Density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal as well as preparation method and application of carboxymethyl chitosan Schiff base

Abstract

The invention belongs to the technical field of supramolecular chemistry, and particularly relates to a density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal as well as a preparation method and application of the density-controllable carboxymethyl chitosan Schiff base. The preparation method comprises the following steps: carrying out nucleophilic addition-elimination reaction on amino compound carboxymethyl chitosan and different moles of trans-2-hexenal to prepare Schiff bases with different densities; the Schiff base can be used for preparing insecticide and bactericide products.

Description

Technical Field

[0001] The invention belongs to the technical field of supramolecular chemistry, and in particular relates to a carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density, and a preparation method and application thereof. Background Art

[0002] Trans-2-hexenal is one of the main volatile substances in fresh fruits, and has high antibacterial and insecticidal activities. In addition to achieving the purpose of antibacterial by damaging the cell membrane integrity and cell wall of pathogenic bacteria, the high electrophilicity of trans-2-hexenal also enables it to consume the nucleophilic molecules of pathogenic bacteria cells through Michael addition reaction, thereby indirectly causing the consumption of reducing agents in cells and further causing oxidative stress, thus playing an antibacterial effect. Trans-2-hexenal may act on the sensory organs of pests. As the treatment time increases, it can penetrate the protective epidermis and enter the insect body, destroy its body wall structure, and dissolve the cuticle, which is conducive to the penetration of essential oils, enhances the penetration, absorption, transport and entry of active components into stomata, and hinders the normal physiological and biochemical metabolism of pests, and significantly enhances the inhibitory effect. Therefore, based on the high antibacterial and insecticidal activities of trans-2-hexenal, trans-2-hexenal may be a good natural antibacterial and insecticide, and has broad application potential in disease control.

[0003] However, the boiling point of trans-2-hexenal is only 47°C, and it is very easy to evaporate in the natural environment, making it difficult to provide long-term and stable antibacterial protection for crops. The existing common treatment methods include short-term closed fumigation treatment and cyclodextrin embedding. Among them, the use scenarios of short-term closed fumigation technology are limited, and the operation requirements are high, and additional processing time is added; although the cyclodextrin embedding method can delay the release rate of trans-2-hexenal, it cannot accurately control its release amount, and it is still insufficient in environmental response. In addition, the effect of trans-2-hexenal on pathogens is closely related to its dosage. If the content is too low, the antibacterial effect is not obvious, and if the content is too high, it will damage the fruit. In summary, the development of a responsive trans-2-hexenal sustained-release agent has great application value.

[0004] As a typical active electrophilic substance, trans-2-hexenal can undergo nucleophilic addition-elimination reaction with substances containing amino groups (amino acids, chitosan, etc.) or methylene amino groups to form a class of organic compounds containing dynamic imine bonds. 2 The addition reaction of the compound is reversible and pH-dependent, so the prepared Schiff base derivative has pH-responsive properties. 2Among the compounds, carboxymethyl chitosan, as a widely available high-molecular compound, contains a large number of active amino groups and has good imine cross-linking reactivity. The substance is biodegradable and has certain biological activities such as antifungal, antibacterial, antiviral and nematicidal activities, and can improve the tolerance of plants to abiotic stresses such as drought, high temperature, salt and heavy metals.

[0005] Therefore, the present invention selects carboxymethyl chitosan as a safe and effective representative substance containing amino compounds to react with trans-2-hexenal, and uses dynamic imine bonds to prepare three trans-2-hexenal Schiff bases with reaction molar ratios. The three prepared Schiff base substances have different internal structural densities, which can control the internal cross-linking degree of the Schiff base product, thereby controlling the release rate of the aldehyde. The products with different reaction molar ratios are used in combination to achieve the effect of "preventing before the disease occurs and curing the disease quickly". The prepared pH-responsive trans-2-hexenal Schiff base sustained-release agent has good indoor toxicity effects on wheat fusarium and pepper phytophthora; at the same time, it also shows good effects on small pests thrips and cinnabarinus spider mites. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density, and a preparation method and application thereof.

[0007] The present invention is achieved through the following technical solutions: A density-controlled carboxymethyl chitosan Schiff base based on trans-2-hexenal, the structural formula of which is as follows: It has the structural formula shown in formula (1): (1).

