WO2023240679A1 - 一对具有抗炎活性的聚酮类化合物及其制备方法和应用 - Google Patents

一对具有抗炎活性的聚酮类化合物及其制备方法和应用 Download PDF

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WO2023240679A1
WO2023240679A1 PCT/CN2022/101720 CN2022101720W WO2023240679A1 WO 2023240679 A1 WO2023240679 A1 WO 2023240679A1 CN 2022101720 W CN2022101720 W CN 2022101720W WO 2023240679 A1 WO2023240679 A1 WO 2023240679A1
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methanol
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卢轩
唐小渊
唐川
冯伟星
冯宝民
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大连大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
    • C07D313/02—Seven-membered rings
    • C07D313/06—Seven-membered rings condensed with carbocyclic rings or ring systems
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • the invention belongs to the field of medical technology, and specifically relates to a pair of new skeleton polyketide epimers with anti-inflammatory activity and their preparation methods and applications.
  • Medicinal plant endophytes have diverse characteristics, and each endophyte has abundant secondary metabolites. Many biologically active alkaloids, polypeptides, and polyketides have been discovered from the secondary metabolites of endophytes. These secondary metabolites not only have the same or similar biological activities as those of the host plant, such as antibacterial, anti-inflammatory, anti-tumor, anti-hyperglycemia, anti-parasitic, etc., but also have many new biological activities. Therefore, the secondary metabolites of medicinal plant endophytes are a huge resource library of potential medicinal substances and have good development prospects.
  • the purpose of the present invention is to provide a pair of polyketides with anti-inflammatory activity and their preparation methods and applications.
  • the present invention fully utilizes plant endophyte resources and collects them from The endophytic fungus Alternaria sp.
  • HJT-Y7 was isolated from the leaves of Rhodiola tibetica in Mongolia. It was deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on May 6, 2022, with the deposit number It is CGMCC No. 40166, and the depository address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the obtained Alternaria sp. HJT-Y7 was solid fermented and used a series of separation and purification methods. The method separated and obtained a pair of polyketide epimers with novel structures, which provided new lead compounds for drug development.
  • the present invention provides a pair of polyketide compounds or pharmaceutically acceptable salts thereof.
  • the structure of the polyketide compound is as follows:
  • the present invention provides an endophytic fungus strain of the genus Alternaria sp. HJT-Y7, which was deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on May 6, 2022, and the deposit number is CGMCC No. 40166, the address of the depository is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • Another aspect of the present invention provides a method for preparing the polyketide compound, which mainly includes the following steps:
  • components 7-1 to 7-10 were obtained in sequence; component 7-6 was loaded onto the Sephadex LH-20 gel column.
  • the mobile phase is a mixture of methylene chloride/methanol with a volume ratio of 1:1.
  • Components 7-6-1 ⁇ 7-6-6 are obtained in sequence.
  • Component 7-6-3 is loaded into the Agilent C18 high performance liquid phase. Chromatography, the mobile phase was 55% methanol aqueous solution, and compound 1 and compound 2 were obtained.
  • step (1) the shaking culture conditions described in step (1) are 20-30°C, 100-200 r/min.
  • the rice solid culture medium described in step (1) is prepared from rice and purified water at a mass-to-volume ratio of 80:80 to 120g/ml.
  • composition of the fungal No. 4 culture medium described in step (1) is: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH 2 PO 4 , 0.03% MgSO 4 ⁇ 7H 2 O, 0.1% corn steep liquor, deionized water.
  • volume ratio of chloroform/methanol during the gradient elution process described in step (2) is specifically 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 0:100 .
  • volume ratio of petroleum ether/ethyl acetate during the gradient elution process described in step (2) is specifically 10:1, 10:3, 10:5, 10:6, 10:7, and 10:9.
  • Another aspect of the present invention provides a pharmaceutical composition, which contains the polyketide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
  • the present invention also provides the use of the polyketide compound or its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of anti-inflammatory drugs.
  • the present invention makes full use of plant endophyte resources, conducts research on plant endophytes, searches for new polyketide active compounds, and conducts anti-inflammatory activity experiments on them.
  • Compound 1 and Compound 2 show significant differences in the concentrations of Compound 1 and Compound 2 at each concentration. Both of them inhibited the release of NO to varying degrees, and at a concentration of 5 ⁇ M, they all showed better inhibitory capabilities than the positive control group, indicating that compound 1 and compound 2 have excellent anti-inflammatory effects.
