CN113201082A - Chitosan-chlorin e6 antibacterial agent and preparation method thereof - Google Patents
Chitosan-chlorin e6 antibacterial agent and preparation method thereof Download PDFInfo
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Abstract
本发明提供了一种壳聚糖‑二氢卟吩e6抗菌剂的制备方法，其中二氢卟吩e6占总重量的13.48％～22.04％；壳聚糖‑二氢卟吩e6由壳聚糖与二氢卟吩e6通过酰胺键结合得到，壳聚糖‑二氢卟吩e6中二氢卟吩e6对壳聚糖的氨基取代度为6.44％～11.68％。本发明利用壳聚糖作为光敏剂二氢卟吩e6的载体，制备得到了一种壳聚糖‑二氢卟吩e6抗菌剂。与二氢卟吩e6相比，本发明制剂可有效提高二氢卟吩e6对金黄色葡萄球菌和大肠杆菌的抗菌活性，在农业、医药等多种领域都具有应用价值。
The invention provides a preparation method of chitosan-chlorin e6 antibacterial agent, wherein chlorin e6 accounts for 13.48%-22.04% of the total weight; chitosan-chlorin e6 is composed of chitosan It is obtained by combining with chlorin e6 through an amide bond, and the amino substitution degree of chlorin e6 to chitosan in chitosan-chlorin e6 is 6.44% to 11.68%. The invention uses chitosan as the carrier of the photosensitizer chlorin e6 to prepare a chitosan-chlorin e6 antibacterial agent. Compared with chlorin e6, the preparation of the invention can effectively improve the antibacterial activity of chlorin e6 on Staphylococcus aureus and Escherichia coli, and has application value in agriculture, medicine and other fields.
Description
Technical Field
The invention belongs to the field of antibacterial agent preparation, and particularly relates to a preparation method of a chitosan-chlorin e6 antibacterial agent.
Background
Photodynamic sterilization is a method for generating active oxygen sterilization by using a photosensitizer, light and oxygen. Photodynamic antibacterial agents are substances which can kill bacteria under the irradiation of light with specific wavelength, and have been widely studied in recent years due to their controllability and high efficiency.
Chlorin e6 is a highly effective photosensitizer, and is mainly prepared from chlorophyll II extract. Chlorin e6 has excellent active oxygen generation efficiency, can be excited by near infrared light, and has low dark toxicity. However, the low water solubility and bacterial affinity of chlorin e6 limit its application and a suitable carrier needs to be found.
Chitosan is a polysaccharide with wide source, no toxicity and antibacterial property, is the only cationic polysaccharide existing in large quantity in the biological world, and can be prepared by chitosan deacetylation. Chitosan has a certain antibacterial property, but the antibacterial property is poor compared with the antibacterial substance on the market, so in order to expand the application range of chitosan and develop a new compound with excellent performance, various modification researches on chitosan are needed, and chemical modification is mostly researched and applied at present.
At present, no research report on the combination of chitosan and chlorin e6 as an antibacterial agent exists.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or technical gaps in the art of existing antibacterial agents.
Accordingly, one of the objects of the present invention is to solve the disadvantages of the prior art and to provide a chitosan-chlorin e6 antibacterial agent.
In order to solve the technical problems, the invention provides the following technical scheme: a chitosan-chlorin e6 antibacterial agent, wherein the chemical structural formula of the chitosan-chlorin e6 antibacterial agent is shown as the following I-A:
wherein X is a group of formula I-B below, and is linked to the nitrogen of chitosan at point d;
the amino substitution degree of the chitosan-chlorin e6 is 6.44% -11.68%, and the chlorin e6 accounts for 13.48% -22.04% of the total weight of the antibacterial agent.
As a preferable embodiment of the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: the molecular weight of the chitosan is 50kDa, and the deacetylation degree is more than 90 percent.
Therefore, it is still another object of the present invention to solve the problems of the prior art and to provide a method for preparing chitosan-chlorin e6 antibacterial agent.
In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing chitosan-chlorin e6 antibacterial agent comprises,
dissolving chitosan in an acetic acid solution, and adjusting the pH value of the solution to 5.8-6.0 to obtain a chitosan solution;
dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS in morpholine ethanesulfonic acid MES buffer to obtain an EDC/NHS MES solution;
dissolving chlorin e6 in dimethyl sulfoxide, adding into MES solution of EDC/NHS, and stirring at room temperature in dark place;
and dropwise adding the mixed solution obtained after stirring into the chitosan solution, stirring at room temperature in a dark place, dialyzing the obtained mixed solution with ultrapure water, pre-freezing the obtained solution at-80 ℃ overnight after dialysis is finished, and freeze-drying to obtain a product, and fully grinding the product into powder to obtain the chitosan-chlorin e6 antibacterial agent.
