KR19990045704A - Method of low molecular weight and oligomerization of water soluble hydroxypropyl chitosan? - Google Patents

Abstract

(Objective) Low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan that does not cause water redness, coagulates in organic solvents, precipitates, penetrates into skin, absorbs well, and is not water-insoluble even when used as an animal therapeutic or added to cosmetics. To provide a way.

(Composition) Put chitosan and isopropanol in a beaker and alkali-slurry it, add propylene oxide to it, warm it for a certain time, adjust the pH to weak neutral after stirring, filter it, wash the residue with methanol, and dry it at low temperature While obtaining oxypropyl chitosan powder,

After dissolving the hydroxypropyl chitosan powder in deionized water in a beaker, the cellulase or chitosanase was added and stirred while warming to 55 ~ 60 ℃, and then fermented by a certain time at about 80 ℃ to remove, precipitate It is a method of low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan, characterized in that the obtained enzyme is filtered off and the filtrate is concentrated and freeze-dried.

Description

Method for Low Molecularization and Oligomerization of Water Soluble Hydroxypropyl Chitosan

The present invention relates to low molecular weight and oligomerization methods of high purity hydroxypropyl chitosan.

General chitosan is a prerequisite for the preparation of molecular weight and oligosaccharides. It is an essential prerequisite to dissolve using organic acid. On the other hand, in the case of a derivative in which chitosan is solubilized, it has excellent solubility in water. There is no need for pretreatment. When using such water-soluble chitosan, as the properties of each substituent have their own characteristics, their use can be applied very differently. Therefore, various studies on the preparation method of the introduction of such substituents have been conducted. It is done at home and abroad. In addition, while general chitosan is easily dissolved in acid, precipitation occurs under basic solution conditions, and general water-soluble chitosan derivatives are easily dissolved in general water, but this is a disadvantage that precipitation occurs under organic solvents such as methanol, acetone, and ethanol. As a result, its use is extremely limited. Therefore, it is necessary to develop stable chitosan derivatives according to pH and organic solvent conditions. Also, it is necessary to establish a method for preparing high purity hydroxypropyl chitosan. have. Therefore, in the manufacturing method of hydroxypropyl chitosan which has been known so far, 10 weight of sodium hydroxide and 45 weight of water are added to chitosan 100 as a general manufacturing method, and 250 weight of n-hexane and t-butanol are administered as an organic solvent, followed by nitrogen. Substitution was performed under atmosphere and reduced pressure. After stirring for 2 hours at 25 ° C., infiltrating alkali into chitosan, and then adding 272 weights of propyl oxide, the reaction was carried out at 80 ° C. for 5 hours to obtain a reactant, and the reactant was dealkalized with acetic acid. It is dissolved in hot water, purified and lyophilized to obtain powdered hydroxypropyl chitosan. However, the above-mentioned manufacturing method has a problem for its purification as a large amount of insoluble matter occurs, and also its removal by using a carcinogenic organic solvent has also been raised as a problem. Accordingly, Japanese Patent Laid-Open No. 94-216801 discloses a method for producing "hydroxypropylated deacetylated chitin". In other words, as a method for preparing general chitosan and preparing hydroxypropyl chitosan without using atmospheric pressure and organic solvent using water as a reaction solvent, chitosan having a deacetylation degree of 70 and 82% was dispersed in 120 g of tertiary deionized water. Next, 4.5g-12g of propylene oxide was added, and then it heated up to 57-85.5 degreeC over 1 hour 30 minutes. The reaction was carried out for 3 hours, and when the temperature was increased to 94.5 ° C, it was left to cool overnight. Then, 4.5 g to 13 g of propylene oxide was added to the third, and 12 g of the third was repeated, and the temperature increase reaction was repeated. Only the supernatant was separated, and excess isopropyl alcohol was added, followed by stirring and filtering. This was dried to obtain 12-13 g of hydroxypropyl. Since the intermediate was insoluble, a small amount of acetic acid was added to dissolve it, and then neutralized with sodium hydroxide solution and filtered to obtain hydroxypropyl chitosan in the final liquid state. Prepared. However, this manufacturing method solves this problem by using water and isopropyl alcohol instead of using the hazardous organic solvent in the above-described method, but there is a disadvantage in that excessive insoluble content is generated due to low substitution degree of the hydroxypropyl group.

