CN115583896A

CN115583896A - Vanilamide compound, preparation method and application

Info

Publication number: CN115583896A
Application number: CN202211082697.2A
Authority: CN
Inventors: 田向荣; 黄丽丽; 胡子龙; 齐银银; 赵龙; 郝楠; 叶生伟
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2023-01-10
Anticipated expiration: 2042-09-06
Also published as: CN115583896B

Abstract

The invention discloses a vanillic amide compound, a preparation method and application thereof, wherein the vanillic amide compound can be obtained by separating and purifying henbane, and also can be synthesized by adopting vanillic acid, gamma-aminobutyric acid and n-butyl alcohol, thereby laying a foundation for the industrial development of the vanillic amide compound. The henbane neoalkaloid shows good immunity induction activity on tobacco by activating defensive enzyme and resistance genes, and stimulates the disease resistance of plants to different fungi, bacteria and virus diseases by generating anaphylactic reaction on various plants. On the other hand, the henbane neoline can obviously promote the growth of crops, promote the germination of seeds and the growth of root length under low concentration, inhibit the germination of seeds and the growth of root length under high concentration, can be widely applied to the preparation of plant immunity inducer and plant growth regulator as a lead compound or an effective component, and has wide development prospect in the aspects of promoting the yield and harvest of crops and disease resistance.

Description

Vanilamide compound, preparation method and application

Technical Field

The invention belongs to the technical field of plant protection, agriculture and pharmacy and traditional Chinese medicine agriculture, and particularly relates to a vanillic amide compound, and a preparation method and application thereof.

Background

The plant immunity inducer is a green plant protection prevention and control strategy developed in recent years, and can stimulate plants to generate immune response reaction, so that the aims of preventing and resisting diseases are fulfilled. On the other hand, plant growth regulators have become one of the main measures for realizing high-yield and high-quality agricultural products in modern agriculture at present. Many plant immunity inducers have the functions of promoting plant growth and improving plant stress resistance besides broad-spectrum disease resistance. The traditional Chinese medicine agriculture applies the traditional Chinese medicine theory to the agriculture field and is the cross-border fusion of the traditional Chinese medicine and the ecological agriculture. Matrine found from traditional Chinese medicine radix sophorae flavescentis is used as a bactericide for preventing and controlling various crop diseases, has the effect of increasing the immune yield, and related products have been subjected to international organic certification. It is seen that the traditional Chinese medicine resources are the main components of traditional Chinese medicine agriculture, are important sources for creating novel green pesticides, and the elicitors with novel structures are found from the traditional Chinese medicine resources, are used in the field of plant protection by taking the elicitors as effective components, and are the key for creating novel plant immunity elicitors and plant growth regulators from the source.

The traditional Chinese medicine henbane (Hyoscyamus niger) is a plant of henbane of Solanaceae, and is widely distributed in Europe, asia, north America and the like. The main medicinal part of henbane is seeds, has the effects of relieving spasm and pain, eliminating dampness and relieving itching, soothing nerves and arresting convulsion in the field of medicine, and has no specific application in the field of plant protection.

Disclosure of Invention

Based on the above purposes, the invention extracts a novel vanillic amide compound from henbane, and realizes complete synthesis in experiments, the novel vanillic amide compound has remarkable plant immunity inducing activity and plant growth promoting function, and can be widely used as a lead compound or an effective component for preparing plant immunity inducing agent and plant growth regulator, so the invention provides a novel vanillic amide compound, a preparation method and an application.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention aims to disclose a vanillic amide compound which has a chemical structural formula shown as the following formula (I):

the compound has the structural characteristics that vanillic acid and gamma-aminobutyric acid form amide (core structure), and carboxyl terminal of the gamma-aminobutyric acid and butanol form ester group.

