阅读说明：本技术 一种免疫增强组合物及其应用 (Immunity enhancing composition and application thereof ) 是由 王建东 郭延生 祁燕蓉 梁小军 郭亚男 唐玉林 高海慧 侯鹏霞 王秀琴 宣小龙 于 2021-06-28 设计创作，主要内容包括：本申请提供一种免疫增强组合物及其应用,其中免疫增强组合物包括枸杞糖肽LbGp4和辅料；所述枸杞糖肽和辅料质量比为5:1,所述枸杞糖肽的纯度≥90％。本申请采用CY诱导雏鸡产生免疫抑制状态,研究LbGp4对免疫抑制的拮抗作用,结果表明LbGp4可以显著提高CY诱导产生免疫抑制雏鸡的生长性能,显著促进脾脏的发育,加快脾脏因CY带来损伤的恢复过程,极显著提高了CY诱导产生免疫抑制雏鸡外周血淋巴细胞的增殖能力,以LbGp4中剂量组(5mg/kg·d)效果最好。由上述结果可知,本申请提供的免疫增强组合物可以作为制备提高雏鸡免疫方面药物中的应用。(The application provides an immune enhancement composition and application thereof, wherein the immune enhancement composition comprises lycium barbarum glycopeptide LbGp4 and auxiliary materials; the mass ratio of the lycium barbarum glycopeptide to the auxiliary materials is 5:1, and the purity of the lycium barbarum glycopeptide is more than or equal to 90%. According to the application, CY is adopted to induce the chicken to generate an immunosuppression state, and the antagonistic effect of LbGp4 on immunosuppression is researched, and the result shows that LbGp4 can obviously improve the growth performance of the chicken induced to generate immunosuppression, obviously promote the development of spleen, accelerate the recovery process of spleen due to damage caused by CY, and greatly improve the proliferation capacity of peripheral blood lymphocytes of the chicken induced to generate immunosuppression, and the best effect is achieved by using a dose group (5 mg/kg. d) in LbGp 4. From the above results, the immune enhancing composition provided by the present application can be used for preparing a medicine for improving chicken immunity.)

1. An immune enhancing composition, which is characterized by comprising lycium barbarum glycopeptide LbGp4 and auxiliary materials; the mass ratio of the lycium barbarum glycopeptide LbGp4 to the auxiliary materials is 5:1, and the purity of the lycium barbarum glycopeptide LbGp4 is more than or equal to 90%.

2. The immunopotentiating composition according to claim 1, wherein the adjuvant comprises deer blood polypeptide, and the mass ratio of the deer blood polypeptide to the adjuvant is 0.4: 1.

3. The immunopotentiating composition of claim 1, for use as a cyclophosphamide antagonist.

4. Use of an immunoenhancing composition according to any of claims 1 or 2 for the manufacture of a product for enhancing chicken immunity, said immunoenhancing composition comprising a pharmaceutically effective amount of the glycopeptide lycium barbarum LbGp 4.

5. The use according to claim 4, wherein the pharmaceutically effective amount is 90-95% by weight.

6. The use of claim 5, wherein the immune enhancing composition is used in the preparation of a medicament or feed for increasing peripheral blood lymphokine of chicken.

Technical Field

The application relates to the technical field of animal health care, in particular to an immune enhancing composition and application thereof.

Background

Immunosuppression refers to the condition that the animal body has immune dysfunction, so that the immunity is reduced, and the animal body is easily invaded by pathogens. The brooding period of the chicks generally refers to the period from birth to the seventh week, and the chicks have the characteristics of high growth speed and poor resistance in the period. Therefore, in addition to providing a good growth environment in the early stages, it is also important to enhance early rearing management and biosafety measures in order to have good productivity in the egg laying period. Although maternal antibodies can protect chicks from early infection, as maternal antibodies gradually diminish in protection, there is a period of immune washout. Early immunity in chicks is also an essential link in biological safety. The time node for early immunization is also critical, and the immunoprotection obtained by the active immunization mode is generated as long as possible at 14-28 d.

At present, the prohibition of adding antibiotics in the feed becomes the normal state of the breeding industry, so that an immune enhancing composition which has small side effect and is beneficial to improving the immunity and the production performance of livestock and poultry is needed to be found, and a large number of researches show that polysaccharide extracted from plants has the effects of enhancing the immunity and promoting the growth.

Disclosure of Invention

The application provides an immune enhancement composition, which aims to solve the problem that the immune enhancement composition which has no small side effect and is helpful for improving the immunity and the production performance of livestock and poultry in the prior art does not exist.

In one aspect, the present application provides an immune enhancing composition comprising lycium barbarum glycopeptide LbGp4 and an adjuvant; the mass ratio of the lycium barbarum glycopeptide LbGp4 to the auxiliary materials is 5:1, and the purity of the lycium barbarum glycopeptide LbGp4 is more than or equal to 90%.

