Chinese wolfberry fruit polysaccharide with effects of resisting oxidation, enhancing immunity and promoting growth and application of Chinese wolfberry fruit polysaccharide in healthy breeding of poultry

Abstract

The invention belongs to the technical field of plant polysaccharides, and particularly relates to Chinese wolfberry fruit polysaccharides with the effects of resisting oxidation, enhancing immunity and promoting growth and application of the Chinese wolfberry fruit polysaccharides in healthy breeding of poultry. Tests prove that the fructus lycii polysaccharide is prepared by taking fructus lycii as a raw material and adopting water extraction and alcohol precipitation, and in-vivo and in-vitro experiments prove that the fructus lycii polysaccharide simultaneously has the effects of resisting oxidation, enhancing immunity and promoting growth and has good health-care and treatment values. Meanwhile, the extraction method of the wolfberry fruit polysaccharide in the technical scheme is simple and low in cost, has universality, is easy for large-scale production, and has a good development prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharides, and specifically relates to a wolfberry polysaccharide with antioxidant, immune-enhancing and growth-promoting effects and an application thereof in healthy poultry breeding. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In the process of poultry farming, factors such as viruses, stress, mycotoxins, and drugs often damage the immune system of poultry, resulting in immunosuppression, increasing the susceptibility of poultry to pathogens and the incidence rate, leading to the occurrence of various poultry diseases. In severe cases, it can cause a large number of poultry deaths and cause economic losses. In addition, various peroxides can damage animal cells and cause diseases such as inflammation, which also affects the development of livestock and poultry farming.

[0004] A large number of studies have shown that Chinese herbal extracts can improve animal health through antioxidant and immune-enhancing pathways. Lycium barbarum (Lycium barbarum L.), a perennial woody plant, is a commonly used medicinal and edible resource that nourishes the liver and kidneys and clears the liver and improves eyesight. Lycium barbarum polysaccharide (LBP) is one of the main active ingredients of Lycium barbarum. Studies have shown that Lycium barbarum polysaccharide has antioxidant, anti-tumor, anti-inflammatory, immune-enhancing, reproductive protection and neuroprotective effects. Existing reports have shown that LBP can affect immune regulation by regulating immune cells such as dendritic cells (DCs), NK, T and B lymphocytes. LBP can promote DCs maturation by mediating Notch signals, enhance the phagocytic ability of macrophages, and promote the release of TNF-α and NO by macrophages, suggesting that Lycium barbarum polysaccharide has an immune-enhancing effect. At the same time, LBP can improve the intestinal immunity and intestinal flora of immunocompromised mice induced by cyclophosphamide, and exert an in vitro antioxidant effect by removing DPPH and ABTS. However, the structural composition and efficacy of Lycium barbarum polysaccharides obtained by different extraction methods are different, so the research on Lycium barbarum polysaccharides still needs to be further promoted and strengthened. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a Lycium barbarum polysaccharide with antioxidant, immune-enhancing and growth-promoting effects and its application in healthy poultry breeding. Specifically, the present invention adopts a water extraction and alcohol precipitation method to extract Lycium barbarum polysaccharide from Lycium barbarum, and in vitro and in vivo experiments prove that the Lycium barbarum polysaccharide has the effects of antioxidant, immune-enhancing and growth-promoting. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides the use of wolfberry polysaccharide in the following (a)-(c):

[0008] (a) Anti-oxidation or preparation of anti-oxidation products;

[0009] (b) enhancing immunity or preparing products that enhance immunity;

[0010] (c) Growth promotion or the preparation of growth promotion products.

[0011] Among them, in the application (a), the antioxidant effect is specifically manifested in that the wolfberry polysaccharide has good DPPH and ABTS free radical scavenging ability and high total antioxidant capacity (T-AOC).

[0012] In (b), the immunity enhancement is specifically manifested as antagonizing immunosuppression, and the immunosuppression is specifically cyclophosphamide-induced immunosuppression; the antagonizing immunosuppression is specifically manifested as effectively improving the body weight, spleen index and peripheral lymphocyte transformation rate of immunosuppressed animals, and improving spleen pathological damage; more specifically, the antagonizing immunosuppression includes regulating (alleviating) transcriptional disorders and metabolic disorders under immunosuppressive conditions.

[0013] The product can be used by humans or non-human animals, and the non-human animals can be birds and mammals, especially poultry, such as chickens, ducks, geese, quails, pigeons, etc. The product can be a medicine or a food, and as mentioned above, the medicine and food can be used by humans or non-human animals.

[0014] The wolfberry polysaccharide in the present invention can be prepared by water extraction and alcohol precipitation. Specifically, it is prepared by the following method:

[0015] The wolfberry is crushed, soaked in water and subjected to microwave-enhanced treatment, and then subjected to heating and reflux extraction to obtain an extract. After the extract is concentrated, ethanol is added for alcohol precipitation, and the extract is allowed to stand for filtration and purified to obtain the extract.

