CN108892658B - Compound lithocarpinol B and its preparation method and application in the preparation of antifungal drugs - Google Patents

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

Compound lithocarpinol B and its preparation method and in the preparation of antifungal drugs applications in

technical field

The invention belongs to the technical field of biomedicine, and particularly relates to a compound lithocarpinol B, a preparation method thereof, and an application in the preparation of antifungal drugs.

Background technique

Aspergillus flavus, a common saprophytic fungus, is mostly found on moldy grains, grain products and other moldy organic matter. A highly toxic substance. Aflatoxin B1 is the most common toxin produced by Aspergillus flavus, and its toxicity and carcinogenicity are also the strongest. Therefore, it is of great significance to find new anti-Aspergillus flavus drugs.

The ocean accounts for about 71% of the earth's surface area and contains abundant microbial resources. The special marine environment (low temperature, high pressure, high salinity, high cold, oligotrophy, etc.) and the ecological interaction between marine species endow marine microorganisms with different terrestrial microorganisms. Special products, including antibacterial, anti-plant pathogenic bacteria, anti-tumor, anti-virus and other active substances. The secondary metabolites of marine microorganisms have unique structural types and rich and diverse biological activities. These new chemical entities can not only reveal new biosynthetic pathways, but also inspire the study of biomimetic synthesis and bioactivity of these chemical entities. Therefore, marine microbial secondary metabolites have important scientific research and application value, and are important resources for drug development.

The deep-sea fungus Phomopsis lithocarpus FS508 was isolated from the bottom sediments of the Indian Ocean (16°50.508'N, 111°53.335'E, water depth 3606 m) in May 2016. Literature review shows that so far there is no relevant research report on the antifungal activity of Phomopsis lithocarpus FS508 metabolite.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a novel compound lithocarpinol B which has inhibitory activity against fungi.

The compound lithocarpinol B of the present invention, its structure is shown in formula (I):

The second object of the present invention is to provide a preparation method of a compound lithocarpinol B, the compound lithocarpinol B described in the preparation method is prepared from the fermentation culture of the deep-sea fungus Phomopsis lithocarpus FS508, which specifically includes the following steps:

(1) prepare the fermentation culture of Phomopsis lithocarpus FS508, extract the fermentation culture with ethyl acetate, and obtain the extract after the ethyl acetate extract is concentrated by distillation; dissolve the extract with methanol aqueous solution (volume fraction is 85%), Then extract with petroleum ether to remove low-polar fatty acid substances, and the remaining methanol aqueous solution is partially distilled and concentrated to obtain brown-black extract;

(2) Pass the brown-black extract through a normal phase silica gel column, first use petroleum ether-ethyl acetate from 10:1, 7:1, 9:2, 3:1, 2:1, 1:1, 1:2v /v gradient elution, then ethyl acetate, and finally dichloromethane: methanol = 5:1, 1:1 v/v gradient elution; the collected extract was washed with petroleum ether: ethyl acetate in a volume ratio of 3:1 The eluted fraction F6, fraction F6 was passed through a Sephadex LH-20 column, with dichloromethane: methanol = 1: 1 v/v as the eluent, collected by TLC thin layer chromatography with petroleum ether: ethyl acetate = 2: 1 v /v developed to obtain a component with Rf=3.0/3.8, which was then subjected to normal phase silica gel column chromatography, using petroleum ether-ethyl acetate from 6:1, 3:1, 2:1, 1:1, 1:2, 0:1 v/v gradient elution, collect TLC thin layer chromatography and develop with dichloromethane: methanol = 20: 1 v/v to obtain a fraction with Rf=3.3/3.6, and then separate this fraction by full preparative HPLC. The chromatographic conditions of HPLC were: YMC ODS-A column, the mobile phase was methanol/water with a volume ratio of 85:15, the flow rate was 8 mL/min, the eluted fractions were collected at the retention time of 14.8 min, and finally the group was purified by semi-preparative HPLC. The chromatographic conditions of semi-preparative HPLC were: YMC-pack ODS-A/AQ column, mobile phase was acetonitrile/water with a volume ratio of 82:18, flow rate was 3 mL/min, retention time was 9.4 min, and compound lithocarpinol B was obtained.

The described fermentation culture of Phomopsis lithocarpus FS508 is prepared by inserting the activated Phomopsis lithocarpus FS508 strain into potato glucose liquid medium at 28° C., 120 rpm, and culturing for 5 days to obtain seed liquid, and the seed liquid is prepared at a rate of 10% mL/g. The inoculum amount of the fermented rice was inserted into the solid fermentation medium of rice, and cultured at room temperature for one month to obtain a fermentation culture.

The potato glucose liquid culture medium is obtained by the following methods: washing and peeling the potatoes, cutting them into small pieces of about ¹ cm in size, weighing 200 g, putting them in 1 L of water and boiling for 20 to 30 minutes, filtering with double-layer gauze, Take the filtrate, add 15 g of sea salt and 20 g of glucose to the filtrate, add water to make up 1 L, and prepare by sterilization.

