TWI846045B - Uses of auricularia polytricha polyomycoid extract - Google Patents

Abstract

A use of auricularia polytricha polyomycoid extract for preparing pharmaceutical compositions intended to enhance the neutralizing antibody titer against Avian Infectious Bronchitis Virus (IBV). The Auricularia Polytricha polysaccharide extract is derived from an aqueous extract of Auricularia Polytricha stipes. This extract contains (1,3)-β-D-glucan and mannan, primarily composed of mannose. Accordingly, the Auricularia Polytricha polysaccharide extract facilitates the proliferation of IBV-specific lymphocyte populations and enhances the humoral immune response, thereby increasing antibody levels.

Description

Uses of Auricularia auricula polysaccharide extract

The present invention relates to a polysaccharide extract of Auricularia auricula, and in particular to a polysaccharide extract made from the pedicel of Auricularia auricula for use in enhancing humoral immunity and increasing antibodies.

Auricularia polytricha is the main cultivated fungus in Taiwan. It is rich in polysaccharides. In recent years, the functional studies of Auricularia polytricha have shown that it can activate macrophages to produce TNF-α and IL-6 to inhibit cancer cell growth, inhibit the cell cycle of human lung cancer cell line A549, induce cancer cell apoptosis, and inhibit the activity of tumor cells. In addition, the study also found that giving Auricularia polytricha powder to mice can enhance the activity of natural killer cells and strengthen the immune function of mice.

It is worth noting that during the harvesting and processing of Auricularia auricula, the fruiting bodies are mostly eaten fresh in the market, or dried and preserved by sun exposure or mechanical drying. The pedicles are hard in texture, have a bad taste, and are inedible. They are agricultural by-products. Most businesses remove the pedicles containing sawdust from the mushroom packaging manually and do not use them further. In addition, the aforementioned studies have almost all focused on the edible part of the fruiting body of Auricularia auricula or the mycelium, ignoring the pedicles that connect the mycelium of Auricularia auricula and the edible part of Auricularia auricula.

However, according to the patent document No. TWI744971B, "The use of Auricularia auricula extract for preparing a composition for inducing non-polarized immune cells to differentiate into anti-inflammatory immune cells", it is known that the Auricularia auricula pedicles contain a considerable amount of polysaccharides, but they have not been utilized.

In view of the above problems, the purpose of the present invention is to provide a use of a polysaccharide extract of Auricularia auricula for preparing a pharmaceutical composition for enhancing humoral immunity and increasing antibodies; wherein the polysaccharide extract of Auricularia auricula is a water extract of Auricularia auricula extracted from Auricularia auricula.

Among them, the Auricularia auricula polysaccharide extract promotes humoral-mediated immune response by increasing IgM antibodies.

Furthermore, the Auricularia auricula polysaccharide extract promotes the production of IgM antibodies by increasing the expression of IL-10 genes in lymphocytes.

Among them, the Auricularia auricula polysaccharide extract promotes humoral-mediated immune response by increasing IgG antibodies.

Among them, the Auricularia auricula polysaccharide extract increases the expression of TGF-β gene in lymphocytes to promote the increase of IgA antibodies and accelerate wound healing.

Among them, the pharmaceutical composition is used to enhance the neutralizing antibody titer of infectious bronchitis virus (IBV) and enhance IgM antibodies.

Among them, the Auricularia auricula polysaccharide extract promotes humoral immune response and increases antibodies by enhancing the proliferation capacity of B cells and IBV-specific lymphocyte populations.

Among them, the Auricularia auriculariae polysaccharide extract promotes humoral mediated immune response by increasing the IL-10 gene expression of lymphocytes and reducing the IFN gene expression.

The composition of the Auricularia auriculariae water extract includes mannan, and the Auricularia auriculariae polysaccharide extract promotes humoral mediated immune response through the specific receptor CD206 of mannan.

The polysaccharide content of the Auricularia auriculariae polysaccharide extract is 164.2 to 662.1 mg; the (1,3)-β-D-glucan content of the Auricularia auriculariae polysaccharide extract is 3.5 to 8.4 meq; the molecular weight of the polysaccharide is 3.6×10 ⁴ to 8.1×10 ⁵ Da; and the (1,3)-β-D-glucan content of the polysaccharide is 20.5 to 22.0 meq.

