CN109053758B - Ailanthus altissima kusnezoff, crystal form, derivative, preparation method and application as antiviral drug - Google Patents

Ailanthus altissima kusnezoff, crystal form, derivative, preparation method and application as antiviral drug Download PDF

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CN109053758B
CN109053758B CN201811085633.1A CN201811085633A CN109053758B CN 109053758 B CN109053758 B CN 109053758B CN 201811085633 A CN201811085633 A CN 201811085633A CN 109053758 B CN109053758 B CN 109053758B
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ailanthone
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ailanthinone
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王四旺
王静
王文
行利
何巧燕
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Hainan Yunze Pharmaceutical Technology Co ltd
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Abstract

The invention relates to ailanthinone, a crystal form, a derivative, a preparation method and application as an antiviral drug. The invention utilizes the microbial fermentation technology to ensure that the extraction rate of the ailanthinone reaches more than 2.5 percent, and discloses antiviral pharmacological data of the ailanthinone, the crystal form and the preparation thereof on hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enteroadenovirus and influenza virus, the action of the ailanthinone, the crystal form and the preparation thereof is obviously superior to that of the first-line medicament lamivudine and entecavir for treating hepatitis B and the Xiyanping injection for treating viral gastroenteritis which are widely applied in international clinical practice, and the ailanthinone, the Xiyanping injection and the preparation thereof are expected to be developed as antiviral medicaments.