The rice solid fermentation medium is obtained by weighing 3 g of sea salt and 160 g of rice respectively, adding 200 mL of water, stirring and mixing, and sterilizing.

The third object of the present invention is to provide the application of the deep-sea fungus Phomopsis lithocarpus FS508 in the preparation of the compound lithocarpinol B.

According to the antifungal activity test of the present invention, the compound lithocarpinol B has obvious inhibitory activity on the fungus Aspergillus flavus, the MIC value for Aspergillus flavus is 20 μg/mL, and the MIC value of the positive control drug nystatin for Aspergillus flavus is 15 μg/mL. The results show that the compound lithocarpinol B of the present invention has obvious inhibitory activity on Aspergillus flavus, and can be used in the preparation of antifungal drugs.

The fourth object of the present invention is to provide the application of the compound lithocarpinol B in the preparation of an antifungal drug, and the antifungal drug is preferably a drug against Aspergillus flavus.

The fifth object of the present invention is to provide an antifungal drug containing the compound lithocarpinol B as an active ingredient; the antifungal drug is preferably an anti-Aspergillus flavus drug.

Compared with the prior art, the advantages of the present invention are:

The present invention obtains a new 2,3-dihydro-1H-indene compound with antifungal activity through the fermentation and culture of the deep-sea fungus Phomopsis lithocarpus FS508, the separation and purification of the fermentation product and the structure identification, which is named lithocarpinol B. The compound has good inhibitory activity against Aspergillus flavus and can be used for preparing antifungal drugs. Therefore, the present invention provides candidate compounds for the research and development of new antifungal drugs and scientific basis for the development and utilization of marine fungal resources.

The deep-sea fungus Phomopsis lithocarpus FS508 of the present invention was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 15, 2018, and the address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, and the preservation number is: GDMCC NO : 60433.

Description of drawings

FIG. 1 is the ¹ H-NMR spectrum of compound 1 (lithocarpinol B).

Fig. 2 is a ¹³ C-NMR spectrum of compound 1 (lithocarpinol B).

Figure 3 is the HSQC spectrum of compound 1 (lithocarpinol B).

Figure 4 is the ¹ H- ¹ H COSY spectrum of compound 1 (lithocarpinol B).

Figure 5 is the HMBC spectrum of compound 1 (lithocarpinol B).

Figure 6 is the NOESY spectrum of compound 1 (lithocarpinol B).

Figure 7 is the HRESIMS spectrum of compound 1 (lithocarpinol B).

Figure 8 is the CD spectrum of compound 1 (lithocarpinol B).

Figure 9 is an ECD fitting curve of compound 1 (lithocarpinol B).

Figure 10 is the UV spectrum of compound 1 (lithocarpinol B).

Figure 11 is the IR spectrum of compound 1 (lithocarpinol B).

Detailed ways

The following examples are further illustrations of the present invention, rather than limitations of the present invention.

Example 1

(1) insert the activated deep-sea fungus Phomopsis lithocarpus ^FS508 into the potato glucose liquid medium (the preparation method is: wash and peel the potato, cut into small pieces about 1cm in size, weigh 200g, put it into 1L water Boil for 20-30 minutes, filter with double-layer gauze, take the filtrate, add 15g of sea salt and 20g of glucose to the filtrate, add water to make up 1L, sterilize and prepare), at 28°C, 120rpm, cultivate for 5d to obtain seed liquid, put the seeds The liquid is connected to the rice solid fermentation medium with an inoculation amount of 10% mL/g (the preparation method is: respectively weigh 3g sea salt and 160g rice, add 200mL water, stir and mix, and sterilize to prepare), and stand for culture at room temperature. One month, a fermentation culture of Phomopsis lithocarpus FS508 was prepared.

(2) Extract the fermented culture obtained in step (1) with ethyl acetate, extract 4 times, obtain the extract after the ethyl acetate extract is concentrated by distillation, dissolve the extract with an appropriate volume fraction of 85% methanol aqueous solution, and then Extracted with petroleum ether three times to remove low-polar fatty acids, and the remaining methanol aqueous solution was partially concentrated by distillation to obtain brown-black extract.