Regarding the technology, means and other effects adopted by the present invention to achieve the above-mentioned purpose, the preferred feasible embodiments are given below with detailed descriptions in conjunction with drawings.

FIG. 1 shows the IgM content of the seven groups of mice in the animal experiment 1 of the present invention after the animal experiment, which are NC group, pedicle low dose group, pedicle high dose group, pedicle small molecule group, pedicle large molecule group, microauricular low dose group, and microauricular high dose group from left to right; FIG. 2 shows the IgG content of the seven groups of mice in the animal experiment 1 of the present invention after the animal experiment, which are NC group, pedicle low dose group, pedicle high dose group, pedicle small molecule group, pedicle large molecule group, microauricular low dose group, and microauricular high dose group from left to right; FIG. 3 shows the paw pad swelling results of the seven groups of mice in the animal experiment 1 of the present invention after the DTH reaction test, and each group is 0 from left to right. 1 hour, 24 hours, 48 hours, 72 hours of foot pad swelling results; Figure 4 is the IL-10 gene expression test results of the seven groups of mice in the animal test one of the present invention, from left to right, they are NC group, pedicle low dose group, pedicle high dose group, pedicle small molecule group, pedicle large molecule group, small ear low dose group, small ear high dose group; Figure 5 is the TGF-β gene expression test results of the seven groups of mice in the animal test one of the present invention, from left to right, they are NC group, pedicle low dose group, pedicle high dose group, pedicle small molecule group, pedicle large molecule group, small ear low dose group, small ear high dose group; Figure 6 is the five groups of quails in the animal test two of the present invention after oral administration of Figure 7 is the IBV virus hemagglutination inhibition titer test results of four groups of quails (NC+VC group, PS1 group, PS2 group, PS3 group) in the animal experiment II of the present invention, the upper half of each group is the IgG test result, and the lower half is the IgM test result; Figures 8, 9, and 10 are the IBV virus hemagglutination inhibition titer test results of four groups of quails (NC+VC group, PS1 group, PS2 group, PS3 group) in the animal experiment II of the present invention. The results of lymphocyte proliferation were measured after adding polysaccharide products (Auricularia auricula water extract PS1 group, commercial yeast cell wall polysaccharide PS2 group, and chitosan PS3 group) to the drinking water of the control group, PS1 group, PS2 group, and PS3 group (Figure 8 is T cells, Figure 9 is B cells, and Figure 10 is IBV-specific lymphocyte populations); Figure 11 is the peripheral blood mononuclear cell (PBMC) cytokine gene expression of the four groups of quails in the animal experiment II of the present invention (from left to right, NC+VC group, PS1 group, PS2 group, and PS3 group); Figure 12 is the average weight change of the quails in the control group, PS1 group, PS2 group, and PS3 group in the animal experiment II of the present invention.

Some typical embodiments of the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various variations in different forms, but they do not deviate from the scope of the present invention, and the descriptions and drawings are essentially for illustrative purposes rather than for limiting the present invention.

The present invention provides a polysaccharide extract of Auricularia auricula and its new use for preparing a pharmaceutical composition for enhancing humoral immunity and increasing antibodies. The following will further describe the extraction method of the polysaccharide extract of Auricularia auricula and its efficacy test.

The Auricularia auricula polysaccharide extract of the present invention refers to the Auricularia auricula polysaccharide aqueous extract extracted from the pedicle and/or fruiting body of Auricularia auricula; wherein, according to the part of Auricularia auricula used, the Auricularia auricula polysaccharide aqueous extract includes the Auricularia auricula fruiting body polysaccharide aqueous extract and the Auricularia auricula pedicle polysaccharide aqueous extract (hereinafter referred to as the Auricularia auricula fruiting body aqueous extract and the Auricularia auricula pedicle aqueous extract, respectively), the extraction steps of the above two aqueous extracts are the same, but the specific operating parameters (such as raw material ratio, temperature, pressure, etc.) can be adjusted according to the actual situation; wherein, the Auricularia auricula fruiting body can be further divided into large ears and small ears according to the size of the fruiting body; large ears refer to the fruiting bodies with a larger volume harvested in the early stage after each period of Auricularia auricula planting; small ears refer to the fruiting bodies with a smaller volume harvested in the later stage.