[0008] The present invention also provides a method for preparing the carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density, comprising the following steps: S1, dissolving carboxymethyl chitosan in water, heating and stirring to dissolve the carboxymethyl chitosan, to obtain a carboxymethyl chitosan aqueous solution, wherein the mass ratio of carboxymethyl chitosan to water is 2-4% (w / w); S2, dissolving an appropriate amount of trans-2-hexenal in ethanol, and adding the solution to the carboxymethyl chitosan solution obtained in step (1), followed by heating and stirring for 5 h to 12 h at a reaction temperature of 30° C. to 60° C.; S3. The product obtained in step (2) is filtered, Soxhlet extracted and vacuum freeze-dried in sequence to obtain trans-2-hexenal Schiff base.

[0009] Furthermore, in step (1), the mass ratio of carboxymethyl chitosan to distilled water is 2% (w / w).

[0010] Furthermore, the reaction molar ratio of the carboxymethyl chitosan and trans-2-hexenal is 1: 1-4.

[0011] Furthermore, the ethanol used in step (2) is analytically pure, the reaction time is 5 h, and the reaction temperature is 60° C. Furthermore, the molecular weight of the carboxymethyl chitosan is 240 kDa.

[0012] The present invention also provides application of the carboxymethyl chitosan Schiff base based on trans-2-hexenal in preparing an antibacterial agent.

[0013] The present invention also provides application of the carboxymethyl chitosan Schiff base based on trans-2-hexenal in preparing insecticides.

[0014] Compared with the prior art, this application has the following beneficial effects: (1) The present invention utilizes the reversible Michael addition reaction of the natural volatile substance trans-2-hexenal in the fruit and the natural product carboxymethyl chitosan, thereby overcoming the application difficulty of the pure product being extremely volatile, and achieving the long-term stable antibacterial effect of trans-2-hexenal; (2) The Schiff base substance provided by the present invention has the property of pH stimulus-responsive release, achieving the effect of slow release of trans-2-hexenal, overcoming the shortcomings of existing cyclodextrin encapsulation technologies such as inability to accurately control release, and improving the antibacterial effect; (3) Carboxymethyl chitosan reacts with different moles of trans-2-hexenal to prepare Schiff base materials with different internal densities and various aldehyde release rates. The density can be controlled to achieve the desired application effect; (4) The present invention utilizes amino compound carboxymethyl chitosan and different moles of trans-2-hexenal to carry out nucleophilic addition-elimination reaction to prepare Schiff bases with different densities. The preparation method is simple, easy to operate, easy to implement, mild reaction conditions, and good reproducibility; (5) The Schiff base substance prepared by the present invention has a good antibacterial effect in the prevention and control of diseases represented by wheat fusarium and pepper phytophthora; (6) The Schiff base substance prepared by the present invention has a good insecticidal effect in the prevention and control of pests represented by small pests such as thrips and cinnabarinus spider mites.

[0015] (7) In view of the problem that trans-2-hexenal is very easy to evaporate in the natural environment, the carboxymethyl chitosan Schiff base modified with trans-2-hexenal was used to prolong the antibacterial effect of trans-2-hexenal on Fusarium graminearum, breaking through the limitation of the use environment. Trans-2-hexenal and carboxymethyl chitosan were combined by Schiff base reaction. In addition to the reaction between trans-2-hexenal and carboxymethyl chitosan, carboxymethyl chitosan also has certain effects on promoting plant growth and resisting adversity. The synergistic effect of the two makes Schiff base more effective than trans-2-hexenal, especially against Fusarium graminearum (F. graminearum) and Phytophthora capsici (P. capsici). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Infrared characterization diagram of the Schiff base obtained in Example 1-3; Figure 2 The scanning electron micrographs of the Schiff bases obtained in Examples 1-3 are (a) 1:1, (b) 1:2, and (c) 1:4; Figure 3 Thermogravimetric analysis of Examples 1-3; Figure 4 The dynamic release of Examples 1-3 in different pH buffers; Figure 5 This is a diagram of the dual culture dish system setup; Figure 6 The figure shows the growth results of Fusarium graminearum on the plate culture medium after being treated with three Schiff base substances for 3 days; Figure 7 The indoor control effect of three types of carboxymethyl chitosan / trans-2-hexenal on pepper phytophthora: Figure 8 To determine the inhibition time of three carboxymethyl chitosan / trans-2-hexenal on Fusarium graminearum; Fig. 9 To test the control effect of trans-2-hexenal emulsifiable concentrate and three carboxymethyl chitosan / trans-2-hexenal addition products on small pest thrips; Fig.10 The control effects of trans-2-hexenal emulsifiable concentrate and three carboxymethyl chitosan / trans-2-hexenal addition products on the small pest Tetranychus cinnabarinus. DETAILED DESCRIPTION

