Biphenyl compound as well as preparation method and application thereof

Abstract

The invention discloses a biphenyl compound as well as a preparation method and application thereof. The biphenyl compound disclosed by the invention is obtained by being separated from a fermentation solution of a fungus Aspergillus candidus HNNU0546 derived from marine cold spring sediments. The biphenyl compound disclosed by the invention has relatively remarkable activity of resisting alternaria solani and pineapple black heart pathogen, so that the biphenyl compound has a good application prospect in the aspect of preparing pesticides for resisting crop diseases and fungi.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing a biphenyl compound and its application in developing pesticides for resisting fungi of crop diseases. Background Art

[0002] Marine fungi can produce novel secondary metabolites such as alkaloids, peptides, polyketides, steroids and terpenes, many of which have significant antibacterial, antitumor, antiviral and antifouling activities. Finding active compounds from marine fungi is a hot topic in the development of natural drugs. Biphenyl compounds are a class of substances with multiple biological activities that exist in nature. Whether they are naturally occurring or artificially synthesized, they have attracted widespread attention from chemists.

[0003] For example, Chinese patent document CN 108727169 A (application number 201710275997.5) discloses a method for preparing a diphenyl ether compound derived from marine fungi and its application as an antibacterial agent. The marine fungi are first cultured in a bacterial culture medium. Food sp., and then the marine fungus is fermented in a fermentation medium to obtain a fermented product, which is then extracted with ethyl acetate for 2 to 6 times. The ethyl acetate extracts are combined and concentrated under reduced pressure to obtain a crude extract, which is then chromatographically separated to obtain two diphenyl ether compounds. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a biphenyl compound and a preparation method and application thereof, wherein the biphenyl compound has the effect of inhibiting the growth of crop disease fungi.

[0005] The technical solution to achieve the first object of the present invention is a class of biphenyl compounds having the structures shown in Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6: The technical solution to achieve the second purpose of the present invention is a method for preparing the above-mentioned biphenyl compound, wherein the above-mentioned compound 1, compound 2, compound 3, compound 4, compound 5 and compound 6 are obtained from strain Aspergillus candidus Isolated from the fermentation product of HNNU0546; Aspergillus candidus HNNU0546 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 65074.

[0006] The specific steps include: ① Bacterial activation and fermentation; Aspergillus candidus HNNU0546 was activated, cultured and fermented to obtain a fermentation product.

[0007] ② Extract the fermented product with ethyl acetate or macroporous adsorption resin, and then concentrate it to obtain a crude extract.

[0008] ③ The crude extract of step ② was segmented by normal pressure silica gel column chromatography, with petroleum ether-ethyl acetate, petroleum ether-acetone or chloroform-methanol solvent system as eluent, and gradient elution was performed from a volume ratio of 100:0 to 0:100, and the combined components were tracked by thin layer chromatography TLC, and the components that could be developed on thin layer chromatography with a volume ratio of 8:2 petroleum ether-ethyl acetate or a volume ratio of 9:1 chloroform-acetone solvent system were further subjected to silica gel column chromatography to obtain a crude product, which was then purified by high performance liquid chromatography to obtain compound 1-6.

[0009] In step ①, the strain Aspergillus candidus HNNU0546 was inoculated on PDA agar medium to obtain a plate with cultured bacteria; then a single colony was selected from the plate with an inoculation loop or a bamboo stick and inoculated into a conical flask of PDB seed liquid medium, and cultured on a shaker to obtain seed liquid; then the seed liquid was inoculated into bottles containing PDB fermentation medium, and static culture was performed to obtain fermentation products.

[0010] As an option, in step ①, the strain Aspergillus candidus HNNU0546 was inoculated into PDA agar medium supplemented with 3% artificial sea salt, and cultured at 28°C for 3 days for activation to obtain a plate containing the strain; the strain was then inoculated into a sterile fresh PDB seed liquid medium supplemented with 3% sea salt, and cultured on a shaker at 200 rpm and 28°C for 3 days to obtain a seed liquid; the seed liquid was then inoculated into a PDB fermentation medium supplemented with 3% sea salt, and cultured at room temperature for 28 days.

[0011] Optionally, when preparing the PDA agar medium, 200 g of potatoes are washed and peeled, cut into slices and boiled in water, boiled for 30 minutes after the water boils, filtered with gauze to remove the residue, 20 g of glucose, 30 g of sea salt and 20 g of agar are added to the filtrate, and finally the volume is adjusted to 1 liter.

