083 ± 0.003 -0.13 + 0.09 25 0.086 ± 0.005 -0.19 ± 0.10 12.5 0.083 ± 0.006 -0.30 ± 0.10 5 0.084 ± 0.002 -0.25 ± 0.08 2.5 0.079 ± 0.004 -0.58 ± 0.22

The effect of AMA on the proliferation of splenocytes stimulated by mitogen (ConA: concanavalin A, LPS: lipid polysaccharide) and OVA was examined in rats immunized with ovalbumin (OVA) RESULTS: Immunogenicity of OVA and AMA-0 (50, 100 ㎍ / ㎖) and OVA and QuilA (10, 20 ㎍ / ㎖) The cell proliferation index was higher than that of the OVA - immunoreactive cells and the splenocyte proliferation index stimulated with OVA. The proliferation of OVA-stimulated spleen cells in the OVA-immunized mice was clearly increased when 100 μg of AMA-0 and 10 μg of QuilA were treated, whereas those of OVA alone or immunized with OVA and Alum Could not observe a clear difference (Fig. 4).

To test the effect of AMA-0 on the induction of immune response to OVA in rats, the level of OVA-specific IgG, IgG1, and IgG2b antibodies in serum after 2 weeks of the final immunization was assayed by ELISA and immunostimulated with OVA IgG and IgG1 antibody levels in rats were increased when treated with 10 μg and 20 μg alum, QuilA and AMA-0 increased to 100 μg and 200 μg higher than control (OVA alone) Respectively. IgG and IgG1 antibody levels were similar to those of Alum, QuilA (10 μg, 20 μg) and AMA-0 (100 μg, 200 μg), respectively. , It was confirmed that the immune assistant effect can be obtained as in QuilA treatment (Fig. 5). In the IgG2b antibody level, the mice treated with AMA-0 (50 μg, 100 μg) or QuilA immunoreactivity were found to be higher than the control group, and these results were higher than those of Alum-treated group . In addition, IgG2b antibody levels were not different between AMA-0 and QuilA, and there was no significant difference between Alum and OVA alone. These results indicate that IgG and IgG1-expressing antibody levels can be increased by AMA-0 or QuilA in the induction of the immune response, and the effect of Alum was not confirmed (FIG. 5).

Example 4. Biochemical Indicative Analysis in the Intestinal tract by Oral Administration of the Polystyrene Fragments

In SD rats (Sprague-Dawley rats) induced with hemorrhagic shock, there was no difference in body weight, body temperature, and blood pressure of the animals treated with intraperitoneal administration of the hyperkeratotic polysaccharide fraction in the hemorrhagic shock-induced animals Table 8).

The body weight, body temperature and blood pressure of the animal model in each experimental group in which hemorrhagic shock was induced Group n Weight (g) Ambient temperature (℃) Bloodletting volume (ml) Normal 8 272.0 + - 18.0 26.51 ± 2.09 - Model
(HS, Hemorrhagic
shock group) 8 268.0 + - 11.0 26.43 + 1.84 4.85 ± 1.74 Low Dose 8 273.0 + - 14.0 25.99 ± 1.10 5.26 ± 1.68 High Dose 8 272.0 + - 12.0 26.74 ± 1.45 5.13 + 1.44

The intestinal mucosal endothelial villi and mammary gland were observed in the normal state and no inflammatory cell infiltration was observed in the normal group (Fig. 6A) The intestinal mucosal endothelial layer of the hemorrhagic shock-induced animal model showed edema in the small intestine villus due to perfusion damage caused by ischemia and reperfusion and hemorrhagic shock, and the villi were destroyed, and the flow of the fluid was observed and inflammatory cell infiltration was observed (Fig. 6B). In the low-dose group of Hwanggi-terrestrial polysaccharide, edema was reduced in small intestinal villi induced by hemorrhagic shock, inflammatory cell infiltration was weakened in weak endothelial cells, and the area of damage was reduced (Fig. 6C) No edema or destruction of the treated group of villi was observed and the mammary gland was in a healthy condition and no lesion of inflammatory cell infiltration was observed (Fig. 6D). These results suggest that intraperitoneal injection of polysaccharide in the aerial part of the hull has the function of preventing damage to the intestinal mucosa induced by hemorrhagic shock. In addition, the Chiu score, which can be used to determine the damage of intracoronary tract, showed the lowest value in the normal group and the highest value in the model group that induced hemorrhagic shock. On the other hand, in the high-dose group administered with intraperitoneal injection of 20 mg / kg of erythrocyte polysaccharide compared with the model group, the value was clearly low (Table 9).

