CN113667039B - Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof - Google Patents

Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof Download PDF

Info

Publication number
CN113667039B
CN113667039B CN202111039635.9A CN202111039635A CN113667039B CN 113667039 B CN113667039 B CN 113667039B CN 202111039635 A CN202111039635 A CN 202111039635A CN 113667039 B CN113667039 B CN 113667039B
Authority
CN
China
Prior art keywords
hinokitiol
natural
modified chitosan
chitosan
nano
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111039635.9A
Other languages
Chinese (zh)
Other versions
CN113667039A (en
Inventor
王莉莉
刘子涵
罗雅培
吴明华
赵小敏
余德游
王懿佳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Sci Tech University ZSTU
Original Assignee
Zhejiang Sci Tech University ZSTU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Sci Tech University ZSTU filed Critical Zhejiang Sci Tech University ZSTU
Priority to CN202111039635.9A priority Critical patent/CN113667039B/en
Publication of CN113667039A publication Critical patent/CN113667039A/en
Application granted granted Critical
Publication of CN113667039B publication Critical patent/CN113667039B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
    • D06M11/83—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with metals; with metal-generating compounds, e.g. metal carbonyls; Reduction of metal compounds on textiles
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
    • D06M15/03—Polysaccharides or derivatives thereof
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/02—Natural fibres, other than mineral fibres
    • D06M2101/04—Vegetal fibres
    • D06M2101/06—Vegetal fibres cellulosic
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/02—Natural fibres, other than mineral fibres
    • D06M2101/10—Animal fibres
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/02—Natural fibres, other than mineral fibres
    • D06M2101/10—Animal fibres
    • D06M2101/12—Keratin fibres or silk
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides nano-silver complexed natural hinokitiol modified chitosan and a preparation method and application thereof. The preparation method comprises the following steps: (1) Dissolving chitosan in a solvent I to prepare a chitosan solution; (2) Respectively adding polyformaldehyde and hinokitiol into the chitosan solution for reaction, and after the reaction is finished, performing post-treatment to obtain a natural hinokitiol modified chitosan stock solution; (3) Adding a solvent II into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution; (4) Adding silver nitrate and a reducing agent into the natural hinokitiol modified chitosan solution for reaction, and performing post-treatment to obtain the nano-silver complex natural hinokitiol modified chitosan. The nano-silver complexed natural hinokitiol modified chitosan has excellent antibacterial performance and antibacterial durability; and the preparation method is simple and convenient, has low requirement on treatment equipment and good reproducibility, and is beneficial to industrial production.

