CN115746076A - 一种紫芝发酵菌丝体中三萜化合物及其抗肿瘤应用 - Google Patents
一种紫芝发酵菌丝体中三萜化合物及其抗肿瘤应用 Download PDFInfo
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Abstract
本发明提供了一种紫芝发酵菌丝体中三萜化合物，该三萜化合物的结构式为结构式2、4、5、8、10、11和12。本发明还提供了紫芝菌丝体中三萜化合物作为预防或治疗肿瘤药物的应用；所述的紫芝发酵菌丝体中三萜化合物为结构式1‑12的化合物中的一种或一种以上。本发明首次对紫芝发酵菌丝体中分离纯化的三萜化合物进行了抗肿瘤活性测试，发现结构式1‑12的化合物具有不同程度的抑制人白血病细胞K562、人肺癌细胞A549、人肝癌细胞HepG2增殖的作用，特别是化合物7、8、9抑制肿瘤细胞的增殖作用具有突出的效果。
Description
技术领域
本发明涉及食用菌应用领域，具体的说涉及一种紫芝发酵菌丝体中三萜化合物及其抗肿瘤应用。
背景技术
“灵芝”是一类著名的药用真菌的统称，在我国利用历史悠久，也被称作“仙药”、“瑞草”。从科学的角度来看，“灵芝”是指灵芝属Ganoderma P.Karst.真菌，隶属于担子菌门Basidiomycota，蘑菇纲Agaricomycetes，多孔菌目Polyporales，灵芝科Ganodermataceae。目前，该属在全世界有95个物种，其中40个种在中国有发现，紫芝是其中的一种。
灵芝三萜是灵芝中主要的活性成分之一，具有抗肿瘤、抗炎、保肝护肝、抗氧化、降血糖、降血脂、改善睡眠等诸多功效。灵芝的发酵与其子实体的栽培相比，具有发酵时间短、条件可控、发酵产物独特的特性。近年来研究发现，灵芝发酵产物中会产生完全不同于灵芝子实体的三萜酸类化合物，此类化合物抗肿瘤活性远远优异于子实体三萜酸，有开发为抗肿瘤药的潜力(Lai HH,Huang CP,Chen DH,Chen KD,Lin SB,2009.Ganodermatriterpenoid ganoderic acid T inhibits growth and metastasis of A549 lungadenocarcinoma in vitro and in vivo.Journal of the Chinese Chemical Society,25(6),413-420；Tang W,2013.Ganoderic acid Me improves the sensitivity ofmultidrug resistant KB-A-1/Dox cells to doxorubicin.Science and Technology ofFood Industry,34(23):97-100)。
因此，从更多丰富的灵芝物种的发酵产物中寻找抗肿瘤药物或先导化合物，是灵芝资源开发的一个具有前景的方向。
发明内容
本发明首先提供了一种紫芝发酵菌丝体中三萜化合物，其结构式为如下结构式2、4、5、8、10、11和12的化合物或其药用盐、晶体或水合物：
本发明还提供了一种用于抗肿瘤的制剂，其含有结构式1-12所示的一种或一种以上化合物或其药用盐以及医学上可接受的载体；
其中所述的结构式1-12的化合物为：
结构式1-12的化合物其化学命名如下：
化合物1：3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid
化合物2：(22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oicacid
化合物3：(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid
化合物4：(22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid
化合物5：(22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid
化合物6：(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid
化合物7：(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid
化合物8：(22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid
化合物9：lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid
化合物10：(22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid
化合物11：(24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid
化合物12：(22S,24Z)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oicacid
本发明还提供了紫芝发酵菌丝体中三萜化合物作为预防或治疗肿瘤的药物的应用；其中所述的紫芝发酵菌丝体中三萜化合物为上述结构式1-12的化合物中的一种或一种以上；
本发明所提供的结构式1-12的化合物还可以作为用于预防和/或治疗肿瘤、或在肿瘤治疗过程之中或之后起辅助作用、或抑制癌细胞生长和/或增殖作用的制剂或食品；