In order to solve the conventional problems, the chitosan of the present invention causes redness on the skin even when used as an animal therapeutic agent or added to cosmetics, or penetrates and absorbs into the skin without coagulation and precipitation in an organic solvent. An object of the present invention is to provide a method for low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan free from water insolubles.

1 is hydroxypropyl chitosan formed over time after enzyme treatment [Hydroxypropyl

chitosan (HPCC)].

Figure 2 is a table of GPC molecular weight distribution analysis showing the molecular weight of hydroxypropyl chitosan reduced by time after the enzyme treatment.

Figure 3 is a graph of the molecular weight distribution, average molecular weight of chitosan first used.

Figure 4 is a graph of the average molecular weight after 24 hours of the enzyme reaction.

Chitosan and isopropanol were added to the beaker, followed by alkali slurrying, propylene oxide was added thereto, warming and stirring for a certain time, the pH was adjusted to moderately neutral. The filtrate was separated and the residue was washed with methanol and dried at low temperature. On the other hand,

After dissolving the hydroxypropyl chitosan powder in deionized water in a beaker, the cellulase or chitosanase was added while warming to 55-60 ° C., stirred and fermented, followed by removal of enzymatic activity at about 80 ° C. for a period of time. A method of low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan, characterized by removing the enzyme and filtering and concentrating the filtrate.

Best Mode for Carrying Out the Invention

The present invention is exemplified by the following examples, and the viscosity, which is a decrease value of the molecular weight in all the examples, was measured at 100 rpm using a Brookfield viscometer (USA), and the chitosan solution was measured to be 600 ml in the measurement of chitosan used in the present invention. 0.5% (W / W) chitosan was completely dissolved in 1% acetic acid solution to remove bubbles. Measurement of hydroxypropyl chitosan was carried out at 600 ml of hydroxypropyl chitosan powder without powder insoluble content. 0.5% (W / W) chitosan was completely dissolved in tertiary deionized water to remove bubbles, and the measuring spindle was measured using Nos. 1 to 4. In addition, the molecular weight distribution of the prepared hydroxypropyl chitosan oligosaccharides was assayed through GPC analysis. In addition, the deacetylation degree for confirming the purity of chitosan used for the first time was measured according to the colloid titration method by applying the Terayama method (J. Polym. Sci., 8. 243, 1952). In order to prepare hydroxypropyl chitosan and test its solubility, the mixture was stirred for 1 hour while slowly adding 10 g of HPCC powder to 90 ml of tertiary deionized water, and then filtered using a standard filter paper to measure the remaining amount of insoluble matter. The amount of each HPCC was also measured by pH meta when 100 g of deionized water was administered with 1 g graded water-soluble chitosan, and then sufficiently dissolved after stirring. In order to confirm the safety under acidic and basic conditions and organic solvents, 10g of hydroxypropyl chitosan powder was dissolved in 90g of tertiary deionized water, and then precipitated according to pH change while adding 2N-HCl solution and 5% NaOH solution. In addition, the stability in the organic solvent was also measured by checking the precipitation using ethanol, acetone, and the like.

For example, it will be described in detail as follows. However, this embodiment does not limit the technical scope of the present invention.

<Production of Water-soluble Chitosan>

Example 1-1

Chitosan for preparing hydroxypropyl chitosan used in the present invention was prepared according to the present invention by preparing chitosan having a deacetylation degree of 79.06% and a molecular weight (viscosity) of 235.8 cps. That is, 200 ml of isopropanol was added to 10 g of chitosan and stirred for 1 hour and 30 minutes. Subsequently, 2 equivalents of a 30% NaOH (w / w) solution was prepared with respect to the amine group equivalent standard of chitosan, a uniform amount was slowly administered for 1 hour, and stirred for 2 hours to alkalize the chitosan. The propylene oxide solution was administered with 2 equivalents of chitosan, sealed and stirred for 21 hours, and then the temperature was raised to 35 ° C and stirred for 16 hours. The pH was adjusted to 7.5 using acetic anhydride and filtered. The filtered intermediate was repeatedly removed three times using 150 ml of 70% (v / v) methanol, and dried at 69 ° C. to obtain hydroxypropyl chitosan powder.

