KR20020037241A - Synthesis and characterization of new chitosan derivatives by use of amino acid - Google Patents

Abstract

The present invention relates to a method for producing a natural polymer antimicrobial amino acid using chitosan represented by the following general formula (I), which is useful for food, as a chitosan derivative, and to its efficacy and use as an application method thereof.

Description

Synthesis and characterization of new chitosan derivatives by use of amino acid}

The present invention relates to a novel chitosan derivative of the following structural formula [I] as a chitosan derivative which is a natural antimicrobial agent currently used, and a method for producing the same.

In the above formula, the compounds of structural formulas [I] and [II] have a structure in which amino acids and organic acids are bonded to chitosan. In recent years, the creation of new materials in medicine and food is one of the most urgent tasks to be solved among the problems of science and technology. Meanwhile, the effects of pesticide residue on the human body due to excessive addition of pesticides to foods are serious, and as reported recently in the media, the use of pesticides greatly exceeds the limit of the standard of use. Health damage is a serious threat to health from newborns to fetuses as well as secondary infections as well as adults. However, antioxidants and excessive pesticides are still used, and various residual pesticides are a major threat to health due to formalin spraying. In the meantime, various studies using the antibacterial and antifungal efficacy of chitin chitosan, a natural product, have been actively conducted.

In particular, chitosan is a substance that is present in a wide range of shells such as shrimp and crabs, and the cell walls of insects, mushrooms, and bacteria. This chitosan is obtained by hydrolyzing chitin by a substance that forms an exoskeleton of an organism like cellulose of a plant, and supports or protects the chitin, and β- 1 by N-acetyl-D-glucosamine where the amino group is acetylated. , 4-linked polymer polysaccharides. First, demineralization using NaOH or KOH and then removing calcium-containing inorganic substances from 5% HCl solution leaves insoluble substances. This is chitin. Chitosan is a substance that hydrolyzes chitin in concentrated alkaline aqueous solution. According to the degree of deacetylation during hydrolysis, the chitosan derivatives that the team intends to develop are also biologically active substances, and similar drugs are the only ones that have superior antimicrobial activity as natural products due to the characteristic effect of the positive charge of amino groups in chitosan itself. Therefore, it is a pure natural material that can be put to practical use beyond the scope of the research academic data, which is a common chitosan antimicrobial derivative reported in order to synthesize NH ₂ -Chitosan derivative which has a specific chemical structure but has an active effect excellent in antimicrobial activity. In addition, it attempts to synthesize by using a natural amino acid and to try to couple it by chemical method. Therefore, by using chitosan and new compounds by using natural materials or amino acids and physiologically active substances, which are mass materials that can be used as a special product of this region, we tried to develop an antimicrobial agent as a food additive as a new lead compound with increased antibacterial activity. Therefore, the final candidates were obtained through the preclinical experiments on the separation of antimicrobial antimicrobial active substance with chemically crosslinking potential from fruit, the synthesis of active substance using natural chitosan and chitin, and the synthesis of Amino acid and chitosan and chitin. We have decided to apply for the discovery of a substance and the uniqueness of water dissolving in chitosan.

.

1 shows a method of binding to chitosan using amino acids

2 shows a method of binding to chitosan using an organic acid containing acetyl group.

3 shows a method of binding to chitosan using an organic acid.

Example 1

2. Materials and Methods (Research Methods)

2-1. material

2-1-1. The chitin used in this experiment was extracted from keratin (Chinoecetes japonicus) keratin from Biotech Co., Ltd., Mokpo-si, Jeonnam, and ground to a constant size (50mesh) and stored in a refrigerator at 0-5 ℃ for use in the experiment.

2-1-2. Measurement of Minimum Inhibitory Concentration (MIC in vitro ) on Synthesized Benzimidazole-Substituted Chitosan Derivatives

2-1-2-1.Use strain

The microorganisms used in this experiment were Escherichia coli 0157: H7 and fungi Candida utilis , Candida albicans ATCC 10231, Saccharomyces cerevisiae ATCC9763 and Aspergillus niger 9029.

