Patent Application
20200181292
The invention relates to the field of chitosan preparation, in particular to a novel water-soluble natural polysaccharide antibacterial material and preparation method thereof.
Chitosan, whose chemical name is polyglucosamine (1-4)-2-amino-BD glucose, is a natural alkaline polysaccharide obtained by deacetylating chitin contained in shells of crustaceans such as shrimp and crab and fungal cell walls. Chitosan has excellent bioaffinity and biodegradability, and can be easily made into various derivatives. Because it has extremely abundant sources, and can be dissolved in hydrochloric acid, acetic acid and other organic acids, the chitosan has been widely used in industrial and medical fields. Because the chitosan has characteristics of biodegradability, biocompatibility, biological non-toxicity and antibacterial activity, chitosan has been made as one of the research hotspots in the development of natural antibacterial agents in recent years. However, because there are large number of hydrogen bonds in and between chitosan molecules, and chitosan has high crystallinity, is hardly soluble in water, and is only soluble in some diluted acid solutions, chitosan has lower antibacterial activity than traditional antibacterial agents, thereby greatly limiting the promotion and application of chitosan as an antibacterial agent.
In order to improve the water solubility of chitosan, various methods have been adopted. For example, water-soluble chitosan or water-soluble derivatives can be obtained by controlling the degree of deacetylation of chitosan between 50-60%, preparing chitosan into various inorganic or organic acid salts, and chemically modifying chitosan. Although these methods have solved the problem of water-solubility of chitosan, the antibacterial performance thereof has not been improved significantly. Chitosan molecules contain reactive hydroxyl and amino groups, other groups can be introduced into chitosan molecules by controlling reaction conditions with hydroxyl or amino groups to perform reactions such as acylation, carboxylation, etherification, NH2 alkylation, esterification, hydrolysis, or the like [J. Adv. Drug. Deliv. Rev., 2001, 50, 591.1], so as to make a series of water-soluble chitosan derivatives, thereby changing physicochemical properties of chitosan and giving chitosan more specific functions to meet the needs of more fields to further expand the application scope of chitosan.
Guanidyl is the most positively-charged biologically active organic basic group currently found in nature, it can be protonated in physiological pH media and can form positively-charged groups under neutral, acidic, and basic conditions. Guanidine compounds are widely present in natural products, have strong solubility, and are strongly basic and electropositive. Guanidyl has biological activities such as anti-inflammatory, antihypertensive and hypolipidemic activities, antiviral activities, antitumor, etc., and also has strong alkalinity, strong stability, and good biological activity. The guanidyl compounds are easy to form hydrogen chains, so they have good antibacterial performance, and is widely used in medicine, agriculture, construction, clothing, chemical and other fields. Guanidyl is in a fully protonated state under normal conditions, and maintains an electropositive property [Wei Changmei, Synthesis of guanidine compounds and research on crystal structure thereof, PhD dissertation, 2004.]. Guanidyl can act on receptors and ligands through electrostatic interaction or hydrogen bonding, so the guanidyl can have good drug effects. Guanidyl compounds, as drugs, are mainly used as antihypertensive drugs, hypoglycemic drugs and antiviral drugs. The amino group of chitosan has higher reactive activity, so guanidination modification is performed for chitosan by means of amino groups to give chitosan similar properties to guanidyl compounds, thereby improving the antibacterial and antibacterial properties of chitosan. Hu et al. obtain guanidyl chitosan bisulfite by reacting thiourea trioxide with chitosan [Hu Y., et. al., Carbohyd. Polym., 2007, 67, 66.]. Sun et al. synthesize guanidinylated chitosan by using sodium tripolyphosphate as a cross-linking agent and polyhexamethyleneguanidine phosphate as a guanidinated reagent [Bioresour. Technol., 2010, 101, 5693.]. Zhai et al. obtain monoguanidine chitosan by reacting mononitrile ammonia, as a guanidinated reagent, with chitosan [Zhai X., et. al., J. Appl. Polym. Sci., 2011, 121, 3569.].
