A highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool and its preparation method.

Abstract

This invention discloses a method for preparing a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool. The method comprises: S1, synthesis of chloroacetate; S2, preparation of a ditertiary amine; and S3, synthesis of the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool. Using the ditertiary amine obtained in S2, the chloroacetate obtained in S1, and a solvent, the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool is obtained. This invention introduces plant-derived linalool into the geminal quaternary ammonium salt structure through chemical bonding. The nitrogen ions on the geminal quaternary ammonium salt adsorbed onto the negatively charged bacterial surface, combined with the hydrophobic interaction of the plant-derived linalool, produce an excellent "adhesion-killing" highly efficient synergistic antibacterial effect, providing a new approach and technology for the high-value resource utilization of natural products.

Description

Technical Field

[0001] This invention belongs to the technical field of leather specialty chemicals, specifically relating to a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool and its preparation method. Background Technology

[0002] The raw materials for the leather industry come from natural animal hides. Raw hides are rich in 30%–35% protein and 60%–75% water, providing abundant nutrients for the growth and reproduction of microorganisms. Under suitable conditions, raw hides are highly susceptible to decay due to microbial activity and the action of autolytic enzymes on the hide. During storage and processing, they are easily attacked by various harmful bacteria, leading to spoilage. To inhibit the growth and reproduction of harmful bacteria, bactericides are often added. Furthermore, finished leather products can also be contaminated by microorganisms during storage and use, resulting in mold growth and mildew.

[0003] Currently, most leather companies use salting as a sterilization and preservation method in the raw hide soaking process, but this involves a large amount of NaCl. In recent years, limiting the use of NaCl has become an important indicator for evaluating clean production in leather tanning. On the other hand, the bactericides and fungicides used for finished leather are mostly commercially available bactericides and fungicides. The most commonly used one is 2-thiocyanomethylthiobenzothiazole (TCMTB), but this type of fungicide is a sulfur-containing compound and has high toxicity.

[0004] Taking the soaking process as an example, the bactericide must not only inhibit all harmful bacteria on the original skin, but also be used in small quantities, be non-toxic to humans, maintain its antibacterial effect when used in the same bath as other additives, and not cause environmental pollution after being discharged into the water body, and be biodegradable. Therefore, the bactericide used must be highly effective, broad-spectrum, low in toxicity, have good compatibility with other additives, be non-polluting, and be easily biodegradable.

[0005] Gemini quaternary ammonium cationic surfactants are recognized as highly effective bactericides. Their molecular structure, with two long-chain hydrophobic groups and two positively charged N+ ions, facilitates adsorption of the bactericide molecules onto bacterial surfaces, increasing cell wall permeability and causing bacterial rupture. Furthermore, after adsorption onto the bacterial surface, the hydrophobic and hydrophilic groups can respectively enter the lipid and protein layers of the bacterial cell, leading to enzyme inactivation and protein denaturation. This synergistic effect results in extremely significant bactericidal activity of quaternary ammonium cationic Gemini surfactants. Due to their unique molecular structure, Gemini quaternary ammonium bactericides possess unique surface activity, high efficiency and broad-spectrum bactericidal properties, good water solubility and permeability, enabling them to rapidly penetrate the skin and kill microorganisms. Moreover, quaternary ammonium bactericides are low-toxicity chemicals and safe for human health. Existing literature reports their widespread use in industrial water treatment, wastewater treatment, and petrochemicals, and they are gradually being adopted in the leather industry. Quaternary ammonium salt bactericides effectively inhibit bacterial growth during the soaking process in leather tanning. As a type of cationic surfactant, they also accelerate soaking, remove dirt, degrease, and wash, without negatively impacting subsequent tanning processes. Among them, the ester group in the ester-type geminal quaternary ammonium salt molecule not only imparts good biodegradability but also, as a strong electron-withdrawing group, enhances the positive charge density on the nitrogen atom. This facilitates electrostatic attraction, allowing the bacteria to adsorb onto the negatively charged bacterial surface, resulting in superior bactericidal performance. Furthermore, based on the functional requirements of practical applications, precise design of the linking and hydrophobic groups is necessary to achieve the preparation of high-performance geminal quaternary ammonium salts.

[0006] Plant-derived antibacterial agents have attracted much attention due to their environmentally friendly properties. Linalool, as a monoterpene compound, is widely found in various plant essential oils. In addition to imparting special aromas to food, its broad-spectrum antibacterial activity has gradually become a research hotspot. Existing studies have shown that this component can effectively inhibit the formation of biofilms of various pathogenic bacteria through mechanisms such as inhibiting bacterial growth, weakening motility, and interfering with quorum sensing, thereby achieving antibacterial effects. Summary of the Invention

