Preparation method and application of monoterpene Trichodesmic acid

Abstract

The invention discloses a preparation method and application of monoterpene Trichodesmic acid, and relates to the technical field of medicines and phytochemistry, the structural formula of the Trichodesmic acid is # imgabs0 #, and the molecular formula of the Trichodesmic acid is C10H16O5; the invention relates to application of Trichodesmic acid in prevention and treatment of meloidogyne incognita. Trichodesmic acid is separated from janapan seeds, and the compound has relatively strong nematicidal activity on meloidogyne incognita.

Description

Technical Field

[0001] The invention relates to the technical field of medicine and plant chemistry, and in particular to a preparation method of monoterpene Trichodesmic acid and application thereof. Background Art

[0002] Crotalaria juncea L. is an upright shrubby herb of the Leguminosae family, Crotalaria genus, native to India, and distributed in Fujian, Taiwan, Guangdong, Guangxi, Sichuan, and Yunnan, China. It grows in sparse forests on roadsides and hillsides of wild wasteland at an altitude of 50-2000 meters. It can be used for medicinal purposes and is often used as an effective drug for detoxification and anesthesia. The stems and branches can be made into various ropes, sacks, etc., and its fiber can be used as a raw material for papermaking; because it is rich in nitrogen, phosphorus, and potassium, it can also be used as a green manure, which has the effect of improving soil. In addition, juncea is a poor host for many important plant parasitic nematodes, including southern root-knot nematodes, Javan root-knot nematodes, peanut root-knot nematodes, reniform reniform nematodes, and root-rot nematodes. On the one hand, its root and leaf secretions contain allelopathic compounds, such as monocrotaline and pyrrolizidine alkaloids, which are toxic to nematodes. When added to the soil, juncea residues can enhance the activity of certain nematode antagonistic microorganisms. On the other hand, sisal can improve and reduce the population density of nematodes and plays an important role in soil nutrient cycling. At present, there are not many literatures on the research of sisal active ingredients against root-knot nematodes. Foreign scholars applied sisal leaf extracts at a ratio of 40 grams of leaf tissue / liter of water to plants infected with southern root-knot nematodes. The results showed that the development and reproduction of nematodes can be prolonged, thereby inhibiting their population accumulation. Domestic scholars conducted research on the intercropping of flue-cured tobacco and sisal against tobacco root-knot nematodes. The results of field tests showed that the 1:1 intercropping of flue-cured tobacco and sisal has a good control effect on root-knot nematode disease and can significantly increase tobacco leaf yield and output value.

[0003] As an important member of the leguminous plant family, sisal can not only provide rich nutrients for the growth of Paecilomyces lilacinus in the soil, but also can be used as a biocontrol factor for root-knot nematodes. At present, there is no clear report on the use of sisal active ingredients to control southern root-knot nematodes. Therefore, it is necessary to study its chemical composition and biological activity, and then obtain compounds with development and utilization value from it. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of monoterpene Trichodesmic acid. Trichodesmic acid is separated from sisal seeds and has strong nematicidal activity against southern root-knot nematodes.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

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

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

[0008] Further: the preparation method comprises the following steps:

[0009] Step 1) pre-treating sunflower seeds to obtain an extract, dissolving the extract with hydrochloric acid and adjusting the pH, then extracting with ethyl acetate for multiple times, collecting the ethyl acetate layer, and then evaporating to dryness to obtain an ethyl acetate layer;

[0010] Step 2) The ethyl acetate layer is subjected to silica gel column chromatography, eluted with petroleum ether-acetone solution, and then decolorized by MCI column to collect the eluate I;

[0011] Step 3) The eluate I is subjected to reverse phase column chromatography, eluted with methanol-distilled water, and the eluate II is collected;

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

[0013] Further: the pretreatment process is: the sunflower seeds are dried, crushed and sieved to obtain raw materials, the raw materials are soaked and extracted with methanol, and the extract is obtained by vacuum distillation and concentration.

