Natural sesquiterpene compound with anti-agricultural pathogenic bacteria effect and preparation method and application thereof

Abstract

The application discloses a natural sesquiterpene compound with an agricultural pathogenic bacteria resisting effect and a preparation method and application thereof. A novel natural sesquiterpene compound is separated from stems of diseased Gordonia aculeata. Activity experiments show that the natural sesquiterpene compound has the agricultural pathogenic bacteria resisting effect, has good antibacterial activity on plant pathogenic bacteria Rhizopus stolonifer, Verticillium dahliae Kleb, Thanatephorus cucumeris and Fusarium oxysporum, and can be applied to plant disease prevention and treatment as an agricultural fungicide.

Description

Technical Field

[0001] This invention belongs to the fields of phytochemistry and agricultural disease control technology, and specifically relates to a natural sesquiterpene compound with anti-agricultural pathogen activity, its preparation method and application. Background Technology

[0002] Plant diseases caused by pathogens pose a significant threat to the security and stability of global agricultural production and can lead to severe declines in crop yield and quality. Chemical control using synthetic fungicides or antibiotics is one of the most common methods for managing these agricultural plant diseases. However, the widespread use and misuse of synthetic chemicals have already caused potential harm to humans, animals, and the environment. Furthermore, resistance to major fungicides is developing rapidly, necessitating new, highly effective, low-toxicity, and environmentally friendly fungicides. Therefore, there is an urgent need to develop new and effective fungicides to prevent these agricultural diseases.

[0003] Secondary metabolites, as a typical defense strategy, are specialized metabolites produced by this complex defense system. These metabolites have low molecular weight and directly antagonize pathogen attacks. They are a valuable resource for discovering molecules of significant economic importance, providing chemists with a valuable resource for developing green fungicide industries and replacing existing commercial products. Therefore, identifying defensive, specialized metabolites with fungicide potential produced by the immune system of diseased plants has significant scientific value and socio-economic benefits.

[0004] Hibiscus tiliaceus, a semi-mangrove plant related to cotton, is widely distributed in tropical and subtropical coastal ecosystems. In China, it is used as an ornamental tree and traditionally as a medicinal herb for clearing heat and detoxifying, resolving blood stasis, and reducing swelling. According to the inventors' previous investigations, infected Hibiscus stems produce large amounts of defensive, specialized metabolites called sesquiterpenes, which are distinctly different from natural products isolated from healthy plants. Therefore, it is speculated that these substances may have anti-agricultural pathogen activity. To obtain such active substances, the inventors isolated and identified 10 novel natural sesquiterpenes, named hibisceusins ​​IR (1-10). These compounds exhibit significant and broad-spectrum anti-agricultural pathogen activity, and have important application value in crop production and the development of novel pesticides. The compounds hibisceusins ​​IR (1-10), their isolation methods, and their applications in combating agricultural pathogens have not yet been reported. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a natural sesquiterpene compound with anti-agricultural pathogen activity, its preparation method and application.

[0006] The technical solution of the present invention is as follows:

[0007] One objective of this invention is to provide a class of natural sesquiterpene compounds with anti-agricultural pathogen activity, the chemical structural formula of which is shown in any one of the following formulas (I) to (V):

[0008]

[0009] In formula (Ⅰ), R1 is selected from C=O or C(OCH3)2;

[0010] In formula (Ⅳ), R1 is selected from CH3, CHO or COOH, R2 is selected from OH or H, R3 is selected from isopropyl or isopropenyl, R4 is selected from CH3 or CH(OCH3)2, R5 is selected from OH or H, and R6 is selected from methoxy, OH or H.