The invention also aims to disclose a preparation method of the vanillic amide compound, which is to separate and purify the henbane to obtain the vanillic amide compound, and the method comprises the following steps:

step 1, collecting fresh henbane seeds, drying, heating and refluxing the fresh henbane seeds by using ethanol, replacing the ethanol solvent once every 2-4 hours, extracting for 4-5 times, combining extracting solutions, and concentrating to obtain an extract;

step 2, dispersing the extract in water, preparing into a suspension water solution, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and concentrating the n-butanol extract to obtain an n-butanol layer extract;

and 3, carrying out normal phase gradient elution on the n-butanol layer extract, wherein the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:1 to 1:1 to obtain fraction 1;

step 4, carrying out reverse phase column chromatography elution on the fraction 1, wherein an eluent is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 40-100;

and 5, performing gradient elution on the fraction 2 by adopting a liquid chromatography, wherein an eluent is a mixed solution of methanol and water or a mixed solution of acetonitrile and water, and the volume ratio of the methanol or the acetonitrile to the water is 35:65 to 65:35; obtaining the vanillic amide compound.

Preferably, the volume ratio of the henbane seeds to the ethanol solvent in the step 1 is 1.

Preferably, the ethanol solvent is 70-95% ethanol; the heating reflux temperature of the ethanol is 70-90 ℃.

Preferably, the extraction process in the step 2 specifically comprises the following steps: extracting with petroleum ether for 3-4 times, extracting the lower liquid phase after petroleum ether extraction with ethyl acetate for 3-4 times, and extracting the lower liquid phase after ethyl acetate extraction with n-butanol for 3-4 times.

Preferably, the volume ratio of the petroleum ether, the ethyl acetate and the n-butanol in the step 2 is 1.

Preferably, the normal phase gradient elution in the step 3 adopts a normal phase silica gel column; the reversed phase column chromatography elution in the step 4 adopts a C-18 reversed phase column;

preferably, the liquid chromatography conditions in step 5 are: the elution time is 50-60 min; the flow rate of the eluent is 8-10 mL/min; the column temperature is 20-35 ℃; the detection wavelength is 210-254 nm.

The invention also discloses a preparation method of the vanillic amide compound, which adopts vanillic acid, gamma-aminobutyric acid and n-butyl alcohol to synthesize the vanillic amide compound, and the synthetic route is as follows:

wherein a represents gamma-aminobutyric acid, b represents N-butyl alcohol, c represents vanillic acid, d represents intermediate gamma-aminobutyric acid butyl ester, HATU is a condensing agent and represents 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate; DIPEA represents diisopropylethylamine and is used as an acid-binding agent; nigeroate represents the final synthetic vanilloid compound.

The specific synthesis steps comprise the following steps:

step 1, mixing gamma-aminobutyric acid, n-butanol and thionyl chloride under an ice bath condition, then stirring the mixture at the temperature of 10-35 ℃ for reaction for 8-15 h, and removing a solvent after the reaction is finished to obtain an intermediate product; wherein the molar ratio of gamma-aminobutyric acid, n-butanol and thionyl chloride is 1;

step 2, adding the intermediate product obtained in the step 1, vanillic acid, HATU and DIPEA into thionyl chloride, stirring and reacting for 8-15 h at 10-35 ℃, concentrating and eluting the filtrate to obtain a vanillic amide compound; wherein the molar ratio of the intermediate product to the vanillic acid to the HATU to the DIPEA is 10:10:13:20.

the ice bath of the present invention is generally referred to as-4 ℃ to 4 ℃.

Obviously, the invention can also be used for preparing the derivative containing the vanillyl gamma-aminobutyric acid by using different aliphatic alcohols such as methanol, ethanol, propanol and the like instead of n-butyl alcohol. The n-butanol of the invention is substituted by other fatty acids such as methanol, ethanol, propanol and the like, and can be used for synthesizing the vanillic amide compound structural analogue of the invention and carrying out the application related to the invention.

