CN117659226A - Chitosan containing sulfhydryl group and preparation method and application thereof - Google Patents
Chitosan containing sulfhydryl group and preparation method and application thereof Download PDFInfo
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- 125000003396 thiol group Chemical group [H]S* 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- 239000003814 drug Substances 0.000 claims abstract description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 22
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000003431 cross linking reagent Substances 0.000 claims description 12
- WYKHFQKONWMWQM-UHFFFAOYSA-N 2-sulfanylidene-1h-pyridine-3-carboxylic acid Chemical compound OC(=O)C1=CC=CN=C1S WYKHFQKONWMWQM-UHFFFAOYSA-N 0.000 claims description 11
- IZMCGGDHSRRCEI-UHFFFAOYSA-N 6-hydroxy-6-sulfanylcyclohexa-2,4-diene-1-carboxylic acid Chemical compound SC1(C(C(=O)O)C=CC=C1)O IZMCGGDHSRRCEI-UHFFFAOYSA-N 0.000 claims description 11
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- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 8
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 8
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- 150000001875 compounds Chemical class 0.000 claims description 6
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- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 4
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
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Abstract
The invention relates to the technical field of ocean chemical engineering, in particular to chitosan containing sulfhydryl groups and a preparation method and application thereof. The structural formula of the chitosan containing the sulfhydryl group is as follows; the invention has the advantages that the sulfhydrylation chitosan is directly substituted in the chitosan skeleton, so that the antioxidation activity of the improved chitosan is obviously improved. Meanwhile, the chitosan derivative is subjected to microwave reaction, so that the reaction time is greatly shortened. Proved by antioxidant experimental study, the obtained chitosan derivative has good antioxidant activity. Can be widely researched and applied as an antioxidant in the fields of medicine, food preservation, agriculture and the like.
Description
Technical Field
The invention relates to the technical field of ocean chemical engineering, in particular to chitosan containing sulfhydryl groups and a preparation method and application thereof.
Background
Chitosan is the only natural polysaccharide with positive charges in nature, and is the deacetylation product of chitin. Because of the existence of amino on the chitosan C-2, the chitosan has better activity compared with chitin, such as biocompatibility, antioxidation activity, antibacterial activity, no toxicity and the like. However, the chitosan has poor water solubility due to the large molecular weight, so that the research and application of the chitosan are greatly limited.
Disclosure of Invention
The invention aims to provide chitosan containing sulfhydryl groups, and a preparation method and application thereof.
In order to achieve the above purpose, the specific technical scheme of the invention is as follows:
a chitosan containing sulfhydryl group has the structural formula as follows:
wherein n has an average value ranging from 35 to 3000.
The preparation method of the chitosan containing the sulfhydryl group has the reaction formula of
Firstly, utilizing microwave reaction to replace hydroxyl on C-6 position on chitosan to obtain sulfhydrylation chitosan; then the chitosan containing sulfhydryl group shown in the formula I is obtained through a cross-linking agent.
Further, the method comprises the following steps:
(1) Dissolving raw material chitosan in acetic acid solution, dropwise adding thiourea solution into the obtained chitosan solution, regulating a system to be acidic, stirring and mixing, and placing the mixed solution into a microwave reactor for sulfhydryl substitution to obtain sulfhydryl chitosan;
(2) Adding a cross-linking agent into an aqueous solution of a sulfhydryl compound containing carboxyl, mixing, adding the sulfhydryl chitosan obtained in the step (1) for acylation reaction, and obtaining the chitosan containing sulfhydryl groups shown in the formula I.
Dissolving raw material chitosan in acetic acid solution, dropwise adding thiourea solution into the obtained chitosan solution, regulating the pH of the system to 4-5, stirring and mixing, and placing the mixed solution in a microwave reactor for sulfhydryl substitution to obtain sulfhydryl chitosan; wherein the thiourea solution is prepared by dissolving thiourea in hydrochloric acid, wherein 1-2.5g of thiourea is used per 1-2g of chitosan, and 1.5-3mL of hydrochloric acid is used.
Placing the mixed solution in a microwave reactor, carrying out reflux treatment at 640W power, and reacting for 2min; after the solution is restored to room temperature, adjusting the pH value of the solution to be 8, continuously reacting for 90 seconds with 640W power, taking out, cooling to room temperature, and adjusting the pH value of the solution to be neutral; and finally precipitating the product by using an ethanol solution to obtain the sulfhydrylation chitosan.
