CN110403929B

CN110403929B - Selenolonic acid M derived from penicillium oxalicum and application thereof in inhibiting human cancer cell proliferation

Info

Publication number: CN110403929B
Application number: CN201910344793.1A
Authority: CN
Inventors: 刘沁颖; 郑秋红; 刘施佳; 陈立; 鲁志浩
Original assignee: Fujian Tumour Hospital (fujian Tumour Institute Fujian Cancer Control And Prevention Center)
Current assignee: Fujian Tumour Hospital (fujian Tumour Institute Fujian Cancer Control And Prevention Center)
Priority date: 2019-04-26
Filing date: 2019-04-26
Publication date: 2022-10-14
Anticipated expiration: 2039-04-26
Also published as: CN110403929A

Abstract

The invention relates to a seclenic acid M derived from penicillium oxalicum and application thereof in inhibiting human cancer cell proliferation. The compound has effect in inhibiting human cancer cell proliferation. The structural formula is as follows:

. Culturing Penicillium oxalicum (Penicillium oxalicum) IBPT-6, obtaining a fermentation product, and separating and purifying the compound from the fermentation product. Experiments prove that the compound has better anti-tumor activity on human cancer cells. Can be used for preparing human cancer cell proliferation inhibition drugs or antitumor drugs for research.

Description

Penicillium oxalicum-derived seclenolonic acid M and application thereof in inhibition of human cancer cell proliferation

Technical Field

The invention relates to a Secalonic acid M derived from penicillium oxalicum and an application thereof in inhibiting human cancer cell proliferation, belonging to the field of biological medicines.

Background

Secalonic acids (Secalonic acids) belong to the Ergochrome (Ergochrome) secondary metabolite and are xanthone dimers. Since Stoll et al isolated Secalonic acid A from fungi in 1952, the series of compounds Secalonic acid A-I was continuously discovered and studied. Seclenic acid compounds have various physiological activities, for example Seclenic Acid D (SAD), 5 mg/ml of SAD is added into physiological saline, and the SAD is within the range of 5-20 mg, namely, the seclenic acid D can treat early bladder cancer, and is within the range of 50-100 mg, so that the seclenic acid D has curative effect on more serious bladder cancer and has no side effect. Researches show that some marine fungi can generate seclenic acid compounds with novel structures and good activity in the secondary metabolic process, and have good medicinal and industrial prospects.

The present inventors have studied and found that Penicillium oxalicum (Penicillium oxalicum) IBPT-6 (having been deposited in China center for type culture Collection in 2013, 12, 25, month and month, address: wuhan university, wuhan, with the deposit number: CCTCC NO: m2013714), the crude extract of the fermentation product has a good cell proliferation inhibitory activity, so that the active ingredients thereof are studied. Researches show that the seclenone acid compound has anticancer activity, and no report of the compound on the proliferation inhibition activity of cancer cells exists at present, so that no medicine related to the compound exists in the market.

Disclosure of Invention

The invention aims to provide a Secalonic acid M derived from penicillium oxalicum and a Secalonic acid M compound, and application thereof in inhibiting human cancer cell proliferation. The compound has effects in inhibiting cancer cell proliferation and resisting cancer. The structural formula is as follows:

。

the preparation method of the compound is to culture the penicillium oxalicum ((C))Penicillium oxalicum) IBPT-6, obtaining a fermentation product, and separating and purifying the compound from the fermentation product. The method comprises the following specific steps:

1 fermentation production

A conventional method for culturing microorganisms comprises collecting Penicillium oxalicum (B) ((R))Penicillium oxalicum) IBPT-6 is inoculated on a PDA solid slant culture medium and cultured in an incubator at 28 ℃ for 2 to 3 days, then is inoculated in a culture solution and is statically cultured at 28 ℃ for 30 days to obtain mycelium and fermentation liquor; the culture solution comprises the following components: each liter of water contains 20.0 g of mannitol, 3.0 g of yeast extract, 20.0 g of maltose, 10.0 g of monosodium glutamate, 10.0 g of glucose and KH ₂ PO ₄ 0.5 g、MgSO ₄ ·7H ₂ O 0.3 g、NaCl 15.0 g。

2. Obtaining extract

The mycelium and the fermentation broth were separated with gauze. Continuously performing ultrasonic wall breaking on the mycelium for 3 times by using an acetone solution (containing 20-30% of water), and filtering to remove residues to obtain a mycelium crude extract containing acetone and water. Concentrating under reduced pressure to remove acetone to obtain water solution of crude extract, extracting with ethyl acetate at volume ratio of 1.

