阅读说明：本技术 一种高纯度硫酸乙酰肝素的制备方法 (Preparation method of high-purity heparan sulfate ) 是由 尹怀君 张岩岩 于 2021-11-26 设计创作，主要内容包括：本发明公开了一种高纯度硫酸乙酰肝素的制备方法,包括有以下步骤：S1,准备清洗干净的猪肠粘膜、牛肺,并加入氯化钠溶液；S2,加入蛋白酶进行酶解；S3,将酶解后的物料依次进行离心、过滤,然后采用树脂离子交换吸附；S4,使用稀盐酸对树脂进行洗脱；S5,将洗脱液进行过滤,然后在滤液中加入乙醇,过滤,得到沉淀物；S6,对硫酸乙酰肝素粗品进行乙醇的二次沉淀；S7,再将固体物质加水溶解；S8,将洗脱液进行膜超滤浓缩；S9,将沉淀物再次溶解、洗脱,将洗脱液进行旋转蒸发浓缩后冻干,得到高纯度硫酸乙酰肝素,本发明涉及硫酸乙酰肝素技术领域。本发明,解决,硫酸乙酰肝素的纯化难度大,进而造成硫酸乙酰肝素的加工纯度不高的问题。(The invention discloses a preparation method of high-purity heparan sulfate, which comprises the following steps: s1, preparing cleaned pig intestinal mucosa and cattle lung, and adding a sodium chloride solution; s2, adding protease for enzymolysis; s3, sequentially centrifuging and filtering the materials subjected to enzymolysis, and then performing ion exchange adsorption by using resin; s4, eluting the resin by using dilute hydrochloric acid; s5, filtering the eluent, adding ethanol into the filtrate, and filtering to obtain a precipitate; s6, carrying out secondary precipitation of ethanol on the heparan sulfate crude product; s7, adding water to dissolve the solid matter; s8, performing membrane ultrafiltration concentration on the eluent; s9, dissolving and eluting the precipitate again, carrying out rotary evaporation and concentration on the eluent, and freeze-drying to obtain the high-purity heparan sulfate, and the invention relates to the technical field of heparan sulfate. The invention solves the problem that the processing purity of the heparan sulfate is not high due to the high purification difficulty of the heparan sulfate.)

1. The preparation method of the high-purity heparan sulfate is characterized by comprising the following steps of:

s1, preparing cleaned pig intestinal mucosa and cattle lung, and adding a sodium chloride solution;

s2, adding protease for enzymolysis, and heating;

s3, sequentially centrifuging and filtering the materials subjected to enzymolysis, and then performing ion exchange adsorption by using resin;

s4, eluting the resin by using dilute hydrochloric acid to obtain an eluent;

s5, filtering the eluent, adding ethanol into the filtrate, and filtering to obtain precipitate and obtain a heparan sulfate crude product;

s6, carrying out secondary ethanol precipitation on the heparan sulfate crude product, and then centrifuging to obtain a solid substance;

s7, adding water to dissolve the solid matter, then performing exchange adsorption by using strong base anion exchange resin, and performing elution;

s8, performing membrane ultrafiltration concentration on the eluent, and then precipitating by using ethanol;

s9, dissolving and eluting the precipitate again, carrying out rotary evaporation and concentration on the eluent, and freeze-drying to obtain the high-purity heparan sulfate.

2. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: in the step S1, the pork intestine mucosa and the beef lung are minced by a meat grinder, the concentration of the sodium chloride solution is 3ml/L, and the pork intestine mucosa, the beef lung and the sodium chloride solution are stirred and mixed according to the weight proportion of 50-60 parts of the pork intestine mucosa, 50-60 parts of the beef lung and 42-55 parts of the sodium chloride solution.

3. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: and in the step S2, stirring, heating to 30-38 ℃, and carrying out enzymolysis for 1-2h, wherein the weight part of the protease is 15-25.

4. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: and in the S3, centrifugate is obtained in a centrifuge, then the centrifugate is filtered to obtain filtrate, and the filtrate is absorbed by resin ion exchange.

5. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: and in the S4, diluted hydrochloric acid is used for washing the resin, and the washing time is 30-50 min.

6. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: in the S5, the weight portion of the ethanol is 20-50, and the reaction time is 2-3 h.

7. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: in the S6, after the heparan sulfate crude product is dissolved by adding water, 30-50 parts by weight of ethanol is added, the solution is heated to 3-10 ℃, and after standing for 1.5-2.5h, all the re-solution is poured into a centrifuge for centrifugation.

8. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: in the S7, preparing the solid matter into a 2% solution, and eluting with 1mol/L sodium chloride solution for 30-50 min.

9. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: in S8, the cut-off value of the membrane ultrafiltration concentration is 3000, and the concentration of ethanol is 60%.

10. The method according to claim 1, wherein the preparation method of high-purity heparan sulfate comprises the following steps: in the S9, the precipitate is prepared into an aqueous solution, the ratio of the precipitate to water is 2/15, and after the aqueous solution passes through a chromatographic column, the aqueous solution is eluted by using 1.5mol/L sodium chloride solution.

Technical Field

The invention relates to the technical field of heparan sulfate, in particular to a preparation method of high-purity heparan sulfate.

Background

Heparan sulfate is a kind of glycosaminoglycan widely existing on the surface of animal cells, and proteoglycan composed of it and protein mainly includes adhesive proteoglycan, fibrinoglycan glycosyl phosphatidylinositol proteoglycan, CD44, etc. They have important biological functions in the aspects of signal transduction, proliferation and differentiation of animal cells, immunoregulation, cell adhesion, information transmission of nerve cells and the like.

In the prior art, the difficulty in purifying heparan sulfate is high, and further the processing purity of the heparan sulfate is not high, so that a preparation method of high-purity heparan sulfate is provided.

Disclosure of Invention

In order to solve the problem that the processing purity of the heparan sulfate is not high due to the high purification difficulty of the heparan sulfate, the invention aims to provide a preparation method of the high-purity heparan sulfate.

In order to achieve the purpose, the invention adopts the following technical scheme: a preparation method of high-purity heparan sulfate comprises the following steps:

s1, preparing cleaned pig intestinal mucosa and cattle lung, and adding a sodium chloride solution;

s2, adding protease for enzymolysis, and heating;

s3, sequentially centrifuging and filtering the materials subjected to enzymolysis, and then performing ion exchange adsorption by using resin;

s4, eluting the resin by using dilute hydrochloric acid to obtain an eluent;

s5, filtering the eluent, adding ethanol into the filtrate, and filtering to obtain precipitate and obtain a heparan sulfate crude product;

s6, carrying out secondary ethanol precipitation on the heparan sulfate crude product, and then centrifuging to obtain a solid substance;

s7, adding water to dissolve the solid matter, then performing exchange adsorption by using strong base anion exchange resin, and performing elution;

s8, performing membrane ultrafiltration concentration on the eluent, and then precipitating by using ethanol;

s9, dissolving and eluting the precipitate again, carrying out rotary evaporation and concentration on the eluent, and freeze-drying to obtain the high-purity heparan sulfate.

Preferably, in S1, the pork intestine mucosa and the beef lung are minced with a meat grinder, the concentration of the sodium chloride solution is 3ml/L, and the pork intestine mucosa, the beef lung and the sodium chloride solution are stirred and mixed, and the weight ratio of the mixture is 50-60 parts of the pork intestine mucosa, 50-60 parts of the beef lung and 42-55 parts of the sodium chloride solution.

Preferably, in the step S2, the mixture is stirred and heated to 30-38 ℃, the enzymolysis time is 1-2h, and the weight part of the protease is 15-25.

Preferably, in S3, the centrifugate is obtained in a centrifuge, and then the centrifugate is filtered to obtain a filtrate, and the filtrate is adsorbed by resin ion exchange.

Preferably, in the step S4, the resin is washed by dilute hydrochloric acid for 30-50 min.

Preferably, in the S5, the weight portion of the ethanol is 20-50, and the reaction time is 2-3 h.

Preferably, in the step S6, after the heparan sulfate crude product is dissolved by adding water, 30 to 50 parts by weight of ethanol is added, the solution is heated to 3 to 10 ℃, and after standing for 1.5 to 2.5 hours, all the re-solution is poured into a centrifuge for centrifugation.

