AU2015403788B2 - Chito-oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and preparation method thereof - Google Patents
Chito-oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and preparation method thereof Download PDFInfo
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Abstract
A chito-oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and preparing method thereof. The method comprises the following steps: (1) mixing N-Methylpiperazine with a chloro kojic acid and reacting the same to obtain a product of a chloro kojic acid-Mannich base; (2) mixing a chito-oligosaccharide Schiff base with the chloro kojic acid-Mannich base and reacting and processing the same to obtain a chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative, and performing an amine deprotection reaction and passivation to obtain a chito-oligosaccharide-O-kojic acid-Mannich base derivative.
Description
Chito-Oligosaccharide-O-Kojic Acid-Mannich Base Derivative as Antibacterial Agent and Preparation Method thereof
This application claims the priority to Chinese Patent Application No. 2015104438518, filed on July 24, 2015 and entitled “chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent and preparation method thereof’, which is incorporated herein by reference in its entirety.
Technical Field of the Invention
The present invention relates to the technical field of food additives, and particularly to a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent, and preparation method thereof.
Background of the Invention
In recent years, food safety has become one of the global focus issues preferentially concerned by the public in the whole world. The food may be contaminated with some harmful micro-organisms during the production, transportation, storage and sale process. These harmful microorganisms can contaminate and decay the food, and this causes not only serious waste of food resources, but also serious harm to the body of a consumer eating the food. Therefore, preservation of various kinds of food is always an urgent problem to be solved. The natural preservative and antimicrobial agents such as chitosan and kojic acid are preferred due to its good characteristics such as good safety and non-toxicity, as well as good thermal stability and other. It is a focus in research to develop a natural food preservative having more potent antimicrobial activity and broader antibacterial spectrum by chemical modification. Chito-oligosaccharide is a low molecular weight alkaline amino oligosaccharide obtained by physical, chemical or enzymatic cleavage of the backbone of chitosan. Chito-oligosaccharide is biodegradable, biocompatible, biologically non-toxic, and chemically reactive, and has inhibiting effects on a range of micro-organisms including bacteria and fungi, thus it is regarded as a desirable material for developing novel natural food preservatives.
However, the chito-oligosaccharide is a natural macromolecule, when used as a preservative and antibacterial agent for food, the chito-oligosaccharide still suffers from the defects such as low antibacterial activity compared with the conventionally used common chemical preservatives. Therefore, the use in food industry is not prevalent at present. In this regard, chito-oligosaccharide has chemically reactive amino and hydroxyl groups on the molecular chain, and these sites are desirable sites for chemical modification of chito-oligosaccharide, thus, the structure of chito-oligosaccharide may be chemically modified, to further enhance the antibacterial activity. This is an effective approach extensively studied in the world.
In view of the above shortcomings, the present inventor contemplates to provide a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent and preparation method thereof after active research and innovation, thereby allowing the chito-oligosaccharide to have great application value in industry.
Summary of the Invention
To solve the above technical problems, an object of the present invention is to provide a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent and preparation method thereof. The active antibacterial group, i.e. γ-pyrone group, in kojic acid and N-methyl piperazine are incorporated into the chain of the chito-oligosaccharide molecules and generate synergize effects with the chito-oligosaccharide molecules, so that the antibacterial activity of chito-oligosaccharide is enhanced. Therefore, a chito-oligosaccharide derivative having a good solubility, high degree of substitution, high antibacterial activity, high stability, and a synergistic effect is obtained.
For the above purpose, the invention utilizes the following technical solutions.
The invention provides a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent, which has a chemical structural formula as shown below:
wherein n is 6-20, and the degree of substitution is 1.21-1.78.