[0017] Example 1 (1) dissolving carboxymethyl chitosan in distilled water, wherein the ratio of carboxymethyl chitosan to distilled water is 2% (w / w), and heating and stirring to dissolve the carboxymethyl chitosan, wherein the molecular weight of the carboxymethyl chitosan is 240 kDa; (2) dissolving trans-2-hexenal in ethanol and adding the solution to the carboxymethyl chitosan in step (1), reacting for 5 hours at a temperature of 60° C., wherein the reaction molar ratio of the carboxymethyl chitosan to trans-2-hexenal is 1:1; (3) The liquid after the reaction in step (2) was filtered and washed, and the obtained product was freeze-dried under vacuum for 24 h at a cold trap temperature of -52 °C and a vacuum degree of 10 Pa.

[0018] The reaction equation is as follows: .

[0019] Example 2 (1) dissolving carboxymethyl chitosan in distilled water, wherein the ratio of carboxymethyl chitosan to distilled water is 2% (w / w), and heating and stirring to dissolve the carboxymethyl chitosan, wherein the molecular weight of the carboxymethyl chitosan is 240 kDa; (2) dissolving trans-2-hexenal in ethanol and adding the solution to the carboxymethyl chitosan in step (1), reacting for 5 hours at a temperature of 60° C., wherein the reaction molar ratio of the carboxymethyl chitosan to trans-2-hexenal is 1:2; (3) The liquid after the reaction in step (2) was filtered and washed, and the obtained product was freeze-dried under vacuum for 24 h at a cold trap temperature of -52 °C and a vacuum degree of 10 Pa.

[0020] Example 3 (1) dissolving carboxymethyl chitosan in distilled water, wherein the ratio of carboxymethyl chitosan to distilled water is 2% (w / w), and heating and stirring to dissolve the carboxymethyl chitosan, wherein the molecular weight of the carboxymethyl chitosan is 240 kDa; (2) dissolving trans-2-hexenal in ethanol and adding the solution to the carboxymethyl chitosan in step (1), reacting for 5 hours at a temperature of 60° C., wherein the reaction molar ratio of the carboxymethyl chitosan to trans-2-hexenal is 1:4; (3) The liquid after the reaction in step (2) was filtered and washed, and the obtained product was freeze-dried under vacuum for 24 h at a cold trap temperature of -52 °C and a vacuum degree of 10 Pa.

[0021] The obtained Schiff base was characterized and analyzed (1) Infrared characterization Figure 1 The infrared characterization diagrams of the Schiff bases obtained by the three reaction molar ratios of Examples 1-3 are shown in FIG. Figure 1 It can be seen that the carboxymethyl chitosan in the a line is at 3411cm -1 There is a strong absorption peak at 1597cm, which is the result of the partial overlap of the characteristic absorption peaks of -OH and -NH. -1 -NH 2This indicates that there are a large number of -NH 2 b, c, and d are FT-IR images of three reaction molar ratio Schiff base derivatives, 1650 cm -1 The appearance of C=N peak at the position of 1650 cm-1 confirms the formation of imine group. At the same time, the absorption peaks are basically consistent, indicating that the products have consistent chemical structures; only at 1650 cm-1 -1 The intensities of the C=N characteristic peaks at the positions are different, and the difference is consistent with the trend of the degree of substitution among the derivative samples.

[0022] (2) Elemental analysis of products Table 1 shows the elemental analysis results of three carboxymethyl chitosan / trans-2-hexenal addition products. As shown in Table 1, Schiff base derivatives with different degrees of substitution can be prepared according to the molar ratio of carboxymethyl chitosan repeating units to the aldehyde functional groups in trans-2-hexenal of 1:1, 1:2, and 1:4, respectively. The degrees of substitution are 27.97%, 47.72%, and 65.94%, respectively. When the feed ratio of the raw material trans-2-hexenal increases, the relative content of nitrogen in the derivative decreases, the relative content of carbon increases, and the degree of substitution of the derivative gradually increases.

[0023] Table 1 is the elemental analysis results of three carboxymethyl chitosan / trans-2-hexenal addition products in Examples 1-3 (3) Scanning electron microscopy Figure 2 is a scanning electron microscope image of three carboxymethyl chitosan / trans-2-hexenal addition products in Examples 1-3, Figure 2 (a) is a scanning electron microscope image of the product obtained in Example 1 with a molar ratio of 1:1, Figure 2 (b) is a scanning electron microscope image of the product obtained in Example 2 with a molar ratio of 1:2, Figure 2 (c) is a scanning electron microscope image of the product obtained in Example 3 with a molar ratio of 1:4. It can be seen that the internal structure of the Schiff base derivative is a network structure, and as the feed ratio of the raw materials increases, the internal network structure becomes denser.