[0007] The purpose of this invention is to provide a highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt based on plant-derived linalool and its preparation method. Plant-derived linalool is chemically bonded to the gemini quaternary ammonium salt structure. The nitrogen ions on the gemini quaternary ammonium salt adsorbed onto the negatively charged bacterial surface, and the hydrophobic interaction with the plant-derived linalool produces an excellent synergistic antibacterial effect. Furthermore, the four ester groups in the gemini quaternary ammonium salt molecule, on the one hand, act as electron-withdrawing groups, enhancing the nitrogen ion charge density and thus imparting superior antibacterial properties; on the other hand, they act as readily degradable groups, endowing it with excellent biodegradability. The preparation method of this invention produces safe, environmentally friendly, efficient, low-toxicity, biodegradable gemini quaternary ammonium salts with excellent bactericidal effects. These gemini quaternary ammonium salts can be used not only for the preservation of raw hides in leather tanning and for sterilization and preservation in wet processing, but also, due to the presence of the active C=C group in their molecular structure, their chemical activity is increased. By reacting with mercaptopropylene glycol, dihydroxy gemini quaternary ammonium salts are obtained, which can then undergo esterification with the carboxyl groups on leather collagen to chemically bond them to the leather raw materials. They can also react with diisocyanates to chemically bond them to polyurethane coating materials. In addition, the presence of the active C=C group can react with acrylate monomers and be chemically bonded to acrylate coating materials, giving them long-lasting bactericidal properties and showing great application prospects.

[0008] The objective of this invention is achieved as follows:

[0009] A method for preparing a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool includes the following steps:

[0010] S1. Synthesis of chloroacetate: Chloroacetyl chloride was added to a flask containing solvent. Then, a certain amount of linalool was weighed in a beaker, and a certain amount of solvent was added to the beaker. After stirring evenly, the first mixture was obtained. The first mixture was poured into a constant pressure dropping funnel, and the first mixture in the constant pressure dropping funnel was added dropwise to the flask. The mixture was stirred at room temperature for 3 hours to obtain the second mixture.

[0011] The reaction was monitored by thin-layer chromatography until linalool was completely reacted, at which point the reaction was stopped. Then, 20 mL of distilled water was added to the second mixture and the mixture was stirred for 0.5 h to remove unreacted chloroacetyl chloride. The aqueous phase was then extracted twice with dichloromethane, and the first organic phase was collected through a separatory funnel. The first organic phase was then subjected to reduced pressure rotary evaporation on a rotary evaporator to obtain the first crude product. Finally, the first crude product was purified by rapid column chromatography to obtain the target product, chloroacetate.

[0012] Synthesis of S2, ditertiary amine: Itaconic acid, N,N-dimethylethanolamine, and the catalyst p-toluenesulfonic acid were added to a certain amount of solvent and reacted at 140 °C to obtain a reaction solution. The reaction was monitored by thin-layer chromatography until itaconic acid was completely reacted and then the reaction was stopped. After cooling to room temperature, brine was added to the reaction solution for washing, and the aqueous phase was extracted with ethyl acetate to obtain a second organic phase. The second organic phases were then combined and dried with anhydrous sodium sulfate for 3 hours until the aqueous phase completely disappeared. The mixture was filtered and concentrated under reduced pressure until no liquid dripped from the funnel to obtain a second crude product. Finally, the second crude product was purified by rapid column chromatography to obtain the target product, ditertiary amine.

[0013] S3. Synthesis of a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool: The ditertiary amine obtained in S2 and the chloroacetic acid ester obtained in S1, along with a solvent, were added to a reactor and refluxed at 100°C for 8-12 h to obtain the synthesized product.

[0014] Excess solvent was removed by rotary evaporation under reduced pressure to obtain a viscous substance. The viscous substance was then washed 2-3 times with anhydrous diethyl ether. The washed viscous substance was purified by recrystallization (ethanol:acetone = 1:9, V / V) to obtain a third crude product. Finally, the third crude product obtained by recrystallization was placed in a vacuum drying oven (50 °C) and dried for 12 h to obtain a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool.

[0015] In S1, the molar ratio of linalool to chloroacetyl chloride is 1.0:(1.1~1.5), the solvent is either dichloromethane or ethyl acetate, and the eluent in the rapid column chromatography is a mixture of petroleum ether and ethyl acetate with a volume ratio of 10:1.

[0016] The synthetic route for chloroacetate in S1 is as follows: .

[0017] In S2, the molar ratio of itaconic acid to N,N-dimethylethanolamine is 1.0:(2.1~2.5), the solvent is either xylene or isopropanol, and the eluent in the rapid column chromatography is a mixture of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 1:1.

[0018] The synthetic route for the ditertiary amine in S2 is as follows:

[0019] .

[0020] In step S3, the molar ratio of the ditertiary amine to chloroacetic acid ester is 1.0:(2.1~2.5), and the solvent is one or more of acetone, isopropanol, and acetonitrile.

[0021] In S3, the synthetic route for the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool is as follows:

[0022] .