The present invention also provides a method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent, comprising the following steps: (1) dissolving chito-oligosaccharide Schiff base and chlorinated kojic acid-Mannich base respectively in an organic solvent, mixing and stirring; and after reaction, adding an organic solvent to precipitate the product, filtering, separating the precipitate by soxhlet extraction with an organic solvent, and freeze drying, to obtain the chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative; and (2) adding 0.25 mol/L of hydrochloric acid/ethanol mixture (V/V=l:4) to the chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative obtained in the step (1), mixing and stirring; and after reaction, adjusting pH value of the reaction solution to neutral, washing with an organic solvent, performing suction filtration and freeze drying, to obtainchito-oligosaccharide-O-kojic acid-Mannich base derivative. Preferably, the method also comprises a purification step after the step (2): dialyzing the dried product, adding an organic solvent to precipitate, performing suction filtration, and freeze drying to finally obtain a purified chito-oligosaccharide-O-kojic acid-Mannich base derivative.
Preferably, the weight ratio of the chito-oligosaccharide Schiff base to the chlorinated kojic acid-Mannich base is 2 : (1-5).
Preferably, the chito-oligosaccharide has a molecular weight of 1000-5000 Da and a degree of deacetylation of 90-95%.
Preferably, in the step (1), the organic solvent for dissolving the chlorinated kojic acid-Mannich base is dimethyl formamide, and more preferably, the amount of dimethyl formamide is 2-4 folds of the weight of the chlorinated kojic acid-Mannich base.
Preferably, the organic solvent for dissolving the chito-oligosaccharide Schiff base is dimethyl formamide and pyridine, more preferably, the amount of dimethyl formamide is 2-4 folds of the weight of the chito-oligosaccharide Schiff base, and the amount of pyridine is 2-6 folds of the weight of the chito-oligosaccharide Schiff base.
Preferably, in the step (1), the reaction temperature is -35°C-40°C, and the reaction time is 2-6 hrs.
Preferably, in the step (2), the reaction temperature is room temperature, and the reaction time is 12-24 hrs.
Preferably, in the step (1), the preparation method of chlorinated kojic acid-Mannich base comprises steps of: dissolving N-methylpiperazine and chlorinated kojic acid in a solution of methanol and formalin, and stirring vigorously at room temperature, to produce a tan precipitate; collecting, filtering, and washing the precipitate several times with anhydrous methanol, recrystallizing the precipitate in anhydrous methanol, and drying, to obtain the chlorinated kojic acid-Mannich base.
More preferably, the weight ratio of the N-methyl piperazine to the chlorinated kojic acid is 1 : (2-3), and the volume ratio of methanol to formalin is (10-20) : 1.
By adopting the above solutions, the present invention has at least the following advantages. In the present invention, based on the conception of substructure connection, the active group, i.e. γ-pyrone group, in kojic acid and N-methyl piperazine are incorporated into the chain of the chito-oligosaccharide molecules, such that the three have synergistic antibacterial activity. Therefore, a new antibacterial agent for food preservation is developed.
The foregoing description is merely summary of the technical solution of the present invention. To make the technical means of the present invention clearer and practicable in accordance with the disclosure of the present invention, the present invention is described in detail by way of preferred examples with reference to accompanying drawings.
Brief Description of Drawings
Fig. 1 is an IR spectrum of chito-oligosaccharide;
Fig. 2 is an IR spectrum of a chito-oligosaccharide derivative prepared in Embodiment 1 of the present invention;
Fig. 3 is a ’H-NMR spectrum of the chito-oligosaccharide derivative prepared in Embodiment 1 of the present invention;
Fig. 4 is a C-NMR spectrum of the chito-oligosaccharide derivative prepared in Embodiment 1 of the present invention;
Fig. 5 is an IR spectrum of a chito-oligosaccharide derivative prepared in Embodiment 2 of the present invention; and
Fig. 6 is an IR spectrum of a chito-oligosaccharide derivative prepared in Embodiment 3 of the present invention.
