WO2023197247A1 - 一种qf-1化合物及其制备方法 - Google Patents

一种qf-1化合物及其制备方法 Download PDF

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WO2023197247A1
WO2023197247A1 PCT/CN2022/086840 CN2022086840W WO2023197247A1 WO 2023197247 A1 WO2023197247 A1 WO 2023197247A1 CN 2022086840 W CN2022086840 W CN 2022086840W WO 2023197247 A1 WO2023197247 A1 WO 2023197247A1
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aspergillus terreus
fermentation
preparation
compound
crude extract
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PCT/CN2022/086840
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English (en)
French (fr)
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汤熙翔
薛晶晶
孙坪
黄文�
徐广鑫
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厦门千夫海洋生物科技有限公司
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Priority to PCT/CN2022/086840 priority Critical patent/WO2023197247A1/zh
Publication of WO2023197247A1 publication Critical patent/WO2023197247A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/58—One oxygen atom, e.g. butenolide

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  • the invention belongs to the field of organic synthesis and relates to a QF-1 compound and a preparation method thereof.
  • QF-1 compound has the structure shown in formula (1), which is one of the main secondary metabolites of the fungus Aspergillus terreus. It is a natural CDK (cyclin-dependent stimulating enzyme)-specific inhibitor. Discovered by KIRIYAMA in 1977, anti-tumor activity was discovered in 2003. This compound can selectively inhibit CDK2 and CDK1 kinases, which play an important role in endowment of cells from the G1 to S phase and G2 to M phase of cell division.
  • QF-1 compounds also have anti-inflammatory, antiviral, antibacterial, antioxidant, treatment of diabetes, inhibition of tyrosine kinases, inhibition of meiosis and inhibition of H1 histone.
  • the purpose of the present invention is to overcome the shortcomings of using existing methods to prepare QF-1 compounds, which have low purity and yield and are not conducive to industrial production, and to provide a QF that can improve the purity and yield of the product and is easy for industrial production.
  • -1 compound and preparation method thereof are not conducive to industrial production.
  • the present invention provides a method for preparing a QF-1 compound, which has a structure represented by formula (1), wherein the method includes the following steps:
  • step S2 Extract the Aspergillus terreus fermentation product obtained in step S1 with an ester solvent, and obtain an ester-soluble Aspergillus terreus crude extract after concentration of the obtained extract;
  • step S3 Separate the ester-soluble Aspergillus terreus crude extract obtained in step S2 by silica gel chromatography, and then remove the solvent in the obtained QF-1 enriched solution to obtain the QF-1 crude extract;
  • step S4 Heat the QF-1 crude extract obtained in step S3 to prepare a methanol-water supersaturated solution, slowly cool down, add distilled water to the obtained supernatant and then centrifuge.
  • the solid product obtained by the centrifugal separation is the QF-1 compound;
  • the fungal culture medium is a liquid fungal fermentation medium or a solid fungal fermentation medium.
  • the fermentation conditions include: the fermentation temperature is 25-30°C, and the fermentation time is 7-15 days when the fungus culture medium is a liquid fungus fermentation culture medium.
  • the fermentation time is 20 to 60 days.
  • the extraction method is to use an equal volume of ester solvent to ultrasonically extract the Aspergillus terreus fermentation product 2 to 4 times, each time to extract independently for 20 to 40 minutes, and then combine the extractions.
  • the liquid was evaporated to dryness to obtain an ester-soluble crude extract of Aspergillus terreus.
  • the ester solvent is selected from at least one of methyl acetate, ethyl acetate, butyl acetate and phenyl acetate.
  • step S3 the particle size of the silica gel filler used for the silica gel chromatographic separation is 200 to 300 mesh.
  • the elution solvent used for the silica gel chromatographic separation is a mixed solvent of dichloromethane and acetone in a volume ratio of 100: (1-10).
  • step S4 the QF-1 crude extract is dissolved in methanol, and then the resulting QF-1 methanol solution is heated while adding water to form a methanol-water supersaturated solution, and then placed in Slowly cool to 0-5°C in a low-temperature environment, absorb the supernatant, add an equal volume of distilled water, and centrifuge.
