Preparation method and application of spitmol compound

Abstract

The invention relates to the technical field of microbial metabolites, in particular to a preparation method and application of a spitol compound, the spitol compound is separated from a co-culture fermentation product of rosa roxburghii endophytic fungus Epicoccum laussicola GUCC 191049.1 and helminthosporium bombycis, the molecular formula of the spitol compound is C13H20O3, and the structure of the spitol compound is shown in the specification. According to the scheme, a new material basis is provided for the use of the bulitol compound in the agricultural field.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial metabolites, and particularly relates to a preparation method of a trans-pinocarve compound and application thereof. BACKGROUND

[0002] Plant endophytic fungi are a kind of fungi living in plants, which do not cause obvious disease symptoms of plants, and can produce a variety of secondary metabolites with biological activities, and have important development value in the fields of agriculture, medicine and the like. Rosehip is a kind of Rosaceae plant with medicinal and edible properties, and its endophytic fungi contain rich metabolic product resources.

[0003] At present, although certain progress has been made in the research on the metabolic products of the rosehip endophytic fungus Epicoccum latusicollum GUCC 191049.1, a large number of unknown compounds still need to be explored. Trans-pinocarve compounds have potential effects on plant growth regulation and physiological metabolism, however, the specific trans-pinocarve compound obtained from the co-culture system of the rosehip endophytic fungus has no related reports on its structure, preparation method and application. In the prior art, there is no method for efficiently obtaining the compound, and the application potential of the compound in the related fields is not clear, which cannot meet the needs of actual production and research. SUMMARY

[0004] The present application aims to provide a preparation method of a trans-pinocarve compound and application thereof, so as to provide a new material basis for using the trans-pinocarve compound in the field of agriculture.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solution: a trans-pinocarve compound is obtained from the co-culture fermentation product of the rosehip endophytic fungus Epicoccum latusicollum GUCC 191049.1 and Exserohilum vermiculatum, and has a molecular formula of C 13 H 20 O3, and a structure as follows: .

[0006] A preparation method of a trans-pinocarve compound comprises the following steps: S1, strain preparation: selecting Epicoccum latusicollum GUCC 191049.1 and Exserohilum vermiculatum as co-culture strains; S2, culture medium selection: selecting potato glucose liquid medium; S3, co-culture fermentation: inoculate the seed liquid of the two strains of Epicoccum latusicollum GUCC 191049.1 and Dictyosporium macrospilum in a volume ratio of 1:1 into the medium selected in step S2, and ferment at 25-30°C and 130-160 r / min for 3-10 days; S4, extraction and separation: centrifuge the fermentation liquor in S3, take the supernatant, enrich by punching adsorption resin, perform silica gel column chromatography and preparative high performance liquid chromatography purification, and obtain a pure product of the tricholoma compound.

[0007] Preferably, as an improvement, the seed liquid in step S3 is cultured at 28°C and 150 r / min for 3-5 days on a shaking table.

[0008] Preferably, as an improvement, D101 macroporous adsorption resin is used for enrichment in step S4, and 70% ethanol solution is used for elution; petroleum ether-ethyl acetate gradient elution is used for silica gel column chromatography; and a C18 column is used for preparative high performance liquid chromatography, with a mobile phase of methanol-water in a volume ratio of 3:2.

[0009] The tricholoma compound is applied to the preparation of a plant growth regulator.

[0010] Advantages of the present scheme: 1. The tricholoma compound is isolated from the co-culture fermentation product of the roxburgh rose endophytic fungus Epicoccum latusicollum GUCC 191049.1 and Dictyosporium macrospilum, and its preparation method is determined, thereby providing a feasible technical route for obtaining the compound.

[0011] 2. The preparation method provided by the present application is simple and easy to operate, has good repeatability, and can efficiently extract and purify the tricholoma compound from the co-culture system, thereby meeting the needs of subsequent research and application.

[0012] 3. The present scheme determines the application direction of the tricholoma compound, lays a foundation for its application in the field of plant growth regulation research, and expands the application range of roxburgh rose endophytic fungal metabolites. DETAILED DESCRIPTION

[0013] The following will be further described in detail through specific embodiments: A tricholoma compound isolated from the co-culture fermentation product of the roxburgh rose endophytic fungus Epicoccum latusicollum GUCC 191049.1 and Dictyosporium macrospilum, having a molecular formula of C 13 H 20 O3, and a structure as follows: .

[0014] Example 1 Preparation method of fructose compound: S1, strain preparation: Epicoccum latusicollum GUCC 191049.1 and Epicoccum latusicollum were selected as co-culture strains; they were inoculated onto potato dextrose agar (PDA) plates and cultured in a constant temperature incubator at 28℃ for 5-7 days to activate the strains; single colonies of activated strains were picked and inoculated into 250mL Erlenmeyer flasks containing 100mL of LPDB medium and cultured in a shaker at 28℃ and 150r / min for 4 days to obtain seed culture.

