CN113248369A - Preparation and application of polyketide compound with anti-new coronavirus activity - Google Patents
Preparation and application of polyketide compound with anti-new coronavirus activity Download PDFInfo
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Abstract
The invention discloses a preparation method and application of polyketone compounds with anti-new coronavirus activity, and belongs to the field of compound synthesis. The invention provides a solid fermentation of Alternaria sp, an endophytic fungus of Alternaria, and a series of pure separation methodsThe polyketide monomer compound with a novel structure is obtained from the fermentation product by the chemical method. The structure is shown as formula (I):
Description
Technical Field
The invention belongs to the field of compound synthesis, and particularly relates to preparation and application of polyketone compounds.
Background
The endophyte of the medicinal plants has diversity characteristics, each endophyte has abundant secondary metabolites, and a plurality of alkaloids, polypeptides, polyketides, terpenoids and other substances with biological activity are found from the endophyte secondary metabolites at present, and the secondary metabolites not only have the same or similar biological activity as the host plants, such as antibacterial, anti-inflammatory, anti-tumor, anti-hyperglycemia, anti-parasite and the like, but also have a plurality of new biological activities. Therefore, the secondary metabolite of the endophyte of the medicinal plant is a huge potential medicinal substance resource library and has good development prospect.
Disclosure of Invention
In order to make up for the blank of the prior art, the invention aims to provide a polyketide compound and an active application thereof. The invention relates to a compound which is separated from a solid fermentation product of endophyte of plant origin and has inhibitory activity to SARS-CoV-2 virus and a novel structure and application thereof. Specifically, the Alternaria sp, endophytic fungi of Alternaria, is subjected to solid fermentation, and a polyketide monomer compound with a novel structure is obtained from a fermentation product by using a series of separation and purification methods. The structure of the compound is determined by adopting technologies such as mass spectrum, nuclear magnetic resonance and the like, and the structure is as follows:
the structure is shown as formula (I):
the invention also claims a preparation method of the polyketone compound.
(1) Fermenting strains: solid fermentation of Alternaria endophytic fungus Alternaria sp with rice and purified water.
(2) Extraction and separation of metabolites: carrying out ultrasonic extraction on a solid fermentation product of endophytic fungi Alternaria sp by using methanol with the same volume, filtering by 8 layers of gauze, separating an extracting solution from mycelium and rice, concentrating the extracting solution to be dry, carrying out gradient elution on the extract by using medium-pressure column chromatography, using 300-plus 400-mesh silica gel as a stationary phase and chloroform/methanol as a mobile phase, and sequentially carrying out gradient elution on chloroform by volume: methanol 100:0, 100:1, 100: 2. 100, and (2) a step of: 3. 100, and (2) a step of: 5. 100, and (2) a step of: 10. 0: 100, eluent flow rate of 30ml/min, collecting eluent (every 500ml collection), suspending the eluent, and combining every five parts of eluent to obtain a component 1-11; passing the component 4 through a silica gel column, taking 200-300-mesh silica gel as a stationary phase, taking petroleum ether/ethyl acetate as a mobile phase, and sequentially taking petroleum ether as a gradient by volume: ethyl acetate 10: 1. 10: 3. 10: 5. 10: 6. 10: 7. 10: 9, collecting eluent (every 500 ml) at eluent flow rate of 30ml/min, and mixing with every five parts of eluent to obtain components 4-1 to 4-16; putting the components 4-11 on a Sephadex LH-20 gel column, wherein the mobile phase is dichloromethane/methanol 1: 1, eluent flow rate is 0.3ml/min, eluent is collected (every 10ml is collected), and every four parts of eluent are combined to obtain components 4-11-1 to 4-11-6; the components 4-11-3 are prepared by high performance liquid chromatography and 48% methanol water to obtain monomer compound, and the retention time is 11 min.
(3) And (3) structural identification: and (3) taking deuterated dimethyl sulfoxide as a solvent, measuring the nuclear magnetic spectrum of the separated compound sample by using a Bruker Avance II 500M nuclear magnetic resonance instrument, deducing the molecular formula by using mass spectrum, and finally characterizing the structure of the product.
Further, the fermentation conditions of the step (1) are as follows: carrying out shake culture on the bacterial strain Alternaria sp by adopting a No. 4 fungal culture medium under the culture condition of 28 ℃ and 180r/min, inoculating fermentation liquor and mycelium subjected to shake culture into a conical flask filled with a rice solid culture medium, and carrying out static fermentation culture for 40 days at the culture temperature of 28 ℃.