The following uses the water extract of the Auricularia auricula stem as an example to illustrate the extraction method. The extraction method is to prepare the Auricularia auricula stem by the following steps:

The material collection step comprises providing a frozen sample of the pedicle of Auricularia auricula prepared through a pretreatment procedure of the pedicle of Auricularia auricula, and mixing the frozen sample of the pedicle of Auricularia auricula with sterile water at a weight ratio of 1:1 to 1:10; the hot extraction step comprises performing high-pressure steaming on the material ratio in the material collection step for 10 to 60 minutes in a high pressure range of 1.0 to 1.2 psi and a high temperature range of 90 to 121° C. to form a primary extraction product of the pedicle of Auricularia auricula; the solid-liquid separation step comprises centrifuging the primary extraction product of the pedicle of Auricularia auricula to separate the solid and liquid, thereby forming a water extract of the pedicle of Auricularia auricula and a residue; wherein the water extract of the pedicle of Auricularia auricula comprises a top solution layer formed after centrifugation and a viscous layer (viscosity layer) in the middle. layer), the polysaccharide concentration of the water extract of Auricularia auricula-sinensis pedicles is 2.5 to 9.0 mg/mL; the residue is the bottom layer product formed after centrifugation.

The water extract of Auricularia auricula pedicles of the present invention contains (1,3)-β-D-glucan ((1-3)-bD-glucan); wherein the polysaccharide content in each gram of the water extract of Auricularia auricula pedicles is 164.2 to 662.1 mg; the (1,3)-β-D-glucan content in each gram of the water extract of Auricularia auricula pedicles is 3.5 to 8.4 meq; the molecular weight of the polysaccharide is 3.6×10 ⁴ to 8.1×10 ⁵ Da; and the (1,3)-β-D-glucan content in the polysaccharide is 20.5 to 22.0 meq.

The same preparation steps as the above-mentioned method for extracting the water extract of Auricularia auricula pedicles were performed on the fruiting bodies of Auricularia auricula to obtain the water extract of Auricularia auricula fruiting bodies. As shown in Table 1, the water extract prepared from the Auricularia auricula pedicles contained a significantly higher (1,3)-β-D-glucan content than the water extract prepared from the fruiting bodies of Auricularia auricula; in addition, analysis showed that the upper layer of the water extract of the Auricularia auricula pedicles had a higher polysaccharide content and (1,3)-β-D-glucan content.

In the embodiment of the present invention, the Auricularia auricula polysaccharide extract is a polysaccharide containing glucose and mannose polymers, wherein the sugar/sugar % (w/w) in the upper liquid is 89%, and the monosaccharide content per gram of polysaccharide in the upper liquid includes 41.5% (w/w) glucose and 58.5% (w/w) mannose; the sugar/sugar % (w/w) in the viscous layer is 87%, and the monosaccharide content per gram of polysaccharide in the viscous layer includes 56.9% (w/w) glucose and 43.1% (w/w) mannose.

The following describes the animals and experimental design used in the present invention to test the efficacy of Auricularia auriculariae polysaccharide extract in enhancing humoral-mediated immunity and increasing antibodies.

Animal test 1

Balb/c mice aged 6-8 weeks (purchased from the National Laboratory Animal Center) were used and divided into seven groups, each group consisting of 10 mice, half male and half female; the seven groups of mice were fasted overnight for 12 hours before the test, and then given different oral solutions and doses once a day for two consecutive weeks (14 days). They were divided into the control group (NC group, Negative Control), the low-dose pedicle group, the high-dose pedicle group, the small molecule pedicle group, the large molecule pedicle group, the low-dose micro-auricle group, and the high-dose micro-auricle group according to the oral solution and dose. Among them, the control group refers to a normal daily diet, but no oral liquid is given; the low-dose pedicle group refers to daily oral administration of 50 mg/kg of Auricularia auricula pedicle water extract; the high-dose pedicle group refers to daily oral administration of 1000 mg/kg of Auricularia auricula pedicle water extract; the pedicle small molecule group refers to daily oral administration of 1000 mg/kg of Auricularia auricula pedicle water extract with a molecular weight between 10 ⁴ and 10 ⁵ Da; the pedicle large molecule group refers to daily oral administration of 1000 mg/kg of Auricularia auricula pedicle water extract with a molecular weight between 10 ⁵ and 10 ⁶ Da; the low-dose small ear group refers to daily oral administration of 50 mg/kg of Auricularia auricula fruiting body water extract, and the fruiting body used is the oral liquid of small ear; the high-dose small ear group refers to daily oral administration of 1000 mg/kg of Auricularia auricula fruiting body water extract, and the fruiting body used is the oral liquid of small ear.

