CN108892658B - Compound lithocarpinol B, preparation method thereof and application thereof in preparation of antifungal drugs - Google Patents
Compound lithocarpinol B, preparation method thereof and application thereof in preparation of antifungal drugs Download PDFInfo
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- C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
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Abstract
The invention discloses a compound litocarpinol B, a preparation method thereof and application thereof in preparing antifungal medicines. The invention obtains 1 new compound of 2,3-dihydro-1H-indene with antifungal activity, named as litocarpinol B, through the fermentation culture of deep sea fungus Phomopsis lithocarpus FS508, the separation and purification of the fermentation product and the structural identification. The compound has good inhibition activity on aspergillus flavus, and can be used for preparing antifungal medicaments. Therefore, the invention provides a candidate compound for the research and development of new antifungal medicines and provides a scientific basis for the development and utilization of marine fungus resources.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a compound lithocarpinol B, a preparation method thereof and application thereof in preparation of antifungal medicines.
Background
Aspergillus flavus, a common saprophytic fungus, is commonly found in mildewed grains, grain products and other mildewed organic matters, and the generated toxin is classified as a class 1 carcinogen by cancer research institutions of the World Health Organization (WHO), so that the Aspergillus flavus is a highly toxic substance. Aflatoxin B1 is the most common toxin produced by aspergillus flavus, has the strongest toxicity and carcinogenicity, has destructive effect on human and animal liver tissues, and can cause liver cancer and even death in severe cases. Therefore, the search for a novel anti-aspergillus flavus medicament is of great significance.
Oceans account for about 71% of the surface area of the earth, contain rich microbial resources, special marine environments (low temperature, high pressure, high salt, high cold, oligotrophy and the like) and ecological effects among marine species, and endow marine microorganisms with special products different from land microorganisms, including active substances such as antibacterial substances, anti-plant pathogenic bacteria substances, anti-tumor substances, anti-virus substances and the like. The secondary metabolites of marine microorganisms have a unique structural pattern and a rich variety of biological activities. These new chemical entities not only reveal new biosynthetic pathways, but also inspire biomimetic synthesis and bioactivity studies of these chemical entities. Therefore, the secondary metabolites of the marine microorganisms have important scientific research and application values and are important resources for drug development.
The deep sea fungus Phomopsis lithocarpus FS508 was isolated in 2016 5 months from surface sediments on the seafloor from the Indian ocean (16 ° 50.508'N,111 ° 53.335' E, water depth 3606 meters). The literature review shows that up to now there are no relevant studies reported on the antifungal activity of the metabolite Phomopsis lithocarpus FS 508.
Disclosure of Invention
It is a first object of the present invention to provide a novel compound, lithocarpinol B, having inhibitory activity against fungi.
The structure of the compound lithocarpinol B is shown as the formula (I):
the second purpose of the invention is to provide a preparation method of a compound litocarpinol B, wherein the compound litocarpinol B is separated and prepared from a fermentation culture of deep sea fungus Phomopsis litocarpus FS508, and the preparation method specifically comprises the following steps:
(1) preparing a fermentation culture of Phomopsis lithocarpus FS508, extracting the fermentation culture by using ethyl acetate, and distilling and concentrating an ethyl acetate extract to obtain an extract; dissolving the extract with methanol water solution (volume fraction of 85%), extracting with petroleum ether to remove low-polarity fatty acid substances, and distilling and concentrating the residual methanol water solution to obtain brownish black extract;
(2) passing the brownish black extract through a normal phase silica gel column, eluting with petroleum ether-ethyl acetate gradient from 10:1,7:1,9:2,3:1,2:1,1:1,1:2v/v, then eluting with ethyl acetate, and finally eluting with dichloromethane: methanol-5: 1,1:1v/v gradient elution; collecting extractum by petroleum ether: fraction F6 eluted at a ethyl acetate volume ratio of 3:1, fraction F6 was subjected to Sephadex LH-20 column, purified with dichloromethane: methanol 1:1v/v as eluent, TLC thin layer chromatography was collected with petroleum ether: developing with ethyl acetate at 2:1v/v to obtain a component with Rf at 3.0/3.8, performing normal phase silica gel column chromatography, eluting with petroleum ether-ethyl acetate gradient from 6:1,3:1,2:1,1:1,1:2,0:1v/v, collecting TLC, performing thin layer chromatography with dichloromethane: developing with methanol at 20:1v/v to obtain a component with Rf at 3.3/3.6, and separating the component by using full preparative HPLC, wherein the chromatographic conditions of the full preparative HPLC are as follows: YMC ODS-A column with mobile phase of methanol/water at volume ratio of 85:15, flow rate of 8mL/min, collecting eluate fraction at retention time of 14.8min, and purifying the fraction by semi-preparative HPLC with chromatographic conditions: YMC-pack ODS-A/AQ column with mobile phase of acetonitrile/water in volume ratio of 82:18, flow rate of 3mL/min, retention time of 9.4min to obtain compound litocarpinol B.