[0016] The mass volume ratio of wolfberry powder to water is 1:40-60, g / mL, preferably 1:50;

[0017] The soaking time is 1-60 min, preferably 30 min;

[0018] The heating reflux extraction can be repeated 2 to 3 times, specifically, the extract after reflux extraction is filtered under reduced pressure, the filter residue is extracted again 2 to 3 times, and the extracts obtained each time are combined to obtain the total extract for subsequent steps.

[0019] The extract is concentrated to 1 / 5-1 / 2 of the original volume, preferably 1 / 4;

[0020] The amount of ethanol added is 2-10 times, preferably 5 times, the volume of the concentrated extract; the ethanol is high-concentration ethanol, specifically 95% ethanol.

[0021] The purification step includes the steps of removing protein by Sevag method, concentration dialysis and washing with organic solvent.

[0022] The organic solvent includes anhydrous ethanol, acetone and ether.

[0023] The second aspect of the present invention provides a product comprising the above-mentioned wolfberry polysaccharide.

[0024] The product has the following effects (a)-(c):

[0025] (a) Antioxidant;

[0026] (b) enhance immunity;

[0027] (c) Growth promotion.

[0028] Among them, in the application (a), the antioxidant effect is specifically manifested in that the wolfberry polysaccharide has good DPPH and ABTS free radical scavenging ability and high total antioxidant capacity (T-AOC).

[0029] In (b), the immunity enhancement is specifically manifested as antagonizing immunosuppression, and the immunosuppression is specifically cyclophosphamide-induced immunosuppression; the antagonizing immunosuppression is specifically manifested as effectively improving the body weight, spleen index and peripheral lymphocyte transformation rate of immunosuppressed animals, and improving spleen pathological damage; more specifically, the antagonizing immunosuppression includes regulating (alleviating) transcriptional disorders and metabolic disorders under immunosuppressive conditions.

[0030] The product can be used by humans or non-human animals, and the non-human animals can be birds and mammals, especially poultry, such as chickens, ducks, geese, quails, pigeons, etc. The product can be a medicine or a food, and as mentioned above, the medicine and food can be used by humans or non-human animals.

[0031] Beneficial technical effects of one or more of the above technical solutions:

[0032] The above technical solution provides a wolfberry polysaccharide and a specific extraction and preparation method thereof. Experiments have shown that the wolfberry polysaccharide is prepared by water extraction and alcohol precipitation using wolfberry as raw material. In vitro and in vivo experiments have shown that the wolfberry polysaccharide has the effects of anti-oxidation, enhancing immunity and promoting growth, and has good health and therapeutic value. At the same time, the extraction method of wolfberry polysaccharide in the above technical solution is simple, low-cost, universal, easy to mass-produce, and has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 The in vitro antioxidant activity of the Lycium barbarum polysaccharide of the present invention: (A) total antioxidant capacity (T-AOC); (B) DPPH free radical scavenging rate; (C) ABTS free radical scavenging rate.

[0035] Figure 2 The effect of LBP of the present invention on cyclophosphamide immunosuppressed chicks. (A) Experimental design; (B) Body weight; (C) Spleen index; (D) Lymphocyte metastasis.

[0036] Figure 3 This is a spleen pathology diagram of the present invention.

[0037] Figure 4 The results of transcriptome sequencing of the present invention are analyzed. (A) Experimental design; (B) PCA analysis results; (C) Volcano plot of up-regulated and down-regulated genes in the NC-CY group; (D) Volcano plot of up-regulated and down-regulated genes in the CY-LbGp group; (E) Intersection Venn diagram of up-regulated genes in CY vs NC and down-regulated genes in LbGp vs CY; (F) Intersection Venn diagram of down-regulated genes in CY vs and up-regulated genes in LbGp vs CY.

[0038] Figure 5 Differential gene analysis for callback of the present invention. (A) GO analysis of the intersection genes of CY vs NC up-regulated genes and LbGp vs CY down-regulated genes; (B) GO analysis of the intersection genes of CY vs down-regulated genes and LbGp vs CY up-regulated genes; (C) KEGG analysis of the intersection genes of CY vs NC up-regulated genes and LbGp vs CY down-regulated genes; (D) KEGG analysis of the intersection genes of CY vs down-regulated genes and LbGp vs CY up-regulated genes. (E) PPI network diagram of callback genes.

[0039] Figure 6 Metabolomics analysis of the present invention; (A) PCA analysis; (B) PLS-DA analysis; (C) Permutation testing. (D) CY vs NC vs LbGp differential metabolite analysis; (E) KEGG enrichment analysis of differential metabolite analysis.