  • Figure 1 is a 1 H-NMR pattern of compound 1 isolated in Example 1;
  • Figure 2 is a 13 C-NMR chart of compound 1 isolated in Example 1;
  • Figure 3 is the HSQC chart of Compound 1 isolated in Example 1;
  • Figure 4 is the HMBC chart of compound 1 isolated in Example 1;
  • Figure 5 is a NOESY diagram of compound 1 isolated in Example 1;
  • Figure 6 is a 1 H-NMR pattern of compound 2 isolated in Example 1;
  • Figure 7 is a 13 C-NMR pattern of compound 2 isolated in Example 1;
  • Figure 8 is the HSQC chart of compound 2 isolated in Example 1;
  • Figure 9 is the HMBC chart of compound 2 isolated in Example 1;
  • Figure 10 is a NOESY diagram of compound 2 isolated in Example 1;
  • Figure 11 is a CD chart of compound 1-2 isolated in Example 1;
  • Figure 12 is a DP4+ diagram of compound 1-2 isolated in Example 1;
  • Figure 13 is a graph showing the anti-inflammatory effect of compound 1-2 isolated in Example 1.
  • the preparation method of a pair of polyketide compounds mainly includes the following steps:
  • Bacterial fermentation Alternaria sp. HJT-Y7, an endophytic fungus collected from the leaves of Rhodiola rosea in Vietnam, was used in fungus No. 4 culture medium (2% mannitol, 2% glucose, 0.5% Yeast extract, 1% peptone, 0.05% KH 2 PO 4 , 0.03% MgSO 4 ⁇ 7H 2 O, 0.1% corn steep liquor, deionized water) was used for shaking culture.
  • the culture conditions were 28°C, 180r/min, and then the shake cultured
  • the fermentation broth and mycelium are inoculated into an Erlenmeyer flask containing rice solid culture medium (rice and purified water are prepared at a mass-volume ratio of 80g:110mL) for static fermentation and culture for 40 days.
  • the culture temperature is 28°C to obtain a solid fermentation product. ;
  • step (2) Extraction and separation of metabolites:
  • the solid fermentation product obtained in step (1) is ultrasonically extracted with an equal volume of methanol, filtered through 8 layers of gauze, the extract is separated from mycelium and rice, and the extract is concentrated. Load the sample to a silica gel chromatographic column for separation. Use 300-400 mesh silica gel as the stationary phase and chloroform/methanol as the mobile phase for gradient elution.
  • RAW 264.7 macrophages were inoculated in high-glucose DMEM medium containing 10% fetal calf serum ( ⁇ 0.5EU/mL), 1% penicillin and streptomycin, and placed at 37 °C, 5% CO2 incubator. When the cell coverage reaches about 90%, discard the supernatant and add 37°C medium directly for passage by pipetting. Cells in logarithmic phase were harvested for subsequent experiments.
  • RAW 264.7 cells growing in the logarithmic phase were seeded into a 96-well cell culture plate at 1 ⁇ 10 4 cells per well. After the cells adhered for 24 hours, a normal control was set group, LPS model group, positive control group and compound group, each group had 6 duplicate holes. The cells in the normal control group and LPS model group were first incubated with serum-free DMEM medium. The compound group was incubated with serum-free DMEM containing compounds 1 and 2 at final concentrations of 40.00, 20.00, 10.00, and 5 ⁇ M.
  • the cells were incubated in serum-free DMEM with 200 ⁇ M L-NAME (nitric oxide synthase inhibitor); after incubating the cells for 4 hours according to the above treatment method, except for the normal control group, which continued to be incubated with serum-free DMEM, the final concentration was added to the remaining groups.
  • For the supernatant add 150 ⁇ L DMSO to each well, shake for 10 min, and measure the absorbance of each well at 570 nm.