Further, the fungus No. 4 culture medium comprises the following components in percentage by mass: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH2PO4，0.03％MgSO4·7H2O, 0.1 percent of corn steep liquor and deionized water.
Further, the solid nutrient of the rice is as follows: adding 80g of rice into 110ml of deionized water, and filling into a conical flask; each flask of rice medium was inoculated with 20ml of seed solution.
The invention also aims to protect the application of the polyketide compound, and the polyketide compound has inhibitory activity on SARS-CoV-2 virus and can be used for preparing medicines for inhibiting SARS-CoV-2 virus.
Has the advantages that: the invention fully utilizes plant endophyte resources, researches the plant endophytes, searches for new polyketone active compounds, performs an anti-new coronavirus activity experiment on the polyketone active compounds, and takes host-oriented medicine clofazimine which is approved by FDA and has anti-SARS-CoV-2 virus activity as a control group to explore the antiviral activity of the compounds. Experimental results show that the SI index of clofazimine is 1.5, the compound SI index separated by the method is 3, the inhibition rate of the compound on viruses is higher than that of a contrast medicament, the compound has the activity of inhibiting novel coronavirus, and a new lead compound is searched for medicament development.
Drawings
FIG. 1 is a chart of the NMR spectra of example 1.
FIG. 2 is the carbon-13 NMR spectrum of example 1.
FIG. 3 is the HSQC spectrum of example 1.
FIG. 4 is the HMBC spectrum of example 1.
Detailed Description
The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources. Alternaria endophytic fungus Alternaria sp is a known strain in the art, and for illustration, the Alternaria endophytic fungus Alternaria sp collected from the leaf parts of Rhodiola tibetica of Rhodiola sachalinensis is exemplified in the following examples.
Example 1
(1) Fermenting strains: carrying out shake culture on the bacterial strain Alternaria sp by adopting a No. 4 fungal culture medium under the culture condition of 28 ℃ and 180 r/min; the fungus No. 4 culture medium comprises: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH2PO4，0.03％MgSO4·7H2O, 0.1 percent of corn steep liquor and deionized water.
Then inoculating the fermentation liquor and mycelium obtained by shake culture into conical flasks filled with rice solid culture medium (formula of rice solid culture medium: 80g rice and 110ml deionized water), inoculating 20ml seed solution into each flask of rice culture medium, and standing for fermentation culture for 40 days at 28 ℃.
(2) Extraction and separation of metabolites: carrying out ultrasonic extraction on a solid fermentation product of endophytic fungi Alternaria sp by using methanol with the same volume, filtering by 8 layers of gauze, separating an extracting solution from mycelium and rice, concentrating the extracting solution to be dry, carrying out gradient elution on the extract by using medium-pressure column chromatography, using 300-plus 400-mesh silica gel as a stationary phase and chloroform/methanol as a mobile phase, and sequentially carrying out gradient elution on chloroform by volume: methanol 100:0, 100:1, 100: 2. 100, and (2) a step of: 3. 100, and (2) a step of: 5. 100, and (2) a step of: 10. 0: 100, eluent flow rate of 30ml/min, collecting eluent (every 500 ml); suspending the eluate, and mixing each five eluents to obtain component 1-11; passing the component 4 through a silica gel column, taking 200-sand 300-mesh silica gel as a stationary phase, taking petroleum ether/ethyl acetate as a mobile phase, and sequentially taking petroleum ether as a gradient by volume: ethyl acetate 10: 1. 10: 3. 10: 5. 10: 6. 10: 7. 10: 9, collecting eluent (every 500 ml) at eluent flow rate of 30ml/min, and mixing with every five parts of eluent to obtain components 4-1 to 4-16; putting the components 4-11 on a Sephadex LH-20 gel column, wherein the mobile phase is dichloromethane/methanol 1: 1, eluent flow rate is 0.3ml/min, eluent is collected (every 10ml is collected), and every four parts of eluent are combined to obtain components 4-11-1 to 4-11-6; the components 4-11-3 are prepared by high performance liquid chromatography and 48% methanol water to obtain monomer compound, and the retention time is 11 min.
(3) And (3) structural identification: and (3) taking deuterated dimethyl sulfoxide as a solvent, measuring the nuclear magnetic spectrum of the separated compound sample by using a Bruker Avance II 500M nuclear magnetic resonance instrument, deducing the molecular formula by using mass spectrum, and finally characterizing the structure of the product.
