CN115245168A - Long-acting antibacterial agent suitable for various substrates and preparation method and application thereof - Google Patents
Long-acting antibacterial agent suitable for various substrates and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
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- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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Abstract
The invention discloses a long-acting antibacterial agent suitable for various substrates and a preparation method and application thereof. The long-acting antibacterial agent is a water-insoluble aggregate formed by co-assembling a polyphenol compound and a quaternary ammonium salt surfactant under non-covalent interaction. The long-acting antibacterial agent can effectively enhance the long-acting antibacterial property of the quaternary ammonium surfactant, improves the using recyclability of the quaternary ammonium surfactant, and has a simple preparation method. The antibacterial agent can form a stable antibacterial film on different types of substrates, the antibacterial film can still maintain high-efficiency and broad-spectrum antibacterial performance on gram-negative bacteria and gram-positive bacteria after being recycled for 20 times and soaked in water for 30 days, and meanwhile, the antibacterial agent has low toxicity on normal cells and better selectivity.
Description
Technical Field
The invention belongs to the technical field of antibiosis, and particularly relates to a long-acting antibacterial agent suitable for various substrates, and a preparation method and application thereof.
Background
Heretofore, various quaternary ammonium salt type surfactants of different structures have been designed as antibacterial agents. The quaternary ammonium salt surfactant can be assembled to form an aggregate, and can increase the local concentration and the cationic charge density thereof, thereby exhibiting excellent antibacterial activity. Unlike conventional antibiotics, the appearance of bacteria is basically maintained after bacteria are inhibited or killed, after the quaternary ammonium surfactant is contacted with the bacteria, firstly, the positively charged head group part of the quaternary ammonium surfactant is combined on the surface of the bacteria through electrostatic interaction, and then, the hydrophobic chain of the surfactant is inserted into the bacterial membrane through hydrophobic interaction with the bacterial membrane, so that the bacterial membrane is ruptured and the bacteria are killed, and therefore, the generation of bacterial drug resistance can be effectively inhibited.
However, the quaternary ammonium salt surfactant has good water solubility and is not resistant to water washing in actual production and life, so that the antibacterial stability and the long-acting property are poor. Most of quaternary ammonium salt surfactant is wasted along with the discharge of production and domestic sewage after being used once. However, the recycling of the quaternary ammonium salt surfactant is limited by high recycling cost, so that the quaternary ammonium salt surfactant is accumulated in a large amount in the environment, and a great threat is caused to the ecological environment. Therefore, there is an urgent need to develop a novel antibacterial agent with high efficiency and long-lasting effect, which can prolong the sterilization time of quaternary ammonium surfactant and improve the recyclability.
Disclosure of Invention
In order to improve the technical problem, the invention provides an aggregate which is formed by co-assembling a polyphenol compound and a quaternary ammonium surfactant through non-covalent interaction;
the quaternary ammonium salt surfactant is at least one of a compound with a structure shown in a formula I and a compound with a structure shown in a formula II,
wherein R is 1 、R 1 ’、R 1 ”、R 2 、R 2 ' and R 2 "identical or different, independently of one another, from the group consisting of unsubstituted or substituted by one, two or more C 6-14 Aryl substituted C 1-8 An alkyl group;
R 3 、R 3 ' and R 3 "identical or different, independently of one another, from C 1-18 An alkyl group;
n is an integer from 2 to 8;
X - is selected from Br - 、F - 、Cl - 、I - 、SO 4 2- 、HCOO - Or HSO 4 - 。
According to an embodiment of the present invention, the non-covalent interaction may be at least one of an electrostatic interaction, a hydrophobic effect, a hydrogen bonding effect, a van der waals force effect, and a pi-pi conjugation effect.
According to an embodiment of the present invention, the polyphenol compound may be selected from at least one of gallic acid, tannic acid, epigallocatechin gallate, anthocyanin, catechin, quercetin, ellagic acid, arbutin, protocatechuic acid, tea polyphenol, chlorogenic acid, and the like.
