Euphorbia lathyris spirocyclic diterpenoid compound and preparation method and application thereof in prevention and treatment of plant fungal diseases

Abstract

The invention relates to the field of natural medicines. In particular to a novel skeleton compound spirolathyrisin A which is obtained by taking roots of euphorbia lathyris as a raw material and extracting and separating through a natural product chemical method, the skeleton of the spirolathyrisin A belongs to euphorbia lathyris spiro-diterpenoid, and the spirolathyrisin A has a relatively strong inhibition effect on plant pathogenic fungi and can provide a novel lead compound for screening of prevention and treatment of plant fungal diseases.

Description

Technical Field

[0001] The present invention relates to the field of natural products. It is characterized in that taking the root of Euphorbia lathyris L. as raw material, through the methods of natural product chemistry for extraction and separation, a new compound is obtained, the skeleton of which belongs to a novel Euphorbia spiro diterpene. This compound has significant antibacterial effects on Botryosphaeria dothidea, Diaporthe phaseolorum, Fusarium oxysporum and Alternaria alternata, and can be used for preparing pesticides for preventing and controlling plant fungal diseases. Background Art

[0002] Euphorbia lathyris L. is a biennial herbaceous plant of the genus Euphorbia in the Euphorbiaceae family. It is widely distributed in Europe, North Africa, Central Asia, East Asia, and North and South America. It is cultivated in Jilin, Liaoning, Inner Mongolia, Hebei, Shaanxi, Gansu, Xinjiang, Shandong, Jiangsu, Anhui, Zhejiang and other places in China (Editorial Committee of Flora Reipublicae Popularis Sinicae, Chinese Academy of Sciences. Flora of China [M]. Beijing: Science Press, 1997: 069.). The dried and mature seeds of Euphorbia lathyris are the traditional Chinese medicine Qianjinzi, and its processed product Qianjinzi Frost has the effects of treating constipation, edema, phlegm retention, stagnation and fullness, blood stasis amenorrhea, and treating stubborn skin diseases and verrucae outside the body (Pharmacopoeia Commission of the People's Republic of China. Chinese Pharmacopoeia [M]. Beijing: China Medical Science and Technology Press, 2020: 36-7.). Euphorbia lathyris is also an important oil crop. Its seeds have a high oil content and good oil quality, and are excellent raw materials for preparing high-quality biodiesel. In addition, the organic matter contained in the Euphorbia lathyris plant is 35%, rich in terpenoid compounds and monosaccharides, which can be converted into gasoline-like substances and fermented into alcohol (NEMETHY E K, OTVOS J W, CALVIN M. Hydrocarbons from Euphorbia lathyris [J]. Pure and Applied Chemistry, 1981, 53(6): 1101-8.), and the rich triterpenoid compounds can be directly converted into fuels by the methods of the petroleum industry and can be used as substitutes for fossil fuels (GASTALDO C, LIPKO A, MOTSCH E, et al. Biosynthesis of Isoprene Units in Euphorbia lathyris Laticifers vs. Other Tissues: MVA and MEP Pathways, Compartmentation and Putative Endophytic Fungi Contribution [J]. Molecules (Basel, Switzerland), 2019, 24(23): 4322.). The diterpenoid chemical components in Qianjinzi have various biological activities, such as anti-inflammatory, anti-tumor, anti-viral and antibacterial activities, etc. (CHEN Yegao. New progress in the study of diterpenoid chemical components and pharmacological activities of Euphorbia plants [J]. Chinese Wild Plant Resources, 2020, 39(6): 8.). In summary, Euphorbia lathyris has rich plant resources. Its seeds can be used for medicine and oil, and the above-ground part can be used as biomass fuel; however, the roots of Euphorbia lathyris have not been effectively utilized and are worthy of further research and utilization.