The fermentation culture for preparing the Phomopsis lithocarpus FS508 is prepared by inoculating activated Phomopsis lithocarpus FS508 strain into a potato glucose liquid culture medium, culturing at 28 ℃,120 rpm for 5d to prepare a seed solution, inoculating the seed solution into a rice solid fermentation culture medium by an inoculation amount of 10% mL/g, and standing and culturing at room temperature for one month to prepare the fermentation culture.
The potato dextrose broth is obtained by: cleaning potato, peeling, and cutting into pieces of about 1cm3Weighing 200g of the small blocks, putting the small blocks into 1L of water, boiling for 20-30 minutes, filtering by using double-layer gauze, taking filtrate, adding 15g of sea salt into the filtrate,Adding glucose 20g, adding water to make up 1L, and sterilizing.
The rice solid fermentation medium is obtained by the following steps: respectively weighing 3g of sea salt and 160g of rice, adding 200mL of water, stirring, mixing, and sterilizing.
The third object of the present invention is to provide the use of the deep sea fungus Phomopsis lithocarpus FS508 for the preparation of the compound lithocarpinol B.
According to the invention, antifungal activity tests show that the compound lithocarpinol B has obvious inhibition activity on Aspergillus flavus, the MIC value on the Aspergillus flavus is 20 mug/mL, and the MIC value on the Aspergillus flavus of a positive control drug nystatin is 15 mug/mL. The results show that: the compound lithocarpinol B has obvious inhibition activity on aspergillus flavus, and can be used for preparing antifungal medicaments.
The fourth object of the present invention is to provide the use of the compound litocarpinol B in the preparation of an antifungal agent, preferably an anti-Aspergillus flavus agent.
A fifth object of the present invention is to provide an antifungal agent comprising the compound litocarpinol B as an active ingredient; the antifungal medicine is preferably an anti-aspergillus flavus medicine.
Compared with the prior art, the invention has the advantages that:
the invention obtains 1 new compound of 2,3-dihydro-1H-indene with antifungal activity, named as litocarpinol B, through the fermentation culture of deep sea fungus Phomopsis lithocarpus FS508, the separation and purification of the fermentation product and the structural identification. The compound has good inhibition activity on aspergillus flavus, and can be used for preparing antifungal medicaments. Therefore, the invention provides a candidate compound for the research and development of new antifungal medicines and provides a scientific basis for the development and utilization of marine fungus resources.
The deep sea fungus Phomopsis lithocarpus FS508 of the present invention was deposited in Guangdong province culture Collection (GDMCC) at 8.8.15.2018, with the following addresses: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC NO: 60433.
drawings
FIG. 1 is a scheme of Compound 1 (litocarpinol B)1H-NMR spectrum.
FIG. 2 is a scheme of Compound 1 (litocarpinol B)13C-NMR spectrum.
FIG. 3 is the HSQC spectrum of Compound 1 (lithacarpinol B).
FIG. 4 shows the preparation of Compound 1 (lithacarpinol B)1H-1H COSY spectra.
FIG. 5 is an HMBC spectrum of compound 1(lithocarpinol B).
FIG. 6 is a NOESY spectrum of Compound 1(lithocarpinol B).
FIG. 7 is a HRESIMS spectrum of Compound 1(lithocarpinol B).
