JPH0577681B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing low molecular weight hyaluronic acid. [Conventional technology] Hyaluronic acid is widely distributed in living tissues such as cock's comb, umbilical cord, cartilage, skin, vitreous body, and joint fluid, and hyaluronic acid extracted from these is used to smooth human skin. It has traditionally been used as an ingredient in cosmetics because it has the effect of smoothing out skin wrinkles. In addition, since such effects are more pronounced in hyaluronic acid, which has higher molecular weight and higher viscosity, conventional research in this field has mainly focused on high molecular weight hyaluronic acid. By the way, unlike high-molecular hyaluronic acid, low-molecular hyaluronic acid is easily soluble in water and has a low viscosity.
Cosmetics containing this product have been found to have the effect of not giving the skin any unpleasant sensations such as stickiness or tightness, and expectations have begun for the use of low-molecular-weight hyaluronic acid as a cosmetic raw material. [Problems to be solved by the invention] Conventionally, when a hyaluronic acid-containing raw material, such as a cock's comb, is left at room temperature for a long time, the hyaluronidase contained in the cock's comb decomposes the hyaluronic acid and converts it into a low-molecular-weight material. It is known to do. However, since hyaluronic acid extracted from such raw materials has been reduced to a low molecular weight by bacterial hyaluronidase produced by bacterial growth, it not only has microbiological problems, but also has problems with microbiology. It has been recognized that the method used for producing low-molecular-weight hyaluronic acid has problems such as difficulty in controlling the degree of decomposition and low yield. On the other hand, hyaluronic acid has poor stability, and in addition to treatment with hyaluronidase as described above, heat treatment,
It is known that it can also be reduced in molecular weight by treatment with strong acids, strong alkalis, etc. However, to the best of the present inventors' knowledge, there have been no research reports on how to efficiently produce low-molecular-weight hyaluronic acid from the viewpoint of production of low-molecular-weight hyaluronic acid on an industrial scale, its quality, yield, etc. Not yet. The present invention aims to solve precisely such problems. [Means for solving the problem] As a result of extensive research in accordance with the above-mentioned objective, the present inventors performed an alkali decomposition treatment under certain conditions on a paste made from a hyaluronic acid-containing raw material. In this case, it was discovered that low-molecular-weight hyaluronic acid with an average molecular weight of about 200,000 to 800,000 could be obtained in high yield. In addition, the present inventors discovered that if the alkaline decomposition treatment is applied until the average molecular weight becomes less than 200,000, the yield of the final product not only decreases, but also the product becomes brown, and conducted further research. However, if acid decomposition treatment is performed under certain conditions on a product that has been subjected to alkaline decomposition treatment to have an average molecular weight of 200,000 to 800,000, the quality of low-molecular-weight hyaluronic acid with an average molecular weight of 10,000 to 200,000 will be lost. It has been found that it can be produced in high yield without any problems. The present inventors further found that when hyaluronic acid-containing raw materials that have not been subjected to alkaline decomposition treatment are directly subjected to acid decomposition treatment, the degree of decomposition is low, probably because decomposition occurs mainly at the terminal end of hyaluronic acid; It was also found that even when alkali decomposition treatment is applied, the degree of molecular weight reduction is almost the same as when the alkali treatment is performed alone, leading to the completion of the present invention. The present invention involves adding an alkali to a pasted hyaluronic acid-containing raw material to decompose the hyaluronic acid in the raw material with an alkali, and at this time, the treatment is carried out at a final alkali concentration of 0.05 to 0.1M, a treatment temperature of 50 to 70°C, and a treatment temperature of 50 to 70°C. The present invention provides a method for producing low-molecular-weight hyaluronic acid, which is characterized by carrying out the method for 60 to 180 minutes, and then extracting hyaluronic acid from the raw material. The hyaluronic acid-containing raw material used in the method of the present invention is made into a paste. Here, the hyaluronic acid-containing raw material is not particularly limited as long as it contains animal tissues that contain hyaluronic acid, and includes, for example, a cock's comb, umbilical cord, joint fluid, vitreous body, and skin. Although it varies depending on the tissue, a cock's comb contains about 1 to 2% hyaluronic acid. Furthermore, the average molecular weight of hyaluronic acid is approximately 2 million to 8 million. It is optional to perform a heat treatment to thermally denature the proteins contained in the raw materials prior to making them into a paste. Furthermore, if the raw material is finely ground using a shredder, chopper, etc. before or after this heat treatment, there is an advantage that it becomes easier to make a paste. The paste can be made by any method as long as the raw material can be crushed and made into fine particles to form a paste. For example, fresh water may be added and the material may be homogenized using a homogenizer, a high-speed stirrer, a crusher, or the like. The reason why the hyaluronic acid-containing raw material is made into a paste in this way is to efficiently carry out the alkaline decomposition treatment of the hyaluronic acid in the raw material. According to the method of the present invention, the pasted hyaluronic acid-containing raw material is then subjected to alkaline decomposition treatment under certain conditions. The certain conditions here are:
Final alkali addition concentration 0.05-0.1M, treatment temperature 50
~70 °C, and treatment time 60-180 minutes. By alkaline decomposition treatment under such conditions, low molecular weight hyaluronic acid with an average molecular weight of about 200,000 to 800,000 can be obtained in a high yield, generally about 0.9 to 1.20% of the raw material structure. For example, sodium hydroxide
Hyaluronic acid with an average molecular weight of about 800,000 was obtained under treatment conditions of 0.05M, 50℃ and 120 minutes, and hyaluronic acid with an average molecular weight of about 200,000 was obtained under treatment conditions of 0.1M sodium hydroxide, 60℃ and 120-150 minutes. ~300,000 hyaluronic acid can be obtained. If the processing conditions exceed the upper limits of each of the above ranges, even if low-molecular-weight hyaluronic acid with an average molecular weight of less than 200,000 can be produced, the yield of the final product will not only decrease, but it will also turn brown. Furthermore, if the treatment conditions do not fall within the lower limits of each of the above ranges, it will not only be difficult to sufficiently reduce the molecular weight of hyaluronic acid, but also the yield will be poor. The alkali used is not particularly limited, but typically potassium hydroxide, calcium hydroxide, etc. in addition to sodium hydroxide are used. According to the method of the present invention, hyaluronic acid is then extracted from the raw material subjected to alkaline decomposition treatment. At this time, prior to extraction, existing proteins may be removed by decomposition treatment for the purpose of increasing the extraction efficiency of hyaluronic acid. Incidentally, this proteolytic treatment may be carried out according to a conventional method. Extraction of hyaluronic acid can be carried out using conventional methods. For example, after removing solids by passing through a protein-decomposed paste, a cetylpyridinium chloride (CPC) solution is added to the resulting liquid to convert hyaluronic acid into CPC. It may be adsorbed and precipitated. The separated precipitate may be dissolved in saline and then subjected to the acid decomposition treatment described below for the purpose of further reducing the molecular weight of hyaluronic acid, or by adding ethanol to the above saline to further reduce the molecular weight of hyaluronic acid. Molecular hyaluronic acid may be precipitated. Furthermore, the crude hyaluronic acid thus obtained may be purified according to a conventional method to produce a low-molecular-weight hyaluronic acid product. According to the method of the present invention, in order to obtain hyaluronic acid with a lower molecular weight, the extracted hyaluronic acid is
Acid decomposition treatment under the conditions of PH2.8~4.0, 0.70~90℃ and 30~180 minutes. By acid decomposition treatment under such conditions, low molecular weight hyaluronic acid having an average molecular weight of about 10,000 to 200,000 can be obtained in a high yield, generally at about 0.75 to 0.95% of the raw material structure. Processing conditions are PH
If it is lower than the lower limit above for other factors and exceeds the upper limit of each range above for other factors, it is possible to produce low molecular weight hyaluronic acid with an average molecular weight of less than 10,000, but not only will the yield of the final product decrease; This stuff starts to turn brown. Furthermore, if the treatment conditions are higher than the upper limit for pH than the above-mentioned upper limit, and lower than the lower limits of each of the above-mentioned ranges for other factors, it is difficult to predict further reduction in the molecular weight of hyaluronic acid. Although the acid used is not particularly limited, hydrochloric acid, sulfuric acid, acetic acid, etc. are typically used. Particular attention should be paid to the material of the equipment when carrying out acid decomposition under conditions of strong acidity, high temperature, and long periods of time. As described above, according to the method of the present invention, desired low molecular weight hyaluronic acid can be treated with alkaline decomposition alone,
Alternatively, by selecting the treatment conditions within the above-mentioned specific range in combination with acid decomposition treatment, high yield production can be achieved. [Effects of the Invention] The following Test Examples 1 to 3 show how low-molecular-weight hyaluronic acid can be effectively produced according to the method of the present invention. Test Example 1 This test example shows that low molecular weight hyaluronic acid having an average molecular weight of 200,000 to 800,000 can be obtained in high yield by alkaline decomposition treatment under certain conditions. still,
In the present invention, % is % by weight. Each 1 kg of chicken comb is immersed in hot water at 80°C for 20 minutes, heat-treated, and then finely chopped with a shredder.