And (2) adding 2-4g of the compound containing the sulfhydryl group into 1-1.5g of the sulfhydryl chitosan in the step (2).
The cross-linking agents 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; the crosslinker was added in an amount of 1.15g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 0.69g of N-hydroxysuccinimide per millimole of thiolated chitosan; the sulfhydryl group-containing compound is respectively 2-mercaptonicotinic acid, 2-mercaptosalicylic acid, 3-mercaptopropionic acid or L-cysteine.
In the step (2), a sulfhydryl compound containing carboxyl is added into deionized water, the pH of the solution is adjusted to be=5, and a cross-linking agent is added into the solution.
A preparation method of the chitosan containing the sulfhydryl group is disclosed, and the chitosan containing the sulfhydryl group shown in the formula I is applied to the preparation of antioxidants in the fields of biological medicines, food health products and the like.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) The water solubility of the chitosan is extremely poor, and the sulfhydrylation chitosan obtained by chemical modification has good water solubility, so that the application range of the chitosan is enlarged.
(2) The preparation process of the invention utilizes microwave reaction, is simple and quick, and greatly shortens the reaction time. The obtained sulfhydrylation chitosan and the derivative have good antioxidation activity and no obvious biotoxicity, a feasible experimental route is provided for the subsequent research and application of chitosan, and the application value of chitosan is greatly improved.
Drawings
FIG. 1 is an infrared spectrum of chitosan.
FIG. 2 is an infrared spectrum of thiolated chitosan.
FIG. 3 is an infrared spectrum of chitosan containing 2-mercaptonicotinic acid provided in example 1 of the present invention;
FIG. 4 is an infrared spectrum of chitosan containing 2-mercaptosalicylic acid provided in example 2 of the present invention;
FIG. 5 is an infrared spectrum of chitosan containing 3-mercaptopropionic acid provided in example 4 of the present invention;
FIG. 6 is an infrared spectrum of chitosan containing L-cysteine according to example 5 of the present invention;
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
The invention replaces the hydroxyl on C-6 with the sulfhydryl through the chitosan with low molecular weight, thus having good water solubility and biological activity, and particularly having obvious promotion effect on the antioxidant activity. On the basis, a cross-linking agent is utilized to introduce a small molecular compound containing a sulfhydryl group into chitosan, so that the sulfhydryl content of the chitosan is further improved, a compound with more excellent antioxidant activity is obtained, and ideas and methods are provided for further research and utilization of the chitosan.
Example 1
The structural formula of the chitosan containing 2-mercaptonicotinic acid is shown as the formula (1), wherein the average value of n is 35-3000.
Preparation of chitosan:
2g of chitosan (molecular weight of chitosan in this experiment is 8000, see FIG. 1) was weighed and dissolved in 100mL of 1% by volume acetic acid solution. 2.2g of thiourea was weighed out and dissolved in 6mL of hydrochloric acid solution (HCl: H) 2 O=1:1), the thiourea solution was added dropwise to the chitosan solution, ph=5 was adjusted, and stirring was continued for 10min. The mixed solution was placed in a microwave reactor and reacted at 640W power for 2min under reflux. After the solution was returned to room temperature, ph=8 was adjusted, the reaction was continued at 640W power for 90s, and the solution was cooled to room temperature and pH was adjusted to neutral. The product was precipitated with an ethanol solution, and the precipitate was repeatedly washed three times and vacuum freeze-dried to obtain thiolated chitosan (see fig. 2).
2.79g of 2-mercaptonicotinic acid was added to 50mL of deionized water, the pH of the solution was adjusted=5, and 6.9g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4.14-g N-hydroxysuccinimide were added to the solution as a crosslinking agent for the reaction. 1.06g of the above-mentioned obtained thiolated chitosan was further added to react for 24 hours. The product was transferred to a dialysis bag with a molecular weight cut-off of 100, dialyzed in deionized water for 48 hours, and lyophilized in vacuo to give chitosan containing 2-mercaptonicotinic acid (see FIG. 3).
FIG. 3 is an infrared spectrum of chitosan containing 2-mercaptonicotinic acid provided in example 1 of the present invention, as seen from FIG. 3, at 757cm -1 、725cm -1 The vibration absorption peak of pyridine ring in 2-mercaptonicotinic acid appears at 1646cm -1 The vibration absorption peak of the amide bond appears, thereby proving that the chitosan containing 2-mercaptonicotinic acid is successfully synthesized.
Example 2
The structural formula of the chitosan containing 2-mercaptosalicylic acid is shown as the formula (2), wherein the average value of n is 35-3000.
The difference from example 1 is that:
2g of chitosan (molecular weight of chitosan in this experiment is 8000, see FIG. 1) was weighed and dissolved in 100mL of 1% by volume acetic acid solution. 2.2g of thiourea was weighed out and dissolved in 6mL of hydrochloric acid solution (HCl: H) 2 O=1:1), the thiourea solution was added dropwise to the chitosan solution, ph=5 was adjusted, and stirring was continued for 10min. The mixed solution was placed in a microwave reactor and reacted at 640W power for 2min under reflux. After the solution was returned to room temperature, the pH of the solution was adjusted=8, the reaction was continued at 640W power for 90s, and the solution was taken out and cooled to room temperature, and the pH of the solution was adjusted to neutrality. And (3) precipitating the product by using an ethanol solution, repeatedly washing the precipitate for three times, and carrying out vacuum freeze-drying to obtain the sulfhydrylation chitosan.
2.78g of 2-mercaptosalicylic acid was added to 50mL of deionized water, the ph=5 of the solution was adjusted, and 6.9g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4.14-g N-hydroxysuccinimide were added to the solution as a crosslinking agent for the reaction. 1.06g of thiolated chitosan was added for reaction for 24h. The product was transferred to a dialysis bag with a molecular weight cut-off of 100, dialyzed in deionized water for 48 hours, and lyophilized in vacuo to give chitosan containing 2-mercaptosalicylic acid (see fig. 4).
FIG. 4 is an infrared spectrum of chitosan containing 2-mercaptosalicylic acid according to example 2 of the present invention, as can be seen from FIG. 4, at 744cm -1 The vibration absorption peak of benzene ring in 2-mercaptosalicylic acid appears at 1658cm -1 The vibration absorption peak of the amide bond appears, thereby proving the successful synthesis of chitosan containing 2-mercaptosalicylic acid.
Example 3
The structural formula of the chitosan containing 3-mercaptopropionic acid is shown as formula (3), wherein the average value of n is 35-3000.
The difference from example 1 is that:
2g of chitosan (molecular weight of chitosan in this experiment is 8000, see FIG. 1) was weighed and dissolved in 100mL of 1% by volume acetic acid solution. 2.2g of thiourea was weighed out and dissolved in 6mL of hydrochloric acid solution (HCl: H) 2 O=1:1), the thiourea solution was added dropwise to the chitosan solution, the pH of the solution was adjusted to=5, and stirring was continued for 10min. The mixed solution was placed in a microwave reactor and reacted at 640W power for 2min under reflux. After the solution was returned to room temperature, the pH of the solution was adjusted=8, the reaction was continued at 640W power for 90s, and the solution was taken out and cooled to room temperature, and the pH of the solution was adjusted to neutrality. And (3) precipitating the product by using an ethanol solution, repeatedly washing the precipitate for three times, and carrying out vacuum freeze-drying to obtain the sulfhydrylation chitosan.
1.56mL of 3-mercaptopropionic acid was added to 50mL of deionized water, the solution ph=5 was adjusted, and 6.9g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4.14g of n-hydroxysuccinimide were added to the solution as a crosslinking agent for the reaction. 1.06g of thiolated chitosan was added for reaction for 24h. The product was transferred to a dialysis bag with a molecular weight cut-off of 100, dialyzed in deionized water for 48 hours, and lyophilized in vacuo to give chitosan containing 3-mercaptopropionic acid (see fig. 5).
FIG. 5 is an infrared spectrum of chitosan containing 3-mercaptopropionic acid according to example 3 of the present invention, as can be seen from FIG. 5, at 1645cm -1 The vibration absorption peak of the amide bond appears, thereby proving that the chitosan containing 3-mercaptopropionic acid is successfully synthesized.
Example 4
The structural formula of the chitosan containing L-cysteine is shown as formula (4), wherein the average value of n is 35-3000.
The difference from example 1 is that:
2g of chitosan (molecular weight of chitosan in this experiment is 8000, see FIG. 1) was weighed and dissolved in 100mL of 1% by volume acetic acid solution. 2.2g of thiourea was weighed out and dissolved in 6mL of hydrochloric acid solution (HCl: H) 2 O=1:1), the thiourea solution was added dropwise to the chitosan solution, the pH of the solution was adjusted to=5, and stirring was continued for 10min. The mixed solution was placed in a microwave reactor and reacted at 640W power for 2min under reflux. After the solution was returned to room temperature, the pH of the solution was adjusted=8, the reaction was continued at 640W power for 90s, and the solution was taken out and cooled to room temperature, and the pH of the solution was adjusted to neutrality. And (3) precipitating the product by using an ethanol solution, repeatedly washing the precipitate for three times, and carrying out vacuum freeze-drying to obtain the sulfhydrylation chitosan.
2.18g L-cysteine was added to 50mL of deionized water, the pH of the solution was adjusted to=5, and 6.9g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4.14g N-hydroxysuccinimide were added to the solution as a crosslinking agent for the reaction. 1.06g of thiolated chitosan was added for reaction for 24h. The product was transferred to a dialysis bag with a molecular weight cut-off of 100, dialyzed in deionized water for 48 hours, and lyophilized in vacuo to give chitosan containing L-cysteine (see fig. 6).
FIG. 6 is an infrared spectrum of L-cysteine-containing chitosan according to example 4 of the present invention, which shows that the chitosan is prepared at 1644cm -1 The vibration absorption peak of the amide bond appears, thereby proving that the chitosan containing L-cysteine is successfully synthesized.
Application example
Antioxidant Activity assay
(1) DPPH radical scavenging ability assay: the DPPH radical scavenging ability of chitosan, thiolated chitosan and thiol group-containing chitosan prepared in each of the above examples was measured and compared (table 1): the chitosan for experiments, the thiolated chitosan, the chitosan containing 2-mercaptonicotinic acid, the chitosan containing 2-mercaptosalicylic acid, the chitosan containing 3-mercaptopropionic acid and the chitosan containing L-cysteine in the examples are subjected to vacuum freeze drying treatment to prepare two groups of solutions to be tested with concentration gradients of 0.1, 0.2, 0.4, 0.8 and 1.6mg/mL respectively. DPPH-ethanol reagent (35.5 mg 1, 1-diphenyl-2-trinitrobenzene, ethanol was added to volume 1000 mL) was prepared. To the sample group, 2mL of DPPH-ethanol reagent was added, the control group was added with 2mL of ethanol solution, and the blank group replaced the sample solution with deionized water. All samples were reacted at room temperature in the absence of light for 20min and the absorbance of the solution was measured at 517 nm. All samples were tested three times and the results averaged. The DPPH radical scavenging ability of the test sample was calculated as follows:
clearance (%) = [1- (a) Sample of -A Control )/A Blank space ]×100
Wherein A is Sample of : absorbance of the sample group; a is that Control : absorbance of control group; a is that Blank space : blank absorbance.
The measurement results of the DPPH radical scavenging ability are shown in table (1):
TABLE 1 free radical scavenging Capacity of Chitosan, thiolated Chitosan, thiol group-containing Chitosan (%)
(2) Superoxide anion radical clearance assay: the superoxide anion radical scavenging capacity of chitosan, thiolated chitosan and thiol group-containing chitosan prepared in each of the above examples was measured and compared (table 2): the chitosan for experiments, the thiolated chitosan, the chitosan containing 2-mercaptonicotinic acid, the chitosan containing 2-mercaptosalicylic acid, the chitosan containing 3-mercaptopropionic acid and the chitosan containing L-cysteine in the examples are subjected to vacuum freeze drying treatment to prepare two groups of solutions to be tested with concentration gradients of 0.1, 0.2, 0.4, 0.8 and 1.6mg/mL respectively, and the concentration gradients of the solutions to be tested are 1.5mL. To the experimental group, 0.5mL NADH (0.03657 g of reduced coenzyme I, buffer to 100 mL), 0.5mL NBT (0.02453 g of nitrotetrazolium blue, buffer to 100 mL), 0.5mL PMS (0.001838 g of phenazine methosulfate, buffer to 100 mL) were added sequentially. The control group replaced NADH solution with Tris-HCl buffer (0.9697 g of Tris, 0.4mL of concentrated hydrochloric acid, added water to a volume of 500 mL); the blank group replaced the sample solution with deionized water. The three groups of solutions were mixed uniformly and then allowed to stand for 10min, and the absorbance of the solutions was measured at 560 nm. All samples were tested three times and the average of the test results was taken. The superoxide anion radical scavenging capacity of the test sample was calculated as follows:
clearance (%) = [1- (a) Sample of -A Control )/A Blank space ]×100
Wherein A is Sample of : absorbance of the sample group; a is that Control : absorbance of control group; a is that Blank space : blank absorbance.