[0012] When preparing the PDB culture medium, 200 g of potatoes were washed and peeled, cut into slices and boiled in water for 30 minutes after the water boiled, and the residue was removed by filtering with gauze, 20 g of glucose and 30 g of sea salt were added to the filtrate, and the volume was finally adjusted to 1 L.

[0013] In the above step ③, the component that can be developed on thin layer chromatography with a petroleum ether-ethyl acetate solvent system with a volume ratio of 8:2 is subjected to silica gel column chromatography and gradient eluted with a petroleum ether-ethyl acetate system of 90:10-50:50 to obtain two subfractions Fr.1-Fr.2.

[0014] Alternatively, the components that can be developed on thin layer chromatography with a chloroform-acetone solvent system in a volume ratio of 9:1 are subjected to silica gel column chromatography and gradient eluted with a chloroform-acetone system of 90:10-0:100 to obtain two subfractions Fr.1-Fr.2.

[0015] Fraction Fr.1 was chromatographed on a silica gel column using a pure chloroform solvent system to obtain seven subfractions Fr.1-1, Fr.1-2, Fr.1-3, Fr.1-4, Fr.1-5, Fr.1-6 and Fr.1-7. Fr.1-3 was further chromatographed on a silica gel column using a 8:2 petroleum ether-ethyl acetate solvent system to obtain three subfractions Fr.1-3-1, Fr.1-3-2 and Fr.1-3-3.

[0016] Fr.1-3-2 was subjected to high pressure liquid semi-preparative purification operation, and compounds 1, 2 and 5 were collected at peak times of 24 minutes, 35 minutes and 45 minutes, respectively. The mobile phase was methanol-water with a volume ratio of 65:35.

[0017] Fr.1-4 was purified by ODS column chromatography using methanol-water 30:70~0:100 as the solvent system for gradient elution to obtain four subfractions Fr.1-4-1, Fr.1-4-2, Fr.1-4-3 and Fr.1-4-4.

[0018] Fr.1-4-3 was purified by HPLC with the mobile phase being methanol-water in a volume ratio of 70:30, and compound 4 was collected at the peak elution time of 16 minutes.

[0019] Fr.1-5 was purified by ODS column chromatography and eluted with methanol-water 30:70~80:20 solvent system to obtain four subfractions Fr.1-5-1, Fr.1-5-2, Fr.1-5-3 and Fr.1-5-4. Fr.1-5-3 was prepared by HPLC with acetonitrile-water in a volume ratio of 36:64 as the mobile phase, and compounds 3 and 6 were collected at elution times of 30 minutes and 33 minutes, respectively.

[0020] The technical solution to achieve the third object of the present invention is the use of compound 2, compound 5 or compound 6 in the preparation of anti-crop disease fungal pesticides. Among them, biphenyl compound 2 is effective against potato early blight fungus ( Alternaria sp.) showed antifungal activity, and biphenyl compound 5 had antifungal activity against potato early blight fungus ( Alternaria sp.) had more obvious antifungal activity, and compound 6 had a stronger antifungal activity against potato early blight ( Alternaria sp.) and pineapple blackheart fungus ( Curvularia australiensis ) had a significant inhibitory effect.

[0021] The technical solution for achieving the fourth object of the present invention is a pesticide composition comprising one or more of active ingredients Compound 2, Compound 5 or Compound 6, and an acceptable carrier.

[0022] The present invention has positive effects: the present invention uses a variety of column chromatography and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy to obtain marine fungi. Aspergillus candidus A new type of biphenyl compound was isolated from the fermentation broth of HNNU0546. This type of biphenyl compound can significantly inhibit the activity of crop disease fungi and can be used to prepare pesticides against crop disease fungi.

[0023] Specifically, biphenyl compound 2 had an effect on early blight of potato ( Alternaria sp.) showed antifungal activity, and biphenyl compound 5 had antifungal activity against potato early blight fungus ( Alternaria sp.) had more obvious antifungal activity, and compound 6 had a stronger antifungal activity against potato early blight ( Alternaria sp.) and pineapple blackheart fungus ( Curvularia australiensis ) had a significant inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 NMR of compound 1 1 H spectrum.

[0025] Figure 2 NMR of compound 1 13 C spectrum.

[0026] Figure 3 This is the high-resolution mass spectrum of compound 1.

[0027] Figure 4 NMR of compound 2 1 H spectrum.

[0028] Figure 5 NMR of compound 2 13 C spectrum.

