JPS5884801A - Preparation of hyaluronic acid - Google Patents

Abstract

PURPOSE:To obtain hyaluronic acid of high purity without containing different kinds of proteins and different types of materials, by washing a connective tissue having the hyaluronic acid bonded thereto previously with a hydrous organic solvent containing an alkali (earth) metallic salt, and collecting the resultant hyaluronic acid. CONSTITUTION:An animal connective tissue containing hyaluronic acid, e.g. a human navel cord, is minced and previously washed with a mixed solvent of a 0.5-3M aqueous solution of an alkali (earth) metallic salt, e.g. sodium acetate, with an organic solvent, e.g. ethanol, at (1/0.1)-(1/3) mixing ratio to remove blood components present in the raw material and soluble components present in the connective tissue. The hyaluronic acid is then collected by the extraction, centrifugation, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for obtaining hyaluronan that is free of foreign proteins and type materials. In detail, animal hyaluronic acid-containing connective tissues are pretreated with aqueous organic solvents containing alkali metal salts or alkaline earth metal rings.
After removing the blood components present in the raw material and the soluble components present in the tissue, the hyaluronic acid is collected using a hyaluronic acid bath to obtain hyaluronic acid that does not contain foreign proteins or type material gold. Regarding the method.

Hyaluronic acid is present as a component in connective tissues such as joints, vitreous body, vagina, cartilage, skin, and the crest of birds, and plays a role in tissue flexibility, structural maintenance, and cellular metabolic regulation.
fulfills an essential function. In addition, hyaluronan is a polymer substance with very large ridges, and its molten g has strong viscoelastic tubes, so it is attracting attention as a treatment for arthritis and as a tissue preservation agent during eye surgery.

By the way, for example, when fractionating hyaluronic acid by precipitating it with an organic solvent such as acetone or ethanol from an aqueous solution containing hyaluronic acid, if the aqueous solution contains a small amount of contaminant proteins, it is necessary to leave the contaminant proteins in the supernatant. Hyaluronic acid can be efficiently separated as a precipitate. However, at this time, if there is a large amount of contaminant proteins in the hyaluronic acid solution, a large amount of protein will precipitate at the same time when hyaluronic acid is precipitated, and no protein removal effect can be expected.

Therefore, when hyaluronic acid is separated into six fractions, it is first digested with a connective tissue protein-degrading enzyme, but through treatment with this proteolytic enzyme, the proteolytic enzyme, which is a foreign protein, is converted into hyaluronic acid. ! 4 There are ongoing concerns and issues of antigenicity arise. Furthermore, when cryopreserved chain bands are used as a raw material, there is a risk that red blood cells and tissue cells will be destroyed by freezing and thawing, and that their membrane components, as well as the type substances present in the membranes, will contaminate the hyaluronic acid.

In view of these circumstances, the present inventors have completed the present invention by repeatedly conducting research to obtain hyaluronic acid that does not contain foreign proteins or heavy substances by using animal hyaluronan-containing connective tissue as a raw material.

An object of the present invention is to provide a method for producing hyaluronic acid that does not contain foreign proteins and type substances.
), such objects can be achieved by the method described in the claims; by applying such a method, protein removal operations such as ethanol fractionation, cetylpyridinium chloride fractionation, etc. is carried out efficiently], and hyaluronic acid containing no foreign proteins or type substances can be obtained.

As the raw material used in the present invention, that is, the hyaluronic acid-containing bonding compound prepared in J, preferred examples include the crest of V4 emperor (% hominid) birds.

It is preferable to use the raw material immediately after cutting tlr-59*' or to freeze it immediately after cutting and store it in a frozen state. It is preferable that the raw material is chopped into minced pieces and then washed with solvent A.

As the metal salt for solvent A, salts of alkali metals such as sodium and potassium, alkaline earth metals such as calcium and barium, and mineral acids (such as hydrochloric acid) and organic acids (such as acetic acid) are preferably used. Preferably, sodium acetate, calcium acetate, and sodium chloride are used. Examples of organic solvents for solvent A include alcohols (preferably ethanol) and ketones (preferably acetone 9).

Solvent A is usually prepared by first dissolving the metal salt in water to prepare a metal salt aqueous solution, and then mixing this with an organic solvent. It is preferable to adjust the concentration so that it is 0.5 to 3M11, and the mixing ratio of the metal salt aqueous solution and the organic solvent is generally 1:0.1 to 1:3 (ma/ma). 0 solution] [A specifically includes ti2M sodium chloride water bath solution: ethanol = 1:l, 0.5M calcium acetate aqueous solution: acetone = 1:Q, 5.1M chloride) (lium: ethanol = t:O, Examples include a.

By washing the raw material with solvent A, foreign proteins and product substances are transferred to the washing liquid.Thus, solvent A
By collecting hyaluronic acid from the washed raw material using a method known per se, hyaluronic acid that does not contain foreign proteins or product substances is produced.

