Preparation method and application of organic compound dimorine A

Abstract

The invention provides a preparation method and application of an organic compound dimorine A, and relates to the technical field of organic compounds. The preparation method of the organic compound dimorine A specifically comprises the following steps: S1, eluting an extract; s2, gradient elution; s3, collecting and grouping; s4, further separation; s5, eluting with methanol; s6, dotting analysis; s7, purifying and eluting; and S8, collecting a target compound. Through contrast tests of different extraction solvents, the ethyl acetate is finally selected as the optimal extraction solvent, the target compound can be efficiently separated and purified, the purity and the yield are remarkably improved, the extractum extracted by the ethyl acetate is moderate in polarity and suitable for separation and excavation of novel skeleton compounds, and the method is suitable for industrial production. Secondary metabolites of strains obtained after rice is fermented for 35 days are richer, the biological activity of the compound is improved, and the method is easy and convenient to operate, short in experimental period, low in cost and high in detection sensitivity, has remarkable environmental and economic benefits and is suitable for large-scale production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of organic compounds, and specifically provides a preparation method and application of an organic compound dimorine A. Background Art

[0002] Organic compounds are compounds formed by covalent bonds between carbon atoms and hydrogen atoms, usually also containing other elements such as oxygen, nitrogen, sulfur, etc. Such compounds are widely present in nature, from simple molecules such as methane and ethanol to complex biological macromolecules such as proteins and nucleic acids. Organic compounds have important applications in many fields such as medicine, agriculture, and materials science. In particular, organic compounds with specific biological activities are often studied and developed as lead compounds for drugs or pesticides.

[0003] The existing organic compound dimorine A is mainly obtained by extraction and separation from endophytic fungi. The specific methods include preliminary extraction using macroporous resin eluate, and then multi-step separation and purification by methods such as reverse-phase chromatography column and gel column chromatography. Although this method can successfully isolate dimorine A, there are some obvious disadvantages. First, the separation and purification process is complex and requires multiple steps, resulting in a long experimental cycle and high operation cost. Second, the yield of the existing method is low, making it difficult to produce on a large scale. Finally, there is room for improvement in the purity and detection sensitivity of the compound dimorine A, which limits its effectiveness and reliability in practical applications. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the present invention provides a preparation method and application of an organic compound dimorine A, which solves the problems of the existing preparation method of organic compound dimorine A having a long cycle, high cost, and low purity.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A preparation method of an organic compound dimorine A specifically includes the following steps: S1. Eluting the extract The total extract obtained by fermenting type II umbellate is eluted with macroporous resin, and the elution part of 60% methanol-water eluent is concentrated under reduced pressure to dryness to obtain an extract. S2. Gradient elution The extract is separated using a reverse-phase chromatography column, and a methanol-water mixed solvent is used as the eluent, and the elution gradients are set to 30%, 35%, 40%, 45%, 50%, and 60% respectively. S3. Collecting and grouping Collect multiple fractionated components (A-K) separated out. S4. Further separation Further separate the fraction with a relatively high possibility of containing the target compound (H fraction) collected, using LH-20 gel column chromatography for separation; S5. Methanol elution Using methanol as the eluent, multiple sub-fractions (H-1 to H-6) are obtained from the LH-20 gel column; S6. TLC plate analysis Determine the sub-fraction (H-3) containing the target compound dimorine A by TLC plate analysis; S7. Purification elution Use semi-preparative high performance liquid chromatography Pre-HPLC to purify the sub-fraction to be purified, the eluent is acetonitrile: water = 20:80, the elution gradient is isocratic elution, and the elution flow rate is 3.0 mL / min; S8. Collect the target compound The detection wavelengths are 210 nm and 254 nm, and the target compound dimorine A is collected at a retention time of 8 min to obtain the purified compound. The structural characterization of compound dimorine A is shown in Table 1 and Figures 2 - 12 , where Table 1 includes 1H NMR (500 MHz) and 13C NMR (126 MHz) data. The structural characterization data of compound dimorine A are taken as examples of 1H NMR (500 MHz) and 13C NMR (126 MHz) data.

[0006] Preferably, the solvent ratio in the elution gradient can be adjusted to optimize the separation effect of the compound.

[0007] Preferably, the target compound is obtained by strain biosynthesis through an acylation reaction between -COOH (carboxyl group) in 2,4-dihydroxy-3,5,6-trimethylbenzoic acid produced by type II umbellate mold and -HS (mercapto group) in homocysteine, with the elimination of molecular H 2 O.

[0008] Preferably, in the separation process of the reverse phase chromatography column, eluents with different combinations of organic phase and water phase can be used to adapt to the specific conditions of different samples.

