KR19980041691A - Method for producing chondroitin sulfate and its sodium salt from shark bone or cartilage - Google Patents

Abstract

The present invention relates to a method for producing chondroitin sulfate and sodium salt thereof from dried shark bone or cartilage, wherein the shark bone or cartilage is immersed in a hydrochloric acid solution, washed with 95% ethanol, Removing impurities; (2) extracting chondroitin sulfate from shark bone or cartilage using proteolytic enzyme; (3) adjusting the pH of the extract to about 8.5 to remove proteins other than chondroitin sulfate; (4) purifying the extract using an ion exchange resin; (5) adjusting the pH of the extract solution passed through the ion exchange resin to 5.0 to 7.0 and then dissolving it completely by adding sodium salt; And crystallizing chondroitin sulfate or its sodium salt using an alcohol.

Here, n: 75 to 85 M: H or Na

Description

Method for producing chondroitin sulfate and its sodium salt from shark bone or cartilage

The present invention relates to a method for producing chondroitin sulfate and sodium salt thereof from dried shark bone or cartilage, and more particularly to a method for producing chondroitin sulfate or chondroitin sulfate from shark bone or cartilage, And a method for preparing the sodium salt thereof.

Chondroitin sulfate, which is represented by the following formula (1), is one of the mucopolysaccharides containing an N-acetyl group, and is used for stabilizing or stabilizing cells or tissues, . It is also known to control the electrolyte by ion exchange and to maintain moisture.

[Chemical Formula 1]

Here, n: 75 to 85 M: H or Na

Conventionally, chondroitin sulfate is extracted from a bovine bone or a snail as a raw material with 50 mM sodium acetate buffer solution with 0.15 M potassium chloride or 4 M guanidinium chloride solution, centrifuged, dialyzed, However, since the thermal manipulation is performed, chondroitin sulfate becomes unstable and can not be refined with high purity.

Therefore, the inventors of the present invention have investigated substances containing chondroitin sulfate in addition to bovine bone or snail, and found that chondroitin sulfate is present in shark bone or cartilage, and chondroitin sulfate is extracted stably without thermal manipulation, , Cosmetics raw materials, and health food raw materials. Thus, the present invention has been completed.

Further, the present invention has been accomplished based on the finding that sodium hyaluronate can be used as a raw material for medicines, cosmetics, and health foods more easily than sodium salt added to chondroitin sulfate refined at high purity.

Accordingly, an object of the present invention is to provide a method for producing chondroitin sulfate and sodium salt thereof represented by the above formula (1) from shark bone or cartilage.

In order to accomplish the above object, the present invention provides a method for producing chondroitin sulfate and sodium salt thereof from shark bone or cartilage of the present invention, comprising the steps of: (1) immersing shark bone or cartilage in a hydrochloric acid solution and then washing with 95% ethanol to remove impurities step ; (2) extracting chondroitin sulfate from shark bone or cartilage using proteolytic enzyme; (3) adjusting the pH of the extract to about 8.5 to remove proteins other than chondroitin sulfate; (4) purifying the extract using an ion exchange resin; (5) adjusting the pH of the extract solution passed through the ion exchange resin to 5.0 to 7.0 and then completely dissolving it by adding a sodium salt; And crystallizing chondroitin sulfate or its sodium salt using an alcohol.

Other objects, features and operations of the present invention will become apparent to those skilled in the art from the following detailed description.

Hereinafter, the present invention will be described more specifically.

The method for preparing chondroitin sulfate and sodium salt thereof from shark bone or cartilage of the present invention will be described in more detail as follows.

① Shark bone or cartilage is immersed in hydrochloric acid solution and then washed with 95% ethanol to remove impurities

The dried shark bones are immersed in a 0.1 to 2N hydrochloric acid solution to remove the saccharides contained in the shark bone or cartilage. As can be seen from the correlation between the immersion time and the ash content in Table 1, the immersion time is preferably as short as possible for improving the productivity, but is preferably 18 hours as the ash removal effect is reduced if the immersion time is too short. After hydrochloric acid immersion, it is washed with 95% ethanol to remove other impurities and then dried.