The measurement results of the superoxide anion radical scavenging ability are shown in table (2):
TABLE 2 super-oxyanion radical scavenging Capacity of Chitosan, thiolated Chitosan, thiol group-containing Chitosan (%)
The results show that the DPPH free radical scavenging capacity of the synthesized chitosan containing the sulfhydryl group, the sulfhydryl chitosan and the chitosan is shown in the table 1, and the superoxide anion free radical scavenging capacity is shown in the table 2. The modified chitosan has greatly improved antioxidant activity and good water solubility, and the obtained chitosan derivative has wide application prospect in the fields of medical treatment, food preservation, agriculture and the like.
Claims (8)
1. A chitosan containing sulfhydryl groups, characterized in that: the structural formula of the chitosan containing the sulfhydryl group is as follows:
wherein n has an average value ranging from 35 to 3000.
2. A method for preparing the chitosan containing sulfhydryl groups as defined in claim 1, which is characterized in that: the reaction is
Firstly, utilizing microwave reaction to replace hydroxyl on C-6 position on chitosan to obtain sulfhydrylation chitosan; then the chitosan containing sulfhydryl group shown in the formula I is obtained through a cross-linking agent.
3. The method for preparing chitosan containing sulfhydryl group according to claim 2, characterized in that:
(1) Dissolving raw material chitosan in acetic acid solution, dropwise adding thiourea solution into the obtained chitosan solution, regulating a system to be acidic, stirring and mixing, and placing the mixed solution into a microwave reactor for sulfhydryl substitution to obtain sulfhydryl chitosan;
(2) Adding a cross-linking agent into an aqueous solution of a sulfhydryl compound containing carboxyl, mixing, adding the sulfhydryl chitosan obtained in the step (1) for acylation reaction, and obtaining the chitosan containing sulfhydryl groups shown in the formula I.
4. A method for preparing chitosan containing sulfhydryl group as defined in claim 3, characterized in that: dissolving raw material chitosan in acetic acid solution, dropwise adding thiourea solution into the obtained chitosan solution, regulating the pH of the system to 4-5, stirring and mixing, and placing the mixed solution in a microwave reactor for sulfhydryl substitution to obtain sulfhydryl chitosan; wherein the thiourea solution is prepared by dissolving thiourea in hydrochloric acid, wherein 1-2.5g of thiourea is used per 1-2g of chitosan, and 1.5-3mL of hydrochloric acid is used.
5. The method for preparing chitosan containing sulfhydryl group according to claim 4, which is characterized in that: placing the mixed solution in a microwave reactor, carrying out reflux treatment at 640W power, and reacting for 2min; after the solution is restored to room temperature, adjusting the pH value of the solution to be 8, continuously reacting for 90 seconds with 640W power, taking out, cooling to room temperature, and adjusting the pH value of the solution to be neutral; and finally precipitating the product by using an ethanol solution to obtain the sulfhydrylation chitosan.
6. A method for preparing chitosan containing sulfhydryl group as defined in claim 3, characterized in that:
and (2) adding 2-4g of the compound containing the sulfhydryl group into 1-1.5g of the sulfhydryl chitosan in the step (2).
7. The method for preparing chitosan containing sulfhydryl group according to claim 2, characterized in that:
the cross-linking agents 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; the crosslinker was added in an amount of 1.15g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 0.69g of N-hydroxysuccinimide per millimole of thiolated chitosan; the sulfhydryl group-containing compound is respectively 2-mercaptonicotinic acid, 2-mercaptosalicylic acid, 3-mercaptopropionic acid or L-cysteine.
8. A method for preparing the chitosan containing sulfhydryl groups as defined in claim 1, which is characterized in that: the chitosan containing sulfhydryl group shown in the formula I is applied to the preparation of antioxidants in the fields of biological medicines, food health products and the like.
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