As a preferable embodiment of the method for preparing the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: dissolving chitosan in an acetic acid solution, wherein the volume concentration of the acetic acid solution is 1%, and the mass-volume ratio of the chitosan to the acetic acid solution is as follows in mg: the mL is 6: 5.
As a preferable embodiment of the method for preparing the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: and adjusting the pH value of the chitosan solution to 5.8-6.0 to obtain a chitosan solution, wherein the pH regulator is a 1mol/L sodium hydroxide solution, and the concentration of the chitosan solution is 1 mg/mL.
As a preferable embodiment of the method for preparing the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS in morpholine ethanesulfonic acid MES buffer, wherein the mass ratio of EDC to NHS is 55: 66, MES buffer to EDC volume to mass ratio in mL: the mg is 1: 11.
as a preferable embodiment of the method for preparing the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: dissolving chlorin e6 in dimethyl sulfoxide, and adding into MES solution of EDC/NHS, wherein the mass-volume ratio of chlorin e6 to dimethyl sulfoxide is mg: the mL is 14-56: 5, and the volume ratio of the MES solution of the dimethyl sulfoxide to the EDC/NHS is 1: 1.
As a preferable embodiment of the method for preparing the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: the chitosan-chlorin e6 is characterized in that the molar ratio of chlorin e6 to chitosan amino is 5-20: 100, the ultrapure water is dialyzed for 3 days, and the cut-off molecular weight of the dialysis bag is 8000 Da-14000 Da.
As a preferable embodiment of the method for preparing the chitosan-chlorin e6 antibacterial agent of the present invention, wherein: the room temperature is 25 ℃, the freeze-drying temperature is-80 ℃, the overnight time is 12 hours, and the freeze-drying is carried out in a freeze-dryer for 48 hours.
The invention also aims to solve the defects in the prior art and provides application of chitosan-chlorin e6 as an antibacterial active ingredient in the antibacterial field.
The invention has the beneficial effects that:
the invention provides a preparation method of a novel substance chitosan-chlorin e6, the preparation method is simple, the cost is low, the purification method is simple and convenient, the obtained chitosan derivative has good thermal stability and antibacterial activity, has good inhibition effect on staphylococcus aureus, escherichia coli and the like, and has good application prospects in the fields of agriculture, medicines and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is an infrared spectrum of chitosan.
FIG. 2 is an infrared spectrum of chitosan-chlorin e6 prepared in the example of the present invention.
FIG. 3 is a 1H NMR spectrum of chitosan.
FIG. 4 is the 1H NMR spectrum of chitosan-chlorin e6 prepared in the examples of the present invention.
FIG. 5 is the 1H NMR spectrum of the product of example 4.
FIG. 6 is a graph (TG) showing the thermogravimetric percentage of chitosan, chlorin e6, and chitosan-chlorin e 6.
FIG. 7 is a first derivative graph (DTG) of the thermogravimetry of chitosan, chlorin e6, chitosan-chlorin e 6.
FIG. 8 is a graph showing the degradation of 1, 3-Diphenylisobenzofuran (DPBF) by chitosan and chitosan-chlorin e 6.
FIG. 9 is a graph comparing the inhibitory effect of chitosan and chitosan-chlorin e6 on Staphylococcus aureus and Escherichia coli, wherein (A) Staphylococcus aureus; (B) escherichia coli.
FIG. 10 is a synthetic scheme of chitosan-chlorin e 6.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The preparation method of the chitosan-chlorin e6 comprises the following steps: at pH 6.0, the carboxyl group of chlorin e6 was first activated using EDC/NHS chemistry and then reacted with the amino group of chitosan. The final product chitosan-chlorin e6 is obtained after dialysis purification and freeze-drying, and the synthetic route is shown in figure 10.
Example 1:
the preparation method of the chitosan-chlorin e6 comprises the following steps:
the chitosan-chlorin e6 was synthesized using the EDC/NHS method.
First, chitosan (120mg) was dissolved in 100mL of acetic acid solution (1%, v/v), the pH was adjusted to about 6.0 with 1mol/L sodium hydroxide solution, and ultrapure water was added to 120mL to obtain a 1mg/mL chitosan solution.