Relationship between immersion time and ash content Immersion time 6 hours 12 hours 18 hours 24 hours Ash content (%) 38.6 31.5 28.9 24.5

② Extraction of chondroitin sulfate from shark bone or cartilage using proteolytic enzyme

In step (1), chondroitin sulfate is extracted with about 0.3% of water and protease such as pepsin or papain in order to separate chondroitin sulfate which is strongly binding to collagen in shark bone or cartilage from which impurities have been removed. As can be seen from Table 2, after adjusting the pH to 2 for pepsin and pH 7 for papain, the yield is increased when the extract is extracted for 24 hours or more, And the value of the end product is lost. Therefore, the yield of chondroitin sulfate is the best to extract for 24 hours.

Extraction time and yield of chondroitin sulfate Extraction time 6 hours 12 hours 18 hours 24 hours yield(%) 4.2 5.0 6.7 7.6

③ Adjust the pH of the extract to about 8.5 to remove proteins other than chondroitin sulfate

In addition to chondroitin sulfate, proteins such as collagen and proteolytic enzymes are present as impurities in the extract obtained in step 2). Therefore, precipitate is induced by adjusting the pH to 8.5, which is slightly alkaline, using 2N sodium hydroxide, and the extract is filtered.

(4) Purifying the extract using an ion exchange resin

In step 3, the pH was adjusted to be slightly alkaline to remove the impurities. However, in addition to chondroitin sulfate, many salts and proteolytic products exist in the form of amines. Therefore, in order to obtain high purity chondroitin sulfate, cationic salts By adsorbing an amine and adsorbing an anionic salt using an anion exchange resin, highly purified purified chondroitin sulfate can be obtained without thermal manipulation.

The ion exchange resins used for the purification of the extract are porous polymeric materials composed of copolymers of styrene and divinylbenzene. SK-1B, WK-30, WK-20, WK-10, WA-30, WA-20, WA-10 manufactured by Dion Inc., MP-65 manufactured by Bayer, etc. may be used as the anion exchange resin. In the case of cation exchange resin, the ion exchange resin is activated by passing H ⁺ through a resin filling column with 6% hydrochloric acid solution and then by water washing with purified water. In the case of anion exchange resin, 4% sodium hydroxide solution After passing through a resin filling tower, it is washed with purified water and an active group is prepared as OH ^- and used.

As shown in Table 3, the volume ratio of the cation exchange resin and the anion exchange resin was determined by adding the final product of chondroitin sulfate The purification is carried out at a yield of 9: 1 which is the maximum.

Relationship between volume ratio and yield of cation exchange resin and anion exchange resin Resin ratio (cation: negative ion) 50:50 30:70 60:40 70:30 80:20 90:10 yield 3.6% 2.5% 4.0% 6.4% 7.1% 7.5%

(5) If necessary, adjust the pH of the extract solution passed through the ion exchange resin to 5.0 to 7.0, then add sodium salt to completely dissolve

Pure chondroitin sulfate is present in the extract solution passed through the ion exchange resin. To increase the solubility in water and to use it more easily as a raw material for pharmaceuticals, cosmetics, health food, etc., sodium salt is added, 1 < / RTI > M is sodium. ≪ RTI ID = 0.0 > Sodium < / RTI >

The sodium salt used to provide the sodium salt of chondroitin sulfate is not particularly limited, and examples thereof include sodium chloride, monohydrogenphosphate sodium salt and sodium dihydrogenphosphate, and when the amount is less than 6% by weight When the sodium group is not bonded to the chondroitin sulfate structure well, the sodium group is precipitated at the precipitation and is present as an impurity when it is 20 wt% or more, so that it is preferably added in an amount of 6 to 20 wt%.

Residual heat of sodium salt of sodium chondroitin sulfate depending on the content of sodium chloride The content of sodium chloride (for the extraction tablet) 2% 4% 6% 8% 12% 16% 20% 23% The standard value (23 to 31%) 21.0 21.2 23.1 24.6 26.4 28.9 30.2 31.8

⑥ Crystallization of chondroitin sulfate or its sodium salt using alcohol

The purified extract or the sodium salt added extract using an ion exchange resin is diluted with alcohol to obtain crystallized chondroitin sulfate or its sodium salt. Crystallized chondroitin sulfate or its sodium salt is again washed with alcohol and dried to give white chondroitin sulfate or its sodium salt. In this case, the alcohol is not particularly limited, but one or a mixture of two or more of methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol and isomers thereof may be used.