[0011] Furthermore, the natural sesquiterpene compound with anti-agricultural pathogen activity includes at least one of compounds 1 to 10, whose chemical structural formulas are as follows:

[0012]

[0013]

[0014] The second objective of this invention is to provide a method for preparing the aforementioned natural sesquiterpene compound with anti-agricultural pathogen activity, comprising the following steps:

[0015] S1: The stems of the infected Hibiscus syriacus were dried and pulverized, extracted with dichloromethane at room temperature, and concentrated under vacuum to obtain the extract; the extract was subjected to column chromatography on silica gel, eluted with a gradient of petroleum ether-CH2Cl2 / CH2Cl2-MeOH, and similar components were combined by TLC thin-layer chromatography to obtain the fractions Fr A-Fr G with increasing polarity;

[0016] S2: Fraction Fr.C was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 and CH2Cl2-MeOH to obtain Fr.C1-Fr.C11 with increasing polarity; Fr.C5 was eluted by semi-preparative liquid chromatography with methanol-water solution to obtain Fr.C5-1 and C5-2; Fr.C5-2 was eluted by semi-preparative liquid chromatography with methanol-H2O solution to obtain compounds 5 and 8; Fr.C6 was eluted by semi-preparative liquid chromatography with methanol-water solution to obtain compound 7.

[0017] S3: Fraction Fr.D was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 and CH2Cl2-MeOH to obtain Fr.D1-Fr.D15 with increasing polarity; Fr.D2 was eluted by semi-preparative liquid chromatography with 80 v / v% acetonitrile-H2O to obtain compound 4; Fr.D3 was eluted by semi-preparative liquid chromatography with 75 v / v% MeOH-H2O to obtain compound 9; Fr.D4 was eluted by semi-preparative liquid chromatography with methanol-water to obtain compound 6.

[0018] S4: Fraction Fr.E was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 and CH2Cl2-MeOH to give Fr.E1-E7 with increasing polarity; Fr.E1 was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 and CH2Cl2-MeOH to give Fr.E1-1-Fr.E1-9 with increasing polarity; Fr.E1-2 was eluted by semi-preparative liquid chromatography with methanol-H2O to give compound 1; Fr.E1-4 was eluted by... Compound 2 was obtained by semi-preparative liquid chromatography using methanol-H2O as the eluent; Fr.E1-5 was separated by semi-preparative liquid chromatography using methanol-water as the eluent to obtain compound 3; Fr.E2 was separated by silica gel column chromatography using gradient elution with petroleum ether / CH2Cl2 and CH2Cl2 / MeOH to obtain fractions Fr.E2-1-E2-6; Fr.E2-4 was separated by semi-preparative liquid chromatography using 65 v / v% MeOH-H2O as the eluent to obtain compound 10.

[0019] The semi-preparative liquid chromatography method uses an Agilent SB-Phenyl column (9.4 × 250 mm) as the stationary phase.

[0020] Furthermore, in step S1 of the preparation method, the diseased Hibiscus stems include Hibiscus stems infected with Fusarium, the volume ratio of petroleum ether to CH2Cl2 is 100:0 to 0:100, and the volume ratio of CH2Cl2 to MeOH is 50:1 to 1:1.

[0021] Furthermore, step S2 of the preparation method is as follows:

[0022] Fraction Fr.C was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) and CH2Cl2-MeOH (v / v) to obtain Fr.C1-Fr.C11, with increasing polarity. Fr.C5 was eluted by semi-preparative liquid chromatography with 80 v / v% methanol-water solution to obtain Fr.C5-1 and C5-2. Fr.C5-2 was eluted by semi-preparative liquid chromatography with methanol-H2O solution (v / v) to obtain compounds 5 and 8. Fr.C6 was eluted by semi-preparative liquid chromatography with methanol-water solution (v / v) to obtain compound 7.

[0023] Furthermore, step S3 of the preparation method is as follows:

[0024] S3: Fraction Fr.D was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) and CH2Cl2-MeOH (v / v) to obtain Fr.D1-Fr.D15 with increasing polarity; Fr.D2 was eluted by semi-preparative liquid chromatography with 80 v / v% acetonitrile-H2O to obtain compound 4; Fr.D3 was eluted by semi-preparative liquid chromatography with 75 v / v% methanol-H2O to obtain compound 9; Fr.D4 was eluted by semi-preparative liquid chromatography with 75:25 methanol-water (v / v) to obtain compound 6.