[0023] The beneficial effects of this invention are:

[0024] 1. This invention introduces plant-derived linalool into the structure of gemini quaternary ammonium salts through chemical bonding. The nitrogen ions on the gemini quaternary ammonium salts adsorb onto the negatively charged bacterial surface, and the hydrophobic interaction with the plant-derived linalool produces an excellent "adhesion-killing" highly efficient synergistic antibacterial effect, providing a new idea and technology for the high-value resource utilization of natural products. Furthermore, starting from the source of the raw materials required for synthesis, the natural product plant-derived linalool replaces the traditional petroleum-based chemical long-chain alcohol, effectively reducing the carbon footprint of gemini quaternary ammonium salt products.

[0025] 2. In response to the functional requirements of geminal quaternary ammonium salts in practical application scenarios, this invention provides new ideas and methods for the precise design of efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salts based on molecular structure.

[0026] 3. This invention can shorten the research and development cycle, eliminating the need for research work on synthesizing a series of geminal quaternary ammonium salts to screen for high-performance geminal quaternary ammonium salts.

[0027] 4. The gemini quaternary ammonium salt bactericide prepared by this invention has the characteristics of good water solubility, high efficiency, low toxicity, stable physicochemical properties, and easy biodegradability. It can be used in the same bath as other leather auxiliaries without losing its bactericidal effect, and will not cause cumulative pollution to the environment after being discharged into water bodies.

[0028] 5. The presence of the active group C=C in the molecular structure of the gemini quaternary ammonium salt of the present invention increases the chemical activity of the gemini quaternary ammonium salt, enabling it to be further chemically bonded to the leather raw material, thus endowing it with long-lasting bactericidal properties.

[0029] 6. Compared with existing leather bactericides, the gemini quaternary ammonium salt bactericide prepared in this invention has an inductive effect of four ester groups in its molecular structure that increases the positive charge density on nitrogen atoms, which helps it to adsorb onto the bacterial surface and exhibits a significant bactericidal effect. Attached Figure Description

[0030] Figure 1 The infrared spectrum of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt synthesized in this invention;

[0031] Figure 2 The graph shows the results of the determination of the minimum inhibitory concentration (MIC) of trans-1,4-bis[2-(decyloxymethyl)diethyl]-2-butene dibromide against Escherichia coli in Experimental Example 2 of the present invention.

[0032] Figure 3 The graph shows the results of the determination of the minimum inhibitory concentration (MIC) of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt against Escherichia coli in Experiment Example 2 of this invention.

[0033] Figure 4 This is a graph showing the percentage of biodegradation of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt (10QAC) and sodium benzoate of this invention.

[0034] Figure 5 The image shows the cell morphology of mouse fibroblast L929 cells in a culture medium containing the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt (10QAC) and dodecyl dimethyl benzyl ammonium chloride (1227) of this invention.

[0035] Figure 6 This is a graph showing the cell viability of mouse fibroblast L929 cells in a culture medium containing the highly efficient, low-toxicity, and environmentally friendly Gemini quaternary ammonium salt (10QAC) and dodecyl dimethyl benzyl ammonium chloride (1227) of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Example 1:

[0038] Preparation of S1 and chloroacetic acid ester

[0039] 2.71 g (24 mmol) of chloroacetyl chloride was weighed and added to a flask containing 30 mL of dichloromethane. Then, 3.08 g (20 mmol) of linalool was weighed using a beaker and 30 mL of dichloromethane was added to the beaker. After stirring, a first mixture was obtained. The first mixture was poured into a constant pressure dropping funnel, and then the first mixture in the constant pressure dropping funnel was added dropwise to the flask. The mixture was stirred at room temperature for 3 hours, and the reaction was monitored by thin-layer chromatography until the linalool was completely reacted. The reaction was then stopped. 20 mL of distilled water was added to the reaction system and stirred for 0.5 h to remove unreacted chloroacetyl chloride. The aqueous phase was then extracted twice with dichloromethane, and the first organic phase was collected through a separatory funnel. The first organic phase was subjected to rotary evaporation under reduced pressure to obtain the crude product. Finally, the crude product was purified by rapid column chromatography (petroleum ether: ethyl acetate = 10:1, V / V) to obtain the target product chloroacetate.

[0040] Preparation of S2 and ditertiary amines

[0041] 2.6 g (20 mmol) of itaconic acid, 3.92 g (44 mmol) of N,N-dimethylethanolamine, and 0.52 g (3 mmol) of p-toluenesulfonic acid catalyst were added to 60 mL of xylene and reacted at 140 °C to obtain a reaction solution. The reaction was monitored by thin-layer chromatography until the itaconic acid was completely reacted, and then the reaction was stopped. After cooling to room temperature, the reaction solution was washed with brine, and the aqueous phase was extracted with ethyl acetate to obtain a second organic phase. The second organic phases were then combined and dried with anhydrous sodium sulfate for 3 hours until the aqueous phase completely disappeared. The solution was filtered and concentrated under reduced pressure until no liquid dripped from the funnel to obtain a second crude product. Finally, the second crude product was obtained by rapid column chromatography (petroleum ether:ethyl acetate = 1:1, V / V). The second crude product was purified to obtain the target product, ditertiary amine.