On the other hand, the solubility of chondroitin sulfate and sodium salt thereof provided by the present invention was confirmed by dissolving chondroitin sulfate and its sodium salt prepared according to the present invention in deionized water and measuring the conductivity. the results are as follow.

conductivity density One% 2% 3% 4% Chondroitin sulfate 9.8 9.1 8.4 7.9 Chondroitin sulfate sodium salt 10.1 12.6 14.4 14.3 The conductivity of desalted water was set at 10.0.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited to these Examples.

[Example 1]

100 g of shark bone or cartilage, which had been pretreated with an aqueous hydrochloric acid solution, was mixed with 1000 ml of purified water and 0.3 g of pepsin, and then extracted for 24 hours to adjust the pH to 8.5. After about 10 minutes, it was confirmed that a precipitate was formed, and filtration was performed using diatomaceous earth. Thereafter, the pH of the purified extract obtained by passing the extract through a top of an ion exchange resin column packed with a cation exchange resin and an anion exchange resin at a flow rate of 20 ml per minute was adjusted to 5.7 to 6.0. The precipitate of chondroitin sulfate was obtained while slowly diluting with methyl alcohol. The precipitated chondroitin sulfate was washed with methyl alcohol and dried to obtain about 14 g of white chondroitin sulfate.

[Example 2]

And extracted and purified in the same manner as in Example 1 except that ethyl alcohol was used instead of methyl alcohol to obtain about 14 g of white chondroitin sulfate.

[Example 3]

And extracted and purified in the same manner as in Example 1 except that propyl alcohol was used instead of methyl alcohol to obtain about 14 g of white chondroitin sulfate.

[Example 4]

And extracted and purified in the same manner as in Example 1, except that butyl alcohol was used instead of methyl alcohol to obtain about 14 g of white chondroitin sulfate.

[Example 5]

100 g of shark bone or cartilage, which had been pretreated with an aqueous hydrochloric acid solution, was mixed with 1000 ml of purified water and 0.3 g of papain, and then extracted for 24 hours to adjust the pH to 8.5. After about 10 minutes, it was confirmed that a precipitate was formed, and filtration was performed using diatomaceous earth. Thereafter, the pH of the purified extract obtained by passing the extract through a top of an ion exchange resin column packed with a cation exchange resin and an anion exchange resin at a flow rate of 20 ml per minute was adjusted to 5.7 to 6.0. The precipitate of chondroitin sulfate was obtained while slowly diluting with methyl alcohol. The precipitated chondroitin sulfate was washed with methyl alcohol and dried to obtain about 14 g of white chondroitin sulfate.

[Example 6]

And extracted and purified in the same manner as in Example 5 except that ethyl alcohol was used instead of methyl alcohol to obtain about 14 g of white chondroitin sulfate.

[Example 7]

Extraction and purification were carried out in the same manner as in Example 5 except that propyl alcohol was used instead of methyl alcohol to obtain about 14 g of white chondroitin sulfate.

[Example 8]

And extracted and purified in the same manner as in Example 5, except that butyl alcohol was used instead of methyl alcohol to obtain about 14 g of white chondroitin sulfate.

[Example 9]

100 g of shark bone or cartilage, which had been pretreated with an aqueous hydrochloric acid solution, was mixed with 1000 ml of purified water and 0.3 g of pepsin, and then extracted for 24 hours to adjust the pH to 8.5. After about 10 minutes, it was confirmed that a precipitate was formed, and filtration was performed using diatomaceous earth. Then, the pH of the purified extract obtained by passing the extract through a top of an ion exchange resin column packed with a cation exchange resin and an anion exchange resin at a flow rate of 20 ml per minute was adjusted to 5.0 to 7.0. Thereafter, 80 g of sodium monohydrogenphosphate (Na ₂ HPO ₄ ) was dissolved, followed by slow dilution with methyl alcohol to obtain a precipitate of chondroitin sulfate sodium salt. The precipitated chondroitin sulfate sodium salt was washed with methyl alcohol and dried to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 10]

And extracted and purified in the same manner as in Example 9, except that ethyl alcohol was used instead of methyl alcohol to obtain about 16 g of sodium chondroitin sulfate sodium salt.