Detailed Description of the Preferred Embodiments
The invention will be further illustrated in more detail with reference to accompanying drawings. It is noted that, the following embodiments are intended for purposes of illustration only and are not intended to limit the scope of the invention. A chito-oligosaccharide-O-kojic acid-Mannich base derivative having good water solubility and antibacterial activity is prepared by the following methods. N-methylpiperazine and chlorinated kojic acid are used as raw materials, and chlorinated kojic acid-Mannich base is obtained after Mannich reaction, which is then subjected to alkylation reaction with N-protected chito-oligosaccharide Schiff base, then deprotected, and purified, to obtain a chito-oligosaccharide-O-kojic acid-Mannich base derivative having active amino groups retained. The synthesis route is shown below.
Embodiment 1:
The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent includes the following steps. 1. Preparation of chlorinated kojic acid-Mannich base N-methylpiperazine and chlorinated kojic acid were dissolved at a weight ratio of 1:2 in a solution of methanol (100 mL) and formalin (10 mL), and stirred vigorously at 25°C, to produce a tan precipitate. The precipitate was collected, filtered, washed 5 times with anhydrous methanol, recrystallized in anhydrous methanol, and dried, to obtain chlorinated kojic acid-Mannich base. 2. Preparation of chito-oligosaccharide-O-kojic acid-Mannich base derivative
Under vigorous stirring, 0.5 g of chlorinated kojic acid-Mannich base was dissolved in 2 mL dimethyl formamide., and 1 g of chito-oligosaccharide Schiff base (molecular weight: 1000 Da, degree of deacetylation: 90%) was dissolved in a mixture of dimethyl formamide (2 mL) and pyridine (2 mL). Then, the chito-oligosaccharide Schiff base dissolved in the mixture of dimethyl formamide and pyridine was added dropwise to the chlorinated kojic acid-Mannich base dissolved in dimethyl formamide. The reaction mixture was magnetically stirred for 2 hrs in a water bath at -35°C, and then an excessive amount of acetone was poured into the resulting solution to precipitate the product. The precipitate was filtered, separated by soxhlet extraction with ethanol and acetone respectively, to remove the dimethyl formamide and pyridine, and then freeze dried, to obtain 1 g of chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative. 20 mL of hydrochloric acid/ethanol mixture (V/V=l:4, 0.25 mol/L) was added to the prepared chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative, after stirring for 12 hrs at room temperature, pH of the mixture was adjusted to neutral with 10% Na2C03, then the resulting solution was washed 5 times with acetone, filtered under suction, and freeze dried. 1 g of the crude product was suspended in 5 mL ultrapure water, transferred to a dialysis bag and dialyzed for 24 hrs. The solution was concentrated, and sufficient acetone was added to precipitate the product, then the precipitate was filtered under suction and freeze dried to finally obtain a purified tan chito-oligosaccharide-O-kojic acid-Mannich base derivative.
The chito-oligosaccharide derivative has a yield of 62.1%, and is characterized by infrared (IR) spectroscopy and Nuclear Magnetic Resonance (NMR) spectroscopy.
Fig. 1 is an IR spectrum of chito-oligosaccharide. In the spectrum, the peak at 3422.36 cm'1 is a O-H and N-H stretching vibration absorption peak, the peak at 2923.83 cm'1 is a C-H stretching vibration absorption peak, the peak at 1628.76 cm'1 is a NH2 bending vibration absorption peak, the peaks at 1155.97 cm'1 and 1071.52 cm'1 are a C-0 stretching vibration absorption peak, and the peak at 893.24 cm'1 is a ring stretching vibration absorption peak.
Fig. 2 is an IR spectrum of a chito-oligosaccharide derivative prepared in this embodiment. The peak at 890.44 cm 1 is a characteristic absorption peak of β-pyran type glycosidic bond, the peak at 1519.37 cm-1 is a C=C stretching vibration absorption peak in the derivative, the peak at 1223.02 cm'1 is a C-O-C stretching vibration absorption peak in the derivative, and the peak at 970.39 cm'1 is an absorption peak of a covalent bond binding the chlorinated kojic acid-Mannich base to the chito-oligosaccharide. The existence of the peaks at 1223.02 cm'1 and 970.39 cm'1 confirms the generation of the target product.