所述肿瘤为白血病、肝癌或肺癌；所述肿瘤细胞可为人白血病细胞、人肝癌细胞、人肺癌细胞。
本发明提供的从紫芝菌丝体中分离出的12个三萜类化合物均具有抑制多种癌细胞增殖的作用，特别是化合物7、8、9抑制部分肿瘤细胞增殖的效果突出，具有广阔的应用前景。
附图说明
图1、Fr3液相色谱图
图2、Fr5液相色谱图
图3、Fr8液相色谱图
图4、Fr9液相色谱图
图5、Fr12液相色谱图
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
紫芝Ganoderma sinense，菌种现保存于北京林业大学生态与自然保护学院微生物所(菌种保藏编号:Dai20076)。
人白血病细胞K562购自中国科学院上海生命科学研究院细胞资源中心，产品目录号为SCSP-5054
人肝癌细胞HepG2购自中国科学院上海生命科学研究院细胞资源中心，产品目录号为SCSP-510。
人肺癌细胞A549购自中国科学院上海生命科学研究院细胞资源中心，产品目录号为SCSP-503。
DMEM培养基、胎牛血清(fetal bovineserum，FBS)：购自美国Gibco公司；
青霉素和链霉素：购自美国Amresco公司；
葡萄糖马铃薯琼脂(PDA)：购自北京索莱宝科技有限公司；
葡萄糖、蔗糖、琼脂、MgSO4·H2O、KH2PO4、CaSO4以及分析级甲醇、氯仿、石油醚、丙酮、乙醇、乙醇、乙腈、冰醋酸：均购自国药集团化学试剂有限公司；
色谱级甲醇、乙腈：购自上海安谱实验科技股份有限公司；
氘代甲醇：购自Sigma公司；
YMC*GEL C18硅胶(50μm，12nm)：北京元宝山色谱科技有限公司；
Zorbax Eclipse Plus C18色谱柱(5μm,4.6mm×250mm)：美国Agilent公司；
Zorbax Eclipse Plus-C18半制备色谱柱(5μm,21.2mm×250mm)：美国Agilent公司；
GF254硅胶板(0.2-0.25mm)：青岛海洋化工厂；
LC3000型高效液相色谱仪：北京创新通恒科技有限公司；
Agilent LC1290 infinity II超高效液相色谱仪：美国安捷伦公司；
Agilent 6495三重四级杆质谱仪：美国安捷伦公司；
BruBer AB II-600核磁共振波谱仪：德国布鲁克公司；
Synergy HT多功能酶标仪：美国BIO-TEK公司；
Thermo Scientific Q Exactive HF Orbitrap-FTMS高分辨质谱仪：美国赛默飞公司
实施例1紫芝菌丝体发酵
培养基配方(重量比)：可溶性淀粉3％，葡萄糖1.2％，酵母粉1％，MgSO4·7H2O0.15％，KH2PO4 0.3％，pH自然。
保存在4℃冰箱中的紫芝菌种，经过活化，黑暗条件下在摇瓶中进行一级振荡培养7d(150r/min，25℃)，后转接到二级摇瓶中静态培养21d，获得菌丝体。
实施例2紫芝菌丝体的提取和萃取
将干燥的菌丝体用95％乙醇的水溶液浸泡提取3次，每次24小时，合并提取液，浓缩得到醇提物。
进一步将醇提物用等体积的石油醚萃取三次，弃去石油醚相，再用等体积乙酸乙酯萃取剩余相三次，合并乙酸乙酯萃取液，减压浓缩干燥得到乙酸乙酯相。
实施例3化合物的制备
乙酸乙酯相通过液相系统分离，以YMC*GEL C18硅胶(50μm，12nm)为填料，以乙腈-0.01％冰醋酸水梯度洗脱，共得到60个流分，通过薄层色谱分析，合并薄层色谱板上具有相同比移值(Rf)的流份，共得到13个组分：Fr1-Fr13。
60个流分经薄层色谱分析后，合并为13个组分：Fr1(1-10，5.02g)、Fr2(11-14，1.14g)、Fr3(15-16，1.65g)、Fr4(17-18，2.68g)、Fr5(19-20，3.89g)、Fr6(21-22，3.95g)、Fr7(23-24，4.3g)、Fr8(25-33，24.88g)、Fr9(34-36，2.78g)、Fr10(36-38，2.46g)、Fr11(39-41，1.33g)、Fr12(42-46，1.72g)、Fr13(47-60，3.17g)。
将Fr1-Fr13进行HPLC检测，根据检测结果可知，Fr3、Fr5、Fr8、Fr9、Fr12富含三萜化合物的特征吸收峰，因此将Fr3、Fr5、Fr8、Fr9、Fr12进一步的分离纯化。
Fr3的分离纯化
选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈(A)-0.01％冰醋酸水进行梯度洗脱(0.0-18.0min，A：50％-50％；18.0-18.5min，A：50％-100％；18.5-23.0min，A：100％-100％)，240nm波长进行检测。反复制备多次，收集8.0min的色谱峰，得到Fr3-3；收集15.5min的色谱峰，得到Fr3-8。
Fr3-3用乙醇重结晶多次，获得化合物1(20.30mg)。Fr3-8选择Zorbax EclipsePlus-C18半制备色谱柱，以乙腈(A)-0.01％冰醋酸水梯度洗脱(0.0-12.0min，A：52％-52％；12.0-12.5min，A：52％-75％；12.5-20.0min，A：75％-75％；20.0-21.0min，A：75％-100％；21.0-26.0min，A：100％-100％)，240nm波长进行检测，反复制备多次，收集8.2min的色谱峰，得到化合物2(100.00mg)；收集18.0min的色谱峰，得到化合物3(140.00mg)。
Fr5的分离纯化