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

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

[0016] Further: the reduced pressure distillation is carried out until there is no alcohol taste.

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

[0018] Further: placing a suspension of southern root-knot nematodes into a well plate to obtain an insect liquid;

[0019] Then, dissolve Trichodesmic acid in methanol and add Trichodesmic acid to the insect solution;

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

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

[0022] Furthermore: the concentration of the insect liquid added with Trichodesmic acid is 6.25 μg / mL-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 monoterpene Trichodesmic acid isolated from the seeds of the present invention has a strong nematicidal activity against the southern root-knot nematode, which provides important inspiration for further studying the leguminous plant Sunflower as a plant resource for drugs against southern root-knot nematode disease, and can be used for the research and development and preparation of drugs for controlling southern root-knot nematodes.

[0025] (2) The raw materials of the invention are easily available, the preparation method is simple, and the preparation cost is low, which will increase the economic and social benefits of sisal and has broad application prospects. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Example 1

[0028] A method for preparing monoterpene Trichodesmic acid comprises the following steps:

[0029] 1) collecting sisal seeds and drying them in the air, crushing 10 kg of the dried sisal seeds, and passing through a 40-mesh sieve as a raw material;

[0030] 2) Soak the obtained raw material in 10 times analytically pure methanol solution for 10 hours for extraction, then add 10 times analytically pure methanol solution for extraction, repeat the operation of adding 10 times analytically pure methanol solution for extraction once, distill under reduced pressure until no alcohol smell is odorless, and concentrate to obtain 800g of extract;

[0031] 3) The obtained extract was dissolved with 2% hydrochloric acid solution, the pH was adjusted to 2, and extracted with an equal volume of ethyl acetate for 6 times, and the solvent was recovered until no ethyl acetate smell was detected, and 100 g of ethyl acetate layer was obtained;

[0032] 4) The ethyl acetate layer is subjected to silica gel column chromatography (column chromatography silica gel is 100-200 mesh), and then eluted with a petroleum ether-acetone solution with a volume ratio of 8:2, and then decolorized by an MCI column, and the eluted eluate I is collected;

[0033] 5) The eluate I is subjected to reverse phase column chromatography, eluted with methanol-distilled water in a volume ratio of 1:1, and the eluate II obtained is collected;

[0034] 6) The eluate II was purified by high performance liquid chromatography (HPLC) 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 the petroleum ether-acetone solution to acetone is 20:1.

[0037] Example 3

[0038] The difference from Example 1 is that the volume ratio of the 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] By analyzing the compounds obtained in Examples 1-5, the compound obtained under the conditions of Example 1 was identified as Trichodesmic acid.

[0044] Structural identification of the compounds of the present invention

[0045] The compound prepared in Example 1 was taken and its structure was determined.

[0046] The compound of the present invention is a colorless oil, ESI-MS m / z: 217 [M + H] +, and the molecular formula is 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 Popper data, the compound was identified as Trichodesmic acid.

[0048] Anti-southern root-knot nematode activity test of the compounds of the present invention:

[0049] Use pure methanol to fully dissolve each monomer compound to a concentration of 1 mg / mL. Place 360 ​​μL, 380 μL, 390 μL, 395 μL, and 397.5 μL of the southern root-knot nematode suspension into a 24-well plate, so that the number of nematodes is about 40. Add 40 μL, 20 μL, 10 μL, 5 μL, and 2.5 μL of 1 mg / mL monomer compound solution to the above worm solution, respectively, and the corresponding final concentrations are 100.0 μg / mL, 50.0 μg / mL, 25.0 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. Set up a control group of methanol-water solution, and repeat each treatment 3 times. After 24h, 48h, and 72h in a constant temperature incubator at 28°C, check the death of southern root-knot nematodes under a microscope, calculate the corrected mortality rate, and evaluate the nematicidal activity. At different concentrations, the LC values ​​at 24h, 48h and 72h were calculated based on the corrected mortality rate. 50 .