[0042] S3. Preparation of highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salts

[0043] 2.72 g (10 mmol) of ditertiary amine, 5.07 g (22 mmol) of chloroacetate, and 60 mL of isopropanol were added to the reactor. The mixture was refluxed at 100 °C for 12 h to obtain the synthesized product. Excess solvent was removed by rotary evaporation under reduced pressure to obtain a viscous substance. The viscous substance was then washed 2-3 times with anhydrous diethyl ether. The washed viscous substance was further purified into a third crude product by recrystallization (ethanol:acetone = 1:9, V / V). Finally, the third crude product obtained by recrystallization was dried in a vacuum drying oven (50 °C) for 12 h to obtain the target product, a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt.

[0044] Example 2

[0045] Preparation of S1 and chloroacetic acid ester

[0046] 2.37 g (21 mmol) of chloroacetyl chloride was weighed and added to a flask containing 30 mL of ethyl acetate. Then, 2.31 g (15 mmol) of linalool was weighed using a beaker and added to the beaker along with 30 mL of ethyl acetate. After stirring, a first mixture was obtained. The first mixture was poured into a constant pressure dropping funnel, and then the first mixture in the constant pressure dropping funnel was added dropwise to the flask. The mixture was stirred at room temperature for 3 hours to obtain a second mixture. The reaction was monitored by thin-layer chromatography until the linalool was completely reacted, and then the reaction was stopped. Subsequently, 20 mL of distilled water was added to the second mixture and stirred for 0.5 h to remove unreacted chloroacetyl chloride. Then, the aqueous phase was extracted twice with ethyl acetate, and the first organic phase was collected through a separatory funnel. The organic phase was then subjected to rotary evaporation under reduced pressure to obtain the first crude product. Finally, the first crude product was purified by rapid column chromatography (petroleum ether: ethyl acetate = 10:1, V / V) to obtain the target product chloroacetate.

[0047] Preparation of S2 and ditertiary amines

[0048] 3.9 g (30 mmol) of itaconic acid, 5.88 g (66 mmol) of N,N-dimethylethanolamine, and 0.77 g (4.5 mmol) of p-toluenesulfonic acid catalyst were added to 60 mL of xylene and reacted at 140 °C to obtain a reaction solution. The reaction was monitored by thin-layer chromatography until the itaconic acid reacted completely, at which point the reaction was stopped. After cooling to room temperature, the reaction solution was washed with brine, and the aqueous phase was extracted with ethyl acetate to obtain a second organic phase. The second organic phases were then combined and dried with anhydrous sodium sulfate for 3 hours until the aqueous phase disappeared completely. The solution was filtered and concentrated under reduced pressure until no liquid dripped from the funnel to obtain a second crude product. Finally, the crude product was purified by rapid column chromatography (petroleum ether:ethyl acetate = 1:1, V / V) to obtain the target product, ditertiary amine.

[0049] S3. Preparation of highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salts based on plant-derived linalool

[0050] 4.09 g (15 mmol) of ditertiary amine, 7.6 g (33 mmol) of chloroacetate, and 60 mL of acetonitrile were added to the reactor. The mixture was refluxed at 100 °C for 12 h to obtain the synthesized product. Excess solvent was removed by rotary evaporation under reduced pressure to obtain a viscous substance. The viscous substance was then washed 2-3 times with anhydrous diethyl ether to obtain the third crude product. The third crude product was further purified by recrystallization (ethanol:acetone = 1:9, V / V). Finally, the third product obtained by recrystallization was dried in a vacuum drying oven (50 °C) for 12 h to obtain the target product, a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool.

[0051] Experimental Example 1:

[0052] Infrared spectral characterization of the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt of this invention

[0053] The samples were tested using the KBr pellet method, with other conditions including a resolution of 4 cm⁻¹. -1 The number of samples collected was 32, and the collection range was 500~4000cm. -1 The high-efficiency, low-toxicity, and environmentally friendly gemini quaternary ammonium salt sample of the present invention is a solid powder. Approximately 1 mg of the sample is weighed and thoroughly ground with KBr. After being mixed evenly, it is pressed into a dense thin sheet using a tablet press before testing.

[0054] Figure 1 The infrared spectrum of the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt synthesized in this patent is shown, with the value at 2918 cm⁻¹. -1 The absorption peak at 2851 cm⁻¹ is the stretching vibration absorption peak of -CH₃. -1 The absorption peak at 1740 cm⁻¹ is the stretching vibration absorption peak of -CH₂.-1 The peak at 1617 cm⁻¹ represents the stretching vibration absorption peak of the -C=O group in the ester group. -1 The absorption peak at 1221 cm⁻¹ is the C=C stretching vibration absorption peak. -1 The absorption peak at 726 cm⁻¹ corresponds to the stretching vibration of the -CO group in the ester group. -1 The absorption peak is due to the in-plane rocking vibration when four or more -CH2 groups form a straight chain. This suggests that chloroacetate and the ditertiary amine undergo a quaternization reaction to generate the target product, a highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt.