  • the solid product obtained by the centrifugal separation is the QF-1 compound.
  • step S4 the heating conditions are such that the temperature of the obtained supersaturated solution is 50-55°C.
  • step S4 the amount of methanol used is just enough to completely dissolve the QF-1 crude extract.
  • the present invention also provides the QF-1 compound prepared by the above method.
  • the key to the present invention is to use Aspergillus terreus as a strain for fermentation and culture, and simultaneously use extraction, silica gel chromatographic separation, and hot alcohol water supersaturated impurity removal phase.
  • QF-1 is used and related impurities in the methanol-water supersaturated solution.
  • the mixture is heated to form a methanol-water supersaturated solution, and then the impurities are removed after slowly cooling the temperature.
  • QF-1 is easy to enrich, and the resulting QF-1 compound According to HPLC analysis, the purity can reach more than 95%, and the total yield can reach more than 23.3%.
  • the preparation process provided by the invention has cheap and easily available starting materials, simple post-processing, is environmentally friendly, and is very conducive to industrial production.
  • the inventor of the present invention discovered after in-depth and extensive research that the QF-1 compound can promote sperm development, protect sperm motility, and has excellent effects in the treatment of oligoasthenozoospermia.
  • Figure 1 is the HPLC spectrum of the QF-1 solid obtained in Example 1;
  • Figure 2 is the 1 H NMR spectrum of the QF-1 compound obtained in Example 1;
  • Figure 3 is the 13 C NMR spectrum of the QF-1 compound obtained in Example 1;
  • FIG. 4 is the HPLC spectrum of the QF-1 solid obtained in Example 2.
  • Figure 5 is the HPLC spectrum of the QF-1 solid obtained in Example 3.
  • step S1 Aspergillus terreus is fermented in a fungus culture medium to obtain an Aspergillus terreus fermentation product.
  • the fermentation conditions are not particularly limited as long as they can produce the QF-1 compound.
  • the fermentation temperature is preferably 25 to 30°C, such as 25°C, 26°C, 27°C, 28°C, 29°C, 30°C ° C, etc.;
  • the fermentation time is preferably 7 to 60 days.
  • the culture time of liquid fungal fermentation medium is preferably 7 to 15 days, such as 7, 8, 9, 10, 11, 12, 13, 14, 15 days, etc.
  • the culture time of solid fungal fermentation medium is preferably 20 to 60 days, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days wait.
  • the Aspergillus terreus can be obtained in various existing ways, usually through commercial purchase.
  • the type of the fungal culture medium is not particularly limited and can be conventionally selected in the art.
  • it can be based on cereal starch (corn, potato, tapioca starch), glucose, molasses, ammonia, ammonium salt or nitrate, At least one of commonly used nitrogen sources and carbon sources such as urea, corn steep liquor, soybean cake powder, peanut cake powder, fish meal, yeast extract, etc. is used as the culture medium for carbon and nitrogen sources.
  • cereal starch corn, potato, tapioca starch
  • glucose molasses
  • ammonia ammonium salt or nitrate
  • At least one of commonly used nitrogen sources and carbon sources such as urea, corn steep liquor, soybean cake powder, peanut cake powder, fish meal, yeast extract, etc. is used as the culture medium for carbon and nitrogen sources.
  • step S2 the Aspergillus terreus fermentation product obtained in step S1 is extracted with an ester solvent, and then the solvent in the obtained QF-1 enriched liquid is removed to obtain a QF-1 crude extract.
  • the extraction method only needs to be able to separate the QF-1 compound from most impurities, with QF-1 entering the extraction liquid and the impurities entering the raffinate.
  • the extraction method is to use an equal volume of ester organic solvent to ultrasonically extract the Aspergillus terreus fermentation product 2 to 4 times (such as 2, 3, 4 times), and extract 20 times each time independently.
  • ester organic solvent include, but are not limited to: at least one of methyl acetate, ethyl acetate, butyl acetate and phenyl acetate.