[0024] (4) Thermogravimetric analysis Figure 3The thermogravimetric analysis results of carboxymethyl chitosan and three kinds of Schiff base products of embodiments 1-3. At 500 ℃, the residual amount of carboxymethyl chitosan is about 49%, the residual amount of 1:1 Schiff base is about 43%, the residual amount of 1:2 Schiff base is about 40%, and the residual amount of 1:4 Schiff base is about 36%. The reason for this result may be that the denser the internal structure, the more trans-2-hexenal that participates in the reaction, the more polymer decomposes under high temperature conditions, the volatiles volatilize, and the less residual amount. However, the working environment of the product in actual application is usually at room temperature, which is far below its thermal decomposition temperature. Therefore, the thermal stability of chitosan after chemical modification is slightly reduced, but its performance is not affected.

[0025] (5) Dynamic release in different pH buffers Figure 4 The dynamic release of the product obtained in Example 1-3 in different pH buffers. The different release of trans-2-hexenal (T2H) under different pH conditions verifies that the Schiff base material has pH-responsive release characteristics. As the pH value increases, the growth rate of the cumulative release concentration gradually slows down. Under pH=3, the release rate of the substance is the fastest, and under pH=7, it is the slowest. Under the same pH conditions, the release of T2H of 1:1 is the highest, followed by 1:2 and 1:4, which shows that 1:4 has good stability in water. The phenomenon of this release can be attributed to the different densities of the internal spatial structures of the three. The internal structure of 1:1 is loose, and the imine bond is easy to break. The internal structure of 1:4 is dense, which will prevent the solution from entering the interior, thereby reducing the chance of the acid-sensitive imine bond contacting the acid signal molecule, and the amount of bond breaking is reduced, thereby showing a lower amount of released aldehyde.

[0026] Example 2 In this example, wild-type isolated bacteria Fusarium graminearum (F. graminearum) and Phytophthora capsici (P. capsici) were used for sensitivity determination and inoculation test. They were inoculated on potato dextrose agar (PDA) medium and cultured in the dark at 25°C for 3 days before use. Under neutral conditions and pH=5.8, the antibacterial effects of the three Schiff base products on Fusarium graminearum and Phytophthora capsici were verified. In order to promote the release of aldehydes in the headspace of the culture dish, a double-plate system was used. For this purpose, 50 mg of each Schiff base was taken for the test. They were placed in the gap between an empty culture dish with a diameter of 150 mm and a height of 17 mm and a culture dish with a diameter of 90 mm and a height of 15 mm (see Figure 5), where the PDA medium was inoculated with a mycelial plug (3 mm). Afterwards, about 20 ml of a buffer solution of pH=7 or pH=5.8 was poured on the Schiff base substance. Then, the lid of a 150 mm culture dish was placed and sealed with Parafilm®. The colony diameter was measured after 3 days of incubation in the dark at 25°C. Each treatment had 4 technical replicates, and all experiments were repeated 3 times independently. Since carboxymethyl chitosan was not in direct contact with the bacteria, there was only a blank control without the addition of product.

[0027] Figure 6 This is a graph showing the growth results of Fusarium graminearum on a plate culture medium after being treated with three Schiff base products in Examples 1-3 for 3 days; Figure 7 The results of the indoor control of pepper phytophthora by three Schiff base products in Examples 1-3 show that the growth of fungi is significantly affected after treatment with Schiff base products, especially when the trans-2-hexenal carboxymethyl chitosan Schiff base material is in an acidic medium. The degree of substitution of 1:2 is in the middle, but its internal structure is looser than 1:4, so its effect is better than 1:1 and 1:4.

[0028] Inhibition time determination The double-dish system was used to test trans-2-hexenal and Schiff's base (the concentration of trans-2-hexenal was 8 μL / L). The bacteria were inoculated on days 0, 2, 4, and 8 of treatment.

