A trans-2-hexenal-based carboxymethyl chitosan schiff base with controllable compactness and a preparation method and application thereof

Abstract

The application belongs to the technical field of supramolecular chemistry, and particularly relates to a dense controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal and a preparation method and application thereof. The Schiff base with different densities is prepared by using the nucleophilic addition-elimination reaction of an amino compound carboxymethyl chitosan and different moles of trans-2-hexenal. The Schiff base can be used for preparing insecticide and fungicide products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of supramolecular chemistry, and particularly relates to a carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable compactness, a preparation method and application thereof. BACKGROUND

[0002] Trans-2-hexenal is one of the main volatile substances in fresh fruits, and has high antibacterial and insecticidal activity. In addition to damaging the cell membrane integrity and cell wall of pathogenic bacteria to achieve the purpose of inhibiting 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 the cells and further causing oxidative stress, thereby achieving the effect of inhibiting bacteria. Trans-2-hexenal can act on the sensory organs of pests, and with the extension of treatment time, it can penetrate the protective cuticle layer into the insect body, destroy the body wall structure, and dissolve the cuticle layer, thereby facilitating the penetration of essential oils, enhancing the penetration, absorption, transport and entry of active components into the stomata, causing the normal physiological and biochemical metabolism of pests to be blocked, and the inhibitory effect to be obviously enhanced. Therefore, based on the high antibacterial and insecticidal activity of trans-2-hexenal, trans-2-hexenal can be a good natural bacteriostatic and insecticidal agent, and has broad application potential in disease control.

[0003] However, the boiling point of trans-2-hexenal is only 47℃, which is easily evaporated in the natural environment, and it is difficult to provide long-term and stable bacteriostatic protection to crops. The existing common treatment methods include short-time closed fumigation treatment and cyclodextrin embedding. Among them, the short-time closed fumigation technology is limited in use, and in addition, it has high operation requirements and increases the additional processing time; the cyclodextrin embedding method can delay the release rate of trans-2-hexenal, but cannot accurately control the release amount, and still has deficiencies in environmental response. In addition, the effect of trans-2-hexenal on pathogenic bacteria is closely related to its dosage, and if the content is too low, the antibacterial effect is not obvious, and if the content is too high, it will cause damage to the fruits. In summary, the development of a responsive trans-2-hexenal slow-release agent has great application value.

[0004] As a typical active electrophilic substance, trans-2-hexenal can react with substances containing amino groups (amino acids, chitosan, etc.) or methylene amino groups to form a class of organic compounds containing dynamic imine bonds. The addition reaction of trans-2-hexenal with amino (-NH2) compounds is reversible and related to pH, so the prepared Schiff base derivatives have pH response characteristics. Among the -NH2-containing compounds, carboxymethyl chitosan is a widely sourced polymer compound containing a large number of active amino groups, has good imine crosslinking reactivity, and is biodegradable, has certain antifungal, antibacterial, antiviral and nematicidal biological activities, and can improve the tolerance of plants to abiotic stresses such as drought, high temperature, salt and heavy metals.

[0005] Therefore, the present application selects carboxymethyl chitosan as a representative safe and effective amino-containing compound to react with trans-2-hexenal, and uses dynamic imine bonds to prepare three kinds of trans-2-hexenal Schiff bases with different reaction molar ratios. The internal structure of the three kinds of Schiff base substances prepared has differences in density, the crosslinking degree in the Schiff base product can be controlled, and the release speed of aldehyde can be controlled. Different reaction molar ratio products are used in combination to achieve the effect of "preventing disease before disease occurs and treating disease quickly after disease occurs". The prepared pH-responsive trans-2-hexenal Schiff base slow-release agent has good indoor virulence effect on wheat scab and pepper Phytophthora; at the same time, it also shows good effect on small pests such as thrips and Tetranychus cinnabarinus. SUMMARY

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

[0007] The present application is realized by the following technical solutions:

[0008] A carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density, the structural formula of which is as follows:

[0009] has a structural formula as shown in formula (1):

[0010] (1).