[0003] Plant fungal diseases are the most harmful diseases in plant production, causing huge economic losses globally every year. Currently, the commonly used control method is to use chemical fungicides. However, the long-term use of chemical fungicides has caused certain harm to the environment, led to the increasing resistance of plant pathogenic fungi year by year, and the control effect of fungicides has gradually declined. Therefore, it is urgent to develop new green fungicides for alternative use. Plant-derived active ingredients have the characteristics of rich sources, specific action modes, low resistance generation, environmental friendliness, and relatively safe for non-target organisms (Zhang Xing, Ma Zhiqing, Feng Juntao, et al. Research progress of plant-derived pesticides [J]. Chinese Journal of Biological Control, 2015, 31: 685-698.), and are an important source for developing new control agents for plant fungal diseases. The medicinal plant Euphorbia lathyris contains novel macrocyclic diterpenoid active ingredients with special structures, making it an ideal object for exploring new active antibacterial molecules. Summary of the Invention

[0004] The object of the present invention is to provide a Euphorbia lathyris spiroditerpenoid compound with a new skeleton, its preparation method, and its use in the preparation of drugs for controlling plant fungal diseases.

[0005] The technical solution adopted by the present invention is: a Euphorbia lathyris spiroditerpenoid compound with a new skeleton of the following structural formula, named Spirolathyrisin A, which is characterized in that its chemical name is (1R,2R,5R,6R,9S,13R,14R)-13-isopropyl-1,5-dimethyl-10-methylene-octahydrospiro[cyclohexane-6,6-indene]-1,2-diol, and its chemical structural formula is:

[0006] Furthermore, the Euphorbia lathyris diterpenoid compound Spirolathyrisin A is prepared as follows: using the seeds of Euphorbia lathyris as raw materials, refluxing and extracting with 5-6 times the volume of alcohol or a mixture of alcohol and water 2-3 times, each time for 1-2 hours, combining the extracts, concentrating under reduced pressure to 0.5-1.5 times the volume of the raw material, and adding petroleum ether for extraction at a volume ratio of 1:1-3; the petroleum ether extract is obtained by column chromatography separation.

[0007] Furthermore, the half-inhibitory concentration (EC 50They are respectively: 13.75±0.91 μg / mL, 7.73±0.36 μg / mL, 14.81±0.83 μg / mL, 13.72±0.85 μg / mL. The present invention can provide a new leading compound for screening the prevention and control of plant fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 are the key 1 H- 1 H COSY correlation and HMBC correlation of compound Spirolathyrisin A.

[0009] Figure 2 are the key NOESY and single crystal structure of compound Spirolathyrisin A. DETAILED DESCRIPTION OF THE INVENTION

[0011] Example 1: 2.3 kg of dried Euphorbia roots were crushed, added with methanol at 10 times the volume of the crude drug, soaked and extracted at room temperature for 3 times, 7 days each time. The crude extracts were combined and concentrated under reduced pressure at 50 °C to 0.5 times the volume of the medicinal material. Then, petroleum ether was added in a volume ratio of 1:1 and extracted 3 times in sequence. The petroleum ether extracts were combined and concentrated under reduced pressure to obtain 33.4 g of petroleum ether extract. The petroleum ether extract was fractionated by silica gel column chromatography and eluted with petroleum ether, petroleum ether-ethyl acetate (20:1), petroleum ether-ethyl acetate (10:1), petroleum ether-ethyl acetate (4:1), petroleum ether-ethyl acetate (2:1) and ethyl acetate solutions. The eluates with similar components were combined and concentrated to obtain a total of 6 parts, F1 - F6. Then, by comprehensively using methods such as silica gel column chromatography, gel LH-20 column chromatography, and recrystallization, compound Spirolathyrisin A (132 mg) was separated from part F2, and the yield was 0.0057%. Then, by comprehensively using various spectroscopic techniques (MS, NMR, UV, IR), the structure of compound Spirolathyrisin A was identified.

[0011] Physical and chemical properties and spectroscopic data of compound Spirolathyrisin A: Compound Spirolathyrisin A is a colorless transparent needle crystal (petroleum ether - ethyl acetate), and TLC shows a purple-red color when heated with vanillin - concentrated sulfuric acid test solution. It is poorly soluble in water and easily soluble in chloroform - methanol mixed solution. It is speculated that Spirolathyrisin A is a terpene. Optical rotation: [c 0.22, methanol]. Melting point: 134 - 137 °C. HR-ESI-MS (m / z): 329.2457 [M+Na] + (calculated: 329.2450 [C 20 H 34 O2 Na] + ), combined with 1 H-NMR and 13 C-NMR spectra (Table 1), the molecular formula was deduced to be C 20 H 34 O 2 , with a molecular weight of 306 and an unsaturation degree of 4.