[0029] Figure 6 This is the high-resolution mass spectrum of compound 2.

[0030] Figure 7 NMR of compound 3 1 H spectrum.

[0031] Figure 8 NMR of compound 3 13 C spectrum.

[0032] Figure 9 This is the high-resolution mass spectrum of compound 3.

[0033] Figure 10NMR of compound 4 1 H spectrum.

[0034] Figure 11 NMR of compound 4 13 C spectrum.

[0035] Figure 12 This is the high-resolution mass spectrum of compound 4.

[0036] Figure 13 NMR of compound 5 1 H spectrum.

[0037] Figure 14 NMR of compound 5 13 C spectrum.

[0038] Figure 15 NMR of compound 6 1 H spectrum.

[0039] Figure 16 NMR of compound 6 13 C spectrum. DETAILED DESCRIPTION

[0040] Introduced below are some of the multiple possible embodiments of the present invention, which are intended to provide a basic understanding of the present invention, and are not intended to confirm the key or decisive elements of the present invention or to limit the scope of protection. It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, a person of ordinary skill in the art can propose other mutually replaceable implementations. Therefore, the following specific embodiments are only exemplary descriptions of the technical solution of the present invention, and should not be regarded as the whole of the present invention or as a limitation or restriction to the technical solution of the present invention.

[0041] (Example 1) ① Bacteria activation and fermentation.

[0042] The bacteria used for fermentation to produce biphenyl compounds in this example are strains isolated and identified from deep-sea cold seep sediments. Aspergillus candidus HNNU0546 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC) with the deposit number GDMCC NO: 65074.

[0043] Aspergillus candidusThe gene sequence SEQ ID No.1 of HNNU0546 is as follows:.

[0044] During fermentation, Aspergillus candidus HNNU0546 is inoculated into a culture medium suitable for Aspergillus fungi, and a fermentation broth is prepared under normal fermentation conditions.

[0045] Preferably, the fermentation broth is prepared according to the following method: The culture medium includes PDA agar culture medium, PDB seed liquid culture medium and PDB fermentation culture medium. The preparation method of the PDA agar culture medium is as follows: 200 g of potatoes are washed and peeled, cut into slices and boiled in water, boiled for 30 minutes after the water boils, filtered through gauze to remove the residue, 20 g of glucose, 30 g of sea salt and 20 g of agar are added to the filtrate, and the volume is finally adjusted to 1 liter. Sterilize with high pressure steam at 121°C for 25 minutes and set aside.

[0046] The preparation method of the PDB seed liquid culture medium is as follows: 200 g of potatoes are washed and peeled, cut into slices and boiled in water, boiled for 30 minutes after the water boils, filtered through gauze to remove the residue, 20 g of glucose and 30 g of sea salt are added to the filtrate, and the volume is finally fixed to 1 L. The PDB culture medium is filled into 1-3 500 mL conical bottles, each bottle is about 150 mL, and sterilized by high pressure steam at 121° C. for 25 minutes.

[0047] The preparation method of the PDB fermentation medium is as follows: 6000 g of potatoes are washed and peeled, sliced ​​and boiled in water, boiled for 30 minutes after the water boils, filtered through gauze to remove the residue, 20 g of glucose and 30 g of sea salt are added to the filtrate, and the volume is finally fixed to 30 L. The PDB medium is placed in 150 1000 mL conical flasks, each containing about 200 mL, and sterilized by high pressure steam at 121° C. for 25 minutes.

[0048] Use an inoculation loop or bamboo stick to inoculate the fungi on the slope Aspergillus candidus The HNNU0546 strain was inoculated on PDA agar medium and cultured at 28°C for 3 days to obtain a plate with the strain. A single colony was then selected from the plate using an inoculation loop or a bamboo stick and inoculated into a cone containing 150 mL of PDB seed liquid medium. The culture was performed on a shaker at 200 rpm and 28°C for 3 days to obtain seed liquid. 5 mL of the seed liquid was then inoculated into each bottle containing 200 mL of PDB fermentation medium. After static culture at room temperature (28°C) for 28 days, the bottles were collected to obtain the fermentation product.

[0049] ② The fermented product was extracted three times with 50 L of ethyl acetate, each time for 2-3 days, and the combined extracts were concentrated under reduced pressure to obtain 20 g of crude extract.

[0050] Alternatively, the extract can be extracted using a macroporous adsorption resin and then concentrated to obtain a crude extract.

