阅读说明：本技术 一种基于中药提取物的鸭天然免疫增强剂的制备方法 (Preparation method of duck natural immunopotentiator based on traditional Chinese medicine extract ) 是由 李国勤 卢立志 顾天天 田勇 陈黎 曾涛 沈军达 陶争荣 杜雪 徐坚 于 2019-10-12 设计创作，主要内容包括：本发明公开了一种基于中药提取物的鸭天然免疫增强剂的制备方法,包括以下步骤：(1)芦丁标准曲线的建立；(2)黄芪黄酮的提取；(3)黄芪黄酮的浓度计算。本发明提供的基于中药提取物的鸭天然免疫增强剂,可以显著升高鸭血清细胞因子IL-1β、IFN-α和IFN-β水平,上调鸭免疫器官肝脏、脾脏、胸腺和法氏囊中天然免疫相关基因RIG-I、TLR3、TLR7、IFN-α、IFN-β、IL-1β和TNF-α、炎症相关基因COX2和iNOS、细胞凋亡相关基因Caspase3的组织表达水平,增强鸭天然免疫反应和抗病性能。(The invention discloses a preparation method of a duck natural immunopotentiator based on a traditional Chinese medicine extract, which comprises the following steps: (1) establishing a rutin standard curve; (2) extracting astragalus flavone; (3) and (4) calculating the concentration of the astragalus flavone. The duck natural immunopotentiator based on the traditional Chinese medicine extract provided by the invention can obviously increase the levels of duck serum cytokines IL-1 beta, IFN-alpha and IFN-beta, up-regulate the tissue expression levels of natural immunity related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha, inflammation related genes COX2 and iNOS, and apoptosis related gene Caspase3 in duck immune organs liver, spleen, thymus and bursa of fabricius, and enhance the natural immune response and disease resistance of ducks.)

1. A preparation method of a duck natural immunopotentiator based on a traditional Chinese medicine extract is characterized by comprising the following steps:

(1) establishment of rutin standard curve

Accurately weighing 5.0mg of rutin standard, diluting with 70% ethanol to different concentrations, measuring light absorption values of rutin solutions with different concentrations at 510nm, and establishing a linear regression equation;

(2) extraction of astragalus flavone

Crushing astragalus, sieving with a 40-mesh sieve, accurately weighing 10g, adding 10 times of 90% ethanol by mass, carrying out hot reflux extraction in a water bath, cooling, filtering, fixing the volume of the filtrate in a volumetric flask, and measuring the absorbance of the filtrate;

(3) calculation of the concentration of Astragalus flavone

And calculating the concentration of the flavone according to the established linear regression equation.

2. The method for preparing a natural duck immunopotentiator based on Chinese herbal medicine extracts as claimed in claim 1, wherein the temperature of the water bath in step 2 is 75 ℃.

3. The method for preparing a natural duck immunopotentiator based on Chinese herbal medicine extracts as claimed in claim 1, wherein the time for reflux extraction in step 2 is 2 hours.

Technical Field

The invention belongs to the technical field of agriculture, and relates to a preparation method of a duck natural immunopotentiator based on a traditional Chinese medicine extract.

Background

With the rapid development of the large-scale and intensive breeding mode of the animal husbandry in China, the harm of animal diseases is becoming more serious day by day, and the animal diseases become the biggest obstacle influencing the healthy development of the animal husbandry in China. At present, immunoprophylaxis and drug therapy are the main means for preventing and controlling animal epidemic diseases, and play an important role, but with the increasing enhancement of drug residues and environmental awareness of people on animal products in recent years, the traditional animal epidemic disease prevention and control means are greatly challenged, and people are promoted to continuously explore new prevention and control technologies.

The immune system of animals is divided into two broad categories, the adaptive immune system and the innate immune system. The adaptive immune system specifically recognizes and eliminates invading pathogens primarily by T and B lymphocytes. The natural immune system recognizes pathogens mainly through pattern recognition receptors, and then generates a series of cytokines to resist pathogen invasion. Among these, the innate immune system plays a very important role in the overall immune response as a first line of defense against pathogenic invasion and infection and as a prerequisite for the activation of subsequent adaptive immunity. In recent years, people have paid more and more attention to the first line of anti-infection defense of natural immunity and the effect of a pattern recognition receptor in anti-pathogenic natural immunity, particularly to the effect of enhancing a certain link in a natural immune system of an animal body and a signal path of the pattern recognition receptor by adopting an immunopotentiator, and the improvement of the natural disease resistance of animals becomes a research hotspot in the field of prevention and treatment of animal epidemic diseases at home and abroad.