  • step S3 the ester-soluble Aspergillus terreus crude extract obtained in step S2 is separated by silica gel chromatography, and then the solvent in the obtained QF-1 enriched solution is removed to obtain the QF-1 crude extract.
  • the particle size of the silica gel filler packed in the silica gel chromatography column used for the silica gel chromatography separation can be 70-230 mesh, 230-400 mesh, 60-100 mesh, 100-200 mesh or 200-300 mesh, preferably 200 ⁇ 300 mesh.
  • the elution solvent used can be used as long as it can separate QF-1 from impurities, preferably dichloromethane and acetone in a volume ratio of 100: (1 to 10) (such as 100:1, 100:2, 100:3, 100 :4, 100:5, 100:6, 100:7, 100:8, 100:9, 100:10) mixed solvents.
  • the method of removing the solvent may be, for example, rotary evaporation, freeze-drying, etc., and is not particularly limited.
  • step S4 the QF-1 crude extract obtained in step S3 is heated to form a methanol-water supersaturated solution, the temperature is slowly cooled, the obtained supernatant is added with distilled water and centrifuged.
  • the solid product obtained by the centrifugal separation is QF-1. compound.
  • the QF-1 crude extract is dissolved in methanol, and then the resulting QF-1 methanol solution is heated while adding water to form a methanol-water supersaturated solution, and then slowly cooled to 0 to 5°C and absorbed.
  • the supernatant is added with an equal volume of distilled water and then centrifuged.
  • the solid product obtained by the centrifugal separation is the QF-1 compound.
  • This step utilizes the principle of different solubilities of QF-1 compounds and related impurities in good solvent methanol and poor solvent water.
  • the QF-1 crude extract is heated to make a methanol-water supersaturated solution. After slowly cooling down, the solubility of the impurities decreases. Precipitate out to further separate QF-1 from impurities, thereby improving the purity of QF-1 product.
  • the heating conditions are preferably such that the temperature of the obtained supersaturated solution is 50°C to 55°C.
  • the amount of methanol used is preferably just enough to completely dissolve the QF-1 crude extract, so that the purity of the QF-1 product obtained is better.
  • the slow cooling is performed in a low temperature environment, for example, at 0 to 5°C.
  • the total yield of the product actual yield of the target product/theoretical yield of the target product ⁇ 100%, where the theoretical yield of the target product is derived from the HPLC spectrum content ratio.
  • Ferment Aspergillus terreus purchased from Shanghai Preservation Biotechnology Center, ATCC 34571 in rice culture medium (composed of rice and water according to a mass ratio of 1:1.5) at 25°C for 45 days to obtain Aspergillus terreus fermentation product.
  • step S3 Separate the ester-soluble Aspergillus terreus crude extract obtained in step S2 through 200-300 mesh silica gel chromatography, use a dichloromethane-acetone mixed solution with a volume ratio of 100:3 as the eluent, and collect QF-1 The enriched liquid was evaporated to dryness using a rotary evaporator to obtain a crude extract of QF-1. After HPLC analysis, the QF-1 crude extract contained QF-1 compound with a purity of 70%.
  • step S4 Dissolve 1g of QF-1 crude extract obtained in step S3 in 1ml of methanol, add 1.6ml of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 50°C, and then place it in a 4°C refrigerator to cool slowly for 30 minutes. Then suck out the supernatant; continue to add 200 ⁇ l of methanol to the lower layer precipitate, and add 400 ⁇ l of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 50°C. Place it in a 4°C refrigerator to slowly cool down. After 30 minutes, suck out the supernatant and precipitate the lower layer.
  • Aspergillus terreus purchased from China Microorganism Collection Center, TS342803
  • potato glucose broth culture medium composed of 5g potato powder, 10g peptone, 15g glucose, 5g sodium chloride and 1L Distilled water. After fermentation at 25°C for 14 days, Aspergillus terreus fermentation product was obtained.