According to an embodiment of the invention, said R 1 、R 1 ’、R 1 ”、R 2 、R 2 ' and R 2 "identical or different, independently of one another, from C unsubstituted or substituted by one, two or more phenyl radicals 1-4 An alkyl group; r is 3 、R 3 ' and R 3 "identical or different, independently of one another, from C 1-12 Alkyl, n is selected from an integer between 2 and 6; x - Is selected from Br - 、F - 、Cl - 、I - 、SO 4 2- 、HCOO - Or HSO 4 - 。
According to an embodiment of the invention, said R 1 、R 1 ’、R 1 ”、R 2 、R 2 ' and R 2 "identical or different, independently of one another, from C unsubstituted or substituted by one, two or more phenyl radicals 1-4 An alkyl group; r is 3 、R 3 ' and R 3 "identical or different, independently of one another, from C 1-12 Alkyl, n is selected from an integer between 2 and 6; x - Is selected from Br - 、F - 、Cl - 、I - 、SO 4 2- 、HCOO - Or HSO 4 - 。
According to an embodiment of the present invention, the quaternary ammonium surfactant may be selected from gemini quaternary ammonium surfactants, such as trimethylene-1, 3-bis-dodecyldimethylammonium bromide [ C ] 12 H 25 N(CH 3 ) 2 (CH 2 ) 3 (CH 3 ) 2 NC 12 H 25 Br 2 ，12-3-12(Br) 2 ]、[C 12 H 25 N(CH 3 ) 2 (CH 2 ) 6 (CH 3 ) 2 NC 12 H 25 Br 2 (i.e., 12-6-12 (Br) 2 ) (ii) a Single head double tail quaternary ammonium surfactants such as didodecyldimethylammonium bromide (DDAB); or an oligomeric quaternary ammonium salt surfactant having a polymerization degree of 3 to 6, for example, an oligomeric quaternary ammonium salt surfactant having a polymerization degree of 3 (Br) 12-3-12-3-12 3 、12-6-12-6-12(Br) 3 The molecular formula is shown as formula III:
the invention further provides a preparation method of the aggregate, which comprises the following steps:
(1) Dissolving a quaternary ammonium salt surfactant in water and then adjusting the pH value to obtain a quaternary ammonium salt surfactant aqueous solution;
(2) Dissolving a polyphenol compound in water, and adjusting the pH value to obtain a polyphenol compound aqueous solution;
(3) Mixing the quaternary ammonium salt surfactant aqueous solution and the polyphenol compound aqueous solution to obtain the aggregate;
the quaternary ammonium surfactant and the polyphenol compound have the definitions described above.
According to an embodiment of the present invention, in step (1), the pH of the aqueous quaternary ammonium salt surfactant solution may be 6 to 8, illustratively 6, 7, 8;
according to an embodiment of the invention, in step (1), the concentration of the aqueous quaternary ammonium surfactant solution may be 0.1 to 15mM, such as 0.1 to 5mM, illustratively 0.1mM, 0.2mM, 0.5mM.
According to an embodiment of the present invention, in step (2), the pH of the aqueous solution of the polyphenol compound may be 4 to 11, for example 4 to 9, exemplified by 4, 5, 6, 7, 8, 9;
according to an embodiment of the invention, in step (2), the concentration of the aqueous solution of polyphenolic compound may be in the range of 1 to 10mM, such as 1 to 5mM, exemplarily 1mM, 1.5mM, 2mM, 2.5mM, 3mM.
According to an embodiment of the present invention, in step (3), the volume ratio of the quaternary ammonium salt surfactant aqueous solution and the polyphenol compound aqueous solution may be (0.1-10) to (0.1-10), for example (1-8) to (1-8), exemplified by 1;
according to an embodiment of the invention, in step (3), the temperature of the mixing may be 0 to 60 ℃, for example 20 to 40 ℃, exemplary 25 ℃; the mixing time may be 10 seconds to 5 minutes, for example 0.5 to 3 minutes, with 1 minute being exemplary.
According to an embodiment of the present invention, the step (3) further comprises a post-treatment step, wherein the post-treatment step can be a step of sequentially centrifuging, washing and drying the aggregate to obtain a dried aggregate.
The invention also provides the use of the above-described aggregates in the field of antibacterials, for example for antibacterials, preferably long-acting antibacterials.
The invention also provides an antimicrobial agent comprising the aggregate. Preferably, the antimicrobial agent is a long-acting antimicrobial agent.
The invention also provides an antibacterial film comprising the aggregate. Preferably, the antimicrobial agent is a long-acting antimicrobial film.
The invention also provides a preparation method of the antibacterial film, which comprises the steps of dissolving the aggregate in a solvent, spraying or coating the aggregate on the surface of a substrate, and drying to obtain the antibacterial film on the surface of the substrate.