[0040] Figure 7 This is the correlation analysis between transcriptome and metabolome of the present invention. (A) Correlation between differential genes and differential metabolites between CYvs control group NC. (B) Correlation between differential genes and differential metabolites between CYbGp vs CY. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0043] In a typical embodiment of the present invention, the use of wolfberry polysaccharide in the following (a)-(c) is provided:

[0044] (a) Anti-oxidation or preparation of anti-oxidation products;

[0045] (b) enhancing immunity or preparing products that enhance immunity;

[0046] (c) Growth promotion or the preparation of growth promotion products.

[0047] Among them, in the application (a), the antioxidant effect is specifically manifested in that the wolfberry polysaccharide has good DPPH and ABTS free radical scavenging ability and high total antioxidant capacity (T-AOC).

[0048] In (b), the immunity enhancement is specifically manifested as antagonizing immunosuppression, and the immunosuppression is specifically cyclophosphamide-induced immunosuppression; the antagonizing immunosuppression is specifically manifested as effectively improving the body weight, spleen index and peripheral lymphocyte transformation rate of immunosuppressed animals, and improving spleen pathological damage; more specifically, the antagonizing immunosuppression includes regulating (alleviating) transcriptional disorders and metabolic disorders under immunosuppressive conditions.

[0049] The product can be used by humans or non-human animals, and the non-human animals can be birds and mammals, especially poultry, such as chickens, ducks, geese, quails, pigeons, etc. The product can be a medicine or a food, and as mentioned above, the medicine and food can be used by humans or non-human animals.

[0050] The wolfberry polysaccharide in the present invention can be prepared by water extraction and alcohol precipitation. Specifically, it is prepared by the following method:

[0051] The wolfberry is crushed, soaked in water and subjected to microwave-enhanced treatment, and then subjected to heating and reflux extraction to obtain an extract. After the extract is concentrated, ethanol is added for alcohol precipitation, and the extract is allowed to stand for filtration and purified to obtain the extract.

[0052] The mass volume ratio of wolfberry powder to water is 1:40-60, g / mL, preferably 1:50;

[0053] The soaking time is 1-60 min, preferably 30 min;

[0054] The microwave treatment conditions are: 300-700 W (preferably 500 W) for 10-30 min (preferably 20 min).

[0055] The specific conditions of heating reflux extraction are: heating and boiling reflux extraction, and the reflux extraction time is 0.1 to 3 hours;

[0056] The heating (boiling) reflux extraction can be repeated 2 to 3 times, specifically, the extract after reflux extraction is filtered under reduced pressure, and the filter residue is repeatedly heated (boiling) reflux extraction 2 to 3 times, and the extract obtained each time is combined to obtain the total extract for subsequent steps. The mass volume ratio of the filter residue to water is 1:20 to 50, g / mL, preferably 1:30.

[0057] The extract is concentrated to 1 / 5-1 / 2 of the original volume, preferably 1 / 4;

[0058] The amount of ethanol added is 2-10 times, preferably 5 times, the volume of the concentrated extract; the ethanol is high-concentration ethanol, specifically 95% ethanol.

[0059] The purification step includes the steps of removing protein by Sevag method, concentration, dialysis and washing with organic solvent.

[0060] The organic solvent includes anhydrous ethanol, acetone and ether.

[0061] The molecular weight cut-off of the dialysis is 2000-5000Da, preferably 3000Da.

[0062] In another specific embodiment of the present invention, a product is provided, wherein the product comprises the above-mentioned wolfberry polysaccharide.

[0063] The product has the following effects (a)-(c):

[0064] (a) Antioxidant;

[0065] (b) enhance immunity;

[0066] (c) Growth promotion.

[0067] Among them, in the application (a), the antioxidant effect is specifically manifested in that the wolfberry polysaccharide has good DPPH and ABTS free radical scavenging ability and high total antioxidant capacity (T-AOC).

[0068] In (b), the immunity enhancement is specifically manifested as antagonizing immunosuppression, and the immunosuppression is specifically cyclophosphamide-induced immunosuppression; the antagonizing immunosuppression is specifically manifested as effectively improving the body weight, spleen index and peripheral lymphocyte transformation rate of immunosuppressed animals, and improving spleen pathological damage; more specifically, the antagonizing immunosuppression includes regulating (alleviating) transcriptional disorders and metabolic disorders under immunosuppressive conditions.

[0069] The product can be used by humans or non-human animals, and the non-human animals can be birds and mammals, especially poultry, such as chickens, ducks, geese, quails, pigeons, etc. The product can be a medicine or a food, and as mentioned above, the medicine and food can be used by humans or non-human animals.

