阅读说明：本技术 一种Pyrophen化合物的制备方法 (Preparation method of Pyrophen compound ) 是由 杨晓艳 梁应忠 娄灯吉 牛婉蓉 郭敏 邵林娇 李银飞 于 2020-12-28 设计创作，主要内容包括：本发明公开一种Pyrophen化合物的制备方法,取滑菇(Pholiota nameko)菌种,采用液体培养基制备种子液,通过固体发酵进行扩大发酵,发酵产物用乙酸乙酯萃取3次,所得萃取液浓缩得浸膏,浸膏经RP-18柱层析、硅胶柱层析和Sephedex LH-20柱层析采用不同的溶剂进行洗脱分离,得Pyrophen化合物；本发明从大型真菌滑菇发酵液中提取该化合物,该化合物及其药物组合物在制备治疗和预防肿瘤及致病菌感染药物方面的应用。(The invention discloses a preparation method of a Pyrophen compound, which comprises the steps of taking Pholiota nameko (Pholiota nameko) strains, preparing seed liquid by adopting a liquid culture medium, carrying out expanded fermentation through solid fermentation, extracting a fermentation product for 3 times by using ethyl acetate, concentrating an obtained extract liquid to obtain an extract, carrying out elution separation on the extract through RP-18 column chromatography, silica gel column chromatography and Sephedex LH-20 column chromatography by adopting different solvents to obtain the Pyrophen compound; the compound is extracted from the fermentation liquor of the large-scale fungus pholiota nameko, and the application of the compound and the pharmaceutical composition thereof in the preparation of the medicines for treating and preventing tumors and pathogenic bacteria infection.)

1. A preparation method of a Pyrophen compound is characterized by comprising the following specific steps:

taking Pholiota nameko (Pholiota nameko) strains, preparing a seed solution by adopting a liquid culture medium, carrying out expanded fermentation through solid fermentation, extracting a fermentation product by using ethyl acetate for 3 times, concentrating an obtained extract liquid to obtain an extract, and eluting and separating the extract through RP-18 column chromatography, silica gel column chromatography and Sephedex LH-20 column chromatography to obtain a Pyrophen compound.

2. The method for producing a Pyrophoen compound according to claim 1, wherein the Pholiota nameko (Pholiota nameko) species is purchased from Beiner's organism, which is owned by the institute of Biotechnology, Beijing Beiner's Association.

3. The process according to claim 1, wherein the medium is PD medium or GP medium.

4. The process according to claim 1, wherein the conditions for the extended fermentation are as follows: the culture medium is prepared from water and rice according to the volume mass ratio of mL to g of water to rice of 2.6:1, the mixture is sterilized at the high temperature of 121 ℃ for 30min, and after cooling, the mixture is inoculated with pholiota nameko seed liquid and is subjected to static culture at the room temperature for 60 days.

5. The method for preparing a Pyrophen compound according to claim 1, wherein the eluent used for RP-18 column chromatography is methanol/water solution, wherein the volume ratio of methanol to water is 1: 1-9: 1.

6. The method according to claim 1, wherein the eluent for the silica gel column chromatography is a petroleum ether/ethyl acetate solution, and the volume ratio of petroleum ether to ethyl acetate is 15:1 to 1: 1.

7. The process for the preparation of Pyrophen compounds according to claim 1, wherein the eluent used for Sephedex LH-20 column chromatography is a dichloromethane/methanol solution, wherein the volume ratio of dichloromethane to methanol is 1: 1.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a Pyrophen compound.

Background

Fungi exist in various ecological environments, the structure and the function of the fungi are selected and optimized in a long-term evolution process, a series of natural products with unique structures can be generated, and the products not only can play roles in defense and physiological regulation of organisms, but also make important contribution to human health. Pholiota nameko (Pholiota nameko) is an edible large fungus belonging to the phylum Eumycota, Basidiomycetes, and Lepidium. The edible fungus not only tastes delicious, but also contains rich nutrient substances, realizes artificial cultivation at present, and is one of five high-quality edible fungi artificially cultivated in the world. Although the pholiota nameko is wide in source and convenient to obtain, the research of scholars at home and abroad is only limited to the biological characteristics of hyphae, deep culture, artificial cultivation, extraction and purification of polysaccharide and protein, activity research and the like, and no study on secondary metabolites by other scholars is found. Therefore, the search of natural products with complex structures and remarkable activities from pholiota nameko is of great significance to the discovery of medicines.