Fig. 3 is a 'H-NMR spectrum of the chito-oligosaccharide derivative prepared in this embodiment. The peak at a chemical shift of 1.968 ppm corresponds to a proton peak of -CH3 on the acetamido residue. The peak at 2.447-2.796 ppm corresponds to a proton peak of H2 on the glucosamine residue and the acetamido residue. The peak at 3.089-3.837 ppm corresponds to a proton peak of H3, H4, H5, and H6 on the glucosamine and the acetylglucosamine. The peak at 4.7 ppm is a solvent peak. The two new chemical shifts at 4.26 and 6.82 ppm may be attributed to the chemical shifts of -CH2(H-7’) and H-3’ on the 5-hydroxypyrone backbone of kojic acid respectively; and the chemical shift of H2 on the glucosamine residue at δ 2.98 ppm still exists. The result is consistent with that obtained from the IR spectrum. The new chemical shifts confirm that alkylation occurs between the 5-hydroxypyrone in kojic acid and the amino group of chito-oligosaccharide. The chemical shifts at 2.88, 3.07, and 3.12 ppm are the chemical shifts of the N-methyl piperazine ring.
Therefore, by the attribution of the above chemical shifts, it is confirmed that the chito-oligosaccharide-O-kojic acid-Mannich base derivative is successfully prepared by the O-alkylation reaction of the chlorinated kojic acid-Mannich base with the chito-oligosaccharide Schiff base.
Fig. 4 is a C-NMR spectrum of the chito-oligosaccharide derivative prepared in this embodiment. The chemical shifts at δ 22.74, 56.33, 60.46, 71.03, 75.11, 76.95, 98.63, and 174.18 ppm are attributed respectively to the chemical shifts of -CH3, C2, C6, C3, C5, C4, Cl, and -C=0 in the chito-oligosaccharide molecules. The chemical shifts at δ 45.83, 47.08, and 56.38 ppm are attributed respectively to C in -CH2 and -CH3 on the N-methylpiperazine ring of the derivative. The chemical shifts at δ 60.44, 114.64, 139.82, 145.87, 159.35 and 176.99 ppm are attributed respectively to the chemical shifts of C-7’, C-3’, C-6’, C-5’, C-2’, and C-4’ in the 5-hydroxypyrone in the derivative. This suggests that the chito-oligosaccharide-O-kojic acid-Mannich base derivative is successfully prepared.
Embodiment 2:
The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent includes the following steps. 1. Preparation of chlorinated kojic acid-Mannich base N-methylpiperazine and chlorinated kojic acid were dissolved at a weight ratio of 1:2.5 in a solution of methanol (150 mL) and formalin (10 mL), and stirred vigorously at 25°C, to produce a tan precipitate. The precipitate was collected, filtered, washed 5 times with anhydrous methanol, recrystallized in anhydrous methanol, and dried, to obtain chlorinated kojic acid-Mannich base. 2. Preparation of chito-oligosaccharide-O-kojic acid-Mannich base derivative
Under vigorous stirring, 1.5 g of chlorinated kojic acid-Mannich base was dissolved in 3 mL dimethyl formamide, and 1 g of chito-oligosaccharide Schiff base (molecular weight: 3000 Da, degree of deacetylation: 93%) was dissolved in a mixture of dimethyl formamide (3 mL) and pyridine (4 mL). Then, the chito-oligosaccharide Schiff base dissolved in the mixture of dimethyl formamide and pyridine was added dropwise to the chlorinated kojic acid-Mannich base dissolved in dimethyl formamide. The reaction mixture was magnetically stirred for 4 hrs in a water bath at -5°C, and then an excessive amount of acetone was poured into the resulting solution to precipitate the product. The precipitate was filtered, separated by soxhlet extraction with ethanol and acetone respectively, to remove the dimethyl formamide and pyridine, and then freeze dried under vacuum, to obtain 1.8 g product. 36 mL of hydrochloric acid/ethanol mixture (V/V=l:4, 0.25 mol/L) was added to the prepared chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative, after stirring for 12 hrs at room temperature, pH of the mixture was adjusted to neutral with 10% Na2CC>3, then the resulting solution was washed 5 times with acetone, filtered under suction, and freeze dried. 1 g of the crude product was suspended in 5 mL ultrapure water, transferred to a dialysis bag and dialyzed for 24 hrs. The solution was concentrated, and sufficient acetone was added to precipitate the product, then the precipitate was filtered under suction and freeze dried to finally obtain a purified tan chito-oligosaccharide-O-kojic acid-Mannich base derivative.