In addition, Xiao et al. also obtain guanidinated chitosan by using arginine as a guanidination reagent, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinyl imine (NHS) as catalysts and allowing arginine to react with chitosan in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution at normal temperature [Xiao B., et. al., Carbohyd. Polym. 2011, 83, 144.]. Leucine, isoleucine, and lysine, similar to arginine, are all essential amino acids in the human body. The carboxyl groups contained in these three types of amino acids all have certain chemical activities, can react with the amino group on the chitosan molecule, and are suitable for functional modification of chitosan.
The first object of the present invention is to provide a novel water-soluble natural polysaccharide antibacterial material.
The molecular structural formula of the natural polysaccharide antibacterial material is shown in Formula 1:
where R1 is:
and
wherein x, y, and n are natural numbers, 0<x≤107, 0<y≤107, 102≤n≤107.
The novel water-soluble natural polysaccharide antibacterial material provided by the present invention contains both amino acids and guanidyl, improves the bacteriostatic effect and application range of the chitosan derivatives, and at the same time, compared with monoguanidine or biguanide hydrochloride derivatives of chitosan, reduces cytotoxicity and improves biological safety thereof.
Another object of the present invention is to provide a method for preparing the novel water-soluble natural polysaccharide antibacterial material.
To achieve the above object, the present invention adopts the following technical solution:
which include the following steps:
1) dissolving chitosan in a diluted acid solution to obtain a diluted acid aqueous solution of chitosan;
2) adding cyanamide or dicyandiamide into the diluted acid aqueous solution of chitosan obtained in step 1) for reaction:
3) adding an amino acid activation solution into the reaction system in the step 2) for amidation;
4) adding hydroxylamine hydrochloride to terminate the reaction; and
5) filtering the reaction solution and then dialyzing the solution with deionized water, and performing microwave vacuum-drying to obtain the novel water-soluble natural polysaccharide antibacterial material.
Preferably, the number average molecular weight of the chitosan in step 1) is 102-107, and the degree of deacetylation is 50-100%; preferably, the diluted acid is hydrochloric acid or acetic acid, and the acid concentration is 0-0.5 mol/L; the concentration of the diluted acid aqueous solution of the chitosan is 0.001-0.1 g/mL.
Preferably, the dissolving condition in step 1) is stirring at constant temperature between 60-110° C.
Preferably, the molar ratio of cyanamide or dicyandiamide to chitosan in step 2) is 0.5-5:1; and the reaction condition is stirring for 6-48 hours at constant temperature between 60-110° C.
Preferably, in step 3), the amino acid activation solution is obtained by the following method:
amino acids, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are dissolved in 2-(N-morpholino) ethanesulfonic acid buffer solution, and stirring is performed for activation at constant temperature between 0-35° C. for 0.5-3 hours; and
the concentration of the 2-(N-cyanamide morpholino) ethanesulfonic acid buffer solution is 30 mmol/L, and has the pH value of 5.0±0.5:
wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to amino acid is 0.5-5:1, and the molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.
Preferably, the amino acids are leucine, isoleucine or lysine.
Preferably, the molar ratio of chitosan to amino acids is 1-50:1; and the amidation reaction temperature in step 3) is 0-35° C.
Preferably, in step 5), during the dialysis by deionized water, the water is changed every 5-10 hours, and the water is changed for 6-8 times.
The beneficial effects of the present invention are as follows:
In the present invention, a new cellulose material never found before having flaky micromorphology is obtained by enabling cellulose powder and a solid high-molecule grinding material to be subjected to mechanical grinding. The flaky cellulose material has a function for blocking ultraviolet transmittance.
The specific embodiments of the present invention are described in detail below in combination with the figures.
The present invention is described in detail below through examples. It should be noted that the examples are only used to further explain the present invention, and should not be construed as limitations on the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments according to the summary of the invention.