The seven groups of mice were subjected to basic hematological analysis, serum immunoglobulin analysis, spleen cell separation, lymph node cell proliferation response, and blood immunoglobulin production capacity tests.

Among them, basic hematological analysis is to collect blood from the mouse vein, place the blood sample in a test tube and wait for it to coagulate, then centrifuge it at 15℃ and 1620×g for 15 minutes, and then draw the upper layer of liquid, divide it into microcentrifuge tubes, and store it in a -20℃ freezer for analysis. The white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet, etc. in the blood were measured using a fully automatic blood biochemistry analyzer (Trace Datapro Plus, Thermo, Australia) with the aid of an analysis kit (Reagent kit; Pointe Scientific, INC.). In addition, the blood biochemical values such as total protein (TP), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), serum urea nitrogen (SUN), total cholesterol concentration (TC), glutamic acid oxaloacetate transaminase (AST), glutamic acid pyroglucose transaminase (ALT), total glucose concentration (TG) and alkaline phosphatase activity (ALP) were measured by blood biochemical analyzer (Roche Cobas Mira 2865-35Automatic Analyzer) for detection.

Among them, serum immunoglobulin analysis is based on enzyme-linked immunosorbent assay (ELISA), supplemented by immunoglobulin analysis kit (Bethyl Lab. Inc., USA) to analyze the content of immunoglobulins IgA, IgG and IgM in serum. ELISA is a method developed based on the fluorescent antibody method. Specifically, the enzyme is linked to the antigen or antibody molecule to detect the antigen-antibody reaction and express it according to the color intensity. For example, in the determination of antibodies, the antigen is linked to a fixed substance, the sample is added first, and then the enzyme-linked immunoglobulin is added. The measured enzyme activity represents the level of antibody content.

In the above basic hematological analysis, there was no significant difference among the seven groups of mice, indicating that the test substance treatment did not cause toxicity or affect the basic health status. On this safe basis, further comparison was made to see whether there was an impact on immune traits, and the immune regulation did not affect the basic immune physiological status.

As shown in Figures 1 and 2, the IgM and IgG levels of seven groups of mice measured after animal testing are shown in sequence. From the comparison of Figures 1 and 2, it can be seen that except for the anti-ovalbumin IgM antibody of the low-dose group of Ditou and the high-dose group of Microauricularia, which is not higher than that of the NC group, the average IgM antibody concentration of the other groups is higher than that of the NC group; the IgG antibody production of the high-dose group of Ditou is significantly higher than that of the NC group. This result supports that Ditou polysaccharide can promote humoral immune response.

Among them, in the spleen cell isolation and lymph node cell proliferation reaction, the T cell stimulator can be selected from concanavalin A (con A) or phytohaemagglutinin (PHA) mitogen, or use cross-linking anti-CD3 antibodies as T cell stimulators; lipopolysaccharide (LPS) is used to stimulate B cells.

The steps of performing the spleen cell isolation and lymph node cell proliferation reaction include first placing 2×10 ⁶ /mL spleen cells in a 96-well plate, and then adding RPMI culture medium (Roswell Park Memorial Institute culture medium) or a culture medium supplemented with cell mitogens such as lipopolysaccharide (LPS), concanavalin A (con A), and phytohemagglutinin (PHA). After culturing for two days at 37°C in CO ₂ , fluorescent dye (Alamar Blue) was added and cultured for another 20 hours. Cells were then collected on filter paper using a cell harvester. After the filter paper was air-dried, the thymidine (3H-thymidine) content was measured using a flash counter to calculate the cell proliferation ability.

Delayed type hypersensitivity response (DTH response), also known as Type IV hypersensitivity, is a cell-mediated response. The pathogenesis is caused by the human body's immune response to antigenic substances in food. DTH response is considered the best in vivo test for evaluating cell-mediated immune response; as shown in Figure 3, the footpad swelling of seven groups of mice after the DTH response test was shown. The results showed that the polysaccharides in the high-dose group of pedicles and the small-molecule group or large-molecule group of pedicles further divided by the high-dose group of pedicles all have good promotion of cell-mediated immune response, while there is no significant difference between the low-dose group of micro-auricularia, the high-dose group of micro-auricularia and the NC group.