FIG. 8 is a CD spectrum of Compound 1 (litocarpinol B).
FIG. 9 is an ECD fit curve of Compound 1(lithocarpinol B).
FIG. 10 shows the UV spectrum of Compound 1(lithocarpinol B).
FIG. 11 is an IR spectrum of Compound 1 (litocarpinol B).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
(1) Inoculating activated deep sea fungus Phomopsis lithocarpus FS508 into potato glucose liquid culture medium (prepared by washing potato, peeling, and cutting into pieces with size of about 1 cm)3Weighing 200g of the small blocks, putting the small blocks into 1L of water, boiling for 20-30 minutes, filtering the small blocks by using double-layer gauze, taking filtrate, adding 15g of sea salt and 20g of glucose into the filtrate, adding water to complement 1L of the filtrate, sterilizing the filtrate, culturing the filtrate for 5 days at 28 ℃ and 120rpm to obtain seed liquid, and inoculating the seed liquid into a rice solid fermentation culture medium in an inoculation amount of 10% mL/g (the preparation method is: respectively weighing 3g of sea salt and 160g of rice, adding 200mL of water, stirring, mixing, sterilizing), and standing at room temperature for one month to obtain a fermentation culture of Phomopsis lithocarpus FS 508.
(2) Extracting the fermentation culture obtained in the step (1) with ethyl acetate for 4 times, distilling and concentrating ethyl acetate extract to obtain an extract, dissolving the extract with a proper amount of 85% methanol aqueous solution by volume fraction, extracting with petroleum ether for three times to remove low-polarity fatty acid substances, and distilling and concentrating the remaining methanol aqueous solution to obtain a brownish black extract.
(3) Passing the brownish black extract obtained in the step (2) through a normal phase silica gel column, eluting with petroleum ether-ethyl acetate in a gradient from 10:1,7:1,9:2,3:1,2:1,1:1,1:2v/v, then eluting with ethyl acetate, and finally eluting with dichloromethane: methanol 5:1,1:1v/v elution; collecting extractum by petroleum ether: fraction F6 eluted at a ethyl acetate volume ratio of 3:1, fraction F6 was subjected to Sephadex LH-20 column, purified with dichloromethane: methanol 1:1v/v as eluent, TLC thin layer chromatography was collected with petroleum ether: developing with ethyl acetate at 2:1v/v to obtain a component with Rf at 3.0/3.8, performing normal phase silica gel column chromatography, eluting with petroleum ether-ethyl acetate gradient from 6:1,3:1,2:1,1:1,1:2,0:1v/v, collecting TLC, performing thin layer chromatography with dichloromethane: the fraction having Rf of 3.3/3.6 developed with methanol of 20:1v/v was separated by preparative HPLC (chromatographic conditions for preparative HPLC: YMC ODS-A column, mobile phase of methanol/water of 85:15 volume ratio, flow rate of 8mL/min), and the fraction was collected at retention time of 14.8min and finally purified by semi-preparative HPLC (chromatographic conditions for semi-preparative HPLC: YMC-pack ODS-A/AQ column, mobile phase of acetonitrile/water of 82:18 volume ratio, flow rate of 3mL/min, retention time of 9.4min) to obtain Compound 1 (Compound litocarpinol B).
The compound 1 is analyzed by mass spectrum and nuclear magnetic resonance spectrum, and the structure is identified as follows:
as shown in FIGS. 1-11, FIG. 1 is for Compound 11H-NMR spectrum; FIG. 2 is a drawing of Compound 113C-NMR spectrum; FIG. 3 is the HSQC spectrum of Compound 1; FIG. 4 is a drawing of Compound 11H-1H COSY spectrum; FIG. 5 is an HMBC spectrum of compound 1; FIG. 6 is a NOESY spectrum of Compound 1; FIG. 7 is a HRESIMS spectrum of Compound 1; FIG. 8 is a CD spectrum of Compound 1; figure 9 is an ECD fit curve for compound 1; FIG. 10 is a UV spectrum of Compound 1; FIG. 11 is an IR spectrum of a compound.
