阅读说明：本技术 一种海洋真菌来源的聚酮化合物及其制备方法和应用 (Polyketide derived from marine fungi as well as preparation method and application thereof ) 是由 黄锡山 覃小芽 杨瑞云 张文秀 薄相龙 吴福荣 于 2021-10-26 设计创作，主要内容包括：本发明属于抗肿瘤药物技术领域,公开了一种海洋真菌来源的聚酮化合物及其制备方法和应用,聚酮化合物的结构式如式(I)所示,其制备方法包括如下步骤：将海洋真菌Aspergillus sp.GXNU-BG1接入种子培养基,摇床培养,得到种子培养液；将种子培养液接入发酵培养基中,静置培养,获得发酵产物；将发酵产物过滤得到菌体,菌体用甲醇提取三次,浓缩提取液得到浸膏,浸膏用乙酸乙酯萃取得粗提物；将乙酸乙酯粗提物用硅胶正相色谱层析进行分离,再采用柱层析分离技术。本发明所得的聚酮化合物对肿瘤细胞的抑制具有显著活性,具有抗肿瘤治疗的临床应用潜力。(The invention belongs to the technical field of antitumor drugs, and discloses a polyketide derived from marine fungi as well as a preparation method and an application thereof, wherein the structural formula of the polyketide is shown as a formula (I), and the preparation method comprises the following steps: inoculating Aspergillus sp.GXNU-BG1 into seed culture medium, and performing shake culture to obtain seed culture solution; inoculating the seed culture solution into a fermentation culture medium, and standing for culture to obtain a fermentation product; filtering the fermentation product to obtain thallus, extracting the thallus with methanol for three times, concentrating the extract to obtain an extract, and extracting the extract with ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by silica gel normal phase chromatography, and then adopting column chromatography separation technology. The polyketide obtained by the invention has obvious activity on inhibiting tumor cells and has clinical application potential of antitumor treatment.)

1. A polyketide of marine fungal origin, characterized in that: the structural formula of the polyketide is shown as the formula (I):

2. the process for the preparation of polyketides of marine fungal origin according to claim 1, characterized in that: the polyketide is obtained by separating from fermentation liquor of Aspergillus sp.GXNU-BG 1; the marine fungus Aspergillus sp.GXNU-BG1 was deposited at the Guangdong province culture Collection on 04.01.2021 with the deposit number GDMCC No: 60940.

3. the process according to claim 2, characterized in that it comprises the following steps:

s1, inoculating Aspergillus sp.GXNU-BG1 into a seed culture medium, and performing shake culture to obtain a seed culture solution;

s2, inoculating the seed culture solution into a fermentation culture medium, and standing for culture to obtain a fermentation product;

s3, filtering the fermentation product to obtain thalli, extracting the thalli for three times by using methanol, concentrating an extracting solution to obtain an extract, and extracting the extract by using ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by silica gel normal phase chromatography, eluting by petroleum ether/ethyl acetate, collecting 20-40% ethyl acetate/petroleum ether fraction, and obtaining the polyketone compound with the structural formula shown in the formula (I) by adopting column chromatography separation technology.

4. The production method according to claim 3, characterized in that: s1 the seed culture medium comprises the following components per liter on average: 30 g of glucose, 1g of yeast extract, 2-5 g of peptone, 1-3 g of agar, 1-5 g of sodium chloride and 1L of water.

5. The production method according to claim 3, characterized in that: s1 the shaking table culture conditions are that the shaking table rotating speed is 80-150 rpm, and the culture is carried out for 5-15 days at 25-30 ℃.

6. The production method according to claim 3, characterized in that: s2 the fermentation medium is a solid rice fermentation medium which is composed of rice and seawater according to the ratio of 1 g: 1.2ml of the mixture was mixed at a mass-to-volume ratio.

7. The production method according to claim 3, characterized in that: s2, standing for 25-50 days at 25-30 ℃.

8. The production method according to claim 3, characterized in that: s3 the eluting with petroleum ether/ethyl acetate is performed by gradient elution with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 100% ethyl acetate-petroleum ether.

9. The production method according to claim 3, characterized in that: s3, the column chromatography separation technology is silica gel column chromatography, gel column chromatography or C-18 reversed phase column chromatography.

10. The use of a polyketide of marine fungal origin according to claim 1 in the preparation of an anti-neoplastic drug.

Technical Field

The invention relates to the technical field of antitumor drugs, and particularly relates to a polyketide derived from marine fungi as well as a preparation method and application thereof.