Optionally, the auxiliary material comprises deer blood polypeptide, and the mass ratio of the deer blood polypeptide to the auxiliary material is 0.4: 1.

In a second aspect, the present application provides the use of an immunoenhancing composition as described above as a cyclophosphamide antagonist.

In a third aspect, the present application provides the use of an immune enhancing composition comprising a pharmaceutically effective amount of the glycopeptide lycium barbarum LbGp4 in the manufacture of a medicament for enhancing immunity in a chicken.

Optionally, in the above application, the pharmaceutically effective amount is 90-95% by weight.

Optionally, the application comprises the application of the immune enhancement composition in preparing a medicament or feed for increasing the peripheral blood lymphokine of the chicks.

The application provides an immune enhancing composition, which comprises lycium barbarum glycopeptide and auxiliary materials; the mass ratio of the lycium barbarum glycopeptide to the auxiliary materials is 9:1, and the purity of the lycium barbarum glycopeptide is more than or equal to 90%. According to the application, CY (cyclophosphamide) is adopted to induce the chicken to generate an immunosuppression state, and the antagonistic effect of LbGp on immunosuppression is researched, and the result shows that LbGp can obviously improve the growth performance of the chicken induced to generate immunosuppression, obviously promote the development of spleen, accelerate the recovery process of spleen damaged by CY, and greatly improve the proliferation capacity of peripheral blood lymphocytes of the chicken induced to generate immunosuppression by CY, and the best effect is achieved by using a dose group (5 mg/kg. d) in LbGp. From the above results, the immune enhancing composition provided by the present application can be used for preparing a medicine for improving chicken immunity.

Drawings

In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the effect of 7d glycopeptide to spleen tissue structure of chicken in examples of the present application;

FIG. 2 is a graph showing the effect of injection of 14d glycopeptide to spleen tissue structure in chickens according to the present example;

FIG. 3 is a graph showing the effect of injection of 21d glycopeptide of Lycium barbarum on spleen tissue structure of chickens according to the example of the present application;

FIG. 4 is a graph showing the effect of 28d glycopeptide to lycium barbarum on spleen tissue structure in chickens according to the example of the present application.

Detailed Description

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.

Lycium Barbarum Polysaccharide (LBP) is the main effective component of fructus Lycii with bioactive function, and has the characteristics of no drug resistance, no residue, safety, simple manufacturing process, etc. The research shows that LBP has the functions of resisting oxidation, resisting inflammation, protecting nerve, reducing blood sugar and blood fat, regulating immunity, etc.

Lycium barbarum glycopeptide (LbGp) is a glycoconjugate with enhanced immunological activity obtained by further isolation and purification from LBP. The chicken immunosuppression is easily induced by pathogenic microorganisms, nutrition, feeding management, stress, medicines and other factors, so that the chicken immunosuppression is induced by injecting Cyclophosphamide (CY) into the chicken, and then the chicken immunosuppression is orally taken by drinking water, and the growth condition, spleen index and peripheral blood lymphocyte proliferation rate are taken as evaluation indexes, and then spleen histological section analysis is assisted to provide the immunoregulation effect of the LbGp on the CY-induced immunosuppressed chicken.

In one aspect, the present application provides an immune enhancing composition comprising a lycium barbarum glycopeptide and an adjuvant; the mass ratio of the lycium barbarum glycopeptide to the auxiliary materials is 5:1, and the purity of the lycium barbarum glycopeptide is more than or equal to 90%.

In a second aspect, the present application provides the use of an immunoenhancing composition as described above as a cyclophosphamide antagonist.

In a third aspect, the application provides an application of an immune enhancing composition in preparing a medicament for improving the immunity of chicks,

the immune enhancing composition comprises a pharmaceutically effective amount of lycium barbarum glycopeptide LbGp.

Optionally, in the above application, the pharmaceutically effective amount is 90-95% by weight.

Optionally, the application comprises the application of the immune enhancement composition in preparing a medicament or feed for increasing the peripheral blood lymphokine of the chicks.

The following examples are provided to demonstrate the immunological effects of lycium glycopeptides in an immunopotentiating composition.

Examples

1. Preparation work

1.1 materials used in the experiments

Test animals and drugs: the 1d old helan laying hens are purchased from Huadu valley poultry industry Co., Ltd, Beijing. The lycium barbarum glycopeptide is obtained from Ningxia Ribenus lycium biological science and technology GmbH, and is prepared from lycium barbarum glycopeptide and auxiliary materials to obtain the immune enhancing composition.

