WO2015066893A1 - Method of preparing low-molecular chondroitin sulfate for treating myocarditis - Google Patents

Abstract

Disclosed is a method of preparing low-molecular chondroitin sulfate (CS) for treating myocarditis, the steps comprising: fermenting raw material through an arthrobacter sulfonivorans fermentation liquor, and conducting enzymolysis treatment on CS in the raw material to obtain low-molecular CS; the content of the CS disaccharide in the prepared low-molecular CS is greater than 97%, and the molecular weight is 450-480; the content of CS A is 70-95%, and the content of CS C is 5-25%. The preparation method is easy to conduct, and has a short cycle and low production cost, and the low-molecular CS has a good treatment effect and high purity, can be used for large-scale industrial production, and has a significant repair effect on myocardial cells in rat experiments.

Description

 Preparation method of low molecular weight chondroitin sulfate for treating myocarditis

 Technical field

 The invention relates to a preparation method of chondroitin sulfate, in particular to a preparation method of low molecular weight chondroitin sulfate for treating myocarditis.

 Background technique

 Chondroitin sulfate (Chondroitin Sulfate (CS) is an acidic mucopolysaccharide found in connective tissues of humans and animals. It is mainly distributed in cartilage, bone, tendon, sarcolemma and blood vessel walls, and is composed of D-glucuronic acid and N-acetyl-D-amino half. Lactose is linked by 1,3 glycosidic bonds to form disaccharides, and disaccharides are linked by β-1,4 glycosidic bonds. The relative molecular mass (Mr) of CS is generally above 20×103, and when Mr is reduced by less than 10×103, it is often referred to as low molecular weight chondroitin sulfate (LMWCS).

 CS As a component of proteoglycans, it is present in intracellular granules, cell membranes, basement membranes, and extracellular matrices. It binds specifically to cytokines, adhesion factors, or their receptors through specific domains in the sugar chain. Important physiological functions are also closely related to many pathological processes. CS has been used as a clinical drug for nearly half a century. In Europe, the United States, Japan and other developed countries, because of its anti-arthritis, blood fat, anti-coagulation, anti-atherosclerosis, heart disease, anti-virus and anti- It has been widely used for various biological activities such as tumors and reduction of nephrotoxicity of platinum drugs. However, due to the large molecular weight of CS, many hydrophilic groups, and poor fat solubility, the in vivo absorption is incomplete, and the lack of bioavailability has been plaguing the large-scale application of CS drugs. Compared with ordinary CS, LMWCS is more conducive to absorption and utilization by the body due to the decrease of Mr. It has great application potential in medicinal, health care products and cosmetics.

 LMWCS It is generally prepared by degradation of CS products, mainly acid hydrolysis and enzymatic methods. The acid degradation reaction product has many impurities and is difficult to remove, and the treatment takes a lot of time. During the reaction, the sulfate groups on the chondroitin sulfate molecule will fall off to different degrees and cause environmental pollution. In contrast, the enzymatic hydrolysis reaction conditions are mild, the pollution is small, the process is simple and easy to control, and the sulfate group on the CS molecule is not destroyed, which is more suitable for the industrial production of LMWCS.

However, the enzymatic method has high requirements for the enzyme to be used, and requires specific chondroitinase. However, at present, chondroitinase-producing bacteria with independent intellectual property rights have not been developed in China, and can only be imported from abroad at a high price, thereby greatly increasing Production costs.

 Summary of the invention

 The object of the present invention is to overcome the deficiencies of the prior art and to provide a preparation method of low molecular weight chondroitin sulfate for treating myocarditis.

 In order to achieve the above object, the technical solution adopted by the present invention is: a method for preparing a low molecular weight chondroitin sulfate for treating myocarditis, comprising the steps of: passing the raw material through Arthrobacter sulphate Fermentation liquid fermentation, chondroitin sulfate in the raw material is enzymatically decomposed to obtain low molecular weight chondroitin sulfate; the prepared low molecular weight chondroitin sulfate has a chondroitin disaccharide content of 97% or more, a molecular weight of 450-480, sulfuric acid The chondroitin A content is between 70-95%, and the chondroitin sulfate C content is between 5-25%.

 Further, the raw material refers to one or a mixture of chicken cartilage, pig cartilage or bovine cartilage.

 Further, the molecular structure of the chondroitin sulfate disaccharide is as follows:

 Further, the chondroitin sulfate has a disaccharide content of 98.5% or more and a molecular weight of 455-460.

 Further, the chondroitin sulfate A content is between 70-80%, and the chondroitin sulfate C content is between 10-15%.

