JPS6254324B2 - - Google Patents

Description

[Detailed description of the invention]

[B] Object of the Invention The present invention relates to a method for separating and producing hyaluronic acid from umbilical cord or chicken crest. (Industrial application field) Hyaluronic acid has excellent moisturizing effects (water absorption, moisturizing), and for this reason, pyrrolidone carboxylic acid,
Along with substances that have a moisturizing effect such as N-acetylglycine, it has come to be used in the formulation of skin cosmetics and topical preparations, and is attracting attention. In addition to this, applications of hyaluronic acid are being considered in the medical treatment field, including eye drops, blood and serum diseases such as arteriosclerosis, arthritis, and nephritis. (Prior art) Regarding the production method of hyaluronic acid, the conventional method of extracting, separating, and purifying it from the umbilical cord and chicken comb is as follows.
For example, the following [A] to [E] are known. [A] A method in which animal tissue is shredded, defatted using an organic solvent such as acetone, and then eluted and separated using a large amount of water or a salt solution... (Biochem.
Biophys.Acta158, 17-30, 1966). [B] A method in which animal tissue is shredded, defatted using an organic solvent such as acetone, treated with protease, and then eluted, separated, and purified (J.
Biol.chem.186, 495-511, 1950). [C] In the separation process of hyaluronic acid, coexisting chondrointi sulfate is not extracted, so
A method in which thawing is performed at a temperature of 35 to 65°C for 30 to 300 minutes, protease is used, and the reaction temperature is limited to 35 to 60°C... (Public Patent Publication: 522-145594) . [D] Protease reaction temperature: 35-60℃, pH
A method in which the number is limited to 5 to 8... (Public Patent Publication:
(Sho 54-67100). [E] Chicken crest with quaternary ammonium salt
A method in which heat treatment is performed with a 0.02-1.0% solution, followed by reaction at 50°C for 5 hours using protease, followed by fractionation... (Public Patent Publication: 1974-74796)
and so on. (Problems to be Solved by the Invention) In the method [A] shown above, the extraction efficiency of hyaluronic acid is poor and the yield is low. In method [B], when separating other mucopolysaccharides that coexist with hyaluronic acid, complicated fractionation, typified by ammonium sulfate fractionation, etc., is performed in the presence of pyridine, alcohol, or chloroform. operation is required. Furthermore, both [A] and [B] involve a treatment step with an organic solvent such as acetone, and this step requires a large amount of organic solvent. On the other hand, in the method shown in [C], when thawing is carried out by immersion in hot water, hyaluronic acid is eluted at this time, which reduces the yield. It also affects the viscosity of the liquid, often leading to a tendency for the viscosity to decrease. The moisturizing and slippery feeling effect of hyaluronic acid is
The higher the viscosity, the higher the viscosity. Particularly in cosmetics, etc., there is a demand for products with a high slipping effect, so in this respect, it is necessary to further devise processing conditions. Next, in the method shown in [D], the optimal temperature for many enzymatic reactions, not just proteases, is around 37.5°C, which is the same as the in-vivo temperature. If it is a protease, of course it is customary to use it at 5 to 8, and when the reaction is performed at 60°C or lower, it depends on the reaction time at that time, but in the case of a reaction of about 4 to 5 hours, the extraction ineffective. For this reason, the next step of filtration takes too much time. In addition, in the method shown in [E], the treatment is performed by adding a quaternary ammonium salt solution, but when this method is adopted, the elution of hyaluronic acid is faster than in the other methods mentioned above. , slightly less. This is probably
This is thought to be caused by denaturation due to heating during the process. In other words,
The viscosity of the liquid after this heating process is lower compared to other methods, and in the end, even if hyaluronic acid is separated and purified from the liquid by this operation, only hyaluronic acid with a low viscosity can be obtained. There will be no.
Therefore, when considering the use of hyaluronic acid, its low viscosity is a critical factor. on the other hand,
A common method for fractionating hyaluronic acid is column chromatography using ion exchange resin, but due to the high viscosity of hyaluronic acid, it is difficult to fractionate it in good yield. This method is extremely difficult and complicated, and is not suitable for industrial mass production. Therefore, the present inventors investigated ways to easily mass-produce hyaluronic acid on an industrial scale by eliminating the drawbacks of the conventional methods described above, by checking for improvements based on the conventional methods. In addition, we conducted research by making prototypes for various improvements. As a result, we have developed a method that simplifies the processing operation, does not require a large amount of organic solvent unlike conventional methods, has a fast filtration speed, and has an excellent yield. [B] Structure of the Invention The specific main part of the present invention lies in the protein removal treatment operation during the process. In other words, "cut the umbilical cord or chicken crest into strips,
Extract with stirring for 30 minutes at 60-65℃ in twice the volume of 0.03-0.05N sodium hydroxide solution, then