[0070] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0071] Example 1: Extraction and in vitro antioxidant capacity of Lycium barbarum polysaccharides:

[0072] 1 Experimental methods:

[0073] 1.1 Experimental materials and medicinal materials:

[0074] Wolfberry, distilled water

[0075] 1.2 Weigh the Chinese wolfberry medicinal material and crush it, put it into a three-necked flask, soak it for 30 minutes at a certain material-water ratio (1:50, g:mL), microwave it at 500W power for 20 minutes, and after strengthening it, place it in an electric heating mantle to heat and boil it, reflux extract it for 1 hour, and filter the extract under reduced pressure. Add 30 times of deionized water (g:mL) to the residue and repeat the extraction for 2 times, each time for 30 minutes. After combining the 3 extracts, use a rotary evaporator to concentrate the filtrate to 1 / 4 of the original volume. Add 5 times the volume of 95% ethanol to the concentrated liquid, stir it evenly, and let it stand for 24 hours for alcohol precipitation. After the alcohol precipitation is completed, filter it, dissolve the precipitate in deionized water to make a 0.1g / mL solution, and shake it thoroughly with an equal volume of Sevag reagent (chloroform: n-butanol 4:1, v:v) to precipitate and remove the protein in the polysaccharide. Repeat the operation many times until the polysaccharide solution no longer produces precipitation. The polysaccharide solution was concentrated under reduced pressure and dialyzed against deionized water (molecular weight cutoff 3000Da) for 48 hours. The dialyzed polysaccharide solution was concentrated to 0.1 g of the original medicinal material per milliliter. Five times the volume of 95% ethanol was added for precipitation overnight. The precipitate was washed three times with anhydrous ethanol, acetone and ether in sequence. The precipitate was dried, dissolved with deionized water and dialyzed (molecular weight cutoff 3000Da) for 48 hours, concentrated, and freeze-dried to obtain wolfberry polysaccharide.

[0076] 1.3 Determination of the antioxidant capacity of wolfberry polysaccharides in vitro:

[0077] The method for determining the antioxidant activity described in 1.3.1 (T-AOC method) comprises the following steps: preparing a test solution of wolfberry polysaccharide with deionized water at a mass concentration of 1, 2, 3, 4, 5, 6, 7, or 8 mg / mL.

[0078] (1) Drawing of standard curve:

[0079] Establishment of calibration curve: Dilute 40 μmol / mL FeSO4 standard solution with distilled water to the following concentrations: 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 μmol / mL.

[0080] Prepare standard solution: Take 100 μL of each concentration of Fe 2+ Standard solution, add 100 μL FRAP working solution. Mix well at room temperature, react for 10 minutes, and measure the absorbance at 593 nm. Record A standard and A1 blank, and calculate ΔA standard = A standard - A1 blank.

[0081] Drawing a standard curve: Fe 2+ The final concentration (0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 μmol / mL) was used as the abscissa (x), and the ΔA standard was used as the ordinate (y). A standard curve was drawn to obtain the linear regression equation y=kx+b.

[0082] (2) Sample measurement: Add reagents: Add 180 μL of FRAP working solution to each well of a 96-well plate. Add 10 μL of the sample to be measured to the measurement group, and add 10 μL of distilled water to the blank group 2 (only 1-2 measurements are required). Reaction: Mix well at room temperature and react for 10 minutes.

[0083] (3) Absorbance measurement: Measure the absorbance at 593 nm, record A measurement and A2 blank, and calculate ΔA = A measurement - A2 blank.

[0084] (4) Sample concentration calculation: Substitute the ΔA measured value into the standard curve equation y=kx+b to calculate the sample Fe 2+ Concentration x (μmol / mL).

[0085] (5) Calculation of total antioxidant capacity:

[0086] Total antioxidant capacity (umol / ml) = x * V 反 / V 样

[0087] 1.3.2 The method for determining the antioxidant activity (DPPH method) comprises the following steps: preparing a test solution of wolfberry polysaccharide with deionized water at a mass concentration of 1, 2, 3, 4, 5, 6, 7, 8 mg / mL,

[0088] (1) Set up the photometer: Preheat the spectrophotometer or the microplate reader for more than 30 minutes, adjust the wavelength to 515 nm, and zero the reagent.

[0089] (2) Preparation of Trolox positive control (TR):

[0090] If you need to set Trolox as a positive control, it is recommended to use 20mmol / L Trolox solution.

[0091] Prepare Trolox dilutions of different concentrations (1.6, 1.2, 0.8, 0.4, 0.2, 0.1 mmol / L) according to the required final concentration. If the extract concentration is high, it is recommended to use the extract to prepare a Trolox dilution greater than 1.6 mmol / L for reagent 3.

[0092] (3) Preparation of reaction system:

[0093] Add the following reagents to a 96-well plate or centrifuge tube: Assay group: 10 μL sample + 190 μL DPPH working solution. Control group: 10 μL sample + 190 μL DPPH working solution. TR group: 10 μL Trolox diluent + 190 μL DPPH working solution. Blank group: 200 μL DPPH working solution.

[0094] (4) Mixing reaction: After thorough mixing, react at room temperature in the dark for 30 minutes.

[0095] (5) Absorbance measurement: Measure the absorbance of each group at 515 nm and record them as A measurement, A control, ATR and A blank. Each dilution TR group and blank group only need to be measured 1-2 times. A control group should be set up for each sample.

