CN111620888A

CN111620888A - Method for preparing ailanthinone by utilizing biological fermentation technology

Info

Publication number: CN111620888A
Application number: CN202010545693.8A
Authority: CN
Inventors: 王四旺; 王静; 王文; 行利
Original assignee: Shaanxi Hanguang Biotechnology Co ltd
Current assignee: Hainan Yunze Pharmaceutical Technology Co ltd
Priority date: 2018-02-13
Filing date: 2018-09-18
Publication date: 2020-09-04
Anticipated expiration: 2038-09-18
Also published as: CN109053758A; CN111620888B; CN109053758B; CN111675722A; CN111675722B

Abstract

The invention relates to a method for preparing ailanthinone by utilizing a biological fermentation technology, which comprises the following steps: (1) pretreatment: crushing one or more of dried ailanthus fruit, ailanthus bark, ailanthus root bark, ailanthus branch, ailanthus leaf, ailanthus flower, ailanthus seed or Chinese medicinal material ailanthus bark to obtain a substrate; (2) fermentation: mixing a proper amount of biological leavening agent and the substrate prepared in the step (1), adding a proper amount of ethanol solution with the volume fraction of 10-40%, adjusting the pH value to 5.5-8.2, and fermenting at the temperature of 38-65 ℃; fermenting for 8-48 hr, filtering to obtain fermentation liquid, and concentrating to obtain fermented product; (3) extraction: and (3) carrying out chromatographic separation or a method combining chromatographic separation and supercritical carbon dioxide extraction on the fermentation product obtained in the step (2) to obtain a crude ailanthone product.

Description

Method for preparing ailanthinone by utilizing biological fermentation technology

The invention is Chinese patent application number: 201811085633.1, the application date of the original application is 9/18 th 2018, the priority date is 2/13 th 2018, the name of the original application is ailanthone, crystal form, derivative, preparation method and application as antiviral drug.

Technical Field

The invention belongs to the field of medicines, and particularly relates to a method for preparing ailanthone by using a biological fermentation technology.

Background

The plant Ailanthus altissima (Mill.) of Simaroubaceae is distributed in China except Heilongjiang, Jilin, Xinjiang, Qinghai, Ningxia, Gansu and Hainan; the main standard Chinese medicinal materials are Ailanthus bark (alias Ailanthus altissima, Ailanthus bark, Ailanthus root bark, Ailanthus root bark and the like), and are dried root bark or dried bark of Swingle of Ailanthus altissima (Mill.) belonging to the family Simaroubaceae, which can be peeled off all the year round, dried in the sun or scraped off the coarse bark and dried in the sun; cold in nature, bitter and astringent in taste; has the effects of clearing heat, eliminating dampness, astringing, stopping leukorrhagia, relieving diarrhea and stopping bleeding; it is commonly used for leucorrhea with reddish discharge, damp-heat dysentery, chronic diarrhea and dysentery, hematochezia and metrorrhagia. Other parts such as ailanthus altissima fruit (alias ailanthus altissima, fruit of ailanthus altissima (Mill.) of ailanthus in Simaroubaceae), ailanthus bark, ailanthus branch, ailanthus leaf, ailanthus flower also have wide pharmacological action and important medicinal value. Cortex Ailanthi, the root bark, contains ailanthone (also known as ailanthone), toosendanin, tannin, ochrophen, etc.; the Ailanthus altissima bark contains ailanthone, ailanthin lactone, acetyl ailanthin lactone, quassin, neoquassin, etc.; the oil content of the seeds in the fruits is about 35 percent, and 2, 6-dimethoxyquinone, ailanthone lactone, chalcone, quassin and the like are added; the leaves contain isodiadema glycoside, vitamin C, etc.

Ailanthone (Ailantholone), also known as Ailanthone or Ailanthone, belongs to a pentacyclic diterpene lactone compound, and has the structure:

studies show that ailanthone has various biological activities, such as antibiosis, malaria resistance, tumor resistance and the like. At present, the research on ailanthus altissima kutkin is less in the prior art, the reported preparation method is to directly extract ailanthus altissima bark, and the extraction rate is less than 0.1 percent; this greatly limits further studies of the pharmacological activity of ailanthone. Because of the problem of medicine sources, no report about the crystal form, preparation or derivative preparation of the ailanthone is found, in order to overcome the defects in the prior art, the invention discloses a method for extracting and purifying ailanthone from other parts of ailanthone such as fruit, seed, branch, leaf, flower and the like of ailanthone bark and/or ailanthone by a biological fermentation comprehensive technology, the method has simple and convenient process, and the extraction rate of the ailanthone is up to more than 3 percent; on the basis, an ailanthone crystal form I with excellent physicochemical and pharmacological properties is obtained by a recrystallization method; in addition, the invention also discloses preparations of the ailanthone, the crystal form and the derivatives thereof and antiviral activity experiments on viruses such as hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enterovirus, influenza virus and the like, and the results show that the ailanthone, the crystal form I, the derivatives and the preparations thereof are expected to be developed to be used for preparing medicines for treating diseases caused by the viruses.

Disclosure of Invention

The invention firstly provides a method for extracting and purifying ailanthone raw material from other parts of ailanthus plants such as ailanthus bark and/or fruits of ailanthus bark by using a biological fermentation comprehensive technology.

The invention provides a preparation method of ailanthus altissima kutkin, which is characterized by comprising the following steps:

(1) pretreatment: crushing one or more of dried ailanthus fruit, ailanthus bark, ailanthus root bark, ailanthus branch, ailanthus leaf, ailanthus flower, ailanthus seed or Chinese medicinal material ailanthus bark to obtain a substrate;

(2) fermentation: mixing a proper amount of biological leavening agent and the substrate prepared in the step (1), adding a proper amount of ethanol solution with the volume fraction of 10-40%, adjusting the pH value to 5.5-8.2, and fermenting at the temperature of 38-65 ℃; fermenting for 8-48 hr, filtering to obtain fermentation liquid, and concentrating to obtain fermented product;

(3) extraction: and (3) carrying out chromatographic separation or a method combining chromatographic separation and supercritical carbon dioxide extraction on the fermentation product obtained in the step (2) to obtain a crude ailanthone product.