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Abstract

公开了一对具有抗炎活性的聚酮类化合物及其制备方法和应用，属于医药技术领域。具体地，从采集自西藏红景天Rhodiola tibetica的叶片部位分离获得链格孢属内生真菌（Alternaria sp.）HJT-Y7，对获得的链格孢属内生真菌（Alternaria sp.）HJT-Y7进行固体发酵，然后通过一系列分离纯化方法分离获得2个新的聚酮类化合物，这2个聚酮类化合物均具有良好的抗炎活性，为抗炎药物的开发提供了新的途径。

Description

一对具有抗炎活性的聚酮类化合物及其制备方法和应用 技术领域
本发明属于医药技术领域，具体涉及一对具有抗炎活性的新骨架聚酮类差向异构体及其制备方法和应用。
背景技术
药用植物内生菌具有多样性特征，每个内生菌又有丰富的次级代谢产物，目前已从内生菌次级代谢产物中发现了许多具有生物活性的生物碱、多肽、聚酮类、萜类等物质，这些次级代谢物不但具有与宿主植物相同或相似的生物活性，例如抗菌、抗炎、抗肿瘤、抗高血糖、抗寄生虫等，还具有许多新的生物活性。因此药用植物内生菌的次级代谢产物是庞大的潜在药用物质资源库，具有良好的开发前景。
发明内容
鉴于此，本发明的目的是提供了一对具有抗炎活性的聚酮类化合物及其制备方法和应用，本发明通过对植物内生菌进行研究，充分的利用植物内生菌资源，从采集自西藏红景天Rhodiola tibetica的叶片部位分离获得链格孢属内生真菌(Alternaria sp.)HJT-Y7，于2022年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.40166，保藏单位地址为北京市朝阳区北辰西路一号院三号，对获得的链格孢属内生真菌(Alternaria sp.)HJT-Y7进行固体发酵，使用一系列分离纯化方法分离获得一对结构新颖的聚酮类差向异构体，为药物开发提供了新的先导化合物。
本发明目的是通过以下方式实现：
本发明提供一对聚酮类化合物或其药学上可接受的盐，所述的聚酮类化合物的结构如下所示：
Figure PCTCN2022101720-appb-000001
本发明另一方面提供一株链格孢属内生真菌(Alternaria sp.)HJT-Y7，于2022 年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.40166，保藏单位地址为北京市朝阳区北辰西路一号院三号。
本发明另一方面提供所述的聚酮类化合物的制备方法，主要包括如下步骤：
(1)将上述的链格孢属内生真菌(Alternaria sp.)HJT-Y7接种到真菌4号培养基中进行震荡培养，将所得的发酵液连同菌丝体接种于大米固体培养基中，20～30℃下静止发酵10～40天，得到固体发酵产物；
(2)将步骤(1)得到的固体发酵产物使用甲醇进行超声提取，过滤，所得的提取液上样至硅胶色谱柱，梯度洗脱，流动相为氯仿/甲醇，氯仿/甲醇的体积比由100:0梯度降低至0：100，根据TLC结果，依次得到组份1-16；将组份7上样至硅胶色谱柱，梯度洗脱，流动相为石油醚/乙酸乙酯，石油醚/乙酸乙酯的体积比由10：1梯度降低至10：9，根据TLC结果，依次得到组份7-1～7-10；将组份7-6上样至Sephadex LH-20凝胶柱，流动相为二氯甲烷/甲醇体积比为1：1的混合液，依次得到组份7-6-1～7-6-6，将组份7-6-3上样至Agilent C18高效液相色谱，流动相为55％甲醇水溶液，得到化合物1和化合物2。
进一步的，步骤(1)中所述的震荡培养的条件为20～30℃，100～200r/min。
进一步的，步骤(1)中所述的大米固体培养基是由大米和纯净水按质量体积比为80:80～120g/ml配制而成。
进一步地，步骤(1)中所述的真菌4号培养基组成为：2％甘露醇，2％葡萄糖，0.5％酵母膏，1％蛋白胨0.05％KH 2PO 4，0.03％MgSO 4·7H 2O，0.1％玉米浆，去离子水。
