CN109106706B

CN109106706B - Application of 4-4' isomerized seclenic acid D derived from penicillium oxalicum in aspect of gastric cancer

Info

Publication number: CN109106706B
Application number: CN201711372136.5A
Authority: CN
Inventors: 陈立; 程苗苗; 刘沁颖; 伍久林; 鲁志浩; 李亚平; 张其清
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2017-12-19
Filing date: 2017-12-19
Publication date: 2021-11-26
Anticipated expiration: 2037-12-19
Also published as: CN109106706A

Abstract

The invention discloses application of 4-4' isomerized seclenic acid D derived from penicillium oxalicum in gastric cancer. Unlike the 2-2 'linkage of the known seclenonate D, this compound isomerizes to the unusual 4-4' linkage. Experiments prove that the polyphenol compounds have good inhibitory activity on gastric cancer cells, can be used for preparing gastric cancer cell proliferation inhibitory drugs or anti-gastric cancer drugs, and are used for anti-tumor research.

Description

Application of 4-4' isomerized seclenic acid D derived from penicillium oxalicum in aspect of gastric cancer

Technical Field

The invention relates to application of 4-4' isomerized seclenic acid D derived from penicillium oxalicum in gastric cancer.

Background

Secolonic acid is a group of polyphenolic organic compounds produced by the secondary metabolism of microorganisms, and since 1952 secolonic acid a was first discovered from fungi to date, more than half a century has passed. A total of 9 compounds are reported to be found to date, and are respectively seclenic acid A-I. All natural sources of seclenic acid are 2-2 ' linked, except seclenic acid I which is 4-2 ' linked, whereas 4-4 ' linked is very rare. Based on the previous research, the present inventors have conducted extensive studies on Penicillium oxalicum (A)Penicillium oxalicum) IBPT-6 (deposited in the China center for type culture Collection on 25.12.2013, address: wuhan university, the preservation number is: CCTCC NO: m2013714) of crude extracts of fermentation productsThe research shows that a 4-4' linked seclenic acid compound which is isomerized into a rare type has anti-human gastric cancer activity, but no report of the proliferation inhibition activity of the compound on human gastric cancer cells exists at present, and no medicine related to the proliferation inhibition activity is found on the market.

Disclosure of Invention

The invention aims to provide application of 4-4' isomerized seclenonate D derived from penicillium oxalicum in gastric cancer. The compound has effects of inhibiting gastric cancer cell proliferation and resisting human gastric cancer activity, and can be used for preparing anti-gastric cancer medicine.

In order to achieve the purpose, the invention adopts the following technical scheme:

a4-4' isomerized seclenic acid D from Penicillium oxalicum, having the formula:

。

the preparation method of the compound is to culture penicillium oxalicum (A)Penicillium oxalicum) IBPT-6, obtaining the fermentation product, and then separating and purifying 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, cultured in an incubator at 28 ℃ for 2-3 days, then inoculated in a culture solution, and 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 KH₂PO₄ 0.5 g、MgSO₄·7H₂O 0.3 g、NaCl 15.0 g；

2. Obtaining extract

Separating the mycelium from the fermentation liquor by using gauze; continuously ultrasonically breaking the wall of 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, which 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.

3. Separation and purification of Compound

Mixing the mycelium extract with 100-plus 200-mesh silica gel, and performing reduced pressure silica gel chromatographic column chromatography by using petroleum ether-dichloromethane-methanol as gradient eluent; performing simple thin layer chromatography, mixing, and separating into components A-E; performing gel column chromatography (Sephadex LH-20) on the component C (dichloromethane eluate) by using dichloromethane and methanol =1:2 as an eluent, and performing thin-layer chromatography analysis and combination to obtain four sub-components C-1-C-4; subfraction C-3 by semi-preparative liquid chromatography (type 1010 ODS-A, 10X 250 mm, 5 μm; separation flow rate 5 mL/min, mobile phase 55% acetonitrile with 0.1% TFA) gave the compound (t%_R 18.5 min）。

Said penicillium oxalicum (A), (B)Penicillium oxalicum) IBPT-6, which has been deposited at the China center for type culture Collection on 25.12.2013, address: wuhan university, the preservation number is: CCTCC NO: m2013714.

The invention also protects the application of the compound in preparing medicaments for inhibiting the proliferation of human gastric cancer cells and the application of the compound in preparing medicaments for resisting human gastric cancer.

The invention has the following remarkable advantages: the seclenone acid compound obtained by the invention has obvious activity of inhibiting the proliferation of human gastric cancer cells, but no report of the activity of the compound on the proliferation of the human gastric cancer cells exists at present, so that no medicine related to the activity is found in the market.

Drawings

FIG. 1 shows the main COSY, HMBC and NOE signals of 4-4' isomerized seclenonate D obtained in the present invention.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

The compound referred to in the examples below is the 4-4' isomerised seclenonic acid D, of the formula:

。

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 (deposited in the China center for type culture Collection on 25.12.2013, address: wuhan university, the preservation number is: CCTCC NO: m2013714) with proper amount, inoculating the mixture to a PDA solid slant culture medium, and culturing for 2-3 days in an incubator at 28 ℃; then inoculated into a container containing 400mL of culture broth [ culture broth composition (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 KH₂PO₄ 0.5，MgSO₄·7H₂O 0.3，NaCl 15.0]In a 1000mL conical flask, the mycelium and the fermentation broth were obtained after static culture at 28 ℃ for 30 days.

2. Obtaining extract

Separating the mycelium from the fermentation liquor by using gauze; continuously ultrasonically breaking the wall of 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, which 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 crude extractive solution of ethyl acetate, and concentrating under reduced pressure to near dry to obtain mycelium extract 36.5 g.

