Preparation method and application of cortex fraxini extract with activity of killing pinewood nematode

Abstract

The present application relates to a preparation method and application of Cortex Fraxini extract with pine wood nematode killing activity, wherein dry powder of Cortex Fraxini is extracted by ethanol to obtain an extract solution, and the ethanol extract obtained after reduced pressure distillation is extracted by ethyl acetate-water liquid-liquid extraction, the ethyl acetate phase is reduced pressure distilled to obtain the ethyl acetate extract of Cortex Fraxini with strong pine wood nematode killing activity, and the main active ingredient of the extract, aesculin, is separated therefrom. The corrected mortality rate of the ethyl acetate extract of Cortex Fraxini to pine wood nematode is as high as 94.14% at a concentration of 1 mg / mL within 72 h; and the half lethal concentration of aesculin to pine wood nematode is 30.37 μg / mL. The present application develops a new use of Cortex Fraxini extract, and provides an effective plant-derived pine wood nematode killing active substance, the raw material source is convenient, the extraction method is simple, the cost is low, the Cortex Fraxini extract has development potential in controlling pine wood nematode disease, and is applied to the development of an environmentally friendly plant-derived pine wood nematode killing agent.

Description

Technical field:

[0001] The invention belongs to the technical field of forestry pest control and disease prevention, and relates to a preparation method of a Fraxinus fraxinus extract with pine wood nematode killing activity and application thereof. Background technology:

[0002] Pine wilt disease (also known as pine wilt) is a devastating disease of pine trees. It is highly contagious, spreads rapidly, and has a high mortality rate. It has caused serious damage to pine resources in many parts of the world, especially in East Asia and Europe. Pine wilt disease was first discovered in Nanjing, Jiangsu Province, my country in the 1980s and spread rapidly. At present, outbreaks have been found in 19 provinces (autonomous regions and municipalities) in my country, and the infected area exceeds 20 million mu. Pine nematodes are the main pathogens of pine wilt disease and are mainly transmitted by vector insects such as pine alternatants. Currently, there are three main methods for preventing and controlling pine nematodes: physical control, chemical control, and biological control. Chemical control is the most direct and effective way to prevent and control pine nematodes, but conventional synthetic nematicides have defects such as single target, drug resistance, threats to non-target beneficial organisms, and damage to the ecological environment. Therefore, the research and development of green and environmentally friendly nematicides is becoming increasingly popular. There are abundant plant resources in nature, which contain a wealth of active ingredients. Extracting substances with pine wood nematode killing activity from plant secondary metabolites and developing corresponding drugs have attracted much attention from researchers in this field.

[0003] Qinpi is a traditional Chinese medicine made from the dried branch bark or dry bark of the Oleaceae plants Fraxinus quercetinus, Fraxinus chinensis, Fraxinus acuminata or Fraxinus chinensis, which is processed and dried. It grows in Liaoning, Jilin, Hebei, Henan, Inner Mongolia, Shaanxi, Shanxi, Sichuan and other places. It is widely distributed and easy to obtain. It has biological activities such as antidiarrheal and anti-inflammatory, anti-tumor, anti-hyperuricemia, antioxidant and antibacterial.

[0004] The present invention discloses for the first time the application of Fraxinus fraxinus extract in killing pine wood nematodes, in order to provide a certain basis for the development of new natural plant-derived pine wood nematode control drugs. Summary of the invention:

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for preparing a Fraxinus fraxinus extract with the activity of killing pine wood nematodes and its application. It has been verified that the Fraxinus fraxinus extract has strong activity in killing pine wood nematodes, and the main active ingredients in killing pine wood nematodes are determined, which can provide a chemical structure model for the development of environmentally friendly plant-based pine wood nematode-killing agents or biomimetic synthetic plant-derived nematicides with better activity.

[0006] In order to achieve the above-mentioned purpose of the invention, the present invention provides an application of a Fraxinus chinensis extract having the activity of killing pine wood nematodes in killing pine wood nematodes. The Fraxinus chinensis extract is prepared from Fraxinus chinensis dry powder as raw material, and is obtained by ethanol extraction, reduced pressure distillation, ethyl acetate-water liquid extraction and other means to obtain a Fraxinus chinensis ethyl acetate extract having strong activity of killing pine wood nematodes. At a concentration of 1 mg / mL, the corrected mortality rate of pine wood nematodes within 72 hours is as high as 94.14%; the fraxinin isolated therefrom has strong activity of killing pine wood nematodes.

[0007] The present invention also provides the use of quercetin in killing pine wood nematodes, wherein the median lethality concentration of quercetin to pine wood nematodes is 30.37 μg / mL; the corrected mortality rates of quercetin to pine wood nematodes for 72 hours when the concentrations are 50 μg / mL, 100 μg / mL, 150 μg / mL and 200 μg / mL are 71.22%, 80.48%, 92.13% and 97.26%, respectively; quercetin can be obtained by separating the components of a quercetin extract using silica gel column chromatography, or can be purchased from the market, or extracted from other plants containing quercetin.

[0008] The present invention also provides a method for preparing a Fraxinus fraxinus extract having pine wood nematode killing activity, and the specific steps are as follows:

[0009] In the first step, commercially available Fraxinus cortex dry powder was mixed with anhydrous ethanol at a solid-liquid ratio of 200 g:1 L, and ultrasonic-assisted shaking was performed at room temperature for 6 h. The extract was filtered through a Buchner funnel under reduced pressure to obtain a Fraxinus cortex ethanol primary extract. The filter residue was repeatedly extracted and filtered under reduced pressure according to the above method, and the extracts were combined to obtain a Fraxinus cortex ethanol extract.

[0010] In the second step, the ethanol extract of Fraxinus fraxinus is concentrated by vacuum distillation on a rotary evaporator to obtain a brown oily ethanol primary extract of Fraxinus fraxinus;

[0011] In the third step, the primary ethanol extract was extracted three times with ethyl acetate and water at a volume ratio of 1:1, the ethyl acetate phases were combined, and concentrated by distillation under reduced pressure to obtain the ethyl acetate extract of Fraxinus chinensis.

[0012] Take an appropriate amount of the ethyl acetate extract of Fraxinus chinensis obtained above, dissolve it in dimethyl sulfoxide (DMSO) to prepare a 20 mg / mL test stock solution. Take 5 μL of the test stock solution and add it to a 96-well plate, then add 95 μL of a 0.5% TritonX-100 aqueous solution containing about 100 pine wood nematodes and mix thoroughly. A 5% DMSO aqueous solution containing 0.5% Triton X-100 was used as a negative control. Each treatment group was repeated 3 times, and the well plate was placed in an incubator at 25°C for dark culture. Take out the well plate every 24 hours within 72 hours and observe and record the survival status of the nematodes under a stereomicroscope. The nematodes were determined to be dead if they were stiff and had no reaction when touched by a dissecting needle. The corrected mortality rate of the nematodes in each treatment was calculated according to the following formula:

[0013]

[0014] The results showed that the ethyl acetate extract of Fraxinus fraxinus had a corrected mortality rate of 94.14% against pine wood nematodes at a concentration of 1 mg / mL for 72 hours, indicating significant activity in killing pine wood nematodes.

[0015] The components of the ethyl acetate extract of Fraxinus chinensis were separated by silica gel (200-300 mesh) column chromatography, and the eluted components were separated by gradient elution with a solvent system of n-hexane-ethyl acetate-methanol. 254 ) thin-layer chromatography comparative analysis, and similar components were combined and concentrated to obtain a total of seven components: Fr.1-Fr.7. Fr.6 showed strong activity against pine wood nematodes. This component was further isolated and purified using silica gel (200-300 mesh) column chromatography with ethyl acetate-methanol (4:1, v / v) as the eluent to obtain the compound quercetin. The chemical formula of quercetin is:

[0016]

[0017] The characteristic constants of quercetin are: colorless to off-white crystalline powder, EIMS (m / z): 208 [M·] + ; 1 H NMR (600MHz, DMSO-d6): δ7.84(1H,d,J=9.48Hz,H-4), δ6.75(1H,s,H-5), δ6.17(1H,d,J=9.48Hz,H-3), δ3.77(3H,s,OCH3); 13 C NMR(DMSO-d6): δ161.06(C-2), δ145.86(C-6), 145.59(C-4), δ139.89(C-7), 139.80(C-9), 133.38(C-8), δ123.39(C-3), 112.36(C-3), δ110.76(C-10), δ100.83(C-5), δ56.57(OCH3).

[0018] Fraxinus quinquefolius was dissolved in DMSO and prepared into a series of stock solutions (1-4 mg / mL) as test solutions. The activity test was then carried out according to the above test method for the killing of pine wood nematodes by Fraxinus quinquefolius extract to obtain the corrected mortality rate of pine wood nematodes for each test solution at 72 hours. Statistical analysis was performed using SPSS 25.0 data processing software. The results showed that the median lethality concentration of Fraxinus quinquefolius against pine wood nematodes at 72 hours was 30.37 μg / mL.

[0019] Compared with the existing technology, the present invention uses the extract of Fraxinus chinensis for killing pine wood nematodes for the first time, develops a new use of the extract of Fraxinus chinensis, and provides an effective plant-based active substance for killing pine wood nematodes. The raw material source is convenient, the extraction method is simple, and the cost is low. The extract of Fraxinus chinensis has development potential in controlling pine wood nematode disease and can be used to develop environmentally friendly plant-based pine wood nematode agents. Specific implementation method:

[0020] The present invention will be further described below through specific examples.

[0021] Example 1

[0022] This embodiment relates to a method for preparing a Fraxinus fraxinus extract having pine wood nematode killing activity, specifically:

[0023] Take 100g of Qinpi dry powder and mix it with 500mL of 95% ethanol aqueous solution by volume, oscillate the mixture under ultrasonic conditions for 24 hours at room temperature, and obtain the Qinpi ethanol primary extract by vacuum filtration. The filter residue is repeatedly extracted once by the same method, and the extracts are combined to obtain ethanol extract; then, the ethanol extract is concentrated by rotary evaporator to obtain 23.3g of Qinpi ethanol primary extract; the ethanol primary extract is fully suspended in 500mL of distilled water and then added with an equal volume of ethyl acetate for extraction, and allowed to stand for 4h. After stratification, the upper ethyl acetate phase is taken, and the lower aqueous phase is extracted again with an equal volume of ethyl acetate, the ethyl acetate phases are combined, and finally concentrated by reduced pressure distillation on a rotary evaporator to obtain 4.7g of Qinpi ethyl acetate phase extract, which is the Qinpi extract with pine wood nematode killing activity.

[0024] Example 2

[0025] This example relates to the determination of the pine wood nematode killing activity of the Fraxinus Fraxinus extract obtained in Example 1, specifically:

[0026] An appropriate amount of the ethyl acetate extract of Fraxinus quinquefolius was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mg / mL test stock solution. 5 μL of the test stock solution was added to a 96-well plate, and then 95 μL of a 0.5% by volume Triton X-100 aqueous solution containing approximately 100 pine wood nematodes was added and mixed thoroughly. The final concentration of the ethyl acetate extract of Fraxinus quinquefolius was 1 mg / mL. A 5% by volume DMSO aqueous solution (containing 0.5% by volume Triton X-100) was used as a negative control. Each treatment group was repeated 3 times, and the well plate was placed in an incubator at 25°C for dark culture. Every 24 hours within 72 hours, the well plate was removed and placed under a stereomicroscope for observation and recording of the survival status of the nematodes. The nematodes were considered dead if they were stiff and had no reaction when touched by a dissecting needle. The corrected mortality rate of the nematodes in each treatment was calculated according to the following formula:

[0027]

[0028] The results showed that the corrected mortality of pine wood nematode by ethyl acetate extract of Fraxinus fraxinus at a concentration of 1 mg / mL after 72 hours was 94.14%.

[0029] Example 3

[0030] This example relates to the separation and identification of the active ingredients that kill pine wood nematodes in the Fraxinus Fraxinus extract obtained in Example 1, specifically:

[0031] (2) The ethyl acetate extract of the qinpi was separated by silica gel (200-300 mesh) column chromatography and eluted with a gradient of n-hexane-ethyl acetate-methanol solvent system (n-hexane:ethyl acetate = 2:1, 1:1, 1:5, v / v; 100% ethyl acetate; ethyl acetate:methanol = 3:1, 1:1, v / v; 100% methanol). The eluted fractions were filtered through silica gel (GF 254 ) thin-layer chromatography for comparative analysis, and identical fractions were combined and concentrated. Column chromatography of the ethyl acetate extract of the cortex qinpi yielded seven fractions: Fr.1-Fr.7. Activity testing against pine wood nematodes revealed that Fr.6 exhibited significant activity against pine wood nematodes. Fraction Fr.6 was further isolated and purified using silica gel (200-300 mesh) column chromatography with ethyl acetate-methanol (4:1, v / v) as the eluent, yielding 39.1 mg of a compound with high nematocidal activity.

[0032] 1.5 mg of active compound was dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and tetramethylsilane (TMS) was used as internal standard. Nuclear magnetic resonance (NMR) spectra were obtained using a nuclear magnetic resonance spectrometer (NMR, jnm-ecz600r, Joel, Japan). 1 H NMR) and carbon spectroscopy ( 13 C NMR) and electron impact mass spectrometry (EIMS) were performed using gas chromatography-mass spectrometry (GCMS, 7890a-5975c, Agilent, USA). The nematocidal compound was identified as fraxinin.

[0033]

[0034] The characteristic constants of quercetin are: colorless to off-white crystalline powder, EIMS m / z: 216.1[M] + , 1 H NMR (600MHz, DMSO-d6): δ7.84(1H,d,J=9.48Hz,H-4), δ6.75(1H,s,H-5), δ6.17(1H,d,J=9.48Hz,H-3), δ3.77(3H,s,OCH3); 13C NMR(DMSO-d6): δ161.06(C-2), δ145.86(C-6), 145.59(C-4), δ139.89(C-7), 139.80(C-9), 133.38(C-8), δ123.39(C-3), 112.36(C-3), δ110.76(C-10), δ100.83(C-5), δ56.57(OCH3).

[0035] Example 4

[0036] This example relates to the test of the pine wood nematode killing activity of fraxinusin obtained in Example 3, specifically:

[0037] Fraxinus quinquefolius was dissolved in DMSO to prepare a series of stock solutions (1-4 mg / mL) as test solutions. The activity of Fraxinus quinquefolius extract against pine wood nematodes was then tested according to the above test method. The corrected mortality rates of 72 hours against pine wood nematodes at final concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL were 71.22%, 80.48%, 92.13%, and 97.26%, respectively. Statistical analysis using SPSS 25.0 data processing software revealed that the median lethality concentration of Fraxinus quinquefolius against pine wood nematodes at 72 hours was 30.37 μg / mL.

Claims

1. An application of a Fraxinus quinquefolius extract having pine wood nematode killing activity in killing pine wood nematodes, characterized in that: The cortex fraxinus extract is obtained by taking cortex fraxinus dry powder as raw material through anhydrous ethanol extraction, vacuum filtration, ethyl acetate extraction and reduced pressure distillation.

2. The use of the extract of Fraxinus fraxinus having pine wood nematode killing activity according to claim 1 in killing pine wood nematodes, characterized in that: The preparation steps of the Fraxinus fraxinus extract include: The first step is to mix the dried powder of Fraxinus fraxinus with anhydrous ethanol at a material-liquid ratio of 200 g:1 L, perform ultrasonic oscillation-assisted extraction at room temperature for 24 hours, and obtain the Fraxinus fraxinus ethanol extract by vacuum filtration. Repeat the above method for the residue, and combine the Fraxinus fraxinus ethanol extracts; The second step is to concentrate the ethanol extract of the Fraxinus chinensis by vacuum distillation on a rotary evaporator to obtain a primary ethanol extract of the Fraxinus chinensis. In the third step, the obtained primary ethanol extract of the Fraxinus chinensis cortex is subjected to liquid extraction twice with ethyl acetate and water in a volume ratio of 1:1, the ethyl acetate phases are combined, and concentrated by vacuum distillation to obtain a Fraxinus chinensis cortex extract with pine wood nematode killing activity.

3. The use of the extract of Fraxinus fraxinus having pine wood nematode killing activity according to claim 1 in killing pine wood nematodes, characterized in that: The corrected mortality rate of the Fraxinus fraxinus extract against pine wood nematodes at a concentration of 1 mg / mL after 72 hours was 94.14%.

4. The application of fraxinin in killing pine wood nematodes is characterized in that: Fraxinusin has activity against pine wood nematodes, with a median lethal concentration of 30.37 μg / mL.

5. The use of fraxinusin in killing pine wood nematodes according to claim 4, characterized in that The corrected mortality rate of the quercetin against pine wood nematodes at a concentration of 200 μg / mL after 72 hours was 97.26%.

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