EDC (55mg) and NHS (66mg) were dissolved in 5mL MES buffer (MES: 0.05 mol/L; pH adjusted to 6.0 with 1mol/L NaOH solution), chlorin e6(56mg) was dissolved in 5mL dimethylsulfoxide, and the resulting chlorin e6 solution was added to the MES solution of EDC/NHS, and stirred at room temperature for 4h in the absence of light at 400 rpm.
And dropwise adding the mixed solution obtained after stirring into the chitosan solution, stirring for 24 hours at room temperature in a dark place, dialyzing the obtained mixed solution for 3 days against ultrapure water, wherein the cut-off molecular weight of a dialysis bag is 8000 Da-14000 Da. After dialysis, the obtained solution is pre-frozen at-80 ℃ overnight, and is put into a freeze dryer for freeze-drying for 48 hours, so that the product is fully ground into powder, the yield of the product is 65%, and the substitution degree of the product is 11.68%.
FIG. 1 shows an infrared spectrum of chitosan. Among them, the broad peak at 3433cm-1 is caused by O-H and N-H stretching vibrations, the peaks at 2926cm-1 and 1384cm-1 are caused by C-H stretching vibrations, the peak at 1635cm-1 is caused by C ═ O stretching vibrations in chitosan, that is, the peak of amide I, the peak at 1095cm-1 is caused by C-O stretching vibrations, the peak near 1158cm-1 is caused by asymmetric stretching vibrations of an ether oxygen bond (C-O-C), and the peak near 592cm-1 is caused by chitosan ring vibrations.
FIG. 2 is an infrared spectrum of chitosan-chlorin e6 prepared in this example. Wherein, the peak intensity of the amide I at 1635cm < -1 > is obviously enhanced, which primarily shows the successful synthesis of the product.
FIG. 3 is a 1H NMR spectrum of chitosan. The characteristic peak of hydrogen atoms on a glucose ring in the chitosan appears at 3.25ppm to 4.19 ppm; the characteristic peak of the methyl hydrogen atom on the N-acetyl monomer appeared at 2.07 ppm; delta. 5.02ppm represents a solvent peak.
FIG. 4 is the 1H NMR spectrum of chitosan-chlorin e6 prepared in this example. The delta-9.00 ppm, the delta-8.64 ppm and the delta-7.72 ppm are absorption peaks belonging to hydrogen on a chlorin ring of chlorin e6 respectively; the nuclear magnetic results are consistent with the infrared results, indicating the successful synthesis of chitosan-chlorin e 6.
Example 2:
first, chitosan (120mg) was dissolved in 100mL of acetic acid solution (1%, v/v), and ultrapure water was added to 120mL to give a 1mg/mL chitosan solution.
Then, chlorin e6(56mg) was dissolved in 5mL of dimethylsulfoxide, EDC (55mg) and NHS (66mg) were added thereto, and the mixture was stirred at room temperature for 4 hours in the absence of light at 400 rpm.
And dropwise adding the mixed solution obtained after the stirring is finished into the chitosan solution, and stirring for 24 hours at room temperature in a dark place. The product was purified by centrifugation at 14000rpm for 20min, but the target product could not be obtained because the product was soluble in the system.
Example 3:
first, chitosan (120mg) was dissolved in 100mL of acetic acid solution (1%, v/v), and ultrapure water was added to 120mL to give a 1mg/mL chitosan solution.
Then, chlorin e6(56mg) was dissolved in 5mL of dimethylsulfoxide, EDC (55mg) and NHS (66mg) were added thereto, and the mixture was stirred at room temperature for 4 hours in the absence of light at 400 rpm.
And dropwise adding the mixed solution obtained after the stirring is finished into the chitosan solution, and stirring for 24 hours at room temperature in a dark place. The product was precipitated by adding three times the acetone of the reaction system and then purified by centrifugation at 14000rpm for 20 min. However, the system volume is too large after acetone is added, a plurality of centrifuge tubes are needed for centrifugation, and the product yield is only about 20% due to large product loss in the mixing process.
Example 4:
first, chitosan (120mg) was dissolved in 100mL of acetic acid solution (1%, v/v), and ultrapure water was added to 120mL to give a 1mg/mL chitosan solution.
Then, chlorin e6(56mg) was dissolved in 5mL of dimethylsulfoxide, EDC (55mg) and NHS (66mg) were added thereto, and the mixture was stirred at room temperature for 4 hours in the absence of light at 400 rpm.