[0025] Furthermore, step S4 of the preparation method is as follows:

[0026] S4: Fraction Fr.E was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) at a ratio of 1:1 to 0:100 and CH2Cl2-MeOH (v / v) at a ratio of 40:1 to 1:1, yielding Fr.E1-E7 with increasing polarity; Fr.E1 was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) at a ratio of 4:1 to 0:1 and CH2Cl2-MeOH (v / v) at a ratio of 99:1 to 1:1, yielding Fr.E1-1-Fr.E1-9 with increasing polarity; Fr.E1-2 was eluted by semi-preparative liquid chromatography with methanol-H2O (v / v) at a ratio of 75:25, yielding compound 1; Fr Compound 2 was obtained by semi-preparative liquid chromatography (SPLC) of Fr.E1-4 with methanol-H2O (v / v) as the eluent. Compound 3 was obtained by semi-preparative SPLC of Fr.E1-5 with methanol-water (v / v) as the eluent. Compound 3 was obtained by semi-preparative SPLC of Fr.E2 with silica gel column chromatography with gradient elution of petroleum ether / CH2Cl2 (v / v) and CH2Cl2 / MeOH (v / v) as the eluent. Fractions Fr.E2-1-E2-6 were obtained by semi-preparative SPLC of Fr.E2-4 with MeOH-H2O (v / v) as the eluent.

[0027] A third objective of this invention is to provide the application of the aforementioned natural sesquiterpene compounds in inhibiting plant pathogens.

[0028] The plant pathogens include Rhizopus stolonifer, Verticillium dahliae Kleb., Thanatephorus cucumeris, or Fusarium oxysporum.

[0029] The beneficial effects of this invention are:

[0030] The natural sesquiterpene compounds described in this invention have novel structures and exhibit good antibacterial activity against Rhizopus spp., Verticillium wilt, Rhizoctonia solani, and Fusarium wilt, and can be applied to the prevention and control of common crop diseases. Attached Figure Description

[0031] Figure 1 The diagram shows the structures of compounds 1-10.

[0032] Figure 2 The structures of compounds 1-10 are shown, mainly... 1 H- 1 H COSY (arrow) and HMBC (thick line) related diagram.

[0033] Figure 3 ECD spectra of compounds 1-4.

[0034] Figure 4 , 6 8, 10, 12, 14, 16, 18, 20, and 22 are the 1H-NMR (600MHz, CDCl3) spectra of compounds 1-10, respectively.

[0035] Figure 5 , 7 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, 17, 19, 21, and 23 are the 13C-NMR (150MHz, CDCl3) spectra of compounds 1-10, respectively. Detailed Implementation

[0036] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1: Isolation and structural identification of natural sesquiterpenoid compounds from Hibiscus syriacus

[0040] The stems of *Hibiscus syriacus* infected with Fusarium (13.0 kg) were dried and pulverized, extracted with dichloromethane at room temperature for 72 h, and concentrated under vacuum to obtain 405.9 g of extract. The extract was subjected to column chromatography on silica gel (200-300 mesh) using a gradient of petroleum ether-CH2Cl2 (v / v 100:0 to 0:100) / CH2Cl2-MeOH (v / v 50:1 to 1:1). Similar components were combined by TLC to obtain fractions of increasing polarity, namely Fr A-Fr G.

[0041] Fr.C (1.54 g) was separated by silica gel column chromatography, eluted with petroleum ether-CH2Cl2 (v / v 6:1 to 0:100) and CH2Cl2 / MeOH (v / v 40:1 to 1:1), yielding Fr.C1-Fr.C11 with increasing polarity. Fr.C5 was separated by semi-preparative liquid chromatography, eluted with 80 v / v% methanol-water solution, yielding Fr.C5-1 and C5-2. Fr.C5-2 was separated by semi-preparative liquid chromatography, eluted with methanol-H2O (v / v 65:35), yielding compound 5 (15.4 mg, Rt 9.6 min) and compound 8 (2.4 mg, Rt 22.4 min). Fr.C6 was separated by semi-preparative liquid chromatography, eluted with methanol-water (v / v 75:25), yielding compound 7 (1.8 mg, Rt 20.9 min). The semi-preparative liquid chromatography method uses an Agilent SB-Phenyl column (9.4 × 250 mm) as the stationary phase.