The method for collecting hyaluronic acid from washed raw materials is as follows:
For example, after extracting hyaluronic acid from the washed raw material with an aqueous solvent such as Shobu saline, the extract is subjected to ethanol fraction, cetylpyridium chloride fraction, and a combination of these fractions. More specifically, the method is as follows, for example.

First, washed fc dermatitis mince is suspended in physiological saline and gently stirred to perform complete extraction of hyaluronic acid.

After extraction, hyaluronic acid is extracted into the aqueous solution obtained by centrifugation. The extracted hyaluronic acid is recovered as a precipitate by cetylpyridinium chloride or ethanol fractionation. In the case of ethanol fractionation, a fraction that precipitates at about 40 to 60% is collected. At this time, 0.5-3.0M sodium chloride r in the aqueous solution
When fractionating cetylpyridinium chloride, which is preferable for the separation and recovery of hyaluronic acid, adding a very small amount of cetylpyridinium chloride (
cetylpyridinium chloride (twice the amount of hyaluronic acid-containing violet)
Hyaluronic acid is recovered as a precipitate.

Hyaluronic acid is recovered by both fractionation methods such as the ethanol fractionation method, the cetylpyridinium chloride precipitation method, and the ethanol fractionation method.

The hyaluronic acid obtained by the present invention does not contain heteropolar proteins or type substances, and is a highly purified hyaluronic acid that does not have antigenicity as a pharmaceutical product.This product has anti-inflammatory effects against arthritis, etc. It is used as an anti-inflammatory agent. It is also generally administered locally, for example into the joint cavity or into the eyeball.

Preparations for topical administration may take the form of liquid preparations, dry preparations, etc., although liquid preparations are convenient.

During formulation, it is preferable to carry out intermittent sterilization at, for example, 80 to 100°C.

Next, the method of the present invention will be explained in detail with reference to Examples and Experiments, but the present invention is not limited or restricted to the following Examples. , To this minced meat, add 250ml of a mixture of 1ml of 2M salt solution and 250ml of Asanaton's mixture (in addition,
After making the mixture cloudy, it was shredded and washed with a dye. After washing,
The washing solution was completely removed by centrifugation, and the obtained washed minced meat was suspended in 250 d of physiological saline, and the solution was subjected to ethanol precipitation, cetylpyridinium chloride precipitation, and ethanol precipitation according to conventional methods to obtain purified hyaluronic acid 150 d. This purified hyaluronic acid.

The average molecular weight U of the acid was 1.5 million, the protein contamination rate was less than 0.1, and no nucleic acid, chondroitin sulfate, or type substance was detected.

Example 2 Frozen minced meat 450jli was washed with 250d of a mixture of 0.75M calcium acetate solution 31F and 2 volumes of ethanol, extracted and purified in the same manner as in Example 1, and 140IIg of hyaluron a! I met you. Its properties were almost the same as those obtained in Examples.

Example 3 Freezing bandage 4711 was washed in the same manner with a mixture of 2 volumes of 2M saline and 3 ethanol, and extracted and purified in the same manner as in Example 1 to obtain 15490 purified hyaluronic acid.
The purity obtained was almost the same as that of Example 1. Experimental Example 1 The crude extracts of hyaluronic acid obtained in Examples 1 and 3 were assayed for type substances. As a control, membranous minced meat 50jF' which had not been washed according to the present invention was washed with physiological saline 2501d.
Type 0 substance assay iA type 1m using the extract obtained by shredding with a medium mixer! The results are shown in Table 1. The results are shown in Table 1. In the control specimen extracted without washing, it was detected up to a 64-fold dilution, whereas in the specimens of Examples 1 and 3, it was slightly undetectable in the undiluted solution. In other words, the 98% of phosphorus substances can be removed by washing.
1! It was found that the above had been removed0 (blank below) Procedural amendment (voluntary) December 21, 1980 Commissioner of the Japan Patent Office Sir 1, Indication of case 1984 If#h Application No. 184647 3,
Relationship with the case of the person making the amendment Patent applicant 6 Number of inventions increased by amendment None”°
″: 2-car 1! m-&a@J..

8. Contents of the amendment (1) On the 3rd day of the specification, on the 6th page, there is an inscription that says “-ni ej”.
Correct to ``in case''.

(2) Is “-matsuri” written in the last line of the 4th page of the 4F specification? Correct to "preparation".

(3) Details 4! Correct 4 to 17459 J, which is r450.9J in the 8th shell and 12th line.

Claims (1)

[Claims] Animal aluronic acid-containing connective tissue [When producing hyaluronic acid as a raw material, the raw material is washed with a water-containing organic solvent containing an alkaline earth metal salt, A method for producing hyaluronic acid that involves collecting hyaluronic acid after removing blood components and soluble components in connective tissue present in the raw materials.