3. Separation and purification of Compound

Mixing the mycelium extract with 100-plus 200-mesh silica gel, and performing reduced pressure silica gel chromatographic column chromatography by using petroleum ether-dichloromethane-methanol as gradient eluent; by simple thin layer chromatography, combining, and separating into components A-E. And (3) performing gel column chromatography (Sephadex LH-20) on the component C (12.7 g, dichloromethane eluate) by using dichloromethane-methanol =1:2 as an eluent, and performing thin-layer chromatography analysis and combination to obtain four sub-components C-1-C-4. Subcomponent C-3 (4.9 g) was subjected to semi-preparative liquid chromatography (type 1010 ODS-A, 10X 250 mm, 5 μm; separation flow rate5 mL/min, mobile phase 55% acetonitrile with 0.1% TFA) to give the indicated compound (1.1 g, t)_R 18.5 min）。

The compound is yellow powder at normal temperature, and high-resolution electrospray ionization mass spectrum HRESI-MS is performedm/z: 661.1531 shows molecular ion peak [ M + Na]⁺（calcd for C₃₂H₃₀NaO₁₄661.1533); the molecular weight was 638, and the molecular formula was C as presumed from the binding spectrum information₃₂H₃₀O₁₄。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 MHz ¹³C, in DMSO-d ₆)

Example 2 in vitro testing of antitumor Activity

1 Experimental sample and experimental method

1) Preparing a solution of a sample to be detected: an appropriate amount of the compound prepared in example 1 was precisely weighed and prepared into a solution of a desired concentration with DMSO for activity measurement.

2) Preparing cell sap: taking out the frozen cell strain from-80 deg.C ultra-low temperature refrigerator or liquid nitrogen, rapidly melting in 37 deg.C water bath, centrifuging at 1000 rpm for 5 min, removing frozen stock solution in ultra-clean bench, adding 1 mL culture medium, blowing, sucking into culture bottle, adding 10 mL fresh DMEM or RPMI 1640 culture medium, gently shaking to make cells uniformly suspended in the culture medium, placing at 37 deg.C and 5% CO₂Cultured in an incubator. When the cells grow up and need passage, the old culture medium in the bottle is discarded, the cells are washed for 2 times by PBS, 300 mu L of pancreatin is added (the pancreatin can cover the bottom of the bottle), the cells are digested in a 37 ℃ culture box until the cells become round and fall off, the culture medium containing 10% fetal calf serum is added to stop the digestion, the cells are evenly blown and distributed into 2-3 new culture bottles, and the culture medium is supplemented to ensure that the cells continue to grow in the culture box.

3) Cell proliferation inhibitory Activity test (WST-1 method):

the activity evaluation of the antitumor cells adopts a WST-1 kit detection method, and the tumor cells in the logarithmic growth phase are digested to prepare the cell concentration of 3 multiplied by 10⁴And mixing the cell suspension, inoculating 100 mu L of the cell suspension into a 96-well plate, and mixing the cell suspension once every 5 repeated wells to ensure the consistent cell number of each well. Then placing at 37 ℃ and 5% CO₂The culture was carried out overnight in an incubator. The supernatant was aspirated, the drug was diluted with medium to different concentrations and added to the corresponding 96-well plate, and the control group was added with medium containing the same amount of DMSO. After culturing for 72 h, adding WST-1 solution, incubating at 37 deg.C for 2-4 h, gently shaking, mixing, and measuring the absorbance at 450 nm with microplate reader. Taking the average OD value of 5 holes, according to the formula: inhibition rate of cell proliferation = (OD control-OD blank)/(OD experimental-OD blank) × 100% inhibition rate of proliferation of tumor cells by drug at each concentration was calculated, and half inhibition rate IC was calculated using Graphpad Prism 5.0 software₅₀。

2. Results of the experiment

The results of the cell proliferation inhibitory activity test are shown in Table 2.

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

3. Conclusion

The compound has good anti-tumor activity on human gastric cancer cells, can be used for preparing gastric cancer cell proliferation inhibition drugs or anti-tumor drugs, and is used for the research of gastric cancer.

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. A method for separating 4-4' isomerized seclenic acid D from penicillium oxalicum, which is characterized by comprising the following steps:

1) fermentation production

Fermentation culture of producing bacteria: taking penicillium oxalicum (B)Penicillium oxalicum) IBPT-6 (deposited in the China center for type culture Collection on 25.12.2013, address: wuhan university, the preservation number is: CCTCC NO: m2013714) is inoculated on a PDA solid slant culture medium, cultured in an incubator at 28 ℃ for 2-3 days, then inoculated in a culture solution, and 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 KH₂PO₄ 0.5 g、MgSO₄·7H₂O 0.3 g、NaCl 15.0 g；

2) Obtaining extract

Separating the mycelium from the fermentation liquor by using gauze; continuously performing ultrasonic wall breaking on the mycelium for 3 times by using an acetone solution, 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; the acetone solution contains 20% -30% of water;

3) separation and purification of Compound

Mixing the mycelium extract with 100-plus 200-mesh silica gel, and performing reduced pressure silica gel chromatographic column chromatography by using petroleum ether-dichloromethane-methanol as gradient eluent; performing simple thin layer chromatography, mixing, and separating into components A-E; performing gel column chromatography on the component C eluted by the dichloromethane by using dichloromethane methanol =1:2 as an eluent, and performing thin-layer chromatography analysis and combination to obtain four sub-components C-1-C-4; and (3) performing semi-preparative liquid chromatography on the subfraction C-3, eluting by using 55% acetonitrile containing 0.1% TFA as a mobile phase to obtain the 4-4' isomerized seclenonic acid D, wherein the structural formula is as follows:

。