[0096] The calculation formula of the antioxidant activity determination method (DPPH method) is:

[0097] DPPH free radical scavenging rate of the sample (%) = (A 空白 -A 测定 +A 对照 ) / A 空白 ×100%

[0098] Trolox positive control group DPPH free radical scavenging rate (%) = (A 空白 -ATR) / A 空白 ×100%

[0099] 1.3.3 The method for determining antioxidant activity (ABTS method) includes the following steps: Lycium barbarum polysaccharide is prepared into test solutions with mass concentrations of 1, 2, 3, 4, 5, 6, 7, and 8 mg / mL with deionized water.

[0100] (1) Instrument preheating: Preheat the spectrophotometer or microplate reader for at least 30 minutes, adjust the wavelength to 405 nm, and zero it with distilled water.

[0101] (2) Trolox positive control (TR) configuration: If a Trolox positive control curve is required, it is recommended to dilute the 20mmol / L Trolox solution to 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1mmol / L, i.e., Trolox dilutions of different concentrations.

[0102] (3) Sample and reagent preparation: Add the following reagents in the ELISA tubes of the 96-well plate in sequence: Assay group: 10 μL sample, 10 μL distilled water, 170 μL ABTS working solution. Control group: 10 μL sample, 190 μL distilled water, 170 μL ABTS working solution. TR group: 10 μL Trolox diluent, 10 μL distilled water, 170 μL ABTS working solution. Blank group: 190 μL distilled water, 170 μL ABTS working solution. Reaction: After thorough mixing, react at room temperature in the dark for 6 minutes.

[0103] (4) Absorbance determination:

[0104] Measure the absorbance at a wavelength of 405 nm and record it as A determination, A control, ATR and A blank. Note: Each concentration of TR group and blank group only needs to be measured 1-2 times, and a control tube must be set up for each sample.

[0105] (5) Calculation of ABTS free radical scavenging ability:

[0106] The ABTS free radical scavenging rate of the sample (D S %)=(A 空白 -A 测定 +A 对照 ) / A 空白 ×100%

[0107] The free radical scavenging rate of Trolox positive control group (D TR %)=(A 空白 -ATR) / A 空白 ×100%

[0108] The antioxidant capacity of the prepared wolfberry polysaccharide was tested by in vitro antioxidant tests (T-AOC, DPPH, ABTS). Figure 1 As shown, it is confirmed that the polysaccharide has significant antioxidant activity, demonstrating its outstanding value in the fields of health care and anti-aging.

[0109] Example 2: Evaluation of the effect of Lycium barbarum polysaccharide on antagonizing immunosuppression

[0110] 1 Experimental methods:

[0111] 1.1 Experimental Materials

[0112] Chicken peripheral blood lymphocyte separation medium kit was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., neutral formalin fixative was purchased from Beijing Solebow Technology Co., Ltd., and chicken peripheral blood lymphocyte complete culture medium was purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0113] Two hundred one-day-old white-feathered broilers were randomly divided into five groups, each with 40 birds (n=40), after adaptive feeding to 7 days of age, including blank control group (NC), cyclophosphamide group (CY) and Lycium barbarum polysaccharide group (LbGp). LbGp was divided into three dose groups (LbGpH, LbGpM, LbGpL), high, medium and low dose groups (LbGpH, LbGpM, LbGpL). CY and LbGp were injected with 80 mg / kg·d CTX into the pectoral muscle for 3 consecutive days to establish the immunosuppressive model, and the NC group was injected with an equal volume of saline. After modeling, LbGpH, LbGpM and LbGpL were given 10, 5, and 2.5 mg / kg·d LBP through drinking water for 7 consecutive days. On the 7th, 14th, 21st and 28th days after the first administration of LbGpH, 5 chickens were randomly selected from each group for the detection of spleen index, lymphocyte proliferation assay, and spleen pathological damage observation.

[0114] 1.2 Physiological index determination of 200 one-day-old Hy-Line laying hens: The body weight, spleen weight, and spleen index of all laying hens were measured. Lymphocyte proliferation was measured, and the liver index of spleen pathological damage observation was calculated according to the following formula:

[0115]

[0116] 1.3 Hematoxylin-eosin staining: Liver tissue samples of Kunming mice were first stored in polyoxymethylene (4%) for at least two days for hematoxylin-eosin staining, and pathological analysis was performed using a microscope.

[0117] 1.4 Lymphocyte proliferation assay

[0118] Blood was collected from the heart and lymphocytes were subsequently isolated. The MTT method was used to determine the lymphocyte OD490 mm value.

[0119] 1.5 Determination of the number of germinal centers in the spleen: The germinal centers of 60 fields in each group were counted manually.

[0120] 2 Experimental results

[0121] 2.1 As Figure 2 As shown, medium dose of Lycium barbarum polysaccharide can improve the body weight, spleen index, number of peripheral blood lymphocytes and number of spleen germinal centers of immunosuppressed laying hens.