  • step S3 Separate the ester-soluble Aspergillus terreus crude extract obtained in step S2 through 200-300 mesh silica gel chromatography, use a dichloromethane-acetone mixed solution gradient with a volume ratio of 100:5 as the eluent for elution, and collect QF- 1 enriched liquid, which was evaporated to dryness using a rotary evaporator to obtain a crude extract of QF-1. After HPLC analysis, the QF-1 crude extract contained QF-1 compound with a purity of 72%.
  • step S4 Dissolve 1g of QF-1 crude extract obtained in step S3 in 1ml of methanol, add 1.5ml of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 55°C, and then place it in a 4°C refrigerator to cool slowly for 30 minutes. Then suck out the supernatant; continue to add 200 ⁇ l of methanol to the lower layer precipitate, and add 400 ⁇ l of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 55°C. Place it in a 4°C refrigerator to slowly cool down. After 30 minutes, suck out the supernatant and precipitate the lower layer.
  • Aspergillus terreus purchased from China Microorganism Collection Center, TS364823 was cultured in Peotone Glucose Yeast extract Medium PGY medium (composed of 10g Peptone (peptone), 5g Yeast extract (yeast extract), 1g Glucose (glucose), Distilled water (distilled water) )1L composition) was fermented at 25°C for 10 days to obtain an Aspergillus terreus fermentation product.
  • step S3 Separate the ester-soluble Aspergillus terreus crude extract obtained in step S2 through 200-300 mesh silica gel chromatography, use a dichloromethane-acetone mixed solution gradient with a volume ratio of 100:4 as the eluent for elution, and collect QF- 1 enriched liquid, which was evaporated to dryness using a rotary evaporator to obtain a crude extract of QF-1. After HPLC analysis, the QF-1 crude extract contained QF-1 compound with a purity of 72%.