[0009] Preferably, the purification of compound dimorine A can be further optimized by changing the ratio of the eluent and the flow rate conditions.

[0010] Preferably, the dimorine A derivative is a theoretically acceptable homocysteine type compound with a similar skeleton structure.

[0011] Application of dimorine A and its derivatives obtained by a preparation method of an organic compound dimorine A in inhibiting phytopathogenic fungi.

[0012] The present invention provides a preparation method and application of an organic compound dimorine A. It has the following beneficial effects: The present invention provides a preparation method and application of an organic compound dimorine A. The method of the present invention extracts and isolates from endophytic fungi to obtain a compound formed by the acylation reaction of -COOH (carboxyl group) in 2,4-dihydroxy-3,5,6-trimethylbenzoic acid produced by Dichotomomyces type II with -HS (mercapto group) in homocysteine, and one molecule of H 2 O is removed, and the compound dimorine A is formed by strain biosynthesis. Compared with the existing chemical synthesis method, the method of producing secondary metabolites by endophytic fungi fermentation has less environmental pollution and meets the requirements of sustainable development. The extraction and purification steps of the invention are relatively simple, greatly shortening the experimental period and reducing the operation cost. By optimizing the separation conditions of the reversed-phase chromatography column and LH-20 gel column chromatography, the present invention can efficiently separate and purify the target compound dimorine A, significantly improving the purity and yield. The TLC spotting plate and semi-preparative high-performance liquid chromatography techniques adopted by the present invention can accurately detect and purify the target compound. Especially at a retention time of 8 min and using detection wavelengths of 210 nm and 254 nm, the high sensitivity of detection is ensured. The compound dimorine A has significant inhibitory activity against a variety of phytopathogenic fungi, especially against Fusarium oxysporum, Botrytis cinerea and Coriolus versicolor, and the MICs are 31.25 - 15.63 μg / mL and 15.63 - 7.82 μg / mL respectively. This discovery provides an important theoretical basis and practical basis for the development of new antifungal agents. The preparation method of the present invention not only has advantages over the prior art in terms of environmental friendliness and operation simplicity, but also shows significant advantages in terms of purity, yield and detection sensitivity, making the application prospect of the compound dimorine A in agriculture more broad. Brief Description of the Drawings

[0013] Figure 1 It is a technical route map of the preparation method and application of the compound dimorine A of the present invention; Figure 2 For the compound dimorine A of the present invention in methanol 1 1H NMR spectrum; Figure 3 For the compound dimorine A of the present invention in methanol 13 13C NMR spectrum; Figure 4DEPT spectrum of compound dimorine A of the present invention in methanol; Figure 5 For the compound dimorine A of the present invention in methanol 1 H- 1 H COSY spectrum; Figure 6 HSQC spectrum of compound dimorine A of the present invention in methanol; Figure 7 HMBC spectrum of compound dimorine A of the present invention in methanol; Figure 8 NOESY spectrum of compound dimorine A of the present invention in methanol; Figure 9 HR-TOF-ESI-MS spectrum of compound dimorine A of the present invention in methanol; Figure 10 UV spectrum of compound dimorine A of the present invention in methanol; Figure 11 IR spectrum of compound dimorine A of the present invention in methanol; Figure 12 Optical rotation spectrum of compound dimorine A of the present invention; Figure 13 TLC analysis chart of the extracts of rice fermented for 28 days and 35 days of the present invention; Figure 14 Alternative technical route chart of the present invention; Figure 15 Schematic diagram of the molecular structure of the homocysteine - like compound and theoretically acceptable derivatives of the present invention. Detailed implementation manners

[0014] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0015] As Figures 1 - 15 shown, the embodiments of the present invention provide a preparation method and application of an organic compound dimorine A, specifically: Source of raw materials The strain of the endophytic fungus Umbelopsis dimorpha used in the experiment was isolated from the root tissue of healthy Vaccinium dunalianum Wight plants in a certain county in October 2016 and cryopreserved at ultra-low temperature (-80 °C) in the Key Laboratory of Forest Resources Conservation and Utilization in Southwest Mountains, Ministry of Education, a certain university.

[0016] Activation and subculture of Umbelopsis dimorpha strain Take out the Umbelopsis dimorpha strain from the ultra-low temperature refrigerator at -80 °C and place it in the refrigerator at 4 °C to thaw for 6 h. Under the sterile environment of the laminar flow hood, inoculate the strain onto the potato sucrose agar (PSA) medium and incubate it at a constant temperature of 28 °C for 14 days. After the mycelium covers the PSA medium, use a borer to punch the strain on the PSA to obtain a mycelial block with a diameter of 5 mm, and transfer the mycelial block onto the PSA medium for subculture to obtain the second-generation strain.