[0050] Statistical method of results:

[0051]

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

[0053] “-”: indicates that the corrected mortality rate is ≤10% and there is no nematicidal activity;

[0054] “+”: indicates that the corrected mortality rate is 10% to 30% and the nematicidal activity is weak;

[0055] “++”: indicates that the corrected mortality rate is 30% to 50% and the nematicidal activity is moderate;

[0056] “+++”: indicates that the corrected mortality rate is 50% to 80% and the nematocidal activity is relatively strong;

[0057] “++++”: indicates that the corrected mortality rate is greater than 80% and the nematocidal activity is strong.

[0058] Table 1 Nematicidal activity of Trichodesmic acid

[0059]

[0060] As shown in the table, when the concentration is 25.0 μg / mL, the compound of the present invention has strong nematicidal activity after 24 hours and strong nematicidal activity after 48 hours, with a corrected mortality rate of 85.04%, LC 50 When the concentration reached 100.0 μg / mL, it had strong nematicidal activity after 24 hours, and all nematodes died, with a corrected mortality rate of 100.00%.

[0061] The monoterpene Trichodesmic acid isolated from the seeds of the present invention has strong nematicidal activity against southern root-knot nematodes, provides important inspiration for further studying the leguminous plant sissa as a plant resource for resisting southern root-knot nematode disease, and can be used for the research, development and preparation of southern root-knot nematode control drugs.

[0062] Although the present invention is described herein with reference to a number of illustrative embodiments of the present invention, it will be appreciated that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure and claims, a variety of variations and improvements may be made to the components and / or layout of the subject combination layout. In addition to the variations and improvements made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

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

2. A method for preparing monoterpene Trichodesmic acid, characterized in that: The preparation method comprises the following steps: step 1) pre-treating sunflower seeds to obtain an extract, dissolving the extract with hydrochloric acid and adjusting the pH, then extracting with ethyl acetate for multiple times, collecting the ethyl acetate layer, and then evaporating to dryness to obtain an ethyl acetate layer; Step 2) The ethyl acetate layer is subjected to silica gel column chromatography, eluted with petroleum ether-acetone solution, and then decolorized by MCI column to collect the eluate I; Step 3) The eluate I is subjected to reverse phase column chromatography, eluted with methanol-distilled water, and the eluate II is collected; Step 4) The eluate II is separated by high performance liquid chromatography and purified by elution with C2H3N-H2O to obtain the compound Trichodesmic acid.

3. The method for preparing a monoterpene Trichodesmic acid according to claim 2, characterized in that: The pretreatment process is as follows: the sunflower seeds are dried, crushed and sieved to obtain raw materials, the raw materials are soaked and extracted with methanol, and the extract is obtained by vacuum distillation and concentration.

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

2.

5. The method for preparing monoterpene Trichodesmic acid according to claim 2, characterized in that: In the step 3), the volume ratio of methanol to distilled water in the methanol-distilled water solution is 3:7-10:

0.

6. The method for preparing monoterpene Trichodesmic acid according to claim 3, characterized in that: The reduced pressure distillation is carried out until there is no alcohol taste.

7. The method for preparing monoterpene Trichodesmic acid according to claim 2, characterized in that: In the step 3), the silica gel for column chromatography is 100-200 mesh.

8. The monoterpene Trichodesmic acid according to claim 1, characterized in that: Putting the southern root-knot nematode suspension into the well plate to obtain worm liquid; Then, dissolve Trichodesmic acid in methanol and add Trichodesmic acid to the insect solution; After the worm solution with trichodesmic acid added was placed in a constant temperature incubator for 24-72 hours, the death of southern root-knot nematodes was checked under a microscope, the corrected mortality rate was calculated, and the nematicidal activity was evaluated.

9. The monoterpene Trichodesmic acid according to claim 8, characterized in that: The volume of the southern root-knot nematode is 97.5 μL-360 μL.

10. The monoterpene Trichodesmic acid according to claim 8, characterized in that: The concentration of the insect solution to which Trichodesmic acid is added is 6.25 μg / mL-100.0 μg / mL.

Images