Preferably, in the step S7, the solid matter is prepared into a 2% solution, and the solution is eluted by 1mol/L sodium chloride solution, wherein the elution time is 30-50 min.

Preferably, in S8, the retention value of membrane ultrafiltration concentration is 3000, and the concentration of ethanol is 60%.

Preferably, in S9, the precipitate is prepared into an aqueous solution, the ratio of the precipitate to water is 2/15, and after passing through a chromatographic column, the precipitate is eluted by using 1.5mol/L sodium chloride solution.

Compared with the prior art, the invention has the following beneficial effects: the invention can prepare high-purity heparan sulfate by adopting resin ion exchange adsorption, elution and precipitation of different methods for many times; according to the invention, after the chromatographic column is adopted, 1.5mol/L sodium chloride solution is used for elution, and rotary evaporation concentration is adopted simultaneously, so that the purity of the heparan sulfate is further improved.

Drawings

The invention is described in further detail below with reference to the following figures and detailed description:

FIG. 1 is a schematic diagram of the process of the present invention.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.

The invention provides a technical scheme that: a preparation method of high-purity heparan sulfate comprises the following steps:

s1, preparing cleaned pig intestinal mucosa and cattle lung, and adding a sodium chloride solution;

s2, adding protease for enzymolysis, and heating;

s3, sequentially centrifuging and filtering the materials subjected to enzymolysis, and then performing ion exchange adsorption by using resin;

s4, eluting the resin by using dilute hydrochloric acid to obtain an eluent;

s5, filtering the eluent, adding ethanol into the filtrate, and filtering to obtain precipitate and obtain a heparan sulfate crude product;

s6, carrying out secondary ethanol precipitation on the heparan sulfate crude product, and then centrifuging to obtain a solid substance;

s7, adding water to dissolve the solid matter, then performing exchange adsorption by using strong base anion exchange resin, and performing elution;

s8, performing membrane ultrafiltration concentration on the eluent, and then precipitating by using ethanol;

s9, dissolving and eluting the precipitate again, carrying out rotary evaporation and concentration on the eluent, and freeze-drying to obtain the high-purity heparan sulfate.

In the step S1, the pork intestine mucosa and the beef lung are minced by a meat grinder, the concentration of the sodium chloride solution is 3ml/L, and the pork intestine mucosa, the beef lung and the sodium chloride solution are stirred and mixed according to the weight proportion of 50-60 parts of the pork intestine mucosa, 50-60 parts of the beef lung and 42-55 parts of the sodium chloride solution.

And in the step S2, stirring, heating to 30-38 ℃, and carrying out enzymolysis for 1-2h, wherein the weight part of the protease is 15-25.

And in the S3, centrifugate is obtained in a centrifuge, then the centrifugate is filtered to obtain filtrate, and the filtrate is absorbed by resin ion exchange.

And in the S4, diluted hydrochloric acid is used for washing the resin, and the washing time is 30-50 min.

In the S5, the weight portion of the ethanol is 20-50, and the reaction time is 2-3 h.

In the S6, after the heparan sulfate crude product is dissolved by adding water, 30-50 parts by weight of ethanol is added, the solution is heated to 3-10 ℃, and after standing for 1.5-2.5h, all the re-solution is poured into a centrifuge for centrifugation.

In the S7, preparing the solid matter into a 2% solution, and eluting with 1mol/L sodium chloride solution for 30-50 min.

In S8, the cut-off value of the membrane ultrafiltration concentration is 3000, and the concentration of ethanol is 60%.

In the S9, the precipitate is prepared into an aqueous solution, the ratio of the precipitate to water is 2/15, and after the aqueous solution passes through a chromatographic column, the aqueous solution is eluted by using 1.5mol/L sodium chloride solution.

When in use, the invention can prepare the high-purity heparan sulfate by adopting resin ion exchange adsorption, elution and precipitation of different methods for many times; according to the invention, after the chromatographic column is adopted, 1.5mol/L sodium chloride solution is used for elution, and rotary evaporation concentration is adopted simultaneously, so that the purity of the heparan sulfate is further improved.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.