[0017] Large-scale fermentation culture and extraction of Umbelopsis dimorpha strain with rice Punch the cultured second-generation strain according to the above punching method, and then transfer the mycelial blocks to 300 500-mL fermentation flasks, with five mycelial blocks placed in each fermentation flask. Each fermentation flask contains 50 g of rice and 50 mL of water. Before inoculating the mycelial blocks, put the fermentation flasks into the autoclave to be cooked and sterilized, and then culture them at room temperature for 35 days. Then extract with an equal volume of ethyl acetate 4 times and concentrate under reduced pressure at 50 °C to obtain an extract (244.47 g). Adsorb the extract with D101 macroporous resin, and elute the extracted extract with pure water, 30%, 60%, and 90% methanol-water mixed solutions in a gradient manner to divide the extract into three large segments with different polarities for subsequent separation and purification.

[0018] Separation and purification process of dimorine A: ① Concentrate the 60% methanol-water eluate under reduced pressure to obtain 6.5 g of extract, and then perform column chromatography separation with an ODS C18 reversed-phase chromatographic column, using methanol:water as the mobile phase for gradient elution. The elution gradients are methanol:water = 30:70, 35:65, 40:60, 45:55, 50:50, 60:40 in turn, and 11 fractions (A-K) are eluted. 18

[0019] ​② The H fraction (225 mg) in the above 11 fractions was separated by an LH-20 gel chromatography column with methanol as the eluent to obtain 6 sub-fractions (H-1, H-2, H-3, H-4, H-5, H-6). H-3 was separated by semi-preparative high performance liquid chromatography with acetonitrile: water = 20:80 isocratic elution, an elution flow rate of 3.0 mL / min, detection wavelengths of 210 nm and 254 nm. At a retention time of 8 min, 7.3 mg of compound dimorine A was obtained by separation and purification. The preparation and application technical route of compound dimorine A is as Figure 1 shown.

[0020] Experimental Example 1 (1) The 60% methanol-water eluate eluted from the macroporous resin was concentrated under reduced pressure to obtain 6.5 g of an extract. The extract was separated by a reverse-phase chromatography column to obtain 11 fraction components (A-K), and the eluent was a methanol-water mixed solvent with elution gradients of 30%, 35%, 40%, 45%, 50%, and 60% respectively. Then, the components in the A-K segments eluted from the reverse-phase column were further separated and purified.

[0021] Experimental Example 2 (2) The H fraction (225 mg) in the 11 fractions was separated by an LH-20 gel column with methanol as the eluent to obtain 6 sub-fraction components (H-1, H-2, H-3, H-4, H-5, H-6). Further separation and purification were carried out on the components in different segments from H-1 to H-6. After TLC spotting, it was found that the target compound dimorine A was in H-3. Therefore, semi-preparative high performance liquid chromatography was selected for separation with acetonitrile: water = 20:80 as the eluent, isocratic elution as the elution gradient, an elution flow rate of 3.0 mL / min, detection wavelengths of 210 nm and 254 nm. At a retention time of 8 min, the target compound dimorine A was purified with a mass of 7.3 mg. The structural characterization of compound dimorine A is shown in Table 1 and Figures 2 - 12 .

[0022] Experimental Example 3 (3) The compound dimorine A was assayed for its antibacterial activity against five plant pathogenic fungi by the micro-double dilution method in 96-well plates, using thiabendazole and carbendazim as positive controls and an equal-concentration dimethyl sulfoxide solution as the negative control. The five plant pathogenic fungi were Alternaria brassicola, Fusarium graminearum, F. oxysporum, Botrytis cinerea, and Coriolus versicolor. After culturing at 28 °C for 48 hours, the minimum inhibitory concentration (MIC) of the compound was observed. The compound dimorine A showed certain inhibitory activity against the five plant pathogenic fungi. Among them, it had a moderate inhibitory effect on F. oxysporum and B. cinerea, with MICs of 31.25 - 15.63 μg / mL; it had a certain inhibitory effect on C. versicolor, with MICs of 15.63 - 7.82 μg / mL. See Table 2 for details.