Disclosure of Invention

The invention provides a preparation method of a Pyrophen compound, which comprises the steps of taking Pholiota nameko (Pholiota nameko) strains, preparing seed liquid by adopting a liquid culture medium, carrying out expanded fermentation through solid fermentation, extracting a fermentation product for 3 times by using ethyl acetate, concentrating an obtained extract liquid to obtain an extract, carrying out elution separation on the extract through RP-18 column chromatography, silica gel column chromatography and Sephedex LH-20 column chromatography by adopting different solvents and gradients, and obtaining the Pyrophen compound.

The Pholiota nameko (Pholiota nameko) strain is purchased from Beinanyi, which belongs to Beijing Beinanyin institute of Biotechnology.

The culture medium is a PD culture medium or a GP culture medium.

The fermentation conditions of the enlarged fermentation are as follows: the culture medium is prepared from water and rice according to the volume mass ratio of mL to g of water to rice of 2.6:1, the mixture is sterilized at the high temperature of 121 ℃ for 30min, and after cooling, the mixture is inoculated with pholiota nameko seed liquid and is subjected to static culture at the room temperature for 60 days.

An eluant used for RP-18 column chromatography is methanol/water solution, wherein the volume ratio of methanol to water is 1: 1-9: 1.

The eluent used for silica gel column chromatography is a petroleum ether/ethyl acetate solution, wherein the volume ratio of the petroleum ether to the ethyl acetate is 15: 1-1: 1.

An eluent used for the Sephadex LH-20 column chromatography is a dichloromethane/methanol solution, wherein the volume ratio of dichloromethane to methanol is 1: 1.

The invention also provides application of the Pyrophen compound in preparation of anti-tumor and antibacterial medicines.

When the compound is used for preparing a medicament, the compound can be directly used or used in the form of a pharmaceutical composition, wherein the pharmaceutical composition contains 0.1-99.0%, preferably 0.5-90.0% of the Pyrophen compound, and the balance of pharmaceutically acceptable salts, or pharmaceutically acceptable carriers and/or excipients which are nontoxic to human and animals, wherein the excipients comprise fillers, diluents, binders, excipients, absorption promoters, surfactants, stabilizers and the like which are conventional in the pharmaceutical field, and flavoring agents, pigments, sweeteners and the like can be added as necessary.

When used as oral medicine, it can be made into solid or liquid preparation, such as powder, tablet, sugar coated preparation, capsule, solution, syrup, dripping pill, etc.

When used as injection, it can be made into solid or liquid preparations, such as powder for injection, solution injection, etc.

The pholiota nameko is a large-scale fungus in microorganisms, strain separation and identification and large-scale artificial cultivation are realized at present, the nutritive value and the medicinal value are high, and the compound Pyrophen separated from the pholiota nameko has good curative effects on inhibiting tumor cells such as breast cancer and the like and resisting bacteria.

The compound is derived from higher fungus fermentation products, is natural and safe, has small toxic and side effects, can be converted into pharmaceutically acceptable salts or derivatives such as esters, ethers and other derivatives, and can be obtained by a conventional pharmaceutical preparation method by a specific method.

Drawings

FIG. 1 is a scheme of EI-MS of Pyrophen compound;

FIG. 2 is a scheme showing a Pyrophen compound¹H NMR chart;

FIG. 3 is a scheme showing a Pyrophen compound¹³C NMR chart;

FIG. 4 is a projection of the cell stacking in the a-axis direction of the Pyrophen compound.

Detailed Description

In order that the nature of the invention may be better understood, the invention will now be further described with reference to specific examples. The drugs used in the examples are either commercially available or prepared by conventional methods.