3. Separation and purification of Compound

The mycelium extract is stirred by 100-200 meshes of silica gel, and the mixture is prepared by mixing petroleum ether: dichloromethane: methanol is used as gradient eluent, and decompression silica gel chromatographic column chromatography is carried out. By simple thin layer chromatography, mixing, and separating into components A-E. Component D (5.9 g) (dichloromethane: methanol v/v =100 eluate) was purified in dichloromethane: performing silica gel chromatography with methanol as gradient eluent, and mixing to obtain five subfractions D1-D5. Component D3 (1.1 g) was purified with methanol: reverse silica gel chromatography with acid water (containing 0.1% trifluoroacetic acid) as gradient eluent to obtain four subfractions D3-1 to D3-4. Subfraction D3-2 of D3 (168 mg) was isolated by semi-preparative liquid chromatography in the mobile phase 50% acetonitrile (containing 0.1% trifluoroacetic acid) to give the compound (2.6 mg).

Said penicillium oxalicum (A), (B)Penicillium oxalicum) IBPT-6, which has been preserved in China center for type culture Collection in 2013, 12 months and 25 days, addresses Wuhan university, with the preservation number: CCTCC NO: and M2013714.

The invention also protects the application of the compound in preparing a medicament for inhibiting human cancer cell proliferation and the application of the compound in preparing an anti-human cancer medicament.

The invention has the remarkable advantages that: the research shows that the seclenone acid compound is not reported and has obvious activity of inhibiting human cancer cell proliferation, and the activity of inhibiting the human cancer cell proliferation of the compound is not reported at present, so that related medicines are not found in the market.

Drawings

FIG. 1 depicts the major COSY, HMBC and NOE signals of Secalonic acid M.

Detailed Description

The chemical structures of the compounds referred to in the examples below:

EXAMPLE 1 fermentative production and isolation purification of the Compound

1 fermentation production

Fermentation culture of producing bacteria: taking Penicillium oxalicum (B) according to a conventional method for culturing microorganismsPenicillium oxalicum) IBPT-6 (having been deposited in China center for type culture Collection in 2013, 12, 25, month and month, address: wuhan university, wuhan, with the deposit number: CCTCC NO: m2013714) and inoculating the mixture to a PDA solid slant culture medium to culture for 2 to 3 days in an incubator at 28 ℃.

Taking penicillium oxalicum (F) which is cultured on a slant for 2 to 3 daysPenicillium oxalicum) An appropriate amount of IBPT-6 was inoculated into a container containing 400mL of culture broth [ composition of culture broth (g/L): 20.0 parts of mannitol, 3.0 parts of yeast extract, 20.0 parts of maltose, 10.0 parts of monosodium glutamate, 10.0 parts of glucose and KH ₂ PO ₄ 0.5，MgSO ₄ 0.3, 15.0 NaCl, constant volume]And then cultured in a 1000mL conical flask at 28 ℃ for 30 days to obtain mycelium and fermentation broth.

2. Obtaining extract

The mycelium and the fermentation broth were separated with gauze. Continuously performing ultrasonic wall breaking on mycelium for 3 times by using an acetone solution (containing 20-30% of water), and filtering to remove residues to obtain a mycelium crude extract containing acetone and water. Concentrating under reduced pressure to remove acetone to obtain water solution of crude extract, extracting with ethyl acetate at volume ratio of 1.