[0051] ③ The crude extract obtained in step ② was subjected to normal pressure silica gel column chromatography, with the stationary phase being 100-200 mesh normal phase silica gel.

[0052] Gradient elution was performed using petroleum ether-ethyl acetate (90:10-50:50), chloroform-methanol (90:10-0:100), and chloroform-acetone (90:10-0:100) as eluents, and the combined components were tracked by thin layer plate analysis. The initial sample was collected using the petroleum ether-ethyl acetate system (8:2) or the chloroform-acetone system (9:1). TLC analysis showed that when the chloroform-methanol system (volume ratio 20:1) was used for development, the R f The value was 0.4~0.7. When petroleum ether-ethyl acetate (volume ratio 8:2) or chloroform-acetone (9:1) was used for development, the R fThe value is 0.3. After the other systems chloroform-acetone (8:2) or chloroform-methanol (9:1) were used for development, compound 1-6 had obvious tailing phenomenon and poor point formation, which was not suitable for elution. After solvent development, the sample could be observed under 254 nm ultraviolet light, and then 5% concentrated sulfuric acid-vanillin color developer was used for color development, and obvious blue spots were found at the location of the sample. The initial sample containing sample 1-6 was further prepared using a high-pressure liquid chromatography column, and compound 1-6 could be obtained by elution with methanol-water or acetonitrile-water solvent system.

[0053] The separation and purification operation of this embodiment is as follows: The component (12.6 g) that can be developed on thin layer chromatography with a petroleum ether-ethyl acetate solvent system in a volume ratio of 8:2 was subjected to silica gel column chromatography and gradient elution was performed using a petroleum ether-ethyl acetate system (90:10-50:50) as an eluent to obtain two subfractions Fr.1-Fr.2.

[0054] Alternatively, the components that can be developed with a chloroform-acetone solvent system in a volume ratio of 9:1 can be subjected to silica gel column chromatography, and gradient elution can be performed using a chloroform-acetone system (90:10-0:100) as an eluent to obtain two subfractions Fr.1-Fr.2.

[0055] Fraction Fr.1 was chromatographed on a silica gel column using a pure chloroform solvent system to obtain seven subfractions: Fr.1-1, Fr.1-2, Fr.1-3, Fr.1-4, Fr.1-5, Fr.1-6 and Fr.1-7.

[0056] Fr.1-3 was then chromatographed on a silica gel column with a petroleum ether-ethyl acetate (8:2) solvent system to obtain three subfractions: Fr.1-3-1, Fr.1-3-2 and Fr.1-3-3.

[0057] Fr.1-3-2 was purified by high pressure liquid chromatography column and prepared by high performance liquid chromatography (detection wavelength was 210 / 230 / 254 / 280 nm, flow rate was 2 mL / min, chromatographic column was Agilent Eclipse XDB-C18 column (9.4 ×250mm, 5 µm), mobile phase was methanol-water with a volume ratio of 65:35) and compounds 1, 2 and 5 were collected at the elution time of 24 minutes, 35 minutes and 45 minutes, respectively.

[0058] Fr.1-4 was purified by ODS column chromatography using methanol-water (30:70~0:100) as the solvent system for gradient elution to obtain four subfractions (Fr.1-4-1, Fr.1-4-2, Fr.1-4-3 and Fr.1-4-4).

[0059] Fr.1-4-3 repeated the above-mentioned high pressure liquid phase semi-preparative purification operation, the mobile phase was methanol-water with a volume ratio of 70:30, and compound 4 was collected at the peak time of 16 minutes.

[0060] Fr.1-5 was purified by ODS column chromatography and eluted with methanol-water (30:70~80:20) solvent system to obtain 4 subfractions (Fr.1-5-1, Fr.1-5-2, Fr.1-5-3 and Fr.1-5-4). Fr.1-5-3 repeated the high pressure liquid phase semi-preparative operation, and the mobile phase was acetonitrile-water with a volume ratio of 36:64. Compounds 3 and 6 were collected at the elution times of 30 minutes and 33 minutes, respectively.