进一步地，步骤(2)中所述梯度洗脱过程中氯仿/甲醇的体积比具体为100:0、100:1、100：2、100：3、100：5、100：10、0：100。
进一步地，步骤(2)中所述梯度洗脱过程中石油醚/乙酸乙酯的体积比具体为10：1、10：3、10：5、10：6、10：7、10：9。
本发明另一方面提供了一种药物组合物，其包含所述的聚酮类化合物或其药学上可接受的盐与药学上可接受的辅料。
本发明还提供了所述聚酮类化合物或其药学上可接受的盐或所述的药物组合物在制备抗炎药物中的应用。
本发明相对于现有技术具有的有益效果如下：
本发明充分利用植物内生菌资源，对植物内生菌进行研究，寻找新的聚酮类活性化合物，并对其进行抗炎活性实验，与模型组相比，化合物1和化合物2在各浓度下 均对NO的释放有不同程度的抑制，在5μM的浓度下均表现出比阳性对照组更优的抑制能力，表明化合物1与化合物2具有优良的抗炎效果。
附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为实施例1分离的化合物1的 1H-NMR图；
图2为实施例1分离的化合物1的 13C-NMR图；
图3为实施例1分离的化合物1的HSQC图；
图4为实施例1分离的化合物1的HMBC图；
图5为实施例1分离的化合物1的NOESY图；
图6为实施例1分离的化合物2的 1H-NMR图；
图7为实施例1分离的化合物2的 13C-NMR图；
图8为实施例1分离的化合物2的HSQC图；
图9为实施例1分离的化合物2的HMBC图；
图10为实施例1分离的化合物2的NOESY图；
图11为实施例1分离的化合物1-2的CD图；
图12为实施例1分离的化合物1-2的DP4+图；
图13为实施例1分离的化合物1-2的抗炎效果图。
具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。
实施例1
一对聚酮类化合物的制备方法，主要包括如下步骤：
(1)菌种发酵：将采集自西藏红景天叶片部位的链格孢属内生真菌(Alternaria sp.)HJT-Y7采用真菌4号培养基(2％甘露醇，2％葡萄糖，0.5％酵母膏，1％蛋白胨0.05％KH 2PO 4，0.03％MgSO 4·7H 2O，0.1％玉米浆，去离子水配制)进行震荡培养，培养条件28℃，180r/min，再将震荡培养的发酵液连同菌丝体接种于装有大米固体培养 基(大米和纯净水按质量体积比为80g：110mL配制)的锥形瓶中静止发酵培养40天，培养温度为28℃，得到固体发酵产物；
(2)代谢产物的提取分离：将步骤(1)得到的固体发酵产物使用等体积甲醇进行超声提取，经8层纱布过滤，将提取液与菌丝体及大米分离，浓缩提取液，提取液上样至硅胶色谱柱进行分离，以300-400目硅胶为固定相，以氯仿/甲醇为流动相进行梯度洗脱，梯度依次为氯仿：甲醇＝100:0、100:1、100：2、100：3、100：5、100：10、0：100(v/v)，洗脱剂流速为30ml/min，每个梯度洗脱10000mL，收集洗脱液(每500ml收集一瓶)，将洗脱液浓缩，根据TLC结果合瓶，依次得到1-16组份；将组份7上样至硅胶色谱柱，以200-300目硅胶为固定相，流动相为石油醚/乙酸乙酯，梯度依次为石油醚：乙酸乙酯＝10：1、10：3、10：5、10：6、10：7、10：9(v/v)，洗脱剂流速为30ml/min，收集洗脱液(每500ml收集一瓶)，将洗脱液浓缩，根据TLC结果合瓶，依次得到7-1～7-10组份；将组份7-6上样至Sephadex LH-20凝胶柱，流动相为二氯甲烷/甲醇＝1：1(v/v)，洗脱剂流速为0.3ml/min，收集洗脱液(每10ml收集一次)，每四份洗脱液合并，依次得到7-6-1～7-6-6组份，将组份7-6-3上样至Agilent C18高效液相色谱，流动性为55％甲醇水溶液，流速为3mL/min，检测器为210nm紫外光检测器，保留时间59min下，得到单体化合物1，保留时间62min下，得到单体化合物2。