The spectral data are as follows:1H NMR(500Hz，DMSO-d6)δ：9.42(1H,brs,3-OH),6.68(1H,d,J＝8.0Hz,H-4), 7.04(1H,t,J＝8.0Hz,H-5),6.65(1H,d,J＝8.0Hz,H-6),2.57(1H,t,J＝8.0Hz,H-2’), 12.1(1H,brs,3-OH’)。13C NMR(125Hz,DMSO-d6)δ：142.9(C-1),122.6(C-2), 156.4(C-3),113.6(C-4),129.2(C-5),120.1(C-6),
64.9(C-7),57.7(C-8),27.9(C-1’),35.9(C-2’),174.5(C-3’)。
its HMBC is related as follows:
(2) the compound was dissolved, adjusted to a concentration of 10mg/mL, diluted 30-fold in the initial gradient and 3-fold later in 7-fold serial gradients. Culturing in 96-well cell plate, adding DMEM complete culture medium 100 μ l/well into virus control group well, adding DMEM complete culture medium 150 μ l/well into cell control group well, adding 142.5 μ l/well into sample to be tested, and adding culture medium 100 μ l/well into the rest wells. 7.5 mul of the sample to be tested is added into the sample hole to be tested. And (3) gently and repeatedly blowing and sucking the liquid in the sample hole to be detected for 6-8 times, then transferring 50 mu l of liquid to the corresponding following holes, and diluting all the holes by 3 times. SARS-CoV-2 pseudovirus (spike-D614G) was diluted to 1.3X 10 with DMEM complete medium4TCID50/mL, 50. mu.l per well. The 96-well plate was placed in a cell incubator (37 ℃ C., 5% CO)2) Incubation for 1 hour, after 30min incubation, digestion of Huh7 cells was started, and the cell concentration was diluted to 2 × 105cells/mL. After incubation, 100. mu.l of cells were added to each well to give 2X 10 cells per well4cells, at 37 ℃ with 5% CO2Culturing for 24 hours in a cell culture box, removing 150 μ l of supernatant after the culture is finished, adding 100 μ l of Bright-GloTM luciferase detection reagent, reacting for 2min at room temperature in a dark place, repeatedly blowing, and transferring 150 μ l of liquid to a white board. Use ofThe luminescence values (RLU) were read by a PerkinElmer EnSight multifunctional imaging microplate reader. Calculating drug EC by using luminescence value50The value is obtained. CC (challenge collapsar)50The experimental procedure was as above except that no pseudovirus was added to each well, in order to see the effect of the drug on the cell activity.
(3) The above method is repeated, and the inhibitory activity of the compound on SARS-CoV-2 pseudovirus is compared and judged by taking FDA approved host-oriented medicine clofazimine with anti-SARS-CoV-2 virus activity as a control group.
Table 1 results of activity experiments:
| Average EC50(ug/ml) | CC50(ug/ml) | SI(SI＝CC50/average(EC50) | |
| clofazimine | 4 | 6 | 1.5 |
| The compound | 52 | 157 | 3.0 |
Note: SI >3 can be used as medicine
The experimental result shows that the compound has inhibitory activity to SARS-CoV-2 virus, especially has higher inhibitory rate to virus than clofazimine, and can be used for preparing medicine for inhibiting SARS-CoV-2 virus.
The above description is only for the purpose of creating a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
Claims (7)
1. A polyketide compound with anti-new coronavirus activity, which is characterized by having a structure shown as a formula (I):
2. the process for preparing polyketides according to claim 1, comprising the steps of:
(1) fermenting strains: subjecting Alternaria sp to solid fermentation with rice and purified water;
(2) extraction and separation of metabolites: carrying out ultrasonic extraction on a plant endophytic fungus Alternaria sp solid fermentation product by using methanol with the same volume, filtering by using 8 layers of gauze, separating an extracting solution from mycelium and rice, concentrating the extracting solution to be dry, carrying out gradient elution by using medium-pressure column chromatography, using 300-mesh 400-mesh silica gel as a stationary phase and chloroform/methanol as a mobile phase, wherein the gradient is chloroform: methanol 100:0, 100:1, 100: 2. 100, and (2) a step of: 3. 100, and (2) a step of: 5. 100, and (2) a step of: 10. 0: 100, eluent flow rate of 30ml/min, collecting eluent every 500ml, suspending the eluent, and combining every five parts of eluent to obtain a component 1-11; passing the component 4 through a silica gel column, taking 200-300-mesh silica gel as a stationary phase, taking petroleum ether/ethyl acetate as a mobile phase, and sequentially taking petroleum ether as a gradient by volume: ethyl acetate 10: 1. 10: 3. 10: 5. 10: 6. 10: 7. 10: 9, collecting eluent every 500ml at eluent flow rate of 30ml/min, and mixing the eluent with every five parts of eluent to obtain components 4-1 to 4-16; putting the components 4-11 on a Sephadex LH-20 gel column, wherein the mobile phase is dichloromethane/methanol 1: 1, collecting eluent every 10ml at eluent flow rate of 0.3ml/min, and combining every four parts of eluent to obtain components 4-11-1 to 4-11-6; the components 4-11-3 are prepared by high performance liquid chromatography and 48% methanol water to obtain monomer compound, and the retention time is 11 min.