Description

Ailanthus altissima kusnezoff, crystal form, derivative, preparation method and application as antiviral drug
Technical Field
The invention belongs to the field of medicines, and particularly relates to ailanthone, a crystal form, a derivative, a preparation method and application of ailanthone as an antiviral drug.
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:
Figure BDA0001803000110000011
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 disclosesThe results of the preparations of the ailanthone, the crystal form and the derivatives and the antiviral activity experiments on viruses such as hepatitis B virus, rotavirus, Norwalk virus, astrovirus, enterovirus, influenza virus and the like show that the ailanthone, the crystal form I, the derivatives and the preparations thereof are expected to be developed to be used for preparing the medicines for treating the 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 leavening agent and the substrate in the step (2) is preferably one of the following two methods, ① the proper amount of biological leavening agent is mixed with the substrate prepared in the step (1), after stirring uniformly, the substrate is transferred into a fermentation tank, ② the biological leavening agent is uniformly spread on a substrate layer with the thickness of every 5cm in the fermentation tank, wherein the biological leavening agent is selected from one or more of food and drug fermentation enzyme preparations, natural fungi (containing endophyte), biogenic bacteria and the like, the food and drug fermentation enzyme preparations are preferably selected from one or more of amyloglucosidase, α -acetolactate enzyme decarboxylase, cellulase, papain and subtilisin, the natural fungi are preferably selected from one or more of natural food and drug bacteria (such as hericium erinaceus, paecilomyces hepialis mycelium and ganoderma lucidum mycelium), endophytic bacteria (such as endophytic fungi, Pestalotiopsis sp), taxomonas taxus anserina andreanna and the like, the endophytic bacteria is preferably selected from Bacillus subtilis, actinomycelitis sp, the endophytic bacteria and the preferable amount of lactobacillus acidophilus is selected from 20% -80%, the lactobacillus acidophilus, lactobacillus acidophilus and the like, the substrate is preferably selected from lactobacillus bifidulus, and the lactobacillus solution is selected from lactobacillus acidophilus, and the lactobacillus.
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 form I characterized by an X-ray powder diffraction pattern using Cu/K α radiation at 2 θ diffraction angles having 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, 29.502 + -0.200.
Another embodiment of the present invention provides ailanthone crystalline form I characterized by an X-ray powder diffraction pattern substantially as shown in figure 1 using Cu/K α 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
Figure BDA0001803000110000031
Figure BDA0001803000110000041
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,
Figure BDA0001803000110000042
) 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 specification. 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:
Figure BDA0001803000110000061
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
Figure BDA0001803000110000062
Or
Figure BDA0001803000110000063
Figure BDA0001803000110000064
Indicating the key position.
The ailanthone derivative is preferably compound 1-33:
Figure BDA0001803000110000065
Figure BDA0001803000110000071
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 semi-solid) may contain conventional excipients such as binding agents, fillers, diluents, tableting agents, lubricants, disintegrants, coloring agents, flavoring agents and wetting agents, and may be coated as necessary, suitable fillers include 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 sulfate, the solid formulations of the present invention may be prepared by conventional methods of mixing, filling, tableting and the like, the liquid formulations of the present invention may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be a dry product which may be formulated with water or other suitable conventional carriers prior to use.
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:
(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; 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;
(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 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, wherein the biological leaven is 'α -acetolactate enzyme decarboxylase-endophytic fungi Pestalotiopsis sp. (1: 1)' 0.2 part, 30% ethanol solution, pH 6.5, temperature 45 ℃, and 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 ①
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 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; 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 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
[ ACTIVE INGREDIENT ] A therapeutically and/or prophylactically effective amount of ailanthone.
[ 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 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 ⑤
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 ⑥
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: inoculating 200 μ L of virus strain (HBV, RV, NV, ASTV, ADV) on monolayer grown Hep-2 and RD cells, adsorbing for 30min, adding 10mL 2% 1640 and DMEM, maintaining the solution 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, vertically repeating for 5 wells, sequentially inoculating to grown cellsSetting cell control and blank control in 96-well plate 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 TCID50Allowing virus solution with concentration to interact for 2h, sequentially inoculating 100 μ L of each well into 96-well plate of grown Hep-2 cells, arranging 5 multiple wells, cell control well, positive reagent (lamivudine, entecavir and Xiyanping injection) well and blank control well, placing 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 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.
The test results in Table 2 show that ① the pharmaceutical composition (product A, D) and the crystal form I have obvious antiviral activity on hepatitis B virus, rotavirus, Norwalk virus, astrovirus and enterovirus and have stronger effect on HBV and ADV, ② compared with the positive reagent, the pharmaceutical composition and the crystal form I have obvious enterovirus inhibition activity, the effect is obviously stronger than that of a Xiyanping injection and an Ailanthus altissima aqueous extract, the anti-HBV effect is obviously stronger than that of lamivudine and Ailanthus altissima aqueous extract but basically equivalent to that of entecavir, ③ the pharmaceutical composition and the crystal form I have better antiviral superior effect on HBV (compared with lamivudine) and have obvious advantages in safety compared with lamivudine, entecavir and Ailanthus aqueous extract, and the treatment index TI 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)
Figure BDA0001803000110000171
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: (
Figure BDA0001803000110000181
n＝8)
Figure BDA0001803000110000182
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: (
Figure BDA0001803000110000183
n＝8)
Figure BDA0001803000110000184
Figure BDA0001803000110000191
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 (
Figure BDA0001803000110000192
n＝8)
Figure BDA0001803000110000193
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 (23)