Background

According to the 2021 global cancer report issued by the world health organization and the international cancer research agency (WHO/LARC), the number of new cancer cases worldwide in 2021 was 1810 ten thousand, of which 45% occurred in the very high human development index countries (HDI), 36% occurred in the high human development index countries, and 15%, 4% occurred in the middle and low human development index countries, respectively. And it is expected that the number of cancer cases will exceed 2700 ten thousand worldwide in 2024. China, as a high human development index country, has 239 ten thousand cancer cases in china in 2021, with urban cancer cases higher than rural cancer cases and males higher than females. In addition, over 955 million cancer deaths occur worldwide at 2021, with high human development index countries accounting for up to 402 million deaths and china accounting for 209 million. Among the chinese malignant tumor reports in 2021, lung cancer, breast cancer, colon cancer, etc. are the most common cancer types, with lung cancer occurring in men higher than in women. In China, women are the first group with high incidence of breast cancer. In recent years, the fatality rate of several cancers is increased year by year in China, and the health of the China is seriously influenced.

At present, in the treatment of cancer, drug therapy is a very important therapeutic means, and the development of anticancer drugs is crucial to the treatment of cancer. Among the new approved drugs in the united states and europe, there are 12 marketed drugs derived directly from marine microbial metabolites. In the research and development of anticancer drugs, the metabolic products of marine fungi play an important role in reducing the cost of the drugs, improving the curative effect of the drugs and reducing the side effect of the anticancer drugs. The new anticancer medicine from marine sources, especially from microbial metabolites, has the characteristics of sustainability, rich and diverse metabolites and the like, and is a natural treasury for screening the anticancer medicine all the time. The marine fungus metabolite has rich activity, and has multiple activities of antibiosis, antitumor, anti-inflammation, enzyme inhibition and the like. The metabolic products with anti-tumor activity are found from marine fungi, and an important way is provided for marine medicine research.

Disclosure of Invention

The invention aims to: aiming at the existing problems, the invention provides a polyketide from marine fungi as well as a preparation method and application thereof, the polyketide is obtained by using the marine fungi Aspergillus sp.GXNU-BG1 for the first time, and the polyketide has positive significance for development and utilization of the polyketide; the obtained polyketide has obvious activity on inhibiting tumor cells and has clinical application potential of antitumor treatment.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a polyketide of marine fungal origin, the polyketide having the formula (I):

the polyketide protected by the invention is obtained by separating from fermentation liquor of Aspergillus sp.GXNU-BG 1; the marine fungus Aspergillus sp.GXNU-BG1 was deposited at the Guangdong province culture Collection on 04.01.2021 with the deposit number GDMCC No: 60940.

the invention also provides a preparation method of the polyketone compound, which comprises the following steps:

s1, inoculating Aspergillus sp.GXNU-BG1 into a seed culture medium, and performing shake culture to obtain a seed culture solution;

s2, inoculating the seed culture solution into a fermentation culture medium, and standing for culture to obtain a fermentation product;

s3, filtering the fermentation product to obtain thalli, extracting the thalli for three times by using methanol, concentrating an extracting solution to obtain an extract, and extracting the extract by using ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by silica gel normal phase chromatography, eluting by petroleum ether/ethyl acetate, collecting 20-40% ethyl acetate/petroleum ether fraction, and obtaining the polyketone compound with the structural formula shown in the formula (I) by adopting column chromatography separation technology.

In the present invention, preferably, the seed culture medium of S1 comprises the following components per liter on average: 30 g of glucose, 1g of yeast extract, 2-5 g of peptone, 1-3 g of agar, 1-5 g of sodium chloride and 1L of water.

In the present invention, preferably, the shaking table cultivation condition in S1 is that the shaking table rotates at 80-150 rpm and is cultivated at 25-30 ℃ for 5-15 days.

In the present invention, preferably, the fermentation medium of S2 is a solid rice fermentation medium consisting of rice and seawater in a ratio of 1 g: 1.2ml of the mixture was mixed at a mass-to-volume ratio.

In the present invention, the static culture condition of S2 is preferably 25-30 ℃ for 25-50 days.

In the present invention, preferably, the elution with petroleum ether/ethyl acetate described in S3 is a gradient elution with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 100% ethyl acetate-petroleum ether, respectively.

In the present invention, preferably, the column chromatography separation technique of S3 is a silica gel column chromatography technique, a gel column chromatography technique or a C-18 reverse phase column chromatography technique.