[0122] 2.2 As Figure 3 As shown, pathological analysis showed that lymphocytes were evenly distributed in each group on the 7th day after the first administration. On the 14th day, the number of germinal centers in the NC group was about 25-30, and the number of germinal centers in the CY group was about 20. The germinal centers were reduced, the marginal areas were blurred, and the spleen was widened. The number of germinal centers in the LbGp groups was about 40-50, which was significantly higher than that in the NC and CY groups (P<0.05). On the 21st day, the number of germinal centers in the NC and LbGp groups was about 30-40, and the number of germinal centers in the CY group was about 25-30, which was significantly lower than that in the NC and LbGp groups (P<0.05). The germinal centers were reduced and the spleen was widened. On the 28th day, the number of germinal centers in each group was about 30-40, and there was no significant difference between the groups (P>0.05). This shows that the concentration of LbGpM is more appropriate, and the consumption cycle can be extended to 28 days.

[0123] Example 3 Lycium barbarum polysaccharide on transcriptional regulation of immunosuppressed laying hens

[0124] 1 Experimental methods

[0125] 1.1 Drugs and materials

[0126] 120 one-day-old Hy-Line brown laying hens (Huadu Yukou Poultry Co., Ltd., China) were randomly divided into three groups after adaptive feeding to 7 days of age, namely NC, CY and LbGp, with 40 birds in each group. The NC group was intramuscularly injected with normal saline for 3 consecutive days, and the other two groups were intramuscularly injected with 80 mg / kgd cyclophosphamide for 3 consecutive days to establish the model. After the model was established, the CYLbGp group was given 5 mg / kgd of Lycium barbarum polysaccharide through drinking water for 30 consecutive days. On the first day after the end of the administration of the LbGp group, 6 chickens in each group were randomly selected to take the spleen and put it in a cryopreservation tube and stored in an ultra-low temperature refrigerator at -80℃ for transcriptome sequencing.

[0127] 1.2 Detection Method

[0128] Total RNA was extracted from chick spleen tissue samples using Trizol reagent (Invitrogen), and then the concentration, purity and integrity of RNA were detected by Nanodrop2000 and agarose gel electrophoresis, and the RIN value was determined using Agilent2100. After adding bufer, small fragments of mRNA of about 300 bp were separated using Oligo (dT) magnetic beads, and random primers were added to synthesize the first-strand cDNA, followed by second-strand synthesis. The obtained double-stranded cDNA was PCR amplified (15 cycles), and finally bridge PCR amplification was performed on cBot to complete the preparation of the entire library. The constructed library was sequenced using the IlumimnaNovaSeg6000 platform (PE library, read length 2x150 bp).

[0129] 1.3 RNA-Seq quality assessment and sequence alignment, transcriptome data analysis

[0130] Follow the R package instructions.

[0131] 1.4 RT-GPCR validation

[0132] Five differentially expressed genes CD83, CCL4, PSPH, LPIN2 and ULK2 were randomly selected from the differentially expressed genes, and the RT-GPCR method was used to verify the accuracy of the transcriptome sequencing results. B-actin was used as the internal reference gene. The primers used were synthesized by Shanghai Meiji Biotechnology Co., Ltd. The RT-gPCR reaction system was 20L, 2XChamOSYBR Color gPCR MasterMix 10L, 0.8ul of each front and rear primer, 0.4μL of 50XROX Reference Dye 1. CDNA2uL, ddHz06L. The reaction conditions were: 95℃ denaturation for 5s, 55℃ annealing for 30s, 72℃ extension for 40s, and 40 cycles. The 2-ΔΔCT method was used to determine the relative expression of each experimental group compared with the control group.