Optionally comprises the step (4) of refining: recrystallizing the crude ailanthinone product obtained in the step (3) to obtain a pure ailanthinone product.

The pulverization in the step (1) is preferably pulverized to 10-24 mesh.

The mode of mixing the biological fermentation agent and the substrate in the step (2) is preferably one of the following two methods: mixing a proper amount of biological leavening agent with the substrate prepared in the step (1), stirring uniformly, and transferring into a fermentation tank; ② uniformly spreading biological leaven in the fermentation tank every 5cm thickness of substrate layer. The biological leaven is selected from one or more of food and drug fermentation enzyme preparation, natural fungi (containing endophytes), biogenic bacteria and the like; wherein the food and drug fermentation enzyme preparation is preferably one or more of amyloglucosidase, alpha-acetolactate enzyme decarboxylase, cellulose, papain and subtilisin, the natural bacteria are preferably one or more of natural edible and medicinal bacteria (such as Hericium erinaceus, Paecilomyces hepiali mycelium and Ganoderma mycelium), endophytic bacteria (such as endophytic fungi preferably one or more of endophytic fungi Pestalotiopsis sp, Taxus andersonii Taxomyces andreanana and the like, the endophytic bacteria are preferably one or more of Bacillus subtilis, Klebsiella oxytoca and the like, the endophytic actinomycetes are preferably Actinomyces bovis, Streptomyces sp.Tc022 and the like), and the biological source bacteria are preferably one or more of human source lactobacillus acidophilus, plant lactobacillus, lactobacillus bifidus and bifidobacterium longum. The dosage of the biological leavening agent is 1 to 5 percent of the mass of the substrate, and preferably 2 to 4 percent; the dosage of the ethanol solution is 20-80% of the mass of the substrate.

The chromatographic separation in the step (3) is preferably one or combination of normal phase silica gel column chromatography, macroporous resin column chromatography and reverse phase silica gel column chromatography, and the preferable conditions of supercritical carbon dioxide extraction are as follows: optionally, methanol or ethanol is used as an entrainment solvent, the extraction pressure is 40MPa, the temperature is 35 ℃, the pressure of the separation kettle I is 8MPa, the temperature is 35 ℃, the pressure of the separation kettle II is 6MPa, and the temperature is 35 ℃.

And (4) the purity of the pure ailanthus altissima kurotkin product in the step (4) is more than 98%, and the yield of the pure ailanthus altissima kutkin product is more than 2.5% by mass of the substrate in the step (1).

The chromatographic separation operation (stationary phase specification, eluent type proportion), the supercritical carbon dioxide extraction operation (pressure, temperature, entrainer and the like), the recrystallization operation (solvent, temperature and the like) and the like related in the invention all belong to the conventional experimental operation conditions in the field, and a person skilled in the art can reasonably select the chromatographic separation operation according to the actual needs, for example, the type and proportion of the eluent can be determined according to the TLC result, and the type and temperature of the recrystallization solvent can be determined according to the solubility of the product; in addition, the amount of ethanol solution used, the fermentation time, the temperature, the pH, etc. during the fermentation process are all determined and optimized by those skilled in the art according to the characteristics of the substrate and the biological fermentation agent and the variation of the fermentation product, for example, by TLC or HPLC detection, and the types of the biological fermentation agent not specifically indicated in the present invention are the types of the related bacteria or enzymes commonly used in the art.

Another embodiment of the present invention provides ailanthone crystalline form I characterized by: an X-ray powder diffraction pattern expressed by a 2 theta diffraction angle by using Cu/Kalpha radiation has characteristic peaks at 7.435 +/-0.200, 9.659 +/-0.200, 10.358 +/-0.200, 11.848 +/-0.200, 14.402 +/-0.200, 15.362 +/-0.200, 16.978 +/-0.200, 17.835 +/-0.200, 19.346 +/-0.200, 20.120 +/-0.200, 21.309 +/-0.200, 21.555 +/-0.200, 23.271 +/-0.200, 28.255 +/-0.200, 29.193 +/-0.200 and 29.502 +/-0.200. Ailanthone crystalline form I may also have characteristics substantially represented by an X-ray powder diffraction pattern as shown in figure 1.

Another embodiment of the present invention provides ailanthone crystalline form I characterized by: the X-ray powder diffraction pattern of the crystal form is basically as shown in figure 1 by using Cu/Kalpha radiation.

The X-ray powder diffraction data for form I in figure 1 are summarized in table 1 below:

table 1 XRPD data for ailanthinone form I

It will be appreciated by those skilled in the art that the relative intensities of the peak heights of the 2 θ characteristic peaks in the XRPD pattern may vary for various reasons when measured in respect of substantially the same crystal form.

The infrared spectrum of the ailanthus altissima crystal form I is about 3428 +/-5, 3404 +/-5, 3092 +/-5, 1710 +/-5, 1681 +/-5, 1465 +/-5, 1244 +/-5 and 1057 +/-5 cm^-1Has a characteristic absorption peak at the position of (2). Ailanthone crystalline form I may also have characteristics represented by an infrared spectrum substantially as shown in figure 2.

Thermogravimetric analysis shows that the ailanthone crystal form I starts to lose weight at 184 +/-1 ℃ and reaches 93.4 +/-2.0% when the temperature reaches 643 +/-1 ℃. The TGA profile of crystalline form I of ailanthone is substantially as shown in figure 3.

The X-ray powder diffraction (XRPD) analysis of the ailanthone crystal form I is carried out by a Cu/K α source (40kV,40mA,

) And the angle ranges from 2 to 55, and the measurement is completed in each step for 0.1 s. The infrared spectroscopic analysis of the invention is completed by the determination of a Cary 610/670 type microscopic infrared spectrometer by a KBr tablet pressing method under the ambient temperature and the ambient humidity. The thermogravimetric analysis (TGA) of the present invention is measured by a Pyris-1 TGA thermogravimetric analyzer (PerkinElmer, USA) at an ambient temperature and an ambient humidity, with a scanning rate of 10.00 ℃/min, a maximum temperature of 650.00 ℃, a protective gas: nitrogen gas. "ambient temperature" is generally 0-40 ℃; "ambient humidity" is typically 30% to 80% relative humidity.