  • step S4 Dissolve 1g of QF-1 crude extract obtained in step S3 in 1ml of methanol. Add 1.6ml of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 50°C. Then place it in a 4°C refrigerator to cool slowly for 30 minutes. Then suck out the supernatant; continue to add 200 ⁇ l of methanol to the lower layer precipitate, and add 400 ⁇ l of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 50°C. Place it in a 4°C refrigerator to slowly cool down. After 30 minutes, suck out the supernatant and precipitate the lower layer.
  • the starting materials of the preparation process provided by the present invention are cheap and easy to obtain, and the obtained QF-1 is easy to enrich and has simple post-processing. It is environmentally friendly, has high purity and total yield, and is conducive to industrial production.

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Abstract

公开一种QF-1化合物及其制备方法。所述QF-1化合物的制备方法包括：将土曲霉在真菌培养基中进行发酵，所得土曲霉发酵物采用酯类溶剂进行萃取，所得萃取液经浓缩后得到酯溶性土曲霉粗提物；将酯溶性土曲霉粗提物进行硅胶色谱分离，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物；将QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温，将所得上清液加蒸馅水后离心分离。采用所述制备方法，QF-1易于富集，所得QF-1化合物的纯度可达95%以上，总收率可达到23.3%以上。此外，制备工艺起始原料廉价易得，后处理简单，环境友好，非常有利于工业化生产。

Description

一种QF-1化合物及其制备方法 技术领域
本发明属于有机合成领域，涉及一种QF-1化合物及其制备方法。
背景技术
QF-1化合物具有式(1)所示的结构，其为真菌土曲霉的主要次生代谢产物之一，是一种天然的CDK(细胞周期蛋白依赖性刺激酶)特异性的抑制剂，最早在1977年由KIRIYAMA发现，抗肿瘤活性在2003年被发现。该化合物可以选择性地抑制CDK2和CDK1激酶，其在细胞***的G1到S期以及G2到M期的细胞赋予中起到重要的作用。此外，QF-1化合物还具有抗炎、抗病毒、抗菌、抗氧化、治疗糖尿病、抑制酪氨酸激酶、抑制减数***以及对H1组蛋白的抑制作用。
Figure PCTCN2022086840-appb-000001
目前制备QF-1化合物的方法大多采用微生物发酵，之后将所得发酵产物采用HPLC分离纯化获得。然而，该方法由于需要采用HPLC设备，成本较高，制备效率低且对环境不利，不利于工业化生产，且所得产物纯度和收率仍有待提高。
发明内容
本发明的目的是为了克服采用现有的方法制备QF-1化合物存在纯度和收率较低且不利于工业化生产的缺陷，而提供一种能够提高产物的纯度和收率且易于工业化生产的QF-1化合物及其制备方法。
具体地，本发明提供了一种QF-1化合物的制备方法，所述QF-1化合物 具有式(1)所示的结构，其中，该方法包括以下步骤：
S1、将土曲霉在真菌培养基中进行发酵，得到土曲霉发酵物；
S2、将步骤S1所得土曲霉发酵物采用酯类溶剂进行萃取，所得萃取液经浓缩后得到酯溶性土曲霉粗提物；
S3、将步骤S2所得酯溶性土曲霉粗提物进行硅胶色谱分离，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物；
S4、将步骤S3所得QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温，将所得上清液加蒸馏水后离心分离，所述离心分离所得固体产物即为QF-1化合物；
