CN111647094B

CN111647094B - Method for extracting nereis polysaccharide with auxiliary protection effect on chemical liver injury

Info

Publication number: CN111647094B
Application number: CN202010532378.1A
Authority: CN
Inventors: 苏捷; 廖登远; 刘智禹
Original assignee: Fisheries Research Institute Of Fujian (fujian Aquatic Disease Prevention Center)
Current assignee: Fisheries Research Institute Of Fujian (fujian Aquatic Disease Prevention Center)
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2021-11-23
Anticipated expiration: 2040-06-11
Also published as: CN111647094A

Abstract

An extraction method of nereis polysaccharide with auxiliary protection effect on chemical liver injury relates to the field of functional foods. The method comprises the following steps: 1) cleaning clamworm, freeze-drying with a freeze dryer, and pulverizing with a pulverizer to obtain clamworm powder; 2) adding clam worm powder into a phosphate buffer solution and mixing to obtain a mixture A; 3) adding trypsin to the mixture A to obtain a mixture B; 4) sequentially carrying out water bath and leaching on the mixture B, and separating to obtain a supernatant; 5) filtering the supernatant with ceramic membrane, precipitating with ethanol to obtain precipitate, washing the precipitate, and lyophilizing to obtain nereis polysaccharide. Nereid polysaccharide has protective effect on liver injury caused by T cells, and can reduce immune injury caused by T cells by regulating and controlling activity of T cells.

Description

Method for extracting nereis polysaccharide with auxiliary protection effect on chemical liver injury

Technical Field

The invention relates to the field of functional foods, in particular to a method for extracting nereis virens polysaccharide with an auxiliary protection effect on chemical liver injury.

Background

The liver is the most important organ of drug metabolism in the body. Chemical liver injury refers to liver injury caused by chemical hepatotoxic substances. These chemicals include alcohol, environmental chemical poisons and certain drugs. The liver, an important detoxifying organ of the human body, has dual blood supplies of hepatic artery and hepatic vein. Chemical substances can enter the liver through the portal vein of the gastrointestinal tract or systemic circulation for transformation, and thus the liver is vulnerable to toxic substances in the chemical substances. In the production process of both nature and human industry, some substances with toxicity to liver exist, which are called as "hepatotropic poisons", the poisons are generally susceptible and have short incubation period in people, and the pathological process is directly related to the infection dosage, and can cause hepatocyte necrosis, fatty deformation, liver cirrhosis and liver cancer of different degrees of liver. Canavalid protein A (Con A) is a specific T cell mitogen, and can accelerate the proliferation and differentiation of T cells, break the balance between helper T cell subsets, cause immunoregulation dysfunction, trigger uncontrolled immune response of organism, and activate immune cells such as macrophage, neutrophil, natural killer cell, etc. The liver is a main affected organ due to the characteristics of rich blood supply, low blood flow speed and the like, immune cells easily act on the liver, and molecules such as proinflammatory cytokines and reactive oxygen species are released, so that the liver is subjected to inflammatory injury.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a method for extracting nereis polysaccharide with an auxiliary protection effect on chemical liver injury, which researches the auxiliary protection effect of the nereis polysaccharide on Con A-induced chemical liver injury through the full-value development of the nereis, develops instant special food and health-care functional food with the auxiliary protection effect on the chemical liver injury, strengthens the diversified development of nereis rear-end industrial chain products and improves the enriching driving effect of cultivation enterprises on surrounding farmers through the modes of medicine source base cultivation, product research and development, and store sales and production research.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting nereis polysaccharide with auxiliary protection effect on chemical liver injury comprises the following steps:

1) cleaning clamworm, freeze-drying with a freeze dryer, and pulverizing with a pulverizer to obtain clamworm powder;

2) adding clam worm powder into a phosphate buffer solution and mixing to obtain a mixture A;

3) adding trypsin to the mixture A to obtain a mixture B;

4) sequentially carrying out water bath and leaching on the mixture B, and separating to obtain a supernatant;

5) filtering the supernatant with ceramic membrane, precipitating with ethanol to obtain precipitate, washing the precipitate, and lyophilizing to obtain nereis polysaccharide.