According to an embodiment of the present invention, the solvent may be an organic solvent or a mixed solvent of an organic solvent and water; for example, the solvent may be at least one of a 75% ethanol aqueous solution, ethanol, methanol, chloroform, dichloromethane, n-hexane, ethyl acetate, and diethyl ether, and is preferably a 75% ethanol aqueous solution.
According to embodiments of the invention, the concentration of the aggregate after dissolution in the solvent may be 1 to 1000. Mu.g/mL, such as 10 to 800. Mu.g/mL, illustratively 100. Mu.g/mL, 200. Mu.g/mL, 300. Mu.g/mL, 400. Mu.g/mL, 500. Mu.g/mL, 600. Mu.g/mL.
According to an embodiment of the present invention, the substrate may be plastic, rubber, stainless steel, glass, silica, silicon, mica, metal alloy, or cotton cloth; for example, the plastic may be a polypropylene material.
According to an embodiment of the present invention, the amount of the aggregates sprayed or coated on the surface of the substrate may be 2 to 20 μ g/cm 2 For example, 2 to 10. Mu.g/cm 2 Exemplary is 4. Mu.g/cm 2 、6μg/cm 2 、8μg/cm 2 。
According to an embodiment of the invention, the drying may be natural evaporation; the drying time may be from 20 seconds to 30 minutes, for example from 1 to 20 minutes, with 5 minutes being exemplary.
The invention also provides the application of the aggregate, the antibacterial agent or the antibacterial film in inhibiting or inactivating bacteria or fungi.
According to embodiments of the invention, the bacteria may be gram-negative or gram-positive bacteria;
for example, the gram-negative bacterium can be escherichia coli;
for example, the gram-positive bacterium can be staphylococcus aureus, lactic acid bacterium, or streptococcus;
for example, the fungus may be candida albicans, a mold or a yeast.
Advantageous effects
The invention provides a long-acting antibacterial aggregate, an antibacterial agent/film, and a preparation method and application thereof. The antibacterial agent is a water-insoluble aggregate formed by co-assembling a polyphenol compound and a quaternary ammonium salt surfactant (particularly a gemini quaternary ammonium salt surfactant) under the co-driving of non-covalent interaction, such as electrostatic interaction, hydrophobic effect, hydrogen bonding effect, van der Waals force effect and/or pi-pi conjugated effect. The aggregate can be effectively deposited on various substrates, the antibacterial long-acting performance of the aggregate is effectively enhanced, and the using cyclability of the aggregate is improved. The aggregate has simple preparation method and short required time, can form a layer of stable antibacterial film on different types of substrates, can be recycled for 20 times, can still maintain the high-efficiency and broad-spectrum antibacterial performance on gram-negative bacteria, gram-positive bacteria and fungi after being soaked in water for 30 days (see figure 5), and simultaneously has low toxicity on normal cells and better selectivity.
Drawings
FIG. 1 is a particle size distribution diagram of the aggregate of example 1;
FIG. 2 is an electron micrograph of the surface topography of the aggregates of example 1 adsorbed on a substrate;
FIG. 3 is the results of the cyclic sterilization test of the antimicrobial-coated substrate of example 9 for E.coli;
FIG. 4 is the contact angle results of water on the antimicrobial-uncoated substrate and the antimicrobial-coated substrate of example 10;
FIG. 5 shows the results of the stability of the antibacterial agent of example 10 coated on a substrate;
FIG. 6 is a graph of the effect of the application of the antimicrobial agent of example 11 to the substrate on killing of bacteria and fungi;
FIG. 7 is a graph of the change in surface topography of the bacteria and fungi of example 12 before and after interaction with the antimicrobial coated substrate;
FIG. 8 is a graph showing the relationship between the cytotoxicity of the antibacterial agent of example 13 and the coating amount thereof.
Definition and description of terms
The term "C 1-18 Alkyl "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 18 carbon atoms. For example, "C 1-8 Alkyl "denotes straight-chain and branched alkyl groups having 1,2, 3, 4, 5, 6, 7 or 8 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a 1, 2-dimethylpropyl group, a neopentyl group, a 1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3, 3-dimethylbutyl group, a 2, 2-dimethylbutyl group, a 1, 1-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 1, 3-dimethylbutyl group or a 1, 2-dimethylbutyl group, or the like, or isomers thereof.