Figure PCTCN2022086840-appb-000002
在一种优选实施方式中，步骤S1中，所述真菌培养基为液体真菌发酵培养基或固体真菌发酵培养基。
在一种优选实施方式中，步骤S1中，所述发酵的条件包括：发酵温度为25～30℃，当所述真菌培养基为液体真菌发酵培养基时发酵时间为7～15天，当所述真菌培养基为固体真菌发酵培养基时发酵时间为20～60天。
在一种优选实施方式中，步骤S2中，所述萃取的方式为将土曲霉发酵物采用等体积的酯类溶剂超声萃取2～4次，每次各自独立地萃取20～40min，之后合并萃取液并蒸干，得到酯溶性土曲霉粗提物。
在一种优选实施方式中，步骤S2中，所述酯类溶剂选自乙酸甲酯、乙酸乙酯、乙酸丁酯和乙酸苯酯中的至少一种。
在一种优选实施方式中，步骤S3中，所述硅胶色谱分离所采用的硅胶填料的粒径为200～300目。
在一种优选实施方式中，步骤S3中，所述硅胶色谱分离所采用的洗脱溶剂为二氯甲烷和丙酮按照体积比100:(1～10)的混合溶剂。
在一种优选实施方式中，步骤S4中，将所述QF-1粗提物溶于甲醇中，再将所得QF-1甲醇溶液边加热边加入水形成甲醇-水过饱和溶液，之后置于低 温环境中缓慢冷却至0～5℃，吸取上清液加等体积蒸馏水后离心分离，所述离心分离所得固体产物即得QF-1化合物。
在一种优选实施方式中，步骤S4中，所述加热条件使所得过饱和溶液的温度为50～55℃。
在一种优选实施方式中，步骤S4中，所述甲醇的用量恰好使得QF-1粗提物完全溶解。
本发明还提供了由上述方法制备得到的QF-1化合物。
本发明的关键在于以土曲霉作为菌株进行发酵培养，同时将萃取、硅胶色谱分离以及热醇水过饱和除杂相配合使用，其中，在热醇水过饱和除杂过程中，利用QF-1及相关杂质在甲醇-水过饱和溶液中溶解度的不同，将混合物加热制成甲醇-水过饱和溶液，缓慢降温之后再将杂质去除，如此操作，QF-1易于富集，所得QF-1化合物经HPLC分析纯度可达95％以上，总收率可达到23.3％以上。此外，本发明提供的制备工艺起始原料廉价易得，后处理简单，环境友好，非常有利于工业化生产。此外，本发明的发明人经过深入且广泛研究之后发现，QF-1化合物可以促进***发育、保护***活力，在治疗少弱***症上具有优异的效果。
附图说明
图1为实施例1所得QF-1固体的HPLC图谱；
图2为实施例1所得QF-1化合物的 1H NMR图谱；
图3为实施例1所得QF-1化合物的 13C NMR图谱；
图4为实施例2所得QF-1固体的HPLC图谱；
图5为实施例3所得QF-1固体的HPLC图谱。
具体实施方式
步骤S1中，将土曲霉在真菌培养基中进行发酵，得到土曲霉发酵物。其中，所述发酵的条件只要能够生成QF-1化合物即可，没有特别的限定，例如，发酵温度优选为25～30℃，如25℃、26℃、27℃、28℃、29℃、30℃等；发酵 时间优选为7～60天，其中，选用液体真菌发酵培养基的培养时间优选为7～15天，如7、8、9、10、11、12、13、14、15天等；选用固体真菌发酵培养基的培养时间优选为20～60天，如20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60天等。此外，所述土曲霉可以采用现有的各种方式获得，通常通过商购获得。此外，所述真菌培养基的种类没有特别的限定，可以为本领域的常规选择，例如，可以为以谷物淀粉(玉米、马铃薯、木薯淀粉)、葡萄糖、糖蜜、氨水、铵盐或硝酸盐、尿素、玉米浆、豆饼粉、花生饼粉、鱼粉、酵母浸出膏等常用氮源、碳源中的至少一种作为碳氮源的培养基。
步骤S2中，将步骤S1所得土曲霉发酵物采用酯类溶剂进行萃取，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物。其中，所述萃取的方式只要能够将QF-1化合物与大部分杂质分离，QF-1进入萃取液，杂质进入萃余液即可。在一种优选实施方式中，所述萃取的方式为将土曲霉发酵物采用等体积的酯类有机溶剂超声萃取2～4次(如2、3、4次)，每次各自独立地萃取20～40min(如20、22、25、28、30、32、35、38、40min)，之后合并萃取液并蒸干，得到酯溶性土曲霉粗提物。此外，所述酯类有机溶剂的具体实例包括但不限于：乙酸甲酯、乙酸乙酯、乙酸丁酯和乙酸苯酯中的至少一种。