The yield of the chito-oligosaccharide derivative is 65.2%. Fig. 5 is an IR spectrum of the sample. In the spectrum, the peak at 892.24 cm-1 is a characteristic absorption peak of β-pyran type glycosidic bond, the peak at 1518.32 cm'1 is a C=C stretching vibration absorption peak in the derivative, the peak at 1226.23 cm'1 is a C-O-C stretching vibration absorption peak in the derivative, and the peak at 971.91 cm'1 is an absorption peak of a covalent bond binding the chlorinated kojic acid-Mannich base to the chito-oligosaccharide. The existence of the peaks at 1226.23 cm'1 and 971.91 cm'1 confirms the generation of the target product.
Embodiment 3:
The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent includes the following steps. 1. Preparation of chlorinated kojic acid-Mannich base N-methylpiperazine and chlorinated kojic acid were dissolved at a weight ratio of 1:3 in a solution of methanol (200 mL) and formalin (10 mL), and stirred vigorously at 25°C, to produce a tan precipitate. The precipitate was collected, filtered, washed 5 times with anhydrous methanol, recrystallized in anhydrous methanol, and dried, to obtain chlorinated kojic acid-Mannich base. 2. Preparation of chito-oligosaccharide-O-kojic acid-Mannich base derivative
Under vigorous stirring, 2.5 g of chlorinated kojic acid-Mannich base was dissolved in 4 mL dimethyl formamide., and 1 g of chito-oligosaccharide Schiff base (molecular weight: 5000 Da, and degree of deacetylation: 95%) was dissolved in a mixture of dimethyl formamide (4 mL) and pyridine (6 mL). Then, the chito-oligosaccharide Schiff base dissolved in the mixture of dimethyl formamide and pyridine was added dropwise to the chlorinated kojic acid-Mannich base dissolved in dimethyl formamide. The reaction mixture was magnetically stirred for 6 hrs in a water bath at 40°C, and then an excessive amount of acetone was poured into the resulting solution to precipitate the product. The precipitate was filtered, separated by soxhlet extraction with ethanol and acetone respectively, to remove the dimethyl formamide and pyridine, and then freeze dried under vacuum, to obtain 3 g of the product. 60 mL of hydrochloric acid/ethanol mixture (V/V=l:4, 0.25 mol/L) was added to the prepared chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative, after stirring for 12 hrs at room temperature, pH of the mixture was adjusted to neutral with 10% Na2CC>3, then the resulting solution was washed 5 times with acetone, filtered under suction, and freeze dried. 1 g of the crude product was suspended in 5 mL ultrapure water, transferred to a dialysis bag and dialyzed for 24 hrs. The solution was concentrated and sufficient acetone was added to precipitate the product, and then the precipitate was filtered under suction and freeze dried to finally obtain a purified tan chito-oligosaccharide-O-kojic acid-Mannich base derivative.