[0061] The structure of the prepared compound was confirmed by NMR of compound 1. 1 H spectrum see Figure 1 , NMR 13 C spectrum see Figure 2 , high resolution mass spectrum see Figure 3 , NMR of compound 2 1 H spectrum see Figure 4 , NMR 13 C spectrum see Figure 5 , high resolution mass spectrum see Figure 6 , NMR of compound 3 1 H spectrum see Figure 7 , NMR 13 C spectrum see Figure 8 , high resolution mass spectrum see Figure 9 , NMR of compound 4 1 H spectrum see Figure 10 , NMR 13 C spectrum see Figure 11 , high resolution mass spectrum see Figure 12 , NMR of compound 5 1 H spectrum see Figure 13 , NMR 13 C spectrum see Figure 14 , NMR of compound 6 1 H spectrum see Figure 15 , NMR 13 C spectrum see Figure 16 ; The hydrogen spectrum and carbon spectrum data of compounds 1-4 are shown in Tables 1 and 2.

[0062] The structure of the compound prepared above was analyzed as follows.

[0063] The structural analysis of the new compound 1 is as follows: By high resolution mass spectrometry (HRESIMS) m / z 371.1492 [M+H] + The quasi-molecular ion peak is given at the position, and the molecular formula of compound 1 is C 21 H 22 O6. 1 H-NMR showed 1 active proton ( δ H 9.56), 2 sets of equivalent aromatic proton signals ( δ H 7.36, 6.83), 1 olefin proton signal ( δ H 6.58), 2 methoxy signals ( δ H 3.85, 3.68), 2 methylene signals ( δ H 4.13, 3.90), 2 methyl signals ( δ H 2.25, 1.20). 13 C-NMR and DEPT135 showed an ester carbonyl group ( δ C 169.0), 14 unsaturated carbon signals including 5 methines ( δ C 105.2, 130.3×2, 115.1×2,105.2), 2 methylene groups ( δ C 60.8, 31.9), 2 oxygen methyl groups ( δ C 60.4, 55.8) and 2 methyl groups ( δ C 14.1, 9.5). In the HMBC spectrum, OH correlates with C-3" / C-4" / C-5", Me-2 correlates with C-1' / C-1 / C-3, 3'-OMe correlates with C-3', 6'-OMe correlates with C-6', H2-4 correlates with C-1 / C-3 / C-5, and Me-7 correlates with C-6. In the COSY spectrum, H-2" correlates with H-3", H-5" correlates with H-6", and H-6 correlates with H-7. Compound 1 is a pale yellow amorphous powder, which is easily soluble in chloroform, methanol, and DMSO, but poorly soluble in water.

[0064] The structural analysis of compound 2 is as follows: By high resolution mass spectrometry (HRESIMS) m / z 313.1070 [M+H] + The quasi-molecular ion peak is given at the position, and the molecular formula of compound 2 is C 18 H16 O5. 1 H-NMR showed one aldehyde proton ( δ H 9.95), 1 active proton ( δ H 9.66), 2 sets of equivalent aromatic proton signals ( δ H 7.45, 6.87), 1 olefin proton signal ( δ H 6.70), 2 methoxy signals ( δ H 3.93,3.73), 1 methyl signal ( δ H 1.68). 13 C-NMR and DEPT135 showed an aldehyde group ( δ C 179.2), 14 unsaturated carbon signals including 5 methines ( δ C 130.4×2, 115.2×2, 105.9) and 2 oxygen methyl groups ( δ C 60.5, 56.0), 1 methyl ( δ C 9.7). In the HMBC spectrum, OH is correlated with C-3" / C-4" / C-5", Me-2 is correlated with C-1' / C-1 / C-3, 3'-OMe is correlated with C-3', 6'-OMe is correlated with C-6', and H-4 is correlated with C-1 / C-3 / C-4. In the COSY spectrum, H-2" is correlated with H-3", and H-5" is correlated with H-6". Compound 2 is a pale yellow amorphous powder, which is easily soluble in chloroform, methanol, and DMSO, but poorly soluble in water.

[0065] The structural analysis of compound 3 is as follows: By high resolution mass spectrometry (HRESIMS) m / z 359.1125 [M+H] + The quasi-molecular ion peak is given at the position, and the molecular formula of compound 3 is C 19 H 18 O7. 1 H-NMR showed two overlapping active protons ( δ H 8.98), 3 aromatic proton signals ( δ H 6.98, 6.83, 6.78), 1 olefin proton signal ( δ H 6.54), 2 methoxy signals ( δH 3.82, 3.67), 1 methylene signal ( δ H 3.78) and 1 methyl signal ( δ H 2.23). 13 C-NMR and DEPT135 showed a carboxylic acid carbonyl group ( δ C 170.5), 14 unsaturated carbon signals including 4 methines ( δ C 120.2, 116.7, 115.4, 105.2), 2 methoxy groups ( δ C 60.4, 55.7), 1 methylene ( δ C 32.1) and 1 methyl group ( δ C 9.5). In the HMBC spectrum, H-2" is correlated with C-4" / C-6", H-5" is correlated with C-1" / C-3", H-6" is correlated with C-2" / C-4", Me-2 is correlated with C-1' / C-1 / C-3, 3'-OMe is correlated with C-3', 6'-OMe is correlated with C-6', and H2-4 is correlated with C-1 / C-3 / C-5. In the COSY spectrum, H-5" is correlated with H-6". Compound 3 is a pale yellow amorphous powder, which is easily soluble in chloroform, methanol, and DMSO, but poorly soluble in water.