2-1-2-2. Used badge

Bacterial media used in this experiment: Mueller Hinton broth, Mueller Hinton ager and fungal media: Sabouraud Dextrose Broth, Sabouraud Dextrose agar.

2-1-2-3. Reagents and Equipment

Reagents and solvents were used as Aldrich Co., Ltd. The melting point of the instrument used to identify the separated compound was measured by Thomas Hoover melting point Apparatus was not calibrated. The nuclear magnetic resonance spectrometer used Varain T60 and HA-100. Infrared spectroscopy was obtained using a Bio-Red FTS-45A Spectrophotometer. TLC plate uses Merck DC-plastik follen Kieselgsl 60F 254, chitosan has 89% deacetylation, viscosity 20cps, chitosanase derived from Bacillus pumilus BM- (molecular weight: about 30,000 Da, minimum pH: 5.5 ~ 605, optimum temperature) : 30 to 50 ° C., isoelectric point: about 93) was purchased from Wakopure Chemical Co., Ltd., Japan, and D-glucosamine was purchased from Sigma Chemical Co., Ltd. (St. Louis, USA). The amino acids Gly, Ala, Asp Asn, Boc-S- (p-methoxybenzyl) -L-Cys and Boc-L-Met for synthesis of chitosan derivatives and DCC (N, N'-dicyclohexyl-carbodiimide) as a synthesis catalyst are Sigma It was purchased from Chemical Company.

2-2. Way

2-2-1. Preparation of methanol extract and assay medium and determination of antimicrobial activity:

One). To make the fungal diluent, preculture is mixed with a concentration of 10 ⁴ -10 ⁶ CFU / ml.

2). Each sample is dissolved in a suitable solvent and then diluted in 2-fold dilution with medium.

3). Inoculate each test tube with bacterial solution.

4). Bacteria are incubated at 37 degrees for 24 hours and fungi are incubated for 36-48 hours.

5). After each incubation, shake the test tube to check the turbidity of the bacteria. After checking the turbidity, check each of them by re-inoculating each of Mueller Hinton ager and Sabouraud Dextrose agar.

6). After confirming the growth of the bacteria, determine the lowest test tube concentration at which growth is not recognized and determine the MIC by the broth serial dilution method as described above.

2-2-2. Antimicrobial activity against each compound:

Measurement results for the synthetic compound and control compound minimum inhibitory concentration in the i n vitro for the chitosan in (MIC, (㎍ / ml) ) was given in Table 1.

2-2-3. Manufacture of Chitosan

Chitin was extracted from the red snow crab shells and demineralized by adding 35 times of 4N HCl to the crustacean shell weight, followed by 1 hour reaction with 1N NaOH solution for deproteinization. Then, decolorized with 0.5% potassium permanganate, filtered, washed with water, and dried at 60 ° C. using a hot air dryer to prepare chitin.

2-4. Acetylation Measurement

In this study, the mass% results of the chitin and chitosan elemental analysis and the chitin chitosan elemental analysis were compared and calculated in terms of N / C.

Example 2

2-5-1. Benzimidazole Derivative Synthesis Using Benzimidazole

1. Synthesis of Intermediate Derivative Compound (3b)

1.5 g of Compound (2) was dissolved in 50 ml of a three-necked round flask, and 13 ml of sulfuric acid and 10 ml of nitric acid were added thereto for 10 minutes at 5 ° C. or lower and allowed to react for 20 minutes. The mixed solution was dispersed in 30 ml of iced water and left to obtain 1.8 g of the resulting crystal compound. The mixture was dried again and separated by column chromatography to obtain 0.7 g of a yellow crystalline compound substituted at the 2nd position.

Example 3

2. Synthesis of Intermediate Derivative Compound (4)

10.5 g (0.06 mole) of Compound (3b) was dissolved in 30 ml of re-distilled EtOH, and stirred with Pd-C, followed by a reduction reaction until the reaction was completed while passing hydrogen gas. After the reaction was completed, the mixture was filtered, washed with 10 ml of ethanol again, and the filtrate was concentrated under reduced pressure, and the resulting crystal compound was dried to yield 7.72 g.