And dropwise adding the mixed solution obtained after stirring into the chitosan solution, stirring for 24 hours at room temperature in a dark place, dialyzing the obtained mixed solution for 3 days against ultrapure water, wherein the cut-off molecular weight of a dialysis bag is 8000 Da-14000 Da.
After dialysis, the resulting solution was prefrozen overnight at-80 ℃ and lyophilized in a lyophilizer for 48h to give a product which was then ground to a powder.
Fig. 5 is a 1H nuclear magnetic spectrum of the final product in example 4, from which it can be seen that no characteristic peak of chlorin e6 appears, and it is presumed that the reason may be that the pH during the carboxyl activation and the pH during the reaction with the amino group are not controlled, and the success rate of the reaction between the amino group and the carboxyl group due to the degradation of the intermediate product is too low, so that no signal of chlorin e6 is shown in the 1H nuclear magnetic spectrum.
Example 5:
thermal stability assay of chitosan, chlorin e6 and chitosan-chlorin e 6:
the thermal stability of chitosan, chlorin e6 and chitosan-chlorin e6 was analyzed using a TGA2 thermal analysis system.
Precisely weighing about 5mg of sample in a crucible, wherein the measuring conditions are N2 flow rate of 20mL/min, temperature rise rate of 20 ℃/min and measuring temperature range of 25-450 ℃.
FIG. 6 is a TG curve of chitosan-chlorin e6 prepared in this example. It can be seen that chitosan and chitosan-chlorin e6 gradually lost weight with increasing temperature.
FIG. 7 is a DTG curve of chitosan-chlorin e6 prepared in this example. It can be seen that chlorin e6 has a weight loss stage between 25 ℃ and 450 ℃ and two weight loss stages between 140 ℃ and 250 ℃ and between 25 ℃ and 450 ℃ for chitosan. The first-order weight loss occurs at 40-160 ℃, and the second-order weight loss occurs at 230-380 ℃. The modified chitosan-chlorin e6 has two weight loss stages at 25-450 ℃. Wherein the first-order weight loss is between 40 and 160 ℃, the first-order weight loss is the same as that of the chitosan, and the second-order weight loss is between 220 and 390 ℃, and is similar to that of the chitosan. Therefore, the thermal stability of the chitosan-chlorin e6 is similar to that of chitosan, and is improved compared with that of chlorin e 6.
Example 6:
determination of active oxygen production ability of Chitosan-chlorin e6
The capacity of the product to generate reactive oxygen species was determined using DPBF as a reactive oxygen species probe. DPBF is dissolved in dimethyl sulfoxide to prepare DPBF solution with 25 mug/mL. Taking 150 mu L of DPBF solution and 3mL of sample solution (100 mu g/mL) to be uniformly mixed, and then, using a laser source with the wavelength of 13mW/cm under the condition of 660nm2Is irradiated with the optical density of (1). Every 1min of irradiation, the ultraviolet-visible absorption spectrum of the mixed solution was measured. Ultrapure water without any sample added was set as a blank control.
The active oxygen measurement results in this example are shown in fig. 8, and chitosan-chlorin e6 has good active oxygen generating ability.
Example 7: chitosan-chlorin e6 antimicrobial assay
The antibacterial property of the product is measured by measuring the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of chitosan, chlorin e6 and chitosan-chlorin e6 to escherichia coli and staphylococcus aureus.
The MIC and MBC are determined by broth microdilution, which comprises the following steps:
activating strains: the preserved strain in a refrigerator at-80 ℃ was taken out and thawed, and the strain was inoculated in LB liquid medium at an inoculum size of 2% in a safety cabinet. Putting the mixture into a shaker at 37 ℃ for overnight culture and activation, wherein the rotation speed of the shaker is 160rpm, inoculating the activated bacterium solution into a fresh LB culture medium in an inoculation amount of 2%, and performing shake culture until the turbidity of the bacterium solution is equivalent to that of a 0.5 McLeeb turbidimetric tube (5 h-6 h).
Preparing bacterial liquid: diluting the obtained bacterial liquid by 1000 times with MH liquid culture medium to obtain bacterial liquid (the concentration of the bacterial liquid is approximately equal to 1.5 multiplied by 105CFU/mL) for antibacterial experiments.
Antibacterial sample solution: dissolving a sample in 1% (v/v) acetic acid solution, adjusting the pH value to about 6.0 by using 1mol/L sodium hydroxide solution, and respectively preparing a chitosan solution with the concentration of 2.0mg/mL, a chitosan-chlorin e6 solution and a chlorin e6 solution with the concentration of 0.3 mg/mL.