The term "C 6-14 Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring having a monovalent or partially aromatic character with 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (" C 6-14 Aryl group "), in particular a ring having 6 carbon atoms (" C 6 Aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C 9 Aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 Aryl radicals), such as tetralinyl, dihydronaphthyl or naphthyl, or rings having 13 carbon atoms ("C 13 Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C) 14 Aryl), such as anthracenyl. When said C is 6-20 When the aryl group is substituted, it may be mono-or polysubstituted. And, the substitution site thereof is not limited, and may be, for example, ortho-, para-or meta-substitution.
In the present invention, the term "long-acting antibacterial" means that the antibacterial agent/film can be recycled for at least 10 times (preferably 20 times) and/or can still maintain high antibacterial rate and broad-spectrum antibacterial performance after being soaked in water for at least 15 days (preferably 30 days), and the antibacterial rate is maintained to be more than 99%.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the techniques realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
"mM" represents the concentration mmol/L.
Example 1
Preparing 3mM aqueous solution of gallic acid with pH of 4 and 0.5mM 12-6-12 (Br) with pH of 7 2 The aqueous solution of (4) was mixed at room temperature in a volume ratio of 1. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.2 mg/mL. The above clear solution was added at a rate of 20. Mu.g/cm 2 The amount of the antibacterial agent is sprayed on a polypropylene sheet, and after about 10 minutes of natural volatilization of the solvent, a layer of antibacterial film is obtained on the surface of the polypropylene.
The aggregate is taken to be dissolved in 75% ethanol to prepare a solution with the concentration of 0.5mg/mL, the average Z-average diameter of the aggregate formed by the co-assembly of the system is 220nm as shown by the result of dynamic light scattering, and the distribution diagram of the particle size of the aggregate is shown in figure 1.
After the 75% ethanol solution with the concentration of 0.5mg/mL aggregate is coated on a substrate, the surface topography of the substrate is shown in an electron microscope picture 2, which shows that the aggregate can be adsorbed on the surface of the substrate to form a layer of antibacterial film formed by a large number of spherical aggregates which are tightly connected with each other.
Example 2
Preparing 2.5mM aqueous solution of gallic acid with pH of 4 and 0.5mM 12-6-12-6-12 (Br) with pH of 7 3 Mixing the two aqueous solutions at room temperature in a volume ratio of 3And then, vacuum drying to obtain powder. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.2 mg/mL. And spraying the transparent solution on cotton cloth, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the cotton cloth.
Example 3
Preparing 2.5mM aqueous solution of gallic acid having pH of 4 and 0.1mM 12-3-12 (Br) having pH of 7 2 Mixing the two aqueous solutions at room temperature according to a volume ratio of 5. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.5 mg/mL. And spraying the transparent solution on rubber, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the rubber.
Example 4
Preparing 1.5mM aqueous solution of gallic acid with pH of 4 and 0.1mM 12-3-12-3-12 (Br) with pH of 7 2 Mixing the two aqueous solutions at room temperature according to a volume ratio of 2. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.2 mg/mL. And spraying the transparent solution on silicon dioxide, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the silicon dioxide.
Example 5
Preparing 1mM tannic acid aqueous solution at pH 9 and 0.1mM pH 7 of 12-3-12 (Br) 2 Mixing the two aqueous solutions at room temperature according to a volume ratio of 1. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.5 mg/mL. And spraying the transparent solution on a stainless steel sheet, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the stainless steel sheet.
Example 6
Preparing 1mM tannic acid aqueous solution at pH 9 and 0.2mM pH 7 of 12-6-12-6-12 (Br) 3 Mixing the two aqueous solutions at room temperature according to a volume ratio of 1. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.5 mg/mL. And spraying the transparent solution on a mica sheet, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the mica.
Example 7
Preparing 2.5mM epigallocatechin gallate aqueous solution with pH of 9 and 0.5mM 12-6-12 (Br) with pH of 7 2 The aqueous solution of (4) was mixed at room temperature in a volume ratio of 1. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.2 mg/mL. And spraying the transparent solution on a glass sheet, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the glass.
Example 8
Preparing 2mM epigallocatechin gallate aqueous solution with pH of 9 and 0.1mM 12-3-12-3-12 (Br) with pH of 7 2 Mixing the two aqueous solutions at room temperature according to a volume ratio of 1. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.5 mg/mL. And spraying the transparent solution on a silicon wafer, and naturally volatilizing the solvent for about 10 minutes to obtain a layer of antibacterial film on the surface of the silicon wafer.