[0011] The present application also provides a preparation method of the carboxymethyl chitosan Schiff base based on trans-2-hexenal with controllable density, comprising the following steps:

[0012] S1, dissolving carboxymethyl chitosan in water, heating and stirring to dissolve the carboxymethyl chitosan, to prepare a carboxymethyl chitosan aqueous solution, the mass ratio of carboxymethyl chitosan to water being 2-4% (w / w);

[0013] S2, dissolve a proper amount of trans-2-hexenal in ethanol and add to the carboxymethyl chitosan solution obtained in step (1), then heat and stir for 5-12 hours at 30-60℃;

[0014] S3, the product obtained in step (2) is subjected to filtration, Soxhlet extraction and vacuum freeze-drying in sequence to obtain trans-2-hexenal Schiff base.

[0015] Further, the mass ratio of carboxymethyl chitosan to distilled water in step (1) is 2% (w / w).

[0016] Further, the reaction molar ratio of carboxymethyl chitosan to trans-2-hexenal is 1:1-4.

[0017] Further, the ethanol used in step (2) is analytical pure, the reaction time is 5 hours, and the reaction temperature is 60℃.

[0018] Further, the molecular weight of the carboxymethyl chitosan is 240 kDa.

[0019] The application also provides the use of the trans-2-hexenal-based carboxymethyl chitosan Schiff base in the preparation of bacteriostatic agents.

[0020] The application also provides the use of the trans-2-hexenal-based carboxymethyl chitosan Schiff base in the preparation of insecticides.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] (1) The application utilizes the reversible Michael addition reaction between the natural volatile substance trans-2-hexenal in fruits and the natural product carboxymethyl chitosan, overcomes the application difficulty of pure product being easily volatile, and realizes the long-term stable bacteriostatic effect of trans-2-hexenal;

[0023] (2) The Schiff base substance provided by the application has the property of pH stimulus response release, realizes the slow release effect of trans-2-hexenal, overcomes the defect that the existing cyclodextrin embedding technology cannot accurately control the release, and improves the bacteriostatic effect;

[0024] (3) The Schiff base substance prepared by the reaction of carboxymethyl chitosan and different moles of trans-2-hexenal has different internal densities, has various release aldehyde speeds, and the density can be controlled to achieve the ideal application effect;

[0025] (4) The application utilizes the nucleophilic addition-elimination reaction between the amino compound carboxymethyl chitosan and different moles of trans-2-hexenal to prepare Schiff bases with different densities. The preparation method is simple, easy to operate, easy to realize, has mild reaction conditions, and has good repeatability.

[0026] (5) The prepared Schiff base substance has good bacteriostatic effect in disease control represented by wheat scab and pepper Phytophthora;

[0027] (6) The prepared Schiff base substance has good insecticidal effect in pest control represented by small pests, thrips and Tetranychus cinnabarinus.

[0028] (7) In view of the problem that trans-2-hexenal is easily evaporated in the natural environment, the carboxymethyl chitosan Schiff base modified by trans-2-hexenal prolongs the bacteriostatic effect of trans-2-hexenal on F. graminearum, and breaks through the limitation of the use environment. In addition to the reaction of trans-2-hexenal and carboxymethyl chitosan, carboxymethyl chitosan also has the effects of promoting plant growth and resisting adversity. The synergistic effect of the two makes the effect of the Schiff base better than that of trans-2-hexenal, especially for F. graminearum and P. capsici. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The infrared characterization graph of the Schiff base obtained in Examples 1-3;

[0030] Figure 2 The scanning electron microscope graph of the Schiff base obtained in Examples 1-3, (a) 1:1, (b) 1:2, (c) 1:4;

[0031] Figure 3 The thermogravimetric analysis graph of Examples 1-3;

[0032] Figure 4 The dynamic release situation of Examples 1-3 in different pH buffer solutions;

[0033] Figure 5 It is a double culture dish system device diagram;

[0034] Figure 6 It is a growth result diagram of F. graminearum on the plate medium after three kinds of Schiff base substances are treated for 3 days;

[0035] Figure 7 It is an indoor control effect diagram of three kinds of carboxymethyl chitosan / trans-2-hexenal on P. capsici:

[0036] Figure 8 It is an inhibition time determination of three kinds of carboxymethyl chitosan / trans-2-hexenal on F. graminearum;

[0037] Figure 9Trans-2-hexenal emulsion and three kinds of carboxymethyl chitosan / trans-2-hexenal addition products were tested for their effects on the control of small pests, thrips;