Preferably, the step 2 of adding the intermediate product, vanillic acid, HATU and DIPEA comprises the following steps: dissolving vanillic acid and HATU in thionyl chloride, adding the intermediate product, stirring for 10-15min, and then adding DIPEA. The subsequent addition results in complete reaction, stable vanillic amide compound structure and high yield.

Preferably, the step 2 adopts a column chromatography technology for elution, and the eluent is a mixture of petroleum ether and ethyl acetate according to a volume ratio of 5-1.

The invention also discloses the application of the vanillic amide compound in preparing plant immunity inducer and growth regulator.

The prepared plant immunity inducer and related products can be used for improving the self disease resistance of plants, wherein the virus diseases mainly comprise: tobacco mosaic virus, cucumber mosaic virus, tomato mosaic virus, and the like. Fungal diseases include: botrytis cinerea, phytophthora capsici, apple tree rot, poplar rot, tobacco brown spot, tobacco black shank and the like. Bacterial diseases include: kiwifruit canker, tobacco bacterial wilt and the like. But is not limited to the above plant diseases. The growth regulator and the related products thereof are prepared for regulating the growth of plants and promoting or inhibiting the germination of seeds, wherein the plants for promoting the growth include but are not limited to crops such as cucumbers, pumpkin and the like, and the plants for promoting or inhibiting the germination of the seeds include but are not limited to lettuce seeds.

Compared with the prior art, the invention has the beneficial effects that:

the invention extracts a novel vanillic amide compound (hereinafter referred to as 'henbane neoalkali' in the embodiment) from henbane for the first time, and can also use cheap commercial medicaments of vanillic acid, gamma-aminobutyric acid and n-butyl alcohol to realize the total synthesis of the henbane neoalkali. The novel compound shows good therapeutic activity and inductive resistance activity when heart-leaf tobacco (Nicotiana luteinosa) and common tobacco (N.tabacum cv.k326) are infected by tobacco mosaic virus; the bactericidal composition has good protection effect on fungal diseases such as tomato gray mold, apple tree canker and the like and bacterial diseases such as kiwi fruit canker and the like; the compound fertilizer has obvious growth regulation effect on crops such as cucumber, pumpkin and the like, promotes seed germination of lettuce seeds at low concentration and inhibits seed germination at high concentration. The invention provides an important theoretical basis for researching and developing a novel plant source plant immunity inducer and a plant growth regulator by taking henbane neoalkali as an effective component, and provides a new strategy and thought for green prevention and control of crop diseases and quality safety guarantee of agricultural products.

The advantages of the invention will be further described and supplemented by the detailed description and drawings.

Drawings

FIG. 1 is a graph of the effect of henbane alkaloid on SOD (A), CAT (B), POD (C) and PAL (D) defense enzymes on K326 Nicotiana tabacum (data mean. + -. Standard error, different letters indicate significant differences at the 0.05 level between data).

FIG. 2 shows that the henbane alkaloid causes the change of the expression levels of the tobacco disease resistance gene and the salicylic acid synthesis gene in 24h (the data are mean values +/-SD, and different letters indicate that the data have a significant difference at a 0.05 level).

FIG. 3 is a graph of the hypersensitivity responses elicited by henbane neoline at various concentrations (1000, 500,250,125, and 62.5) on tobacco leaf smoke (A, hypersensitivity of tobacco leaf lamina. B, statistical plot of area of necrotic spots, data mean. + -. Standard error, different letters indicate significant differences at the 0.05 level between data).

FIG. 4 is the growth promoting effect of henbane neoalkaloid on cucumber (A) and cucurbita pepo (B).

Detailed Description

The following embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention are within the protection scope of the present invention.

Example 1

In this embodiment, the extraction of the vanillic amide compound from the seeds of henbane, which is a traditional Chinese medicine, specifically includes:

step 1, reflux-extracting dried henbane seeds with 95% ethanol at 78 ℃, wherein the volume ratio of the henbane seeds to the 95% ethanol is 1:3, replacing the solvent every 2 to 4 hours, extracting for 3 to 4 times, filtering dregs of a decoction, combining extracting solutions, and concentrating to obtain the 95 percent ethanol extract.