Experiments prove that the polyketide prepared by the method has antitumor activity, and the invention also protects the application of the polyketide from marine fungi in preparing antitumor drugs.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the polyketide compound shown in the formula (I) is obtained by using the marine fungus Aspergillus sp.GXNU-BG1 for the first time, and the polyketide compound has obvious activity on inhibiting tumor cells, so that the polyketide compound derived from the marine fungus has clinical application potential of antitumor treatment.

2. The preparation method for preparing the polyketide by using the Aspergillus sp.GXNU-BG1 is simple in process and convenient for industrial mass production of the polyketide.

Detailed Description

In order that the invention may be more clearly expressed, the invention will now be further described by way of specific examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

First, preparation example

Example 1 isolation and characterization of the Marine fungus Aspergillus sp.GXNU-BG1

1. Materials: endophytic fungus samples of leaves of Acanthus ilicifolius of mangrove forest collected from coastal parks of northern sea city of Guangxi.

2. Pure endophytic fungi strains are obtained through culture, separation and identification of the strains, are identified as penicillium sp.GXNU-BG1, are preserved in Guangdong province microorganism strain preservation center at 04 th.01.2021, and have the preservation number of GDMCC No: 60940, the address of the depository is No. 59 building 5 of No. 100 Dazhong Jie of Guangzhou city.

Example 2

A preparation method of polyketide from marine fungi comprises the following steps:

s1, seed culture: the seed culture medium comprises the following components per liter on average: 30 g of glucose, 1g of yeast extract, 2 g of peptone, 1g of agar, 1g of sodium chloride and 1L of water; preparing a test tube inclined plane by using a culture medium, selecting an Aspergillus sp.GXNU-BG1 strain of marine fungus, inoculating the strain into the inclined plane, and performing shake culture under the conditions that the rotation speed of a shaking table is 80rpm and the temperature is 25-30 ℃ for 5 days to obtain a seed culture solution;

s2, fermentation culture: inoculating the seed culture solution into a fermentation culture medium, wherein the fermentation culture medium is a solid rice fermentation culture medium which is prepared by mixing rice and seawater according to the weight ratio of 1 g: mixing 1.2ml of the mixture according to the mass-volume ratio, and performing static culture for 25 days under the condition of 25-30 ℃ to obtain a fermentation product;

s3, filtering the fermentation product to obtain thalli, extracting the thalli for three times by using methanol, concentrating an extracting solution to obtain an extract, and extracting the extract by using ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by using silica gel normal phase chromatography, performing gradient elution by using 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70% and 100% of ethyl acetate-petroleum ether respectively, collecting 20% -40% of ethyl acetate/petroleum ether fraction, and obtaining the polyketone compound with the structural formula shown in the formula (I) by adopting a column chromatography separation technology.

Example 3

A preparation method of polyketide from marine fungi comprises the following steps:

s1, seed culture: the seed culture medium comprises the following components per liter on average: 30 g of glucose, 1g of yeast extract, 5 g of peptone, 3 g of agar, 5 g of sodium chloride and 1L of water; preparing a test tube inclined plane by using a culture medium, selecting an Aspergillus sp.GXNU-BG1 strain of marine fungus, inoculating the strain into the inclined plane, and performing shake culture under the conditions that the rotation speed of a shaking table is 150rpm and the temperature is 25-30 ℃ for 15 days to obtain a seed culture solution;

s2, fermentation culture: inoculating the seed culture solution into a fermentation culture medium, wherein the fermentation culture medium is a solid rice fermentation culture medium which is prepared by mixing rice and seawater according to the weight ratio of 1 g: mixing 1.2ml of the mixture according to the mass-volume ratio, and performing static culture for 50 days under the condition of 25-30 ℃ to obtain a fermentation product;

s3, filtering the fermentation product to obtain thalli, extracting the thalli for three times by using methanol, concentrating an extracting solution to obtain an extract, and extracting the extract by using ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by using silica gel normal phase chromatography, eluting with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 100% ethyl acetate-petroleum ether in a gradient manner, collecting 20% -40% ethyl acetate/petroleum ether fraction, and then obtaining the polyketone compound with the structural formula shown in the formula (I) by using gel column chromatography technology or C-18 reverse phase column chromatography technology.