Example 1-2

In Example 1-2, the reaction was carried out under the same conditions as in Example 1-1, except that the amount of propylene oxide solution added to induce substitution of the hydroxypropyl group in chitosan was increased to 3 equivalents.

Example 1-3

In Example 1-3, the reaction was carried out under the same conditions as in 1-2, except that the amount of the propylene oxide solution added to induce the substitution of the hydroxypropyl group in chitosan was increased to 10 equivalents.

Example 1-4

In Example 1-4, 200 ml of isopropanol was added to 10 g of the chitosan used in Examples 1-1 to 1-3, and stirred for 1 hour and 30 minutes. Subsequently, 3 equivalents of a 30% NaOH (w / w) solution was uniformly and slowly administered for 1 hour and 23 minutes based on the amine group equivalent of chitosan, followed by stirring for 1 hour and 30 minutes to make the alkali slurry into chitosan. Then, the propylene oxide solution was administered with 30 equivalents of chitosan standard, sealed, and stirred for 2 hours. Then, the temperature was raised to 36 ° C and stirred for 43 hours. And, the pH was adjusted to 7.3 with acetic anhydride and filtered. During filtration, the intermediate was repeated twice with 150 ml of 70% (v / v) methanol to remove the first produced base. Secondly, the intermediate was further washed with 150 ml of pure ethanol. 17 g of oxypropyl chitosan powder was obtained.

Example 1-5

In Example 1-5, 400 ml of isopropanol was added to 20 g of the chitosan used in Examples 1-1 to 1-4 and stirred for 2 hours. Subsequently, 3 equivalents of a 30% NaOH (w / w) solution was uniformly and slowly administered for 1 hour and 20 minutes with respect to the amine group equivalent standard of chitosan, and stirred for 18 hours to alkalize the chitosan. The propylene oxide solution was administered with 35 equivalents of chitosan, sealed, and stirred for 2 hours and 30 minutes. Then, the temperature was raised to 30 ° C. and stirred for 70 hours. And pH was adjusted to 7.5 with acetic anhydride and filtered. After filtration, the intermediate was repeated twice with 300% solution of 70% (v / v) methanol to remove the produced base, and secondly washed with 300 ml of pure acetone twice each time, and then forced to 60 ° C. Drying gave 35 g of the final hydroxypropyl chitosan powder.

Example 1-6

In Example 1-6, 200 ml of isopropanol was added to 10 g of chitosan used in Examples 1-1 to 1-5 and stirred for 4 hours. Subsequently, 2.5 equivalents of a 30% NaOH (w / w) solution was uniformly and slowly administered for 1 hour with respect to the amine group equivalent standard of chitosan, and stirred for 20 hours to alkalize the chitosan. The propylene oxide solution was administered with 35 equivalents of chitosan, sealed, and stirred for 2 hours and 30 minutes. Then, the temperature was raised to 35 ° C and stirred for 240 hours. And pH was adjusted to 7.5 with acetic anhydride and filtered. During filtration, the intermediate was repeatedly washed twice with 250 ml of 80% (v / v) methanol to remove the first base, and further washed with 200 ml of pure methanol and secondly with 500 ml of pure acetone. Then, forced drying at 70 ℃ to obtain 35g of the final hydroxypropyl chitosan powder.