And 2, dispersing the 95% ethanol extract in water, preparing the mixture into a suspension state, sequentially extracting the suspension state for 3 to 4 times by using petroleum ether, ethyl acetate and n-butyl alcohol with the same volume, and combining and concentrating the n-butyl alcohol extract to obtain the n-butyl alcohol crude extract.

Step 3, normal phase column chromatographic separation: and (3) performing normal phase silica gel column chromatography on the n-butanol crude extract, and performing reaction by using dichloromethane: methanol is mixed according to the volume ratio of 100:1 to 1:1, dichloromethane: the volume ratio of methanol is 25: fraction 1 was obtained by elution of 1.

And 4, reversed phase column chromatographic separation: fraction 1 was passed through a C-18 reverse phase chromatography column eluting with methanol: water, volume ratio 50:50 to 100:0, wherein the ratio of methanol to water is 50: fraction 2 was obtained at 50 ℃.

Step 5, HPLC separation and purification: fraction 2 was separated and purified by preparative HPLC. The column was a C-18 reverse phase column (YMC, 250X 20mm,5 μm) and the eluent was methanol: water, wherein the methanol can be replaced by acetonitrile, and the volume ratio of the methanol to the water is 35:65 to 65:35 carrying out gradient elution; the detection wavelength is 210 nm-254 nm; the column temperature is normal temperature; the flow rate is 9mL/min; the sample injection amount is 500 mu L; the retention time is 30-50 min. The final product is obtained.

The structure of the final product obtained in this example was identified below, specifically by HRESIMS, ¹ H-NMR、 ¹³ C-NMR、 ¹ H- ¹ H COSY, HSQC and HMBC map identification.

The product was an amorphous powder. From HRESIMS (-) m/z 308.1488, M-H] ^– (calcd.for C ₁₆ H ₂₂ NO ₅ 308.1498) determining the compound has the formula C ₁₆ H ₂₃ NO ₅ 。 ¹ H-NMR gives a signal delta of 3 aromatic protons _H 6.83 (1H, d, J =8.3Hz, H-6), 7.34 (1H, dd, J =8.3Hz, H-7) and 7.43 (1H, d, J =2.1Hz, H-3), suggesting the presence of one ortho-and meta-substituted trisubstituted benzene ring in the structure. In addition, the hydrogen spectrum also gives 6 methylene hydrogens delta _H 1.37(2H,m,H ₂ -3″)、1.59(2H,m,H ₂ -2″)、1.91(2H,m,H ₂ -2′)、2.41(2H,m,H ₂ -3′)、3.40(2H,m,H ₂ -1') and 4.05 (2H, t, J=6.5Hz ₂ -1 "), 1 methylhydrogen δ _H 0.93(3H,t,J＝7.5Hz,H ₃ -4 ") and 1 methoxyhydrogens δ _H 3.90(3H,s,4-OCH ₃ )。 ¹³ C NMR spectra gave 16 carbon signals including 6 aryl carbons, 6 methylene carbons, 1 methyl carbon, 1 methoxy carbon, 2 carbonyl carbons delta _C 170.0 (C-1) and 175.2 (C-4'). The above information suggests that the compound contains one vanillyl group, 1 gamma aminobutyryl group and 1 n-butyl group. The vanillyl group obtains H-3, C-1 and C-2 (delta) in HMBC map _C 126.8)、C-4(δ _C 148.8)、C-5(δ _C 151.3 And C-7 (. Delta.) _C 122.0 H-6 with C-2, C-4)C-5 and C-7, and 4-OCH ₃ The signal associated with C-4 is corroborated. The gamma amino butyryl group obtains H in a COSY atlas ₂ -2' and H ₂ -1' and H ₂ -3' correlation signal, and H in HMBC mapping ₂ -2' and H ₂ The correlation signals of-3 'and C-4'. N-butyl represented by H in COSY spectrum ₂ -2' with H ₂ -1' and H ₂ -3 ", and H ₂ -3' with H ₂ -4 "corroboration of the relevant signal. The C-4' of carbonyl carbon in n-butyl and gamma amino butyryl are connected by H in HMBC spectrum ₂ -1 'is determined from the C-4' related signal. The amino group of the gamma amino butyryl group is connected with the C-1 of vanillic acid through H in HMBC map ₂ -1' is determined from the C-1 correlation signal. In combination with the operations of HSQC, HMBC, ¹ H- ¹ the spectral data of H COSY, NOESY, etc., and the spectral data of the compound are shown in Table 1.