Gradient dilution of sample liquid: adding 200 mu L of antibacterial sample liquid into the first hole of each row of a 96-well plate, taking 100 mu L to the second hole, adding 100 mu L of sterile physiological saline into the second hole, uniformly mixing, sucking 100 mu L to the third hole, repeating the steps, continuously diluting to the tenth hole, discarding the last 100 mu L, and finally obtaining the chitosan and chitosan-chlorin e6 solution with the concentration range of 1000-2 mu g/mL and the chlorin e6 solution with the concentration range of 300-0.6 mu g/mL.
Mixing: to each well of the 96-well plate, 100. mu.L of the bacterial solution was added, and 3 sets of each sample were set in parallel with a blank of sterile physiological saline, and the experiment was repeated 3 times. Mixing, and placing in a dark incubator at 37 deg.C for 30 min.
Illumination: and (3) placing the 96-well plate under a 660nm laser source for illumination, wherein the illumination intensity is 200mW/cm2, the illumination time is 5min, placing the 96-well plate in an incubator at 37 ℃ for culturing for 24h after illumination, setting a corresponding lightless group on each plate, and determining the influence of the sample on the bacterial growth under the dark condition.
MIC and MBC determination: the minimum concentration in the visually clear wells was recorded as the MIC value of the sample, and all visually clear wells were pipetted 100. mu.L onto MH agar plates and after incubation at 37 ℃ for 24h, the maximum concentration of colonies grown was recorded as MBC.
The MIC and MBC of chitosan, chlorin e6 and chitosan-chlorin e6 with various degrees of substitution are shown in Table 1.
TABLE 1 MIC and MBC for each product
Wherein, the product 1 is prepared by replacing the content of chlorin e6 with 14mg under the condition of the example 1 and other steps are the same as the example 1; the product 2 was prepared by replacing the chlorin e6 content with 28mg under the conditions of example 1 and performing the same procedures as in example 1.
In this example, the substitution degree of the product is preferably selected, and the target product having the best antibacterial property is selected by comparing the MIC and MBC of 3 chlorin e6 substitution degree products (6.44%, 821%, 11.68%), wherein the substitution degree of amino group of chitosan by chlorin e6 is 11.68%. Meanwhile, chitosan-chlorin e6 has significantly lower MIC and MBC than chitosan and chlorin e6, indicating that chitosan-chlorin e6 has stronger antibacterial effect.
Example 8:
determination of antibacterial Effect of Chitosan-chlorin e6
In order to more clearly understand the difference in antibacterial effect between the products, the antibacterial effect was measured by the plate method using chitosan, chitosan-chlorin e6 and chlorin e6 at initial concentrations of 100. mu.g/mL.
The experimental method is as follows: preparing a bacterial solution with the concentration of about 105CFU/mL, and mixing the bacterial solution and the sample solution according to the ratio of 1:1 for 30min in an incubator at 37 ℃, irradiating for 5min under a 660nm laser source at the optical density of 200mW/cm2, sucking 100 mu L of the mixed solution, coating the mixed solution on an LB agar plate, and observing the growth condition of bacteria after culturing for 24h in the incubator at 37 ℃.
The inhibitory effect of chitosan, chlorin e6 and chitosan-chlorin e6 on staphylococcus aureus and escherichia coli is shown in fig. 9.
The modified material is observed to have good antibacterial property, and the inhibition effect on bacteria is obviously enhanced compared with chitosan and chlorin e 6.
The invention utilizes the principle that the photosensitizer generates active oxygen under the illumination to sterilize (namely the photodynamic principle), the obtained antibacterial agent does not generate drug resistance, has strong sterilization effect, and can control the sterilization effect by controlling the illumination; meanwhile, for the carboxyl activation treatment of Ce6, the synthesis was attempted by directly dissolving EDC and NHS in water and then adjusting pH, but the reaction success rate was not high and the product was not obtained substantially.
The chitosan is provided with three modification sites which are respectively C-2 amino, C-3 hydroxyl and C-6 hydroxyl, the chlorin e6 contains carboxyl, the chlorin e6 can be modified on the chitosan through the reaction between the amino and the carboxyl, the chitosan is chemically modified, the chitosan is used as a carrier of the chlorin e6, and the combination of the two can be used as a novel photodynamic antibacterial agent applied to the sterilization field.