The yield of the chito-oligosaccharide derivative is 63.5%. Fig. 6 is an IR spectrum of the sample. In the spectrum, the peak at 893.34 cm 1 is a characteristic absorption peak of β-pyran type glycosidic bond, the peak at 1514.18cm'1 is a C=C stretching vibration absorption peak in the derivative, the peak at 1223.73 cm'1 is a C-O-C stretching vibration absorption peak in the derivative, and the peak at 970.39 cm'1 is an absorption peak of a covalent bond binding the chlorinated kojic acid-Mannich base to the chito-oligosaccharide. The existence of the peaks at 1226.23 cm'1 and 971.91 cm'1 confirms the generation of the target product.
Detection of Antibacterial Activity 1. Preparation of solutions containing antibacterial agents
The samples in embodiments 1, 2, and 3 were designated as derivative 1, derivative 2, and derivative 3 respectively. The chito-oligosaccharide, kojic acid, derivative 1, derivative 2, and derivative 3 were dissolved in deionized water, and formulated into solutions having gradient concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 mg/mL respectively. The solutions were filtered with a 0.22 pm microporous filter membrane, for later use. 2. Determination of antibacterial activity A bacterial suspension (1 mL) was mixed with a sterilized nutrient broth medium (98 mL), and then various concentrations of the antibacterial agent (1 mL) were added. Deionized water (1 mL) was used as a blank in place of the solution containing the antibacterial agent. The incubation was carried out at 37°C in a shaker. During incubation, the culture (1 mL) was removed after 8 hrs, two-fold diluted, and then plated to calculate the number of colonies. Each sample was triplicated. The antibacterial inhibition rate (I) is calculated according to an equation below: I(%)= N, - N.J . 10{) Λ where N1 is the total number of colonies existing in the initial culture medium; and N2 is the total number of colonies existing in the culture medium containing the antibacterial agent.
The IC50 value is obtained by plotting with the concentration of the antibacterial agent as the horizontal coordinate and the inhibition rate as the longitudinal coordinate.
Table 1. Performance detection of antibacterial agent IC50 (mg/mL)
Sample Staphylococ Streptococ Bacillus Salmonella Shigella Escherichi cus aureus cus subtilis typhimuriu dysenteriae a coli pyogenes m
Chito-oligos 6.25±0.23 6.47±0.15 6.52±0.21 6.68±0.22 7.15±0.18 7.29±0.21 accharide
Kojic acid 6.63±0.13 6.25±0.16 6.43±0.13 6.16±0.21 6.23±0.23 6.04±0.12
Derivative 1 0.02±0.002 0.04±0.001 0.08±0.002 0.09±0.003 0.12±0.004 0.15±0.008
Derivative 2 0.06±0.008 0.09±0.015 0.15±0.008 0.18±0.004 0.21±0.005 0.22±0.007
Derivative 3 0.08±0.007 0.09±0.011 0.12±0.013 0.19±0.019 0.23±0.013 0.28±0.014
It can be seen from Table 1 that the antibacterial agent prepared in the present invention has good antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae, and Escherichia coli, and the antibacterial property of the antibacterial agent of the invention is greatly advantageous over that of chito-oligosaccharide and kojic acid, thus having potential application value in food preservation.
In summary, the principles of the present invention are as follows. Three active sites for chemical modification are present in the molecular structure of chito-oligosaccharide. Depending on the activity of the three sites, the alkylation reaction occurs initially at the amino group of position C-2, then at the primary hydroxyl group of position C-6, and finally at the secondary hydroxyl group of position C-3. Since in an acidic solution, the amino group at position C-2 in the chito-oligosaccharide molecules is an active group having antibacterial effect after protonation, the active amino group at position C-2 is protected with benzaldehyde, and then deprotected in a solution of acid in alcohol after the alkylation reaction, to release the active amino group. The hydroxyl group at position C-3 does not participate in the reaction due to the steric hindrance effect. Therefore, the hydroxyl group at position C-6 is O-alkylated with the chlorinated kojic acid-Mannich base.