1h and13c NMR data; HRESIMS M/z [ M + Na ]]+447.1778(calcd for C25H28NaO6,447.1778)。1H-NMR(500MHz,CDCl3):9.82(1H,s,H-13),7.54(1H,d,J＝8.4Hz,H-5),7.07(1H,s,H-6'),6.87(1H,d,J＝8.4Hz,H-4),6.68(1H,d,J＝1.8Hz,H-4'),4.91(1H,s,H-11b),4.76(1H,s,H-11a),4.41(1H,dd,J＝11.0,2.0Hz,H-8a'),3.79(1H,dd,J＝9.3,2.0Hz,H-9'),3.70(1H,m,H-8b'),3.59(1H,m,H-8),3.23(1H,dd,J＝15.3,9.3Hz,H-7a),2.94(1H,dd,J＝15.3,7.8Hz,H-7b),2.24(1H,s,H-7'),1.62(3H,s,H-10),0.99(3H,s,H-11'),0.75(3H,s,H-12')。13C-NMR(125MHz,CDCl3) 196.6(C-13),162.5(C-3),151.3(C-1),144.5(C-3'),144.2(C-9),138.4(C-2'),135.7(C-1'),134.7(C-6),134.4(C-5),130.7(C-5'),120.3(C-6'),118.3(C-4),117.7(C-4'),117.1(C-2),114.4(C-11),84.5(C-12),80.2(C-9'),70.3(C-10'),65.4(C-8'),57.8(C-8),34.5(C-7),27.5(C-11'),23.9(C-12'),23.5(C-10),21.1 (C-7'). The CD spectral data show that the compound showed a positive cotton effect at 277nm and negative cotton effects at 214, 229, 246nm, consistent with the calculated ECD-fitted curve for 8S,12R,9' S (figure 9). Thus, the absolute configuration of compound 1 can be determined to be 8S,12R,9' S. Through literature search, the compound 1 is a new skeleton compound, belongs to a new compound of 2,3-dihydro-1H-indene class, is named as litocarpinol B, and has a specific structure shown in a formula I.
The isolated compound lithocarpinol B has the structural formula shown in the following formula (I):
example 2
The antibacterial activity of the compound litocarpinol B against Aspergillus flavus was determined by a filter paper sheet method.
Taking Aspergillus flavus in logarithmic growth phaseThe normal saline is added until the number of spores is 1.0 × 106The compound litocarpinol B and the positive control nystatin were diluted to concentrations of 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL, and 100. mu.g/mL, respectively, in a spore suspension. Taking 1mL of spore suspension liquid into a culture dish, pouring a PDA culture medium, uniformly mixing, placing 6mm in a bacteria-containing culture dish, respectively dropwise adding 5 mu L of sample diluent on a filter paper sheet, dropwise adding 5 mu L of DMSO on a blank control, taking nystatin as a positive control, placing a flat plate in a thermostat at 26 ℃ for culturing for 72h, observing the bacteriostatic zone around the filter paper sheet, and taking the lowest concentration of the contained sample with the bacteriostatic zone around the filter paper sheet as the minimum bacteriostatic concentration (MIC value).
The experimental results are as follows: the MIC value of the compound lithocarpinol B to aspergillus flavus is 20 mug/mL, and the MIC value of the positive control drug nystatin to aspergillus flavus is 15 mug/mL. The results show that: the compound lithocarpinol B has obvious inhibition activity on aspergillus flavus, and can be used for preparing antifungal medicaments. Therefore, the invention provides a candidate compound for the research and development of new antifungal medicines and provides a scientific basis for the development and utilization of marine fungus resources.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (7)
1. A compound lithocarpinol B, characterized by the structural formula (І):
formula (І).