Example 1-7

In Example 1-7, 200 ml of isopropanol was added to 10 g of chitosan having a deacetylation degree of 50.5% and a viscosity (molecular weight) of 235.8 cps, and stirred for 2 hours. Subsequently, 3.2 equivalents of a 30% NaOH (w / w) solution was uniformly and slowly administered for 1 hour with respect to the amine group equivalent standard of chitosan, and stirred for 2 hours to make the chitosan alkaline slurry. The propylene oxide solution was uniformly administered with 46 equivalents of chitosan for 30 minutes and sealed, and then the temperature was raised to 34 ° C. and stirred for 48 hours with stirring. And, the pH was adjusted to 7.3 with acetic anhydride and filtered. During filtration, the intermediate was firstly removed using a 500 ml solution of pure methanol, further washed with 500 ml of pure methanol each, and then dried in vacuo to obtain 20 g of a final hydroxypropyl chitosan powder.

In the present invention, the results of Examples 1-1 to 1-7 are as follows.

Table 1. Analysis Table of Physical Properties of Hydroxypropyl Chitosan Prepared by the Invention Examples

Example Classification 1-1 1-2 1-3 1-4 Chitosan Properties Used for the First Time Viscosity (cps) 235.8 235.8 235.8 235.8 DAc (%) 79.06 79.06 79.06 79.06 Chitosan (g) 10 5 5 10 Isopropanol addition amount (ml) 200 100 100 200 Stirring Time 1 hour 30 minutes 1 hour 30 minutes 1 hour 30 minutes 1 hour 30 minutes 30% NaOH addition amount (equivalent) 2 2 2 3 NaOH addition time 50 minutes 1 hours 1 hours 1 hour 23 minutes Stirring Time 1 hour 30 minutes 1 hour 30 minutes 1 hour 30 minutes 1 hour 30 minutes Propylene oxide addition amount (equivalent) 2 3 10 30 Propylene Oxide Addition Time 1 hour 30 minutes 30 minutes 1 hour 30 minutes 2 hours Reaction temperature (℃) 35 80 80 36 Stirring Time 18 hours 18 hours 18 hours 43 pH adjustment 7.5 7.5 7.5 7.3 Solvent Used in Filtering Primary 70% MeOH, 500ml 70% MeOH, 150ml 70% MeOH, 150ml 70% MeOH, 150ml Secondary - - - 70% MeOH, 150ml 3rd - - - Pure Ethanol, 150ml Drying condition (℃) Forced drying, 60 Forced drying, 60 Forced drying, 60 Forced Drying, 40 stability(%) 2N-HCl 100 100 100 100 5% NaOH - 42 16 100 Insoluble content (%) 40 100 100 0.01 HPCC Production (g) 11 < 6 < 7 < 17 < Viscosity (cps) 180 238 238 14.7 pH 7.5 7.4 7.5 7.64 color White Brown Brown White

(Continued in Table 1)

Example Classification 1-5 1-6 1-7 Chitosan Properties Used for the First Time Viscosity (cps) 235.8 432 235.8 DAc (%) 79.06 94.11 50.5 Chitosan (g) 20 10 10 Isopropanol addition amount (ml) 400 200 200 Stirring Time 2 hours 4 hours 1 hour 40 minutes 30% NaOH addition amount (equivalent) 3 2.5 3.2 NaOH addition time 1 hour 20 minutes 1 hours 1 hours Stirring Time 18 hours 20 hours 1 hour 30 minutes Propylene oxide addition amount (equivalent) 35 35 46 Propylene Oxide Addition Time 2 hours 2 hours 30 minutes Reaction temperature (℃) 30 35 34 Stirring Time 70 hours 70 hours 48 hours pH adjustment 7.5 7.5 7.3 Solvent Used in Filtering Primary Pure MeOH, 300ml 80% MeOH, 250ml 70% MeOH, 500ml Secondary Pure MeOH, 300ml Pure MeOH, 200ml 70% MeOH, 500ml 3rd Acetone 300 ml Pure Acetone, 500ml Drying condition (℃) Forced Drying, 60 Forced drying, 69 Forced drying, 40 stability(%) 2N-HCl 100 100 100 5% NaOH 100 100 100 Organic solvent 100 100 100 Insoluble matter (%) <0.001 <0.001 <0.001 HPCC Production (g) 30 15 20 Viscosity (CPS) 9.6 13.2 34.5 pH 7.4 7.5 7.5 color White transparent White and transparent White and transparent