Reagents and instruments: CY is available from Dalian Melam Biotechnology Ltd (cat # A0505A); the kit for separating the chicken peripheral blood lymphocyte is purchased from the third-class ocean biological product science and technology Limited liability company (the product number is LTS 1090C); the sodium citrate anticoagulant as the disposable test animal venous blood sample collection container is purchased from the third ocean biologicals science and technology Limited liability company (cargo number: TBDTM-0001); neutral formalin fixing solution (10% NBF) (cat # G2161) was purchased from Solebao corporation; the complete culture medium of chicken peripheral blood lymphocytes is purchased from Wuhan Punuoise Life technologies, Inc. (Cat. No: CM-C007); 0.9% sodium chloride injection available from san ao, shaxi; the electronic scales are purchased from a second balance instrument factory in Shanghai; CO 2₂The incubator was purchased from Shanghai Li Shen science, Inc.; tissue microtomes were purchased from Leica, germany.

1.2 Experimental grouping and pretreatment

200-feather 1-day-old kalimeris indica laying hens are adaptively raised to 7-day-old kalimeris indica laying hens and are randomly divided into 5 groups, 40 feathers are adopted in each group, namely a blank group (NC), a cyclophosphamide group (CY) and a LbGp4 group, namely a high group (LbGp4H group), a medium group (LbGp4M group) and a low group (LbGp4L group) 3 dose groups, the rest 4 groups are injected with 80 mg/kg-day CY continuously for molding except for the NC group, and 10, 5 and 2.5 mg/kg-day-Gp 4 are respectively given to each group of LbGp4 continuously 7 days after molding. At 7, 14, 21 and 28d after the first dose, 5 chickens were randomly weighed per group, followed by removal of the spleen for weighing, and data were recorded to calculate the spleen index, which was given by the formula: spleen index is spleen weight (g)/body weight (g) × 100.

At 7, 14, 21 and 28d after the first administration, 5 chicken sterile hearts were randomly selected from each group and collected, followed by centrifugation to separate lymphocytes, and the lymphocytes OD490nm values were measured by MTT method after the culture.

At 7, 14, 21 and 28d after the first administration, 5 chickens were randomly selected from each group and sacrificed, spleens were taken, completely placed in 10% NBF, stored at room temperature for 3d, paraffin sections were prepared according to conventional procedures, HE stained, and microscopic examined.

Data were analyzed for significance of differences between groups using the One-Way ANOVA test with SPSS25.0, and results are expressed as mean ± standard error.

2. Results of the experiment

2.1 Effect of Lycium barbarum glycopeptide on CY immunosuppressive Chicken weight

Table 1 shows the effect of lycium barbarum glycopeptide on chicken body weight. As can be seen from Table 1, the weight of the immunosuppressive chicks of the CY group was lower than that of the remaining groups at each of 7 th, 14 th, 21 th and 28 th days. Wherein at 7d, the weight of NC group is significantly higher than that of LbGp4H, LbGp4L and CY (P < 0.05); at 14d, the body weight of the LbGp4M group is significantly higher than that of the CY group and the NC group (P < 0.05); at 21d, LbGp4M group was significantly higher than LbGp4H, LbGp4H was significantly higher than CY group (P <0.05), LbGp4M was significantly higher than LbGp4L, CY and NC groups (P < 0.01); at 27d, the body weight of the LbGp4M group was significantly higher than that of the LbGp4H and LbGp4L groups (P <0.05), and LbGp4M was significantly higher than that of the CY and NC groups (P < 0.01).

TABLE 1 Effect of Lycium barbarum glycopeptides on Chicken weight

Table 1Effect of Lycium Barbarum Glycopeptide on body weight of chickens

2.2 Effect of Lycium chinense glycopeptide on CY immunosuppression Chicken spleen index

Table 2 shows the effect of lycium barbarum glycopeptide on chicken spleen index. As can be seen from Table 2, spleen indices of CY group immunosuppressive chicks were lower than those of the remaining groups at each of 7 th, 14 th, 21 th and 28 th d. At 7d, the NC group is obviously higher than the CY group (P is less than 0.05); at 14, 21d and 278d, LbGp4M was significantly higher in both groups than CY (P < 0.05).

TABLE 2 Effect of Lycium barbarum glycopeptide on Chicken spleen index

Table 2Effect of Lycium Barbarum Glycopeptide on spleen index of chickens

2.3 Effect of Lycium barbarum glycopeptide on immunosuppression of Chicken peripheral blood lymphocyte transformation

Table 3 shows the effect of Lycium barbarum glycopeptide on the transformation of chicken peripheral blood lymphocytes. As is clear from Table 3, the peripheral blood lymphocyte A490nm values of the CY group immunosuppressed chicks were lower at each of the 7 th, 14 th, 21 th and 28 th days than in the remaining groups. Wherein the A490nm value of LbGp4M group peripheral blood lymphocytes at 7d is significantly higher than that of CY group (P < 0.05); the A490nm value of peripheral blood lymphocytes of LbGp4H, LbGp4M and LbGp4L groups is very higher than that of CY group (P <0.01) at 14d, and the NC group is higher than that of CY group (P < 0.05); the peripheral blood lymphocytes A490nm of LbGp4H and LbGp4M group at 21d are significantly higher than those of NC and CY groups (P < 0.01); peripheral blood lymphocytes A490nm of LbGp4H and LbGp4M at 28d were significantly higher than those of LbGp4L, NC and CY (P < 0.01).