Description

Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof
Technical Field
The invention belongs to the technical field of fabric finishing, and particularly relates to nano-silver complexed natural hinokitiol modified chitosan and a preparation method and application thereof.
Background
The natural fiber fabrics (cotton, hemp, silk and wool) have the advantages of good hygroscopicity, skin-friendly softness, easy biodegradation and the like, and are always well loved by consumers. However, natural fiber fabrics are easily damaged by microorganisms such as bacteria and mold, and have problems of discoloration, mold, moth damage, embrittlement, degradation and the like, thereby seriously affecting the wearability. Therefore, the antibacterial finishing is an important way for improving the wearing comfort of the natural fiber fabric.
At present, three major classes of antibacterial agents for textiles on the market are organic antibacterial agents, inorganic antibacterial agents and natural antibacterial agents. With the enhancement of environmental awareness, natural antibacterial agents have attracted great attention and have been effectively developed by relevant scientific research departments at present. The natural antibacterial agent is completely extracted from natural animals and plants or directly used, does not cause harm and pollution to people and livestock or environment in the production and use processes, is a safe, ecological and environment-friendly antibacterial agent, such as aloe, honeysuckle, mugwort, juniper, liquorice, chitosan, tea polyphenol, hinokitiol and the like, and has the problems of short service life, poor heat resistance, narrow application range, limited production conditions and the like. In general, the ideal antibacterial agent should satisfy the following three requirements while satisfying the requirements of killing harmful microorganisms and preventing the spread of diseases: the toxicity to human body and environment must not be too strong; the performance of the finished textile is not adversely affected; the finished textile should be sufficiently durable. Therefore, the development of highly effective, low toxicity, durable, environmentally friendly antimicrobial agents is a major goal of current research and development.
Among a plurality of natural antibacterial agents, chitosan has various excellent characteristics such as bacteriostasis, low cost, film forming property, biocompatibility, biodegradability and the like, and has a structure containing abundant amino and hydroxyl functional groups, so that the chitosan is widely concerned. However, chitosan generally has antibacterial activity only in an acidic medium, and can inhibit only individual bacteria, and the antibacterial performance thereof after film formation is drastically reduced. Therefore, the problem of improving the antibacterial activity and broad spectrum of chitosan is urgently needed to be overcome. Hinokitiol is a natural compound, has good broad-spectrum antibacterial property, strong antibacterial and antifungal effects, moisture retention and pest repellent effect, has no obvious cytotoxicity on human body endothelial cells and epithelial cells, has low price, and has been widely used in the fields of cosmetics, medicines, agriculture, textiles and the like. Silver-based antibacterial agents, which are typical inorganic antibacterial agents, have the characteristics of strong bactericidal activity and long-lasting bactericidal activity.
However, the research on the antibacterial finishing of natural fiber fabrics, which is prepared by combining chitosan, hinokitiol and silver, has not been reported at home and abroad at present.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides nano-silver complexed natural hinokitiol modified chitosan and a preparation method thereof. The nano-silver complexed natural hinokitiol modified chitosan has excellent antibacterial property and antibacterial durability, and the preparation method is simple and convenient, has low requirement on treatment equipment and good reproducibility, and is beneficial to industrial production.
The invention also provides application of the nano-silver complexed natural hinokitiol modified chitosan as an antibacterial agent, and the nano-silver complexed natural hinokitiol modified chitosan is applied to antibacterial finishing of natural fiber fabrics, so that the natural fiber fabrics subjected to the antibacterial finishing have better broad-spectrum antibacterial property and durability, the original physical wearability of the fabrics is not damaged, and the nano-silver complexed natural hinokitiol modified chitosan has a wide application prospect in the antibacterial finishing of the natural fiber fabrics.
The structural formula of the nano-silver complexed natural hinokitiol modified chitosan is as follows:
Figure BDA0003248774910000021
wherein R is 2 Is H,
Figure BDA0003248774910000022
R 3 Is H,
Figure BDA0003248774910000031
All R in the nano-silver complexed natural hinokitiol modified chitosan 2 、R 3 Not H at the same time.
A preparation method of the nano-silver complexed natural hinokitiol modified chitosan comprises the following steps:
(1) Dissolving chitosan in a solvent I to prepare a chitosan solution;
(2) Respectively adding polyformaldehyde and hinokitiol into the chitosan solution for reaction, and after the reaction is finished, performing post-treatment to obtain a natural hinokitiol modified chitosan stock solution;
(3) Adding a solvent II into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution;
(4) Adding silver nitrate and a reducing agent into the natural hinokitiol modified chitosan solution for reaction, and performing post-treatment to obtain the nano-silver complex natural hinokitiol modified chitosan.
In the above preparation method, in the step (1):
preferably, the solvent I is one or more of acetic acid, formic acid, lactic acid, malic acid and ascorbic acid;
in the step (3), the solvent II is water.
More preferably, the solvent is an aqueous acetic acid solution. More preferably, the volume concentration of the aqueous acetic acid solution is 0.2 to 2v/v%.
Preferably, the chitosan has the deacetylation degree of more than or equal to 95 percent, the viscosity of 100-200 mPas and the molecular weight of 12-26 ten thousand.
Preferably, the chitosan is heated and dissolved in the solvent I, and the heating temperature is 70-90 ℃.
Preferably, the concentration of the chitosan solution is 0.01 to 0.03mol/L calculated by the molar weight of the chitosan unit. Further preferably 0.02mol/L.