TABLE 1 of the final product ¹ H (500 MHz) and ¹³ c (125 MHz) NMR spectral data ^a .

^a The attribution of the hydrocarbon is determined by HSQC, ¹ H- ¹ h COSY and HMBC.

^b In CD ₃ OD was measured.

^c In CDCl ₃ The measurement in (1).

In conclusion, the compound is analyzed to be butyl 4- (4-hydroxy-3-methoxybenzamido) -butyrate, named as henbane neoalkali, and is determined to be a new compound through Scifinder database search, and the structure is confirmed to be as follows:

example 2

This example shows the specific procedure for the chemical synthesis of the above henbane neobase:

step 1, gamma-aminobutyric acid (10 mM) was added with 20mL of n-butanol under ice bath (0 ℃ C.), and then 20mM of thionyl chloride was added dropwise. And (3) reacting for 12 hours under the condition of 25 ℃ by magnetic stirring, and removing the solvent from the product by a rotary evaporator to obtain an intermediate product of gamma-butyl aminobutyric acid with the yield of 97%.

Step 2,1mM of vanillic acid was dissolved in 10mL of thionyl chloride, and simultaneously 1.3mM of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) was added, followed by 1mM of intermediate butyl γ -aminobutyric acid, and stirred for 10-15min. Diisopropylethylamine (DIPEA, 2 mM) was added thereto, the reaction was carried out at 25 ℃ with magnetic stirring for 12 hours, and the filtrate was concentrated under reduced pressure to obtain a reaction product. The reaction product was subjected to gradient elution by normal phase silica gel column chromatography (silica gel H, petroleum ether: ethyl acetate =5:1 to 1) under the following conditions: ethyl acetate ratio 3:1 time a colorless solid was obtained in 95% yield.

Warp beam ¹ H and ¹³ C-NMR comparison with the natural product confirmed (Table 1) that the colorless solid was identical to the spectrum data of the henbane base prepared in example 1, indicating the same product.

The henbane new base used in the following examples was prepared for example 1 or example 2.

Example 3: anti-TMV Activity of Galium aparine

The half-leaf withered spot method is adopted, and healthy 5-6 leaf period heart-leaf tobacco with consistent growth vigor is selected as a research object. Mixing henbane neoline and TMV solution in equal volume, standing for 1h, and inoculating to folium Coryli Cantoniensis; the protective activity is that diluted TMV solution is inoculated after 48 hours of spraying henbane alkaloid medicament; therapeutic activity the henbane neobase agent was sprayed 48h after inoculation with TMV solution. Drug concentration settings 500,250,125 μ g/mL,1% dmso and ningnanmycin were blank and positive controls, respectively. 3 leaves were inoculated per treatment, repeated 3 times, and after 3 days, the number of scorched spots was counted to calculate the inhibition rate, wherein the inhibition rate (%) = [ (control number of scorched spots-number of treated scorched spots)/control number of scorched spots ] × 100. The results are shown in Table 2.

TABLE 2. 3 disease-resistant effects of henbane neokaline on TMV

Note: data are mean ± sem, significant differences between 3 activities were tested by Duncan Multiple Range Test (DMRT) in SPSS software, and different letters indicated significant differences at the 0.05 level between data.