[0029] Figure 8 The results of inhibition time determination. At 0 days of treatment, the inhibition rates of the three Schiff base products on mycelial growth were significantly lower than those of T2H. However, after 2 and 4 days of treatment, the inhibition of mycelial growth by Schiff base substances began to be higher or significantly higher than that of T2H. After 8 days of treatment, the inhibition rate of mycelial growth by T2H decreased from 43.12% to 10.50%, indicating that the volatility of T2H greatly reduced its inhibitory effect on Fusarium graminearum. Schiff base products extended the inhibition time of T2H on Fusarium graminearum. The calculation formula of inhibition rate is as follows: Example 3 Select cucumber plants that have been cultivated for two weeks with consistent growth, spray trans-2-hexenal emulsifiable concentrate and three ratios of carboxymethyl chitosan / trans-2-hexenal Schiff base suspension (solvent is water + 1.5% polyethoxy-modified trisiloxane) (concentration of trans-2-hexenal is 200 μL / L) evenly on the surface of cucumber leaves, and then inoculate 100 adult thrips, and then seal transparent plastic bags to keep moisture. For each treatment of six cucumber pots, the mortality rate was counted on the 3rd and 7th days, and the corrected mortality rate was calculated according to the following formula.

[0030] Depend on Fig. 9 It can be seen that on the third day, different proportions of Schiff base products caused a corrected mortality rate of about 60% for thrips, and the highest was 80%, while the corrected mortality rate of trans-2-hexenal emulsifiable concentrate was only 33%. And on the seventh day, the corrected mortality rates of the three materials did not change significantly, but the corrected mortality rate of trans-2-hexenal dropped to about 10%. The insecticidal effect of Schiff base products is the aldehyde released by the cleavage of the imine bond in the Schiff base products. At the same time, the Schiff base products can continuously release aldehydes, which prolongs the effective period of pest control.

[0031] Example 4 Select cucumber plants that have been cultivated for two weeks with consistent growth, and spray trans-2-hexenal emulsifiable concentrate and three ratios of carboxymethyl chitosan / trans-2-hexenal Schiff base suspension (solvent is water + 1.5% polyethoxy-modified trisiloxane) (concentration of trans-2-hexenal is 200 μL / L) evenly on the surface of cucumber leaves, and then inoculate 200 adult cinnabarinus spider mites, and then seal transparent plastic bags to keep moisture. For each treatment of six cucumber pots, the mortality rate was counted on the 3rd and 7th days, and the corrected mortality rate was calculated according to the following formula.

[0032] Depend on Fig.10 It can be seen that on the third day, the Schiff base product caused a corrected mortality rate of about 60% of Tetranychus cinnabarinus, and the highest corrected mortality rate was 80%, but the corrected mortality rate of trans-2-hexenal emulsifiable concentrate was only about 30%. On the seventh day, the corrected mortality rate of Schiff base products did not change significantly, but the insecticidal effect of trans-2-hexenal decreased significantly, only 15%. The insecticidal effects of trans-2-hexenal and Schiff base products were significantly different, showing that Schiff base products increased the utilization rate of aldehydes and enhanced the insecticidal effect.

Claims

1. A carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density, characterized in that: The structural formula is as follows: It has the structural formula shown in formula (1): （1）。 2. A method for preparing a carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density as claimed in claim 1, characterized in that: The steps include: S1, dissolving carboxymethyl chitosan in water, heating and stirring to dissolve the carboxymethyl chitosan, to obtain a carboxymethyl chitosan aqueous solution, wherein the mass ratio of carboxymethyl chitosan to water is 2-4% (w / w); S2, dissolving an appropriate amount of trans-2-hexenal in ethanol, and adding the solution to the carboxymethyl chitosan solution obtained in step (1), followed by heating and stirring for 5 h to 12 h at a reaction temperature of 30° C. to 60° C.; S3. The product obtained in step (2) is filtered, Soxhlet extracted and vacuum freeze-dried in sequence to obtain trans-2-hexenal Schiff base.

3. The method for preparing the carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density according to claim 2, characterized in that: The mass ratio of carboxymethyl chitosan to water is 2% (w / w).

4. The method for preparing the carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density according to claim 2, characterized in that: The reaction molar ratio of the carboxymethyl chitosan to trans-2-hexenal is 1: 1-4.

5. The method for preparing the carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density according to claim 2, characterized in that: The ethanol used in step (2) is analytical grade, the reaction time is 5 h, and the reaction temperature is 60° C.

6. The method for preparing the carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density according to claim 2, characterized in that: The molecular weight of the carboxymethyl chitosan is 240 kDa.

7. Use of the density-controlled carboxymethyl chitosan Schiff base based on trans-2-hexenal as claimed in claim 1 in an antibacterial agent.

8. Use of the density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal as claimed in claim 1 in pesticides.

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