The typical X-ray powder diffraction pattern, infrared pattern and thermogravimetric analysis pattern of the ailanthone crystal form I are listed in the attached drawing of the specificationIn (1). The 'representative X-ray powder diffraction pattern or infrared pattern' means that the X-ray powder diffraction characteristics or infrared characteristics of the crystal form basically accord with the overall appearance displayed by the pattern, and it can be understood that in the test process, the peak-appearing positions or peak intensities of the X-ray powder diffraction pattern or infrared pattern measured by the same crystal form have certain differences due to the influence of various factors (such as the granularity of a test sample, the processing method of the test sample, an instrument, test parameters, test operation and the like). The 2 theta values of the X-ray powder diffraction pattern can slightly vary from machine to machine or from sample to sample, and the experimental error of the 2 theta values is generally +/-0.2 degrees; the experimental error of the absorption peak in the infrared spectrum is generally +/-5 cm^-1(ii) a The experimental error of the weight loss temperature in thermogravimetric analysis is generally +/-1 ℃, and the experimental error of the weight loss percentage is generally +/-2.0%.

The invention provides a preparation method of the ailanthinone crystal form I, which comprises the steps of crystallizing a crude ailanthinone product from a proper solvent, separating and drying; wherein the mass content of the ailanthinone in the crude ailanthinone product is more than 75 percent, and the crystallization can be static or stirring; separating comprises filtering or centrifuging to collect the precipitated solid; the drying temperature is 20-50 deg.C, and can be normal pressure drying or reduced pressure drying; suitable solvents are selected from one or more of dichloromethane, chloroform, carbon tetrachloride, THF, dioxane, ethyl acetate, methyl acetate, methanol, ethanol, isopropanol, benzene, toluene, acetic acid, formic acid, N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide, ethylene glycol dimethyl ether, diethyl ether, isopropyl ether, acetonitrile, water, petroleum ether, N-hexane, N-pentane and acetone, wherein one or more of dichloromethane, chloroform, toluene, ethyl acetate, petroleum ether, N-hexane, N-pentane, diethyl ether, ethanol, methanol, isopropanol, acetonitrile, water and acetone is preferably selected.

The method specifically comprises the following steps:

(1) dissolving the ailanthone crude product in a suitable solvent, wherein the suitable solvent is selected from one or a mixture of more of dichloromethane, chloroform, carbon tetrachloride, THF, dioxane, ethyl acetate, methyl acetate, methanol, ethanol, isopropanol, benzene, toluene, acetic acid, formic acid, N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide, ethylene glycol dimethyl ether, diethyl ether, isopropyl ether, acetonitrile, water, petroleum ether, N-hexane, N-pentane and acetone, and preferably one or a mixture of more of dichloromethane, chloroform, toluene, ethyl acetate, petroleum ether, N-hexane, N-pentane, diethyl ether, ethanol, methanol, isopropanol, acetonitrile, water and acetone.

(2) And (3) crystallizing, wherein the crystallizing can be static or stirring.

(3) And separating, including filtering or centrifuging to collect the precipitated solid.

(4) Drying the solid obtained by the separation in the step (3), wherein the drying temperature is selected from 20-50 ℃, and the drying can be carried out under normal pressure or reduced pressure.

Another embodiment of the present invention provides an ailanthone derivative or a pharmaceutically acceptable salt thereof, characterized in that the ailanthone derivative has a structure described by formula II:

wherein R is₁、R₂、R₃、R₄、R₅Each independently selected from H, OH or R₃And R₄Together are O (i.e., ═ O), "- - -" represents a single bond or is absent, and X is selected from

Or

Indicating the key position.

The ailanthone derivative is preferably compound 1-33:

another embodiment of the present invention provides the use of ailanthone or a pharmaceutically acceptable salt thereof, ailanthone crystalline form I or a pharmaceutically acceptable salt thereof, and an ailanthone derivative (formula II) or a pharmaceutically acceptable salt thereof in the preparation of an antiviral medicament. The virus is selected from hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enterovirus and influenza virus.

Another embodiment of the present invention provides the use of ailanthone or a pharmaceutically acceptable salt thereof, ailanthone crystal form I or a pharmaceutically acceptable salt thereof, an ailanthone derivative (formula II) or a pharmaceutically acceptable salt thereof for the treatment and/or prevention of a disease caused by infection with one or more of hepatitis b virus, rotavirus, norwalk virus, astrovirus, enterovirus or influenza virus; the diseases are preferably infectious hepatitis B, gastroenteritis, influenza and the like.

Another embodiment of the present invention provides the use of ailanthone or a pharmaceutically acceptable salt thereof, ailanthone crystal form I or a pharmaceutically acceptable salt thereof, and ailanthone derivative (formula II) or a pharmaceutically acceptable salt thereof in the preparation of antiviral drug lead compounds. The virus is selected from hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enterovirus and influenza virus.

Another embodiment of the present invention provides the use of ailanthone or a pharmaceutically acceptable salt thereof, ailanthone crystalline form I or a pharmaceutically acceptable salt thereof, an ailanthone derivative (formula II) or a pharmaceutically acceptable salt thereof in the preparation of antiviral candidate drugs. The virus is selected from hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enterovirus and influenza virus.

Another embodiment of the present invention provides a use of a pharmaceutical composition for the treatment and/or prevention of a disease caused by infection with one or more of hepatitis b virus, rotavirus, norwalk virus, astrovirus, enterovirus or influenza virus; the pharmaceutical composition takes ailanthone or pharmaceutically acceptable salt thereof, ailanthone crystal form I or pharmaceutically acceptable salt thereof, and ailanthone derivative (formula II) or pharmaceutically acceptable salt thereof as effective components; the pharmaceutical composition may further comprise pharmaceutically acceptable pharmaceutical excipients (e.g. pharmaceutically acceptable carriers, diluents or excipients including solubilizers, surfactants, film forming agents, antioxidants, stabilizers, binders, lubricants, etc.). The dosage form of the pharmaceutical composition can be solid preparation, liquid preparation or semisolid preparation, preferably tablets, capsules, injection (containing powder injection), microemulsion and submicron emulsion, including sustained release tablets, sustained release capsules and sustained release injection.