步骤S3中，将步骤S2所得酯溶性土曲霉粗提物进行硅胶色谱分离，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物。其中，所述硅胶色谱分离所采用的硅胶色谱柱中装填的硅胶填料的粒径可以为70～230目、230～400目、60～100目、100～200目或200～300目，优选为200～300目。所采用的洗脱溶剂只要能够使得QF-1与杂质分离即可，优选为二氯甲烷和丙酮按照体积比100:(1～10)(如100:1、100:2、100:3、100:4、100:5、100:6、100:7、100:8、100:9、100:10)的混合溶剂。此外，将溶剂去除的方式例如可以为旋蒸、冻干等，没有特别的限定。
步骤S4中，将步骤S3所得QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温，将所得上清液加蒸馏水后离心分离，所述离心分离所得固体产物即为 QF-1化合物。具体地，将所述QF-1粗提物溶于甲醇中，再将所得QF-1甲醇溶液边加热边加入水形成甲醇-水过饱和溶液，之后放入缓慢冷却至0～5℃，吸取上清液加等体积蒸馏水后离心分离，所述离心分离所得固体产物即得QF-1化合物。此步骤利用QF-1化合物及相关杂质在良溶剂甲醇和不良溶剂水中溶解度的不同的原理，将QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温后，由于杂质溶解度降低而析出沉淀，以实现QF-1与杂质的进一步分离，从而提高QF-1产物的纯度。其中，所述加热的条件优选使所得过饱和溶液的温度为50～55℃。此外，所述甲醇的用量优选恰好使得QF-1粗提物完全溶解，如此所得QF-1产物的纯度更佳。所述缓慢冷却在低温环境中进行，例如，在0～5℃下进行。
以下将通过实施例对本发明进行详细描述。
以下各实施例和对比例中，产物的总收率＝目标产物实际产量/目标产物理论产量×100％，其中，目标产物理论产量由HPLC谱图含量比例得出。
实施例1
S1、将土曲霉(购自上海保藏生物技术中心，ATCC 34571)在大米培养基(由大米和水按照质量比1:1.5组成)中于25℃下发酵45天，得到土曲霉发酵物。
S2、将步骤S1所得土曲霉发酵物采用等体积的乙酸乙酯超声萃取三次，每次30min，所得萃取液合并后用旋转蒸发仪蒸干，得到酯溶性土曲霉粗提物。
S3、将步骤S2所得酯溶性土曲霉粗提物经200～300目的硅胶色谱分离，以体积配比为100:3的二氯甲烷-丙酮混合溶液作为洗脱剂进行洗脱，收集QF-1富集液，将其用旋转蒸发仪蒸干，得到QF-1粗提物。经HPLC分析，该QF-1粗提物中含有纯度为70％的QF-1化合物。
S4、将步骤S3所得QF-1粗提物1g溶于1ml甲醇中，边加热边加入1.6ml蒸馏水形成温度为50℃的甲醇-水过饱和溶液，之后放入4℃冰箱中缓慢冷却，30min后吸出上清液；下层沉淀继续加入200μl甲醇，边加热边加入400μl蒸馏水形成温度为50℃的甲醇-水过饱和溶液，放入4℃冰箱缓慢降温，30min 后吸出上清液，将下层沉淀采用该步骤重复操作三次；将所有上清液合并，加等体积蒸馏水后离心分离，得到240mg浅黄色的沉淀物，即为QF-1固体，经HPLC检测，如图1所示，其纯度为95.53％，收率为34.29％。该QF-1固体的 1H NMR图谱和 13C NMR图谱分别如图2和图3所示，从图2和图3可以看出，QF-1化合物确实具有式(1)所示的结构。
实施例2
S1、将土曲霉(购自中国微生物保藏中心，TS342803)在马铃薯葡萄糖肉汤培养基(由马铃薯浸粉5g、蛋白胨10g、葡萄糖15g、氯化钠5g和Distilled water(蒸馏水)1L组成)中于25℃下发酵14天，得到土曲霉发酵物。
S2、将步骤S1所得土曲霉发酵物采用等体积的乙酸乙酯超声萃取三次，每次30min，所得萃取液合并后用旋转蒸发仪蒸干，得到酯溶性土曲霉粗提物。
S3、将步骤S2所得酯溶性土曲霉粗提物经200～300目的硅胶色谱分离，以体积配比为100:5的二氯甲烷-丙酮混合溶液梯度作为洗脱剂进行洗脱，收集QF-1富集液，将其用旋转蒸发仪蒸干，得到QF-1粗提物。经HPLC分析，该QF-1粗提物中含有纯度为72％的QF-1化合物。
S4、将步骤S3所得QF-1粗提物1g溶于1ml甲醇中，边加热边加入1.5ml蒸馏水形成温度为55℃的甲醇-水过饱和溶液，之后放入4℃冰箱中缓慢冷却，30min后吸出上清液；下层沉淀继续加入200μl甲醇，边加热边加入400μl蒸馏水形成温度为55℃的甲醇-水过饱和溶液，放入4℃冰箱缓慢降温，30min后吸出上清液，将下层沉淀采用该步骤重复操作三次；将所有上清液合并，加等体积蒸馏水后离心分离，得到245mg浅黄色的沉淀物，即为QF-1固体，经HPLC检测，如图4所示，其纯度为96.34％，收率为34.03％。
实施例3