[0066] The structural analysis of compound 4 is as follows: By high resolution mass spectrometry (HRESIMS) m / z 373.1281 [M+H] + The quasi-molecular ion peak is given at the position, and the molecular formula of compound 4 is C 20 H 20 O7. 1 H-NMR showed three aromatic proton signals ( δ H 6.98, 6.83, 6.80), 1 olefin proton signal ( δ H 6.56), 3 methoxy signals ( δ H 3.84, 3.68, 3.66), 1 methylene signal ( δ H 3.92) and 1 methyl signal ( δ H 2.25). 13 C-NMR and DEPT135 showed an ester carbonyl group ( δC 169.4), 14 unsaturated carbon signals including 4 methines ( δ C 120.2, 116.7, 115.4, 105.2), 3 methoxy groups ( δ C 60.4, 55.7, 52.1), 1 methylene ( δ C 31.6) and 1 methyl group ( δ C 9.5). In the HMBC spectrum, H-2" is correlated with C-4" / C-6", H-5" is correlated with C-1" / C-3", H-6" is correlated with C-2" / C-4", Me-2 is correlated with C-1' / C-1 / C-3, 3'-OMe is correlated with C-3', 6'-OMe is correlated with C-6', H2-4 is correlated with C-1 / C-3 / C-5, and Me-5 is correlated with C-5. In the COSY spectrum, H-5" is correlated with H-6". Compound 4 is a pale yellow amorphous powder, which is easily soluble in chloroform, methanol, and DMSO, but poorly soluble in water.

[0067] The structural analysis of compound 5 is as follows: 1 H-NMR showed 1 active proton signal ( δ H 9.53), 2 groups of equivalent aromatic proton signals ( δ H 7.38, 6.84), 1 aromatic proton signal ( δ H 6.59), 1 methylene proton signal ( δ H 3.93), 3 methoxy signals ( δ H 3.85, 3.69, 3.66) and 1 methyl signal ( δ H 2.25). 13 C-NMR and DEPT135 showed an ester carbonyl group ( δ C 169.4), 2 sets of equivalent aromatic methylene carbon signals ( δ C 130.3, 115.1), 1 methine ( δ C 105.2), 3 methoxy groups ( δ C 60.3, 55.7, 52.1), 1 methylene ( δ C 31.6) and 1 methyl group ( δ C 9.4).

[0068] The structural analysis of compound 6 is as follows: 1 H-NMR shows two groups of equivalent aromatic proton signals ( δ H 7.43, 6.86), 3 olefin proton signals ( δ H 7.38,7.08, 6.71), 2 methoxy signals ( δ H 3.97, 3.75). 13 C-NMR and DEPT135 showed 20 carbon signals, including 11 unsaturated quaternary carbons and 2 groups of equivalent aromatic methine carbon signals ( δ C 130.4, 115.1), 3 unsaturated methine groups ( δ C 114.1, 107.1, 98.5), 2 methoxy groups ( δ C 60.6, 55.9).

[0069] In summary, the structural formula of compound 1-6 is determined as follows: In the above chemical formula, the main structures of Compound 1 and Compound 5 are the same. When the substituent at the 5th position is ethyl, it is Compound 1; and when the substituent at the 5th position is methyl, it is Compound 5.

[0070] The main structures of compound 3 and compound 4 are the same. When the 5-position substituent is a hydroxyl group, it is compound 3; and when the 5-position substituent is a methoxy group, it is compound 4.

[0071] (Example 2, dispersible oil of compound 5) 40% dispersible oil of compound 5: 40% of compound 5, 6% of phosphate, 4% of alkylbenzene polyoxyethylene ether, 8% of calcium dodecylbenzene sulfonate, 0.5% of organic bentonite, and methyl oleate is supplemented to 100%, and a dispersible oil with a mass percentage of 40% is prepared according to a conventional preparation method.