(3)结构鉴定：以氘代二甲基亚砜为溶剂，使用Bruker AvanceⅡ500M核磁共振仪测定分离得到的化合物1-2的核磁图谱并利用质谱推断分子式；使用圆二色谱仪J-810-150S检测化合物1-2的圆二色光谱；使用DP4+软件对化合物1-2进行空间结构计算，结合核磁图谱、CD结果与计算最终对产物结构进行表征。
化合物1的光谱数据如下：
1H-NMR(500Hz,DMSO-d 6)δ：3.14(1H,d,J＝13.5Hz,H-1a),4.17(1H,d,J＝13.5Hz,H-1b),9.45(1H,s,H-3OH),6.63(1H,d,J＝8.0Hz,H-4),7.00(1H,t,J＝8.0Hz,H-5),6.72(1H,d,J＝8.0Hz,H-6),6.56(1H,d,J＝12.5Hz,H-8),6.01(1H,d,J＝12.5Hz,H-9),3.72(1H,q,J＝6.0Hz,H-11),0.87(3H,d,J＝6.0Hz,H-12),5.04(1H,d,J＝12.5Hz,H-1'a),5.23(1H,d,J＝12.5Hz,H-1'b),9.67(1H,s,H-3'OH),6.83(1H,d,J＝8.0Hz,H-4'),7.05(1H,t,J＝8.0Hz,H-5'),6.76(1H,d,J＝8.0Hz,H-6'),4.04(1H,d,J＝10.0Hz,H-8'),4.54(1H,d,J＝10.0Hz,H-9'),3.66(1H,s,H-10'),2.17(3H,s,H-12')。
13C-NMR(125Hz,DMSO-d 6)δ：59.5(C-1),126.8(C-2),153.3(C-3),114.4(C-4),128.3(C-5),121.2(C-6),137.4(C-7),131.1(C-8),137.1(C-9),93.7(C-10),81.7(C-11), 12.9(C-12),61.0(C-1'),122.8(C-2'),156.1(C-3'),115.3(C-4'),128.6(C-5'),123.3(C-6'),139.3(C-7'),59.8(C-8'),79.3(C-9'),79.7(C-10'),209.5(C-11'),28.0(C-12')。
化合物1的HMBC谱结果如下：
Figure PCTCN2022101720-appb-000002
化合物1的NOESY谱结果如下：
Figure PCTCN2022101720-appb-000003
化合物2的光谱数据如下：
1H-NMR(500Hz,DMSO-d 6)δ：4.33(1H,d,J＝13.5Hz,H-1a),5.11(1H,d,J＝13.5Hz,H-1b),9.62(1H,s,H-3OH),6.83(1H,d,J＝8.0Hz,H-4),7.16(1H,t,J＝8.0Hz,H-5),6.85(1H,d,J＝8.0Hz),6.63(1H,d,J＝12.5Hz,H-8),5.85(1H,d,J＝12.5Hz,H-9),4.31(1H,q,J＝6.5Hz,H-11),1.31(3H,d,J＝6.5Hz,H-12),4.33(1H,d,J＝13.5Hz,H-1'a),5.27(1H,d,J＝13.5Hz,H-1'b),9.40(1H,s,H-3'OH),6.58(1H,d,J＝7.8Hz,H-4'),6.53(1H,t,J＝7.8Hz,H-5'),6.82(1H,d,J＝7.8Hz,H-6'),3.84(1H,d,J＝10.0Hz,H-8'),3.75(1H,d,J＝10.0Hz,H-9'),4.01(1H,s,H-10'),2.14(3H,s,H-12')。
13C-NMR(125Hz,DMSO-d 6)δ：59.1(C-1),126.0(C-2),154.0(C-3),115.2(C-4),128.6(C-5),122.1(C-6),137.5(C-7),131.6(C-8),132.9(C-9),92.5(C-10),84.6(C-11),20.7(C-12),64.0(C-1'),126.4(C-2'),154.4(C-3'),113.8(C-4'),127.9(C-5'),119.8(C-6'),136.7(C-7'),52.5(C-8'),78.7(C-9'),91.5(C-10'),205.3(C-11'),27.7(C-12').