The Nereid comprises at least one of Perinereis aibuhitensis and Nereis schlegeli.

In the step 2), the volume ratio of the weight of the silkworm powder to the phosphate buffer is 1: 5-20.

The pH value of the phosphate buffer solution is 7.0-10.0.

In the step 3), the weight ratio of the trypsin to the silkworm powder is 1: 300-3000.

The enzymatic activity of the trypsin is 1000-5000U/mg.

In the step 4), the temperature of the water bath is 35-50 ℃, the time of the water bath is 1-8 hours, the leaching temperature is 50-90 ℃, and the leaching time is 1-5 hours.

In the step 4), the separation method for separating and obtaining the supernatant can adopt suction filtration or centrifugation.

In the step 5), the precipitate is washed by absolute ethyl alcohol, acetone and ether respectively.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

nereid polysaccharide has protective effect on liver injury caused by T cells, can reduce immune injury caused by T cells by regulating and controlling activity of T cells, and has protective effect on liver injury caused by canavalin A and CCl by liver histopathological examination₄The acute liver injury caused by the drug also has the auxiliary protection effect.

Drawings

FIG. 1 is a graph showing the data of the AST content in the serum of mice in each experimental group;

FIG. 2 is a data graph of ALT content in serum of mice of each experimental group.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

Example 1

1) cleaning perinereis aibuhitensis, freeze-drying with a freeze dryer, and pulverizing with a pulverizer to obtain powder;

2) adding the clam worm powder into a phosphate buffer solution with the pH value of 8.0 and mixing to obtain a mixture A, wherein the volume ratio of the weight of the clam worm powder to the phosphate buffer solution is 1: 10;

3) adding trypsin (enzyme activity 2500U/mg) into the mixture A to obtain a mixture B, wherein the weight ratio of the trypsin to the silkworm powder is 1: 300;

4) carrying out water bath at 45 ℃ for 5h, leaching at 80 ℃ for 2h, centrifuging or carrying out suction filtration, and taking supernatant;

5) filtering the supernatant with ceramic membrane, precipitating with ethanol to obtain precipitate, washing the precipitate with anhydrous ethanol, acetone and diethyl ether, and lyophilizing to obtain Nereis Japonica polysaccharide.

Example 2

1) cleaning Nereid wartii, lyophilizing, and pulverizing to obtain Nereis Japonica powder;

2) adding the clam worm powder into a phosphate buffer solution with the pH value of 10.0 and mixing to obtain a mixture A, wherein the volume ratio of the weight of the clam worm powder to the phosphate buffer solution is 1: 20;

3) adding trypsin (with the enzyme activity of 5000U/mg) into the mixture A to obtain a mixture B, wherein the weight ratio of the trypsin to the silkworm powder is 1: 3000;

4) carrying out 35 ℃ water bath on the mixture B for 8h, leaching at 90 ℃ for 1h, centrifuging or carrying out suction filtration, and taking supernatant;

Example 3

2) adding the clam worm powder into a phosphate buffer solution with the pH value of 7.0 and mixing to obtain a mixture A, wherein the volume ratio of the weight of the clam worm powder to the phosphate buffer solution is 1: 5;

3) adding trypsin (enzyme activity is 1000U/mg) into the mixture A to obtain a mixture B, wherein the weight ratio of the trypsin to the silkworm powder is 1: 1500;

4) carrying out water bath at 50 ℃ for 1h, leaching at 50 ℃ for 5h, centrifuging or carrying out suction filtration, and taking supernatant;

The nereis polysaccharide prepared in example 1 was subjected to relevant experimental tests, as follows:

1. selecting adult male mice (18-22 g), wherein each group comprises 10-15 mice, and performing experiments in 6 groups, namely a control group, a model group, a No. 1 sample group, a No. 2 sample group, a No. 3 sample group and a positive group;

2. the mice of the control group and the model group are normally fed with feed and purified water; feeding nereis polysaccharide with different dosages for 30 days in 3 groups of sample groups, and feeding for 2 times every day, wherein the sample group 1 is 200mg/kg, the sample group 2 is 400mg/kg, and the sample group 3 is 600 mg/kg; the positive group is fed with prednisone acetate 7.8mg/kg every day;

3. the model group, the positive group and each sample group are administered with the canavalin A with the dose of 20mg/kg through the tail vein once at the end of the experiment, after 8 hours of fasting, the canavalin A is anesthetized by intraperitoneal injection of 60mg/kg BW pentobarbital sodium solution, the abdominal aorta is subjected to blood collection, and the liver tissue is taken for detection of each index and pathological histological examination. Detecting the body weight, alanine Aminotransferase (ALT) in serum, aspartate Aminotransferase (AST) in serum, Lactate Dehydrogenase (LDH) in serum and liver histopathological changes of the mice.

As shown in fig. 1 and fig. 2, the nereis polysaccharide can significantly reduce the content of AST and ALT in the serum of the mice with liver injury induced by the sword bean protein, which indicates that the nereis polysaccharide has liver protection effect on the mice with liver injury induced by the sword bean protein.

As shown in Table 1, the influence of nereis polysaccharide on the serum INF-gamma and TNF-alpha of mice with acute liver injury induced by jack bean protein A is shown. Compared with a normal control group, the level of INF-gamma and TNF-alpha in the serum of the mouse in the model group is obviously increased; compared with the model group, the levels of INF-gamma and TNF-alpha in the serum of the mice of the positive group and each sample group are obviously reduced.

TABLE 1

Group of	INF-γ(ng/L)	TNF-α(ng/L)
			Control group	3.8±0.12	28.2±2.2
Model set	81.2±6.5	151.5±12.5
			Sample set No. 1	50.6±5.3	98.7±6.2
Sample set No. 2	37.2±4.2	84.3±5.7
			Sample set No. 3	23.5±4.3	61.8±4.2
Positive group	22.3±5.2	63.2±5.1

The results show that the nereis polysaccharide has a protective effect on liver injury caused by T cells, the nereis polysaccharide can reduce immune injury caused by the T cells by regulating and controlling the activity of the T cells, and in addition, the nereis polysaccharide has a protective effect on liver injury caused by the canavalin A through liver pathological histology examination.

Claims

1. A method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury is characterized by comprising the following steps:

3) adding trypsin to the mixture A to obtain a mixture B;

4) sequentially carrying out water bath and leaching on the mixture B, and separating to obtain a supernatant; the temperature of the water bath is 35-50 ℃, the time of the water bath is 1-8 h, the leaching temperature is 50-90 ℃, and the leaching time is 1-5 h;

2. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: the Nereid comprises at least one of Perinereis aibuhitensis and Nereis schlegeli.

3. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: in the step 2), the volume ratio of the weight of the silkworm powder to the phosphate buffer is 1: 5-20.

4. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: the pH value of the phosphate buffer solution is 7.0-10.0.

5. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: in the step 3), the weight ratio of the trypsin to the silkworm powder is 1: 300-3000.

6. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: the enzymatic activity of the trypsin is 1000-5000U/mg.

7. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: in the step 4), the separation method for separating and obtaining the supernatant can adopt suction filtration or centrifugation.

8. The method for extracting nereis polysaccharide with auxiliary protection effect on immunological liver injury according to claim 1, which comprises the following steps: in the step 5), the precipitate is washed by absolute ethyl alcohol, acetone and ether respectively.

9. The nereis polysaccharide extracted by the extraction method of nereis polysaccharide as claimed in any one of claims 1-8 is applied to preparation of products with auxiliary protection effect on immunological liver injury.