Neutralize using 30w/w% trichloroacetic acid, and after neutralization, add trichloroacetic acid to 3 to 5% w/w to remove protein. (Means for solving the problem) The main part of extracting and purifying highly viscous hyaluronic acid with good operability and good yield is the method of deproteinization. . In other words, the purification of hyaluronic acid and the quality of its workability are basically related to how the raw materials, proteins present in the umbilical cord and chicken comb, are broken down and removed. It's always no exaggeration. As a solution to this problem, the present inventors obtained hyaluronic acid by using a combination of alkali and acid to achieve high yield and good operability (filtration), and on an industrial scale. This made production possible. Of course, as in the present invention, the two combinations of alkali and acid can be used on an industrial scale with good yield.
There are no other examples of successful production of high purity. In other words, one of the conditions for extracting hyaluronic acid on an industrial scale is the separation, extraction, and purification of hyaluronic acid, as well as the hydrolysis of proteins coexisting in the raw material organs. Although the viscosity is greatly affected, in this case, the previously disclosed hydrolysis using acids or alkalis is insufficient, and recently, proteases have been used exclusively as a means to solve these problems. It was believed that this could be done easily using protease. That is, an important key to methods using alkali or acid is that filtration after protein hydrolysis is poor. Furthermore, methods using alkalis or acids cause fluctuations in viscosity, making it difficult to obtain a constant level of hyaluronic acid. The present inventors therefore discovered conventional methods [A] to [E]
Based on the known technical means disclosed in et al., various conditions were set, extraction experiments were repeated, and various devices were devised based on the hydrolysis method using a combination of alkali and acid. We have been making improvements and considering improvements. As a result, as shown in the following example, under the conditions of caustic soda as the alkali and trichloroacetic acid as the acid, hyaluronic acid with excellent operability can be obtained by setting the relationship between the concentration, reaction time, and temperature. They discovered that it is possible to manufacture That is, according to the present method, the protein removal operation is facilitated, and the method is integrally connected to this operation.
Filtration, which is one of the steps for purification, has been made easier, and high-purity purification can now be produced efficiently. [Example-1] After washing 5 kg of fresh human umbilical cord with water, finely chop it with a mince cutter, add twice the amount of 0.03N sodium hydroxide solution, and stir and extract with a homomixer at 60°C for 30 minutes. If this extract is noticeably colored,
Add hydrogen peroxide and stir gently for another 30 minutes at 60°C. Next, it is neutralized using 30w/w% trichloroacetic acid, and residual hydrogen peroxide is decomposed with catalase at 60°C for 2 hours. Thereafter, trichloroacetic acid is added to remove protein to a concentration of 3 w/w%, the mixture is filtered, and then neutralized with dilute alkali. Leave this in running water (tap water) overnight.
After dialysis, the filtrate (crude extract) obtained by filtration is approx.
15, add 4% cetylpyridinium chloride solution to about 2
Collect the precipitate that is added, and collect this precipitate.
Dissolve in 0.3M sodium chloride solution and filter. Furthermore, sodium chloride was added to this filtrate to make a 0.5M solution, and the pre-regenerated Dowex
-1 (manufactured by Dow Chemical) by 5-10%, about 30%
After stirring for a minute, twice the amount of ethanol was added to the filtrate obtained by filtration, and the resulting precipitate was collected and dried to obtain about 25 g of hyaluronic acid. [Example-2] After washing 5 kg of fresh chicken crest, crush it with a mince cutter, add twice the amount of 0.05N sodium hydroxide solution, and stir and extract at 65°C for 30 minutes with a homomixer. If the extract is noticeably colored, add hydrogen peroxide and stir gently at 60°C for 30 minutes. Next, it is neutralized using 30 w/w % trichloroacetic acid, and residual hydrogen peroxide is decomposed with catalase at 55° C. for 1 hour. Thereafter, trichloroacetic acid is added to remove protein to a concentration of 5 w/w%, the mixture is filtered, and then neutralized with dilute alkali. After dialyzing this in running water (tap water) overnight, the filtrate obtained by filtration (crude extract)
Add 5% cetylpyridinium chloride solution to approx.
1.6. Collect the resulting precipitate, dissolve in 0.4M sodium chloride solution, and filter. Take the filtrate, add sodium chloride to this filtrate to make a 0.5M solution, add 5-10% of DEAE cellulose (manufactured by Pharmacia), which has been regenerated in advance, and add about 30% of DEAE cellulose (manufactured by Pharmacia).
After stirring for a minute, twice the amount of ethanol was added to the filtrate obtained by filtration, and the resulting precipitate was collected and dried to obtain about 30 g of hyaluronic acid. The viscosity of the hyaluronic acids obtained in Examples 1 and 2 was measured using an Ubbelohde viscometer, and as shown in Table 1, the viscosity was sufficiently satisfactory. Table 1 also shows the measured values for the N content and S content. Further, electrophoresis using a cellulose acetate membrane and toluidine blue staining were performed, but all the results showed monospots, indicating that the product was obtained in a state close to pure.