[0038] Figure 10 Trans-2-hexenal emulsion and three kinds of carboxymethyl chitosan / trans-2-hexenal addition products were tested for their effects on the control of small pests, thrips; DETAILED DESCRIPTION

[0039] Example 1

[0040] (1) Carboxymethyl chitosan was dissolved in distilled water at a ratio of 2% (w / w) of carboxymethyl chitosan to distilled water, and the carboxymethyl chitosan was dissolved by heating and stirring, the molecular weight of the carboxymethyl chitosan being 240 kDa;

[0041] (2) Trans-2-hexenal was dissolved in ethanol and added to the carboxymethyl chitosan solution of step (1), the reaction being carried out for 5 h at a temperature of 60°C, the reaction molar ratio of carboxymethyl chitosan to trans-2-hexenal being 1:1;

[0042] (3) The liquid after the reaction of step (2) was filtered and washed, and the resulting product was vacuum freeze-dried for 24 h at a cold trap temperature of -52°C and a vacuum degree of 10 Pa.

[0043] The reaction equation is as follows:

[0044] .

[0045] Example 2

[0046] (1) Carboxymethyl chitosan was dissolved in distilled water at a ratio of 2% (w / w) of carboxymethyl chitosan to distilled water, and the carboxymethyl chitosan was dissolved by heating and stirring, the molecular weight of the carboxymethyl chitosan being 240 kDa;

[0047] (2) Trans-2-hexenal was dissolved in ethanol and added to the carboxymethyl chitosan solution of step (1), the reaction being carried out for 5 h at a temperature of 60°C, the reaction molar ratio of carboxymethyl chitosan to trans-2-hexenal being 1:2;

[0048] (3) The liquid after the reaction of step (2) was filtered and washed, and the resulting product was vacuum freeze-dried for 24 h at a cold trap temperature of -52°C and a vacuum degree of 10 Pa.

[0049] Example 3

[0050] (1) Carboxymethyl chitosan was dissolved in distilled water at a ratio of 2% (w / w) of carboxymethyl chitosan to distilled water, and the carboxymethyl chitosan was dissolved by heating and stirring, the molecular weight of the carboxymethyl chitosan being 240 kDa;

[0051] (2) trans-2-hexenal was dissolved in ethanol and added to the carboxymethyl chitosan solution of step (1), and reacted for 5 h at 60℃, the molar ratio of carboxymethyl chitosan and trans-2-hexenal was 1:4;

[0052] (3) the liquid after step (2) was filtered and washed, and the obtained product was vacuum freeze-dried for 24 h, the cold trap temperature was -52℃, and the vacuum degree was 10 Pa.

[0053] The obtained Schiff base was characterized and analyzed

[0054] (1) infrared characterization

[0055] Figure 1 The infrared characterization graphs of the Schiff bases obtained in three reaction molar ratios of Examples 1-3 were as follows: Figure 1 It can be seen that the carboxymethyl chitosan in the a line has a strong absorption peak at 3411 cm -1 , which is the result of the partial overlap of the characteristic absorption peaks of -OH and -NH. The characteristic absorption peak of -NH2 at 1597 cm -1 indicates that there are a large number of -NH2 on the molecular chain of carboxymethyl chitosan. The FT-IR of the Schiff base derivatives of three reaction molar ratios b, c, and d shows that the C=N peak at 1650 cm -1 appears, confirming the formation of the imine group. At the same time, the absorption peaks are basically consistent, indicating that the products have consistent chemical structures; only the intensity of the C=N characteristic peak at 1650 cm -1 is different, and the difference changes are consistent with the trend of the degree of substitution between the derivative samples.

[0056] (2) elemental analysis of the product

[0057] Table 1 is the elemental analysis results of the three carboxymethyl chitosan / trans-2-hexenal addition products. As shown in Table 1, according to the molar ratio of 1:1, 1:2, and 1:4 of the repeat units of carboxymethyl chitosan to the aldehyde functional groups in trans-2-hexenal, Schiff base derivatives with different degrees of substitution can be prepared, and 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.