Comparative example 1
Preparing a 3mM aqueous solution of gallic acid having pH 9 and a 0.1mM aqueous solution of DTAB (dodecyl trimethyl ammonium bromide) having pH 7, mixing the two aqueous solutions at a volume ratio of 5 at room temperature to form a water-insoluble gallic acid-monoquaternary ammonium salt surfactant complex, centrifuging to remove the supernatant, washing the aggregate with three times of water for 3 times, and vacuum-drying to obtain a powder. Dissolving the dried powder in 75% ethanol to obtain a transparent solution with a concentration of 0.5 mg/mL. And spraying the transparent solution on a silicon wafer, and naturally volatilizing the solvent for about 10 minutes to obtain a film coated with a gallic acid-mono-quaternary ammonium salt surfactant compound-containing layer on the surface of the silicon wafer.
Comparative example 2
Preparing 12-3-12 (Br) with pH of 5.8 and 0.5mg/mL 2 The solution is sprayed on a silicon wafer, and after the solvent naturally volatilizes for about 10 minutes, a film coated with a quaternary ammonium salt surfactant is obtained on the surface of the silicon wafer.
Example 9 Long-acting antibacterial Properties of antibacterial agent
Experimental groups: mu.L of E.coli suspension (OD = 0.2) was allowed to act on the PBS solution coated with 0.5mg/mL of the antibacterial agent-coated substrate (2 cm. Times.2 cm) obtained in examples 1 to 8 at 37 ℃ for 30min, and the antibacterial activity of the above antibacterial film against E.coli in the first use was evaluated by the plate method. And then cleaning the substrate subjected to the escherichia coli action by using water for three times at the rotating speed of 350rpm, cleaning for 30min, then drying the substrate in vacuum, and continuously inspecting the recycled antibacterial activity of an antibacterial film formed by coating the antibacterial agent on the substrate after multiple times of water washing according to the experimental conditions.
Control group 1: also, 25. Mu.L of E.coli suspension (OD = 0.2) was allowed to act on a PBS solution coated with 0.5mg/mL of the substrate (2 cm. Times.2 cm) of the complex of comparative example 1 at 37 ℃ for 30min, and the antibacterial activity of the complex against E.coli at the first use was evaluated by the plate method. And then, washing the substrate acted by the escherichia coli by using water for three times at the rotating speed of 350rpm, washing for 30min, then, drying the substrate in vacuum, and continuously inspecting the recycled antibacterial activity of the compound after being washed for multiple times according to the experimental conditions.
Control group 2: mu.L of E.coli suspension (OD = 0.2) was allowed to act on the above PBS solution having a surface coated with only 0.5mg/mL of the substrate (2 cm. Times.2 cm) of the gemini quaternary ammonium surfactant of comparative example 2 at 37 ℃ for 30min, and the antibacterial activity of the gemini quaternary ammonium surfactant on E.coli in the first use was evaluated by the plate method. And then, washing the substrate subjected to the escherichia coli action by using tertiary water at the rotating speed of 350rpm for 30min, then, drying the substrate in vacuum, and continuously inspecting the circularly used antibacterial activity of the gemini quaternary ammonium salt surfactant after being washed for multiple times according to the experimental conditions.
The results of the antibacterial activities of the experimental group and the control group are shown in fig. 3.
As can be seen from fig. 3, the experimental group coated with the antibacterial agent on the substrate still showed 99.9% antibacterial activity against e.coli after 10 cycles of use under the condition of consistent external conditions of the control experiment; the control group 1 coated with the gallic acid-single-chain quaternary ammonium salt surfactant complex on the substrate has greatly reduced antibacterial activity after the second cycle use, the antibacterial activity is reduced to 51.3%, and the antibacterial activity to escherichia coli is completely lost after the 6 th cycle use; the antibacterial activity of the control group 2 coated with the gemini quaternary ammonium salt surfactant on the substrate is greatly reduced after the second cycle use, the antibacterial activity is reduced to 43.5 percent, and the antibacterial activity to escherichia coli is completely lost after the 5 th cycle use.
Example 10 stability of antibacterial film formed by coating antibacterial agent on substrate
Three water drops were dropped on the substrate which was not coated with the antibacterial agent and which was coated with the antibacterial agent prepared in example 2 at 0.5mg/mL, respectively, and the contact angle sizes of water on the above two substrates were tested. As shown in fig. 4, the contact angle of water molecules on the antimicrobial-free substrate was 62.2 ° ± 0.9 °, while the contact angle of water molecules on the substrate coated with the antimicrobial was increased to 88.2 ° ± 0.6 °, indicating that the aggregates were adsorbed on the substrate surface and the hydrophobicity of the substrate surface was increased.