II, confirmation of the Compound

The polyketide obtained above is subjected to structural test analysis, and the following test data are obtained:

polyketide a: c₂₅H₃₀O₉；HRESIMS：473.1816[M-H]^-(theory of the invention)Calculated value 474.1889); one-dimensional nuclear magnetic resonance data:¹HNMR 3.67(s,3H),3.73(s,2H),2.50(s,2H),2.10(s,3H),2.07(s,3H).¹³CNMR 207.9,173.8,157.5,155.3,130.6,123.1,118.7,112.5,52.5,36.5,32.5,12.1,9.0.

through identification, the compound is a polyketide, and the structural formula of the polyketide is shown as the formula (I):

anti-lung cancer experiment of polyketone compounds

Five cell lines were tested for cytotoxic activity against the novel polyketides of examples 2 and 3:

1. materials:

1.1 tetrazolium compound (MTS):

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium,CellTiter 96Aqueous One Solution Reagent,Promega,USA)。

1.2 preparation of target cells: reviving and culturing human lung cancer cell strain A549, human liver cancer cell strain HepG2, cervical cancer cell strain Hela, human breast cancer cell strain MCF-7 and human normal cell HEK 293T.

a. Taking out a cryopreservation tube of a human lung cancer cell strain A549, a human liver cancer cell strain HepG2, a cervical cancer cell strain Hela, a human breast cancer cell strain MCF-7 and human normal cells HEK293T from a liquid nitrogen tank, quickly placing the cryopreservation tube into a water bath box at 37 ℃, continuously shaking to quickly dissolve the cryopreservation tube, and transferring the cryopreservation tube into a centrifuge tube under aseptic operation;

b. adding DMEM complete culture solution into human lung cancer cell strain A549, human liver cancer cell strain HepG2, cervical cancer cell strain Hela and human breast cancer cell strain MCF-7 to 10mL, centrifuging at 1000rmp for 5min, and removing supernatant; adding the human normal fine HEK293T into the RPMI-1640 culture solution to 10mL, centrifuging for 5min at 1000rmp, and discarding the supernatant;

c. repeating the above operations once;

d. completely culturing human lung cancer cell strain A549, human liver cancer cell strain HepG2, cervical cancer cell strain Hela and human breast cancer cell strain MCF-7 in DMEMMixing the cells by liquid-blowing, transferring into culture flask, and adding 5% CO₂Culturing at 37 ℃; while the HEK293T normal human cells are added into RPMI-1640 culture solution to blow and beat the cells, the cells are evenly mixed and then are transferred into a culture bottle, and 5% CO is added₂Culturing at 37 ℃;

e. and (5) observing the growth condition of the cells, replacing the culture solution in time and separating the culture solution into bottles.

1.3 cell count:

a. selecting cells in logarithmic phase, digesting with pancreatin, stopping culture medium, transferring into a centrifuge tube, and adding the culture medium to 10 mL;

b. dripping 10 mul of cell suspension into a groove at one side of a counting plate, counting the total number of cells of four lattices under a microscope, dividing the total number by 4, multiplying by 104 to obtain the number of cells contained in each milliliter of culture solution;

c. adjusting the cell count to 1X 10⁵/mL。

1.4 formulation of Compound 1:

adding the compound prepared by the invention into a DMEM complete culture medium, adjusting the concentration to 500 mu mol/mL, performing ultrasonic emulsification, performing filtration sterilization, and storing at 4 ℃.

2. Test method

a.96 well plate, adding human lung cancer cell strain A549, human liver cancer cell strain HepG2, cervical cancer cell strain Hela, human breast cancer cell strain MCF-7 and human normal cell HEK293T, 50 uL (1 × 105/mL), 5% CO₂Cultured at 37 ℃ for 12 hours.

b. mu.L of the different concentrations of the subjects were added, 50. mu.L of the control DMEM complete medium was added, and the culture was continued for 72 hours.

c. 10. mu.L each of MTS (CellTiter,96Aqueous, One, Solution, Reagent, Promega, USA) was added and the culture was continued for 1 hour.

d. OD values were measured at 490nm in a microplate reader (TECAN, Switzerland).

e. Calculating an inhibition rate: the tumor cell killing rate [ (% of average OD value measured in control group-average OD value measured in drug-added group)/average OD value measured in control group ] × 100%.

f. The IC was determined by plotting the inhibition versus the logarithm of the drug concentration₅₀A value; IC was determined using lg c as the abscissa and the suppression ratio as the ordinate₅₀The value is obtained.

3. Test results

Test results show that the polyketide can obviously and effectively inhibit a human lung cancer cell strain A549, and has certain selectivity, a cervical cancer cell strain Hela, a human breast cancer cell strain MCF-7, a human normal cell HEK293T, and tumor inhibition IC of the polyketide₅₀The values (. mu.M) are shown in Table 1.

TABLE 1 in vitro cytotoxic assay results (IC) of polyketides₅₀μM)

Therefore, the polyketide derived from marine fungi provided by the invention has significant activity on inhibition of tumor cells and has clinical application potential of antitumor treatment.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.