[0012] Determination of the planar structure of compound spirosequoia A. Analysis of the 1 H- 1 H COSY spectrum ( Figure 1 ) showed that H-3 / H-4 / H-5 / H-16 were correlated, suggesting the presence of fragment a; H-7 / H-8 / H-9 / H-14 / H-13 / H-12 / H-11 were correlated, suggesting the presence of fragment b; H-18 / H-19 / H-20 were correlated, suggesting the presence of fragment c. From the HMBC spectrum ( Figure 1 ), it was found that H-15 was correlated with C-1, C-2, and C-6; H-3, H-4 were correlated with C-1; H-15 was correlated with C-6; H-9, H-11 were correlated with C-10. Thus, the planar structure of spirosequoia A was determined to be a spiro ring structure formed by the fusion of a 5-membered ring B and a 6-membered ring A, and then combined with a 6-membered ring C. From the correlation of H-5, H-7, H-14 with C-6, it was known that the spiro carbon atom was C-6; from the correlation of H-17 with C-9, C-11, it was speculated that the exocyclic double bond was located at C-10 of ring C; from the correlation of H-19, H-20 with C-13, and H-18 with C-12, C-13, C-14, it was speculated that the isopropyl group was located at C-13 of ring C. Combining the above speculations, spirosequoia A was a spiro ring structure formed by a 6-membered ring (ring A) and a nonacarbocyclic indene ring (ring B / C).

[0013] Determination of the relative and absolute configurations of compound spirosequoia A. The relative configuration of spirosequoia A was determined by key signals in the ROESY spectrum ( Figure 2 ), as follows: The correlation signals of H-9 / H-8β and H-8β / H-7β determined that H-9, H-8β, and H-7β were all on one side of the B / C indene ring; the correlation signals of H-14 / H-12α and H-12α / H-11α determined that H-14, H-12α, and H-11α were all on the other side of the B / C indene ring; the correlation signals of H-5 / H-4β and H-4β / H-3β determined that H-5, H-4β, and H-3β were all on one side of ring A. The single crystal of spirosequoia A was obtained by recrystallization from a mixed solvent of ethyl acetate - methanol (4:1), and analyzed by X-ray single crystal diffraction with a copper target ( Figure 2) It was determined that the absolute configurations of the seven chiral carbons of the compound spirolathyrisin A were 1R, 2R, 5R, 6R, 9S, 13R, and 14R [Flack value was 0.03(3)]. According to the systematic nomenclature, it was named (1R,2R,5R,6R,9S,13R,14R)-13-isopropyl-1,5-dimethyl-10-methylene-octahydrospiro[cyclohexane-6,6-indene]-1,2-diol (Spirolathyrisin A).

[0014] Table 1. NMR (CDCl 3 as solvent) spectral data of the compound spirolathyrisin A a Data were measured at 400MHz for 1 H and 100MHz for 13 C in CDCl 3 , δ in ppm, J in Hz.

[0015] The purity of the compound spirolathyrisin A was determined by UPLC-HRESI-MS. The chromatographic conditions were as follows: chromatographic column C 18 (Agilent Poroshell C18, 4.6×100mm, 2.7μm), column temperature: 30°C, mobile phase: methanol-water (containing 0.1% formic acid) gradient elution (methanol concentration was 40 - 75% from 0 to 5 min, 75% from 5 to 15 min, 75 - 100% from 15 to 30 min, 100% from 30 to 40 min), flow rate: 0.5 mL min -1 , injection volume: 5 μL; the mass spectrometry detector was 6530ESI Q-TOF MS (Agilent, USA), the detection mode was cation, and the detection range was m / z 100–1700. The retention time of the compound spirolathyrisin A was 28.02 min, and the purity of the compound spirolathyrisin A was calculated to be 98.9% by the area normalization method.