The reaction described in the present invention is as follows:
where R1 is
wherein x, y, and n are natural numbers, 0<x≤107, 0<y≤107, 102≤n≤107.
0.5 g chitosan was added to 100) ml 0.1 mol/L diluted hydrochloric acid, and was mechanically stirred for half an hour in an oil bath at 60° C., so that the chitosan was completely dissolved, so as to obtain a homogeneous solution with a chitosan concentration of 0.005 g/mL; the oil bath was heated to 110° C., 1.3 g dicyandiamide was added to the chitosan solution in one portion, the molar ratio of dicyandiamide to chitosan was 5:1, keeping constant temperature stirring for 6 hours; the reaction solution was cooled to room temperature, and then added with a 20 mL mixed solution activated at room temperature for 2 hours of lysine, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (the solvent was a 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution at a concentration of 30 mmol/L with a pH value of about 5.0), the reaction was performed with continuous stirring at room temperature for 24 hours, wherein the molar ratio of chitosan, lysine, NHS, and EDC was 5:1:2:2: the reaction solution was filtered and loaded into a dialysis bag, the two ends of the dialysis were tied tightly, the bag was placed in deionized water for dialysis, the water was changed every four hours, after the water was changed for eight times, and the dialysate was placed in a microwave vacuum dryer for treatment, thereby obtaining the novel water-soluble antibacterial material.
The statistical data result for the antibacterial rate of Staphylococcus aureus detected by using the pour plate method on the product prepared in this example is as follows:
The results of the above data show that the novel water-soluble natural polysaccharide antibacterial material not only has good antibacterial performance, but also can lead to normal cell growth under effective bacteriostatic concentrations, having good biological safety.
1.0 g chitosan was added to 100 ml diluted hydrochloric acid with a concentration of 0.1 mol/L, and mechanically stirred for one hour in an oil bath at 60° C. so that the chitosan was completely dissolved to obtain a homogeneous solution with a chitosan concentration of 0.01 g/mL; the oil bath was heated to 105° C., 1.05 g cyanamide was added to the chitosan solution system in one portion, and the molar ratio of cyanamide to chitosan was 4:1, keeping stirring for 6 hours at constant temperature; then the reaction solution on the oil bath was cooled to room temperature, and was added with 20 ml mixed solution activated in a mixed ice-water bath for 3 hours of leucine, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC.HCl) (the solvent was a 30 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution), reaction was continued with stirring for 10 hours at room temperature, wherein the molar ratio of chitosan, leucine, NHS, and EDC was 50:1:5:5; the reaction solution was filtered and loaded into a dialysis bag, the two ends of the dialysis bag were tied tightly, the bag was put into deionized water for dialysis, the water was changed once every four hours, the dialysat was placed into a microwave vacuum dryer after changing the water for eight times, thereby obtaining the novel soluble natural polysaccharide antibacterial material.
2.0 g chitosan was added to 100 ml diluted hydrochloric acid with a concentration of 0.15 mol/L, and mechanically stirred for one hour in an oil bath at 60° C. so that the chitosan was completely dissolved to obtain a homogeneous solution with a chitosan concentration of 0.02 g/mL; the oil bath was heated to 100° C., 2.08 g dicyanamide was added to the chitosan solution system in one portion, and the molar ratio of dicyanamide to chitosan was 2:1, keeping stirring for 12 hours at constant temperature of 100° C.; then the reaction solution on the oil bath was cooled to room temperature, and was added with 20 ml mixed solution activated in a mixed ice-water bath for 3 hours of isoleucine, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC.HCl) (the solvent was a 30 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution with a pH value of about 5), reaction was continued with stirring for 24 hours at room temperature, wherein the molar ratio of chitosan, isoleucine, NHS, and EDC was 20:1:4:4; the reaction solution was filtered and loaded into a dialysis bag, the two ends of the dialysis bag were tied tightly, the bag was put into deionized water for dialysis, the water was changed once every four hours, the dialysat was placed into a microwave vacuum dryer after changing the water for eight times, thereby obtaining the novel soluble natural polysaccharide antibacterial material.