Disclosure of Invention

The invention aims to provide a preparation method of a duck natural immunopotentiator based on a traditional Chinese medicine extract.

The specific technical scheme is as follows:

a preparation method of a duck natural immunopotentiator based on traditional Chinese medicine extract comprises the following steps:

(1) establishment of rutin standard curve

Accurately weighing rutin standard substance 5.0mg, diluting with 70% ethanol solution of different mass concentration to different concentrations, measuring light absorption values of rutin solutions of different concentrations at 510nm, and establishing linear regression equation.

(2) Extraction of astragalus flavone

Crushing astragalus, sieving with a 40-mesh sieve, accurately weighing 10g, adding 10 times of 90% ethanol by mass, carrying out hot reflux extraction in a water bath, cooling, filtering, fixing the volume of the filtrate in a volumetric flask, and measuring the absorbance of the filtrate.

(3) Calculation of the concentration of Astragalus flavone

And calculating the concentration of the flavone according to the established linear regression equation.

Further, in step 2, the temperature of the water bath was 75 ℃.

Further, in step 2, the time for reflux extraction was 2 hours.

Compared with the prior art, the invention has the beneficial effects that:

the duck natural immunopotentiator based on the traditional Chinese medicine extract provided by the invention can obviously increase the levels of duck serum cytokines IL-1 beta, IFN-alpha and IFN-beta, up-regulate the tissue expression levels of natural immunity related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha, inflammation related genes COX2 and iNOS, and apoptosis related gene Caspase3 in duck immune organs liver, spleen, thymus and bursa of fabricius, and enhance the natural immune response and disease resistance of ducks.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific embodiments.

1. Preparation of duck natural immunopotentiator

The pharmacological active ingredients are extracted and identified from certain traditional Chinese medicines with definite chemical compositions and pharmacological effects, and the extracted pharmacological active ingredients are used for preparing the duck natural immunopotentiator.

2. Grouping and handling of test animals

Selecting 1 day old ducklings which are taken out of shells in the same batch, adaptively feeding the ducklings for 7d, and then randomly dividing the ducklings into a test group and a control group, wherein each group comprises 20 ducklings. Wherein the duckling of the test group is injected with synthetic duck natural immunopotentiator (2.0 mg/feather) subcutaneously, and the duckling of the control group is injected with normal saline with the same volume subcutaneously for 3 days continuously. During the experiment, each group of ducklings can eat and drink water freely.

3. Sample collection

On day 18 after the start of the test, blood was collected from the jugular vein, and serum was separated at room temperature and stored frozen at-20 ℃; the test duck is killed, and tissues and organs such as liver, spleen, thymus, bursa of Fabricius and the like are collected and immediately put into liquid nitrogen for preservation.

4. Detection of duck natural immune response indexes

(1) Detection of serum cytokines and immunoglobulin levels

Serum cytokine IL-1 beta/12 p40, IFN-alpha and IFN-beta and immunoglobulin IgG, IgA and IgM levels were determined by ELISA.

(2) Detection of native immune-related genes in immune tissue organs

The mRNA expression levels of the natural immune related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in liver, spleen, thymus and bursa of Fabricius tissues are detected by a qRT-PCR method.

(3) Detection of inflammation-associated genes in immune tissue organs

The mRNA expression levels of inflammation related genes COX2 and iNOS in liver, spleen, thymus and bursal disease tissues are detected by adopting a qRT-PCR method; the levels of COX2 and iNOS proteins were measured using the Western Blot method.

(4) Detection of apoptosis-related genes in immune tissue and organs

The mRNA expression levels of apoptosis related genes Bcl2 and Caspase3 in tissues of liver, spleen, thymus and bursa of Fabricius are detected by adopting a qRT-PCR method; the levels of Bcl2 and Caspase3 proteins were measured using the Western Blot method.

5. Test results

(1) Detection of serum cytokines and immunoglobulin levels

The duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta and the levels of immunoglobulins IgG, IgA and IgM in the test and control groups are shown in tables 1 and 2.