As shown in Figure 4, the results of interleukin 10 (IL-10) gene expression detection of seven groups of mice are shown. Specifically, as can be seen from the figure, the IL-10 production of lymphocytes in the large molecule group and the low-dose group of microtia was significantly higher than that in the NC group. This result supports that these two groups have the highest IgM production.

As shown in Figure 5, the results of the gene expression test of Transforming Growth Factor Beta (TGF-β) in seven groups of mice are shown. Specifically, the figure shows that the lymphocyte TGF-β production in the large molecule group and the low-dose group of Microauricularia is significantly higher than that in other groups, which has the effect of promoting the increase of IgA antibodies and accelerating wound healing.

Among them, the ability to produce immunoglobulins in the blood is to use antibody titers to represent the concentration of immunoglobulins. Specifically, as shown in Table 2 below, the titers of IgM and IgG in the production of primary and secondary antibodies are evaluated by using the non-stained stimulating antigen sheep red blood cells (SRBC) as antigens to induce antibody production. As can be seen from Table 2, two weeks after the oral solution was administered (W2), the two groups of mice administered with the water extract of Auricularia auricula prepared by the present invention (prepared with the pedicle and the small ear, respectively) showed significant differences in the titers of IgG compared with the NC group and the CD group; among them, the difference in male mice was more significant than that in female mice. From the above, it can be seen that the antibody of mice administered with the Auricularia auriculariae polysaccharide water extract prepared by the present invention increases, that is, the Auricularia auriculariae polysaccharide water extract prepared by the present invention has the effect of promoting the improvement of mouse antibody value.

Table 2:

Animal experiment 2

Forty Japanese quails of half male and half female, four weeks old, were selected and kept in a Specific Pathogen Free (SPF) experimental animal house with negative pressure air filtration and controlled environment. In this animal experiment, the quails were divided into five groups, each with 8 quails. The five groups of quails were given a basic complete feed and reverse osmosis filtered water before the test, and then given different oral solutions and dosages every day for two consecutive weeks (14 days). According to the oral solution and dosage, they were divided into three groups, which were given vaccines and basic commercial layer feed, a control group (NC group, Negative Control) and a vaccine control group (VC group, Vaccine Control); the three groups given vaccines and basic commercial laying hen feed were all given New World Disease (ND)/Infectious Bronchitis Virus (IBV) vaccines, and the Auricularia auricula-derived water extract PS1, yeast cell wall polysaccharide PS2 or chitosan PS3 of the present invention were added to the drinking water, and they were defined as PS1 group, PS2 group and PS3 group in sequence.

The five groups of quails were subjected to growth trait analysis, blood biochemistry analysis, antibody production, vaccine efficiency and immune assessment tests.

Among them, the growth trait analysis is to measure and record the weight, weight gain, feed intake and feed efficiency of the five groups of quails. As shown in Figure 12, the average weight changes of the control group, PS1 group, PS2 group and PS3 group are shown. As can be seen from the figure, there is no significant difference in the weight changes of the quails in each group, indicating that the growth traits of the quails in the PS1 group, PS2 group and PS3 group are not affected by oral additives.

Among them, blood biochemical analysis is to draw blood samples from the jugular/wing vein of five groups of quails for blood analysis and immune assessment tests. Specifically, the levels of alkalinephosphatase (ALP), aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and γ-glutamyl transferase (γ-GT) were measured in the quails to understand whether there are adverse effects on fiber and polysaccharide decomposition.

Among them, the antibody production vaccine efficiency and immunity evaluation were conducted by giving Nobilis ^® MA5+Clone 30 vaccine to all quails through nasal administration at the beginning of animal trial 2, and giving the same vaccine as a booster 3 weeks later.

More specifically, the antibody production vaccine efficiency and immune assessment include serum vaccine specific antibody titer analysis, anti-sheep erythrocyte antibody titer determination, flow cytometer analysis of phagocytic ability, measurement of intracellular peroxide concentration of phagocytes (Analysis of respiratory burst), lymphocyte proliferation test and cytokine expression analysis.

Among them, the serum vaccine specific antibody titer analysis is to detect the specific antibody concentration of the quail serum sample according to the ELISA test technology of the IDEXX IBV antibody test kit and the IDEXX ND antibody test kit.