TABLE 3 Effect of Lycium barbarum glycopeptides on Chicken peripheral blood lymphocyte transformation (A490 nm)

Table 3Effect of lycium Barbarum Glycopeptide on lymphocyte transformation in chicken peripheral blood(A490nm)

2.4 Effect of Lycium barbarum glycopeptide on immunosuppression of Chicken spleen tissue slices

FIG. 1 shows the effect of 7d glycopeptide of Lycium barbarum on spleen tissue structure of chickens, with lymphocytes in 7d groups distributed uniformly after the first administration and no significant difference between groups (P > 0.05). FIG. 2 shows the effect of 14d glycopeptides of Lycium barbarum on spleen tissue structure of chicken, at 14d, the number of center of hair growth of NC group is about 25-30, the number of center of hair growth of CY group is about 20, the center of hair growth is reduced, the marginal zone is fuzzy, the spleen sinus is widened, and the number of center of hair growth of each group of LbGp4 is about 40-50, which is significantly higher than that of NC and CY groups (P < 0.05). FIG. 3 shows the effect of the glycopeptide of Lycium barbarum at 21d on the spleen tissue structure of chicken, the number of germinal centers of NC and LbGp4 at 21d was about 30-40, and the number of germinal centers of CY was about 25-30, which was significantly lower than that of NC and LbGp4 (P <0.05), and the germinal centers were reduced and the spleen sinuses were widened. FIG. 4 shows the effect of 28d glycopeptides of Lycium barbarum on spleen tissue structure in chickens, with approximately 30-40 germinal centers in each group at 28d, and no significant difference between groups (P > 0.05).

The results show that the CY group has growth performance inhibition, spleen index reduction and peripheral blood lymphocyte transformation weakening, and spleen histological sections can show that the center of hair growth is reduced, the marginal zone is blurred, and the spleen sinus is widened, which indicates that the test chick immunosuppression construction is successful. The application result also suggests that the LbGp4 has the effect of improving the growth performance and spleen index of CY-induced immunosuppressed laying hens, wherein the dose concentration in the LbGp4 has the best effect (5 mg/kg. d). The application result proves that LbGp4 can improve the peripheral blood lymphocyte transformation effect of the laying hens. According to the application, through observation of histological changes of chicken spleens, CY can damage the chicken spleens and reduce the number of germinal centers and the number of lymphocytes, and LbGp4 can promote recovery of spleen damage to a certain extent.

The lycium barbarum glycopeptide (LbGp4) in the immune enhancing composition provided by the application has a regulating effect on Cyclophosphamide (CY) induced immunosuppression of chicks. The 200-feather 7-day-old halan laying hens are randomly divided into 5 groups, 40 feathers are taken in each group, namely a blank group (NC), a cyclophosphamide group (CY) and LbGp4 high (LbGp4H), medium (LbGp4M) and low (LbGp4L)3 dose groups, the NC group is injected with physiological saline, the other 4 groups are injected with 80 mg/kg-d CY continuously for molding, and 10 mg/kg-d LbGp4 is respectively given to LbGp4H, LbGp4M and LbGp4L groups which are continuously 7d after molding. At 7, 14, 21 and 28d after the first dose, 5 chickens were randomly selected for body weight, spleen index, peripheral blood lymphocyte transformation, and spleen histological analysis. The results show that the LbGp4H can obviously improve the weight of CY-induced immunosuppressed chickens (P is less than 0.05), and the LbGp4M group can obviously improve the weight of the CY-induced immunosuppressed chickens (P is less than 0.01); the LbGp4M group remarkably improves spleen index (P is less than 0.05) of CY induced immunosuppression chicks; the LbGp4H and LbGp4M groups extremely remarkably improve the CY-induced immunosuppressed chick peripheral blood lymphocyte conversion rate (P < 0.01); spleen histology showed that the number of germinal centers was significantly higher in groups 14 and 21d LbGp4 than in CY (P <0.05) after the first dose. In conclusion, the lycium barbarum glycopeptide has a remarkable antagonistic effect on cyclophosphamide-induced immunosuppression of chicks, and the effect is the best in a medium-dose group.

The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.