In view of the above, the present inventors have devised a method for preparing a chitosan-chlorin e6 antibacterial agent, and have improved the application value of chitosan and chlorin e6 in the antibacterial field. The invention provides a preparation method of a novel substance chitosan-chlorin e6, the preparation method is simple, the cost is low, the purification method is simple and convenient, the obtained chitosan derivative has good thermal stability and antibacterial activity, has good inhibition effect on staphylococcus aureus, escherichia coli and the like, and has good application prospects in the fields of agriculture, medicines and the like.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A chitosan-chlorin e6 antimicrobial agent, comprising: the chitosan-chlorin e6 antibacterial agent has a chemical structural formula shown as the following I-A:
wherein X is a group of formula I-B below, and is linked to the nitrogen of chitosan at point d;
the amino substitution degree of the chitosan-chlorin e6 is 6.44% -11.68%, and the chlorin e6 accounts for 13.48% -22.04% of the total weight of the antibacterial agent.
2. The chitosan-chlorin e6 antimicrobial agent of claim 1, wherein: the molecular weight of the chitosan is 50kDa, and the deacetylation degree is more than 90 percent.
3. The method for preparing a chitosan-chlorin e6 antibacterial agent as claimed in claim 1 or 2, wherein: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
dissolving chitosan in an acetic acid solution, and adjusting the pH value of the solution to 5.8-6.0 to obtain a chitosan solution;
dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS in morpholine ethanesulfonic acid MES buffer to obtain an EDC/NHS MES solution;
dissolving chlorin e6 in dimethyl sulfoxide, adding into MES solution of EDC/NHS, and stirring at room temperature in dark place;
and dropwise adding the mixed solution obtained after stirring into the chitosan solution, stirring at room temperature in a dark place, dialyzing the obtained mixed solution with ultrapure water, pre-freezing the obtained solution at-80 ℃ overnight after dialysis is finished, and freeze-drying to obtain a product, and fully grinding the product into powder to obtain the chitosan-chlorin e6 antibacterial agent.
4. The method of preparing a chitosan-chlorin e6 antimicrobial agent of claim 3, wherein: dissolving chitosan in an acetic acid solution, wherein the volume concentration of the acetic acid solution is 1%, and the mass-volume ratio of the chitosan to the acetic acid solution is as follows in mg: the mL is 6: 5.
5. The method of preparing a chitosan-chlorin e6 antimicrobial agent of claim 3, wherein: and adjusting the pH value of the chitosan solution to 5.8-6.0 to obtain a chitosan solution, wherein the pH regulator is a 1mol/L sodium hydroxide solution, and the concentration of the chitosan solution is 1 mg/mL.
6. The method of preparing a chitosan-chlorin e6 antimicrobial agent of claim 3, wherein: dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS in morpholine ethanesulfonic acid MES buffer, wherein the mass ratio of EDC to NHS is 55: 66, MES buffer to EDC volume to mass ratio in mL: the mg is 1: 11.
7. the method of preparing a chitosan-chlorin e6 antimicrobial agent of claim 3, wherein: dissolving chlorin e6 in dimethyl sulfoxide, and adding into MES solution of EDC/NHS, wherein the mass-volume ratio of chlorin e6 to dimethyl sulfoxide is mg: the mL is 14-56: 5, and the volume ratio of the MES solution of the dimethyl sulfoxide to the EDC/NHS is 1: 1.
8. The method of preparing a chitosan-chlorin e6 antimicrobial agent of claim 3, wherein: the chitosan-chlorin e6 is characterized in that the molar ratio of chlorin e6 to chitosan amino is 5-20: 100, the ultrapure water is dialyzed for 3 days, and the cut-off molecular weight of the dialysis bag is 8000 Da-14000 Da.
9. The method of preparing a chitosan-chlorin e6 antimicrobial agent of claim 3, wherein: the room temperature is 25 ℃, the freeze-drying temperature is-80 ℃, the overnight time is 12 hours, and the freeze-drying is carried out in a freeze-dryer for 48 hours.
10. Use of chitosan-chlorin e6 as an antibacterial active ingredient in the antibacterial field as claimed in any one of claims 1 to 9.
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| CN116918820A (en) * | 2023-06-06 | 2023-10-24 | 江苏省农业科学院 | A special cold-resistant regulator for cotton seedling stage and its preparation method and application method |
| CN119461689A (en) * | 2024-07-01 | 2025-02-18 | 湖南金益环保股份有限公司 | A process for treating organic wastewater in a chemical park |
| CN119461689B (en) * | 2024-07-01 | 2025-10-28 | 湖南金益环保股份有限公司 | A process for treating organic wastewater in a chemical park |
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