In the above preparation method, in the step (2):
preferably, the molar ratio of chitosan to hinokitiol is 1 (1-2.5) in terms of the molar amount of chitosan unit. More preferably 1 (1.5 to 2.5).
Preferably, the molar ratio of hinokitiol to paraformaldehyde is 1 (0.5 to 2) in terms of the molar amount of formaldehyde monomer. More preferably 1 (0.8 to 1.3). Still more preferably 1.
Preferably, the reaction temperature is 80 to 100 ℃. More preferably 85 to 95 ℃.
Preferably, the progress of the reaction is monitored to the end point by thin layer chromatography during the reaction.
Further preferably, when the progress of the reaction is monitored by thin layer chromatography, the completion of the reaction is determined by observing whether the hinokitiol raw material spot disappears or remains stable.
Preferably, the paraformaldehyde is added into the chitosan solution to be dissolved, and then the hinokitiol is added to carry out reaction.
More preferably, the paraformaldehyde is added into the chitosan solution and stirred until the chitosan solution is dissolved, wherein the stirring time is 0.5 to 2 hours, and the stirring speed is 150 to 300r/min.
Preferably, after the reaction is completed, the following post-treatment is performed:
and cooling the reaction liquid, adding diethyl ether for extraction to remove unreacted hinokitiol, and removing residual diethyl ether to obtain the natural hinokitiol modified chitosan stock solution.
More preferably, in the extraction with diethyl ether, the volume ratio of diethyl ether to the reaction mixture is (3 to 5): 1.
Preferably, the grafting ratio of the natural hinokitiol modified chitosan is 35-85%.
More preferably, the grafting ratio of the natural hinokitiol-modified chitosan is 39 to 82%.
In the above preparation method, in the step (3):
preferably, the volume concentration of the natural hinokitiol modified chitosan solution is 10-50 v/v%. The volume concentration of the natural hinokitiol modified chitosan solution is the volume percentage of the natural hinokitiol modified chitosan stock solution in the natural hinokitiol modified chitosan solution.
More preferably, the volume concentration of the natural hinokitiol-modified chitosan solution is 30 to 50v/v%. More preferably 35 to 45v/v%.
In the above preparation method, in the step (4):
preferably, the concentration of silver nitrate in the natural hinokitiol modified chitosan solution is 6-15 mmol/L. More preferably, the concentration of silver nitrate in the natural hinokitiol modified chitosan solution is 8-12 mmol/L.
Preferably, after adding the silver nitrate, stirring the mixture for 15 to 30min at the rotating speed of 100 to 200 r/min.
Preferably, the reducing agent is one or more of aloe extract, vitamin C and glucose. More preferably, the reducing agent is aloe extract; the aloe extract is added dropwise.
Preferably, the preparation method of the aloe extract comprises the following steps:
washing aloe leaves, smashing the aloe leaves into paste, preparing aloe slurry, heating the mixture of the aloe slurry and distilled water in a volume ratio of 1 (4-5) at 90-100 ℃ for 10-15 min, cooling, and filtering through a pore to remove solid particles to obtain the aloe extracting solution.
More preferably, the volume concentration of the aloe extract is 23 to 25v/v%.
Preferably, the reaction temperature is 80-100 ℃; the reaction time is 4-7 h. More preferably, the reaction temperature is 85 to 95 ℃; the reaction time is 5.5 to 6.5 hours.
Preferably, after the reaction is completed, the following post-treatment is performed:
and (3) cooling the reaction liquid, centrifuging at the speed of 6000r/min for 5min, and filtering to obtain the nano-silver complexed natural hinokitiol modified chitosan.
As specific optimization, the preparation method of the nano-silver complexed natural hinokitiol modified chitosan comprises the following steps:
1. dissolving chitosan in acetic acid solution, and stirring at 70-90 ℃ until the chitosan is completely dissolved to obtain chitosan solution; adding paraformaldehyde, stirring to dissolve, adding hinokitiol, reacting at 80-90 ℃, and monitoring the reaction process to the end point by using a thin layer chromatography; and cooling, adding diethyl ether for extraction to remove unreacted hinokitiol, separating to obtain a pre-product, and removing residual diethyl ether by rotary evaporation at normal temperature to obtain the natural hinokitiol modified chitosan.
2. Heating the natural hinokitiol modified chitosan solution to 80-90 ℃, adding silver nitrate, stirring, dropwise adding an aloe extracting solution for reaction, and performing post-treatment to obtain the nano-silver complex hinokitiol grafted chitosan.
An antibacterial agent comprises the nano-silver complexed natural hinokitiol modified chitosan.
Preferably, the antibacterial agent is applied to an antibacterial finishing process of natural fiber fabrics.
Specifically, the antibacterial finishing process of the natural fiber fabric is as follows:
pretreating the natural fiber fabric by citric acid, padding by a nano silver complex natural hinokitiol modified chitosan solution (water is used as a solvent), drying and baking to obtain the antibacterial natural fiber fabric.
The obtained nano-silver complexed natural hinokitiol modified chitosan antibacterial finishing natural fiber fabric has excellent antibacterial property and antibacterial durability.
More preferably, the citric acid is contained in an amount of 0.3 to 0.5wt%.
As a further preference, the pretreatment process is:
padding the natural fiber fabric with citric acid solution at room temperature, wherein the bath ratio is 1-40, the rolling residual rate is 82-86%, the drying temperature is 60-80 ℃, the drying time is 4-6 min, the baking temperature is 120-140 ℃, and the baking time is 2-4 min.
More preferably, the mass concentration of the nano silver complexed natural hinokitiol modified chitosan solution is 0.025-0.1 wt%.
As a further preference, the padding process is:
the dipping temperature is 20-40 ℃, the dipping time is 30-300 s, the bath ratio is 1 (20-30), and the rolling residual rate is 83-86%.
More preferably, the drying temperature is 60-80 ℃, the drying time is 4-6 min, the baking temperature is 120-140 ℃, and the baking time is 2-4 min.
According to the invention, hinokitiol is grafted onto chitosan through a Mannich reaction ingeniously, and then hydroxyl and carbonyl complex silver nano particles in a hinokitiol structure are utilized to prepare the nano-silver complex hinokitiol modified chitosan antibacterial agent. The antibacterial agent can exert the comprehensive advantages of three antibacterial agents, and endows natural fiber fabrics with excellent broad-spectrum antibacterial property and antibacterial durability through durable antibacterial finishing.