After the substrate coated with the antibacterial agent was further soaked in three times of water for one month, the contact angle of water molecules on the substrate was continuously tested and the long-lasting antibacterial activity of the antibacterial agent was evaluated, as shown in fig. 5.
As can be seen from fig. 5, the contact angle of water molecules of the antimicrobial-coated substrate after one month immersion in three times of water was 83.6 ° ± 0.2 °, still exhibiting a higher contact angle than the substrate without the aggregate coating, demonstrating that the antimicrobial film formed on the substrate by the antimicrobial agent has better stability. And the antibacterial film after being soaked for one month can still keep the antibacterial activity of more than 99 percent to the escherichia coli.
Example 11 correlation of antimicrobial coating amount with the killing effect of bacteria and fungi
mu.L of E.coli suspension (OD = 0.2), staphylococcus aureus suspension (OD = 0.2), and Candida albicans suspension (OD = 0.4), respectively, and the above substrate surface-coated with the antibacterial agent prepared in example 2 in different amounts were subjected to the PBS solution at 37 ℃ for 30min, and the effect of the coating amount of the antibacterial agent on the killing effect of the three bacteria and fungi was evaluated by the plate method, as shown in FIG. 6.
As can be seen from fig. 6, as the mass of the antibacterial agent coated on the substrate increases, the antibacterial activity thereof also increases. The coating amount of the antibacterial agent on the substrate is 0.26 mu g/cm 2 99.9 percent of antibacterial activity to staphylococcus aureus can be achieved; the coating amount of the antibacterial agent on the substrate was 0.41. Mu.g/cm 2 99.9 percent of antibacterial activity to escherichia coli can be achieved; the coating amount of the antibacterial agent on the substrate was 2.04. Mu.g/cm 2 99.9 percent of antibacterial activity to the candida albicans can be achieved.
Example 12 changes in the morphology of bacteria and fungi before and after interaction with antimicrobial agents
The bacterial solution before and after the action of the antibacterial agent prepared in example 2 was centrifuged at 7100rpm for 5min, the supernatant was removed, and then the cells were resuspended in sterilized water. 5 mu L of the bacteria are dropped on a clean silicon wafer, the silicon wafer is immediately immersed in 0.1 percent glutaraldehyde aqueous solution after being dried in an ultra-clean bench, and the silicon wafer is fixed in a refrigerator at 4 ℃ overnight. After washing the sample 2 times with sterile water, the sample was dehydrated with a gradient of 50%, 70%, 90% and 100% ethanol for 6min each time. After the sample is naturally dried, the sample is dried for 2 hours in vacuum, and then the gold spraying treatment is carried out. The surface topography of E.coli, S.aureus and C.albicans membranes before and after the action of the antibacterial agent was directly observed by SEM, as shown in FIG. 7.
As can be seen in fig. 7, the sem images of the control group showed the integrity of the cells of escherichia coli, staphylococcus aureus and candida albicans, with clear edges, before contact with the antimicrobial coated substrate. Upon contact with the substrate coated with the aggregate antimicrobial agent, the bacterial and fungal membranes collapse, rupture, and the internal cytoplasm leaks causing bacterial and fungal death.
EXAMPLE 13 cytotoxicity of antibacterial Agents
Experimental groups: different masses of the antimicrobial agent prepared in example 2 were coated on 96-well cell culture plates, each set of which was made into 6 parallel wells. Hela cells in logarithmic growth phase were then counted by cell counting plate at a cell density of 8X 10 3 Per well with a final volume of 100. Mu.L per well, seeded on the above antimicrobial-treated 96-well cell culture plate, and then put into 5% CO 2 And culturing in a constant-temperature incubator at 37 ℃ for 24 hours.
Control group: hela cells in logarithmic growth phase were counted by cell counting plate at a cell density of 8X 10 3 One/well, seeded in 96-well cell culture plates without any treatment, each well having a final volume of 100. Mu.L, then put in 5% CO 2 And culturing in a constant-temperature incubator at 37 ℃ for 24 hours to make the wall adhere.