[0023] Table 1 Compound dimorine A in CD 3 OD 1 H (500 MHz) and 13 C NMR (126 MHz) data

[0024] Table 2 Inhibitory activity of compound dimorine A against five plant pathogenic fungi

[0025] Taking the control experiment as a comparative example, such as when the reaction temperature is 20 - 50 °C, test schemes corresponding to reaction temperatures outside the range of 20 - 50 can be provided. Comparative Example 1: (1) In the large-scale fermentation culture and extraction of the Aspergillus bisporus strain in rice, after the large-scale fermentation of rice was completed, the fermentation products were extracted 4 times with petroleum ether and ethyl acetate. Using dichloromethane:methanol = 10:1 as the developing agent, after TLC spotting and analysis, it was found that the compounds extracted with petroleum ether had a relatively small polarity and a large difference in polarity from most new skeleton compounds. Therefore, it was not suitable for the separation and purification of compounds with novel skeletons.

[0026] Comparative Example 2: (2) In the large-scale fermentation culture and extraction of the Ustilago maydis strain in rice, after the large-scale fermentation of rice was completed, the fermented product was extracted 4 times with ethyl caproate of industrial grade. Using dichloromethane:methanol = 10:1 as the developing agent, after TLC spotting and analysis, it was found that the extract obtained by ethyl acetate extraction was closer in polarity to most of the reported active lead compounds. Therefore, the extract obtained by ethyl acetate extraction has greater potential in the separation and discovery of new skeleton compounds.

[0027] Comparative Example 3: In the large-scale fermentation culture and extraction of the Ustilago maydis strain in rice, after the large-scale fermentation of rice was completed, the fermented product was extracted 4 times with methanol of industrial grade. Using dichloromethane:methanol = 10:1 as the developing agent, after TLC spotting and analysis, it was found that the extract obtained by methanol extraction had a relatively large polarity, and the possibility of discovering novel skeleton compounds was small. Therefore, the extract obtained by methanol extraction has less potential compared to the extract obtained by ethyl acetate extraction.

[0028] Comparative Example 4: (4) Based on the above analysis, ethyl acetate was finally selected as the solvent to extract the fermented rice. The production of secondary metabolites on the 28th day and the 35th day of the rice fermentation extract was compared. The rice at two different time periods was extracted with ethyl acetate. After TLC spotting and analysis, it was found that the secondary metabolites of the strain after 35 days of rice fermentation were more abundant than those after 28 days of fermentation, as Figure 13 shown.

[0029] Figure 15 homocysteine-like compounds and theoretically acceptable derivatives thereof, having any one of the structures shown in Formulas I and II.

[0030] When R 1 is OH and R 2 is A, or when R 1 is A and R 2 is OH, it is denoted as Formula II-1; When R 1 is OH and R 2 is B, or when R 1 is B and R 2 is OH, it is denoted as Formula II-2; When R 1 is OH and R 2 is C, or when R 1 is C and R 2 is OH, it is denoted as Formula II-3; When R 1 is OH and R 2 is D, or when R 1 is D and R 2 is OH, it is denoted as Formula II-4; When R 1For OH, R 2 When E or R 1 For E, R 2 When OH is, it is expressed as formula II-5; When R 1 For OH, R 2 When OAc or R 1 For OAc, R 2 When OH is, it is expressed as formula II-6; When R 1 For OAc, R 2 When it is OAc, it is represented by Formula II-7.

[0031] In the technical solution, the endophytic fungus Agaricus type II strain was fermented with rice on a large scale, and the rice fermentation can be changed to potato sucrose (PSB) liquid medium fermentation. Like rice solid fermentation, PSB liquid medium fermentation can also provide nutrients for the growth and development of endophytic fungi, and can be used as another fermentation method to obtain the compound dimorine A. Before using ethyl acetate extraction, petroleum ether can be used for extraction 4 times. This operation can first extract the fatty acids in the fermentation, and then continue to extract with ethyl acetate, which is convenient for the subsequent separation and purification of the target compound, saving experimental costs and separation and purification time.

[0032] Biopesticides mainly refer to agricultural preparations developed from secondary metabolites produced by natural biological resources such as animals, plants, and microorganisms. This study proposed a compound dimorine A produced by plant endophytic fungi with a novel skeleton and good antibacterial activity. In recent years, the development of microbial metabolites as biopesticides has been very rapid, accounting for about 90% of the total biopesticides. They have the characteristics of safety, low toxicity, easy degradation, no residue, strong specificity, and not easy to develop drug resistance. According to statistics, the proportion of new active compounds discovered from endophytic fungi is 51%, far exceeding the proportion of 38% of soil microorganisms. Their molecular skeletons are of many types and large variations, with rich structural diversity. Many active lead compounds with novel structures can be screened from them. They have huge research, development and utilization potential and are one of the effective ways to solve agricultural diseases.