Fr5选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈(A)-0.01％冰醋酸水进行梯度洗脱(0.0-19.0min，A：80％-80％；19.0-19.5min，A：80％-83％；19.5-25.0min，A：83％-83％；25.0-25.5min，A：83％-100％；25.5-30.0min，A：100％-100％)，240nm波长进行检测。收集15.0min的色谱峰，得到Fr5-4；收集20.8min的色谱峰，得到化合物4(101.00mg)，收集23min的色谱峰，得到Fr5-6。
Fr5-4选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈(A)-0.01％冰醋酸水进行梯度洗脱(0.0-18.0min，A：53％-53％；18.0-18.5min，A：53％-79％；18.5-27.0min，A：79％-79％；27.0-28.0min，A：79％-100％；28.0-32.0min，A：100％-100％)，240nm波长进行检测，反复制备多次，收集12.2min的色谱峰，得到化合物5(100.00mg)，收集25.0min的色谱峰，得到化合物6(110.00mg)。
Fr5-6选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈(A)-0.01％冰醋酸水进行梯度洗脱(0.0-17.0min，A：89％-89％；17.0-17.5min，A：89％-100％；17.5-22.0min，A：100％-100％)，240nm波长进行检测。反复制备多次，收集15.0min的色谱峰，得到化合物7(280.00mg)。
Fr8分离纯化
Fr8选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈(A)-0.01％冰醋酸水进行梯度洗脱(0.0-20.0min，A：80％-80％；20.0-20.5min，A：80％-85％；20.5-32.0min，A：85％-85％；32.00-40.5min，A：85％-100％；40.50-45.0min，A：100％-100％)，240nm波长进行检测。反复制备多次，收集35.0min色谱峰，得到化合物8(612.00mg)，收集40.0min色谱峰，得到化合物9(109.00mg)。
Fr9分离纯化
Fr9选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈-0.01％冰醋酸水进行梯度洗脱(0.0-21.0min，A：82％-82％；21.0-21.5min，A：82％-87％；21.5-27.5min，A：87％-87％；27.5-28.0min，A：87％-100％；28.0-35.0min，A：100％-100％)，240nm波长进行检测。反复制备多次，收集14.9min色谱峰，得到Fr9-5。Fr9-5用乙醇重结晶，获得化合物10(12.30mg)。
Fr12分离纯化
Fr12选择Zorbax Eclipse Plus-C18半制备色谱柱，以乙腈-0.01％冰醋酸水进行梯度洗脱(0.0-22.0min，A：84％-84％；22.0-22.5min，A：84％-100％；22.5-35.0min，A：100％-100％)，240nm波长进行检测。反复制备多次，收集24.0min色谱峰，得到Fr12-3。Fr12-3利用制备薄层色谱分离，展开剂选择氯仿和甲醇，比例为96：4，得到化合物11(29.8mg)和化合物12(11.2mg)。
实施例3化合物1-12的确认。
利用质谱以及核磁检测等手段，对紫芝菌丝体中得到12个化合物进行结构解析，确定其精准的化学结构。具体的12个化合物的确认信息如下：
化合物1[3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid]
分子式C30H46O5，白色无定形粉末，易溶于甲醇。负离子ESI MS m/z:485.00[M-H]–,1H-NMR(CD3OD,500MHz)δ:6.83(1H,m,H-24),5.90(1H,d,J＝5.0Hz,H-7),5.35(1H,d,J＝10.0Hz,H-11),4.23(1H,dd,J＝10.0,10.0Hz,H-15),3.73(1H,m,H-22),3.15(1H,dd,J＝5.0,10.0Hz,H-3),2.42(1H,m,H-23),2.41(1H,m,H-1),2.34(1H,m,H-12),2.14(1H,m,H-6),2.08(1H,m,H-6),2.07(1H,m,H-17),2.06(1H,m,H-12,16),2.02(1H,m,H-1),1.87(1H,m,H-16),1.83(3H,s,H-27),1.69(1H,m,H-1),1.41(1H,m,H-2),1.37(1H,m,H-20),1.28(1H,m,H-23),1.08(1H,dd,J＝5.0,10.0Hz,H-5),1.00(3H,s,H-19),0.99(3H,s,H-28),0.95(3H,s,H-30),0.89(3H,d,J＝5.0Hz,H-21),0.87(3H,s,H-29),0.64(3H,s,H-18)；13C-NMR(CD3OD,125MHz)δ:171.6(C-26),117.1(C-11),147.7(C-9),142.4(C-8),141.1(C-24),130.1(C-25),122.6(C-7),79.6(C-3),75.2(C-15),73.4(C-22),50.6(C-5),53.3(C-14),46.4(C-17),45.3(C-13),42.3(C-20),39.9(C-16),39.8(C-12),38.6(C-4),38.6(C-10),37.2(C-1),35.8(C-23),28.9(C-28),28.5(C-2),24.1(C-6),23.4(C-19),18.0(C-30),16.5(C-18),16.5(C-29),12.8(C-27),12.2(C-21)。