Molecular Weight Control Using Cellulase Enzyme

Example 2-1

Example 2-1 was carried out to check the enzyme dosage range that effectively induces a molecular weight reduction of the hydroxypropyl chitosan prepared in Example 1-7 by the enzyme. Cellulase (Brand name: Bio-ACE, Antarctic)

Amalgamated Resoures Limited. USA.), And the enzyme had a value of 24,733 units / g.

2.75 L of tertiary deionized water was added to a 3 L beaker, and the solution was completely dissolved while slowly injecting 20 g of HPCC prepared in Example 1-7 while stirring at 150 rpm. After the temperature was raised to 55-60 ° C., the cellulase was separately divided into 11 hydrolysates from 1 hour to 24 hours with stirring at 150 rpm after 20 ml of administration of cellulase, which was then stirred at 80 ° C. for 30 minutes. After removing the activity of the precipitate, the precipitated enzyme was centrifuged (15,000 rpm, 15 minutes) to completely remove the precipitate. This was concentrated and lyophilized to prepare a water-soluble hydroxypropyl chitosan having a molecular weight of powdery grade.

Example 2-2

Example 2-2 of the present invention was carried out in the same manner as in Example 2-1 except that the amount of cellulase added in Example 2-1 was 10 ml.

Example 2-3

Example 2-3 of the present invention was carried out in the same manner as in Example 2-1 except that the amount of cellulase added in Example 2-1 was 5 ml.

Example 2-4

Example 2-4 of the present invention was carried out in the same manner as in Example 2-1 except that the amount of cellulase added in Example 2-1 was 2.5 ml.

Example 2-5

Example 2-5 of the present invention was carried out in the same manner as in Example 2-1 except that the amount of cellulase added in Example 2-1 was 1.5 ml.

The results of Examples 2-1 to 2-5 are as follows.

Table 2. Analysis table confirming the molecular weight decrease of hydroxypropyl water-soluble chitosan with time using cellulase.

Example 2-1 2-2 2-3 2-4 2-5 HPCC solution (ml) 250 Temperature (℃) 55 to 60 Cellulose addition amount (ml) 20 10 5 2.5 1.5 result first Viscosity (cps) 34.5 34.5 34.5 34.5 34.5 1 hour after reaction 33.4 28.4 12.3 34.3 34.5 After 2 hours reaction 28.3 12.5 6.8 33.5 34.5 After 3 hours reaction 25.6 6.3 4.6 28.5 34.5 After 4 hours reaction 18.5 4.8 2.5 27.9 32.7 5 hours after reaction 15.9 2.4 1.8 26.7 30.5 6 hours after reaction 13.4 1.8 1.6 20.4 29.6 9 hours after reaction 12.6 1.4 1.2 * 18.5 26.5 12 hours after reaction 6.0 1.2 * 1.2 * 16.3 25.6 15 hours after reaction 4.4 1.2 * 1.2 * 15.8 24.3 18 hours after reaction 2.8 1.2 * 1.2 * 8.9 20.5 After 24 hours reaction 1.8 1.2 * 1.2 * 5.8 18.6

*: Oligosaccharide formation (content of less than 10 sugars is 80% or more)

<Molecular weight control using chitosanase>

Example 3-1

In Example 3-1, chitosanase (Japan) was used. Chitosanase had a purity of 3,000-20,000 U / g and was a chitosan degrading enzyme produced by Bacillus pumilus BN-262. In addition, an enzyme having a physical property of about 31,000 (SDS-PAGE method) was used.

