CN108558817B - Isocoumarin derivative and preparation method and application thereof - Google Patents

Isocoumarin derivative and preparation method and application thereof Download PDF

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CN108558817B
CN108558817B CN201810288455.6A CN201810288455A CN108558817B CN 108558817 B CN108558817 B CN 108558817B CN 201810288455 A CN201810288455 A CN 201810288455A CN 108558817 B CN108558817 B CN 108558817B
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methanol
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dichloromethane
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丁立建
何山
黄立明
崔巍
斯拉瓦·爱泼斯坦
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Shenzhen Dragon Totem Technology Achievement Transformation Co ltd
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Abstract

The invention discloses an isocoumarin derivative and a preparation method and application thereof, and is characterized in that the structural formula of the isocoumarin derivative is shown as I, the preparation method comprises the steps of obtaining a fermentation product containing the isocoumarin derivative through fermentation culture of marine fungi with the preservation number of CCTCC No: M2014086, then adding methanol for soaking, carrying out ultrasonic filtration, drying by distillation, extracting by ethyl acetate, drying by distillation to obtain a crude extract, carrying out reduced pressure silica gel column chromatography and reversed phase medium pressure column chromatography on the extract, and finally separating and purifying by reversed phase semi-preparative high performance liquid chromatography.

Description

Isocoumarin derivative and preparation method and application thereof
Technical Field
The invention relates to an isocoumarin derivative, in particular to an isocoumarin derivative and a preparation method and application thereof.
Background
The isocoumarin derivatives have anticancer, antibacterial, protease inhibiting and antioxidant effects. The inventor carries out an antioxidation experiment on an ethyl acetate extract obtained by solid fermentation of sponge epiphyte Aspergillus ustus (preserved in China center for type culture Collection, with the preservation number of CCTCC No: M2014086), finds that the sponge epiphyte Aspergillus ustus has antioxidation activity, and researches the active ingredients of the sponge epiphyte Aspergillus ustus to separate out a novel isocoumarin derivative. At present, the chemical structure and the antioxidant activity of the compound are not reported, so that the related medicines are not seen in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing an isocoumarin derivative with antioxidant activity and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: an isocoumarin derivative, the structural formula of which is shown as I:
Figure GDA0001676989820000011
the preparation method of the isocoumarin derivative specifically comprises the following steps:
(1) fermentation production: marking off Aspergillus niger (Aspergillus ustus) with the preservation number of CCTCC No. M2014086 for rejuvenation, selecting a single colony, inoculating into a PDB liquid culture medium, and culturing at 28 ℃ and 150rpm for 5 days to obtain a seed solution; inoculating the seed liquid into a rice culture medium according to the volume ratio of 5-20%, standing and fermenting at 28 deg.C for 30 days;
(2) obtaining a crude extract: adding methanol into the conical flask after fermentation, immersing the solid, performing ultrasonic treatment for 15 minutes, standing overnight, and filtering the methanol extract with gauze to remove solid residues; leaching the solid residue for 3-5 times, mixing the methanol extractive solutions, evaporating to remove methanol, dissolving with water, adding ethyl acetate with the same volume as the water solution, extracting for 5 times, mixing extractive solutions, and concentrating under reduced pressure to remove ethyl acetate to obtain crude extract;
(3) separation and purification of compounds: dissolving the crude extract by using a mixed solvent of dichloromethane and methanol, adding 200-mesh 300-mesh silica gel for sample mixing, performing reduced pressure silica gel column chromatography on the crude extract by using petroleum ether/ethyl acetate with a volume ratio of 2:1 as an eluent, and collecting an elution component; performing gel column chromatography (LH-20) on the eluted component by using a mixed solvent of dichloromethane and methanol as an eluent, and collecting the eluted component; separating the eluted components by reverse phase medium pressure column chromatography, adopting methanol and water as eluent, linearly eluting the components from 30% methanol in volume fraction to 100% methanol in gradient, eluting for 120 minutes at a flow rate of 20mL/min, collecting the fraction with peak time of 80-90 minutes, and finally separating and purifying the fraction by semi-preparative reverse phase high performance liquid chromatography to obtain the isocoumarin derivative, wherein the structure of the isocoumarin derivative is shown as formula I:
Figure GDA0001676989820000021
the PDB liquid culture medium has the following formula: 200g of potatoes, 20g of glucose, 35g of sea crystals and 1L of distilled water; the formula of the rice culture medium is as follows: 100g of rice, 3.5g of sea crystal and 100mL of distilled water.
The volume ratio of the dichloromethane to the methanol in the dichloromethane and methanol mixed solvent is 1: 1.
the eluent of the semi-preparative reverse phase high performance liquid chromatography is prepared by mixing acetonitrile and water according to the volume ratio of 28: 72.
The application of the isocoumarin derivative and the application of the isocoumarin derivative in the aspect of preparing an antioxidant.
Compared with the prior art, the invention has the advantages that: the invention relates to an isocoumarin derivative and a preparation method and application thereof, wherein a fermentation product containing a chlorine aromatic compound is obtained through microbial fermentation culture, then the fermentation product is soaked by methanol and extracted by ethyl acetate to obtain a crude extract, and the crude extract is separated and purified by reduced pressure silica gel column chromatography, medium pressure column chromatography and reversed phase semi-preparative high performance liquid chromatography; the isocoumarin derivative is obtained by microbial fermentation, and the method has the characteristics of simple operation process, short production period, low product cost and no pollution to the environment.