化合物2[(22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oicacid]
分子式为C34H50O7，淡黄色无定型粉末，易溶于甲醇、氯仿。负离子ESI MS m/z:569[M-H]-。1H-NMR(CD3OD,500MHz)δ:6.83(1H,m,H-24),4.24(1H,dd,J＝5.0,10.0Hz,H-15),3.75(1H,m,H-22),2.80(1H,m,H-2),2.77(1H,m,H-6),2.50(2H,m,H-11),2.40(2H,m,H-2,6),2.28(1H,m,H-23),2.19(1H,m,H-1),2.18(1H,m,H-5),2.05(1H,m,H-17),2.04(1H,m,H-16),1.84(1H,m,H-1),1.83(1H,m,H-23),1.81(1H,m,H-12),1.95(1H,m,H-12),1.92(1H,m,H-16),1.83(3H,s,H-27),1.41(1H,m,H-20),1.40(3H,s,H-19),1.14(3H,s,H-29),1.08(3H,s,H-28),0.95(3H,s,H-30),0.92(3H,d,J＝5.0Hz,H-21),0.78(3H,s,H-18)；13C-NMR(CD3OD,125MHz)δ:217.0(C-3),203.6(C-7),171.9(C-26),171.3(C-9),141.2(C-24),140.1(C-8),130.5(C-25),74.0(C-15),73.4(C-22),53.0(C-14),51.7(C-5),48.5(C-4),47.2(C-13),46.4(C-17),42.7(C-20),41.5(C-10),38.1(C-6),37.6(C-16),36.4(C-1),36.0(C-23),35.6(C-2),32.3(C-12),25.8(C-28),25.5(C-11),22.2(C-29),19.0(C-30),16.9(C-18),18.1(C-19),13.1(C-27),12.5(C-21)。
化合物3[(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid]
分子式C30H44O5，淡黄色无定型粉末，易溶于甲醇。负离子ESI MS m/z:483.0[M-H]–。1H-NMR(CD3OD,500MHz)δ:6.83(1H,m,H-24),5.95(1H,d,J＝10.0Hz,H-7),5.46(1H,d,J＝5.0Hz,H-11),4.24(1H,dd,J＝5.0,10.0Hz,H-15),3.75(1H,m,H-22),2.85(1H,m,H-2),2.43(1H,m,H-23),2.35(1H,m,H-1),2.35(1H,m,H-12),2.28(1H,m,H-2),2.28(1H,m,H-23),2.23(1H,m,H-6),2.12(1H,m,H-6),2.09(1H,m,H-12),2.09(2H,m,H-16,17),1.89(1H,m,H-16),1.83(3H,s,H-27),1.71(1H,m,H-1),1.54(1H,dd,J＝5.0,10.0Hz,H-5),1.38(1H,m,H-20),1.22(3H,s,H-19),1.13(3H,s,H-29),1.07(3H,s,H-28),0.96(3H,s,H-30),0.90(3H,d,J＝5.0Hz,H-21),0.67(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:219.4(C-3),171.8(C-26),146.5(C-9),142.9(C-8),141.3(C-24),130.4(C-25),122.5(C-7),118.5(C-11),75.3(C-15),73.6(C-22),53.5(C-14),52.5(C-5),48.8(C-4),46.7(C-17),45.5(C-13),42.5(C-20),40.0(C-11,16),38.8(C-10),38.1(C-1),36.0(C-2,23),26.2(C-28),24.9(C-6),23.2(C-29),22.8(C-19),18.2(C-30),16.8(C-18),13.1(C-27),12.5(C-21)。