3. Separation and purification of Compound

The mycelium extract is stirred by 100-200 meshes of silica gel, and the mixture is prepared by mixing petroleum ether: dichloromethane: methanol is used as gradient eluent, and decompression silica gel chromatographic column chromatography is carried out. By simple thin layer chromatography, mixing, and separating into components A-E. Component D (5.9 g) (dichloromethane: methanol v/v =100 eluate) was purified in dichloromethane: performing silica gel chromatography with methanol as gradient eluent, and mixing to obtain five subfractions D1-D5. Component D3 (1.1 g) was replaced with methanol: reverse silica gel chromatography with acid water (containing 0.1% trifluoroacetic acid) as gradient eluent, and separating to obtain four subcomponents D3-1 to D3-4. Subfraction D3-2 (168 mg) of D3 was isolated by semi-preparative liquid chromatography in the presence of mobile phase 50% acetonitrile (containing 0.1% trifluoroacetic acid) to give the compound (2.6 mg).

The compound is yellow oily matter at normal temperature, and the high resolution electrospray ionization mass spectrum HRESI-MS is carried out on the conditions that m/z:661.1535 shows the molecular ion peak [ M + Na]+（calcd for C ₃₂ H ₃₀ NaO ₁₄ 661.1533) of formula C ₃₂ H ₃₀ O ₁₄ 。 ¹ H and ¹³ the C-NMR data are shown in Table 1, and the main COSY, HMBC and NOE signals are shown in FIG. 1.

Of the compounds of Table 1 ¹ H and ¹³ C-NMR data (500 MHz) ¹ H and 125 MHz ¹³ C，in DMSO d6）

^ab LetterThe numbers may be interchanged.

Example 2 in vitro test of antitumor Activity

1. Experimental sample and experimental method

Preparation of test sample solution the test sample was the purified compound isolated and purified in example 1 above. Accurately weighing a proper amount of sample, and preparing a solution with a required concentration by using methanol for measuring the activity.

The cell line and subculture of the cells were tumor cell line cultured in DMEM medium containing 10% FBS at 37 deg.C under 5% CO ₂ Subculturing in the incubator of (1).

Cell proliferation inhibition activity test method

Tetrazolium salt (MTT) method takes tumor cells in logarithmic growth phase, and adjusts the cell density to 2X 10/ml ⁵ The cells were seeded at 200. Mu.l/well in 96-well cell culture plates and 5% CO was passed through the plates at 37 ℃ ₂ Was cultured in the incubator of (1) for 4 hours. After 24 hours of incubation, 2. Mu.l of sample solution or blank solution was added to each well, 10. Mu.l of MTT solution (5 mg/ml of MTT in physiological saline) was added to each well, incubation was continued for 4 hours, centrifugation was carried out at 37 ℃ and 2000 rpm for 8 minutes, and the supernatant was aspirated. DMSO was added in an amount of 100. Mu.l per well, and the mixture was shaken on a micro-shaker for 15 minutes until the crystals were completely dissolved, and then the absorbance (OD) at 570 nm was measured in each well using a SPECTRAMAX Plus type microplate reader manufactured by MD. Three wells are provided for each concentration of sample in the same 96-well plate, and three additional wells are provided for a blank control and a cell-free withered well (if the drug is colored, cell-free wither is performed for the corresponding drug concentration). The OD value of each well is firstly subjected to corresponding cell-free withering, and then the average OD value of three wells is taken according to IR (%) = (OD) _{Blank control} -OD _{Sample (I)} )/OD _{Blank control} X 100% proliferation inhibition (IR%) of cells at each concentration was calculated.

2. Results of the experiment

Results of cell proliferation inhibitory Activity test

In MTT method test, SPSS16.0 software is used for data processing and calculating half inhibition concentration IC according to the tumor cell proliferation inhibition rate of the compound at different concentrations ₅₀ The value is obtained. The results are shown in Table 2.

TABLE 2 inhibitory Activity of Compounds on human cancer cell proliferation (IC) ₅₀ ，μM)

3. Conclusion

The compound has better anti-tumor activity on human cancer cells. Can be used for preparing cancer cell proliferation inhibiting drugs or antitumor drugs for research.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. The application of the compound in preparing the medicine for inhibiting the proliferation of the human cancer cells is characterized in that: the compound is

。

2. The application of the compound in preparing anti-human cancer drugs is characterized in that: the compound is

。