[0133] 2 Experimental results

[0134] In order to understand the expression of differentially expressed genes between samples and groups, PCA analysis was performed using DESeq2. The results showed that the gene expression profiles of the CYLbGp group and the NC group overlapped, indicating that there was no significant difference in the gene expression levels between the two groups, while the CY group had a larger difference between the NC and CYLbGp groups, and the gene expression level changed significantly ( Figure 4 , B), indicating that CTX changed the gene expression in the spleen of chickens, while LBP had a significant improvement on the effect of CTX stimulation. Differentially expressed genes were screened using P-adjust < 0.05 and log2FC| ≥ 0 as the criteria. Compared with the NC group, 408 differentially expressed genes were screened in the CY group, of which 193 were upregulated and 215 were downregulated. Compared with the CY group, a total of 896 differentially expressed genes were screened in the CYLbGp group, of which 408 were upregulated and 488 were downregulated ( Figure 4 , C&D). To identify the main target genes of LBP in antagonizing the immunosuppressive effect of CTX, the intersection of differentially regulated genes upregulated in the CY group compared with the NC group and differentially regulated genes downregulated in the CYLbGp group compared with the CY group was selected, and 108 differentially regulated genes were obtained. The intersection of differentially regulated genes downregulated in the CY group compared with the NC group and differentially regulated genes upregulated in the CYLbGp group compared with the CY group was selected, and 70 differentially regulated genes were obtained ( Figure 4 ,E&F). The 108 genes downregulated after intervention with Lycium barbarum polysaccharide were significantly enriched in 6 GO terms enriched in biological processes (BP) (Padiust < 0.05), including regulation of biological quality, cellular response to chemical stimulus, biological regulation, regulation of multicellular organismal process, homeostatic process, and response to chemical ( Figure 5, A). The 70 genes that were downregulated after CTX stimulation and upregulated after intervention with Lycium barbarum polysaccharide were also significantly enriched in 23 GO BP items (Padiust < 0.05). Many of these items are related to immune regulation, including positive regulation of immune system process, lymphocyte activation, immune system process, regulation of immune system process, leukocyte activation, leukocyte differentiation, regulation of leukocyte cell-celadhesion and other biological processes ( Figure 5 ,B). KEGG pathway enrichment analysis was performed on 108 differentially expressed genes and 70 up-regulated genes after intervention with Lycium barbarum polysaccharide. The results showed that 13 pathways were significantly enriched among the 108 differentially expressed genes after CTX stimulation, mainly involving mitochondria, mTOR signaling pathway, AMPK signaling pathway and other pathways related to cell autophagy ( Figure 5 C). The 70 differentially expressed genes significantly enriched in pathways that were upregulated after intervention with Lycium barbarum polysaccharides were mainly involved in immune-related signaling pathways such as chemokine signaling pathway, C-type lectin receptor signaling pathway, B cell receptor signaling pathway, platelet activation, natural killer cell-mediated cytotoxicity, Th1 and Th2 cell differentiation ( Figure 5 ,D). A total of 12 differentially expressed genes were enriched in the above pathways. PPI network diagram of differentially expressed genes after intervention with Lycium barbarum polysaccharide ( Figure 5 ,E). The results showed that PIK3CD, JAK3, GATA3, HIFIA, NKX2-5 and INPPL1 were the key genes, and except for NKX2-5, they were all enriched genes in the above-mentioned differential pathways, which may be used as Lycium barbarum polysaccharides to antagonize immune suppression in chicks.

[0135] The results of RT-qPCR validation showed that CD83 and CCL4 showed an up-regulation trend in the prognosis of Lycium barbarum polysaccharide, while PSPH, LPIN2, and ULK2 showed a down-regulation trend, which was basically consistent with the trend of transcriptomics data ( Figure 5 F&G), indicating that the transcriptome data have high reliability.

[0136] Example 4 Regulatory effect of Lycium barbarum polysaccharide on spleen metabolism in immunosuppressed laying hens

[0137] 1 Experimental methods

[0138] 1.1 Drugs and materials

[0139] Metabolomics was performed by Meiji Biotechnology Co., Ltd.

[0140] 1.2 Detection Method

[0141] The sample is from Example 3. Chromatographic conditions: AB SCIEXExionLCAD System ultra-high performance liquid chromatography was used for analysis, and the chromatographic column was selected as ACOUITYUPLC HSS T3 (100 mm x2.1 mm id, 1.8 um) of Waters Company of the United States, and the column temperature was set to 40°C: mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), and the gradient elution program was set to (0-2.5 min, 0%-25% B; 2.5-9 min, 25%-100% B; 9-13 min, 100% B: 13-13.1 min, 100-0% B: 13.1-16 min, 0% B): flow rate was 0.40 mL / min, and the injection volume was 10 μL.

[0142] Mass spectrometry conditions: AB SCIEX-Triple TOF 5600 mass spectrometer was used for mass spectrometry analysis. The point spray ion source temperature was 550°C, the positive and negative electrode voltages were 5000 V and -4000 V, respectively, the ion source gases G1, G2 and the curtain gas were 50, 50 and 30 psi, respectively, and the ion pairs in the triple quadrupole (Qtrap) were scanned and detected (range 50-1000, cycle time 510 ms) according to the optimized declustering voltage (80 V) and collision energy (40+20).

[0143] ProgenesisQ (Waters, USA) software was used to remove eigenvalues ​​with missing values ​​> 20% in each group, and the missing values ​​were filled with the minimum value and normalized according to the sum. Then the variables with relative standard deviation > 30% in OC samples were removed and log10 logarithmization was performed to obtain the preprocessed data for subsequent analysis. Ropls (R packages) was used for principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discrimination analysis (OPLS-DA). Differential metabolites were screened based on the results of t-test and OPLS-DA (P < 0.05, VIP > 1). The pathway analysis module and joint pathway analysis module in the MateboAnanlyst 5.0 online platform were used to investigate the effects of Lycium barbarum polysaccharides on the spleen metabolic pathways in immunosuppressed chicks.