[Example 11]

And extracted and purified in the same manner as in Example 9 except that propyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 12]

And extracted and purified in the same manner as in Example 9, except that butyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 13]

100 g of shark bone or cartilage, which had been pretreated with an aqueous hydrochloric acid solution, was mixed with 1000 ml of purified water and 0.3 g of papain, and then extracted for 24 hours to adjust the pH to 8.5. After about 10 minutes, it was confirmed that a precipitate was formed, and filtration was performed using diatomaceous earth. Then, the pH of the purified extract obtained by passing the extract through a top of an ion exchange resin column packed with a cation exchange resin and an anion exchange resin at a flow rate of 20 ml per minute was adjusted to 5.0 to 7.0. Thereafter, 80 g of sodium chloride was dissolved, followed by slow dilution with methyl alcohol to obtain a precipitate of chondroitin sulfate sodium salt. The precipitated chondroitin sulfate sodium salt was washed with methyl alcohol and dried to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 14]

And extracted and purified in the same manner as in Example 13 except that ethyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 15]

Was extracted and purified in the same manner as in Example 13 except that propyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 16]

And extracted and purified in the same manner as in Example 13, except that butyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 17]

100 g of shark bone or cartilage, which had been pretreated with an aqueous hydrochloric acid solution, was mixed with 1000 ml of purified water and 0.3 g of papain, and then extracted for 24 hours to adjust the pH to 8.5. After about 10 minutes, it was confirmed that a precipitate was formed, and filtration was performed using diatomaceous earth. Then, the pH of the purified extract obtained by passing the extract through a top of an ion exchange resin column packed with a cation exchange resin and an anion exchange resin at a flow rate of 20 ml per minute was adjusted to 5.0 to 7.0. Thereafter, 80 g of sodium dihydrogenphosphate (NaH ₂ PO ₄ ) was dissolved and diluted slowly with methyl alcohol to obtain a precipitate of chondroitin sulfate sodium salt. The precipitated chondroitin sulfate sodium salt was washed with methyl alcohol and dried to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 18]

And extracted and purified in the same manner as in Example 17 except that ethyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 19]

And extracted and purified in the same manner as in Example 17 except that propyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

[Example 20]

And extracted and purified in the same manner as in Example 17 except that butyl alcohol was used instead of methyl alcohol to obtain about 16 g of white chondroitin sulfate sodium salt.

As described above, the method of producing chondroitin sulfate and sodium salt thereof from shark bone or cartilage of the present invention is firstly purified by using an ion exchange resin, so that it is possible to obtain a high-purity sulfosuccinate with a stable condehyde, In addition, the addition of sodium salt increases the solubility in water and can be easily used in various fields such as raw materials for pharmaceuticals, raw materials for cosmetics, and raw materials for health food.

Claims (4)

A method for producing chondroitin sulfate and its sodium salt represented by the following formula (1) from shark bone or cartilage, (1) immersing shark bone or cartilage in a hydrochloric acid solution, and then washing with 95% ethanol to remove impurities; (2) extracting chondroitin sulfate from shark bone or cartilage using proteolytic enzyme; (3) adjusting the pH of the extract to about 8.5 to remove the protein of chondroitin sulfate; (4) purifying the extract using an ion exchange resin; ⑤ Completely dissolving, if necessary, adding sodium salt to the extract which has passed through the ion exchange resin; And (6) crystallizing chondroitin sulfate or its sodium salt using alcohol; ≪ / RTI > wherein the chondroitin sulfate and chondroitin sulfate are sodium chondroitin sulfate. [Chemical Formula 1] Here, n: 75 to 85 M: H or Na The ion exchange resin according to claim 1, wherein the ion exchange resin is a mixed ion exchange resin filled with a cation exchange resin and an anion exchange resin mixed at a volume ratio of 9: 1 to produce chondroitin sulfate and sodium salt thereof from shark bone or cartilage Way. The method according to claim 1, wherein the sodium salt is added in an amount of 6 to 20% by weight of the extracted purified liquid, wherein the sodium salt is at least one selected from sodium chloride, sodium monohydrogen phosphate salt and sodium dihydrogen phosphate salt. A method for producing chondroitin sulfate and its sodium salt from cartilage. The method according to claim 1, wherein the alcohol is selected from the group consisting of methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, and a mixture of two or more of the isomers thereof to produce chondroitin sulfate and sodium salt thereof from shark bone or cartilage Way.