[Table] Measurement of N content and S content was carried out in accordance with the Japanese Bureau's General Test Methods. N amount: semi-micro Kjeldahl method. S amount: Measured after acid decomposition. On the other hand, the results obtained by the organic trace element analyzer are as follows.
The hyaluronic acids obtained in Examples 1 and 2 were approximately as shown in Table 2, and although the C content was slightly lower than the theoretical value, it was confirmed that they were almost pure products. The ash content was 8.3%.

[Table] As shown in Examples 1 and 2 above, the method for producing hyaluronic acid according to the present invention includes the following basic operations:
A crude extract mainly composed of mucopolysaccharides is obtained using an alkaline solution, and a dialysis step → cetylpyridinium chloride solution step → an anion exchange resin or anion exchanger is used to produce hyaluronic acid. It consists of a combination of sequences, and the main part is based on the conventional method, the optimal conditions for the chemicals used in each step, their concentrations, the relationship between temperature and reaction time, the yield and processing steps, and the protease It is obtained by a method that does not use , and is the best method as an industrial production method. That is, according to the present invention, there is an advantage that highly viscous hyaluronic acid can be obtained in high yield. The reason why we were able to achieve this viscosity and high yield is the setting of the conditions for the treatment step using a combination of alkali and acid when obtaining the crude extract in the first step. [C] Effect of the invention The main part of the production method according to the present invention is to add 0.03 to 2 times the amount of umbilical cord or chicken crest as the starting material.
The feature is that a 0.05N sodium hydroxide solution is used, and the extraction temperature is 60-65°C. Furthermore, the extraction time is 30
It is best to keep it to about a minute. In this case, efficient elution of the acidic mucopolysaccharide is not sufficient under conditions other than the combining conditions. In other words, although conventional crude extraction methods using alkali or acid include simple heating extraction with water or alkali, it is difficult to determine whether sufficient acidic mucopolysaccharide has been transferred to the crude extract. Alternatively, the alkali or acid and heating conditions were different, and as a result, a significant decrease in the viscosity of the acidic mucopolysaccharide was observed. On the other hand, if this method is used, the acidic mucopolysaccharide contained can be smoothly separated even in the next step (protein removal operation). In addition, in the known method, there was no procedure to perform decolorization under alkaline extraction conditions, but the present inventors used hydrogen peroxide in advance to solve the problem, since pigments are also eluted into the extract. We adopted a method that includes a decolorization process. Of course, the remaining hydrogen peroxide is decomposed using catalase. This decolorization operation makes the subsequent protein removal operation even smoother. Particularly when the umbilical cord is used as a starting material, it is advantageous in removing hemoglobin, which coexists with blood-derived proteins contained in large amounts in the umbilical cord. Next, in the protein removal operation, trichloroacetic acid is used, but in the present invention,