After removing the culture medium from the experimental group and the control group, 100. Mu.L of 0.5mg/mL MTT was added to each well, the culture was continued at 37 ℃ for 4 hours, the supernatant was removed, 100. Mu.L DMSO was added to each well, and then the 96-well plate was placed in a microplate reader, followed by shaking for 5 minutes to dissolve the produced blue-violet formazan particles sufficiently, and the absorbance at 520nm was measured. Cell viability was calculated according to the following formula: cell viability (%) = A/A 0 X 100, wherein A 0 As the absorbance value of the control group, A is the absorbance value of the experimental group. The cell viability was correlated with the amount of antimicrobial coating, as shown in FIG. 8.
As is known from FIG. 6, when the content of the antibacterial agent coated on the substrate reached 2.04. Mu.g/cm 2 It has 99.9% antibacterial activity against Escherichia coli, staphylococcus aureus and Candida albicans. However, as can be seen from FIG. 8, the content of the antibacterial agent as high as 3.02. Mu.g/cm when coated on the substrate 2 However, no significant cytotoxicity was shown on HeLa cells. This is probably because the negative charge on the surfaces of bacteria and fungi is stronger than that on mammalian cells, and the static electricity between the antibacterial agent and the bacteria is strongerThe effect is stronger than that between HeLa cells, so that the antibacterial agent shows excellent antibacterial activity without significant cytotoxicity.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An aggregate formed by the co-assembly of a polyphenolic compound and a quaternary ammonium surfactant by non-covalent interactions;
the quaternary ammonium salt surfactant is at least one of a compound with a structure shown in a formula I and a compound with a structure shown in a formula II,
wherein R is 1 、R 1 ’、R 1 ”、R 2 、R 2 ' and R 2 "identical or different, independently of one another, from the group consisting of unsubstituted or substituted by one, two or more C 6-14 Aryl substituted C 1-8 An alkyl group;
R 3 、R 3 ' and R 3 "identical or different, independently of one another, from C 1-18 An alkyl group, which is a radical of an alkyl group,
n is an integer of 2 to 8;
X - is selected from Br - 、F - 、Cl - 、I - 、SO 4 2- 、HCOO - Or HSO 4 - 。
2. The aggregate according to claim 1, wherein the polyphenolic compound is selected from at least one of gallic acid, tannic acid, epigallocatechin gallate, anthocyanins, catechins, quercetin, ellagic acid, arbutin, protocatechuic acid, tea polyphenols, and chlorogenic acid;
preferably, the non-covalent interaction is at least one of an electrostatic interaction, a hydrophobic effect, a hydrogen bonding effect, a van der waals force effect, and a pi-pi conjugation effect.
3. Aggregate according to claim 1 or 2, characterized in that said R is 1 、R 1 ’、R 1 ”、R 2 、R 2 ' and R 2 "identical or different, independently of one another, from C which is unsubstituted or substituted by one, two or more phenyl radicals 1-4 An alkyl group; r is 3 、R 3 ' and R 3 "identical or different, independently of one another, from C 1-12 Alkyl, n is selected from an integer between 2 and 6; x - Is selected from Br - 、F - 、Cl - 、I - 、SO 4 2- 、HCOO - Or HSO 4 - 。
4. An aggregate according to any of claims 1 to 3, characterised in that the quaternary ammonium surfactant is selected from gemini quaternary ammonium surfactants, such as trimethylene-1, 3-bis-dodecyldimethylammonium bromide [ C [ ] 12 H 25 N(CH 3 ) 2 (CH 2 ) 3 (CH 3 ) 2 NC 12 H 25 Br 2 ，12-3-12(Br) 2 ]、[C 12 H 25 N(CH 3 ) 2 (CH 2 ) 6 (CH 3 ) 2 NC 12 H 25 Br 2 (i.e., 12-6-12 (Br) 2 ) (ii) a Single head double tail quaternary ammonium surfactants such as didodecyldimethylammonium bromide (DDAB); oligomeric quaternary ammonium salt surfactant with polymerization degree of 3-6, such as oligomeric quaternary ammonium salt surfactant with polymerization degree of 3 12-3-12-3-12 (Br) 3 、12-6-12-6-12(Br) 3 The molecular formula is shown as the formula III,
5. a method for producing aggregates according to any of claims 1-4, characterized in that it comprises the following steps:
(1) Dissolving a quaternary ammonium salt surfactant in water and then adjusting the pH value to obtain a quaternary ammonium salt surfactant aqueous solution;
(2) Dissolving a polyphenol compound in water, and adjusting the pH value to obtain a polyphenol compound aqueous solution;
(3) Mixing the quaternary ammonium salt surfactant aqueous solution and the polyphenol compound aqueous solution to obtain the aggregate;
wherein the quaternary ammonium surfactant and the polyphenolic compound have the definitions set forth in any one of claims 1 to 4;
preferably, in the step (1), the pH value of the quaternary ammonium salt surfactant aqueous solution is 6-8;
preferably, in step (1), the concentration of the aqueous quaternary ammonium surfactant solution is from 0.1 to 15mM, for example from 0.1 to 5mM;
preferably, in step (2), the pH value of the aqueous solution of the polyphenol compound is 4 to 11, for example, 4 to 9;
preferably, in step (2), the concentration of the aqueous solution of the polyphenol compound is 1 to 10mM, such as 1 to 5mM;
preferably, in the step (3), the volume ratio of the quaternary ammonium salt surfactant aqueous solution to the polyphenol compound aqueous solution is (0.1-10) to (0.1-10), such as (1-8) to (1-8);
preferably, in step (3), the temperature of the mixing is 0 to 60 ℃, for example 20 to 40 ℃; the mixing time is 10 seconds to 5 minutes, for example 0.5 to 3 minutes;
preferably, the step (3) further comprises a post-treatment step, wherein the post-treatment step is to sequentially perform centrifugation, washing and drying on the aggregate to obtain a dried aggregate.