化合物4[(22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C32H48O6，淡黄色无定型粉末，易溶于甲醇。HRTOFMS m/z:551.3350[M+Na]+(C32H48O6Na,计算值551.3343)。1H-NMR(CD3OD,500MHz)δ:6.83(1H,m,H-24),5.91(1H,d,J＝10.0Hz,H-7),5.37(1H,d,J＝5.0Hz,H-11),4.45(1H,m,H-3),4.23(1H,dd,J＝10.0,10.0Hz,H-15),3.73(1H,m,H-22),2.41(1H,m,H-23),2.35(1H,m,H-12),2.28(1H,m,H-23),2.13(1H,m,H-6),2.08(1H,m,H-17),2.07(1H,m,H-6),2.07(2H,m,H-12,16),2.04(1H,m,H-1),2.04(3H,s,OCOCH3-3),1.87(1H,m,H-16),1.83(3H,s,H-27),1.72(1H,m,H-2),1.47(2H,m,H-1,2),1.37(1H,m,H-20),1.18(1H,m,H-5),1.03(3H,s,H-19),0.99(3H,s,H-29),0.97(3H,s,H-30),0.90(3H,s,H-28),0.90(3H,d,J＝5.0Hz,H-21),0.64(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:173.0(OCOCH3-3),171.9(C-26),147.6(C-9),142.7(C-8),141.3(C-24),130.4(C-25),122.5(C-7),117.7(C-11),82.6(C-3),75.4(C-15),73.6(C-22),53.6(C-14),51.0(C-5),46.7(C-17),45.6(C-13),42.5(C-20),40.1(C-16),40.0(C-12),38.9(C-4),38.8(C-10),36.9(C-1),36.0(C-23),28.9(C-28),25.5(C-2),24.1(C-6),23.7(C-19),21.4(OCOCH3-3),18.3(C-30),17.7(C-29),16.8(C-18),13.1(C-27),12.5(C-21)。
化合物5[(22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid]
分子式为C34H44O8，黄色晶体，易溶于甲醇。HRTOFMS m/z:557.3114[M+H]+(C32H45O8,计算值557.3109)。1H-NMR(CD3OD,500MHz)δ:6.68(1H,m,H-24),5.07(1H,m,H-22),4.33(1H,m,H-15),2.93(1H,m,H-12),2.87(1H,m,H-1),2.80(1H,m,H-6),2.68(1H,m,H-2),2.54(1H,m,H-23),2.54(1H,m,H-12),2.47(1H,m,H-6),2.45(1H,m,H-2),2.40(1H,m,H-23),2.39(1H,m,H-5),2.04(3H,s,OCOCH3-22),1.98(1H,m,H-17),1.97(1H,m,H-16),1.86(1H,m,H-1),1.84(3H,s,H-27),1.62(1H,m,H-20),1.27(3H,s,H-19),1.16(3H,s,H-30),1.12(3H,s,H-28),1.11(3H,s,H-29),0.99(1H,m,H-16),0.99(1H,d,J＝5.0Hz,H-16),0.91(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:218.3(C-3),206.6(C-7),202.5(C-11),171.2(C-26),153.9(C-9),152.0(C-8),138.3(C-24),131.5(C-25),75.9(C-22),73.5(C-15),54.0(C-14),52.8(C-12),50.1(C-5),48.7(C-13),47.8(C-4),46.3(C-17),40.6(C-20),40.4(C-10),37.7(C-6),36.8(C-16),36.3(C-1),35.0(C-2),32.5(C-23),27.6(C-28),20.7(C-29),20.4(C-30),18.2(C-19),17.5(C-18),13.1(C-21),12.7(C-27)。
化合物6[(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C32H46O6，淡黄色无定型粉末，易溶于甲醇。负离子ESI MS m/z:525.1[M-H]–。1H-NMR(CD3OD,500MHz)δ:6.69(1H,m,H-24),5.95(1H,d,J＝10.0Hz,H-7),5.45(1H,d,J＝5.0Hz,H-11),5.07(1H,m,H-22),4.24(1H,d,J＝5.0,10.0Hz,H-15),2.84(1H,m,H-2),2.57(1H,m,H-23),2.40(1H,m,H-23),2.34(1H,m,H-1),2.33(1H,m,H-12),2.27(1H,m,H-2),2.23(1H,m,H-6),2.11(1H,m,H-6),2.10(1H,m,H-12),2.04(3H,s,OCOCH3-22),1.95(1H,m,H-16),1.86(1H,m,H-16),1.84(3H,s,H-27),1.80(1H,m,H-17),1.71(1H,m,H-1),1.55(1H,m,H-20),1.53(1H,m,H-5),1.22(3H,s,H-19),1.13(3H,s,H-29),1.06(3H,s,H-28),1.00(3H,d,J＝5.0Hz,H-21),0.91(3H,s,H-30),0.67(