KR20020071772A - Manufacturing method of hyaluronic acid to have sterilization activity - Google Patents

Abstract

PURPOSE: A process of preparing hyaluronic acid by imparting antimicrobial properties to hyaluronic acid during the production of hyaluronic acid is provided. Whereby, the product has antimicrobial activity and can be effectively used in the treatment of diseases. CONSTITUTION: A crest of chickens is ground, extracted in water two or three times, added with trichloro acetic acid and then filtered. The precipitate is washed with an organic solvent, filtered, added with a tea extract in a weight ratio of 1:0.2 to 0.4, left alone at 40 to 60deg.C for 1hr or more and dried after filtering. The tea extract is obtained by mixing 1 parts by weight of dried tea leaves with 80 to 100 parts by weight of water at 95 to 100deg.C and then extracting.

Description

Manufacturing method of hyaluronic acid to have sterilization activity

The present invention relates to a method for producing hyaluronic acid having bactericidal activity, and more specifically, it is possible to prepare hyaluronic acid added with antimicrobial activity, so that it is used not only for the use of conventional hyaluronic acid but also for the treatment of diseases caused by pathogen infection of the human body. It relates to a method for producing hyaluronic acid having an antimicrobial activity so that it can be.

Hyaluronic acid is a linear polymer consisting of beta bonds of D-glucuronic acid and N-acetyl-D-glucosamine. Cellular membranes, the synovial fluid of joints, the intraocular fluid of the eye, It can be found in human umbilical cord tissue and chicken clans.

Hyaluronic acid has characteristics such as high viscosity, moisturizing property, and bioadaptability, and thus is widely used in various fields including ophthalmic surgery, the treatment of racing horse knee joints, and cosmetic moisturizers.

The hyaluronic acid has been commonly obtained through a biochemical method or a chemical method obtained by using a strain or a method of extracting from plants and animals, many techniques are known.

In particular, a method of obtaining hyaluronic acid is known by pulverizing chicken crustaceans in a method of obtaining hyaluronic acid from animals and plants, extracting hyaluronic acid with water several times, and then using this extract through a series of purification and separation processes.

However, hyaluronic acid obtained from the above-described conventional methods can be suitably used for ophthalmic surgery or cosmetic moisturizers due to its high viscosity, but its use is limited. Is lifting up.

Accordingly, an object of the present invention is to provide a method for preparing hyaluronic acid having antimicrobial activity that can be effectively used for treating diseases by providing antimicrobial activity to hyaluronic acid in the process of obtaining hyaluronic acid.

1 is a block diagram showing a hyaluronic acid production process of the present invention.

The present invention to achieve the above object

In the method of pulverizing chicken crust and extracting with water two or three times, adding trichloroacetic acid to the extract and filtering to obtain a precipitate, washing the precipitate with an organic solvent and drying to obtain hyaluronic acid. ,

The precipitate washed with the organic solvent was dissolved in water, filtered, and the mixture was added and mixed with the tea extract, and then left at 40 to 60 ° C. for at least 1 hour, and the filtrate was filtered and dried. It can be achieved by providing a method for producing hyaluronic acid having an antimicrobial activity.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings, which are presented to aid the understanding of the present invention, but the present invention is not limited thereto.

1 is a block diagram showing a hyaluronic acid production process of the present invention, as shown in the present invention to obtain a hyaluronic acid-containing extract (110); Adding trichloro acetic acid to the extract and then filtering to obtain a precipitate (120); Washing the precipitate with an organic solvent (130); The washed precipitate is dissolved in water and then filtered (140); Adding the tea extract to the filtrate and mixing (150); Filtering the mixed solution (160); The hyaluronic acid having antibacterial activity is obtained through the process of drying the filtrate (170).

Looking at the process in more detail in the present invention is first obtained by extracting the hyaluronic acid-containing extract by grinding the chicken shackles and extracted 2-3 times with water (110). In this process, hyaluronic acid contained in chicken crust is eluted and dissolved in water.

At this time, the water used to extract the hyaluronic acid from the ground chicken rice is not necessarily limited to the amount used, it is irrelevant as long as the amount of hyaluronic acid can be sufficiently eluted. In the present invention, 400 to 600 parts by weight of water at 20 ° C. was added to 100 parts by weight of crushed chicken crust, and the mixture was left to stand for 2 to 4 hours to perform hyaluronic acid extraction three times.

Trichloro acetic acid is added to the extract thus obtained and then filtered to obtain a precipitate (120). In this process, hyaluronic acid contained in the extract precipitates.

When trichloroacetic acid is slowly added to the extract, it is possible to obtain a hyaluronic acid precipitate in the form of a jelly, which can be easily obtained by filtration. It is not necessary to necessarily limit the amount of the trichloroacetic acid added, in the present invention, trichloroacetic acid in an amount corresponding to 1 to 2% by volume is added to the extract, and trichloroacetic acid is added to the extract, followed by 1 at 20 ° C. It was left for hours.