The formulations of the present invention (solid, liquid or semisolid) may contain conventional excipients such as binders, fillers, diluents, tabletting agents, lubricants, disintegrants, coloring agents, flavoring agents and wetting agents, and the tablets may be coated if necessary. Among the suitable fillers are cellulose, mannitol, lactose and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate. The solid preparation of the present invention can be prepared by a conventional method such as mixing, filling, tabletting and the like. The liquid formulation of the present invention is in the form of, for example: aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous carriers (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerol, propylene glycol or ethyl alcohol; preservatives, for example p-hydroxybenzyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents. The injection of the present invention may contain any of the usual pharmaceutical carriers and/or excipients, stabilizers, antioxidants, complexing agents, and may also contain pharmaceutically acceptable preservatives, buffers, local anesthetics, and the like. The preparation method adopts a common method. The pharmaceutically acceptable pharmaceutic adjuvants of the invention are preferably mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, EDTA disodium and EDTA calcium sodium, carbonate, acetate, phosphate or an aqueous solution thereof of a monovalent alkali metal, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, an amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, a silicon derivative, cellulose and a derivative thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, tween-80, agar, calcium carbonate, calcium bicarbonate, a surfactant, polyethylene glycol, cyclodextrin, β -agar cyclodextrin, a phospholipid material, kaolin, talc, calcium stearate, magnesium stearate, and the like.

The term "pharmaceutically acceptable salts" according to the present invention refers to non-toxic inorganic or organic acid and/or base addition salts. See, e.g., "Salt selection for basic drugs", int.J. pharm. (1986),33, 201-.

The various dosage forms related to the pharmaceutical composition of the present invention can be prepared into preparations meeting the requirements of clinical treatment and/or prevention of viral infection and various suitable specifications and dosage forms (including solid preparations, liquid preparations and semi-solid preparations) such as sustained-release, controlled-release and enteric-soluble capsules, tablets, microemulsions, submicron emulsions or various injections containing the same single or multi-component combinations according to the technical specifications and requirements of the pharmaceutical field (for example, the requirements and specifications of Chinese pharmacopoeia (2015 edition) preparation general rules, specifications or other methods in the prior art, and also according to similar methods described in patent application No. 201710594462.4).

Compared with the prior art, the invention has the advantages that: (1) the invention utilizes the microbial fermentation technology, so that the extraction rate of the ailanthus altissima kurotkin reaches more than 2.5 percent, and the highest extraction rate of the ailanthus altissima kutkin exceeds 3.1 percent and is far better than the extraction rate of the ailanthus altissima kutkin the prior art (less than 0.1 percent); (2) the invention discloses the antiviral pharmacological data of ailanthone, crystal form and preparation thereof on hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enterovirus and influenza virus for the first time, and the action of the ailanthone and the crystal form is obviously superior to that of the first-line medicines for treating hepatitis B, lamivudine and entecavir which are widely applied in the international clinical field and the Xiyanping injection for treating viral gastroenteritis; (3) the invention discloses a new crystal form of ailanthone, which is different from the ailanthone (which is tested to be in an amorphous state) obtained in Chinese patent application No. 201310197365.3 and the commercially available ailanthone; (4) the ailanthinone crystal form I and the preparation provided by the invention have lower toxicity.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of crystalline form I of ailanthone of the present invention;

FIG. 2 is an infrared spectrum of crystalline form I of ailanthone of the present invention;

FIG. 3 is a thermogravimetric analysis of crystalline form I of ailanthone of the present invention;

FIG. 4 is a HPLC analysis chart of the pure ailanthone of example 1 of the present invention.

Detailed Description

In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.

The general preparation process comprises the following steps:

(2) fermentation: mixing a proper amount of biological leavening agent and the substrate prepared in the step (1), adding a proper amount of ethanol solution with the volume fraction of 10-40%, adjusting the pH value to 5.5-8.2, and fermenting at the temperature of 38-65 ℃; fermenting for 8-48 hr, filtering to obtain fermentation liquid, and concentrating to obtain fermented product; wherein the dosage of the ethanol solution is suitable for fully soaking the substrate, and the ethanol solution can be reasonably selected by a person skilled in the art according to the dosage of the substrate;

The conditions not specified in examples 1 to 8 below are referred to the general preparation process described above, 1 part representing 1.0 kg.

Example 1

(1) Pretreatment: 5 parts of Chinese toon bark.

(2) Fermentation: the biological leaven is 'amyloglucosidase-cellulase (1: 1)' 0.1 part, 10% ethanol solution, pH 5.5, temperature 38 deg.C, fermentation time 48 hours.

(3) Extraction: collecting ailanthic ketone flow by normal phase silica gel column chromatography (silica gel mesh number 200-;

(4) refining: and (3) recrystallizing the crude ailanthinone product obtained in the step (3) by ethyl acetate to obtain 0.1585 parts of pure ailanthinone product, wherein the yield reaches 3.170%, the chemical purity (HPLC method) is 99.44%, and the crude ailanthinone product has the characteristics shown in figures 1-3, namely the ailanthinone crystal form I.

Example 2

(1) Pretreatment: 5 parts of dried ailanthus bark.

(2) Fermentation: the biological leaven is 'alpha-acetolactate enzyme decarboxylase-endophytic fungi Pestalotiopsissp. (1: 1)' 0.2 parts, 30% ethanol solution, pH 6.5, temperature 45 ℃, fermentation time 36 hours.

(3) Extraction: collecting ailanthin kurton flow by normal phase silica gel column chromatography (silica gel mesh number 200-;

(4) refining: recrystallizing the crude ailanthinone product obtained in the step (3) with acetonitrile to obtain 0.1450 parts of pure ailanthinone product, wherein the yield reaches 2.900%, the chemical purity (HPLC method) is 98.46%, and the crude ailanthinone product has the characteristics shown in figures 1-3, namely the ailanthinone crystal form I.

Example 3

(1) Pretreatment: 5 parts of Chinese toon bark.

(2) Fermentation: the biological leaven is food and drug leaven, natural edible and drug strain and biological source compound strain combination, wherein the combination comprises 0.05 part of amyloglucosidase-endophytic fungi Pestalotiopsis sp. -Lactobacillus plantarum (1:1:1), 20% ethanol solution, pH 7.5, temperature 55 ℃, and leavening time 24 hours.