2L of tertiary deionized water was added to a 3L beaker and stirred at 150rpm to completely dissolve the HPCC prepared in Example 1-7 while slowly adding 20g of the HPCC. The pH was adjusted to 6 ～ 6 using a dilute hydrochloric acid solution and NaOH dilution solution. After adjusting to 7, put into a 0.5L three-necked round pilsk in 11 pieces of 0.25L, the temperature was raised to 50 ℃, 0.011g of enzyme to 1g of water-soluble chitosan, and then stirred at 150rpm for 1 hour After hydrolysis into 11 parts up to 24 hours, the mixture was stirred at 80 ° C. for 30 minutes to remove the activity of the enzyme, followed by standing for 12 hours to centrifuge the precipitated enzyme (15,000 rpm, 15 minutes) to completely remove the precipitate. It was. The filtrate was concentrated and lyophilized to produce a water soluble chitosan graded in powder form.

Example 3-2

Example 3-2 of the present invention was carried out in the same manner as in Example 3-1, except that the input amount of chitosanase was 0.022 g in Example 3-1.

Example 3-3

Example 3-3 of the present invention was carried out in the same manner as in Example 3-1 except that the amount of cellulase added in Example 3-1 was 0.044 g.

Example 3-4

Example 3-4 of the present invention was applied in the same manner as in Example 3-1 except that the dose of cellulase was 0.066 g.

The results of Examples 3-1 to 3-4 are as follows.

Table 3. Analysis table confirming the decrease in molecular weight of the water-soluble chitosan (HPCC) over time using chitosanase.

Example 3-1 3-2 3-3 3-4 HPCC solution (ml) 250 Temperature (℃) 55 to 60 Amount of chitosan added (g) 0.011 0.022 0.044 0.066 result first Viscosity (cps) 34.5 34.5 34.5 34.5 1 hour after reaction 34.4 34.4 34.5 28.5 After 2 hours reaction 34.4 33.2 29.6 26.3 After 3 hours reaction 34.3 30.8 23.5 16.7 After 4 hours reaction 34.0 30.2 22.4 12.4 5 hours after reaction 33.3 29.4 19.5 10.8 6 hours after reaction 34.0 28.7 16.8 7.5 9 hours after reaction 33.6 26.3 13.5 4.5 12 hours after reaction 32.6 25.3 8.4 3.5 15 hours after reaction 32.0 22.4 2.4 2.8 18 hours after reaction 31.3 20.5 1.2 * 1.2 After 24 hours reaction 30.4 18.5 1.2 * 1.2

*: Oligosaccharide formation (content of less than 10 sugars is 80% or more)

When used as a therapeutic agent for animals or added to cosmetics, it does not cause redness on the skin, coagulates or precipitates in organic solvents, penetrates and absorbs skin well, and has low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan without water insoluble content.

Claims (5)

Chitosan and isopropanol were put in a beaker, alkali slurry was added, and propylene oxide was added thereto. After stirring for a certain period of time, the pH was adjusted to mild neutrality. On the other hand, After dissolving the hydroxypropyl chitosan powder in deionized water in a beaker, the cellulase or chitosanase was added and stirred while warming to 55-60 ° C., followed by stirring at about 80 ° C. to remove enzymatic activity for a certain time and precipitated enzyme. The method of low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan, characterized in that the filtrate is removed by filtration and the filtrate is concentrated and freeze-dried. The method of low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan according to claim 1, wherein the degree of deacetylation is at least 50% chitosan. The low molecular weight of the water-soluble hydroxypropyl chitosan according to claim 1, wherein propylene oxide is added to the alkali slurry and the reaction is carried out by adding 2 to 3.5 equivalents of a solution of 20 to 35 DEG C and 30% caustic soda based on the amine group of the chitosan. And methods of oligomerization. The water-soluble hydroxypropyl according to claim 1, wherein the amount of propylene oxide added to chitosan is 30 to 46 equivalents, based on the amino group of chitosan, and reacted for 18 to 70 hours at 30 to 120 minutes and 20 to 35 ° C. Method of low molecular weight and oligomerization of chitosan. The method of low molecular weight and oligomerization of water-soluble hydroxypropyl chitosan according to claim 1, wherein the water-soluble hydroxypropyl chitosan oligosaccharide contains 80 wt% or more.