3. The process for producing polyketides according to claim 2, further comprising a structural identification step (3):
and (3) measuring the nuclear magnetic spectrum of the separated compound sample by using a Bruker Avance II 500M nuclear magnetic resonance instrument by using deuterated dimethyl sulfoxide as a solvent, deducing the molecular formula by using mass spectrum, and finally characterizing the structure of the product.
4. The process for preparing polyketides according to claim 2, wherein the fermentation conditions in step (1): carrying out shake culture on the bacterial strain Alternaria sp by adopting a No. 4 fungal culture medium under the culture condition of 28 ℃ and 180r/min, inoculating fermentation liquor and mycelium subjected to shake culture into a conical flask filled with a rice solid culture medium, and carrying out static fermentation culture for 40 days at the culture temperature of 28 ℃.
5. The method for preparing polyketides as claimed in claim 4, wherein the fungal No. 4 medium consists of, in mass percent: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH2PO4，0.03％MgSO4·7H2O, 0.1 percent of corn steep liquor and deionized water.
6. The method for preparing polyketides according to claim 4, wherein the rice solid medium is: adding 80g of rice into 110ml of deionized water, and filling into a conical flask; each flask of rice medium was inoculated with 20ml of seed solution.
7. Use of a polyketide according to claim 1 in the manufacture of a medicament for inhibiting SARS-CoV-2 virus.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110592143.6A CN113248369A (en) | 2021-05-28 | 2021-05-28 | Preparation and application of polyketide compound with anti-new coronavirus activity |
| PCT/CN2022/091616 WO2022247618A2 (en) | 2021-05-28 | 2022-05-09 | Preparation of polyketone compound having anti-novel coronavirus activity, and use thereof |
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| CN202110592143.6A CN113248369A (en) | 2021-05-28 | 2021-05-28 | Preparation and application of polyketide compound with anti-new coronavirus activity |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023240679A1 (en) * | 2022-06-13 | 2023-12-21 | 大连大学 | Pair of polyketide compounds having anti-inflammatory activity, preparation method therefor, and use thereof |
| WO2023240678A1 (en) * | 2022-06-13 | 2023-12-21 | 大连大学 | Polyketone compound with anti-inflammatory activity, and preparation method therefor and use thereof |
-
2021
- 2021-05-28 CN CN202110592143.6A patent/CN113248369A/en active Pending
-
2022
- 2022-05-09 WO PCT/CN2022/091616 patent/WO2022247618A2/en unknown
Non-Patent Citations (4)
| Title |
|---|
| XUAN LU 等: "Polyketone metabolites isolated from Rhodiola tibetica endohytic fungus Alternaria sp. HJT-Y7 and their SARS-CoV-2 virus inhibitory activitives", 《BIOORGANIC CHEMISTRY》 * |
| XUAN LU 等: "Quinones from endophytic fungus Fusarium sp. HJT-P-5 of Rhodiola angusta Nakai", 《PHYTOCHEMISTRY LETTERS》 * |
| 卢轩 等: "长白红景天内生微生物Fusarium sp. HJT-P-5次级代谢产物的研究", 《中国化学会第30届学术年会摘要集-第九分会：有机化学》 * |
| 魏磊 等: "长白红景天内生真菌Fusarium sp. HJT-P-5次级代谢产物研究", 《沈阳药科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023240679A1 (en) * | 2022-06-13 | 2023-12-21 | 大连大学 | Pair of polyketide compounds having anti-inflammatory activity, preparation method therefor, and use thereof |
| WO2023240678A1 (en) * | 2022-06-13 | 2023-12-21 | 大连大学 | Polyketone compound with anti-inflammatory activity, and preparation method therefor and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2022247618A2 (en) | 2022-12-01 |
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Application publication date: 20210813 |