The present invention has the following beneficial effects. (1) The active antibacterial group, i.e. γ-pyrone group, in kojic acid and N-methylpiperazine are incorporated into the chain of the chito-oligosaccharide molecules by the O-alkylation of the hydroxyl group at position C-6 of chito-oligosaccharide with the chlorinated kojic acid-Mannich base, and has synergistic effect with the chito-oligosaccharide molecules, whereby the antibacterial activity of chito-oligosaccharide is greatly enhanced. (2) Such a new type of derivative has a low toxicity and high water solubility, and can be dissolved in a variety of organic and inorganic solvents, thus avoiding the use of organic solvents. This is beneficial to environmental protection and broadens the application areas, and the derivative is of great application value in the fields of medicine, food, cosmetics, agriculture and the like. The above preferred embodiments are described for illustration only, and are not intended to limit the scope of the invention. It should be understood, for a person skilled in the art, that various improvements or variations can be made therein without departing from the spirit and scope of the invention, and these improvements or variations should be covered within the protecting scope of the invention.
Claims (10)
- What is claimed is:1. A chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent, having a chemical structural formula as shown below:wherein n is 6-20, and the degree of substitution is 1.21-1.78.
- 2. A method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 1, comprising steps of: (1) dissolving chito-oligosaccharide Schiff base and chlorinated kojic acid-Mannich base respectively in an organic solvent, mixing and stirring; and after reaction, adding an organic solvent to precipitate the product, filtering, separating the precipitate by soxhlet extraction with an organic solvent, and freeze drying to obtain the chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative; and (2) adding 0.25 mol/L of hydrochloric acid/ethanol mixture (V/V=l:4) to the chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative obtained in the step (1), mixing and stirring; and after reaction, adjusting pH value of the reaction solution to neutral, washing with an organic solvent, performing suction filtration and freeze drying, to obtain chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent.
- 3. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 2, wherein the method further comprises a purification step after the step (2): dialyzing the dried product, adding an organic solvent to precipitate, performing suction filtration and freeze drying, to finally obtain a purified chito-oligosaccharide-O-kojic acid-Mannich base derivative.
- 4. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 2, wherein the weight ratio of the chito-oligosaccharide Schiff base to the chlorinated kojic acid-Mannich base is 2 : (1-5).
- 5. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 4, wherein the chito-oligosaccharide has a molecular weight of 1000-5000 Da and a degree of deacetylation of 90-95%.
- 6. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 2, wherein in the step (1), the organic solvent for dissolving the chlorinated kojic acid-Mannich base is dimethyl formamide, and the amount of dimethyl formamide being 2-4 folds of the weight of the chlorinated kojic acid-Mannich base; and the organic solvent for dissolving the chito-oligosaccharide Schiff base is dimethyl formamide and pyridine, the amount of dimethyl formamide being 2-4 folds of the weight of the chito-oligosaccharide Schiff base, and the amount of pyridine being 2-6 folds of the weight of the chito-oligosaccharide Schiff base.
- 7. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 2, wherein in the step (1), the reaction temperature is -35°C-40°C, and the reaction time is 2-6 hrs.
- 8. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 2, wherein in the step (2), the reaction temperature is room temperature, and the reaction times is 12-24 hrs.
- 9. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 2, wherein in the step (1), the preparation method of chlorinated kojic acid-Mannich base comprises steps of: dissolving N-methylpiperazine and chlorinated kojic acid in a solution of methanol and formalin, and stirring vigorously at room temperature, to produce a tan precipitate; collecting, filtering, and washing the precipitate several times with anhydrous methanol, recrystallizing the precipitate in anhydrous methanol and drying, to obtain the chlorinated kojic acid-Mannich base.
- 10. The method for preparing a chito-oligosaccharide-O-kojic acid-Mannich base derivative as antibacterial agent according to claim 9, wherein the weight ratio of the N-methylpiperazine to the chlorinated kojic acid is 1 : (2-3), and the volume ratio of methanol to formalin is (10-20) : 1.
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