TABLE 1 serum cytokine levels

TABLE 2 serum immunoglobulin levels

As can be seen from tables 1 and 2, the levels of the duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta in the experimental group are all significantly higher than those in the control group, while the levels of the serum cytokines IL-1 beta/12 p40 and the immunoglobulins IgA, IgG and IgM are not significantly different. The duck natural immunopotentiator based on the traditional Chinese medicine extract provided by the invention can obviously increase the levels of the duck serum cytokines IL-1 beta, IFN-alpha and IFN-beta, and has no obvious influence on the levels of the serum cytokines IL-1 beta/12 p40 and the immunoglobulin IgA, IgG and IgM.

(2) Detection of native immune-related genes in immune tissue organs

The mRNA expression levels of the natural immune related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in the tissues of the duck liver, spleen, thymus and bursa of Fabricius in the test group and the control group are shown in tables 3-6.

TABLE 3 expression levels of innate immunity-related genes in liver tissue

TABLE 4 expression levels of innate immunity-related genes in spleen tissue

TABLE 5 expression levels of innate immunity-related genes in thymus tissue

TABLE 6 expression levels of native immune-related genes in bursal tissue

As can be seen from tables 3 to 6, the mRNA expression levels of the natural immunity related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in the liver, spleen, thymus and bursa of Fabricius tissues of the duck in the test group are mostly higher or remarkably higher than those of the control group, which indicates that the duck natural immunopotentiator based on the traditional Chinese medicine extract provided by the invention can up-regulate or remarkably up-regulate the expression level of the natural immunity related genes in the immune organ tissues of the duck.

(3) Detection of inflammation-associated genes in immune tissue organs

The expression levels of inflammation-related genes COX2 and iNOS mRNA and protein in tissues of liver, spleen, thymus and bursa of Fabricius of the duck in the test group and the control group are shown in tables 7-10.

TABLE 7 expression levels of mRNA and protein of inflammation-related genes COX2 and iNOS in liver tissue

TABLE 8 expression levels of mRNA and protein of inflammation-related genes COX2 and iNOS in spleen tissue

TABLE 9 expression levels of mRNA and protein of COX2 and iNOS genes, genes involved in inflammation, in thymus tissue

TABLE 10 expression levels of inflammation-associated genes COX2 and iNOS mRNA and protein in bursal tissue of Fabricius

As can be seen from tables 7 to 10, the expression levels of inflammation-related genes COX2 and iNOS mRNA and protein in tissues of liver, spleen, thymus and bursa of Fabricius of the experimental group duck (except iNOS mRNA in spleen and thymus) are almost all higher or remarkably higher than those of the control group, which indicates that the duck natural immunopotentiator based on the traditional Chinese medicine extract provided by the invention can up-regulate or remarkably up-regulate the expression levels of inflammation-related genes COX2 and iNOS mRNA and protein in immune organ tissues of the duck.

(4) Detection of apoptosis-related genes in tissue and organ

The expression levels of mRNA and protein of apoptosis-related genes Bcl2 and Caspase3 in liver, spleen, thymus and bursa of Fabricius tissues of the duck in the test group and the control group are shown in tables 11-14.

TABLE 11 mRNA and protein expression levels of apoptosis-related genes Bcl2 and Caspase3 in liver tissue

TABLE 12 expression levels of mRNA and protein of apoptosis-related genes Bcl2 and Caspase3 in spleen tissue

mRNA and protein expression levels of apoptosis-related genes Bcl2 and Caspase3 in thymus tissue of TABLE 13

TABLE 14 expression levels of mRNA and protein of apoptosis-related genes Bcl2 and Caspase3 in bursal tissue

As can be seen from tables 11 to 14, the expression levels of Caspase3 gene mRNA and protein in the liver, spleen, thymus and bursa of Fabricius of the duck in the test group are all significantly higher than those of the control group, while the difference between the expression levels of Bcl2 gene mRNA and protein of the duck in the test group and the duck in the control group is not significant. The natural duck immunopotentiator based on the traditional Chinese medicine extract can up-regulate the expression levels of Caspase3 gene mRNA and protein related to apoptosis in immune organ tissues of ducks, and has no obvious influence on the expression levels of Bcl2 gene mRNA and protein.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.