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:219.1(C-3),171.2(C-26),146.3(C-9),142.4(C-8),138.5(C-24),131.3(C-25),122.5(C-7),118.1(C-11),76.2(C-22),74.9(C-15),53.2(C-14),52.2(C-5),48.6(C-4),46.8(C-17),45.3(C-13),40.6(C-20),40.0(C-16),39.7(C-12),38.5(C-10),37.9(C-1),35.8(C-2),32.6(C-23),25.9(C-28),24.6(C-6),22.9(C-29),22.5(C-19),17.9(C-30),16.4(C-18),13.2(C-21),12.7(C-27)。
化合物7[(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C34H48O7，白色固体，易溶于甲醇。负离子ESI MS m/z:567.1[M-H]–。1H-NMR(CD3OD,500MHz)δ:6.67(1H,m,H-24),5.58(1H,d,J＝5.0Hz,H-7),5.49(1H,d,J＝5.0Hz,H-11),5.07(1H,dd,J＝5.0,10.0Hz,H-15),5.03(1H,m,H-22),2.84(1H,m,H-2),2.56(1H,m,H-23),2.39(1H,m,H-23),2.36(1H,m,H-16),2.35(1H,m,H-12),2.29(1H,m,H-2),2.23(1H,m,H-6),2.15(1H,m,H-12),2.14(1H,m,H-16),2.11(1H,m,H-6),2.07(3H,s,OCOCH3-15),2.04(3H,s,OCOCH3-22),1.84(3H,s,H-27),1.83(1H,m,H-17),1.82(1H,m,H-1),1.70(1H,m,H-1),1.61(1H,m,H-20),1.52(1H,dd,J＝5.0,10.0Hz,H-5),1.22(3H,s,H-19),1.13(3H,s,H-29),1.06(3H,s,H-28),1.01(3H,d,J＝5.0Hz,H-21),1.01(3H,s,H-30),0.72(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:218.9(C-3),173.0(OCOCH3-15),172.6(OCOCH3-22),171.5(C-26),146.0(C-9),141.7(C-8),138.2(C-24),131.6(C-25),122.5(C-7),118.4(C-11),78.4(C-15),76.1(C-22),52.7(C-14),52.1(C-5),48.5(C-4),46.9(C-17),45.1(C-13),40.8(C-20),39.2(C-12),38.5(C-10),37.8(C-16),37.7(C-1),35.7(C-2),32.6(C-23),25.9(C-28),24.6(C-6),22.9(C-29),22.5(C-19),21.2(OCOCH3-15),20.9(OCOCH3-22),18.8(C-30),16.3(C-18),13.1(C-21),12.8(C-27)。
化合物8[(22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C34H50O7，白色无定型粉末，易溶于甲醇。HRTOFMS m/z:569.3485[M-H]-(C34H49O7,计算值569.3484)。1H-NMR(CD3OD,500MHz)δ:6.71(1H,m,H-24),5.94(1H,d,J＝5.0Hz,H-7),5.38(1H,d,J＝5.0Hz,H-11),5.08(1H,t,J＝5.0Hz,H-22),4.47(1H,m,H-3),4.24(1H,dd,J＝5.0,10.0Hz,H-15),2.58(1H,m,H-23),2.41(1H,m,H-23),2.34(1H,m,H-12),2.16(1H,m,H-6),2.10(1H,m,H-6),2.08(1H,m,H-1),2.09(1H,m,H-12),2.06(3H,s,OCOCH3-3),2.06(3H,s,OCOCH3-22),1.94(1H,m,H-16),1.86(3H,s,H-27),1.85(1H,m,H-16),1.82(1H,m,H-17),1.74(1H,m,H-2),1.62(1H,m,H-20),1.49(1H,m,H-1),1.49(1H,m,H-2),1.19(1H,m,H-5),1.05(3H,s,H-19),1.01(3H,s,H-29),1.01(3H,d,J＝5.0Hz,H-21),0.94(3H,s,H-30),0.92(3H,s,H-28),0.67(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:172.8(OCOCH3-3),172.5(OCOCH3-22),171.2(C-26),147.4(C-9),142.3(C-8),138.5(C-24),131.3(C-25),122.5(C-7),117.3(C-11),82.3(C-3),76.2(C-22),75.0(C-15),53.3(C-14),50.7(C-5),46.8(C-13,17),40.6(C-20),40.0(C-16),39.6(C-12),38.7(C-4),38.6(C-10),36.6(C-1),32.6(C-23),28.7(C-28),25.2(C-2),23.8(C-6),23.4(C-19),21.2or20.9(OCOCH3-3),20.9or 21.2(OCOCH3-22),18.0(C-30),17.4(C-29),16.4(C-18),13.2(C-21),12.7(C-27)。