[0055] Experimental Example 2:

[0056] The minimum inhibitory concentration determination of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt and other gemini quaternary ammonium salts (trans-1,4-bis[2-(decyloxymethyl)diethyl]-2-butene dibromide) of this invention.

[0057] Preparation of bacterial suspension: 200 μL of bacterial inoculum was inoculated into 100 mL of sterile nutrient broth and incubated with shaking at 37 ℃ and 120 r / min for 12–15 h. The concentration of the bacterial suspension at this point was 10. 10 ~10 11 CFU / mL.

[0058] The highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt synthesized in this invention was prepared with sterile water to a concentration of 1.2 × 10⁻⁶. -3 Solution M. Take a sterilized 12-well bacterial culture plate, numbering the wells 0-11. Well 0 contains only 4 mL of nutrient broth as a negative control. Wells 1-10 contain nutrient broth, the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt solution synthesized in this invention, and E. coli suspension as the experimental group. Add 2 mL of nutrient broth to wells 1-10, then add 2 mL of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt solution synthesized in this invention to well 1. Stir well, then use a pipette to add 2 mL of the mixture from well 1 to well 2, and so on, serially diluting until well 10. After mixing, discard the 2 mL mixture. Thus, the concentration of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt synthesized in this invention decreases in a two-fold gradient in each well. Next, add 0.2 mL of bacterial suspension to each of wells 1-10, and finally add nutrient broth to bring the total to 4 mL. Add 3.8 mL of nutrient broth and 0.2 mL of E. coli suspension to well 11. mL of bacterial suspension was used as a positive control, at which point the bacterial concentration in the mixture was 10. 7 ~10 8CFU / mL. Finally, the 12-well bacterial culture plate was placed in a constant temperature incubator at 37 ℃ for 24 h, and the results were observed and photographed. There should be no bacterial growth in the negative control wells, and the bacteria should grow well in the positive control wells. The experimental groups were observed by turbidimetry, starting from well number 10 and comparing sequentially. The concentration of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt synthesized in this invention corresponding to the first well that became clear and transparent was the minimum inhibitory concentration (MIC).

[0059] Other geminal quaternary ammonium salts (trans-1,4-bis[2-(decyloxymethyl)diethyl]-2-butene dibromide) were prepared with sterile water to a concentration of 1.2 × 10⁻⁶. -3 Solution M. Take a sterilized 12-well bacterial culture plate, numbering the wells 0-11. Well 0 contains only 4 mL of nutrient broth as a negative control. Wells 1-10 contain nutrient broth, trans-1,4-bis[2-(decoxymethyl)diethyl]-2-butene dibromide solution, and Escherichia coli suspension as the experimental group. Add 2 mL of nutrient broth to wells 1-10, then add 2 mL of trans-1,4-bis[2-(decoxymethyl)diethyl]-2-butene dibromide solution to well 1. Mix well, then pipette 2 mL of the mixture from well 1 to well 2, and so on, serially diluting until well 10 is reached. Discard the diluted solution. The concentration of trans-1,4-bis[2-(decoxyylmethylene)diethyl]-2-butene dibromide in each well decreased in a two-fold gradient after dilution. Then, 0.2 mL of bacterial suspension was added to each of wells 1 through 10, and finally, nutrient broth was added to bring the total volume to 4 mL. Well 11 was added with 3.8 mL of nutrient broth and 0.2 mL of bacterial suspension as a positive control. At this point, the bacterial concentration in the mixture was 10⁻⁶. 7 ~10 8 CFU / mL. Finally, the 12-well bacterial culture plate was placed in a constant temperature incubator at 37 ℃ for 24 h, and the results were observed and photographed. There should be no bacterial growth in the negative control wells, and the bacteria should grow well in the positive control wells. The experimental groups were observed by turbidimetric method, starting from well number 10 and comparing sequentially. The concentration of trans-1,4-bis[2-(decyloxymethyl)diethyl]-2-butene dibromide corresponding to the first well that became clear and transparent was the minimum inhibitory concentration (MIC).

[0060] Depend on Figure 2 and Figure 3It was observed that the bacteria in the positive control (nutrient broth medium and bacterial suspension) grew well, and the mixture was turbid, while the negative control (nutrient broth medium only) was clear, indicating that the bacteria were biologically active and that there was no contamination during the operation. The MIC values ​​of the two geminal quaternary ammonium salts (the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt of this invention and trans-1,4-bis[2-(decoxyylmethylene)diethyl]-2-butene dibromide) against *Escherichia coli* were determined using turbidimetric assay. The minimum inhibitory concentration (MIC) of trans-1,4-bis[2-(decoxyylmethylene)diethyl]-2-butene dibromide against *Escherichia coli* was 18.75 × 10⁻⁶. -6 The minimum inhibitory concentration (MIC) of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt of this invention against Escherichia coli is 2.34 × 10⁻⁶ mol / L. -6 mol / L. The smaller the MIC value, the better the antibacterial performance of the antibacterial agent. Therefore, the above test results show that the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt synthesized in this invention has stronger antibacterial performance than trans-1,4-bis[2-(decoxyylmethylene)diethyl]-2-butene dibromide.