Among them, the determination of anti-sheep red blood cell antibody titer was carried out by collecting blood samples three times during the trial. The first blood collection was carried out three weeks after the animals were given the test feed at the beginning of the trial. This first blood sample was taken before the animals were injected with sheep red blood cells as a pre-treatment antibody titer assessment (pre-challenged period); after the first sampling, the quail was immediately injected with 0.5mL of 2.5% sheep red blood cell (SRBC) solution through the wing vein. One week later, the second blood collection was carried out. The blood sample contained anti-SRBC specific antibodies. This was the primary antibody response (primary response). The second dose of SRBC was injected into the wing vein immediately after the blood sample was collected; one week later, the third blood collection was carried out, which was the secondary antibody response (secondary response). After the blood samples are collected, they are naturally agglutinated and precipitated, or centrifuged at 3000RPM for 5 minutes, and the serum is collected in a -20℃ refrigerator to wait for the antibody value analysis test.

The antibody titer analysis test method follows the literature described by Wegmann and Smithies (1966). In brief, first, 25 μL of physiological saline is placed in each well of a 96-well plate, 25 μL of sample serum containing antibodies is added to the first well, and then serial dilution is performed in the next well; after the serial dilution is completed, 25 μL of 7.5% SRBC solution is added to each well of the 96-well plate and placed in a 37°C incubator for 4 hours. The lowest dilution concentration agglutination point produced by the antibody and blood cells is visually inspected and observed, and the last agglutination reaction point is recorded as the antibody titer.

Among them, flow cytometry analysis of phagocytic ability is to separate heterophils from peripheral blood and count the number of live cells using an AutoT4 (USA) automatic cell counter. Live cells were suspended in RPMI1640 medium containing 10% fetal bovine serum (FBS) and added to a 96-well plate at a concentration of 1×10 ⁶ /mL. Fluorescently labeled E. coli were added to the 96-well plate at a ratio of bacteria to cells MOI = 10:1 and incubated in a 37°C CO ₂ incubator for 3 hours to allow heterophils to phagocytose fluorescently labeled Salmonella or E. coli. The phagocytic ability was analyzed by flow cytometry (FACscan, BD, USA) to analyze the fluorescence intensity of cells after phagocytosis. The method followed Fan et al. 2011.

Among them, the measurement of intracellular peroxide concentration in phagocytes is a method that uses the production of peroxides in phagocytes as the main method for killing phagocytic Salmonella. Measuring the intracellular peroxide concentration can understand the bactericidal ability of phagocytes. The analysis method follows the literature of Fan et al. in 2011, using dichlorofluorescein (DCFH) (Sigma, Co. Ltd) to co-culture with cells for 10 minutes. If peroxide reactive oxygen species (ROS) are produced, DCFH will be oxidized to produce fluorescent substances. Then, the intensity of intracellular fluorescence production is analyzed by flow cytometer as an analysis to evaluate the ROS production ability (respiratory burst). 200ng/mL of phorbol 12-myristate 13-acetate (PMA) (PMA, Sigma Co. Ltd) was used as a substance to stimulate the production of ROS.

Among them, the lymphocyte proliferation test uses vaccines or mitogens PHA (phytohemagglutinin) or LPS to stimulate the proliferation of T or B lymphocyte populations respectively. The method is based on the literature of Weng et al. 2011. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated and purified by gradient density separation. The number of live cells in total PBMCs was determined by an automatic cell counter. The cells were suspended in RPMI1640 medium containing 10% fetal bovine serum (FBS) and seeded into 96-well plates at a density of 1×10 ⁶ /mL. The cells were cultured three times at 37°C in a humidified incubator with 5% CO _2. After 24 hours of overnight culture, mitogens were added. After 24 hours of culture, fluorescent dye (Alamar Blue dye) (Serotec Co., Oxford, UK) as a cell proliferation indicator. After 72 hours of culture, the 96-well plates were transferred and the absorption spectrum was measured at an optical density (OD) of 583/30 using a full-band multi-well spectrophotometer (FLX800, Bio-Tek Instruments, Inc. Winooski, VT, USA). The proliferation ability of lymphocytes was evaluated by the difference in the absorption value between the mitogen-stimulated group and the unstimulated control group.

