CN107298671B

CN107298671B - Selenolonic acid H from penicillium oxalicum and application thereof in preparing medicine for resisting human colon cancer

Info

Publication number: CN107298671B
Application number: CN201710460067.7A
Authority: CN
Inventors: 陈立; 鲁志浩; 夏其文; 刘沁颖; 毕延雪; 伍久林; 张其清
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2017-06-17
Filing date: 2017-06-17
Publication date: 2020-05-08
Anticipated expiration: 2037-06-17
Also published as: CN107298671A

Abstract

The invention relates to a seclenic acid H from penicillium oxalicum and application thereof in preparing a medicine for resisting human colon cancer. The compound has effect in inhibiting proliferation of human colon cancer cell. 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 colon cancer cells HCT 116. Can be used for preparing human colon cancer cell proliferation inhibition drugs or antitumor drugs for the research of human colon cancer.

Description

Selenolonic acid H from penicillium oxalicum and application thereof in preparing medicine for resisting human colon cancer

Technical Field

The invention relates to a seclenic acid H from penicillium oxalicum and application thereof in preparing a medicine for resisting human colon cancer, belonging to the field of 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 (secalonic acid A) from fungi in 1952, the series of compounds secalonic acid A, B, C, D, E, F, G have been discovered and studied continuously. 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, 12/25/12/h, address Wuhan university, with the deposit number: CCTCC NO: m2013714) has a good cell proliferation inhibitory activity, and the active ingredients thereof were investigated. Researches show that the seclenone acid compound has activity of resisting human colon cancer, and no report of the proliferation inhibition activity of the compound on human colon cancer cells exists at present, so that no medicine related to the proliferation inhibition activity is found on the market.

Disclosure of Invention

The invention aims to provide seclenic acid H derived from penicillium oxalicum and application of the seclenic acid H in preparing a medicine for resisting human colon cancer. The compound has the effect of inhibiting colon cancer cell proliferation, and has anti-human colon cancer activity. The structural formula is as follows:

。

the preparation method of the compound is to culture penicillium oxalicum (A)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

Culturing microorganism by conventional method, collecting Penicillium oxalicum (B) ((B))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 KH₂PO₄0.5 g、MgSO₄·7H₂O 0.3 g、NaCl 15.0 g；

2 obtaining of 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 crude extract of the mycelium, wherein the crude extract contains 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 1:2 for 3 times to obtain ethyl acetate crude extractive solution, and concentrating under reduced pressure to near dry to obtain mycelium extract 36.5 g.

3 separation and purification of Compound

The mycelium extract is mixed with 100-plus 200-mesh silica gel, and the mixture is mixed with petroleum ether: dichloromethane: methanol is used as gradient eluent, and decompression silica gel chromatographic column chromatography is carried out. By simple thin layer chromatography, combining, and separating into components A-E. Component D (5.9 g) (dichloromethane: methanol v/v =100:1 eluate) was purified in dichloromethane: performing silica gel chromatography with methanol as gradient eluent, and mixing to obtain five subfractions D1-D5 after thin layer chromatography. Component D2 (1.2 g) was calculated as chloroform: and (3) taking methanol =1:2 as a gradient eluent, performing gel column chromatography (Sephadex LH-20), and combining to obtain four sub-components D2-1-D2-4 after thin-layer chromatography analysis. Subfraction D2-3 (212 mg) of D2 by semi-preparative liquid chromatography (type 1010 ODS-A, 10X 250 mm, 5 μm): the isolation flow was 5 mL/min and the mobile phase was 55% acetonitrile with 0.1% TFA to give the indicated compound (2.4 mg, t)_R8.1 min)。

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: m2013714.

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

The invention has the following remarkable advantages: researches show that the seclenone acid compound is not reported and has remarkable activity of inhibiting the proliferation of human colon cancer cells, and the compound is not reported to have the activity of inhibiting the proliferation of the human colon cancer cells at present, so that a related medicine is not seen in the market.

Drawings

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

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, address: Wuhan university, the accession number is: CCTCC NO: m2013714) and inoculating to PDA solid slant culture medium and culturing in 28 deg.C incubator for 3 days.

Taking penicillium oxalicum cultured on slant for 2 to 3 days (Penicillium 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 KH₂PO₄0.5，MgSO₄·7H₂O0.3, NaCl 15.0, constant volume]And then cultured in a 1000mL conical flask at 28 ℃ for 30 days to obtain mycelium and fermentation broth.

2 obtaining of extract

The mycelium and the fermentation broth were separated with gauze. Continuously breaking cell wall of mycelium with acetone solution (containing 30% water) by ultrasonic wave for 3 times, filtering to remove residue to obtain crude extract containing acetone and water of mycelium. Concentrating under reduced pressure to remove acetone to obtain water solution of crude extract, extracting with ethyl acetate at volume ratio of 1:2 for 3 times to obtain ethyl acetate crude extractive solution, and concentrating under reduced pressure to near dry to obtain mycelium extract 36.5 g.