[0061] Experimental Example 3:

[0062] Biodegradability test of the highly efficient, low-toxicity, and environmentally friendly geminal quaternary ammonium salt prepared by this invention

[0063] 3.1 Preparation of culture medium

[0064] (1) Phosphate buffer: Accurately weigh 28.50 g dipotassium hydrogen phosphate (K2HPO4·3H2O), 8.50 g potassium dihydrogen phosphate (KH2PO4), 67.20 g disodium hydrogen phosphate (Na2HPO4·12H2O) and 0.50 g ammonium chloride (NH4Cl), dissolve in distilled water and bring to a final volume of 1 L. The pH of the solution is 7.4.

[0065] (2) Magnesium sulfate solution: Accurately weigh 22.50 g of magnesium sulfate heptahydrate (MgSO4·7H2O), dissolve it in distilled water and bring the volume up to 1 L.

[0066] (3) Ferric chloride solution: Accurately weigh 0.25 g of ferric chloride hexahydrate (FeCl3·6H2O), dissolve it in distilled water, and make up to 1 L. Add 0.05 mL of concentrated hydrochloric acid for storage.

[0067] (4) Calcium chloride solution: Accurately weigh 27.50 g of anhydrous calcium chloride (CaCl2), dissolve it in distilled water and bring the volume up to 1L.

[0068] Take 1 mL of phosphate buffer, calcium chloride solution, magnesium sulfate solution and ferric chloride solution respectively, and dilute to 1 L with 800 mL of distilled water.

[0069] 3.2 Treatment of inoculum

[0070] The inoculum used in the experiment was derived from the secondary effluent of the leather tanning plant at Shandong Zibo Dahuan Jiubaoen Leather Group Co., Ltd., which underwent biochemical treatment. (This secondary effluent, after primary treatment, uses biochemical methods to remove dissolved organic matter, resulting in lower organic matter content and no large suspended solids, thus avoiding interference from physical adsorption.) The inoculum was aerated at room temperature for 3 days, filtered through filter paper, and the resulting filtrate was used as the experimental inoculum. (The microorganisms involved in biodegradation mainly included *Xanthomonas*, *Pseudomonas*, and *Aeromonas*.)

[0071] 3.3 Preparation of the reference solution

[0072] Weigh 1 g (accurate to 0.001 g) of sodium benzoate, a readily degradable chemical, and dissolve it in the test culture medium to prepare a 1 g / L sodium benzoate stock solution. Before conducting the sealed bottle test, dilute the 1 g / L sodium benzoate stock solution to a 2 mg / L sodium benzoate solution using the test culture medium.

[0073] 3.4 Preparation of test solution

[0074] Weigh 1 g (accurate to 0.001 g) of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt solid powder synthesized in this patent and dissolve it in the test culture medium to prepare a 1 g / L stock solution. Before conducting the sealed bottle test, dilute the 1 g / L stock solution to a 2 mg / L solution using the test culture medium.

[0075] 3.5 Closed Bottle Test

[0076] (1) Prepare 30 cleaned and dried 250 mL stoppered BOD culture flasks (sodium benzoate group, the high-efficiency, low-toxicity and environmentally friendly gemini quaternary ammonium salt group synthesized in this invention (10QAC group), blank group, and 2 parallel samples in each group), and number them in order.

[0077] (2) Add 350 μL of inoculum to all stoppered BOD culture flasks. Place one end of the tubing into the test culture medium and the other end into the bottom of the stoppered BOD culture flask of the blank group. Use a siphon method to draw the test culture medium into the BOD culture flask (to prevent interference from oxygen in the air), so that the test culture medium fills the BOD culture flask. In addition, fill the reference group BOD culture flask with 2 mg / L sodium benzoate solution and the test group culture flask with 2 mg / L of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt solution synthesized in this invention. Stopper the flasks, ensuring that no air bubbles adhere, and form a liquid seal at the flask opening. o Incubate at C under light for 28 days, and regularly replenish the test culture medium at the bottle opening to prevent O2 from entering from the air.

[0078] (3) During the 28-day culture period, two parallel samples were taken at fixed time intervals (1 time / week) and their dissolved oxygen concentration was measured by a dissolved oxygen meter.

[0079] Depend on Figure 4 As shown in the graph, the biodegradation rate of the reference material (sodium benzoate) was 77.56% (not less than 60%) after 14 days, meeting the quality assurance requirements of the national standard GB / T 21831-2008, "Test Method for Rapid Biodegradability of Chemicals in Closed Bottles". According to the OECD's chemical testing guidelines, a chemical is considered readily biodegradable if its biodegradation percentage reaches 60% within 28 days. The graph also shows that the biodegradation rate of the high-efficiency, low-toxicity, and environmentally friendly gemini quaternary ammonium salt group (10QAC group) exceeded 60% on day 21, indicating that the high-efficiency, low-toxicity, and environmentally friendly gemini quaternary ammonium salt is indeed readily biodegradable.