(3) Extraction: performing AB-8 type macroporous resin column chromatography (eluent: water/methanol 1:1), collecting ailanthone fluid fraction, performing normal phase silica gel column chromatography (silica gel mesh number 200-.

Example 4

(1) Pretreatment: 5 parts of Chinese toon bark.

(2) Fermentation: the biological leaven is 0.2 part of bifidobacterium, 15 percent ethanol solution, pH 7.0, temperature 40 ℃ and fermentation time 48 hours.

(3) Extraction: 0.1439 parts of ailanthone are obtained in the same way as in example 3, the yield reaches 2.878 percent, and the HPLC purity is 92.63 percent.

Example 5

(1) Pretreatment: 5 parts of dried ailanthus bark.

(2) Fermentation: the biological leaven is 0.25 parts of Hericium erinaceus-paecilomyces hepiali mycelium (1:1), 40% ethanol solution, pH 8.2, temperature 65 deg.C, and fermentation time 8 hr.

(3) Extraction and refining: 0.1555 parts of ailanthone are obtained in the same way as in example 1, the yield reaches 3.110%, and the chemical purity (HPLC method) is 98.20%.

Example 6

(1) Pretreatment: 5 parts of Chinese toon bark.

(2) Fermentation: the biological leaven is 'amyloglucosidase' 0.1 part, 15% ethanol solution, pH 6.0, temperature 38 deg.C, fermentation time 40 hours.

(3) Extraction and refining: 0.1483 parts of ailanthone are obtained in the same way as in example 2, the yield reaches 2.966%, and the chemical purity (HPLC method) is 98.15%.

Example 7

(1) Pretreatment: 5 parts of dried ailanthus bark.

(2) Fermentation: the biological leaven is biological strain combination 'human acidophilic lactobacillus-plant lactobacillus (1: 1)' 0.2 parts, 20% ethanol solution, pH 6.5, temperature 38 deg.C, fermentation time 48 hours.

(3) Extraction and refining: 0.1562 parts of ailanthone are obtained in the same way as in example 2, the yield reaches 3.124%, and the chemical purity (HPLC method) is 98.47%.

Example 8

The method of example 2 was followed to replace the bark of ailanthus altissima with the same weight parts of dried bark, branches, fruits, leaves and flowers, respectively, to obtain 0.0450, 0.0351, 0.0311, 0.0198 and 0.0091 parts of ailanthone, respectively, with yields of 0.900%, 0.702%, 0.622%, 0.396% and 0.182%, respectively.

Examples 1-8 above illustrate that ailanthinone can be purified from root bark (bark), bark, branches, fruits, leaves and flowers of plants of the genus ailanthus according to a comprehensive biofermentation technique, wherein the content in root bark is the highest, the contents in bark, branches and fruits are sequentially reduced, and the contents in leaves and flowers are low; the NMR and MS structural confirmation data of the ailanthone prepared in examples 1-8 are consistent with those reported in the prior art.

Example 9

Dissolving ailanthone (1.0G) prepared in example 3 in 12mL ethanol at 60 ℃, naturally cooling to room temperature, crystallizing overnight, filtering, and drying to obtain a white solid (0.78G), namely ailanthone crystal form I (hereinafter referred to as product G); the X-ray powder diffraction pattern is shown in figure 1, the infrared pattern is shown in figure 2, and the thermogravimetric analysis pattern is shown in figure 3.

Example 10

Dissolving the crude ailanthinone product (with the purity of more than 75 percent and 10g) in 100mL of mixed solution of ethyl acetate and petroleum ether (the volume ratio is 1:1), controlling the temperature to be between 20 ℃ below zero and 15 ℃ below zero, standing for crystallization for 48 hours, centrifugally collecting precipitated solid, and drying at the temperature of 30 ℃ to 40 ℃ to obtain an ailanthinone crystal form I (6.9g), wherein XRPD, IR and TGA diagrams of the ailanthinone crystal form I are consistent with those of example 9.

Example 11

Dissolving the crude ailanthinone product (with the purity of more than 90 percent and 1.0g) in 8mL of acetone, dropwise adding water while stirring until the solution is turbid, standing for crystallization for 24 hours, filtering and drying to obtain a white solid (0.87g), namely ailanthinone crystal form I, wherein XRPD, IR and TGA graphs of the crystal form I are consistent with those of example 9.

Example 12

Dissolving ailanthone (1.0g) prepared in example 4 in 10mL of methanol at 50 ℃, naturally cooling to room temperature, crystallizing overnight, filtering, and drying to obtain a white solid (0.80g), namely ailanthone crystal form I; the X-ray powder diffraction pattern is shown in figure 1, the infrared pattern is shown in figure 2, and the thermogravimetric analysis pattern is shown in figure 3.

To facilitate a further understanding of the present invention, the dosage forms of example 13 provided below were prepared according to the general guidelines for the preparation of the "Chinese pharmacopoeia"; however, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.

Example 13

Injection preparation

[ ACTIVE INGREDIENT ] A therapeutically and/or prophylactically effective amount of ailanthone.

[ Main adjuvants ] solubilizer, excipient, surfactant, etc.

[ PROCEDURE AND PRINTION ] an injection refers to a sterile preparation for injection into the body, which is prepared from active ingredients or appropriate excipients; the injection can be prepared into injection, sterile powder for injection and the like according to actual needs.

[ PREPARATION EXAMPLES ] according to the content of the effective components, respectively weighing each effective component, mixing with solubilizer, excipient, surfactant and the like which are allowed to be added in the main auxiliary materials in the amount below the industry regulation allowance, adding proper amount of water to fully dissolve, filtering, sterilizing, subpackaging, and freeze-drying to obtain the sterile powder for injection.

Preparation example (I)

5g of ailanthone prepared in example 1 is uniformly mixed with citric acid with equal mole number, dissolved in 200ml of distilled water, the pH value is about 5.0, activated carbon with the volume of 0.1 percent of the solution is added, and the mixture is filtered, filled, sealed, sterilized, checked and packaged under the aseptic condition to obtain an injection (hereinafter referred to as a product A, and each milliliter contains 20mg of ailanthone).