(3) Pass the brown-black extract obtained in step (2) through a normal phase silica gel column, first use petroleum ether-ethyl acetate from 10:1, 7:1, 9:2, 3:1, 2:1, 1 : 1, 1: 2 v/v gradient elution, then eluted with ethyl acetate, and finally eluted with dichloromethane: methanol = 5: 1, 1: 1 v/v; the collected extract was washed with petroleum ether: ethyl acetate Fraction F6 eluted with a volume ratio of 3:1, fraction F6 was passed through a Sephadex LH-20 column, with dichloromethane: methanol = 1:1 v/v as the eluent, collected by TLC thin layer chromatography with petroleum ether: ethyl acetate Ester = 2:1 v/v to obtain the fraction of Rf = 3.0/3.8, and then subjected to normal phase silica gel column chromatography with petroleum ether-ethyl acetate from 6:1, 3:1, 2:1, 1:1 , 1:2, 0:1 v/v gradient elution, TLC thin layer chromatography was developed with dichloromethane: methanol = 20: 1 v/v to obtain the Rf = 3.3/3.6 components, and then the whole preparative HPLC was used to separate the Components (chromatographic conditions for full preparative HPLC are: YMC ODS-A column, mobile phase is methanol/water with a volume ratio of 85:15, flow rate is 8mL/min), collect elution fractions at retention time 14.8min, and finally use This fraction was purified by semi-preparative HPLC (chromatographic conditions of semi-preparative HPLC were: YMC-pack ODS-A/AQ column, mobile phase was acetonitrile/water with a volume ratio of 82:18, flow rate was 3 mL/min, retention time was 9.4 min), Compound 1 (compound lithocarpinol B) was obtained.

Compound 1 was analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy, and its structure was identified as follows:

As shown in Figures 1-11, Figure 1 is the ¹ H-NMR spectrum of Compound 1; Figure 2 is the ¹³ C-NMR spectrum of Compound 1; Figure 3 is the HSQC spectrum of Compound 1; Figure 4 is the ¹ H-NMR spectrum of Compound 1 ¹ H COSY spectrum; Figure 5 is the HMBC spectrum of compound 1; Figure 6 is the NOESY spectrum of compound 1; Figure 7 is the HRESIMS spectrum of compound 1; Figure 8 is the CD spectrum of compound 1; Figure 9 is the ECD fitting of compound 1 curve; Figure 10 is the UV spectrum of compound 1; Figure 11 is the IR spectrum of the compound.

UV(MeOH)λ_max(logε)208.8(3.86),264.8(3.09)nm；CD(0.16mg/mL,MeOH)λ_max(Δε)214(–42.07),229(–5.68),246(–1.98),277(+6.34)nm；IRν_max 3360,2943,1645,1479,1286cm^-1。Compound 1 has the following physicochemical and spectral properties: white solid powder;

UV(MeOH) _λmax (logε)208.8(3.86),264.8(3.09)nm; CD(0.16mg/mL,MeOH) _λmax (Δε)214(–42.07),229(–5.68),246(–1.98 ), 277(+6.34) nm; IRν _max 3360, 2943, 1645, 1479, 1286 cm ⁻¹ .

¹ H and ¹³ C NMR data; HRESIMS m/z [M+Na] ⁺ 447.1778 (calcd for C ₂₅ H ₂₈ NaO ₆ , 447.1778). ¹ H-NMR (500MHz, CDCl ₃ ) δ: 9.82 (1H, s, H-13), 7.54 (1H, d, J=8.4 Hz, 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'). ¹³ C-NMR (125MHz, CDCl ₃ )δ: 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 showed that the compound exhibited a positive cotton effect at 277 nm and a negative cotton effect at 214, 229, and 246 nm, which was consistent with the calculated ECD fitting curve of 8S, 12R, 9'S (Figure 9). Therefore, the absolute configuration of compound 1 can be determined as 8S, 12R, 9'S. After literature search, the compound 1 is a new skeleton compound, which belongs to the 2,3-dihydro-1H-indene class of new compounds, named lithocarpinol B, and the specific structure is shown in formula I.

The structural formula of the isolated compound lithocarpinol B is shown in the following formula (I):

Example 2

The antibacterial activity of compound lithocarpinol B against Aspergillus flavus was determined by filter paper method.

Take the logarithmic growth phase of Aspergillus flavus and prepare the spore suspension with the spore number of 1.0×10 ⁶ /mL with normal saline, and dilute the compound lithocarpinol B and the positive control drug nystatin to 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL concentrations. Take 1 mL of spore suspension into a petri dish, pour it into PDA medium, mix evenly, place 6 mm in the culture dish containing bacteria, drop 5 μL of sample diluent on the filter paper, and add 5 μL of DMSO dropwise to the blank control. As a positive control, the plate was incubated in a 26°C incubator for 72 hours, and the inhibition zone around the filter paper was observed.

Experimental results: The MIC value of compound lithocarpinol B against Aspergillus flavus was 20 μg/mL, and the MIC value of positive control drug Nystatin against Aspergillus flavus was 15 μg/mL. The results show that the compound lithocarpinol B of the present invention has obvious inhibitory activity against Aspergillus flavus, and can be used in the preparation of antifungal drugs. Therefore, the present invention provides candidate compounds for the research and development of new antifungal drugs and scientific basis for the development and utilization of marine fungal resources.

The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limitations of the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present 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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