Among them, the cytokine expression analysis is to stimulate the separated white blood cells with phytohemagglutinin (PHA), collect the white blood cells and extract RNA with RNA extraction reagent Trizol, use RT-PCR to amplify the fragments of cytokines such as IFN- and IL-4, and then use colloid coloring to perform semi-quantitative analysis of the expression of cytokine mRNA; use commercial ELISA kits to perform quantitative comparison of cytokines in plasma and cell culture supernatant.

As shown in Figure 6, the IBV vaccine antibody levels of five groups of quails after oral administration of different polysaccharide oral solutions are shown. As can be seen from the figure, the IBV vaccine antibody levels of the five groups of quails increased on the 7th day after administration of the IBV vaccine and maintained the highest antibody level on the 14th day.

As shown in FIG. 7 , the test results of the IBV virus hemagglutination inhibition titer of four groups of quails (NC+VC group, PS1 group, PS2 group, and PS3 group) two weeks after the administration of the polysaccharide oral solution are shown; among them, the NC+VC group refers to the quails in the NC group raised in the same space, which have been infected by the virus of the VC group after two weeks of the test because the vaccine administered to the VC group is an active virus and is infectious. Therefore, the NC group becomes the quail infected after the VC group, and the NC group and the VC group are collectively referred to as the NC+VC group in the data analysis after the end of the test. As shown in the figure, the IBV virus neutralizing antibody titer test results measured on the 14th day after the administration of the IBV vaccine show that the Auricularia auricula water extract (PS1 group) of the present invention increased the IBV virus neutralizing antibody titer in the quail mode and significantly increased the IgM neutralizing antibody. The PS1 group has a better performance than the commercial yeast cell wall polysaccharide (PS2 group) and chitosan (PS3 group).

As shown in Figures 8, 9 and 10, the lymphocyte proliferation results were measured after adding polysaccharide products (Auricularia auricula water extract PS1 group, commercial yeast cell wall polysaccharide PS2 group, chitosan PS3 group) to the drinking water of four groups of quails (NC+VC group, PS1 group, PS2 group, PS3 group). In the acquired immune response, the proliferation of lymphocytes produces a large number of lymphocytes, which is related to the production of humoral antibodies or cell-mediated immune responses. The lymphocyte proliferation activity can be activated by T cell or B cell-specific mitogens, and can also induce the proliferation of antigen-specific lymphocyte populations by producing memory for specific antigens; all lymphocyte proliferation manifestations include T cells, B cells and IBV-specific lymphocyte populations. As shown in the figure, the proliferation capacity of B cells and IBV-specific lymphocyte populations in the PS1 group is higher than that of other polysaccharide product groups (PS2 group, PS3 group) and NC+VC group.

As shown in Figure 11, the peripheral blood mononuclear cell (PBMC) cytokine gene expression of four groups of quails (NC+VC group, PS1 group, PS2 group, PS3 group) is shown. As can be seen from the figure, compared with the control group (PS2 group, PS3 group), the water extract of the pedicle of Auricularia auricula (PS1 group) of the present invention significantly increased the gene expression of IL-10, and it was higher than that of the PS2 group and PS3 group; the IFN gene expression of the PS1 group was lower than that of the PS2 group and PS3 group. It is speculated that the immune response mediated by the polysaccharide of the pedicle of Auricularia auricula is a humoral immunity dominated by Th2.

In summary, the efficacy test of the Auricularia auricula polysaccharide extract of the present invention shows that in addition to β-glucan, the composition of Auricularia auricula polysaccharide also contains a large amount of mannan, which is mainly mannose. The activity of mannan is through its specific receptor CD206. The subsequent immune response induced by mannan is different from that of β-glucan. It deviates from the T cell-mediated killing immune response and tends to the humoral immune response, activating antigen-presenting cells such as macrophages to produce IL-10 and TGF-β, and enhancing the production of antibodies mainly of B cell IgA type.

The results of IBV-specific antibody production showed that all groups produced anti-IBV antibodies. Although the control group (NC group) was not vaccinated, it was kept in the same pen as the vaccine control group (VC group), and each group was tested in a single closed negative pressure space. IBV is a weakened live virus vaccine, and the NC group should have produced antibodies through indirect infection or sharing water and food. However, the average induced antibody production was lower than that of the other vaccine-vaccinated groups. The antibody production of each group was the highest on the 7th day, and the antibody concentration in the plasma decreased on the 14th day but was still higher than the antibody concentration on the 3rd day. In addition, the average antibody production of each group on the 14th day was higher in all polysaccharide-treated groups than in the VC and NC groups. The application of polysaccharide products may require a longer administration period to have a more obvious effect.