6. Use of the aggregate according to any of claims 1-4 in the field of antibacterials, for example in the preparation of antibacterials, preferably in the preparation of long-acting antibacterials.
7. An antimicrobial agent, comprising the aggregate of any of claims 1-4; preferably, the antimicrobial agent is a long-acting antimicrobial agent.
8. An antimicrobial film, characterized in that it comprises the aggregate according to any one of claims 1 to 4; preferably, the antimicrobial agent is a long-acting antimicrobial film.
9. A preparation method of an antibacterial film, which is characterized by comprising the steps of dissolving the aggregate of any one of claims 1 to 4 in a solvent, spraying or coating the aggregate on the surface of a substrate, and drying to obtain the antibacterial film on the surface of the substrate;
preferably, the solvent is an organic solvent or a mixed solvent of an organic solvent and water; the organic solvent is at least one of 75% ethanol-containing water solution, ethanol, methanol, chloroform, dichloromethane, n-hexane, ethyl acetate and diethyl ether, preferably 75% ethanol-containing water solution;
preferably, the concentration of the antibacterial aggregate after dissolving in the solvent is 1 to 1000. Mu.g/mL, for example, 10 to 800. Mu.g/mL;
preferably, the substrate is plastic, rubber, stainless steel, glass, silica, silicon, mica, a metal alloy or cotton cloth; the plastic is a polypropylene material;
preferably, the amount of the antimicrobial aggregate sprayed or coated on the surface of the substrate is 2 to 20 μ g/cm 2 For example, 2 to 10. Mu.g/cm 2 ；
Preferably, the drying is natural volatilization; the drying time is 20 seconds to 30 minutes, for example 1 to 20 minutes.
10. Use of the aggregate of any one of claims 1 to 4, the antimicrobial agent of claim 7 or the antimicrobial film of claim 8 for inhibiting or inactivating bacteria or fungi;
preferably, the bacterium is a gram-negative or gram-positive bacterium;
preferably, the gram-negative bacterium is escherichia coli;
preferably, the gram-positive bacterium is staphylococcus aureus, lactobacillus, or streptococcus;
preferably, the fungus is candida albicans, a mold or a yeast.
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| CN1274026A (en) * | 1999-05-18 | 2000-11-22 | 株式会社伊藤园 | Method of producing antibiotic fiber |
| CN108276820A (en) * | 2018-01-24 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of antimicrobial coating agent and preparation method thereof and a kind of antimicrobial coating |
| CN111153815A (en) * | 2020-03-10 | 2020-05-15 | 北京化工大学 | Antibacterial polyphenol material and application thereof |
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2021
- 2021-04-27 CN CN202110465197.6A patent/CN115245168A/en active Pending
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| CN1274026A (en) * | 1999-05-18 | 2000-11-22 | 株式会社伊藤园 | Method of producing antibiotic fiber |
| CN108276820A (en) * | 2018-01-24 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of antimicrobial coating agent and preparation method thereof and a kind of antimicrobial coating |
| CN111153815A (en) * | 2020-03-10 | 2020-05-15 | 北京化工大学 | Antibacterial polyphenol material and application thereof |
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