Compared with the prior art, the invention has the beneficial effects that:
(1) The phenolic hydroxyl and carbonyl on the hinokitiol are complexed with the nano silver, so that the stability and the dispersibility of nano silver particles are improved; compared with chitosan, the natural hinokitiol modified chitosan serving as an antibacterial agent has the advantages that the minimum inhibitory concentration to staphylococcus aureus and escherichia coli is reduced by about 95%, and the antibacterial agent has good broad-spectrum antibacterial property.
(2) The preparation method is simple and convenient, the reaction condition is mild, the controllability is strong, and the reproducibility is good.
(3) The antibacterial natural fiber fabric subjected to durable antibacterial finishing has the bacteriostatic rate of over 95 percent on staphylococcus aureus and escherichia coli; after 25 times of washing, the antibacterial rate of the antibacterial agent to escherichia coli and staphylococcus aureus can still reach over 90%, and the antibacterial agent has good antibacterial performance and antibacterial durability.
In conclusion, the nano-silver complexed natural hinokitiol modified chitosan has excellent antibacterial performance and antibacterial durability, and is applied to the antibacterial finishing of natural fiber fabrics, so that the natural fiber fabrics subjected to the antibacterial finishing have better broad-spectrum antibacterial performance and durability, the original physical wearability of the fabrics is not damaged, and the nano-silver complexed natural hinokitiol modified chitosan has a wide application prospect in the antibacterial finishing of the natural fiber fabrics; and the preparation method is simple and convenient, has low requirement on treatment equipment and good reproducibility, and is beneficial to industrial production.
Drawings
FIG. 1 shows the growth of Staphylococcus aureus and Escherichia coli on different treated cotton fabrics;
FIG. 2 is the IR spectrum of natural hinokitiol-modified chitosan prepared according to the different mole ratios of hinokitiol to chitosan in example 5.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. After reading the teaching of the present invention, the skilled in the art can make various changes or modifications to the invention, and these equivalents also fall within the scope of the claims appended to the present application.
Example 1
(1) Dissolving chitosan with the unit molar weight of 1mmol in 50mL of 2v/v% acetic acid aqueous solution, and stirring at 80 ℃ until the chitosan is completely dissolved to obtain 0.02mol/L chitosan solution; adding paraformaldehyde with the molar weight of formaldehyde monomer of 2mmol, stirring until the paraformaldehyde is dissolved, then adding 2mmol of hinokitiol, controlling the molar ratio of the paraformaldehyde to the hinokitiol to be 1, reacting at 90 ℃, and monitoring the reaction process to the end point by using a thin-layer chromatography to obtain a reaction solution containing the natural hinokitiol modified chitosan; cooling the reaction liquid, adding 200mL of diethyl ether for extraction to remove unreacted hinokitiol, separating liquid to obtain a pre-product, and removing residual diethyl ether by rotary evaporation at normal temperature to obtain a natural hinokitiol modified chitosan stock solution; adding water (the volume of the added water is 950mL, 450mL, 200mL, 75mL, 33mL, 12.5mL and 0mL respectively) into the natural hinokitiol modified chitosan stock solution to prepare natural hinokitiol modified chitosan solutions with volume concentrations of 5v/v%, 10v/v%, 20v/v%, 40v/v%, 60v/v%, 80v/v% and 100v/v% respectively for later use.
(2) Washing folium Aloe, and mashing into paste to obtain slurry; heating the mixture of the slurry and distilled water at a volume ratio of 1:4 at 90 deg.C for 10min, cooling, and filtering through fine pores to remove solid particles to obtain aloe extractive solution for use.
(3) Heating 50mL of natural hinokitiol modified chitosan solution (the volume concentration is respectively 5v/v%, 10v/v%, 20v/v%, 40v/v%, 60v/v%, 80v/v% and 100 v/v%) to 80 ℃, adding 10mmol/L silver nitrate, stirring for 20min under the condition of the rotation speed of 150r/min, dropwise adding 15mL of the aloe extracting solution, reacting for 6h, cooling the reaction solution, centrifuging for 5min at the speed of 6000r/min, and filtering to obtain seven different nano-silver complex natural hinokitiol modified chitosans.
The antibacterial performance (minimum inhibitory concentration) of the seven different nano-silver complexed natural hinokitiol modified chitosan is researched, and the result is shown in table 1. The minimum inhibitory concentration is the concentration of the antibacterial agent which can inhibit the propagation of bacteria at the minimum, and is an index for effectively judging the antibacterial activity of the antibacterial agent. Since a small amount of the antibacterial agent is necessary in the bacteriostasis process to inhibit the growth and reproduction of bacteria, the lower the minimum bacteriostasis concentration is, the more effective the antibacterial agent is.
TABLE 1 comparison of the minimum inhibitory concentrations of different nano-silver complexed natural hinokitiol-modified chitosans
Figure BDA0003248774910000081
As can be seen from Table 1, the minimum inhibitory concentration of the nano-silver complexed natural hinokitiol-modified chitosan on Staphylococcus aureus and Escherichia coli gradually decreases with the increase of the amount of the natural hinokitiol-modified chitosan (volume concentration of the nano-silver complexed natural hinokitiol-modified chitosan solution). When the dosage of the natural hinokitiol modified chitosan antibacterial agent is 40v/v%, the minimum inhibitory concentration of the nano-silver complexed natural hinokitiol modified chitosan on two bacteria reaches the minimum, and the antibacterial activity is the best. However, when the natural hinokitiol-modified chitosan is used in an excessive amount, the minimum inhibitory concentration increases, which may be caused by excessively strong chelation, thereby preventing reduction reaction and further affecting the antibacterial activity. The result shows that the dosage of the natural hinokitiol modified chitosan is moderately increased, the formation of nano silver can be promoted to be complexed with the natural hinokitiol modified chitosan, the antibacterial activity is effectively improved, and the excellent antibacterial performance on staphylococcus aureus and escherichia coli is shown.