As can be seen from Table 2, the inactivation and protection activity effects of henbane alkaloid on TMV are lower than that of ningnanmycin, but the treatment and prevention effects of henbane alkaloid on tobacco at the concentrations of 250 and 500 mu g/mL are respectively 58.00% and 69.14%, and the effects are obviously better than that of the positive medicament ningnanmycin. The new henbane alkali has better treatment effect on TMV.

Example 4: systemic resistance of henbane neobase to TMV on tobacco

And selecting common tobacco K326 to carry out verification of systemic induced disease resistance activity. The henbane alkaloid with the concentration of 500,250 and 125 mu g/mL is sprayed on the lower leaves of the tobacco with the same growth vigor and the 6-7 leaf stage. TMV was inoculated to the non-sprayed upper leaves after 48 h. The blank control was 1% dmso treatment and the positive control was ningnanmycin solution. Each plant was inoculated with 2 to 3 leaves, each treatment included 10 tobacco plants, and the whole experiment was repeated 3 times. After 3d, the ELISA method is used for extracting viruses according to a kit method, and the formula for calculating the inhibition rate is the same as that in example 3. The results are shown in Table 3.

TABLE 3 systemic resistance of henbane neobase to TMV on regular tobacco K326

As can be seen from table 3, henbane neobase showed good systemic resistance to TMV on regular cigarettes. The curative and preventive effects on tobacco under the concentrations of 250 and 500 mu g/mL are respectively 53.82 percent and 44.21 percent, and the effect is obviously superior to that of the positive medicament ningnanmycin.

Example 5: effect of henbane alkaloid on four defense enzymes of PAL, SOD, CAT and POD on tobacco

The effect of Nigeroate on defensive enzymes on tobacco was tested using a kit of phenylalanine ammonia enzyme (PAL), superoxide dismutase (SOD), catalase (CAT) and Peroxidase (POD). The coating is respectively coated on K326 tobacco leaves at the concentrations of 500,250 and 125 mu g/mL. The corresponding fresh leaves (0.1 g) were collected after 0,1, 3, 5, 7, 9d, respectively, and 1mL of the extract was added and put on an ice bath for milling extraction. The homogenate was centrifuged on a refrigerated centrifuge according to the instructions of the corresponding kit. Finally, OD values of PAL, SOD, CAT and POD at 290, 560, 240 and 470nm were read, respectively, and enzyme activities were calculated according to the formulas in the respective specifications.

The results are shown in FIG. 1. As shown in FIG. 1A, SOD activity reached a maximum on the first day, which was 1.43, 1.95 and 3.94 times (P) that of the blank control at 125, 250 and 500. Mu.g/mL, respectively<0.05). This indicates that excess SOD is catalyzed to form H ₂ O ₂ Activating the burst of active oxygen. CAT and POD frequently participate in H ₂ O ₂ Catabolism of (c). CAT is a major H ₂ O ₂ Enzymes are eliminated, playing an important role in the ROS scavenging system. POD pair elimination H ₂ O ₂ The toxicity of (b) and the metabolism of phenols and amines have a dual role. Changes in CAT and POD As shown in FIGS. 1B and 1C, the CAT and POD activities reached maximum values at 5d, 2.24, 2.88 and 5.12 times as high as those of the blank control at 125, 250 and 500. Mu.g/mL, and 7.00, 8.56 and 11.06 times as high as those of the blank control with POD (P)<0.05). The above results indicate that the new henbane base can consume the accumulated H by activating POD and CAT at relatively later times ₂ O ₂ To avoid oxidative stress damage in tobacco. In addition, PAL is an important restriction enzyme for phenylpropanoid metabolism in plants. As shown in FIG. 1D, PAL activity gradually increased from day 0 to 7, and declined at day 9. PAL activity was dose-dependent at 7d peak, 1.55, 2.00 and 500. Mu.g/mL in the blank at 125, 250 and 500. Mu.g/mL concentrations, respectively2.44 times. The results indicate that the new alkaloid-induced defense response of henbane is associated with the triggering of metabolic pathways.