[0072] Dispersible oils of other compounds 1, 2, 3, 4 and 6 were prepared according to the above formulations.

[0073] In addition to dispersible oils, compounds 1-6 can also be made into other pesticide formulations, such as suspensions, wettable powders or granules, etc. The carriers of each formulation are conventional carriers in the art and are prepared according to conventional formulation methods.

[0074] (Test example) The biphenyl compounds 1-6 prepared in Example 1 were subjected to anti-crop disease fungus pineapple blackheart fungus Curved Australian , potato early blight Alternaria sp., mango anthracnose Colletotrichum asianum HNM408, Colletotrichum spp. Colletotrichum acutatum HNMRC 178, Fusarium wilt of banana Fusarium oxysporum HNM 1003, Pyricularia oryzae HNM 1003, Fusarium moniliforme Fusarium ring-shaped bio-52799 and Fusarium solani Fusarium solani Bio-80814 activity screening experiment: 1. Sample compounds 1-6 and positive control anisole were dissolved in DMSO and prepared to a concentration of 10 mg / L.

[0075] 2. Fungi Aspergillus candidus After HNNU0546 was activated, it was diluted with a 0.02% aqueous solution at a temperature of −80 °C and the spore concentration was about 10 6 -10 7 spores / mL.

[0076] 3. Add 100 µL of diluted bacterial solution to fresh PDA solid medium and spread evenly.

[0077] 4. Place the filter paper on the coated plate and load 5µL of sample onto each filter paper. Perform three parallel tests, using DMSO as the negative control and anisole as the positive control.

[0078] 5. After 3 days of culture, observe and measure the size of the inhibition zone.

[0079] The results of antibacterial experiments on eight crop pathogenic fungi showed that compound 2 was effective against potato early blight pathogen ( Alternaria sp.) with an inhibition zone diameter of 6 mm at 100 µg / disc. Compound 5 had antibacterial activity against potato early blight pathogen ( Alternaria sp.) had a more obvious antifungal activity, with an inhibition zone diameter of 7 mm at 50 µg / disc. Compound 6 had a stronger antifungal activity against potato early blight ( Alternaria sp.) and pineapple blackheart fungus ( Curved Australian ) had a significant inhibitory effect, and the inhibition zone diameters at 50 µg / disc were 11 and 9 mm, respectively. Compound 6 was further tested for its effects on potato early blight pathogen ( Alternariasp.) and pineapple blackheart fungus ( Curvularia australiensis ) 50 The results were all 3 µM, which was at the same level as the positive control anisole (2.5 µM), showing a significant inhibitory effect. In addition, biphenyl molecules are easily degraded in nature and have low toxicity. Therefore, the biphenyl compounds of the present invention can be used to prepare green anti-crop disease fungal pesticides.

[0080] The inhibitory effect data of compounds 2, 5 and 6 are shown in Table 3 below.

[0081] Table 3. Effects of compounds 2, 5 and 6 and the positive control anisofenazole on the crop disease fungus Alternaria sinensis ( Alternaria sp.) and pineapple blackheart fungus ( Curvularia australiensis ) Inhibitory effect

Claims

1. A biphenyl compound having the structures shown in Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6: A method for preparing a biphenyl compound according to claim 1, characterized in that: Compound 1, compound 2, compound 3, compound 4, compound 5 and compound 6 were isolated from strain Aspergillus candidus Isolated from the fermentation product of HNNU0546; Aspergillus candidus HNNU0546 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 65074.

2. The method for preparing a biphenyl compound according to claim 2, characterized in that The following steps are involved: ① Bacterial activation and fermentation; Aspergillus candidus activating, culturing and fermenting HNNU0546 to obtain a fermentate; ② Extract the fermented product with ethyl acetate or macroporous adsorption resin, and then concentrate to obtain a crude extract; ③ The crude extract of step ② was segmented by normal pressure silica gel column chromatography, with petroleum ether-ethyl acetate, petroleum ether-acetone or chloroform-methanol solvent system as eluent, and gradient elution was performed from a volume ratio of 100:0 to 0:100, and the combined components were tracked by thin layer chromatography TLC, and the components that could be developed on thin layer chromatography with a volume ratio of 8:2 petroleum ether-ethyl acetate or a volume ratio of 9:1 chloroform-acetone solvent system were further subjected to silica gel column chromatography to obtain a crude product, which was then purified by high performance liquid chromatography to obtain compound 1-6.