5.0 g chitosan was added to 100 ml diluted hydrochloric acid with a concentration of 0.15 mol/L, and mechanically stirred for one hour in an oil bath at 60° C., so that the chitosan was completely dissolved to obtain a homogeneous solution with a chitosan concentration of 0.05 g/mL; the oil bath was heated to 80° C., 3.91 g cyanamide was added to the chitosan aqueous solution system in one portion, and the molar ratio of cyanamide to chitosan was 3:1, keeping reaction for 24 hours at 80° C.; then the reaction solution on the oil bath was cooled to room temperature, and was added with 20 ml mixed solution activated at room temperature for 2 hours of lysine, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC.HCl) (the solvent was a 30 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution with a pH value of about 5), reaction was continued with stirring for 24 hours at room temperature, wherein the molar ratio of chitosan, lysine, NHS, and EDC was 5:1:2:2; the reaction solution was filtered and loaded into a dialysis bag, the two ends of the dialysis bag were tied tightly, the bag was put into deionized water for dialysis, the water was changed once every four hours, the dialysat was placed into a microwave vacuum dryer after changing the water for eight times, thereby obtaining the novel soluble natural polysaccharide antibacterial material.
7.0 g chitosan was added to 100 ml diluted hydrochloric acid with a concentration of 0.3 mol/L, and mechanically stirred for two hours in an oil bath at 70° C., so that the chitosan was completely dissolved to obtain a homogeneous solution with a chitosan concentration of 0.07 g/mL; under the condition where the oil bath was 70° C., 1.83 g cyanamide was added to the chitosan aqueous solution system in one portion, and the molar ratio of cyanamide to chitosan was 1:1, keeping for 36 hours at constant temperature; then the reaction solution on the oil bath was cooled to room temperature, and was added with 20 ml mixed solution activated at room temperature for 2 hours of leucine, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC.HCl) (the solvent was a 30 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution with a pH value of about 5), reaction was continued with stirring for 24 hours at room temperature, wherein the molar ratio of chitosan, leucine, NHS, and EDC was 5:1:4:4; the reaction solution was filtered and loaded into a dialysis bag, the two ends of the dialysis bag were tied tightly, the bag was put into deionized water for dialysis, the water was changed once every four hours, the dialysat was placed into a microwave vacuum dryer after changing the water for eight times, thereby obtaining the novel soluble natural polysaccharide antibacterial material.
10.0 g chitosan was added to 100 ml diluted hydrochloric acid with a concentration of 0.5 mol/L, and mechanically stirred for two hours in an oil bath at 60° C., so that the chitosan was completely dissolved to obtain a homogeneous solution with a chitosan concentration of 0.1 g/mL; under the condition where the oil bath was 60° C., 2.61 g dicyanamide was added to the chitosan aqueous solution system in one portion, and the molar ratio of dicyanamide to chitosan was 0.5:1, keeping reaction for 48 hours at 60° C.; then the reaction solution on the oil bath was cooled to room temperature, and was added with 30 ml mixed solution activated at room temperature for 2 hours of isoleucine, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC.HCl) (the solvent was a 30 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution with a pH value of about 5), reaction was continued with stirring for 24 hours at 35° C., wherein the molar ratio of chitosan, isoleucine, NHS, and EDC was 4:1:3:3; the reaction solution was filtered and loaded into a dialysis bag, the two ends of the dialysis bag were tied tightly, the bag was put into deionized water for dialysis, the water was changed once every four hours, the dialysat was placed into a microwave vacuum dryer after changing the water for eight times, thereby obtaining the novel soluble natural polysaccharide antibacterial material.
Obviously, the foregoing examples of the present invention are merely examples for clearly explaining the present invention, and are not limitations for the embodiments of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made, and all embodiments cannot be exhaustive herein. Any obvious changes or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201710556550.5 | Jul 2017 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/088173 | 5/24/2018 | WO | 00 |