In the animal test 2, in the experiment of adding polysaccharide products to drinking water, the results of lymphocyte proliferation in each group showed that the proliferation of lymphocyte strains in the acquired immune response to produce a large number of lymphocytes is related to the production of humoral antibodies or cell-mediated immune responses. The lymphocyte proliferation activity can activate cell proliferation through T cell or B cell-specific mitogens, and can also induce antigen-specific lymphocyte population proliferation by producing memory for specific antigens; in the performance of all lymphocyte proliferation, including T cells, B cells and IBV-specific lymphocyte populations, compared with polysaccharide products, the proliferation of B cells and IBV-specific lymphocyte populations in the PS1 group is higher. The activity of PS2 group was higher than that of other polysaccharide groups and NC+VC group. The PS2 group was the best in T cell proliferation activity. The products currently on the market are mainly polysaccharides fermented by yeast or other microorganisms, and their polysaccharide structure is mainly β-(1,3)(1,6)-glucan. In addition, there are also brown algae β-(1,3)(1,6)-glucan polysaccharides extracted from algae and barley β-(1,4)(1,3)-glucan. According to their molecular weight, they all have the function of activating immunity. This type of glucan mainly affects cells expressing this type of receptor, such as macrophages, through the Dectin-1 receptor.

The neutralizing antibody titer test of avian infectious bronchitis virus IBV was determined by the red blood cell competitive inhibition agglutination test. The results showed that the three polysaccharide products all had neutralizing antibodies that were superior to those of the NC+VC group. The polysaccharide in the PS1 group had the highest neutralizing antibody, followed by the PS2 group. The neutralizing antibodies in the PS1 group were mainly IgM, while those in the PS2 group were mainly IgG. This result shows that the two polysaccharide products may have different action mechanisms in the production of activation-specific antibodies. Polysaccharides have different effects and receptor binding sites due to differences in molecular weight and structural composition. In the future, in the development of function-oriented polysaccharide products, polysaccharide components from different sources can be adjusted to achieve the best application effect.

Claims (9)

A kind of Auricularia auriculariae polysaccharide extract is used for preparing a pharmaceutical composition for enhancing the neutralizing antibody value of avian infectious bronchitis virus (IBV); wherein the Auricularia auriculariae polysaccharide extract is an Auricularia auriculariae pedicle water extract prepared by extracting the Auricularia auriculariae pedicle, and the Auricularia auriculariae polysaccharide extract contains (1,3)-β-D-glucan ((1,3)-β-D-glucan) and mannan (mannan) mainly composed of mannose. The use as described in claim 1, wherein the Auricularia auricula polysaccharide extract promotes humoral-mediated immune response by increasing IgM antibodies. The use as described in claim 2, wherein the Auricularia auricula polysaccharide extract promotes the production of IgM antibodies by increasing the expression of IL-10 gene in lymphocytes. The use as described in claim 1, wherein the Auricularia auricula polysaccharide extract promotes humoral-mediated immune response by increasing IgG antibodies. The use as described in claim 1, wherein the Auricularia auricula polysaccharide extract increases the expression of TGF-β gene in lymphocytes to promote the increase of IgA antibodies and accelerate wound healing. The use as described in claim 1, wherein the Auricularia auricula polysaccharide extract promotes humoral-mediated immune response and increases antibodies by enhancing the proliferation capacity of B cells and IBV-specific lymphocyte populations. The use as described in claim 1, wherein the Auricularia auricula polysaccharide extract promotes humoral mediated immune response by increasing the IL-10 gene expression of lymphocytes and reducing the IFN gene expression. The use as described in claim 1, wherein the composition of the Auricularia auricula water extract includes mannan, and the Auricularia auricula polysaccharide extract promotes humoral mediated immune response through the specific receptor CD206 of mannan. The use as described in claim 1, wherein the polysaccharide content in the Auricularia auriculariae polysaccharide extract is 164.2 to 662.1 mg; the (1,3)-β-D-glucan content in the Auricularia auriculariae polysaccharide extract is 3.5 to 8.4 meq; the molecular weight of the polysaccharide is 3.6×10 ⁴ to 8.1×10 ⁵ Da; and the (1,3)-β-D-glucan content in the polysaccharide is 20.5 to 22.0 meq.