Preparation example 2

Mixing 5g of ailanthinone with citric acid with equal mole number, dissolving in 200ml of distilled water, adding active carbon with the volume of 0.1 percent of the solution, and filtering, wherein the pH value is about 5.0; and adding 5g of mannitol as a freeze-dried propping agent, quickly freezing for 8-10h at low temperature, freeze-drying to obtain loose blocks, capping, inspecting and packaging under aseptic conditions to obtain the injection powder injection (hereinafter referred to as product B).

Preparation example 3

Weighing 450mg of PLA (Mw is 20000), 50mg of mPEG-PLGA (PEG Mw is 5000; PLGA Mw is 2400) and 50mg of ailanthone, adding into 3.0mL of ethyl acetate, stirring and dissolving in water bath at 35 ℃ to obtain an organic phase; dissolving F68 as emulsifier in water solution, adding 0.5mL of benzyl alcohol, heating at 35 deg.C, and stirring to obtain water phase. Adding the organic phase into the water phase under shearing condition to obtain primary emulsion, homogenizing under high pressure, quenching in ice water bath (temperature controlled at 5 deg.C), stirring and solidifying for 3-4 hr, adding cold water, removing organic solvent with hollow fiber membrane module, centrifuging, and filtering to obtain nanometer particles of ailanthone; the drug loading rate is 3.0 percent; the encapsulation rate is 92%, and then adding a proper amount of sorbitol as a freeze-drying protective agent for freeze-drying to obtain the ailanthone freeze-dried powder injection (hereinafter referred to as product C).

Tablet formulation

[ MAIN ACCESSORIES ] bulking agent or diluent, including starch, sugar powder, dextrin, milk essence, compressible starch, microcrystalline cellulose, and inorganic salts; adhesives and lubricants including distilled water, ethanol, starch slurry, sodium carboxymethylcellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methyl cellulose; disintegrating agent including dry starch, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose; lubricants, including magnesium stearate, aerosil, talc, hydrogenated vegetable oils, polyethylene glycols and magnesium lauryl sulfate.

[ PREPARATION EXAMPLES ] according to the content of the [ effective ingredients ], respectively weighing each effective ingredient and one or more of the main auxiliary materials, mixing uniformly, and preparing into tablets according to the conventional process.

Preparation example 4

Taking a proper amount of starch, diluting to a concentration of 15%, heating to paste, and preparing into an adhesive; weighing 10g of ailanthone, 6.8g of starch, 0.9g of low-substituted hydroxypropyl cellulose, 0.5g of sodium carboxymethyl starch and 0.2g of magnesium stearate, respectively sieving with a 100-mesh sieve, fully and uniformly mixing the ailanthone, the starch and the low-substituted hydroxypropyl cellulose, pasting with 15% starch to prepare a soft material, sieving with a 14-mesh sieve for granulation, drying at 50-60 ℃, controlling the water content to be 3-5%, sieving with a 14-mesh sieve for granulation, adding the sodium carboxymethyl starch and the magnesium stearate for mixing, and determining the content of the granules; calculating the tablet weight according to the particle content, tabletting and detecting the difference of the tablet weight; packaging after passing inspection (hereinafter referred to as product D, each tablet weighs 100mg and contains 50mg of ailanthone).

Granules

Preparation example C

Weighing 10g of ailanthone, 180g of powdered sugar and 75g of dextrin, sieving with a 120-mesh sieve, uniformly mixing, preparing the uniformly mixed materials into soft materials by using 3% PVP-K30 mucilage, granulating by using a swing granulator, drying at 70 ℃, grading, inspecting, and subpackaging (hereinafter referred to as product E) after passing inspection.

Capsule preparation

Preparation example (c)

Weighing 36g of ailanthus altissima kutkin, 6g of blank pill cores, 3g of polyvinylpyrrolidone, 2g of croscarmellose sodium and 0.5g of magnesium stearate, and preparing the following steps: preparing granules by adopting a centrifugal granulation method, pouring blank pellets into a centrifugal granulator, starting a centrifugal turntable, setting the rotating speed to be 100-120 rpm, starting a peristaltic pump, adjusting the atomization pressure, spraying ethanol, simultaneously spraying ailanthone, polyvinylpyrrolidone and croscarmellose sodium for medicine application, drying the pellets after the medicine application is finished, adding magnesium stearate into the pellets of 16-26 meshes, filling capsules, and then packaging by aluminum plastics (hereinafter referred to as product F).

Submicron emulsion

[ active ingredient ] a therapeutically and/or prophylactically effective amount of ailanthone; selecting 1-7 parts of ailanthone by weight.

[ MAIN ACCESSORIES ] oil, emulsifier, stabilizer, antioxidant, osmotic pressure regulator, and water.

A submicron emulsion (submicron emulsion) is a stable O/W type dispersion system with a particle size of 100-1000nm prepared from an oil phase, an emulsifier (phospholipid) and a water phase by using vegetable oil as a matrix. The drug can be encapsulated in the oil phase and phospholipid interfacial film, and the appearance is opaque, turbid or milky. As a novel drug delivery system, the drug can be selectively accumulated at a target site, so that the concentration of the therapeutic drug at the target site exceeds that of the traditional preparation by several times to hundreds of times, and the curative effect is obviously improved. Meanwhile, the half-life period of the medicine can be prolonged, the distribution amount of the medicine in normal tissues is reduced, adverse reactions are reduced, and the effects of high efficiency and low toxicity are achieved.

[ PREPARATION EXAMPLES ] according to the content of the [ effective ingredients ], respectively weighing each effective ingredient and an emulsifier, adding the effective ingredients and the emulsifier into oil, and stirring to dissolve the effective ingredients to obtain an oil phase; adding a stabilizer, an osmotic pressure regulator and an antioxidant into water, and stirring to dissolve the stabilizer, the osmotic pressure regulator and the antioxidant to obtain a water phase; adding the oil phase into the water phase, and carrying out high-speed shearing dispersion to form primary emulsion; adjusting pH of the primary emulsion to 6.0-7.5, and homogenizing under high pressure to obtain refined emulsion, i.e. submicron emulsion.