The precipitate, such as jelly, thus obtained is washed with an organic solvent (130). This washing process removes fat and water from the sediment. In the present invention, the obtained precipitate was washed with acetone and ether, and when washed with acetone, it is possible to remove the water contained in the precipitate, and when washed with ether, it is possible to remove the fat contained in the precipitate. .

The amount of acetone and ether is not necessarily limited to the amount used to sufficiently wash the precipitate, but in the present invention, acetone and ether corresponds to 150 to 250 parts by weight based on 100 parts by weight of chicken crust, respectively Washing process was repeated three times.

The precipitate thus washed is dissolved in water again and filtered (140). In the present invention, the precipitate was completely dissolved using water in an amount corresponding to 200 to 400 parts by weight based on 100 parts by weight of chicken crust, and then filtered.

The tea extract is added to the filtrate obtained by filtration and mixed (150). Through this process, hyaluronic acid contained in the filtrate shows excellent antibacterial properties by interaction with polyphenols contained in tea extract. At this time, the polyphenol itself exhibits antimicrobial activity, but when mixed with hyaluronic acid, a double antimicrobial activity can be obtained.

The tea extract used in the present invention was used by adding 1 part by weight of dried tea leaves to 80-100 parts by weight of water at 95-100 ° C. for 5 to 10 minutes, and the tea extract obtained as described above was used for the filtrate described above. It added in the ratio of 1: 0.2-0.4.

At this time, if the addition amount of the tea extract is added in less than 1: 0.2 ratio to the filtrate, sufficient antimicrobial effect is not obtained, even if the addition amount of the tea extract is added to exceed the ratio of 1: 0.4 to the filtrate. It is not possible to obtain sufficient antibacterial effect. Therefore, the addition amount of the tea extract is preferably added in a ratio of 1: 0.2 to 0.4 with respect to the filtrate.

If the tea extract is added within the range as described above and then left at 40 to 60 ° C. for at least 1 hour, hyaluronic acid having excellent antimicrobial activity can be obtained.

Thereafter, in the present invention, through the step of filtering the reactant (160), the step of drying the filtrate (170). Through this process, hyaluronic acid powder having excellent pale yellow antibacterial properties can be obtained.

At this time, the drying is a process for evaporating the water of the filtrate can be applied to a variety of methods including a method of completely evaporating the water by applying ordinary heat.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

<Example 1>

Pour 4Kg of water into 1Kg of ground chicken crust and extract for 2 hours at 20 ° C., and then filter and extract the extract three times. 100 g of trichloroacetic acid was added to 10 Kg of the extract thus obtained, and the mixture was stirred at 20 ° C. for 1 hour. It was left to stand and then filtered to obtain a precipitate.

The obtained precipitate was washed three times with acetone 2Kg, and then washed three times with ether 2Kg, and then sufficiently washed, and then completely dissolved in 2Kg of water and filtered.

200 g of the tea extract was added to the filtrate, and the mixture was left at 40 ° C. for 1 hour, after which the filtrate was heated and dried to obtain 51.8 g of a light pale yellow powder. At this time, the tea extract was added to 10g of dried tea leaves (black tea) in 800ml of 95 ℃ water was used for 5 minutes and then filtered.

In order to determine the antimicrobial activity of the hyaluronic acid thus prepared 0.5 g of the obtained hyaluronic acid completely dissolved in 100g of water and then the antimicrobial experiment as follows and the results are shown in Table 1 below.

-Antimicrobial Activity Test-

After cooling to a TSB culture medium sterilized at 121 ℃ 15 minutes and dispensed by coagulation by 15㎖ a Petri dish aseptically, previously cultured broth tested here (in the present test was used for Bacillus subtilis and E. coli) 0.1㎖ Was applied uniformly to make a test plate medium. Separately, the sterilized sample solution was filtered with a 0.45㎛ membrane filter (Milipore, USA) and absorbed 2 mg of each solution into a pre-sterilized 8 mm filter paper disc (Toyo, Japan). After 1 hour of incubation in a refrigerator at 4 ° C., the incubator was incubated for 48 hours at 30 ° C., and then the diameter of the clear zone around the disk was measured.

<Example 2>

Pour 5 Kg of water into 1 Kg of ground chicken crust and extract it for 3 hours at 20 ° C., followed by three times to obtain an extract by filtration. 187.5 g of trichloroacetic acid was added to 12.5 Kg of the extract thus obtained, and 1 at 20 ° C. It was left for 30 minutes and then filtered to obtain a precipitate.

The obtained precipitate was washed three times with acetone 2Kg, and then washed three times with ether 2Kg, and then sufficiently washed, and then completely dissolved in 2Kg of water and filtered.

400 g of the tea extract was added to the filtrate, and the mixture was left at 50 ° C. for 1 hour, after which the filtrate was heated and dried to obtain 52.0 g of a light pale yellow powder. At this time, the tea extract was added to 10g of dried tea leaves (black tea) in 800ml of 95 ℃ water was used for 5 minutes and then filtered.

In order to determine the antimicrobial activity of the hyaluronic acid thus prepared 0.5 g of hyaluronic acid was completely dissolved in 100g of water and then carried out the antimicrobial experiments performed in Example 1 and the results are shown in Table 1 below.