[0042] Fr.D (4.5 g) was separated by silica gel column chromatography, eluted with petroleum ether-CH2Cl2 (v / v 6:1 to 0:100) and CH2Cl2-MeOH (v / v 40:1 to 1:1), yielding Fr.D1-Fr.D15 with increasing polarity. Fr.D2 was separated by semi-preparative HPLC, eluted with 80 v / v acetonitrile-H2O, to give compound 4 (2.5 mg, Rt 7.6 min). Fr.D3 was separated by semi-preparative HPLC, eluted with 75 v / v MeOH-H2O, to give compound 9 (5.2 mg, Rt 33.8 min). Fr.D4 was separated by semi-preparative HPLC, eluted with methanol-water (v / v 75:25), to give compound 6 (2.4 mg, Rt 33.8 min). t 35.0 min).

[0043] Fr.E (66.8 g) was separated by silica gel column chromatography, eluted with petroleum ether-CH2Cl2 (v / v ratio 1:1 to 0:100) and CH2Cl2-MeOH (v / v ratio 40:1 to 1:1), to give Fr.E1-E7 with increasing polarity. Fr.E1 was separated by silica gel column chromatography, eluted with petroleum ether-CH2Cl2 (v / v ratio 4:1 to 0:1) and CH2Cl2-MeOH (v / v ratio 99:1 to 1:1), to give Fr.E1-1-Fr.E1-9 with increasing polarity. Fr.E1-2 was separated by semi-preparative liquid chromatography, eluted with methanol-H2O (v / v ratio 75:25), to give compound 1 (3.3 mg, Rt 11.1 min). Fr.E1-4 was separated by semi-preparative liquid chromatography, eluting with methanol-H2O (v / v) to give compound 2 (5.6 mg, Rt 11.8 min). Fr.E1-5 was separated by semi-preparative liquid chromatography, eluting with methanol-water (v / v) (70:30) to give compound 3 (2.0 mg, Rt 19.1 min). Fr.E2 was separated by silica gel column chromatography, eluting with gradients of petroleum ether / CH2Cl2 (v / v) (2:1 to 0:100) and CH2Cl2 / MeOH (v / v) (40:1 to 1:1) to give fractions Fr.E2-1-E2-6. Fr.E2-4 was separated by semi-preparative liquid chromatography, eluting with 65 v / v% MeOH-H2O to give 10 (4.1 mg, Rt 19.5 min).

[0044] The compounds 1-10 obtained above were named hibisceusins ​​IR(1-10), respectively.

[0045] Structural identification: The structures of isolated compounds 1-10 were identified using modern spectroscopic techniques such as 1H NMR and 13C NMR. The identification results are as follows: Figures 1-23 As shown.

[0046] Experimental Example: Determination of Antimicrobial Activity

[0047] The bacterial strains: Rhizopus stolonifer (R. stolonifer), Verticillium dahliae Kleb. (V. dahliae), Thanatephorus cucumeris (T. cucumeris) and Fusarium oxysporum Schltdl. (F. oxysporum) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, and F. oxysporum was prepared in laboratory HK-27.

[0048] The antifungal effects of five plant pathogenic fungi (R. stolonifer, V. dahliae, T. cucumeris, F. oxysporum, and F. oxysporum HK-27) were evaluated using a two-fold dilution method. Simply put, each compound was sequentially diluted to a suspension containing fungal pathogen spores (approximately 1 × 10⁻⁶). 6 In 96-well plates containing carbendazim (Aladdin Industrial Inc., Shanghai, China) at a final sample concentration of 100–1.25 μg / mL (cfu / mL), the positive control group was determined. The minimum inhibitory concentration (MIC) was then recorded. Results are shown in Table 1.

[0049] Table 1

[0050]

[0051] The results showed that compounds 1-10 exhibited inhibitory activity against plant pathogens Rhizopus stolonifer, V. dahliae Kleb., T. cucumeris, and F. oxysporum. Among them, compounds 1, 2, and 5 showed significant inhibitory effects against Rhizopus stolonifer. Except for compound 5, the other compounds showed significant inhibitory effects against a variety of pathogens, including V. dahliae Kleb., T. cucumeris, and F. oxysporum, with the lowest inhibitory concentration as low as 5.0 μg / mL, indicating that the compounds provided in this invention have certain broad-spectrum antibacterial properties.