[0016] Example 2: 2 kg of dried Euphorbia lathyris roots were pulverized, 5 times the volume of methanol based on the crude drug was added, and extraction was carried out by refluxing 3 times, 1.5 h each time. The crude extracts were combined and concentrated under reduced pressure at 50 °C to 0.5 times the volume of the medicinal material. Then, petroleum ether was added in a volume ratio of 1:1 and extraction was carried out 3 times in sequence. The petroleum ether extracts were combined and concentrated under reduced pressure to obtain 30 g of petroleum ether extract. Then, by comprehensively using methods such as silica gel column chromatography, Sephadex LH-20 column chromatography, and recrystallization, compound spiroescin A (124 mg) was isolated from the petroleum ether extract, with a yield of 0.0062%, and the purity of the product detected by UPLC-HRESI-MS was 97.7%.

[0017] Example 3: 10 kg of dried Euphorbia lathyris seeds were pulverized, 6 times the volume of 90% methanol / water based on the crude drug was added, and extraction was carried out by refluxing 2 times, 2 h each time. The crude extracts were combined and concentrated under reduced pressure at 50 °C to 1.5 times the volume of the medicinal material. Then, petroleum ether was added in a volume ratio of 1:2 and extraction was carried out 4 times in sequence. The petroleum ether extracts were combined and concentrated under reduced pressure to obtain 620 g of petroleum ether extract. Then, by comprehensively using methods such as silica gel column chromatography, Sephadex LH-20 column chromatography, and recrystallization, spiroescin A (563 mg) was isolated from the petroleum ether extract, with a yield of 0.0063%, and the purity of the product detected by UPLC-HRESI-MS was 98.6%.

[0018] Example 4: Determination of the antibacterial activity of compound spiroescin A. Compound spiroescin A was dissolved in DMSO to prepare a stock solution of 10000 μg / mL, and serially diluted with DMSO to obtain 6 concentrations. Different concentrations of spiroescin A solutions were added to PSA medium to prepare drug-containing plates. The PSA plate added with an equal amount of DMSO was used as the solvent control, and thiophanate-methyl was used as the control agent. Four plant pathogenic fungi (Botryosphaeria dothidea, Diaporthe sojae, Fusarium oxysporum, and Alternaria alternata) were pre-cultured in an incubator at 25 °C. Subsequently, agar discs were taken from the edges of the pre-cultured pathogenic fungal colonies and inoculated into the centers of the drug-containing plates, and cultured at 25 °C until the control was nearly fully grown. The colony diameters of each treatment were measured, the inhibition rate was calculated based on the colony diameters, and the EC 50 value was calculated based on the inhibition rate and compound concentration. Three technical replicates were set for each treatment, and the experiment was repeated three times. The measurement results are shown in Table 2. Spiroescin A has good antibacterial activity against Botryosphaeria dothidea and Diaporthe sojae, but is weaker than the control agent thiophanate-methyl, and its antibacterial activity against Fusarium oxysporum and Alternaria alternata is better than that of thiophanate-methyl.

[0019] Table 2. Inhibitory activity of compound spiroescin A against four plant pathogenic fungi (EC 50, μg / mL)

Claims

1. A Euphorbia lathyris L. spirocyclic diterpenoid compound, named Spirolathyrisin A, characterized in that its chemical name is (1R,2R,5R,6R,9S,13R,14R)-13-isopropyl-1,5-dimethyl-10-methylene-octahydrospiro[cyclohexane-6,6-indene]-1,2-diol, and its chemical structural formula is: Spirolathyrisin A.

2. The preparation method of the diterpenoid compound according to claim 1, characterized in that it comprises the following steps: Using the root of Euphorbia lathyris L. as raw material, reflux extracting with alcohol or a mixture of alcohol and water at 5-6 times the volume of the raw material for 2-3 times, 1-2 hours each time, combining the extracts, concentrating under reduced pressure to 0.5-1.5 times the volume of the raw material amount, and adding petroleum ether for extraction in a volume ratio of 1:1-3; the petroleum ether extract is obtained by column chromatography separation.

3. Use of the compound according to claim 1 in the preparation of preventing and treating plant fungal diseases.

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