Example 6: the new alkaloid of henbane causes the change of the expression quantity of the tobacco disease-resistant gene and the salicylic acid synthetic gene

Spraying the new henbane alkali solution on the tobaccos in the leaf stage of 4-6, and collecting samples after processing for 24 hours. Extracting total RNA of tobacco by liquid nitrogen method, and determining expression quantity change of disease-resistant related genes NPR1, PR1 and PR2 and salicylic acid synthetic genes ICS1 and PAL by real-time fluorescent quantitative PCR.

As shown in FIG. 2, the new henbane alkaloid can cause the obvious changes of the transcription level of PR protein and the transcription level of salicylic acid metabolism of disease resistance related genes. The expression levels of NPR1, PR2, ICS1 and PAL are all obviously increased relative to the control group, which shows that the henbane neoline induces the tobacco to generate disease-resistant defense behavior in vivo, and lays a foundation for controlling tobacco diseases such as tobacco bacterial wilt, tobacco brown spot, tobacco black shank and the like.

Example 7: the henbane alkaloid can induce different plants to generate hypersensitivity reaction

Hypersensitivity is the induction of local cell death at the site of pathogen infestation, and is the most significant disease-resistant reaction of plants. To prevent further spread of plant fungal, bacterial, viral and nematode diseases, plants can limit pathogens to the site of infection through hypersensitivity reactions. The novel henbane alkali is used for treating different plant leaves, and the novel henbane alkali can induce the leaves of various plants such as tobacco, apple, kiwi fruit, poplar, tomato, pepper, cucumber, poplar and the like to generate hypersensitivity under the concentration of 125-1000 mu g/mL. Figure 3 shows only hypersensitivity reactions caused by hyoscyamine on tobacco. Therefore, the effect of the henbane neoalkaloid on the hypersensitivity of plants is demonstrated, the prevention and control of the henbane neoalkaloid on different plant diseases are mainly used for inducing the disease resistance of the plants, and the henbane neoalkaloid can be developed as a plant immunity inducer and has great development and application potential.

Example 8: indoor disease-resistant activity of henbane neobase on different plant pathogenic bacteria

In order to verify the disease resistance of henbane neoline to different plant diseases, the protection effect of henbane neoline on different plant tissues is measured indoors by using a living tissue method with tomato botrytis cinerea, phytophthora capsici, apple tree canker, poplar canker, kiwi fruit canker and the like as targets. Cutting sterilized plant tissue into small openings, treating the cut with 250 μ g/mL and 500 μ g/mL henbane neokaline for 3d, smearing different bacterial liquids, calculating control effect by adopting 1% DMSO as blank control and 25 ℃ moisture-preserving culture for 7 d.

TABLE 4 protective Effect of henbane neobase on different plant pathogens

As shown in Table 4, the effect of henbane neoline on the protection of Botrytis cinerea and Phytophthora capsici is the best, and the control effect at 500. Mu.g/mL is 84.3% and 89.2%. Also has certain control effect (56.7-64.6%) on fruit tree diseases such as apple tree canker, poplar canker, kiwi fruit canker and the like.

Example 9: influence of henbane neoline on lettuce seed germination and root length

The lettuce seeds are taken as a representative, and the influence of henbane neoline on seed germination and root length is developed. Lettuce seeds were treated with varying concentrations of henbane neoalkali, blanked with 1% dmso, observed for seed germination after 3d, and root length measured after 7 d. Seed germination inhibition (%) = (germinated seeds/total seeds) × 100.