[0058] Table 1 is the elemental analysis results of the three carboxymethyl chitosan / trans-2-hexenal addition products in Examples 1-3

[0059]

[0060] (3) scanning electron microscopy

[0061] Figure 2 Figure 1 is a scanning electron microscope image of the product obtained in Example 1, Figure 2 (a) is a scanning electron microscope image of the product obtained in Example 1 at a molar ratio of 1:1, Figure 2 (b) is a scanning electron microscope image of the product obtained in Example 2 at a molar ratio of 1:2, Figure 2 (c) is a scanning electron microscope image of the product obtained in Example 3 at a molar ratio of 1:4. It can be seen that the Schiff base derivative product has a network structure inside, and the network structure inside becomes denser as the feed ratio of the raw material increases.

[0062] (4) Thermogravimetric analysis

[0063] Figure 3 Figure 4 is a thermogravimetric analysis result of carboxymethyl chitosan and the three Schiff base products obtained in Examples 1-3. At 500°C, the residual amount of carboxymethyl chitosan is about 49%, the residual amount of the 1:1 Schiff base is about 43%, the residual amount of the 1:2 Schiff base is about 40%, and the residual amount of the 1:4 Schiff base is about 36%. The reason for this result is that the more dense the internal structure, i.e. the more trans-2-hexenal involved in the reaction, the less residual amount under high temperature conditions, because the polymer decomposes and the volatile volatilizes. However, the working environment of the product in practical application is usually at room temperature, which is much lower than its thermal decomposition temperature. Therefore, the thermal stability of the chemically modified chitosan is slightly reduced, but its performance is not affected.

[0064] (5) Dynamic release in different pH buffers

[0065] Figure 4 Figure 5 is a dynamic release of the products obtained in Examples 1-3 in different pH buffers. The different release of trans-2-hexenal (T2H) under different pH conditions verifies that the Schiff base substance has a pH-responsive release characteristic. As the pH value increases, the growth rate of the cumulative release concentration gradually slows down. Under the condition of pH=3, the release speed of the substance is the fastest, and under the condition of pH=7, it is the slowest. Under the same pH condition, the release of T2H is the highest for 1:1, followed by 1:2 and 1:4, which indicates that 1:4 has good stability in water. This release phenomenon can be attributed to the different densities of the internal space structure of the three, the internal structure of 1:1 is loose, the imine bond is easy to break, the internal structure of 1:4 is dense, which prevents the solution from entering the inside, thereby reducing the opportunity for the acid-sensitive imine bond to contact the acid signal molecule, and the amount of bond breakage is reduced, thereby showing a lower amount of released aldehyde.

[0066] Example 2

[0067] The wild type isolated bacteria F. graminearum and P. capsici were used for sensitivity determination and inoculation test, and were inoculated on potato dextrose agar (PDA) medium, and were used after 3 days of culture at 25°C in the dark. The antibacterial effect of the three Schiff base products on F. graminearum and P. capsici was verified under neutral and pH = 5.8 conditions. In order to promote the release of aldehydes in the headspace of the petri dish, a double plate system was used. For this, 50 mg of each Schiff base was tested. It was placed in the gap between a 150 mm in diameter and 17 mm in height empty petri dish and a 90 mm in diameter and 15 mm in height petri dish (see Figure 5 ), in which the PDA medium was inoculated with a mycelium plug (3 mm). Then, about 20 ml of buffer solution at pH = 7 or pH = 5.8 was poured over the Schiff base material. Then, the lid of the 150 mm petri dish was placed and sealed with Parafilm®. The colony diameter was determined after 3 days of culture at 25°C in the dark. There were 4 technical replicates per treatment, and all experiments were independently repeated 3 times. Since the carboxymethyl chitosan did not come into direct contact with the bacteria, only the blank control without product was used.

[0068] Figure 6 The results of the growth of F. graminearum on the plate medium after 3 days of treatment with the three Schiff base products of examples 1-3; Figure 7 The results of the indoor control effect of the three Schiff base products of examples 1-3 on P. capsici: the results show that the growth of the fungus is significantly affected after treatment with the Schiff base products, especially in the case of the trans-2-hexenal carboxymethyl chitosan Schiff base material in an acidic medium. The degree of substitution of 1:2 is intermediate, but its internal structure is looser than that of 1:4, so its effect is better than that of 1:1 and 1:4.

[0069] Inhibition time determination

[0070] The double petri dish system was used for the test with trans-2-hexenal and the Schiff base (the concentration of trans-2-hexenal was 8 μL / L). The bacteria were inoculated at 0, 2, 4, 8 days of treatment.