Comparison of intestinal mucosa MDA, nitric oxide (NO), and endothelin-1 (ET) contents in induced gastric mucosa Group Chiu score MDA
(nmol / 100 mg) NO
(nmol / 100 mg) ET
(pg / 100 mg) Normal 0.90 + 0.78 25.32 + - 2.65 41.27 + - 8.98 285.85 ± 15.66 Model
(HS, Hemorrhagic shock group) 7.45 ± 1.63 68.44 + - 8.22 19.86 + 4.14 498.44 ± 58.28 Low Dose 5.88 ± 2.77 34.08 ± 2.01 26.14 + - 5.33 414.27 ± 95.87 High Dose 4.35 ± 1.16 31.78 + 4.08 39.97 + 4.14 325.25 + - 74.14

The amount of MDA in the intestinal mucosa of the injured model group was 68.44 nmol / 100 mg in the model group, which was higher than that in the normal group or the low dose group and the high dose group in the erythrocyte polysaccharide fraction, The MDA content of the animals injected intraperitoneally with 10 and 20 mg / kg decreased to 34.08 and 31.78 nmol / 100 mg, respectively. In addition, endothelin-1 (ET-1) content was highest in the model group of 498.44 pg / 100mg, and the fraction of erythrocyte sediments with hemorrhagic shock was injected intraperitoneally at 10 and 20 mg / kg The content of endothelin-1 in animals decreased to levels of 414.27 and 325.25 pg / 100 mg. In the case of nitrogen monoxide (NO), it was about 41.27 nmol / 100 mg in the normal group whereas in the model group it decreased to about 19.86 nmol / 100 mg in the case of hemorrhagic shock, and 10 mg / kg of the erythrocyte polysaccharide fraction In the low-dose group injected intraperitoneally to 26.14 nmol / 100 mg and in the high-dose group administered intraperitoneally to 20 mg / kg of the erythrocyte polysaccharide fraction, the level was 39.97 nmol / 100 mg, similar to that of the normal group (Table 9).

The amount of blood supplied to the small intestine from the peritoneal artery of the mesenteric artery constitutes about 20% of the total blood volume of the human body. When hemorrhagic shock is present, the flow of blood rapidly decreases in the intestinal mucosa and the intestinal tract is easily reperfused Resulting in damage caused by Thus, damage due to reperfusion in the intestinal mucosa causes multiple organ dysfunction syndrome (MODS). Therefore, it is very important to protect the intestinal mucosa from hemorrhagic damage. These results suggest that the polysaccharide of Shijiazhuang area has a function to protect the intestinal mucosa against reperfusion injury after concentration-dependent hemorrhagic shock. In addition, reactive oxygen radicals are formed by ischemic perfusion damage, which causes damage to the cell membrane and mitochondrial membrane structure by lipid peroxidation, resulting in destruction of cell structure and loss of function. The results of this study showed that ischemic perfusion injury induced lipid peroxidation in the small intestine and increased MDA production by lipid peroxidation. However, the polysaccharide fraction of the erythrocyte inhibited the production of MDA by controlling lipid peroxidation.

NO and ET are active substrates derived from endothelial cells such as lungs and play an important role in endogenous regulation. Maintaining a balance between NO and ET is the most important factor in blood perfusion in which blood is transported to tissues and organs. In general, during hemorrhagic damage, a rapid increase in NO and ET in plasma and an imbalance between NO and ET lead to damage of the small intestine mucosa. In this study, the level of NO decreased and the level of ET increased due to hemorrhagic injury (Table 9), which means that the microfluidic flow in the small intestine is destroyed after hemorrhagic injury. The intraperitoneal administration of the polysaccharide fraction of the shijiazhuang increased the endogenous NO levels and the ET levels (Table 9), indicating that the erythrocyte polysaccharide fraction had a balance between NO and ET Thereby preventing organ damage caused by hemorrhagic shock and improving the flow of blood flowing into the organ.

Example 5. Influence of vaccine (Vac) combination treatment of macrophage-like polysaccharide fractions on macrophages

 5-1. Effect of vaccine (Vac) combination treatment of macrophage-like polysaccharide fractions on macrophages

The cell growth rates of macrophages were 101.49%, 104.71% and 100.62%, respectively, when combined with the vaccine and various concentrations (100, 200, 500, 1000, 2000 mg / 102.49% and 110.76%, respectively, but it showed significant proliferative effect compared to the control. However, when Alum was treated with vaccine, toxicity to macrophages was confirmed as compared with single treatment, and cell proliferative effect could not be expected. The use of the immunoadjuvant Alum in the Zrotif-Vaccine could indicate toxicity to macrophages but could promote the proliferation of macrophages using the hyperpigmented polysaccharide (Figure 7).