[0052] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A natural sesquiterpene compound with anti-agricultural pathogen activity, characterized in that, The natural sesquiterpene compound is any one of the following compounds 3, 4, and 10: 。 2. The method for preparing the natural sesquiterpene compound with anti-agricultural pathogen activity as described in claim 1, characterized in that, Includes the following steps: S1: The stems of the infected Hibiscus syriacus were dried and pulverized, extracted with dichloromethane at room temperature, and concentrated under vacuum to obtain the extract; the extract was subjected to column chromatography on silica gel, eluted with a gradient of petroleum ether-CH2Cl2 / CH2Cl2-MeOH, and similar components were combined by TLC thin-layer chromatography to obtain the fractions Fr A – Fr G with increasing polarity; S2: Fraction Fr. D was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 and CH2Cl2-MeOH to obtain Fr. D1 – Fr. D15 with increasing polarity; Fr. D2 was eluted by semi-preparative liquid chromatography with 80 v / v% acetonitrile-H2O to obtain compound 4; S3: Fractions Fr. E were separated by silica gel column chromatography, eluted with petroleum ether-CH2Cl2 and CH2Cl2-MeOH, yielding Fr. E1 - E7 with increasing polarity; Fr. E1 was separated by silica gel column chromatography, eluted with petroleum ether-CH2Cl2 and CH2Cl2-MeOH, yielding Fr. E1-1 - Fr. E1-9 with increasing polarity; Fr. E1-5 was separated by semi-preparative liquid chromatography, using methanol-water as the eluent, to obtain compound 3; Fr. E2 was separated by silica gel column chromatography, eluted with a gradient of petroleum ether / CH2Cl2 and CH2Cl2 / MeOH, yielding fractions Fr. E2-1 – E2-6; Fr. E2-4 was separated by semi-preparative liquid chromatography, using 65 v / v% MeOH-H2O as the eluent, to obtain 10.

3. The preparation method according to claim 2, characterized in that, In step S1, the infected hibiscus stems include hibiscus stems infected with Fusarium, the volume ratio of petroleum ether to CH2Cl2 is 100:0 ~ 0:100, and the volume ratio of CH2Cl2 to MeOH is 50:1 ~ 1:

1.

4. The preparation method according to claim 2, characterized in that, Step S2 is as follows: S2: Fraction Fr. D was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) at a ratio of 6:1 to 0:100 and CH2Cl2-MeOH (v / v) at a ratio of 40:1 to 1:1 to obtain Fr. D1 – Fr. D15 with increasing polarity; Fr. D2 was eluted by semi-preparative liquid chromatography with 80 v / v% acetonitrile-H2O to obtain compound 4.

5. The preparation method according to claim 2, characterized in that, Step S3 is: S3: Fraction Fr. E was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) at a ratio of 1:1 to 0:100 and CH2Cl2-MeOH (v / v) at a ratio of 40:1 to 1:1, yielding Fr. E1 - E7 with increasing polarity; Fr. E1 was eluted by silica gel column chromatography with petroleum ether-CH2Cl2 (v / v) at a ratio of 4:1 to 0:1 and CH2Cl2-MeOH (v / v) at a ratio of 99:1 to 1:1, yielding Fr. E1-1 - Fr. E1-9 with increasing polarity; Fr. E1-5 was separated by semi-preparative liquid chromatography with methanol-water (v / v) at a ratio of 70:30 to obtain compound 3; Fr. E2 was eluted by silica gel column chromatography with a gradient elution of petroleum ether / CH2Cl2 (v / v) at a ratio of 2:1 to 0:100 and CH2Cl2 / MeOH (v / v) at a ratio of 40:1 to 1:1, yielding fraction Fr. E2-1 – E2-6 and Fr. E2-4 were separated by semi-preparative liquid chromatography with 65 v / v% MeOH-H2O as the eluent, yielding 10.

6. The application of the natural sesquiterpene compound according to claim 1 in inhibiting plant pathogens, characterized in that, The plant pathogen was selected from Rhizopus stolonifera. Rhizopus stolonifer Verticillium wilt Verticillium dahliae Kleb., the pathogen of sheath blight Thanatephorus cucumeris or wilt pathogen Fusarium oxysporum .

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