1. A preparation method of ailanthinone 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 biological fermentation agent with the substrate prepared in the step (1), adding 10-40% ethanol solution by volume, adjusting the pH value to 5.5-8.2, and fermenting at 38-65 ℃; fermenting for 8-48 hr, filtering to obtain fermentation liquid, and concentrating to obtain fermented product;
(3) extraction: carrying out chromatographic separation or chromatographic separation and supercritical carbon dioxide extraction on the fermentation product obtained in the step (2) to obtain a crude ailanthinone product;
(4) and (3) recrystallizing the crude ailanthus altissima ketone product obtained in the step (3) to obtain a pure ailanthus altissima ketone product, wherein the pure ailanthus altissima ketone product 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 by using Cu/K α radiation and an X-ray powder diffraction pattern represented by a 2 theta diffraction angle.
2. The method of claim 1, wherein said ailanthone pure product has an infrared spectrum of 3428 ± 5, 3404 ± 5, 3092 ± 5, 1710 ± 5, 1681 ± 5, 1465 ± 5, 1244 ± 5, 1057 ± 5cm-1Has a characteristic absorption peak at the position of (2).
3. The method of claim 1, wherein said pure ailanthone has a weight loss of 93.4 ± 2.0% at 184 ± 1 ℃ to 643 ± 1 ℃ as measured by thermogravimetric analysis.
4. The process according to claim 1, wherein the pulverization in the step (1) is selected from pulverization to 10-24 mesh.
5. The method of claim 1, wherein the biofermentation agent is mixed with the substrate in the step (2) in a manner selected from one of ① mixing the biofermentation agent with the substrate prepared in the step (1), stirring the mixture uniformly, and transferring the mixture into the fermenter, and ② dispersing the biofermentation agent uniformly in the fermenter per 5cm thickness of the substrate layer.
6. The preparation method of claim 1, wherein the biological fermentation agent is selected from one or more of food and drug fermentation enzyme preparation, natural bacteria and biological bacteria, wherein the food and drug fermentation enzyme preparation is selected from one or more of amyloglucosidase, α -acetolactate enzyme decarboxylase, cellulase, papain and subtilisin, the natural bacteria is selected from one or more of natural edible fungi and plant endophytes, and the biological bacteria is selected from one or more of human acidophilic lactobacillus, plant lactobacillus, bifidobacterium bifidum and bifidobacterium longum.
7. The method of claim 1, wherein the amount of biofermentation agent is 1% to 5% of the mass of the substrate; the dosage of the ethanol solution is 20-80% of the mass of the substrate.
8. The preparation method of claim 1, 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: 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 ℃.
9. The process according to claim 1, wherein the purity of the pure ailanthone in step (4) is more than 98%.
10. An ailanthinone crystal form I is characterized in that an X-ray powder diffraction pattern expressed by a 2 theta diffraction angle by using Cu/K α 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.
11. The method of claim 10The ailanthinone crystal form I is characterized in that the infrared spectrum of the crystal form I is in the range of 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).
12. Crystalline form I of ailanthone of any one of claims 10-11, characterized in that the crystalline form I has an initial weight loss at 184 ± 1 ℃ and a weight loss of 93.4 ± 2.0% by thermogravimetric analysis to 643 ± 1 ℃.
13. A process for the preparation of crystalline form I of ailanthinone according to any of claims 10-12, characterized in that it comprises the steps of crystallization of crude ailanthinone from a suitable solvent selected from ethyl acetate, methanol, ethanol, acetonitrile, a 1:1 volume ratio of ethyl acetate-petroleum ether mixture, acetone, separation, drying.
14. Use of ailanthone prepared in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of an antiviral medicament; the virus is selected from hepatitis B virus.
15. Use of ailanthone prepared in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment and/or prevention of a disease caused by hepatitis b virus infection.
16. The use according to claim 15, characterized in that said disease is selected from infectious hepatitis b.
17. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment and/or prophylaxis of a disease, wherein said disease is caused by infection with hepatitis B virus; the pharmaceutical composition comprises ailanthone prepared according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
18. The use of claim 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable pharmaceutical excipient.
19. Use according to claim 18, characterized in that the pharmaceutical excipient is selected from pharmaceutically acceptable carriers, diluents or excipients.
20. The use according to claim 18, wherein the pharmaceutical excipient is selected from the group consisting of solubilizers, surfactants, film formers, antioxidants, stabilizers, binders, lubricants.
21. Use according to any one of claims 18 to 20, characterized in that the pharmaceutical composition is in a dosage form selected from the group consisting of a solid formulation, a liquid formulation or a semi-solid formulation.
22. The use according to claim 21, characterized in that the pharmaceutical composition is in a dosage form selected from the group consisting of tablets, capsules, injections, microemulsions, submicroemulsions.
23. The use according to claim 21, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of a sustained release tablet, a sustained release capsule, and a sustained release injection.
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