化合物9[lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid]
分子式为C36H52O8，白色无定型粉末，易溶于甲醇。负离子ESI MS m/z:611.2[M-H]–。1H-NMR(CD3OD,500MHz)δ:6.67(1H,m,H-24),5.53(1H,d,J＝5.0Hz,H-7),5.40(1H,d,J＝5.0Hz,H-11),5.06(1H,m,H-15),5.03(1H,m,H-22),4.45(1H,m,H-3),2.57(1H,m,H-23),2.38(1H,m,H-23),2.35(1H,m,H-12),2.13(1H,m,H-16),2.12(1H,m,H-12),2.09(1H,m,H-6),2.07(3H,s,OCOCH3-15),2.06(1H,m,H-6),2.05(1H,m,H-1),2.07(3H,s,OCOCH3-3),2.07(3H,s,OCOCH3-22),1.83(1H,m,H-16),1.81(1H,m,H-17),1.83(3H,s,H-27),1.73(1H,m,H-2),1.59(1H,m,H-20),1.47(1H,m,H-1),1.47(1H,m,H-1),1.16(1H,m,H-5),1.03(3H,s,H-19),1.00(3H,d,J＝5.0Hz,H-21),1.00(3H,s,H-30),0.98(3H,s,H-29),0.90(3H,s,H-28),0.69(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:173.3(OCOCH3-15),173.1(OCOCH3-3),172.8(OCOCH3-22),171.4(C-26),147.3(C-9),141.8(C-8),138.7(C-24),131.6(C-25),122.8(C-7),117.8(C-11),82.5(C-3),78.8(C-15),76.3(C-22),52.9(C-14),50.9(C-5),47.1(C-17),45.4(C-13),41.0(C-20),39.4(C-12),38.9(C-4),38.8(C-10),38.0(C-16),36.9(C-1),32.9(C-23),28.9(C-28),25.5(C-2),24.1(C-6),23.6(C-19),21.4(OCOCH3-15,22),21.1(OCOCH3-3),19.2(C-30),17.7(C-29),16.6(C-18),13.4(C-21),13.0(C-27)。
化合物10[(22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C34H50O7，白色无定型粉末，易溶于甲醇。HRTOFMS m/z:593.3451[M+Na]+(C34H50O7Na,计算值651.3504)。1H-NMR(CD3OD,500MHz)δ:6.82(1H,m,H-24),5.53(1H,d,J＝5.0Hz,H-7),5.41(1H,d,J＝5.0Hz,H-11),5.06(1H,dd,J＝5,0,10.0Hz,H-15),4.46(1H,m,H-3),3.69(1H,m,H-22),2.41(1H,m,H-23),2.38(1H,m,H-12),2.28(1H,m,H-23),2.14(1H,m,H-17),2.12(3H,m,H-6,12),2.07(3H,s,OCOCH3-15),2.07(1H,m,H-1),2.04(3H,s,OCOCH3-3),1.83(3H,s,H-27),1.80(1H,m,H-16),2.10(1H,m,H-16),1.73(1H,m,H-2),1.47(2H,m,H-1,2),1.40(1H,m,H-20),1.17(1H,m,H-5),1.07(3H,s,H-30),1.04(3H,s,H-19),0.98(3H,s,H-29),0.91(3H,d,J＝10.0Hz,H-21),0.90(3H,s,H-28),0.69(3H,s,H-18)；13C-NMR(CD3OD,125MHz)δ:173.1(OCOCH3-15),172.8(OCOCH3 -3),171.5(C-26),147.0(C-9),141.8(C-8),141.0(C-24),130.1(C-25),122.3(C-7),117.8(C-11),82.3(C-3),78.8(C-15),73.2(C-22),52.7(C-14),50.6(C-5),46.5(C-17),45.1(C-13),42.1(C-20),39.3(C-12),38.7(C-4),38.6(C-10),37.4(C-16),36.6(C-1),35.7(C-23),28.7(C-28),25.2(C-2),23.8(C-6),23.4(C-19),21.3(OCOCH-15),21.2(OCOCH-3),19.0(C-30),17.4(C-29),16.5(C-18),12.8(C-27),12.1(C-21).