Example 1

A preparation method of a Pyrophen compound comprises the following specific steps:

pholiota nameko (Pholiota nameko) species were purchased from a national standards net, with the manufacturer: BeiNaobian belonging to Beijing BeiNai Chuanglian union biotechnology research institute, the strain adopts PB culture medium to prepare seed liquid, the temperature is kept at 24 ℃, the rotation speed is 150rpm, after 5 days of shaking culture, the seed liquid is placed in solid culture medium to carry out enlarged culture (the seed liquid is inoculated according to the volume (mL) of the seed liquid which is about 10 percent of the culture medium mass (mg)), the solid culture medium is obtained by mixing rice and water according to the volume mass ratio mL: g of 2.6:1, sterilizing at the high temperature of 121 ℃ and 30min, after inoculation of the culture medium, standing culture is carried out for 60 days at room temperature to obtain fermentation product, the fermentation product is extracted for 3 times by ethyl acetate, the extraction liquid is combined, and concentrated by a rotary evaporator to obtain extract, the extract is sequentially subjected to RP-18 column chromatography (the eluent is methanol/water solution, the volume ratio of the methanol to the water is 5:5, 6:4, 7:3, 8:2, 5, Gradient elution is carried out at a ratio of 9: 1), silica gel column chromatography (gradient elution is carried out at an eluent which is petroleum ether/ethyl acetate solution, and the volume ratio of the petroleum ether to the ethyl acetate is 15:1, 10:1, 5:1 and 1:1) and Sephadex LH-20 column chromatography (gradient elution is carried out at an eluent which is dichloromethane/methanol solution, and the volume ratio of the dichloromethane to the methanol is 1:1) are carried out for elution separation, so as to obtain a product sample.

Example 2

The chemical structural formula of the product prepared in the method of example 1 is analyzed based on the spectrum data of infrared, optical rotation, mass spectrum, nuclear magnetic resonance, single crystal and the like, and the specific steps are as follows:

FIG. 1 is a graph of EI-MS of Pyrophen compound, mass data: EI-MS (positive) m/z 288, molecular formula C₁₆H₁₈NO₄；

Infrared spectrum data: IR (KBr) v max 2927, 2854, 1697, 1651, 1568, 1455, 1413, 1249, 1145, 1032, 942, 827cm^-1；

Optical rotation data: [ alpha ] to]^24.7 _D-7.1(c 0.11，MeOH)；

FIG. 2 is a scheme showing a Pyrophen compound¹H NMR chart and FIG. 3 are of Pyrophen compound¹³C NMR chart, specific nuclear magnetic data are shown in Table 1:

table 1: NMRsptractatover Pyrophen (1) in CDCl₃

FIG. 4 is a projection of the cell stacking in the a-axis direction of the Pyrophen compound; according to the following steps: c is Crystal data for cu _ lpn3_0m₁₆H₁₇NO₄，M＝287.30，α＝90°，β＝90°，γ＝90°，T＝100(2)K，space group P212121，Z＝4，μ(CuKα)＝0.793mm^-1，8324reflections measured，2580independent reflections(R_int＝0.0348).The final R₁ values were 0.0311(I＞2σ(I)).The final wR(F²)values were 0.0853(I＞2σ(I)).The final R₁ values were 0.0314(all data).The final wR(F²)values were 0.0857(all data).The goodness of fit on F² was 1.071.Flack parameter＝-0.09(5)。

The single crystal structure of the product was determined to be of formula i:

finally, the chemical structural formula of the product is determined to be shown as the following formula II, namely, the Pyrophen compound:

example 3

The Pyrophen compound prepared in example 1 was tested for antitumor activity:

the MTT method was used to examine the effect of the Pyrophen compound on the activity of four tumor cells, namely, hepatocellular cancer SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7 and colon cancer SW480 cell lines.