Example 2
(1) Dissolving chitosan with the unit molar weight of 1mmol in 50mL of 2v/v% acetic acid aqueous solution, and stirring at 80 ℃ until the chitosan is completely dissolved to obtain 0.02mol/L chitosan solution; adding paraformaldehyde with the molar weight of formaldehyde monomer of 2mmol, stirring to dissolve, adding 2mmol of hinokitiol, reacting at 90 deg.C, and monitoring the reaction process by thin layer chromatography to obtain reaction solution containing natural hinokitiol modified chitosan; after the reaction liquid is cooled, adding 200mL of diethyl ether for extraction to remove unreacted hinokitiol, separating liquid to obtain a pre-product, and removing residual diethyl ether by rotary evaporation at normal temperature to obtain a natural hinokitiol modified chitosan stock solution; adding 75mL of water into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution with the volume concentration of 40v/v% for later use.
(2) Washing folium Aloe, mashing into paste, and making into slurry; heating the mixture of the slurry and distilled water at a volume ratio of 1:4 at 90 deg.C for 10min, cooling, and filtering through fine pores to remove solid particles to obtain aloe extractive solution for use.
(3) Heating 50mL of natural hinokitiol modified chitosan solution with volume concentration of 40v/v% to 80 ℃, adding silver nitrate (with the concentration of 0mmol/L, 1mmol/L, 2mmol/L, 4mmol/L, 6mmol/L, 8mmol/L, 10mmol/L and 20mmol/L respectively after the addition), stirring for 20min under the condition of rotation speed of 150r/min, dropwise adding 15mL of the aloe extract, reacting for 6h, cooling the reaction solution, centrifuging at 6000r/min for 5min, and filtering to obtain eight different nano-silver complex natural hinokitiol modified chitosan.
The antibacterial performance (minimum inhibitory concentration) of the eight different nano-silver complexed natural hinokitiol modified chitosan is researched, and the result is shown in table 2.
TABLE 2 comparison of the minimum inhibitory concentrations of eight different nano-silver complex natural hinokitiol-modified chitosans
Figure BDA0003248774910000091
As can be seen from Table 2, with the increase of the amount of silver nitrate, the minimum inhibitory concentration of the nano-silver complexed natural hinokitiol modified chitosan on Staphylococcus aureus and Escherichia coli is gradually reduced, and when the amount of silver nitrate is 10mmol/L, the minimum inhibitory concentration reaches the lowest value, and the antibacterial activity is optimal at the moment. However, when the amount of silver nitrate is excessively large, the minimum inhibitory concentration value increases, which may be due to agglomeration caused by the excessive amount of silver nitrate, which in turn affects the antibacterial activity thereof. The result shows that the dosage of silver nitrate is increased moderately, the formation of nano silver can be promoted to be complexed with the natural hinokitiol modified chitosan, the antibacterial activity is further effectively improved, and the excellent antibacterial performance on staphylococcus aureus and escherichia coli is shown.
Example 3
(1) Dissolving Chitosan (CS) with the monomer molar weight of 1mmol in 50mL of 2v/v% acetic acid aqueous solution, and stirring at 80 ℃ until the Chitosan (CS) is completely dissolved to obtain 0.02mol/L chitosan solution; adding paraformaldehyde with the molar weight of formaldehyde monomer of 2mmol, stirring to dissolve, adding 2mmol of Hinokitiol (HT), reacting at 90 deg.C, and monitoring the reaction process by thin layer chromatography to obtain reaction solution containing natural hinokitiol modified chitosan; after the reaction liquid is cooled, adding 200mL of diethyl ether for extraction to remove unreacted hinokitiol, separating liquid to obtain a pre-product, and removing residual diethyl ether by rotary evaporation at normal temperature to obtain a natural hinokitiol modified chitosan stock solution; adding 75mL of water into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution with the volume concentration of 40 v/v%.
(2) Washing folium Aloe, and mashing into paste to obtain slurry; heating the mixture of the slurry and distilled water at a volume ratio of 1:4 at 90 deg.C for 10min, cooling, and filtering through fine pores to remove solid particles to obtain aloe extractive solution for use.
(3) Heating 50mL of natural hinokitiol modified chitosan solution with volume concentration of 40v/v% to 80 ℃, adding 10mmol/L of silver nitrate, stirring for 20min under the condition of rotation speed of 150r/min, dropwise adding 15mL of the aloe extract, reacting for 6h, cooling the reaction solution, centrifuging at the speed of 6000r/min for 5min, and filtering to obtain the nano-silver complex natural hinokitiol modified chitosan.
(4) Padding the cotton fabric with 0.5wt% of citric acid solution, wherein the soaking bath ratio is 1. Then padding with 0.025wt% nano silver complex natural hinokitiol modified chitosan solution (water as solvent), wherein the dipping temperature is 25 ℃, the dipping time is 30s, the dipping bath ratio is 1. Drying at 60 ℃ for 5min, and baking at 120 ℃ for 2min to obtain the nano-silver complexed natural hinokitiol modified chitosan antibacterial finishing cotton fabric (F-AgNPs @ CSHT).
Respectively finishing chitosan and hinokitiol on the fabrics by the same finishing mode in the step (4) to obtain chitosan finished cotton fabrics (F-CS) and hinokitiol finished cotton fabrics (F-HT).
According to GB/T20944.3-2008, antibacterial rate and washing fastness of the nano-silver complex natural hinokitiol modified chitosan antibacterial finishing cotton fabric (F-AgNPs @ CSHT), untreated common cotton fabric (raw fabric), chitosan finishing cotton fabric (F-CS) and hinokitiol finishing cotton fabric (F-HT) are tested, and experimental results are shown in a figure 1 and a table 3.
FIG. 1 shows the growth of Staphylococcus aureus and Escherichia coli after treatment of common cotton fabric (original fabric), chitosan-finished cotton fabric (F-CS), hinokitiol-finished cotton fabric (F-HT), and nano-silver complex natural hinokitiol-modified chitosan antibacterial-finished cotton fabric (after washing for 25 times) before and after 25 washes.