Yield: 88.2%

Example 4

3. Synthesis of Benzimidazole Derivative

Dissolve 1.5 g (0.014 mole) of compound (5) in 27 ml of PPA in 100 ml three-necked pusque, then react β- PA with 0.014 mole for 6 hours at 90 ° C or more, collect the resulting crystals and dry it. After recrystallization with 60 ml of ethanol to give 1.32 g of compound (6).

Example 5

4.Synthesis of Chitosan Derivatives Using Benzimidazole Derivatives (A)

5.5 g of the compound chitosan was added to 80 ml of re-distilled DMF, followed by adding 1/2 mole of the compound (6) to the chitosan standard and reacting at 80 ° C. or higher for 6 hours. The resultant crystals were collected and 50 ml of distilled water and 60 ethanol were added. After washing with 60 ml of ether and 6.13 g of chitosan derivatives (7) were obtained.

Example 6

5.Synthesis of Chitosan Derivatives Using Benzimidazole Derivatives (B)

1.5 g of the compound chitosan was added to 30 ml of a mixture of glacial acetic acid 3% -EtOH, and then compound (8) was added to 1/3 mole of chitosan standard, stirred for 10 hours, and then the resulting crystal compound was collected and 50 ml of distilled water was collected. After washing with 60 ml of ethanol and 60 ml of ether, 1.2 g of chitosan derivative compound (7) was obtained.

Example 7

6-2). Antimicrobial Activity on Compounds:

Measurement results for the synthetic compound and control compound minimum inhibitory concentration in the i n vitro for the chitosan in (MIC, (㎍ / ml) ) was given in Table 1.

Activity of antimicrobial activity for each compound:

The results of measuring the minimum inhibitory concentrations while culturing the diluted acetic acid solution in which the chitosan derivative was adjusted to pH 4.9-5.0 in a liquid medium are shown in Table 1.

The minimum inhibitory concentration of the synthesized compound (A) was 300 ㎍ / ㎖, 250 ㎍ / ㎖, 600 ㎍ / ㎖, 400 ㎍ / ㎖ with Candida albicans ATCC 10231, Candida utilis, Saccharomyces cerevisiae ATCC 9763, Aspergillus niger 9029, etc. Growth was inhibited at a relatively low concentration compared to chitosan, but E. coli strain was the highest in bacteria at 800 µg / ml. Meanwhile, the compound (B) is relatively free of activity when it is compared with the compound (A) Candida albicans ATCC 10231 is shown the same as compound (A) to 300 ㎍ / ㎖ nateu or Candida utilis, Saccharomyces cerevisiae ATCC 9763, Aspergillus niger 9029 of the fungus Low. However, these chitosan-benzimidazole compounds showed antibacterial activity about 7-10 times higher than chitosan.

.

Claims (6)

Compounds of the formula [I], [II] In the above formula, Substituents represent alginic acid, amino acids (aspartic acid, glutamic acid, other amino acetic acid, succinic acid, etc.), and n represents a degree of polymerization of 0 to 10,000 or more. The degree of acetylation also has 20-100 In addition to the components according to the amino acid binding method shown in the formula also includes a method for binding other amino acids The compound of formula [I], [II] according to claim 1, wherein the acid is protected by an ester method of an amino acid and an organic acid (e.g. succinic acid, etc.) with respect to the chitosan compound, and then prepared using an anhydride and an amino acid. The mixture of formic acid and acetic acid to form an amino group and at the same time to form an anhydride, combined with chitosan in the form of N-formyl amino acid produced by using an alkyl amide or a hydrogenation reaction to remove formyl and benzyl groups to combine amino acids How to According to claim 3, pH 3- using acetic acid, formic acid using at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, isopropyl alcohol (IPA), acetic acid, lactic acid, hydrochloric acid (HCl) as a solvent A composition method having five. The method for preparing oligosaccharide and chitosan-containing antibacterial amino acid and organic acid according to claim 3, wherein the reaction is carried out at 25-90 ° C., pH = 5-7.5. The antimicrobial agent, feed additive, food packaging coating agent, food processing additive, fruit coating agent, textile coating and composition, pesticide substitute in plant breeding, skin ointment, cosmetic raw material, liquid according to any one of claims 1 to 3. Use as a shampoo, liquid soap composition, and skin disease treatment