After the treatment with AMA-0 in macrophages and various concentration ranges (100, 200, 500, 1000, 2000㎎ / ㎖) of the vaccine, NO measurement of the macrophage activation index, Respectively, to 5.12 ± 2.16 μM and 47.07 ± 4.63 μM, respectively. When the vaccine alone was treated with NO, the NO production was 5.12 ± 2.16 μM, whereas when the vaccine was supplemented with Alum, it was 16.12 ± 2.87 μM, indicating that the NO production amount increased 3.15 times. When the vaccine was supplemented with the polysaccharide fractions by the concentration, 7.75 ± 0.84 μM when treated with 100 mg / ㎖ of hyaluronic acid, 9.83 ± 2.13 μM, 500 mg / ㎖ when treated with 200 mg / 12.61 ± 1.26 μM, 14.69 ± 1.52 μM at 1000 mg / ㎖ and 15.66 ± 2.68 μM at 2000 mg / ㎖, respectively, and the vaccine alone produced the appropriate amount of NO When the results of AMA-0 (Vac + AMA-0) treatment group were compared between the single treatment group and the vaccine, the amount of NO production was increased in a concentration dependent manner in the hyaluronic acid polysaccharide fraction. In order to show the vaccine adjuvant efficacy of the polysaccharide fraction of hwanggi-geupyung, it should be applied at a concentration of 500mg / ㎖ or more, and when treated with vaccine, the NO of the macrophage was increased to 10.34 ± 2.63μM It was confirmed that it would be better to apply 2000 mg / ml to replace the effect of Alum which increases the production rate (Fig. 8).

TNF-α promotes adhesion of leukocytes to vascular endothelial cells in the inflammatory response, and acts on mononuclear cells to produce cytokines involved in various inflammatory responses, and stimulates the production of co-stimulators (co -stimulator, and it is known to act on tumor cells to induce apoptosis. As a result of examining the effect of the auxotrophic treatment of macronutrient polysaccharide fraction on vaccine in macrophages, the effect of TNF-α on macrophage secretion was investigated in various concentrations (100, 200, 500, 1000, 2000㎎ / After the polysaccharide fraction supplementation treatment, TNF-α, a cytokine secreted by macrophages, was measured. As a result, TNF-α secretion was 6430.6 ± 615.0 pg / In the case of treatment with 100 and 200 mg / ㎖ of treatment, there was no change, but the concentration of 500 ~ 2000 ㎎ / ㎖ increased the dose-dependency of secretion, and at the level of 2000 ㎎ / ㎖, 9330.6 ± 233.0 pg / ㎖ of TNF- Respectively. The TNF-α secretion potency was found to be 8995.5 ± 201.2 pg / ㎖ when monocotyledonous polysaccharide fraction was treated alone. When the vaccine was supplemented with 1000 and 2000 mg / ㎖ of Shigella topoisomeric polysaccharide fraction, the TNF-α secretion potency was 8936.4 ± 514.2 and 9330.6 ± 233.0 pg / ㎖, respectively. The vaccine was tested at a level similar to that of 9445.7 ± 72.9 pg / ㎖ when the vaccine adjuvant, Alum, was supplemented. Therefore, 1000 and 2000 mg / ㎖ of Shigella topoisomeric polysaccharide fraction (Fig. 9). In addition, it is possible to obtain a vaccine adjuvant effect.

5-2. Effect of Polysaccharide Fraction of Shijiazhuang Area on Splenocytes in Combination with Vac