Table 3 shows the bacteriostatic ratios of chitosan finished cotton fabric (CS), hinokitiol finished cotton fabric (HT), and nano-silver complexed natural hinokitiol modified chitosan antibacterial finished cotton fabric (after F-AgNPs @ CSHT and F-AgNPs @ CSHT25 times of washing) to Staphylococcus aureus and Escherichia coli.
TABLE 3 bacteriostasis rates of different antibacterial finished fabrics
Figure BDA0003248774910000111
As can be seen from fig. 1 and table 3, the antibacterial rate of the nano-silver complexed natural hinokitiol modified chitosan antibacterial finished cotton fabric to staphylococcus aureus and escherichia coli can reach more than 99%, and is improved by about 30% compared with the antibacterial rate of chitosan and about 20% compared with the antibacterial rate of hinokitiol. The antibacterial rate of the nano-silver complexed natural hinokitiol modified chitosan antibacterial finished cotton fabric washed for 25 times to staphylococcus aureus and escherichia coli can still reach over 90%. The nano silver complex hinokitiol grafted chitosan antibacterial finished cotton fabric has better antibacterial activity and washability (antibacterial durability).
Example 4
(1) Dissolving chitosan with the unit molar weight of 1mmol in 50mL of 2v/v% acetic acid aqueous solution, and stirring at 80 ℃ until the chitosan is completely dissolved to obtain 0.02mol/L chitosan solution; adding paraformaldehyde with the molar weight of formaldehyde monomer of 2mmol, stirring to dissolve, adding 2mmol of hinokitiol, reacting at 90 deg.C, and monitoring the reaction process by thin layer chromatography to obtain reaction solution containing natural hinokitiol modified chitosan; after the reaction liquid is cooled, adding 200mL of diethyl ether for extraction to remove unreacted hinokitiol, separating liquid to obtain a pre-product, and removing residual diethyl ether by rotary evaporation at normal temperature to obtain a natural hinokitiol modified chitosan stock solution; adding 75mL of water into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution with the volume concentration of 40 v/v%.
(2) Washing folium Aloe, mashing into paste, and making into slurry; heating the mixture of the slurry and distilled water at a volume ratio of 1:4 at 90 deg.C for 10min, cooling, and filtering through fine pores to remove solid particles to obtain aloe extractive solution for use.
(3) Heating 50mL of natural hinokitiol modified chitosan solution with volume concentration of 40v/v% to 80 ℃, adding 10mmol/L of silver nitrate, stirring for 20min under the condition of rotation speed of 150r/min, dropwise adding 15mL of the aloe extract, reacting for 6h, cooling the reaction solution, centrifuging at the speed of 6000r/min for 5min, and filtering to obtain the nano-silver complex natural hinokitiol modified chitosan.
(4) Respectively padding cotton fabrics, linen fabrics, silk fabrics and wool fabrics with 0.5wt% of citric acid solution, wherein the padding bath ratio is 1. Then padding with 0.025wt% nano silver complex natural hinokitiol modified chitosan solution (solvent is water), wherein the dipping temperature is 25 ℃, the dipping time is 1min, the dipping bath ratio is 1. Drying at 60 ℃ for 5min, and baking at 120 ℃ for 2min to obtain nano-silver complexed natural hinokitiol modified chitosan antibacterial finishing cotton fabric (F-AgNPs @ CSHT), nano-silver complexed natural hinokitiol modified chitosan antibacterial finishing hemp fabric (M-AgNPs @ CSHT), nano-silver complexed natural hinokitiol modified chitosan antibacterial finishing silk fabric (S-AgNPs @ CSHT) and nano-silver complexed natural hinokitiol modified chitosan antibacterial finishing wool fabric (W-AgNPs @ CSHT).
According to GB/T20944.3-2008, the obtained four nano-silver complex natural hinokitiol modified chitosan antibacterial finishing fiber fabrics are subjected to bacteriostasis rate test comparison, and as shown in Table 4:
TABLE 4 antibacterial rate of nano silver complexed natural hinokitiol modified chitosan for antibacterial finishing of different fiber fabrics
Figure BDA0003248774910000121
Figure BDA0003248774910000131
As can be seen from Table 4, the bacteriostatic rates of four different fiber fabrics finished by the nano-silver complex hinokitiol grafted chitosan antibacterial agent on staphylococcus aureus and escherichia coli can reach more than 94%. The nano silver complex hinokitiol grafted chitosan antibacterial agent has wide application prospect in antibacterial finishing of natural fiber fabrics.
Example 5: selection of mole ratio of hinokitiol to chitosan in preparation of natural hinokitiol modified chitosan stock solution
(1) Dissolving chitosan with the unit molar weight of 1mmol in 50mL of 2v/v% acetic acid aqueous solution, and stirring at 80 ℃ until the chitosan is completely dissolved to obtain 0.02mol/L chitosan solution; preparing five parts of the same chitosan solution for later use;
(2) Adding polyformaldehyde (wherein the molar weight of formaldehyde monomer is 0.5mmol, 1mmol, 1.5mmol, 2mmol and 2.5mmol respectively) into the five parts of chitosan solution, stirring until the polyformaldehyde is dissolved, correspondingly adding hinokitiol (the molar weight is 0.5mmol, 1mmol, 1.5mmol, 2mmol and 2.5mmol respectively), controlling the molar ratio of paraformaldehyde to hinokitiol to be 1;
(3) And (3) cooling the reaction liquid, adding 200mL of diethyl ether for extraction to remove unreacted hinokitiol, separating liquid to obtain a pre-product, and removing residual diethyl ether by rotary evaporation at normal temperature to obtain five different natural hinokitiol modified chitosan.
In order to find out the grafting condition of hinokitiol on chitosan, the five different natural hinokitiol modified chitosans are subjected to infrared test and grafting rate calculation, and the results are shown in a figure 2 and a table 5.
TABLE 5 grafting percentage of different kinds of natural hinokitiol-modified chitosans
Figure BDA0003248774910000132
Figure BDA0003248774910000141
As can be seen from Table 5, the grafting rate of hinokitiol on chitosan gradually increased with the increase of hinokitiol amount in the natural hinokitiol-modified chitosan, and the grafting rate reached the maximum when the molar ratio of hinokitiol to chitosan was 2. As can be seen from FIG. 2, with the increase of hinokitiol dosage, hinokitiol 1400-1600cm is corresponding to natural hinokitiol modified chitosan -1 The characteristic peak between the two gradually becomes obvious, and the success of grafting is proved.