[0080] Experimental Example 4:

[0081] In vitro cytotoxicity test of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt prepared by this invention.

[0082] 4.1 Resuscitation of mouse fibroblasts (L929 cells)

[0083] Preheat the prepared serum-containing high-glucose medium (high-glucose medium: fetal bovine serum: antibiotics = 100:10:1) in a 37 °C water bath. Remove the cell cryopreservation tubes from the liquid nitrogen tank (-196 °C) and immediately place them in a 37 °C water bath for thawing. Then, transfer the thawed cell solution to a 15 mL centrifuge tube and add a small amount of serum-containing high-glucose medium to dilute the dimethyl sulfoxide (DMSO) preservation solution in the cell cryopreservation solution. Centrifuge the 15 mL tubes at 1000 rpm for 5 min. After removing the centrifuge tubes, discard the supernatant and add 1 mL of serum-containing high-glucose medium. Gently pipette the tubes to detach the L929 cells. Transfer the entire cell solution to a disposable cell culture dish, add 9 mL of serum-containing high-glucose medium, and incubate in a CO2 incubator (5% CO2, 37 °C) for 12 h. Change the medium afterward to remove any residual DMSO preservation solution.

[0084] 4.2 1:3 passage culture of L929 cells

[0085] Prepare alcohol swabs, an alcohol lamp, high-glucose culture medium, trypsin, antibiotics, fetal bovine serum, PBS, pipettes, pipette tips, and disposable culture dishes. PBS and pipette tips must be autoclaved for 30 minutes before use. Fetal bovine serum and trypsin must be filtered with a disposable 0.22 μm water filter to remove bacteria and impurities before use. All experimental items should be sprayed with alcohol before being placed in a sterile operating table. Reagent bottles must be sterilized by baking with an alcohol lamp before unsealing. Remove the revived L929 cells that have filled the bottom of the culture dish and observe them under an inverted microscope, taking photographs. Place the cell culture solution on a sterile operating table, remove the original culture medium, wash the cells 1-2 times with PBS, then add 1 mL of trypsin digestion solution. After about 10 seconds, swirl the culture dish in a cross shape until the cells shrink inward. Observe it under an inverted microscope; if bright spots appear, it indicates that the cells have been digested. Next, remove the trypsin digestion solution, add 3 mL of serum-containing high-glucose medium to stop digestion, and gently pipette the cells (moving the pipette tip along the bottom of the culture dish to ensure all cells are dislodged) to evenly disperse the cells in the culture medium. Finally, transfer 1 mL of the cell suspension to a new culture dish, add 10 mL of serum-containing medium, gently shake in a cross shape to mix, and incubate in a CO2 incubator (5% CO2, 37 °C) for 2–3 days.

[0086] 4.3 In vitro cytotoxicity test

[0087] First, weigh the samples of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt (10QAC) and dodecyl dimethyl benzyl ammonium chloride (1227) synthesized in this invention, and dissolve them separately in a high-glucose medium containing serum (high-glucose medium: fetal bovine serum: antibiotics = 100:10:1) to prepare a solution with a concentration of 10. -3 M's patented synthesis of highly efficient, low-toxicity, and environmentally friendly Gemini quaternary ammonium salt (10QAC) and dodecyl dimethyl benzyl ammonium chloride (1227) stock solutions was stored at 4 °C for later use. During cytotoxicity testing, the stock solutions were diluted to the required concentration using a serum-containing high-glucose medium, and then bacteria and other impurities were removed using a 0.22 μm filter membrane in a clean bench.

[0088] L929 cells that have been revived and passaged to the logarithmic growth phase were used as experimental cells, at which point the cell density was greater than 1 × 10⁻⁶. 5Cell / mL. 0.1 mL of cell suspension was seeded into 20 wells of a sterile 96-well plate and cultured in a CO2 incubator (37 °C, 5% CO2) for 24 h to allow cell adhesion. Then, the old culture medium was removed from 15 wells, and the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt solution synthesized in this invention was added to each well. The plates were then incubated in a CO2 incubator (37 °C, 5% CO2) for 48 h. The 96-well plate was removed, the culture medium was aspirated, and serum-containing high-glucose medium and CCK-8 reagent were added to the wells at a ratio of 9:1. After incubation for 90 min, the absorbance of each well was measured at 570 nm, and cell viability was calculated. The negative control was complete culture medium, and the positive control was complete culture medium and cells. Cell viability was calculated using the following formula:

[0089]

[0090] Among them, OD t OD p and OD n These represent the absorbance values ​​of the test group, positive control group, and negative control group, respectively. The relationship between cell viability and cytotoxicity grading is based on the United States Pharmacopeia, as shown in the table below. The evaluation criteria are: grade 0 or 1 is considered acceptable; grade 2 should be evaluated in conjunction with cell morphology; and grades 3, 4, and 5 are considered unacceptable.