The experimental method comprises the following steps: preparing single cell suspension by using a culture solution containing 10% fetal bovine serum, inoculating 10000 cells per well into a 96-well plate, inoculating adherent cells for 12 hours in advance, adding a Pyrophohen compound solution (primary screening is carried out at the concentration of 40 mu M, 5 concentrations of 10, 30, 50, 70 and 90 mu M are set for the Pyrophohen compound which inhibits the growth of tumor cells to be near 50% in the concentration, entering a gradient secondary screening), setting 3 secondary wells for each treatment with the final volume of 200 mu L per well, culturing at 37 ℃ for 48 hours, adding 20 mu L of MTT solution into each hole, continuously incubating for 4h, terminating the culture, absorbing and discarding culture supernatant in each hole, adding 200 mu L of DMSO into each hole, incubating overnight at 37 ℃, reading the absorption value of each hole by an enzyme linked immunosorbent assay (ELISA) detector at 490nm wavelength, and calculating the inhibition rate of Pyrophen, wherein the result shows that the compound has better inhibitory activity and IC (integrated circuit) on breast cancer cells MCF-7.₅₀Was 38. mu.M.

Example 4

Pyrophen compounds prepared in example 1 were tested for anti-pathogenic activity:

adopting a filter paper method to carry out four pathogenic bacteria bacteriostasis tests on the Pyrophen compound, wherein the four test strains are respectively as follows: staphylococcus aureus (Staphylococcus aureus CMCC (B)26003), Pseudomonas aeruginosa CMCC (B)10104), Candida albicans (Monilia albicans CMCC (F)98001), and Escherichia coli (Escherichia coli CMCC (B) 44102).

The experimental procedure was as follows:

(1) activating strains: taking out the four pathogenic bacteria from a refrigerator at the temperature of-80 ℃, burning a red inoculating loop in an ultra-clean workbench, cooling, marking on an LB or YPD solid culture medium by using the inoculating loop by adopting a three-line method, and placing in a biochemical incubator at the temperature of 37 ℃ for upright culture for 1 h;

(2) preparing a bacterial suspension: after the strain is activated, adding PBS buffer solution on the inclined plane in an ultraclean workbench in an aseptic operation, slightly shaking the inclined plane on a palm for 80 times to completely wash down the strain, pouring the strain into an aseptic test tube, slightly shaking the strain uniformly, and diluting the bacterial suspension according to test requirements to prepare bacterial suspensions with different concentrations (the test concentration is 10)⁸、10⁶、10⁴CFU/mL)；

(3) And (3) activity test: pouring bacterial suspension into a sterilized culture dish to prepare a bacterial-containing plate, dissolving a Pyrophen compound by DMSO (dimethyl sulfoxide), setting 3 concentrations of 0.5, 1.0 and 2.0mg/mL respectively, soaking filter paper sheets in different Pyrophen solutions for 2 hours respectively, airing in an aseptic environment, pasting the filter paper sheets to the bacterial-containing plate, setting 3 repeated tests for each treatment, placing the filter paper sheets in a biochemical incubator at 37 ℃ for upright culture, culturing for 16-18 hours, observing a bacteriostatic result, measuring a bacteriostatic circle by selecting a uniform and completely aseptically-grown bacteriostatic circle, measuring the diameter by taking the outer edge of the bacteriostatic circle as a boundary, measuring the diameter of the bacteriostatic circle by using a vernier caliper, recording, performing active primary screening by using a filter paper sheet method, and measuring the minimum bacteriostatic concentration of pathogenic bacteria with bacteriostatic activity by using a 96-orifice plate dilution method.

As a result, the Pyrophen compound has certain inhibitory activity on pathogenic fungi Candida albicans and pathogenic bacteria Escherichia coli, and the Minimum Inhibitory Concentration (MIC) of the Pyrophen compound is 85 mu g/mL and 79 mu g/mL respectively.

The compound Pyrophen prepared in example 1 was prepared into capsules according to a conventional capsule preparation method for administration.

The compound Pyrophen prepared in example 1 is added with an excipient according to the weight ratio of the compound to the excipient of 1: 2-1: 4, and the mixture is granulated and tabletted for taking.