Claims (9)

1. The nano-silver complexed natural hinokitiol modified chitosan is characterized in that the structural formula of the nano-silver complexed natural hinokitiol modified chitosan is as follows:
Figure DEST_PATH_IMAGE001
wherein R is 2 Is H,
Figure 979955DEST_PATH_IMAGE002
、
Figure DEST_PATH_IMAGE003
；
R 3 Is H,
Figure 640743DEST_PATH_IMAGE002
、
Figure 127220DEST_PATH_IMAGE003
；
All R in the nano-silver complexed natural hinokitiol modified chitosan 2 、R 3 Not H at the same time;
the preparation method of the nano-silver complexed natural hinokitiol modified chitosan comprises the following steps:
(1) Dissolving chitosan in a solvent I to prepare a chitosan solution;
(2) Respectively adding polyformaldehyde and hinokitiol into the chitosan solution for reaction, and after the reaction is finished, performing post-treatment to obtain a natural hinokitiol modified chitosan stock solution;
(3) Adding a solvent II into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution;
(4) Adding silver nitrate and a reducing agent into the natural hinokitiol modified chitosan solution for reaction, and performing post-treatment to obtain the nano-silver complexed natural hinokitiol modified chitosan;
the molar ratio of the chitosan to the hinokitiol is 1 (1 to 2.5) calculated by the molar weight of the chitosan unit body;
the molar ratio of hinokitiol to paraformaldehyde is 1 (0.5 to 2) calculated by the molar weight of the formaldehyde monomer.
2. The preparation method of the nano-silver complexed natural hinokitiol modified chitosan of claim 1, which is characterized by comprising the following steps:
(1) Dissolving chitosan in a solvent I to prepare a chitosan solution;
(2) Respectively adding polyformaldehyde and hinokitiol into the chitosan solution for reaction, and after the reaction is finished, performing post-treatment to obtain a natural hinokitiol modified chitosan stock solution;
(3) Adding a solvent II into the natural hinokitiol modified chitosan stock solution to prepare a natural hinokitiol modified chitosan solution;
(4) Adding silver nitrate and a reducing agent into the natural hinokitiol modified chitosan solution for reaction, and performing post-treatment to obtain the nano-silver complexed natural hinokitiol modified chitosan.
3. The process according to claim 2, wherein the reaction temperature in the step (2) is from 80 to 100 ℃.
4. The preparation method according to claim 2, wherein the volume concentration of the natural hinokitiol-modified chitosan solution is 10 to 50v/v%;
the solvent I is one or more aqueous solutions of acetic acid, formic acid, lactic acid, malic acid and ascorbic acid;
the solvent II is water.
5. The preparation method according to claim 2, wherein in the step (4), the concentration of silver nitrate in the natural hinokitiol-modified chitosan solution is 6 to 15mmol/L.
6. The preparation method according to claim 2, wherein in the step (4), the reducing agent is one or more of aloe extract, vitamin C and glucose.
7. The production method according to claim 2, wherein the reaction temperature in the step (4) is 80 to 100 ℃.
8. An antibacterial agent comprising the nano silver complexed natural hinokitiol-modified chitosan as set forth in claim 1.
9. Use of the antimicrobial agent according to claim 8 in the antimicrobial finishing of natural fiber fabrics.
CN202111039635.9A 2021-09-06 2021-09-06 Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof Active CN113667039B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111039635.9A CN113667039B (en) 2021-09-06 2021-09-06 Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111039635.9A CN113667039B (en) 2021-09-06 2021-09-06 Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN113667039A CN113667039A (en) 2021-11-19
CN113667039B true CN113667039B (en) 2023-04-14