[0091] from Figure 5 As can be seen, the mouse fibroblast L929 cells in the culture medium of the high-efficiency, low-toxicity and environmentally friendly gemini quaternary ammonium salt (concentrations of 10 μM and 40 μM) synthesized in this invention have the same intact and normal morphology as normally growing L929 cells (blank control), exhibiting a unique cell morphology (spindle-shaped or flattened star-shaped).

[0092] from Figure 6 As can be seen, the survival rate of L929 cells in the culture medium of the highly efficient, low-toxicity, and environmentally friendly gemini quaternary ammonium salt (concentrations of 10 μM and 40 μM) synthesized in this invention is above 90%, indicating that the gemini quaternary ammonium salt at this concentration does not produce toxic effects on cells. The main reason is that at this concentration, the balance between the intracellular oxidative stress effect and the antioxidant defense system of L929 cells is not broken, thus no cytotoxicity is observed. However, when the concentration of dodecyl dimethyl benzyl ammonium chloride (1227) is 40 μM, the cell survival rate is only 9.7%, indicating that it has a significant toxic effect on cells. At this time, the balance between the intracellular oxidative stress effect and the antioxidant defense system of L929 cells is broken, with a tendency towards oxidative stress, leading to the formation of toxic reactive oxygen species, causing the degeneration of normal L929 cell function, and ultimately leading to cell death.

Claims

1. A method for preparing a low-toxicity, environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool, characterized in that, Includes the following steps: S1. Synthesis of chloroacetate: Chloroacetyl chloride was added to a flask containing solvent. Then, a certain amount of linalool was weighed in a beaker, and a certain amount of solvent was added to the beaker. After stirring evenly, the first mixture was obtained. The first mixture was poured into a constant pressure dropping funnel, and the first mixture in the constant pressure dropping funnel was added dropwise to the flask. The mixture was stirred at room temperature for 3 hours to obtain the second mixture. The reaction was monitored by thin-layer chromatography until linalool was completely reacted, at which point the reaction was stopped. Then, 20 mL of distilled water was added to the second mixture and the mixture was stirred for 0.5 h to remove unreacted chloroacetyl chloride. The aqueous phase was then extracted twice with dichloromethane, and the first organic phase was collected through a separatory funnel. The first organic phase was then subjected to rotary evaporation under reduced pressure to obtain the first crude product. Finally, the first crude product was purified by rapid column chromatography to obtain the target product, chloroacetate. Synthesis of S2, ditertiary amine: Itaconic acid, N,N-dimethylethanolamine, and the catalyst p-toluenesulfonic acid were added to a certain amount of solvent and reacted at 140 °C to obtain a reaction solution. The reaction was monitored by thin-layer chromatography until itaconic acid was completely reacted and then the reaction was stopped. After cooling to room temperature, brine was added to the reaction solution for washing, and the aqueous phase was extracted with ethyl acetate to obtain a second organic phase. The second organic phases were then combined and dried with anhydrous sodium sulfate for 3 hours until the aqueous phase completely disappeared. The mixture was filtered and concentrated under reduced pressure until no liquid dripped from the funnel to obtain a second crude product. Finally, the second crude product was purified by rapid column chromatography to obtain the target product, ditertiary amine. S3. Synthesis of low-toxicity and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool: The ditertiary amine obtained in S2 and the chloroacetic acid ester obtained in S1 and the solvent were added to the reactor and refluxed at 100°C for 8-12 h to obtain the synthesized product. Excess solvent was removed by rotary evaporation under reduced pressure to obtain a viscous substance. The viscous substance was then washed 2-3 times with anhydrous diethyl ether. The washed viscous substance was purified by recrystallization to obtain a third crude product. Finally, the third crude product obtained by recrystallization was placed in a vacuum drying oven and dried for 12 h to obtain a low-toxicity and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool. The synthetic route for chloroacetate in S1 is as follows: ; The synthetic route for the ditertiary amine in S2 is as follows: ； In S3, the synthetic route for the low-toxicity and environmentally friendly gemini quaternary ammonium salt based on plant-derived linalool is as follows: .

2. The method for preparing low-toxicity and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool according to claim 1, characterized in that, In S1, the molar ratio of linalool to chloroacetyl chloride is 1.0:(1.1~1.5), the solvent is either dichloromethane or ethyl acetate, and the eluent in the rapid column chromatography is a mixture of petroleum ether and ethyl acetate with a volume ratio of 10:

1.

3. The method for preparing low-toxicity and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool according to claim 1, characterized in that, In S2, the molar ratio of itaconic acid to N,N-dimethylethanolamine is 1.0:(2.1~2.5), the solvent is either xylene or isopropanol, and the eluent in the rapid column chromatography is a mixture of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 1:

1.

4. The method for preparing low-toxicity and environmentally friendly geminal quaternary ammonium salt based on plant-derived linalool according to claim 1, characterized in that, In step S3, the molar ratio of the ditertiary amine to chloroacetic acid ester is 1.0:(2.1~2.5), and the solvent is one or more of acetone, isopropanol, and acetonitrile.

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