<Example 3>

Pour 5 Kg of water into 1 Kg of ground chicken crust and extract it for 3 hours at 20 ° C., followed by three times to obtain an extract by filtration. 187.5 g of trichloroacetic acid was added to 12.5 Kg of the extract thus obtained, and 1 at 20 ° C. It was left for 30 minutes and then filtered to obtain a precipitate.

The obtained precipitate was washed three times with acetone 2Kg, and then washed three times with ether 2Kg, and then sufficiently washed, and then completely dissolved in 2Kg of water and filtered.

800 g of tea extract was added to the filtrate, and the mixture was left at 50 ° C. for 1 hour, after which the filtrate was heated and dried to obtain 52.1 g of a light pale yellow powder. At this time, the tea extract was added 15g of dried tea leaves (black tea) to 1350ml of water at 97 ℃ was stirred for 7.5 minutes and then filtered.

In order to determine the antimicrobial activity of the hyaluronic acid thus prepared 0.5 g of hyaluronic acid was completely dissolved in 100g of water and then carried out the antimicrobial experiments performed in Example 1 and the results are shown in Table 1 below.

<Example 4>

Pour 6Kg of water into 1Kg of crushed chicken crust and extract it for 4 hours at 20 ° C., and then filter and extract the extract three times.Added 300 g of trichloroacetic acid to 15 Kg of the extract thus obtained and for 2 hours at 20 ° C. It was left to stand and then filtered to obtain a precipitate.

The obtained precipitate was washed three times with acetone 2Kg, and then washed three times with ether 2Kg, and then sufficiently washed, and then completely dissolved in 2Kg of water and filtered.

1400 g of tea extract was added to the filtrate, and the mixture was left at 60 ° C. for 2 hours, after which the filtrate was heated and dried to obtain 52.3 g of a light pale yellow powder. At this time, the tea extract was added to 20g of dried tea leaves (black tea) in 2000ml of water at 97 ℃ 10 minutes and used to filter.

In order to determine the antimicrobial activity of the hyaluronic acid thus prepared 0.5 g of hyaluronic acid was completely dissolved in 100g of water and then carried out the antimicrobial experiments performed in Example 1 and the results are shown in Table 1 below.

Comparative Example 1

Pour 5Kg of water into 1Kg of ground chicken crust and extract it for 4 hours at 20 ° C., and then filter and extract the extract three times. 300 g of trichloroacetic acid was added to 12.5Kg of the extract thus obtained, and 2 hours at 20 ° C. It was left for a while and then filtered to obtain a precipitate. The obtained precipitate was washed three times with acetone 2Kg, and then washed three times with ether 2Kg, sufficiently washed and dried to obtain 51.6Kg of hyaluronic acid.

In order to determine the antimicrobial activity of the hyaluronic acid thus prepared 0.5 g of hyaluronic acid was completely dissolved in 100g of water and then carried out the antimicrobial experiments performed in Example 1 and the results are shown in Table 1 below.

Comparative Example 2

To investigate the antibacterial activity of green tea extract, 10 g of dried tea leaves (black tea) was added to 800 ml of 95 ° C. water for 5 minutes, and then 1 ml of the filtered filtrate was mixed with distilled water to prepare 100 ml. Using the antimicrobial experiments conducted in Example 1 using the results are shown in Table 1 below.

division Antimicrobial activity (mm) Bacillus subtilis E. coli Example 1 4 5 Example 2 41 43 Example 3 47 51 Example 4 4 5 Comparative Example 1 Does not appear Does not appear Comparative Example 2 16 19

As shown in Table 1, it can be confirmed that the antibacterial activity of Example 2 and Example 3 obtained by adding a tea extract within the preferred range of the present invention to obtain hyaluronic acid.

As described above, in the present invention, hyaluronic acid having antimicrobial activity was obtained by adding and mixing tea extract in the process of obtaining a conventional hyaluronic acid, and the hyaluronic acid obtained has antimicrobial activity and thus can be effectively used for treating diseases. It is a useful invention to provide a method for preparing hyaluronic acid having antimicrobial activity.

Claims (4)

In the method of pulverizing chicken crust and extracting with water two or three times, adding trichloroacetic acid to the extract and filtering to obtain a precipitate, washing the precipitate with an organic solvent and drying to obtain hyaluronic acid. , The precipitate washed with the organic solvent was dissolved in water and filtered, the tea extract was added to and mixed with the filtrate and left at 40 to 60 ° C. for at least 1 hour, and the filtrate was filtered and dried. Method for producing hyaluronic acid having an antimicrobial activity. The method for preparing hyaluronic acid having antimicrobial activity according to claim 1, wherein the tea extract is added to 1 part by weight of dried tea leaves at 80 to 100 parts by weight of water at 95 to 100 ° C for 5 to 10 minutes. The method of claim 2, wherein the tea extract is added to the filtrate in a ratio of 1: 0.2 to 0.4. The method for preparing hyaluronic acid having antibacterial activity according to any one of claims 1 to 3, wherein acetone and ether are used as organic solvents for washing the precipitate.