The Aspergillus oryzae (Aspergillus oryzae) is DJ003 strain with preservation number of CCTCC No. M2014086, and is preserved in China center for type culture Collection in 2014 at 03 and 14 days, with preservation address of Wuhan university in Wuhan.
Drawings
FIG. 1 shows that compound I scavenges DPPH radicals in a concentration-dependent manner. Compound i or vitamin c (vitc) was added to a 0.2mM DPPH solution at the indicated concentrations for 20 minutes, and the DPPH free radical content was measured by evaluating the absorbance at 517nm, the data representing the mean ± SEM of three separate experiments; p <0.01 relative to control (ANOVA and Duncet test).
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
The structural formula of the isocoumarin derivative is shown as the following formula I:
Figure GDA0001676989820000031
example 2
The preparation method of the isocoumarin derivative shown as the formula I comprises the following steps:
(1) fermentation production: marking off Aspergillus niger (Aspergillus ustus) with the preservation number of CCTCC No. M2014086 for rejuvenation, selecting a single colony, inoculating into a PDB liquid culture medium, and culturing at 28 ℃ and 150rpm for 5 days to obtain a seed solution; inoculating the seed liquid into a rice culture medium according to the volume ratio of 5-20%, standing and fermenting at 28 deg.C for 30 days; the formula of the PDB liquid culture medium is as follows: 200g of potatoes, 20g of glucose, 35g of sea crystals and 1L of distilled water; the formula of the rice culture medium is as follows: 100g of rice, 3.5g of sea crystal and 100mL of distilled water;
(2) obtaining a crude extract: adding methanol into the conical flask after fermentation, immersing the solid, performing ultrasonic treatment for 15 minutes, standing overnight, and filtering the methanol extract with gauze to remove solid residues; leaching the solid residue for 3-5 times, mixing the methanol extractive solutions, evaporating to remove methanol, dissolving with water, adding ethyl acetate with the same volume as the water solution, extracting for 5 times, mixing extractive solutions, and concentrating under reduced pressure to remove ethyl acetate to obtain crude extract;
(3) separation and purification of compounds: dissolving the crude extract by using a mixed solvent of dichloromethane and methanol, adding 200-mesh 300-mesh silica gel for sample mixing, performing reduced pressure silica gel column chromatography on the crude extract by using petroleum ether/ethyl acetate with a volume ratio of 2:1 as an eluent, and collecting an elution component; performing gel column chromatography (LH-20) on the eluted component by using a mixed solvent of dichloromethane and methanol as an eluent, and collecting the eluted component; separating the eluted components by reverse phase medium pressure column chromatography, adopting methanol and water as eluent, linearly eluting the components from 30% methanol in volume fraction to 100% methanol in gradient, eluting for 120 minutes at a flow rate of 20mL/min, collecting the fraction with peak time of 80-90 minutes, and finally separating and purifying the fraction by semi-preparative reverse phase high performance liquid chromatography to obtain the isocoumarin derivative, wherein the structure of the isocoumarin derivative is shown as formula I:
Figure GDA0001676989820000032
wherein the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is 1: 1; the eluent of the semi-preparative reverse phase high performance liquid chromatography is prepared by mixing acetonitrile and water according to the volume ratio of 28: 72.
The compound I is purple red powder with a molecular formula C14H18O5，
Figure GDA0001676989820000033
(c 0.2,CHCl3) Cation HRESIMS m/z: 284.1490[ M + NH4]+，1H and13the C-NMR data are shown in Table 1.
TABLE 1 preparation of compound I1H and13c NMR data (600 and 150MHz, in DMSO-d)6)a
Figure GDA0001676989820000041
Note: the signal attribution of the table is based on DEPT,1H-1H COSY, HSQC and HMBC map analysis results. The hydrogen signals are used for multiple degrees respectivelys (singlet), d (doublet), t (triplet) and m (multiplet) tables.
Example 3
In vitro antioxidant Activity test (free radical scavenging Activity test)
(1) Experimental sample
Preparing a solution of a sample to be detected: the test sample is the pure compound I separated and purified in the above example 2, and an appropriate amount of sample is precisely weighed for measuring the activity.
(2) Experimental methods
The antioxidant properties of compound i were determined by DPPH free radical scavenging assay. The drug was added at various concentrations to a solution of DPPH in methanol (0.2 mM). The mixture (200. mu.L/well) was added to a 96-well plate and shaken at room temperature for 20 minutes. The absorbance was measured at 517nm with a microplate reader. Vitamin C was used as a positive control. DPPH radical scavenging activity was calculated using the formula: DPPH radical scavenging activity (%) (absorbance of control-absorbance of sample)/absorbance of control ] × 100.
(3) Results of the experiment
In the free radical scavenging assay of compound i. Compound I or vitamin C (VitC) was added to a 0.2mM DPPH solution at the indicated concentration for 20 minutes, and the DPPH radical content was measured by evaluating the absorbance at 517 nm. As shown in figure 1, compound i concentration-dependently decreased DPPH free radical content, indicating that compound i may act as an effective antioxidant, and the data represent the mean ± SEM of three separate experiments, p <0.01 relative to control (ANOVA and Duncet test). The potent antioxidant effect of compound i may be due to its hydroxyl group. Many antioxidants, such as vitamin C and vitamin E, may help delay the onset of Alzheimer's Disease (AD) and help alleviate its symptoms. Therefore, the compound I can be used as an isocoumarin derivative with strong free radical scavenging performance and can also be used for treating AD.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions are within the true spirit of the invention.