The effect of vaccine alone on the proliferation of splenocytes after 24 hours of single treatment with the vaccine and ConA and Alum in the spleen cells resulted in the proliferation of spleen cells compared with the control, ConA also promoted the proliferation of lymphocytes and showed proliferative effects. In the spleen cells, the supernatant of polysaccharide fractions of various concentrations (100, 200, 500, 1000, 2000 mg / ㎖) was supplemented and the growth rate of splenic cells was compared after 24 hours. As a result, In the Vac + AMA-0 treatment group, the treated group (Vac + AMA-0) treated with the vaccine alone showed a dose-dependent increase compared to the vaccine treated group. Cell proliferation rate. This efficacy was similar to the efficacy of the Erysipelas polysaccharide fraction itself (AMA-0, 2000), which showed a growth rate of 160.78%. On the other hand, the group treated with 100 mg / ㎖ and the group treated with 200 mg / ㎖ were 269.12 ± 7.59 and 268.06 ± 29.90%, respectively, compared with the group treated with vaccine alone (152.83 ± 4.55% In the low-concentration treatment group, the proliferative effect was remarkable. These results showed a statistically significant level of cell proliferation effect when compared with the efficacy of AMA-0 itself (260.00% of AMA 2000) after 72 hours. These results suggest that the polysaccharide of Shijiazhuang is effective for the proliferation of splenocytes in adjuvant treatment. In the case of Alum alone treatment, the proliferation rate of splenocytes was more than 2 times at 24 hours, but this was confirmed to be a transient cell proliferation effect of Alum itself, not a vaccine adjuvant effect, and the effect of splenocyte proliferation could not be confirmed after 72 hours. In the case of the Vac + Alum treated group, it was confirmed that the vaccine had no cytotoxic effect because the vaccine had cytotoxicity that did not occur in the group treated with the light chain oligosaccharide or in the group treated with Alum alone (FIG. 10) .

Example  6. Hwanggi  Application of vaccine adjuvant composition of fraction containing surface polysaccharide

Animal model was used to evaluate the effect of adjuvant on the treatment of conjunctival hyperplasia. The results showed that the body weight change was the body weight of the final sacrifice day There was no reduction in body weight. The relative spleen weight was not significantly different from the spleen weight in the final sacrifice weight, but the effect of relative spleen weight on weight change was comparable to Vac + AMA-0 (100mg / ㎖) (Fig. 11).

In order to determine whether macrophage-associated polysaccharide-assisted treatment of purified Vi capsular polysaccharide of Salmonella typhi affects macrophages, we measured IFN-γ and IL-2, cytokines produced in Th1 cells Respectively. The IFN-γ level in the control group without any treatment with the vaccine and vaccine adjuvant was 23.04 ng / ml, while the vaccine alone showed no change at 23.10 ng / ml, and the vaccine was supplemented with the immunoadjuvant Alum , But it was not a statistically significant level. However, in the case of treatment with the hyperpigmented polysaccharide (100, 200 mg / ml), the tendency was increased to 29.21 ng / ml and 29.69 ng / ml, respectively (FIG. Changes in IL-2 did not change in the basal production, and the effects of vaccine and AMA-0 on Th1 cells via IL-2 could not be confirmed (FIG. 13). In the case of IgG, the level of IgG was 2393.80 ㎍ / ㎖ in the control, whereas it decreased to 1804.450 ㎍ / ㎖ in the case of vaccine alone and 2226.03 ㎍ / ㎖ when the vaccine was coadministered with 200 mg / (Fig. 14). In the case of adjuvant treatment of vaccine with Alum, there was no difference from the vaccine alone treatment. Therefore, in case of Alum, the immunoadjuvant used does not induce cell-mediated immune response, whereas IFN- And can induce a strong cellular immune response.

Claims (8)

An arabinogalactan complex polysaccharide having a galactose structure of Formula 1 and a sugar chain structure of 3,6-branched galactose alternately separated from the radial basal portion and connected to a galactose-rich monosaccharide, or the polysaccharide Wherein the pharmaceutical composition for immune adjuvant contains an erbotropic topical polysaccharide fraction as an active ingredient and does not induce hemolysis.
[Chemical Formula 1]

Wherein Ara represents arabinose, Gal represents galactose, and f represents forward. delete An arabinogalactan complex polysaccharide having a galactose structure of Formula 1 and a sugar chain structure of 3,6-branched galactose alternately separated from the radial basal portion and connected to a galactose-rich monosaccharide, or the polysaccharide Wherein the functional food for immune adjuvant does not induce a haemolysis phenomenon containing an emulsifier fraction containing the emulsifier as an active ingredient.
[Chemical Formula 1]

Wherein Ara represents arabinose, Gal represents galactose, and f represents forward. A vaccine comprising a pharmaceutical composition for immune supplementation, which does not induce the hemolysis phenomenon of claim 1. The vaccine according to claim 4, which is for oral or mucosal administration. An arabinogalactan complex polysaccharide having a galactose structure of Formula 1 and a sugar chain structure of 3,6-branched galactose alternately separated from the radial basal portion and connected to a galactose-rich monosaccharide, or the polysaccharide Wherein the vaccine adjuvant does not induce a haemolytic phenomenon containing an emulsifier component of an embryonic stem, comprising:
[Chemical Formula 1]

Wherein Ara represents arabinose, Gal represents galactose, and f represents forward. delete delete

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