Family

ID=78548758

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111039635.9A Active CN113667039B (en) 2021-09-06 2021-09-06 Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN113667039B (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000217509A (en) * 1999-01-28 2000-08-08 Sangi Co Ltd Freshness retaining agent
JP2007091686A (en) * 2005-09-30 2007-04-12 Ltt Bio-Pharma Co Ltd Nanoparticle containing hinokitiol
CN101203272A (en) * 2005-06-20 2008-06-18 宝洁公司 A product release system to atomize cosmetic hair and skin cleaning compositions
CN105494430A (en) * 2015-12-16 2016-04-20 河北科技大学 Silver-loaded low-molecular-weight chitosan composite microsphere antibacterial agent and preparation method thereof
CN106259872A (en) * 2016-08-15 2017-01-04 上海市农业科学院 A kind of instant vegetables and fruits fresh-keeping bacteriostatic agent
CN108003440A (en) * 2017-12-29 2018-05-08 成都新柯力化工科技有限公司 A kind of fruit-vegetable plastic wrap and preparation method with antibacterial functions
CN108586215A (en) * 2018-06-08 2018-09-28 山东师范大学 A kind of preparation method of high-purity chamenol and its complex
CN109528549A (en) * 2018-12-21 2019-03-29 上海理工大学 A kind of chitosan anti-bacteria moisturizing Non-water washing hand cleanser and preparation method thereof
CN112160161A (en) * 2020-09-29 2021-01-01 安徽农业大学 Preparation method of active chitosan modified cotton fabric

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000217509A (en) * 1999-01-28 2000-08-08 Sangi Co Ltd Freshness retaining agent
CN101203272A (en) * 2005-06-20 2008-06-18 宝洁公司 A product release system to atomize cosmetic hair and skin cleaning compositions
JP2007091686A (en) * 2005-09-30 2007-04-12 Ltt Bio-Pharma Co Ltd Nanoparticle containing hinokitiol
CN105494430A (en) * 2015-12-16 2016-04-20 河北科技大学 Silver-loaded low-molecular-weight chitosan composite microsphere antibacterial agent and preparation method thereof
CN106259872A (en) * 2016-08-15 2017-01-04 上海市农业科学院 A kind of instant vegetables and fruits fresh-keeping bacteriostatic agent
CN108003440A (en) * 2017-12-29 2018-05-08 成都新柯力化工科技有限公司 A kind of fruit-vegetable plastic wrap and preparation method with antibacterial functions
CN108586215A (en) * 2018-06-08 2018-09-28 山东师范大学 A kind of preparation method of high-purity chamenol and its complex
CN109528549A (en) * 2018-12-21 2019-03-29 上海理工大学 A kind of chitosan anti-bacteria moisturizing Non-water washing hand cleanser and preparation method thereof
CN112160161A (en) * 2020-09-29 2021-01-01 安徽农业大学 Preparation method of active chitosan modified cotton fabric

Also Published As

Publication number Publication date
CN113667039A (en) 2021-11-19

Similar Documents

Publication Publication Date Title
CN102733184B (en) Finishing technology for aromatic anti-bacteria cotton textile
CN102217651A (en) Method for preparing inorganic aromatic nano-antibacterial agent and antibacterial fabrics
CN101368331A (en) Cotton fabric inorganic nanometer antibiotic finishing agent and preparation method thereof
CN106758216A (en) A kind of processing method of antibacterial, uvioresistant bafta
CN103614927A (en) Antibacterial finishing method for textile containing cellulose
CN102605630A (en) Traditional Chinese medicine inclusion compound with antibacterial effect and textile finishing method of traditional Chinese medicine inclusion compound
CN108166259A (en) A kind of non-woven cloth composite antibacterial agent and preparation method thereof
CN102094323A (en) Composite antibacterial textile finishing agent and application thereof
CN107201646A (en) A kind of preparation method of tree cotton anti-bacterial fibre
CN103981701B (en) A kind of shitosan/Ag+Composite antibacterial dressing liquid and its preparation method and application
CN105821653A (en) Method for preparing polyacrylonitrile antibacterial fiber
CN103710993B (en) A kind of preparation technology of the silver-colored silicon complex antimicrobials with permanent antibacterial action
JP6562251B2 (en) Method for producing metal nanoparticle composition
CN103321039B (en) Durable antibacterial ready-made garment post-finishing processing method
CN113863004B (en) Mosquito-repellent antibacterial plant traditional Chinese medicine essential oil composition and preparation method of mosquito-repellent antibacterial functional cotton knitted fabric
CN104532559A (en) A silk/cotton fabric afterfinish method based on tea polyphenol
CN102691214A (en) Antibacterial finishing method for grafting lysozyme to laccase-catalyzed hemp fibers (fabrics)
CN109440454A (en) A kind of method for sorting of antibacterial shirt
CN102493014A (en) Epsilon-polylysine and polyvinyl alcohol composite biological antibacterial fiber and preparation method thereof
CN113667039B (en) Nano-silver complexed natural hinokitiol modified chitosan and preparation method and application thereof
CN105088755A (en) Preparation method and application of silver nano-particles
CN112251835A (en) Olive-containing polyamide fiber and preparation method thereof
CN112590317A (en) Antibacterial spun-bonded non-woven fabric material and preparation method thereof
CN113637095B (en) Natural hinokitiol modified chitosan and preparation method and application thereof
CN103061125A (en) Halamine antibacterial agent containing reactive functional group, as well as preparation method and application for same

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant