A method for preparing the monoterpene trichodesmic acid and its application

Abstract

This invention discloses a method for preparing the monoterpene trichodesmic acid and its application, relating to the fields of pharmaceutical and phytochemistry. The structural formula of trichodesmic acid is: [Insert structural formula here]. Its molecular formula is C[Insert molecular formula here]. 10 H 16 O5; Application of Trichodesmic acid in the control of southern root-knot nematodes. Trichodesmic acid was isolated from sesame seeds, and this compound has strong nematicidal activity against southern root-knot nematodes.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical and phytochemical technology, specifically to a method for preparing the monoterpene trichodesmic acid and its application. Background Technology

[0002] Crotalaria juncea L., a legume belonging to the genus Crotalaria, is an erect, shrubby herb native to India and distributed in Fujian, Taiwan, Guangdong, Guangxi, Sichuan, and Yunnan provinces of China. It grows in wild wastelands, roadsides, and sparse forests on hillsides at altitudes of 50-2000 meters. It has medicinal uses, commonly used as an effective antidote and anesthetic. The stems and branches can be used to make various ropes and sacks, and its fiber can be used as a raw material for papermaking. Rich in nitrogen, phosphorus, and potassium, it can also be used as green manure, improving soil quality. Furthermore, Crotalaria juncea is an unfavorable host for many important plant-parasitic nematodes, including the southern root-knot nematode, the Javan root-knot nematode, the peanut root-knot nematode, the kidney-shaped nematode, and the root-rot nematode. On one hand, its root and leaf secretions contain allelopathic compounds, such as limonene and pyrrolizidine alkaloids, which are toxic to nematodes. When added to the soil, Crotalaria juncea residue can enhance the activity of certain nematode-antagonistic microorganisms. On the other hand, bulrush can improve and reduce nematode population density and plays an important role in soil nutrient cycling. Currently, there is not much literature on the anti-root-knot nematode activity of bulrush active ingredients. Foreign scholars applied bulrush leaf extract at a ratio of 40 grams of leaf tissue / liter of water to plants infected with root-knot nematodes in southern China. The results showed that it could prolong the development and reproduction of nematodes, thereby inhibiting their population accumulation. Domestic scholars conducted research on the anti-tobacco root-knot nematode activity of intercropping flue-cured tobacco and bulrush. Field trial results showed that 1:1 intercropping of flue-cured tobacco and bulrush had a good control effect on root-knot nematode disease and could significantly increase tobacco yield and output value.

[0003] As an important member of the legume family, *Paecilomyces lilacinus* not only provides abundant nutrients for the growth of *Paecilomyces lilacinus* in the soil, but also acts as a biocontrol agent against root-knot nematodes. Currently, there are no clear reports on the use of *Paecilomyces lilacinus*'s active ingredients to control southern root-knot nematodes. Therefore, it is necessary to study its chemical composition and biological activities to obtain compounds with development and utilization value. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing the monoterpene Trichodesmic acid and its application. Trichodesmic acid was isolated from sesame seeds, and this compound has strong nematicidal activity against southern root-knot nematodes.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A monoterpene, trichodesmic acid, has the following structural formula: Its molecular formula is C 10 H 16 O5;

[0007] Application of trichodesmic acid in the control of southern root-knot nematodes.

[0008] Furthermore, the preparation method includes the following steps:

[0009] Step 1) The sesame seeds are pretreated to obtain an extract. The extract is dissolved in hydrochloric acid and the pH is adjusted. Then, it is extracted multiple times with ethyl acetate. The ethyl acetate layer is collected and then evaporated to dryness to obtain the ethyl acetate layer.

[0010] Step 2) The ethyl acetate layer was separated by silica gel column chromatography, then eluted with petroleum ether-acetone solution, and then decolorized by MCI column to obtain eluent I;

[0011] Step 3) Eluent I is subjected to reversed-phase column chromatography and eluted with methanol-distilled water to obtain eluent II;

[0012] Step 4) Eluent II was separated by high performance liquid chromatography and purified by elution with C2H3N-H2O to obtain the compound Trichodesmic acid.

[0013] Furthermore, the pretreatment process is as follows: the sesame seeds are dried, crushed, and sieved to obtain raw materials, which are then extracted by soaking in methanol, vacuum distillation, and concentration to obtain an extract.

[0014] Further: In step 2), the volume ratio of petroleum ether to acetone in the petroleum ether-acetone solution is 40-1:2.

[0015] Furthermore: In step 3), the volume ratio of methanol to distilled water in the methanol-distilled aqueous solution is 3:7-10:0.

[0016] Further: the reduced pressure distillation is continued until no alcohol odor is detected.

[0017] Furthermore: In step 3), the silica gel for column chromatography is 100-200 mesh.

[0018] Further: The suspension of Southern Root-knot Nematodes was placed into the well plate to obtain the nematode solution;

[0019] Next, dissolve the trichodesmic acid in methanol and add it to the insect solution;

[0020] After the insect solution containing trichodesmic acid was placed in a constant temperature incubator for 24-72 hours, the mortality of the southern root-knot nematodes was examined under a microscope, the corrected mortality rate was calculated, and the nematicidal activity was evaluated.

[0021] Furthermore, the volume of the southern root-knot nematode is 97.5 μL-360 μL.

[0022] Furthermore, the concentration of the insect solution containing Trichodesmic acid was 6.25 μg / mL to 100.0 μg / mL.

[0023] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0024] (1) The monoterpenoid Trichodesmic acid isolated from sesame seeds in this invention has strong nematicidal activity against southern root-knot nematodes. This provides important inspiration for further research on sesame, a legume, as a plant resource for drug treatment against southern root-knot nematodes and can be used for the research and development of drugs for the control of southern root-knot nematodes.

[0025] (2) The raw materials of this invention are readily available, the preparation method is simple, and the preparation cost is low. This invention will increase the economic and social benefits of sesame seeds and has broad application prospects. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example 1

[0028] A method for preparing a monoterpene, trichodesmic acid, includes the following steps:

[0029] 1) Collect soybean seeds, dry them, then crush 10kg of dried soybean seeds and pass them through a 40-mesh sieve as raw material;

[0030] 2) Soak the obtained raw material in 10 times the amount of analytical grade methanol solution for 10 hours to extract, then add 10 times the amount of analytical grade methanol solution to extract again. Repeat this extraction operation once. Distill under reduced pressure until no alcohol smell can be detected, and concentrate to obtain 800g of extract.

[0031] 3) Dissolve the obtained extract in 2% hydrochloric acid solution, adjust the pH to 2, extract with an equal volume of ethyl acetate 6 times, and recover the solvent until there is no smell of ethyl acetate, to obtain 100g of ethyl acetate layer;

[0032] 4) The ethyl acetate layer was separated by silica gel column chromatography (the silica gel for column chromatography was 100-200 mesh), then eluted with a petroleum ether-acetone solution with a volume ratio of 8:2, and then decolorized by MCI column. The eluent I was collected.

[0033] 5) Eluent I was subjected to reversed-phase column chromatography and eluted with methanol-distilled water at a volume ratio of 1:1. The resulting eluent II was collected.

[0034] 6) Eluent II was then purified by high performance liquid chromatography using C2H3N-H2O (C2H3N-H2O = 55%:45%, V / V) to obtain the compound Trichodesmic acid.

[0035] Example 2

[0036] The difference from Example 1 is that the volume ratio of petroleum ether-acetone solution to acetone is 20:1.

[0037] Example 3

[0038] The difference from Example 1 is that the volume ratio of petroleum ether-acetone solution to acetone is 1:2.

[0039] Example 4

[0040] The difference from Example 1 is that the volume ratio of methanol to distilled water in the methanol-distilled water solution is 3:7.

[0041] Example 5

[0042] The difference from Example 1 is that the volume ratio of methanol to distilled water in the methanol-distilled water solution is 10:0.

[0043] Analysis of the compounds obtained in Examples 1-5 revealed that the compound obtained under the conditions of Example 1 was identified as Trichodesmic acid.

[0044] Structural identification of the compounds of this invention

[0045] The structure of the compound prepared in Example 1 was determined.

[0046] The compound of this invention is a colorless oil, ESI-MS m / z: 217[M+H]+, with the molecular formula C. 10 H 16O5.1H NMR(600MHz,MeOD)δ3.35(s,1H),2.50(1H,d,J=8.4Hz,H-2),1.95(1H,m,H-8),1.59(3H ,s,H-6),1.31(3H,s,H-7),1.20(3H,d,J=6.7Hz,H-10),1.12(3H,d,J=6.7Hz,H-9).13C NMR(150MHz,MeOD)δ175.42(C-1),173.68(C-5),88.91(C-4),79.44(C-3),55 .00(C-2),26.82(C-8),22.54(C-7),21.18(C-6),19.68(C-9),19.18(C-10).

[0047] Based on its spectral data, the compound was identified as Trichodesmic acid.

[0048] Tests to detect the activity of the compound against southern root-knot nematodes in this invention:

[0049] Each monomer compound was thoroughly dissolved in pure methanol to a concentration of 1 mg / mL. 360 μL, 380 μL, 390 μL, 395 μL, and 397.5 μL of *Symplocos spp.* suspension were placed in 24-well plates, with approximately 40 nematodes per well. 40 μL, 20 μL, 10 μL, 5 μL, and 2.5 μL of the 1 mg / mL monomer compound solution were added to these suspensions, corresponding to final concentrations of 100.0 μg / mL, 50.0 μg / mL, 25.0 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, respectively. A control group of methanol-water solution was included. Each treatment was repeated three times. After incubation at 28°C for 24 h, 48 h, and 72 h, the mortality of *Symplocos spp.* was examined under a microscope, the corrected mortality rate was calculated, and the nematicidal activity was evaluated. LC at 24h, 48h, and 72h was calculated based on the corrected mortality rate at different concentrations. 50 .

[0050] Statistical methods for results:

[0051]

[0052] According to the literature method, the killing activity is divided into five levels:

[0053] "-": indicates a corrected mortality rate of ≤10%, meaning no killing activity;

[0054] "+": indicates a corrected mortality rate of 10% to 30%, indicating weak killing activity;

[0055] "++": indicates a corrected mortality rate of 30% to 50%, and moderate killing activity;

[0056] "+++": indicates a corrected mortality rate of 50% to 80%, indicating strong killing activity;

[0057] "++++": indicates a corrected mortality rate > 80%, indicating strong killing activity.

[0058] Table 1. Thread-killing activity of Trichodesmic acid

[0059]

[0060] As shown in the table, at a concentration of 25.0 μg / mL, the compound of this invention exhibits strong nematicidal activity after 24 hours and intense nematicidal activity after 48 hours, with a corrected mortality rate of 85.04% (LC). 50 The concentration was 11.744 μg / mL. When the concentration reached 100.0 μg / mL, it exhibited strong nematode-killing activity after 24 hours, and all nematodes died, with a corrected mortality rate of 100.00%.

[0061] This invention isolates the monoterpene trichodesmic acid from sesame seeds, which exhibits strong nematicidal activity against southern root-knot nematodes. This provides important insights for further research on sesame, a legume, as a medicinal plant resource for controlling southern root-knot nematodes and can be used in the research and development of drugs for the prevention and control of southern root-knot nematodes.

[0062] Although the invention has been described herein with reference to several illustrative embodiments, it should be understood that many other modifications and implementations can be devised by those skilled in the art, which will fall within the scope and spirit of the principles disclosed herein. More specifically, various variations and modifications can be made to the components and / or layout of the subject matter combination within the scope of the disclosure and claims. Besides variations and modifications to the components and / or layout, other uses will be apparent to those skilled in the art.

Claims

1. The application of a monoterpene, trichodesmic acid, in the control of southern root-knot nematodes, characterized in that: The structural formula of the Trichodesmic acid is: Its molecular formula is C 10 H 16 O5.

2. The application of the monoterpene trichodesmic acid according to claim 1 in the control of southern root-knot nematodes, characterized in that: A suspension of southern root-knot nematodes was placed into a well plate to obtain nematode solution; Dissolve Trichodesmic acid in methanol and add it to the insect solution. After placing the insect solution with added Trichodesmic acid in a constant temperature incubator for 24-72 hours, examine the mortality of the southern root-knot nematodes under a microscope, calculate the corrected mortality rate, and evaluate the nematicidal activity.

3. The application of the monoterpene trichodesmic acid according to claim 2 in the control of southern root-knot nematodes, characterized in that: The volume of the southern root-knot nematode ranged from 97.5 μL to 360 μL.

4. The application of a monoterpene trichodesmic acid according to claim 2 in the control of southern root-knot nematodes, characterized in that: The concentration of the insect solution with added trichodesmic acid was: 6.25 μg / mL - 100.0 μg / mL.

5. A method for preparing a monoterpene, trichodesmic acid, characterized in that: The preparation method comprises the following steps: Step 1) Soybean seeds are pretreated to obtain extract. The pretreatment process is as follows: Soybean seeds are dried, crushed and sieved to obtain raw material. The raw material is extracted by soaking in methanol, vacuum distillation and concentration to obtain extract. The extract is dissolved in hydrochloric acid and the pH is adjusted to 2. Then it is extracted with ethyl acetate 6 times, the ethyl acetate layer is collected and then evaporated to dryness to obtain ethyl acetate layer. Step 2) The ethyl acetate layer was separated by silica gel column chromatography, then eluted with petroleum ether-acetone solution, and then decolorized by MCI column to obtain eluent I; Step 3) Eluent I is subjected to reversed-phase column chromatography and eluted with methanol-distilled water to obtain eluent II; Step 4) Eluent II is separated by high performance liquid chromatography and purified by elution with C2H3N-H2O to obtain the compound Trichodesmic acid; The structural formula of the Trichodesmic acid is: Its molecular formula is C 10 H 16 O5.

6. The method for preparing a monoterpene trichodesmic acid according to claim 5, characterized in that: In step 2), the volume ratio of petroleum ether to acetone in the petroleum ether-acetone solution is 40-1:

2.

7. The method for preparing a monoterpene trichodesmic acid according to claim 5, characterized in that: In step 3), the volume ratio of methanol to distilled water in the methanol-distilled aqueous solution is 3:7-10:

0.

8. The method for preparing a monoterpene trichodesmic acid according to claim 5, characterized in that: The process involves vacuum distillation until no alcohol odor is detected.

9. The method for preparing a monoterpene trichodesmic acid according to claim 5, characterized in that: In step 3), the silica gel for column chromatography is 100-200 mesh.

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