Double the amount of fresh water was added to form a paste using a homogenizer, and sodium hydroxide was added to the paste, which was then subjected to alkaline decomposition treatment under the conditions shown in Table 1 below. Each sample was then subjected to proteolytic treatment, and undissolved solids were removed by filtration, and a cetylpyridinium chloride solution was added to the resulting liquid to adsorb and precipitate hyaluronic acid. Each separated precipitate was purified by washing with ethanol and then dried. The yield and average molecular weight (calculated from the intrinsic viscosity) of each product obtained after drying were determined, and the results are summarized in Table 1.

[Table] Note: Under conditions 4 and 5, the product turned brown.
Test Example 2 This test example shows that low-molecular-weight hyaluronic acid with an average molecular weight of 200,000 or less can be obtained by performing alkaline decomposition treatment under certain conditions and then acid decomposition treatment under certain conditions. . Follow the method of Test Example 1 above from each 1 kg of chicken comb, and perform alkali decomposition treatment at the final alkali addition concentration.
Hyaluronic acid solutions dissolved in 2% saline were obtained by the same treatment except that the treatment was carried out under the conditions of 0.1M NaOH, treatment temperature 60° C., and treatment time 120 minutes. 4N hydrochloric acid was added to these solutions, and each solution was subjected to acid decomposition treatment under the conditions shown in Table 2 below. After cooling, ethanol was added to precipitate hyaluronic acid, and each precipitate was then purified by washing with ethanol, followed by drying. Determine the yield and average molecular weight (calculated from the intrinsic viscosity) of each product obtained after drying,
The results are summarized in Table 2.

[Table] Test Example 3 This test example shows that low molecular weight hyaluronic acid having an average molecular weight of 100,000 or less can be obtained in high yield by appropriately selecting the treatment conditions for acid decomposition. Follow the method of Test Example 1 above from each 1 kg of chicken comb, and perform alkali decomposition treatment at the final alkali addition concentration.
Hyaluronic acid solutions dissolved in 2% saline were obtained by the same treatment except that the treatment was carried out under the conditions of 0.1M NaOH, treatment temperature 60° C., and treatment time 150 minutes. 4N hydrochloric acid was added to these solutions, and each solution was subjected to acid decomposition treatment under the conditions shown in Table 3 below. After cooling, ethanol was added to precipitate hyaluronic acid, and then each separated precipitate was washed with ethanol, purified, and then dried. Determine the yield and average molecular weight (calculated from the intrinsic viscosity) of each product obtained after drying,
The results are summarized in Table 3.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 1 kg of chicken comb was immersed in hot water at 80°C for 20 minutes, heat-treated, and then finely crushed with a shredder. Approximately three times the amount of fresh water was added to this, and the mixture was made into a paste using a homogenizer. Sodium oxide (final alkali addition concentration 0.1M) was added, and alkali decomposition treatment was carried out under stirring conditions at 60°C for 120 minutes. The mixture was then subjected to proteolytic treatment to remove undissolved solids by filtration, and a cetylpyridinium chloride solution was added to the resulting liquid to adsorb and precipitate hyaluronic acid. After the separated precipitate was dissolved in 2% saline, 4N hydrochloric acid was added to this solution to adjust the pH to 3.0, and acid decomposition treatment was performed at 90°C for 40 minutes. After cooling, ethanol was added to precipitate hyaluronic acid, and the separated precipitate was purified by washing with ethanol and dried to obtain 8.4 g of white powder. The average molecular weight of this product was 70,000, and the yield was 0.84%. Example 2 In the method of Example 1 above, acid decomposition treatment was performed at PH
3.8, 9.6 g of white powder was obtained from 1 kg of chicken comb in the same manner except that it was carried out under the conditions of 80°C and 100 minutes.
I got it. The average molecular weight of this product is 160,000, and the yield is
It was 0.96%. Example 3 In the method of Example 1 above, the alkali decomposition treatment was carried out at a final alkali addition concentration of 0.1M, a treatment temperature of 60°C,
The treatment time was 150 minutes, and the acid decomposition treatment was performed at PH.
2.8, 7.9 g of white powder from 1 Kg of chicken comb was carried out in the same manner except that it was carried out under the conditions of 90°C and 150 minutes.
I got it. The average molecular weight of this product is 10,000, and the yield is
It was 0.79%.

Claims (1)

[Claims] 1. Add an alkali to a pasted hyaluronic acid-containing raw material to decompose the hyaluronic acid in the raw material with an alkali, and at this time, the treatment is carried out at a final alkali concentration of 0.05 to 0.1M and a treatment temperature of 50 to 70%. 1. A method for producing low-molecular-weight hyaluronic acid, which is carried out under conditions of temperature and treatment time of 60 to 180 minutes, and then extracting hyaluronic acid from the raw material. 2 Using sodium hydroxide as the alkali,
A method for producing low molecular weight hyaluronic acid according to claim 1.