[0144] 2 Results

[0145] The spleen metabolome of CY, CYLbGp and NC groups changed significantly 30 days after drug administration, indicating that cyclophosphamide had a significant effect on the spleen metabolome of chickens ( Figure 6 , A). There was a certain angle between the metabolic profiles of the CY group and the CYLbGp group, indicating that there were also significant differences in the spleen metabolite profiles between the CYLbGp group and the CY group. In order to further analyze the effectiveness of Lycium barbarum polysaccharide in antagonizing immunosuppression in chicks, PLS-DA analysis was performed on the NC, CY and CYLbGp groups. The results showed that the CYLbGp group was located between the NC and CY groups, indicating that Lycium barbarum polysaccharide had a certain correction effect on the disorder of spleen metabolites in immunosuppressed chicks ( Figure 6 , B). The compounds were identified by HMDB database. The screening results showed that there were 114 metabolites in the CY and NC groups. Then, the differential metabolites that were regulated by the prognosis of wolfberry polysaccharide were selected. Finally, 20 differential metabolites that met the conditions were selected ( Figure 6,D), aminocaproic acid, carnitine PS (18:1 (9Z) / 0:0) were extremely significantly different (P < 0.01), LySOPE (22:4 (7Z, 10Z, 13Z, 16Z) 10:0) and SM (d18:0 / 16:1 (9Z)) were significantly different (P < 0.05). The 20 metabolites that were homologous after the intervention of wolfberry polysaccharides were imported into the MetaboAnalyst5.0 platform for pathway analysis. The pathway enrichment results showed that wolfberry polysaccharides affected important metabolic pathways in immunosuppressive chicks. Lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, carbohydrate metabolism, metabolism of other amino acids, synthesis of other secondary metabolites, xenobiotic biodegradation and metabolism, glycan biosynthesis and metabolism, metabolism, environmental information processing including membrane transport, signal molecules and interactions, signal transduction, translation folding, genetic information processing including translation folding, classification and degradation, cellular programs including cell growth and death, biological systems including sensory systems and metabolic diseases.

[0146] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of Lycium barbarum polysaccharide in the following (a)-(c): (a) Anti-oxidation or preparation of anti-oxidation products; (b) enhancing immunity or preparing products that enhance immunity; (c) Growth promotion or the preparation of growth promotion products.

2. The use according to claim 1, characterized in that: In the application (a), the antioxidant effect is specifically manifested in that the wolfberry polysaccharide has good DPPH and ABTS free radical scavenging ability and high total antioxidant capacity (T-AOC).

3. The use according to claim 1, characterized in that: In (b), the immunity enhancement is specifically manifested as antagonizing immunosuppression, and the immunosuppression is specifically cyclophosphamide-induced immunosuppression.

4. The use according to claim 3, characterized in that: The antagonistic immunosuppression is specifically manifested in effectively improving the body weight, spleen index and peripheral lymphocyte transformation rate of immunosuppressed animals, and improving spleen pathological damage; specifically, the antagonistic immunosuppression includes regulating (alleviating) transcriptional level disorders and metabolic disorders under immunosuppressive conditions.

5. The use according to claim 3, characterized in that: The product is for use by humans or non-human animals, wherein the non-human animals are birds and mammals, and further poultry; The product is a medicine or a food.

6. The use according to claim 1, characterized in that: Lycium barbarum polysaccharide is prepared by water extraction and alcohol precipitation method. Specifically, it is prepared by the following method: The wolfberry is crushed, soaked in water and subjected to microwave-enhanced treatment, and then subjected to heating and reflux extraction to obtain an extract. After the extract is concentrated, ethanol is added for alcohol precipitation, and the extract is allowed to stand for filtration and purified to obtain the extract.

7. The use according to claim 6, characterized in that The mass volume ratio of wolfberry powder to water is 1:40-60, g / mL; Soaking time is 1-60min; Microwave treatment conditions: 300-700W for 10-30min; The specific conditions of heating reflux extraction are: heating and boiling reflux extraction, and the reflux extraction time is 0.1 to 3 hours; Furthermore, the heating reflux extraction is repeated 2 to 3 times, specifically, the extract after reflux extraction is subjected to reduced pressure filtration, the filter residue is heated and refluxed again for 2 to 3 times, and the extracts obtained each time are combined to obtain the total extract for subsequent steps; The extract is concentrated to 1 / 5-1 / 2 of the original volume; The amount of ethanol added is 2-10 times the volume of the concentrated extract; the ethanol is high-concentration ethanol, specifically 95% ethanol.

8. The use according to claim 7, characterized in that The purification step includes the steps of removing protein by Sevag method, concentration, dialysis and washing with organic solvent; The molecular weight cut-off of the dialysis is 2000-5000Da, preferably 3000Da.

9. The use according to claim 8, characterized in that The organic solvent includes anhydrous ethanol, acetone and ether.

10. A product, characterized in that The product comprises the wolfberry polysaccharide used in any one of claims 1 to 9; The product has the following effects (a)-(c): (a) Antioxidant; (b) enhance immunity; (c) Growth promotion.

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