Example 14

Experiment 1: antiviral activity of ailanthinone, crystal form I and preparation thereof

1. Method of producing a composite material

(1) Amplification of the virus: 200 μ L of the viral strains (HBV, RV, NV, ASTV, ADV) were inoculated into Hep-2 and RD which had grown in monolayersAdsorbing on cells for 30min, adding 10mL of 2% 1640 and DMEM solution, standing at 37 deg.C and 5% CO₂Culturing in a virus incubator and setting cell contrast, wherein the cell contrast is the same, changing strain into maintenance liquid, observing once every 4h, taking out when more than 90% of cells have pathological changes, repeatedly freezing and thawing for 3 times, blowing, centrifuging (1000rpm, 20min), quantitatively packaging supernatant into small blue bottles, sealing with adhesive tape, and storing in an ultra-low temperature refrigerator at-80 ℃ for later use.

(2) Determination of viral virulence: digestion of Hep-2, RD cells in cell culture flasks at 10 deg.C⁵Cell concentration per mL was inoculated into 96-well plates at 100. mu.L/well, 37 ℃ and 5% CO₂Culturing in incubator for 24 hr. Diluting the amplified virus with 2% culture solution by 10 times, diluting with 10 gradients, longitudinally repeating for 5 wells, sequentially inoculating to 96-well-grown cells, setting cell control and blank control, standing at 37 deg.C and 5% CO₂Culturing in virus incubator, observing with inverted microscope day by day, stopping culturing when more than 90% of cells have pathological changes, adding 10 μ L CCK-8 per well, staining at 37 deg.C and 5% CO₂The virus was incubated in an incubator for 3 hours and the absorbance (OD) was measured at a wavelength of 540nm using a microplate reader. Calculating half infection concentration TCID of virus liquid according to Reed-Muench formula₅₀。

(3) Measurement of cytotoxicity of pharmaceutical composition and the like: diluting the tested medicine with 2% maintenance solution at twice ratio to 7 concentration gradients, sequentially inoculating to 96-well plate with grown cells, setting 5 multiple wells, cell control well and blank control well, standing at 37 deg.C and 5% CO₂Culturing in virus incubator, observing under inverted microscope, stopping culturing when 90% of cells have pathological changes, adding 10 μ L CCK-8 per well, staining at 37 deg.C and 5% CO₂Culturing in a virus incubator for 3h, and measuring an OD value at a wavelength of 540nm by using an enzyme-labeling instrument; calculating half toxic concentration TC of medicine by applying Reed-Muench formula₅₀And determining the maximum nontoxic concentration TC₀。

(4) The medicine composition has direct virus killing effect: each tested medicine is diluted by 7 concentration gradients in 2 percent maintenance solution according to a twofold ratio from nontoxic concentration to equal volume of 100 times of TCID₅₀Concentration ofThe virus solution is interacted for 2h, each 100 mu L of virus solution is sequentially inoculated in a 96-well plate of grown Hep-2 cells, 5 composite wells, cell control wells, positive reagent (lamivudine, entecavir and Xiyanping injection) wells and blank control wells are arranged, the temperature is 37 ℃, and 5% CO is added₂Culturing in virus incubator, observing under inverted microscope, stopping culturing when 90% of cells have pathological changes, adding 10 μ L CCK-8 per well, staining at 37 deg.C and 5% CO₂Culturing in virus incubator for 3h, measuring absorbance OD value at 540nm wavelength with enzyme labeling instrument, calculating cell survival rate, and calculating half Effective Concentration (EC) of medicine by using Reed-Muench formula₅₀) And a Therapeutic Index (TI).

2. Test results

The antiviral activity of the pharmaceutical composition, the crystal form I, the ailanthus bark aqueous extract and the positive drug is as follows: the test results of the pharmaceutical composition, the crystal form I, the ailanthus bark aqueous extract and the positive drug of the invention on the inhibition effect of the virus strains (HBV, RV, NV, ASTV and ADV) are shown in Table 2.

As can be seen from the test results in Table 2, the pharmaceutical composition (product A, D) and the crystal form I of the invention have significant antiviral activity on hepatitis B virus, rotavirus, Norwalk virus, astrovirus and enterovirus, and have stronger effects on HBV and ADV; compared with positive test drugs, the pharmaceutical composition and the crystal form I have obvious enterovirus inhibition activity, and the effect is obviously better than that of Xiyanping injection and ailanthus altissima aqueous extract; the anti-HBV effect is obviously stronger than that of lamivudine and ailanthus altissima aqueous extracts, but the anti-HBV activity is basically equivalent to that of entecavir; the pharmaceutical composition and the crystal form I have better antiviral efficacy (compared with lamivudine) on HBV, and have obvious advantages in safety compared with lamivudine, entecavir and toona sinensis skin aqueous extracts, and the therapeutic index TI of the pharmaceutical composition and the crystal form I is 5.26-5.71.

TABLE 2 antiviral Activity (TC) of pharmaceutical compositions, Crystal form I, Ailanthi cortex aqueous extract and Positive drugs₅₀And EC₅₀Unit of (2) is μ g/mL)

Note: product A, D is an injection and tablet, respectively, prepared in example 12, product G is form I of ailanthone prepared in example 9, product H is commercially available ailanthone (purity greater than 98%), and product I is according to the Chinese patent application No. 201610349044.4 Specification [0038 ]]The ailanthus altissima aqueous extract prepared by the method described in the paragraph, LMV (LAMIVUDING) namely lamivudine, EDT (ENTECAVIR) namely entecavir positive reagent, XYP namely Xiyanping injection; TC for group of products A, D₅₀And EC₅₀The dose has been converted to μ g/mL of ailanthone (except TI).

Experiment 2: effect of ailanthone crystal form I on HBV transgenic mice

1. Materials and methods

(1) Experimental animals and groups: tg (HBV 1.3genome) Swb mode male mice 32 (purchased from military science and technology ltd, guangzhou), 7 weeks old; animals are randomly divided into 4 groups of a solvent, namely a control group (0.5 percent of sodium carboxymethylcellulose), LMV (30mg/kg), EDT (1mg/kg) and a product G, namely the crystal form I (1mg/kg) of ailanthone, each group contains 8 animals, and the animals are administrated by intragastric administration (each reagent adopts 0.5 percent of sodium carboxymethylcellulose to prepare a suspension solution), 1 time a day, continuously for 50 days, and then stop the administration for 14 days; before administration, serum was taken from mice every 10 days; on day 50 of dosing and day 14 of drug withdrawal, half of the mice were sacrificed (3 hours post-dose) per group and liver sampled.