[0071] Figure 8 The results of the inhibition time determination. At 0 d of treatment, the inhibition rate of mycelial growth by the three Schiff base products was significantly lower than that of T2H, but after 2 d and 4 d of treatment, the inhibition effect of the Schiff base material on mycelial growth began to be higher or significantly higher than that of T2H, and after 8 d 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 F. graminearum. The Schiff base products prolonged the inhibition time of T2H on F. graminearum. The formula for calculating the inhibition rate is as follows:

[0072]

[0073] Example 3

[0074] Select the growth of two weeks of cucumber plants, trans-2-hexenal emulsion and three kinds of proportion of carboxymethyl chitosan / trans-2-hexenal Schiff base suspension agent (solvent is water + 1.5% polyethoxy modified trisiloxane) (trans-2-hexenal concentration is 200 μL / L), uniform spraying on the surface of cucumber leaves, then access to 100 thrips adults, then the transparent plastic bag sealed and humidified. Each treatment six pots of cucumber, 3 d, 7 d statistics mortality, and according to the following formula to calculate the corrected mortality.

[0075]

[0076]

[0077] From Figure 9 It is known that the 3 d, different proportions of Schiff base product to cause thrips 60% or so of the corrected mortality mortality, up to 80%, the corrected mortality of trans-2-hexenal emulsion is only 33%. And the 7 d, the corrected mortality of three kinds of materials did not change significantly, but the corrected mortality of trans-2-hexenal decreased to about 10%. The insecticidal effect of Schiff base product is the effect of aldehyde released by the breaking of imine bond in Schiff base product, at the same time, the Schiff base product can continuously release aldehyde, prolong the effective period of pest control.

[0078] Example 4

[0079] Select the growth of two weeks of cucumber plants, trans-2-hexenal emulsion and three kinds of proportion of carboxymethyl chitosan / trans-2-hexenal Schiff base suspension agent (solvent is water + 1.5% polyethoxy modified trisiloxane) (trans-2-hexenal concentration is 200 μL / L), uniform spraying on the surface of cucumber leaves, then access to 200 thrips adults, then the transparent plastic bag sealed and humidified. Each treatment six pots of cucumber, 3 d, 7 d statistics mortality, and according to the following formula to calculate the corrected mortality.

[0080]

[0081]

[0082] From Figure 10It can be seen that on the 3rd day, the Schiff base product caused about 60% corrected mortality of T. c. leaf and the highest corrected mortality was 80%, but the corrected mortality of trans-2-hexenal EC was only about 30%. On the 7th day, the corrected mortality of the Schiff base product did not change significantly, but the insecticidal effect of trans-2-hexenal decreased greatly, only 15%. The insecticidal effect of trans-2-hexenal and the Schiff base product was obviously different, which showed that the Schiff base product improved the utilization rate of aldehyde and enhanced the insecticidal effect.

Claims

1. A method for preparing a carboxymethyl chitosan Schiff base with controllable density based on trans-2-hexenal, characterized in that: Schiff bases have the structural formula shown in formula (1): （1）； The method for preparing the density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal includes the following steps: S1. Dissolve carboxymethyl chitosan in water, heat and stir to dissolve carboxymethyl chitosan, and prepare an aqueous solution of carboxymethyl chitosan. The mass ratio of carboxymethyl chitosan to water is 2% (w / w). S2. Dissolve an appropriate amount of trans-2-hexenal in ethanol and add it to the carboxymethyl chitosan solution obtained in step (1). Then heat and stir for 5-12 hours at a temperature of 30-60°C. S3. The product obtained in step (2) is subjected to filtration, Soxhlet extraction and vacuum freeze drying to obtain trans-2-hexenal Schiff base. The molar ratio of the carboxymethyl chitosan to trans-2-hexenal is 1:1-4; The application of the density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal in antibacterial agents and insecticides.

2. The method for preparing a density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal according to claim 1, characterized in that: The ethanol used in S2 was of analytical grade, the reaction time was 5 hours, and the reaction temperature was 60°C.

3. The method for preparing a density-controllable carboxymethyl chitosan Schiff base based on trans-2-hexenal according to claim 1, characterized in that: The molecular weight of the carboxymethyl chitosan is 240 kDa.

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