Claims (1)

1. A preparation method of isocoumarin derivatives is characterized by comprising the following steps:
(1) fermentation production: marking off Aspergillus niger (Aspergillus ustus) with the preservation number of CCTCC No. M2014086 for rejuvenation, selecting a single colony, inoculating into a PDB liquid culture medium, and culturing at 28 ℃ and 150rpm for 5 days to obtain a seed solution; inoculating the seed liquid into a rice culture medium according to the volume ratio of 5-20%, standing and fermenting at 28 deg.C for 30 days;
(2) obtaining a crude extract: adding methanol into the conical flask after fermentation, immersing the solid, performing ultrasonic treatment for 15 minutes, standing overnight, and filtering the methanol extract with gauze to remove solid residues; leaching the solid residue for 3-5 times, mixing the methanol extractive solutions, evaporating to remove methanol, dissolving with water, adding ethyl acetate with the same volume as the water solution, extracting for 5 times, mixing extractive solutions, and concentrating under reduced pressure to remove ethyl acetate to obtain crude extract;
(3) separation and purification of compounds: dissolving the crude extract by using a mixed solvent of dichloromethane and methanol, adding 200-mesh 300-mesh silica gel for sample mixing, performing reduced pressure silica gel column chromatography on the crude extract by using petroleum ether/ethyl acetate with a volume ratio of 2:1 as an eluent, and collecting an elution component; performing gel column chromatography on the eluted components by using a mixed solvent of dichloromethane and methanol as an eluent, and collecting the eluted components; separating the eluted components by reverse phase medium pressure column chromatography, adopting methanol and water as eluent, linearly eluting the components from 30% methanol in volume fraction to 100% methanol in gradient, eluting for 120 minutes at a flow rate of 20mL/min, collecting the fraction with peak time of 80-90 minutes, and finally separating and purifying the fraction by semi-preparative reverse phase high performance liquid chromatography to obtain the isocoumarin derivative, wherein the structure of the isocoumarin derivative is shown as formula I:
Figure FDA0002965170920000011
the formula of the PDB liquid culture medium is as follows: 200g of potatoes, 20g of glucose, 35g of sea crystals and 1L of distilled water; the formula of the rice culture medium is as follows: the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is 1: 1, the eluent of the semi-preparative reverse phase high performance liquid chromatography is prepared by mixing acetonitrile and water according to the volume ratio of 28: 72.
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CN110483467B (en) * 2019-07-11 2023-01-03 宁波大学 Dihydroisomerin derivative and preparation method and application thereof
CN111592989B (en) * 2020-06-28 2021-09-17 曲靖师范学院 Aspergillus sp.G12 and method for preparing isocorydine alkali by fermenting Aspergillus sp.G12
CN113061631B (en) * 2021-03-31 2022-09-16 华中科技大学 Isochroman derivative and enzymatic synthesis method and application thereof
CN118165005A (en) * 2021-11-26 2024-06-11 北京中医药大学东直门医院 Application of dihydrothiophene hybrid isocoumarin compound with circular polarized fluorescence in preparation of anti-counterfeiting material or cell imaging material
CN116650475A (en) * 2023-04-24 2023-08-29 福建中医药大学 Application of isocoumarin derivative diaorthothin in preparation of medicine with neuroprotective function

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