(2) Liver tissue HBcAg immunohistochemical analysis, liver tissue HBV DNA quantitative PCR analysis, serum HBV DNA analysis and statistical treatment: all according to the method [7] of Lixiume with appropriate modification.

2. Test results

(1) Immunohistochemistry results for liver tissue HBcAg: the results are shown in Table 3.

TABLE 3 immunohistochemical analysis of HBcAg in liver tissue: (

n＝8)

Note: comparison with the control group:^*P<0.05，^**P<0.05; comparison with LMV group:^#P<0.05。

(2) quantitative PCR analysis of HBV DNA in liver tissue: the results are shown in Table 4.

TABLE 4 quantitative PCR values for HBV DNA in liver tissue: (

n＝8)

Note: comparison with the control group: p < 0.05.

(3) Serum HBV DNA analysis: the results are shown in Table 5.

TABLE 5 serum HBV DNA quantitation values (

n＝8)

Note: comparison with the control group:^*P<0.05。

the number of HBcAg positive cell nucleuses, the average area of the positive cell nucleuses and the optical density ratio of the ailanthone crystal form I group on the 50 th day are all obviously lower than those of a solvent group (P <0.01) and an LMV group (P <0.05), and the ailanthone crystal form I group has obvious inhibition effect on liver tissues and serum HBV DNA (P < 0.05); on the 64 th day (14 days after drug withdrawal), the three indexes have no statistical difference among all the groups.

The combination of the results shows that the ailanthone crystal form I can obviously inhibit the HBcAg expression of the liver tissue of the HBV transgenic mouse, has an effect obviously superior to LMV and is basically equivalent to EDT; but is superior to LMV and EDT in safety.

Experiment No. 3: antiviral activity of ailanthone derivative (compound with structure as shown in formula II)

According to the method described in the prior art (for example, European patent EP0206112A1), compounds 1-16 can be obtained, and compounds 1-16 can be reacted in an organic solvent (for example, tetrahydrofuran or methanol) at-15-0 deg.C for 3-5 hours under the action of sodium borohydride to obtain compounds 17-32 (the mass spectrum of which shows that [ M + H ] of the corresponding ketocarbonyl group is reduced to hydroxyl group) with 56-75% yield]⁺、[M+Na]⁺Peaks consistent with the theoretical values), compounds 1-33 were tested for antiviral activity according to the antiviral activity test method described in section 1 of this example, and the results showed that compounds 1-32 had EC for HBV, RV, NV, ASTV, ADV₅₀All are less than 10.00 mu g/mL, the TI is more than 4.00, the toxicity of the compound 33 is high, and the TI is less than 2.0.

Reference to the literature

1.THOMASv L，LARROCHE C，PANDEY A.Current developments in solid-statefermentation[J].Biochem Eng J，2013，81(4):146-161

2.HUANG Y Y，XIAO X D，CONG L，et al.A fermented tea with high levels ofgallic acid processed by anaerobic solid-state fermentation[J].LWT-Food SciTechnol，2016，71(1):260-267.

3.BEHERA S S，RAY R C.Solid state fermentation for production ofnicrobial cellulases:recent advances and improvement strategies[J].Int J BiolMacromol， 2016，86(6):656-669.

4.CHANG B V，CHANG Y M.Biodegradation of toxic chemicals by Pleurotuseryngii in submerged fermentation and solidfermentation fermentation[J].JMicrobiol Immun Infect，2016，49(2):175-181.

5. Zhang Changwei, Pengsheng, Zhang Linjie, etc. solid fermentation process of preparing phosphate solubilizing biological fertilizer with eucommia leaf dregs as carrier is optimized [ J ]. Nanjing university of forestry academy (Nature science edition), 2014, 38(5): 134-doped 138.

6.NEVES P V，PITARELO A P，RAMOS L P.Production of cellulosic ethanolfrom sugarcane bagasse by steam explosion:effect of extractives content，acidcatalysis and different fermentation technologies[J].Bioresour Technol，2016，208(8):184-194.

7. Plum, beautiful plum, tangerine yangzhou, liu luster, lijing, beautiful silk, zhou army glow, luckiness, and pinking well, pharmacodynamics study of anti-hepatitis B virus new drug Bay41-4109 in HBV transgenic mice [ J ]. liberary and medical journal, 2011; 31(9):893-896

8.EP0206112A1。

Claims

1. A preparation method of ailanthinone is characterized by comprising the following steps:

2. The process according to claim 1, wherein the pulverization in the step (1) is selected from pulverization to 10-24 mesh.

3. The process according to any one of claims 1 to 2, wherein the means for mixing the biofermentation agent with the substrate in the step (2) is selected from one of the following two methods: mixing a proper amount of biological leavening agent with the substrate prepared in the step (1), stirring uniformly, and transferring into a fermentation tank; or ② uniformly spreading biological leaven in the fermentation tank every 5cm thickness of substrate layer.

4. The preparation method according to any one of claims 1 to 3, wherein the biological fermentation agent is selected from one or more of food and drug fermentation enzyme preparations, natural fungi and biogenic bacteria; wherein the food and drug fermentation enzyme preparation is selected from one or more of amyloglucosidase, alpha-acetolactate enzyme decarboxylase, cellulose, papain and subtilisin, the natural bacteria is selected from one or more of natural edible and medicinal bacteria and plant endophytes, and the biological source bacteria is selected from one or more of human source lactobacillus acidophilus, plant lactobacillus, bifidobacterium and bifidobacterium longum.

5. The method according to any one of claims 1 to 4, wherein the amount of the biological fermentation agent is 1% to 5% by mass of the substrate; the dosage of the ethanol solution is 20-80% of the mass of the substrate.

6. The preparation method according to any one of claims 1 to 5, wherein the chromatographic separation in step (3) is selected from one or more of normal phase silica gel column chromatography, macroporous resin column chromatography and reverse phase silica gel column chromatography, and the supercritical carbon dioxide extraction conditions are as follows: optionally, methanol or ethanol is used as an entrainment solvent, the extraction pressure is 40MPa, the temperature is 35 ℃, the pressure of the separation kettle I is 8MPa, the temperature is 35 ℃, the pressure of the separation kettle II is 6MPa, and the temperature is 35 ℃.