[0015] S2, Culture medium selection: Potato glucose liquid medium (PDB) was selected. S3, co-culture fermentation: Prepare 10 1000mL Erlenmeyer flasks containing 500mL of LPDB medium. Add 20mL of seed culture of two strains, Epicoccum latusicollum GUCC191049.1 and Epicoccum latusicollum, at a volume ratio of 1:1 to each Erlenmeyer flask. Ferment for 3 days at 25℃ and 130r / min. S4, Extraction and Separation: After fermentation, the 10 flasks of fermentation broth were combined and centrifuged at 8000 rpm for 20 minutes using a high-speed centrifuge. The supernatant was collected and passed through a D101 macroporous adsorption resin column (50 mm × 600 mm) at a flow rate of 2 BV / h. After loading, the column was eluted with distilled water at a flow rate of 2 BV / h, and the eluent was collected until no absorption was observed at 254 nm. Then, the column was eluted with 70% ethanol solution at a flow rate of 1.5 BV / h, and the 70% ethanol eluent was collected.

[0016] The 70% ethanol eluent was concentrated under reduced pressure at 50°C and a vacuum of 0.08-0.1 MPa to obtain approximately 15 g of extract. The extract was dissolved in 10 mL of methanol and loaded onto a silica gel column (200 mesh silica gel, 30 mm × 500 mm column). Gradient elution was performed using a petroleum ether-ethyl acetate mixture, with gradients of 5:1, 3:1, and 1:1, for each gradient, with 3 BV of elution per gradient. The eluent for each gradient was collected in 200 mL portions.

[0017] The collected eluent was analyzed by TLC with petroleum ether-ethyl acetate (2:1) as the developing solvent and 10% sulfuric acid ethanol solution as the colorimetric reagent. The mixture was heated to 105 °C for color development. Fractions with Rf values ​​of 0.4–0.5 were combined to obtain approximately 2.3 g of fraction A.

[0018] Component A was dissolved in methanol and purified by preparative HPLC. The chromatographic column was a C18 column (250 mm × 10 mm, 5 μm), the mobile phase was methanol-water (60:40), the flow rate was 3 mL / min, the detection wavelength was 254 nm, and the chromatographic peak eluent with a retention time of 12.5-13.5 minutes was collected.

[0019] The collected effluent was concentrated under reduced pressure at 45°C and a vacuum of 0.08-0.1 MPa to obtain approximately 15 mg of pure white powdered fructose compound.

[0020] Example 2 Preparation method of fructose compound: S1, strain preparation: Epicoccum latusicollum GUCC 191049.1 and Epicoccum latusicollum were selected as co-culture strains; they were inoculated onto potato dextrose agar (PDA) plates and cultured in a constant temperature incubator at 28℃ for 5-7 days to activate the strains; single colonies of activated strains were picked and inoculated into 250mL Erlenmeyer flasks containing 100mL of LPDB medium and cultured in a shaker at 28℃ and 150r / min for 4 days to obtain seed culture.

[0021] S2, Culture medium selection: Potato glucose liquid medium (PDB) was selected. S3, co-culture fermentation: Prepare 10 1000mL Erlenmeyer flasks containing 500mL of LPDB medium. Add 20mL of seed culture of two strains, Epicoccum latusicollum GUCC191049.1 and Epicoccum latusicollum, at a volume ratio of 1:1 to each Erlenmeyer flask. Ferment for 10 days at 30℃ and 160r / min. S4, Extraction and Separation: After fermentation, the 10 flasks of fermentation broth were combined and centrifuged at 8000 rpm for 20 minutes using a high-speed centrifuge. The supernatant was collected and passed through a D101 macroporous adsorption resin column (50 mm × 600 mm) at a flow rate of 2 BV / h. After loading, the column was eluted with distilled water at a flow rate of 2 BV / h, and the eluent was collected until no absorption was observed at 254 nm. Then, the column was eluted with 70% ethanol solution at a flow rate of 1.5 BV / h, and the 70% ethanol eluent was collected.

[0022] The 70% ethanol eluent was concentrated under reduced pressure at 50°C and a vacuum of 0.08-0.1 MPa to obtain approximately 15 g of extract. The extract was dissolved in 10 mL of methanol and loaded onto a silica gel column (250 mesh silica gel, 30 mm × 500 mm column). Gradient elution was performed using a petroleum ether-ethyl acetate mixture, with gradients of 5:1, 3:1, and 1:1, for each gradient, with 3 BV of elution per gradient. The eluent for each gradient was collected in 200 mL portions.

[0023] The collected eluent was analyzed by TLC with petroleum ether-ethyl acetate (2:1) as the developing solvent and 10% sulfuric acid ethanol solution as the colorimetric reagent. The mixture was heated to 105 °C for color development. Fractions with Rf values ​​of 0.4–0.5 were combined to obtain approximately 2.3 g of fraction A.

[0024] Component A was dissolved in methanol and purified by preparative HPLC. The chromatographic column was a C18 column (250 mm × 10 mm, 5 μm), the mobile phase was methanol-water (60:40), the flow rate was 3 mL / min, the detection wavelength was 254 nm, and the chromatographic peak eluent with a retention time of 12.5-13.5 minutes was collected.