S1、将土曲霉(购自中国微生物保藏中心，TS364823)在Peotone Glucose Yeast extract Medium PGY培养基(由Peptone(蛋白胨)10g、Yeast extract(酵母膏)5g、Glucose(葡萄糖)1g、Distilled water(蒸馏水)1L组成)中于25℃ 下发酵10天，得到土曲霉发酵物。
S2、将步骤S1所得土曲霉发酵物采用等体积的乙酸乙酯超声萃取三次，每次30min，所得萃取液合并后用旋转蒸发仪蒸干，得到酯溶性土曲霉粗提物。
S3、将步骤S2所得酯溶性土曲霉粗提物经200～300目的硅胶色谱分离，以体积配比为100:4的二氯甲烷-丙酮混合溶液梯度作为洗脱剂进行洗脱，收集QF-1富集液，将其用旋转蒸发仪蒸干，得到QF-1粗提物。经HPLC分析，其该QF-1粗提物中含有纯度为72％的QF-1化合物。
S4、将步骤S3所得QF-1粗提物1g溶于1ml甲醇中，边加热边加入1.6ml蒸馏水形成温度为50℃的甲醇-水过饱和溶液，之后放入4℃冰箱中缓慢冷却，30min后吸出上清液；下层沉淀继续加入200μl甲醇，边加热边加入400μl蒸馏水形成温度为50℃的甲醇-水过饱和溶液，放入4℃冰箱缓慢降温，30min后吸出上清液，将下层沉淀采用该步骤重复操作三次；将所有上清液合并，加等体积蒸馏水后离心分离，得到250mg浅黄色的沉淀物，即为QF-1固体，经HPLC检测，如图5所示，其纯度为95.48％，收率为34.72％。
从以上结果可以看出，本发明提供的制备工艺起始原料廉价易得，所得QF-1易于富集且后处理简单，环境友好，纯度和总收率均较高，利于工业化生产。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (9)

  1. 一种QF-1化合物的制备方法，所述QF-1化合物具有式(1)所示的结构，其特征在于，该方法包括以下步骤：
    S1、将土曲霉在真菌培养基中进行发酵，得到土曲霉发酵物；
    S2、将步骤S1所得土曲霉发酵物采用酯类溶剂进行萃取，所得萃取液经浓缩后得到酯溶性土曲霉粗提物；
    S3、将步骤S2所得酯溶性土曲霉粗提物进行硅胶色谱分离，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物；
    S4、将步骤S3所得QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温，将所得上清液加蒸馏水后离心分离，所述离心分离所得固体产物即为QF-1化合物；
    Figure PCTCN2022086840-appb-100001
  2. 根据权利要求1所述的QF-1化合物的制备方法，其特征在于，步骤S1中，所述真菌培养基为液体真菌发酵培养基或固体真菌发酵培养基；所述发酵的条件包括：发酵温度为25～30℃，当所述真菌培养基为液体真菌发酵培养基时发酵时间为7～15天，当所述真菌培养基为固体真菌发酵培养基时发酵时间为20～60天。
  3. 根据权利要求1所述的QF-1化合物的制备方法，其特征在于，步骤S2中，所述萃取的方式为将土曲霉发酵物采用等体积的酯类溶剂超声萃取2～4次，每次各自独立地萃取20～40min，之后合并萃取液并蒸干，得到酯溶性土曲霉粗提物。
  4. 根据权利要求1所述的QF-1化合物的制备方法，其特征在于，步骤S2中，所述酯类溶剂选自乙酸甲酯、乙酸乙酯、乙酸丁酯和乙酸苯酯中的至少一种。
  5. 根据权利要求1所述的QF-1化合物的制备方法，其特征在于，步骤 S3中，所述硅胶色谱分离所采用的硅胶填料的粒径为200～300目。
  6. 根据权利要求1所述的QF-1化合物的制备方法，其特征在于，步骤S3中，所述硅胶色谱分离所采用的洗脱溶剂为二氯甲烷和丙酮按照体积比100:(1～10)的混合溶剂。
  7. 根据权利要求1～6中任意一项所述的QF-1化合物的制备方法，其特征在于，步骤S4中，将所述QF-1粗提物溶于甲醇中，再将所得QF-1甲醇溶液边加热边加入水形成甲醇-水过饱和溶液，之后置于低温环境中缓慢冷却至0～5℃，吸取上清液加等体积蒸馏水后离心分离，所述离心分离所得固体产物即得QF-1化合物。
  8. 根据权利要求7所述的QF-1化合物的制备方法，其特征在于，步骤S4中，所述加热的条件使所得过饱和溶液的温度为50～55℃；所述甲醇的用量恰好使得QF-1粗提物完全溶解。
  9. 由权利要求1～8中任意一项所述的方法制备得到的QF-1化合物。
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