After considering the concentration used and the state of protein separation, we found that the concentration of the extract was 3w/w%.
The best conditions are to add trichloroacetic acid. Next, as a next step, a 4% cetylpyridinium chloride solution is added to the crude extracts obtained in Examples 1 and 2 at a ratio of 1 to 2 parts per about 10 parts of the crude extracts. The resulting precipitate was collected by filtration,
Next, a method of dissolving in 0.2M sodium chloride solution and filtering was adopted. A method using this quaternary ammonium salt is, for example, using chicken crest as a starting material, heat-treating it in a 0.02 to 1.0% solution, and then reacting with protease at 50°C for 5 hours. Method for carrying out (Public Patent Publication: 1974-74796)
However, in this known method, the final yield of hyaluronic acid is lower than in the method of the present invention. The results of investigating the cause of this are that, along with heat treatment, it seems to have a strong influence on the relationship between protease reaction temperature and time, and the viscosity of the hyaluronic acid finally obtained also decreases. It was thought that this was caused by denaturation, especially due to heating. The present inventors conducted additional tests based on the ability of acidic mucopolysaccharide to form a precipitate using a quaternary ammonium salt solution, selected the most suitable one, and determined the relationship with the concentration used. As a result, 4%
It has been found that acidic mucopolysaccharides can be precipitated smoothly using a cetylpyridinium chloride solution. Then, as a neutral salt, hyaluronic acid forming a complex salt with quaternary ammonium is eluted in a 0.2-0.4M solution of sodium chloride, and then sodium chloride is further added.
When a 0.5M solution was obtained, an anion exchange resin or anion exchanger was added, gently stirred for 30 minutes, and then filtered. Through this operation, acidic mucopolysaccharides other than hyaluronic acid (mostly chondroitin sulfate), which are present in extremely small amounts, are filtered out while bound to an anion exchange resin or anion exchanger. It can be removed as a residue. Therefore, the filtrate obtained by filtration contains only hyaluronic acid, and the rest is precipitated by adding an equal to twice the amount of ethanol to the filtrate, and then the precipitate is dried. Hyaluronic acid can be obtained by The present invention has simpler processing operations than conventional alkaline hydrolysis extraction methods, and in particular, filtration during each step is easy. In addition, it is suitable for industrial production because it requires less use of organic solvents and high viscosity hyaluronic acid can be obtained in high yield.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum of hyaluronic acid obtained in Examples 1 and 2.

Claims (1)

[Claims] 1. When extracting hyaluronic acid from the umbilical cord or chicken comb, it is usually obtained by protein removal treatment, dialysis treatment, cetylpyridinium chloride solution treatment, sodium chloride solution treatment, ion exchange resin treatment, and finally ethanol precipitation. During the process of the method, the conditions for protein removal treatment are as follows: umbilical cord or chicken comb fragments are treated in twice the volume of 0.03 to 0.05N sodium hydroxide solution at 60 to 65°C for 30 minutes. A method for producing hyaluronic acid, which comprises stirring, then neutralizing with 30 w/w % trichloroacetic acid, and further adding 3 to 5 w/w % trichloroacetic acid to perform protein removal treatment.