[0025] The collected effluent was concentrated under reduced pressure at 45°C and a vacuum of 0.08-0.1 MPa to obtain approximately 15 mg of pure white powdered fructose compound.

[0026] Example 3 Preparation method of fructose compound: S1, strain preparation: Epicoccum latusicollum GUCC 191049.1 and Epicoccum latusicollum were selected as co-culture strains; they were inoculated onto potato dextrose agar (PDA) plates and cultured in a constant temperature incubator at 28℃ for 5-7 days to activate the strains; single colonies of activated strains were picked and inoculated into 250mL Erlenmeyer flasks containing 100mL of LPDB medium and cultured in a shaker at 28℃ and 150r / min for 4 days to obtain seed culture.

[0027] S2, Culture medium selection: Potato glucose liquid medium (PDB) was selected. S3, co-culture fermentation: Prepare 10 1000mL Erlenmeyer flasks containing 500mL of LPDB medium. Add 20mL of seed culture of two strains, Epicoccum latusicollum GUCC191049.1 and Epicoccum latusicollum, at a volume ratio of 1:1 to each Erlenmeyer flask. Ferment for 5 days at 27℃ and 150r / min. S4, Extraction and Separation: After fermentation, the 10 flasks of fermentation broth were combined and centrifuged at 8000 rpm for 20 minutes using a high-speed centrifuge. The supernatant was collected and passed through a D101 macroporous adsorption resin column (50 mm × 600 mm) at a flow rate of 2 BV / h. After loading, the column was eluted with distilled water at a flow rate of 2 BV / h, and the eluent was collected until no absorption was observed at 254 nm. Then, the column was eluted with 70% ethanol solution at a flow rate of 1.5 BV / h, and the 70% ethanol eluent was collected.

[0028] The 70% ethanol eluent was concentrated under reduced pressure at 50°C and a vacuum of 0.08-0.1 MPa to obtain approximately 15 g of extract. The extract was dissolved in 10 mL of methanol and loaded onto a silica gel column (300 mesh silica gel, 30 mm × 500 mm column). Gradient elution was performed using a petroleum ether-ethyl acetate mixture, with gradients of 5:1, 3:1, and 1:1, for each gradient, with 3 BV of elution per gradient. The eluent for each gradient was collected in 200 mL portions.

[0029] The collected eluent was analyzed by TLC with petroleum ether-ethyl acetate (2:1) as the developing solvent and 10% sulfuric acid ethanol solution as the colorimetric reagent. The mixture was heated to 105 °C for color development. Fractions with Rf values ​​of 0.4–0.5 were combined to obtain approximately 2.3 g of fraction A.

[0030] Component A was dissolved in methanol and purified by preparative HPLC. The chromatographic column was a C18 column (250 mm × 10 mm, 5 μm), the mobile phase was methanol-water (60:40), the flow rate was 3 mL / min, the detection wavelength was 254 nm, and the chromatographic peak eluent with a retention time of 12.5-13.5 minutes was collected.

[0031] The collected effluent was concentrated under reduced pressure at 45°C and a vacuum of 0.08-0.1 MPa to obtain approximately 15 mg of pure white powdered fructose compound.

[0032] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A compound containing fructose, characterized in that: It was isolated from the fermentation products of co-culture of the endophytic fungus *Epicoccum latusicollum* GUCC 191049.1 and *Epipremnum adenophorum*, and its molecular formula is C. 13 H 20 O3 has the following structure: 。 2. The method for preparing a compound according to claim 1, characterized in that: Includes the following steps: S1, strain preparation: Epicoccum latusicollum GUCC 191049.1 and Epicoccum latusicollum were selected as co-culture strains; S2, Culture medium selection: Potato glucose liquid culture medium was selected; S3, co-culture fermentation: The seed liquids of two strains, Epicoccum latusicollum GUCC 191049.1 and Epicoccum latusicollum, were inoculated into the culture medium selected in step S2 at a volume ratio of 1:1, and fermented for 3 to 10 days at 25-30℃ and 130-160r / min. S4, Extraction and Separation: After centrifuging the fermentation broth in S3, the supernatant was collected, enriched by perforated adsorption resin, purified by silica gel column chromatography and preparative high performance liquid chromatography to obtain pure tebufenol compound.

3. The method for preparing the emamectin compound according to claim 2, characterized in that: The seed culture conditions in step S3 are 28℃, 150r / min, and shaker culture for 3-5 days.

4. The method for preparing the emamectin compound according to claim 3 and its application, characterized in that: In step S4, D101 macroporous adsorption resin was used for enrichment, and 70% ethanol solution was used for elution; silica gel column chromatography was performed using a gradient elution of petroleum ether and ethyl acetate; preparative high performance liquid chromatography was performed using a C18 column, with methanol-water as the mobile phase at a volume ratio of 3:

2.

5. Eupatorol compounds are used in the preparation of plant growth regulators.

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