Further, the composition of the culture medium of the bacterium of the bacterium of the bacterium of the bacterium is: w/v: chondroitin sulfate 0.7-0.9%, KH ₂ PO ₄ 0.01-0.03%, yeast dipping powder 0.5-0.7%, MgSO ₄ ·7H ₂ 0 content 0.7-0.9%, pH value 5-7; fermentation conditions: according to 1.5% v / v inoculum amount of seed bacteria into the fermenter at 20-35 ° C, 100-300r / min oscillatory aeration culture 48- 96h.

 Further, the The conditions for enzymatic hydrolysis of chondroitin sulfate by the fermentation broth are: enzymatic hydrolysis of chondroitin sulfate 0.1-0.6 kg per liter of fermentation broth, enzymatic hydrolysis time 5-8 h, enzymatic hydrolysis temperature 30-45 ° C, and enzymatic hydrolysis pH 6-8.

Further, the low molecular weight chondroitin promotes the growth and proliferation of H9c2 cardiomyocytes at a concentration of 200 μg•mL ^-1 , and plays an important role in the treatment of myocarditis and myocardial damage repair.

The invention has the advantages of convenient operation, short cycle and low production cost, low molecular weight chondroitin sulfate treatment, high purity, and can be used for large-scale industrial production. After rat test, the effect of repairing myocardial cell damage is remarkable.

 detailed description

A preparation method of low molecular weight chondroitin for treating myocarditis, wherein one or several kinds of chicken cartilage, pig cartilage or bovine cartilage are mixed and fermented by the fermentation broth of Arthrobacter sinensis, and the medium composition of the fermentation broth of the bacterium (w/v): chondroitin sulfate 0.7-0.9%, KH ₂ PO ₄ 0.01-0.03%, yeast dipping powder 0.5-0.7%, MgSO ₄ ·7H ₂ 0 content 0.7-0.9%, pH 5-7; fermentation The conditions were as follows: 1.5% (v/v) inoculum was added to the fermenter at 20-35 ° C, and cultured at 100-300 r/min for 48-96 h. The chondroitin sulfate in the raw material is enzymatically decomposed to obtain low molecular weight chondroitin sulfate; the prepared low molecular weight chondroitin sulfate has a chondroitin disaccharide content of 97% or more, a molecular weight of 450-480, and a chondroitin sulfate A content. Between 70-95%, the chondroitin sulfate C content is between 5-25%.

 Said The conditions for enzymatic hydrolysis of chondroitin sulfate by the fermentation broth are: enzymatic hydrolysis of chondroitin sulfate 0.1-0.6 kg per liter of fermentation broth, enzymatic hydrolysis time 5-8 h, enzymatic hydrolysis temperature 30-45 ° C, and enzymatic hydrolysis pH 6-8.

 The molecular structure of the chondroitin sulfate disaccharide is as follows:

 The chondroitin sulfate disaccharide content is above 98.5%, and the molecular weight is between 455-460.

 The chondroitin sulfate A content is between 70-80%, and the chondroitin sulfate C content is between 10-15%.

The low molecular weight chondroitin promotes the growth and proliferation of H9c2 cardiomyocytes at a concentration of 200 μg•mL ^-1 , which plays an important role in the treatment of myocarditis and myocardial injury.

 Example 1

The fermentation conditions of the bacterium of the genus Sulfate were: (w/v): chondroitin sulfate 0.8%, KH ₂ PO ₄ 0.02%, yeast dipping powder 0.6%, MgSO ₄ · 7H ₂ 0 content 0.8%, pH=6.

The optimum fermentation medium conditions were as follows: 1.5 liters of seed broth was placed in a 100 liter fermentor at 25 ° C, and cultured at 200 r/min for 72 hours.

The optimal condition for enzymatic hydrolysis of chondroitin sulfate by fermentation broth is to hydrolyze 30 liters of chondroitin sulfate in a 100 liter fermentation broth, the hydrolysis time is 8 h, the enzymatic hydrolysis temperature is 35 ° C, and the enzymatic hydrolysis pH is 7.

 Spray drying after enzymatic hydrolysis The low molecular weight chondroitin sulfate was prepared, the chondroitin sulfate disaccharide content was 99%, the molecular weight was 458, the chondroitin sulfate A content was 85%, and the chondroitin sulfate C content was 15%.

 Example 2

The fermentation conditions of the bacterium of the genus Sulfate were: (w/v): 10 g of chondroitin sulfate, ₂ g of KH ₂ PO ₄ , 60 g of yeast dip powder, and 80 g of MgSO ₄ ·7H ₂ 0 , pH = 6.

The optimum fermentation medium conditions were as follows: 10 liters of seed broth was connected to a 1000 liter fermenter at 30 ° C, and incubated at 250 r/min for 80 hours.