2. A process for the preparation of the compound lithocarpinol B according to claim 1, characterized in that it comprises the following steps:
(1) preparation ofPhomopsis lithocarpusExtracting the fermentation culture of FS508 by using ethyl acetate, and distilling and concentrating ethyl acetate extract to obtain extract; dissolving the extract with methanol water solution, extracting with petroleum ether, and distilling and concentrating the residual methanol water solution to obtain brownish black extract;
(2) passing the brownish black extract through a normal phase silica gel column, performing gradient elution by using petroleum ether-ethyl acetate from 10:1,7:1,9:2,3:1,2:1,1:1,1:2v/v, collecting petroleum ether: fraction F6 eluted at a ethyl acetate volume ratio of 3:1, fraction F6 was subjected to Sephadex LH-20 column, purified with dichloromethane: methanol =1: 1v/v as eluent, collected TLC thin layer chromatography with petroleum ether: ethyl acetate =2: 1v/v to develop fractions with Rf =3.0/3.8, which are then subjected to normal phase silica gel column chromatography, eluting with a petroleum ether-ethyl acetate gradient from 6:1,3:1,2:1,1:1,1:2,0:1v/v, collecting TLC thin layer chromatography in dichloromethane: methanol =20:1v/v developed fractions Rf =3.3/3.6, which were purified by HPLC to give the compound litocarpinol B;
the HPLC purification is to separate the component by using full-preparative HPLC, and the chromatographic conditions of the full-preparative HPLC are as follows: YMC ODS-A column with mobile phase of methanol/water at volume ratio of 85:15, flow rate of 8mL/min, collecting eluate fraction at retention time of 14.8min, and purifying the fraction by semi-preparative HPLC with chromatographic conditions: YMC-pack ODS-A/AQ column, wherein the mobile phase is acetonitrile/water with volume ratio of 82:18, the flow rate is 3mL/min, and the retention time is 9.4min to obtain A compound litocarpinol B;
saidPhomopsis lithocarpusFS508 deposit number is: GDMCC NO: 60433.
3. the method of claim 2, wherein the preparing is carried out byPhomopsis lithocarpusThe fermentation culture of FS508 is to be activatedPhomopsis lithocarpusInoculating FS508 strain into potato glucose liquid culture medium, culturing at 28 deg.C and 120rpm for 5d to obtain seed solution, inoculating the seed solution into rice solid fermentation culture medium at an inoculum size of 10% mL/g, standing at room temperature for one month to obtain fermentation cultureThe potato dextrose broth is obtained by: cleaning potato, peeling, and cutting into pieces of about 1cm3Weighing 200g of the small blocks, putting the small blocks into 1L of water, boiling for 20-30 minutes, filtering by using double-layer gauze, taking filtrate, adding 15g of sea salt and 20g of glucose into the filtrate, adding water to supplement 1L of the filtrate, and sterilizing to obtain the sea salt cake; the rice solid fermentation medium is obtained by the following steps: respectively weighing 3g of sea salt and 160g of rice, adding 200mL of water, stirring, mixing, and sterilizing.
4. Deep sea fungusPhomopsis lithocarpusUse of FS508 in the preparation of the compound lithocarpinol B according to claim 1; saidPhomopsis lithocarpusFS508 deposit number is: GDMCC NO: 60433.
5. use of the compound lithocarpinol B according to claim 1 for the preparation of a medicament against aspergillus flavus.
6. An antifungal agent characterized by containing the compound litocarpinol B according to claim 1 as an active ingredient.
7. The antifungal agent of claim 6 wherein the antifungal agent is an anti-Aspergillus flavus agent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434783A (en) * | 2016-04-05 | 2017-02-22 | 广东工业大学 | Benzopyrone compound, benzopyrone compound preparation method and application of benzopyrone compound to preparation of antibacterial medicines |
| CN108277164A (en) * | 2017-12-28 | 2018-07-13 | 中山大学 | A kind of indene derivative in sea paint endogenetic fungus source and its application in preparing anti-inflammatory drug |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108277164A (en) * | 2017-12-28 | 2018-07-13 | 中山大学 | A kind of indene derivative in sea paint endogenetic fungus source and its application in preparing anti-inflammatory drug |
Non-Patent Citations (1)
| Title |
|---|
| Lithocarpins A–D: four tenellone-macrolide conjugated [4 + 2] hetero-adducts from the deepsea derived fungus Phomopsis lithocarpus FS508;Jianlin Xu等;《ORGANIC CHEMISTRY FRONTIERS》;20180402;第5卷;1792-1797 * |
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