TABLE 5 influence of henbane neoline on lettuce seed germination and root length

The results are shown in Table 5, and it is clear that the seed germination rate of the henbane neoline is 58.3% -83.3% at the concentration of 1-40 mug/mL, which is obviously higher than that of the negative control 56.1%, but the lettuce seed germination can be obviously inhibited at the concentration of more than 60 mug/mL. The concentration of the henbane neokaline is 1-20 mu g/mL, the length of the cochlear root is 31.3-57.3 mm, which is 30.9mm higher than that of the negative control. The new henbane alkaloid can promote seed germination and root growth under low concentration and inhibit seed germination and root growth under high concentration, and can be used as a good plant growth regulator. For lettuce, the effect of henbane neoline on seed germination and root length is comprehensively considered, and the safe medication concentration is recommended to be 1-20 mu g/mL.

Example 10: growth promoting effect of henbane neoalkaloid on cucumber and pumpkin

Takes cucumber and cucurbita pepo seedlings as representatives, and develops the growth promoting effect of henbane neoalkaloid. Spraying cucumber and Cucurbita pepo L seedlings with hyoscyamine at a concentration of 250. Mu.g/mL, using 1% DMSO as a blank, and observing plant growth after 7 d.

As shown in FIG. 4, the plant height and leaf area of different crops such as cucumber and pumpkin can be obviously improved by the hyoscyamine with the concentration of 250 μ g/mL, and the growth promoting effect is obvious.

While the preferred embodiments of the present invention have been described, variations and modifications of these embodiments may occur to those skilled in the art once they learn of the basic inventive concepts.

It should be noted that, when the present invention relates to numerical ranges, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and in order to prevent redundancy, the present invention describes a preferred embodiment. It should be noted that, when the present invention relates to numerical ranges, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and in order to prevent redundancy, the present invention describes a preferred embodiment.

Claims

1. A vanillic amide compound having a chemical formula as shown in formula (I):

2. the method for preparing the vanillimide compound according to claim 1, which is used for separating and purifying the vanillimide compound from henbane, and comprises the following steps:

and 5, performing gradient elution on the fraction 2 by adopting a liquid chromatography, wherein an eluent is a mixed solution of methanol and water or a mixed solution of acetonitrile and water, and the volume ratio of the methanol or the acetonitrile to the water is 35: 65-65: 35; obtaining the vanillic amide compound.

3. The method for producing vanillylamide based compound according to claim 2, wherein the volume ratio of henbane seed to ethanol solvent in step 1 is 1.

4. The method for producing vanillyl amide based compound according to claim 2, wherein the extraction process in the step 2 comprises: extracting with petroleum ether for 3-4 times, extracting the lower liquid phase after petroleum ether extraction with ethyl acetate for 3-4 times, and extracting the lower liquid phase after ethyl acetate extraction with n-butanol for 3-4 times.

5. The method for producing vanillin amide compounds according to claim 2 or 4, wherein the volume ratio of petroleum ether, ethyl acetate and n-butanol in step 2 is 1.

6. The method for producing vanillin amide compounds of claim 2, wherein in the step 3, a normal phase silica gel column is used for the normal phase gradient elution; and the reverse phase column chromatography elution in the step 4 adopts a C-18 reverse phase column.

7. A method for preparing the vanillimide compound according to claim 1, which comprises the following steps of synthesizing the vanillimide compound by using vanillic acid, gamma-aminobutyric acid and n-butyl alcohol:

step 2, adding the intermediate product obtained in the step 1, vanillic acid, HATU and DIPEA into thionyl chloride, stirring and reacting for 8-15 h at 10-35 ℃, concentrating and eluting the filtrate to obtain vanillic amide compounds; wherein the molar ratio of the intermediate product to the vanillic acid to the HATU to the DIPEA is 10:10:13:20.

8. the method of claim 7, wherein the step of adding the intermediate product, vanillic acid, HATU and DIPEA of step 2 comprises: dissolving vanillic acid and HATU in thionyl chloride, adding the intermediate product, stirring for 10-15min, and then adding DIPEA.

9. The method of claim 7, wherein step 2 is performed by column chromatography, and the eluent is a mixture of petroleum ether and ethyl acetate at a volume ratio of 5-1.

10. Use of vanilloid compound of claim 1 for the preparation of plant immune elicitors and growth regulators.