The optimal condition for enzymatic hydrolysis of chondroitin sulfate by fermentation broth is to hydrolyze 1000 liters of fermented chondroitin in the enzymatic hydrolysis tank, the enzymatic hydrolysis time is 7 h, the enzymatic hydrolysis temperature is 40 ° C, and the enzymatic hydrolysis pH is 7.

 Spray drying after enzymatic hydrolysis 400 kg of low molecular weight chondroitin sulfate was prepared, the chondroitin sulfate disaccharide content was 98.5%, the molecular weight was 461, the chondroitin sulfate A content was 83%, and the chondroitin sulfate C content was 16%.

 Example 3

The effect of chondroitin disaccharide on normal H9c2 cardiomyocytes was determined by microscopic observation and MTT assay. The results showed that chondroitin disaccharide was not cytotoxic and promoted the growth and proliferation of H9c2 cells to a certain extent. The optimal concentration was 200 μg•mL ^-1 .

The protective effects of different molecular weight CS on damaged myocardium were evaluated. The results show that low molecular weight chondroitin sulfate can significantly promote the growth and recovery of damaged myocardial tissue. Chondroitin sulfate and its molecular chondroitin sulfate promote the recovery of the heart, but the effect is not as obvious as the low molecular chondroitin sulfate.

After 7 days of gavage, the damage of myocardial cells in CS group was slightly reduced. The myocardial cells in the middle molecular chondroitin and low-molecular chondroitin sulfate were arranged in myocardial fiber arrangement, muscle fiber atrophy, interval width, myocardial cytoplasm and nucleus. Myocardial tissue damage in the ISO injury control group was significantly reduced.

After 14 days of intragastric administration, the damaged myocardial tissue improved and the number of eosinophils decreased. Compared with the ISO injury control group, the recovery effect of each group was more obvious, and the low molecular weight chondroitin sulfate was better.

The above is only a specific embodiment of the present invention, and is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope of the present invention, and should be covered by the present invention. Within the scope of protection.

Claims (1)

1. What is claimed is: 1. A method for preparing a low molecular weight chondroitin sulfate for treating myocarditis, characterized in that the step comprises: passing the raw material through the bacterium Fermentation liquid fermentation, chondroitin sulfate in the raw material is enzymatically decomposed to obtain low molecular weight chondroitin sulfate; the prepared low molecular weight chondroitin sulfate has a chondroitin disaccharide content of 97% or more, a molecular weight of 450-480, sulfuric acid The chondroitin A content is between 70-95%, and the chondroitin sulfate C content is between 5-25%.

   The method for preparing low molecular weight chondroitin sulfate for treating myocarditis according to claim 1, wherein: The raw material refers to one or a mixture of chicken cartilage, pig cartilage or bovine cartilage.

   The method for preparing low molecular weight chondroitin sulfate for treating myocarditis according to claim 1, characterized in that: The molecular structure of the chondroitin sulfate disaccharide is as follows:

   

   The method for preparing low molecular weight chondroitin sulfate for treating myocarditis according to claim 1 or 3, wherein: The chondroitin sulfate disaccharide content is above 98.5%, and the molecular weight is between 455-460.

   The method for preparing low molecular weight chondroitin sulfate for treating myocarditis according to claim 1, wherein: The chondroitin sulfate A content is between 70-80%, and the chondroitin sulfate C content is between 10-15%.

   The method for preparing low-molecular-weight chondroitin sulfate for treating myocarditis according to claim 1, wherein: the medium component of the fermentation broth of the bacterium of the bacterium of the bacterium of the genus Sulfate is: w/v: chondroitin sulfate 0.7-0.9%, KH ₂ PO ₄ 0.01-0.03%, yeast dipping powder 0.5-0.7%, MgSO ₄ ·7H ₂ 0 content 0.7-0.9%, pH 5-7; fermentation conditions: seeding bacteria at 1.5% v/v inoculum The liquid was connected to the fermenter at 20-35 ° C, and cultured at 100-300 r/min for 48-96 hours.

   The method for preparing low molecular weight chondroitin sulfate for treating myocarditis according to claim 1, wherein: The conditions for enzymatic hydrolysis of chondroitin sulfate by the fermentation broth are: enzymatic hydrolysis of chondroitin sulfate 0.1-0.6 kg per liter of fermentation broth, enzymatic hydrolysis time 5-8 h, enzymatic hydrolysis temperature 30-45 ° C, and enzymatic hydrolysis pH 6-8.

   The method for preparing low molecular weight chondroitin sulfate for treating myocarditis according to claim 1, wherein: the low molecular weight chondroitin promotes growth and proliferation of H9c2 cardiomyocytes at a concentration of 200 μg•mL ^-1 , It plays an important role in the treatment of myocarditis and myocardial damage repair.