3 separation and purification of Compound

The mycelium extract is mixed with 100-plus 200-mesh silica gel, and the mixture is mixed with petroleum ether: dichloromethane: methanol is used as gradient eluent, and decompression silica gel chromatographic column chromatography is carried out. By simple thin layer chromatography, combining, and separating into components A-E. Component D (5.9 g) (dichloromethane: methanol v/v =100:1 eluate) was purified in dichloromethane: ladder made of methanolEluting with eluent, performing pressure column silica gel chromatography, and mixing to obtain five subfractions D1-D5 after thin layer chromatography. Component D2 (1.2 g) was calculated as chloroform: and (3) taking methanol =1:2 as a gradient eluent, performing gel column chromatography (Sephadex LH-20), and combining to obtain four sub-components D2-1-D2-4 after thin-layer chromatography analysis. Subfraction D2-3 (212 mg) of D2 by semi-preparative liquid chromatography (type 1010 ODS-A, 10X 250 mm, 5 μm): the isolation flow was 5 mL/min and the mobile phase was 55% acetonitrile with 0.1% TFA to give the indicated compound (2.4 mg, t)_R8.1 min)。

The compound is yellow oily matter at normal temperature, and high resolution electrospray ionization mass spectrum HRESI-MS is carried outm/z: 659.1370 shows molecular ion peak [ M + Na]⁺（calcd for C₃₂H₂₈NaO₁₄659.1377); the molecular weight is 636, and the molecular formula is C according to the conjuction with the spectrum information₃₂H₂₈O₁₄。¹H and¹³the 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 1¹H and¹³C-NMR data (500 MHz)¹H and 126 MHz¹³C, in DMSO-d ₆)

Example 2 in vitro testing 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 cell subculture adopt tumor cell line, the tumor cell is cultured in DMEM medium containing 10% FBS, and 5% CO is introduced at 37 deg.C₂Subculturing in the incubator.

Cell proliferation inhibitory Activity test method

Tetrazolium salt (MTT) method takes tumor cells in logarithmic growth phase, and adjusts the cell density to 1X 10/ml⁵The cells were seeded at 200. mu.l/well in 96-well cell culture plates and 5% CO was passed through the plates at 37 ℃₂Was 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 the three wells is taken according to IR (%) = (OD)_{Blank control}-OD_{Sample (I)})/OD_{Blank control}X 100% the inhibition of cell proliferation (IR%) was calculated at each concentration.

2. Results of the experiment

Results of cell proliferation inhibitory Activity test

In the 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 concentrations₅₀The value is obtained. The results are shown in Table 2.

TABLE 2 inhibitory Activity of Compounds on human Colon cancer cell proliferation

3. Conclusion

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

Claims

1. Compound (I)

。

2. The process for preparing the compound according to claim 1, which comprises the following steps:

(1) fermentation production

Inoculating the strain to a PDA solid slant culture medium, culturing in an incubator at 28 deg.C for 2-3 days, inoculating to a culture solution, and standing at 28 deg.C for 30 days to obtain mycelium and fermentation broth;

(2) obtaining extract

Separating mycelium from fermentation liquor by using 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 crude extract containing acetone and water of the mycelium; concentrating under reduced pressure to remove acetone to obtain water solution of crude extract, extracting with ethyl acetate at volume ratio of 1:2 for 3 times to obtain ethyl acetate crude extractive solution, and concentrating under reduced pressure to near dry to obtain mycelium extract;

(3) separation and purification of Compound

The mycelium extract is mixed with 100-plus 200-mesh silica gel, and the mixture is mixed with petroleum ether: dichloromethane: performing reduced pressure silica gel column chromatography with methanol as gradient eluent; performing simple thin layer chromatography, mixing, and separating into components A-E; component D was purified as dichloromethane: performing silica gel chromatography with methanol v/v =100:1 as gradient eluent, and mixing to obtain five subfractions D1-D5 after thin layer chromatography analysis; component D2 was calculated as chloroform: performing Sephadex LH-20 gel column chromatography by using methanol =1:2 as a gradient eluent, and combining to obtain four sub-components D2-1-D2-4 after thin-layer chromatography analysis; subfraction D2-3 of D2 by semi-preparative liquid chromatography, type 1010 ODS-A, 10X 250 mm, 5 μm: the separation flow rate was 5 mL/min, the mobile phase was 55% acetonitrile containing 0.1% TFA to give the compound;

the strain is penicillium oxalicum (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: m2013714。

3. Use of a compound of claim 1 for the preparation of a medicament for inhibiting proliferation of human colon cancer cells.

4. Use of a compound according to claim 1 for the preparation of a medicament against human colon cancer.