[0033] Vaccinium dunalianum Wight belongs to the genus Vaccinium of the Ericaceae family. It is mainly produced in Yunnan, Sichuan, Guizhou provinces and the Tibet Autonomous Region. Since the Ming Dynasty, its young tender leaves have been dried and used as a kind of tea until now, and it has certain medicinal value. Umbelopsis dimorpha is the dominant strain isolated from the roots of Vaccinium dunalianum. At present, the research on the strain Umbelopsis dimorpha mainly focuses on promoting the growth of host plants, improving their drought resistance, producing components identical or similar to those of the host, and the ability to transform the main components of the host. There is less research on its secondary metabolites. It is of great significance to develop relatively novel compounds to prevent agricultural pests and diseases with biopesticides.

[0034] The preparation method of the present invention has obtained for the first time a compound dimorine A with a rare skeleton formed by the combination of benzene series compounds and amino acids. This compound is obtained by the acylation reaction of -COOH (carboxyl group) in 2,4-dihydroxy-3,5,6-trimethylbenzoic acid with -HS (mercapto group) in homocysteine, removing one molecule of H 2 O, and several theoretically acceptable compounds of homocysteine structural types are proposed. The skeleton of compound dimorine A has rarely been reported in previous studies. It can be inferred from this that the secondary metabolites produced by endophytic fungi have great potential. Screening for more structurally novel active lead compounds has great potential for research, development and utilization, and is expected to become one of the effective ways to solve agricultural diseases. The antibacterial activity test found that compound dimorine A has inhibitory activity against five plant pathogenic fungi. Among them, it has a moderate inhibitory effect on Fusarium oxysporum and Botrytis cinerea, and the inhibition rate (MICs) is 31.25 - 15.63 μg / mL; it has a certain inhibitory effect on Coriolus versicolor, and the MICs is 15.63 - 7.82 μg / mL. Therefore, such compounds with novel skeletons are expected to become cheap, easily available and environmentally friendly biopesticides. The discovery of compound dimorine A fills the gap in benzene series compounds of homocysteine and provides a very valuable reference for the development of biopesticides.

[0035] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an organic compound dimorine A, characterized in that: The specific steps include: S1. Elution extract The total extract extracted from the fermentation of Amanita phalloides is eluted with a macroporous resin, and the eluted portion of the 60% methanol-water eluate is concentrated to dryness under reduced pressure to obtain an extract; S2. Gradient elution The extract was separated by a reverse phase chromatography column, with a methanol-water mixed solvent as the eluent, and the elution gradient was set to 30%, 35%, 40%, 45%, 50%, and 60% respectively; S3. Collect Groups Collect the separated multiple fractions, AK; S4. Further separation The collected fractions, H components, which are more likely to contain the target compound, were further separated using LH-20 gel column chromatography; S5. Methanol elution Using methanol as the eluent, multiple subfractions, H-1 to H-6, were obtained from the LH-20 gel column; S6. Spot plate analysis The subfraction containing the target compound dimorine A, H-3, was identified by TLC spot plate analysis; S7. Purification and elution The subfraction to be purified was purified using semi-preparative high performance liquid chromatography Pre-HPLC, the eluent was acetonitrile: water = 20:80, the elution gradient was isocratic elution, and the elution flow rate was 3.0 mL / min; S8. Collect target compounds The detection wavelengths were 210 nm and 254 nm, and the target compound dimorine A was collected at a retention time of 8 min to obtain a purified compound.

2. The method for preparing dimorine A according to claim 1, characterized in that: The solvent ratio in the elution gradient can be adjusted.

3. The method for preparing dimorine A according to claim 1, characterized in that: The target compound is obtained by biosynthesis in the strain through an acylation reaction between the -COOH carboxyl group in 2,4-dihydroxy-3,5,6-trimethylbenzoic acid, a compound produced by Agaricus dimorphus, and the -HS thiol group in homocysteine ​​to remove molecular H2O.

4. The method for preparing dimorine A, an organic compound according to claim 1, characterized in that: The reverse phase chromatography column separation process uses an eluent composed of an organic phase and an aqueous phase in different proportions.

5. The method for preparing dimorine A, an organic compound according to claim 1, characterized in that: The purification of the compound dimorine A was further optimized by changing the ratio of the eluent and the flow rate conditions.

6. The method for preparing dimorine A, an organic compound according to claim 1, characterized in that: The dimorine A derivatives are theoretically acceptable homocysteine-type compounds with similar skeleton structures.

7. Use of dimorine A and its derivatives obtained according to the method for preparing dimorine A, an organic compound, according to any one of claims 1 to 6 in inhibiting plant pathogenic fungi.

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