3. The method for preparing a biphenyl compound according to claim 3, characterized in that: In step ①, the strain Aspergillus candidus HNNU0546 was inoculated on PDA agar medium to obtain a plate with cultured bacteria; then a single colony was selected from the plate with an inoculation loop or a bamboo stick and inoculated into a conical flask of PDB seed liquid medium, and cultured on a shaker to obtain seed liquid; then the seed liquid was inoculated into bottles containing PDB fermentation medium, and static culture was performed to obtain fermentation products.

4. The method for preparing a biphenyl compound according to claim 4, characterized in that: In step ①, the strain Aspergillus candidus HNNU0546 was inoculated in PDA agar medium supplemented with 3% artificial sea salt and cultured at 28°C for 3 days for activation to obtain a plate containing the strain; the strain was then inoculated into a sterile fresh PDB seed liquid medium supplemented with 3% sea salt and cultured on a shaker at 200 rpm and 28°C for 3 days to obtain a seed liquid; The seed liquid was then inoculated into PDB fermentation medium supplemented with 3% sea salt and cultured at room temperature for 28 days.

5. The method for preparing a biphenyl compound according to claim 4 or 5, characterized in that: When preparing the PDA agar medium, 200 g of potatoes were washed and peeled, cut into slices and boiled in water for 30 minutes after the water boiled, and the residue was removed by gauze filtration, 20 g of glucose, 30 g of sea salt and 20 g of agar were added to the filtrate, and the volume was finally adjusted to 1 liter; When preparing the PDB culture medium, 200 g of potatoes were washed and peeled, cut into slices and boiled in water for 30 minutes after the water boiled, and the residue was removed by filtering with gauze, 20 g of glucose and 30 g of sea salt were added to the filtrate, and the volume was finally adjusted to 1 L.

6. The method for preparing a biphenyl compound according to claim 3, characterized in that: In step ③, the component that can be developed on thin layer chromatography with a petroleum ether-ethyl acetate solvent system in a volume ratio of 8:2 is subjected to silica gel column chromatography and gradient eluted with a petroleum ether-ethyl acetate system in a ratio of 90:10-50:50 to obtain two subfractions Fr.1-Fr.2; Alternatively, the components that can be developed on thin layer chromatography with a chloroform-acetone solvent system in a volume ratio of 9:1 are subjected to silica gel column chromatography and gradient eluted with a chloroform-acetone system of 90:10-0:100 to obtain two subfractions Fr.1-Fr.

2.

7. The method for preparing a biphenyl compound according to claim 7, characterized in that: Fraction Fr.1 was subjected to silica gel column chromatography using a pure chloroform solvent system to obtain seven subfractions Fr.1-1, Fr.1-2, Fr.1-3, Fr.1-4, Fr.1-5, Fr.1-6 and Fr.1-7; Fr.1-3 was further subjected to silica gel column chromatography using a 8:2 petroleum ether-ethyl acetate solvent system to obtain three subfractions Fr.1-3-1, Fr.1-3-2 and Fr.1-3-3; Fr.1-3-2 was purified by semi-preparative high-pressure liquid phase, and compounds 1, 2, and 5 were collected at elution times of 24 minutes, 35 minutes, and 45 minutes, respectively. The mobile phase was methanol-water with a volume ratio of 65:35; Fr.1-4 was purified by ODS column chromatography using methanol-water 30:70-0:100 as solvent system for gradient elution to obtain four subfractions Fr.1-4-1, Fr.1-4-2, Fr.1-4-3 and Fr.1-4-4; Fr.1-4-3 was purified by HPLC, the mobile phase was methanol-water with a volume ratio of 70:30, and compound 4 was collected at a peak time of 16 minutes; Fr.1-5 was purified by ODS column chromatography and eluted with a methanol-water 30:70~80:20 solvent system to obtain four subfractions Fr.1-5-1, Fr.1-5-2, Fr.1-5-3 and Fr.1-5-4.

8. Fr.1-5-3 was prepared by HPLC with the mobile phase being acetonitrile-water in a volume ratio of 36:

64. Compounds 3 and 6 were collected at elution times of 30 min and 33 min, respectively.

9. Use of Compound 2, Compound 5 or Compound 6 as claimed in claim 1 in the preparation of anti-crop disease fungal pesticides.

10. A pesticide composition comprising at least one of Compound 2, Compound 5 or Compound 6 as claimed in claim 1, and an acceptable carrier.

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