化合物2的HMBC谱结果如下：
Figure PCTCN2022101720-appb-000004
化合物2的NOESY谱结果如下：
Figure PCTCN2022101720-appb-000005
实施例2
实施例1制备的化合物1-2的抗炎活性研究
小鼠RAW 264.7巨噬细胞的培养：将RAW 264.7巨噬细胞接种在含10％胎牛血清(＜0.5EU/mL)、1％青霉素和链霉素的高糖DMEM培养基中，置于37℃、5％CO 2培养箱内培养。当细胞覆盖率达到约90％时，弃掉上清，直接加入37℃培养基吹打传代。取对数期细胞用于后续实验。
LPS诱导的RAW 264.7巨噬细胞炎症模型的建立：取对数期生长的RAW 264.7细胞以每孔1×10 4个细胞接种于96孔细胞培养板中，待24h细胞贴壁后，设置正常对照组、LPS模型组、阳性对照组和以及化合物组，每组6个复孔。正常对照组和LPS模型组先以无血清DMEM培养基孵育细胞，化合物组分别以含终浓度为40.00、20.00、10.00、5μM化合物1、2的无血清DMEM孵育细胞，阳性对照组以含终浓度为200μM的L-NAME(一氧化氮合酶抑制剂)的无血清DMEM孵育细胞；按上述处理方式孵育细胞4h后，除正常对照组采用无血清DMEM继续孵育外，其余各组再加入终浓度为1μg/mL LPS刺激细胞，24h后收集50μL各孔培养基，运用一氧化氮检测试剂盒检测NO的分泌情况，同时在每孔中加入10μl浓度为5g/L的MTT，继续培养4h后弃上清，每孔加入150μL DMSO，震荡10min，在570nm处测量各孔的吸光度。
MTT结果显示实施例1制备的化合物1-2的IC 50>100μM，抗炎实验结果如图13 所示，与模型组相比，化合物1和化合物2在各浓度下均对NO的释放有不同程度的抑制，在5μM的浓度下均表现出比阳性对照组更优的抑制能力，表明化合物1与化合物2具有优良的抗炎效果。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

  1. 一对聚酮类化合物或其药学上可接受的盐，其特征在于，所述的聚酮类化合物的结构如下所示：
    Figure PCTCN2022101720-appb-100001
  2. 一株链格孢属内生真菌(Alternaria sp.)HJT-Y7，于2022年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.40166，保藏单位地址为北京市朝阳区北辰西路一号院三号。
  3. 权利要求1所述的聚酮类化合物的制备方法，其特征在于，主要包括如下步骤：
    (1)将权利要求2所述的链格孢属内生真菌(Alternaria sp.)HJT-Y7接种到真菌4号培养基中进行震荡培养，将所得的发酵液连同菌丝体接种于大米固体培养基中，20～30℃下静止发酵10～40天，得到固体发酵产物；
    (2)将步骤(1)得到的固体发酵产物使用甲醇进行提取，过滤，所得的提取液上样至硅胶色谱柱，梯度洗脱，流动相为氯仿/甲醇，氯仿/甲醇的体积比由100:0梯度降低至0：100，根据TLC结果，依次得到组份1-16；将组份7上样至硅胶色谱柱，梯度洗脱，流动相为石油醚/乙酸乙酯，石油醚/乙酸乙酯的体积比由10：1梯度降低至10：9，根据TLC结果，依次得到组份7-1～7-10；将组份7-6上样至Sephadex LH-20凝胶柱，流动相为二氯甲烷/甲醇体积比为1：1的混合液，依次得到组份7-6-1～7-6-6，将组份7-6-3上样至Agilent C18高效液相色谱，流动相为55％甲醇水溶液，得到化合物1和化合物2。
  4. 根据权利要求3所述的制备方法，其特征在于，步骤(1)中所述的震荡培养的条件为20～30℃，100～200r/min。
  5. 根据权利要求3所述的制备方法，其特征在于，步骤(1)中所述的大米固体培养基是由大米和纯净水按质量体积比为80:80～120g/ml配制而成。
  6. 根据权利要求3所述的制备方法，其特征在于，步骤(1)中所述的真菌4号培养基组成为：2％甘露醇，2％葡萄糖，0.5％酵母膏，1％蛋白胨0.05％KH 2PO 4，0.03％MgSO 4·7H 2O，0.1％玉米浆，去离子水。
  7. 根据权利要求3所述的制备方法，其特征在于，步骤(2)中所述梯度洗脱过程中氯仿/甲醇的体积比具体为100:0、100:1、100：2、100：3、100：5、100：10、0：100。
  8. 根据权利要求3所述的制备方法，其特征在于，步骤(2)中所述梯度洗脱过程中石油醚/乙酸乙酯的体积比具体为10：1、10：3、10：5、10：6、10：7、10：9。
  9. 一种药物组合物，其特征在于，包含权利要求1所述的聚酮类化合物或其药学上可接受的盐与药学上可接受的辅料。
  10. 权利要求1所述的聚酮类化合物或其药学上可接受的盐或权利要求9所述的药物组合物在制备抗炎药物中的应用。
PCT/CN2022/101720 2022-06-13 2022-06-28 一对具有抗炎活性的聚酮类化合物及其制备方法和应用 WO2023240679A1 (zh)

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