化合物11[(24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C34H50O6，白色固体，易溶于甲醇。HRTOFMS m/z:577.3506[M+Na]+(C36H52O9Na,计算值577.3500).1H-NMR(CD3OD,500MHz)δ:6.41(1H,m,H-24),5.52(1H,d,J＝5.0Hz,H-7),5.41(1H,d,J＝5.0Hz,H-11),5.07(1H,dd,J＝5,0,10.0Hz,H-15),4.45(1H,m,H-3),2.36(1H,m,H-12),2.16(1H,m,H-23),2.13(1H,m,H-12),2.11(2H,m,H-6),2.10(1H,m,H-16),2.06(2H,m,H-1,23),2.06(3H,s,OCOCH3-15),2.04(3H,s,OCOCH3-3),1.79(3H,s,H-27),1.74(1H,m,H-1),1.73(1H,m,H-16,17),1.72(1H,m,H-2),1.49(1H,m,H-22),1.47(1H,m,H-2),1.42(1H,m,H-20),1.17(1H,m,H-5),1.12(1H,m,H-22),1.05(3H,s,H-30),1.03(3H,s,H-19),0.98(3H,s,H-29),0.94(3H,d,J＝5.0Hz,H-21),0.90(3H,s,H-28),0.69(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:178.0(C-26),173.0(OCOCH3-15),172.8(OCOCH3-3),147.0(C-9),141.8(C-8),137.1(C-24),134.7(C-25),122.2(C-7),117.8(C-11),82.3(C-3),78.7(C-15),52.6(C-14),50.6(C-5),50.3(C-17),45.3(C-13),39.2(C-12),38.6(C-4,10),38.0(C-16),37.1(C-20),36.6(C-1),36.4(C-22),28.7(C-28),26.3(C-23),25.2(C-2),23.8(C-6),23.4(C-19),21.3(OCOCH3-15),21.1(OCOCH3-3),18.9(C-30),18.7(C-21),17.4(C-29),16.5(C-18),13.9(C-27).
化合物12[(22S,24Z)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oicacid]
分子式为C34H50O6，白色固体，易溶于甲醇。HRTOFMS m/z:577.3505[M+Na]+(C34H50O6Na,计算值577.3500).1H-NMR(CD3OD,500MHz)δ:6.34(1H,m,H-24),5.51(1H,t,J＝5.0Hz,H-7),5.38(1H,d,J＝10Hz,H-11),5.06(1H,m,H-22),4.44(1H,m,H-3),2.44(1H,m,H-23),2.35(1H,m,H-23),2.24(1H,m,H-12),2.12(2H,m,H-6),2.11(1H,m,H-12),2.06(1H,m,H-1),2.04(3H,s,OCOCH3-3,22),2.04(1H,m,H-16),1.83(3H,s,H-27),1.74(1H,m,H-17),1.73(2H,m,H-2),1.65(2H,m,H-15,20),1.46(1H,m,H-1),1.40(1H,m,H-15),1.38(1H,m,H-16),1.17(1H,m,H-5),1.03(3H,s,H-19),1.00(3H,d,J＝5.0Hz,H-21),0.98(3H,s,H-29),0.89(3H,s,H-28),0.88(3H,s,H-30),0.60(3H,s,H-18).13C-NMR(CD3OD,125MHz)δ:177.4(C-26),172.9(OCOCH3-3),172.7(OCOCH3-22),147.1(C-9),143.8(C-8),137.5(C-25),131.3(C-24),121.5(C-7),117.7(C-11),82.4(C-3),76.9(C-22),51.6(C-14),50.8(C-5),48.9(C-17),44.8(C-13),40.0(C-20),39.0(C-12),38.7(C-4),38.5(C-10),36.6(C-1),32.5(C-15),32.2(C-23),28.7(C-28),28.5(C-16),26.1(C-30),25.2(C-2),23.9(C-6),23.3(C-19),21.2(OCOCH3-22),21.1(OCOCH3-3),17.4(C-29),16.1(C-18),14.2(C-27),13.1(C-21).
经过结构解析后，7个新的化合物，5个已知的化合物，均为羊毛甾烷型三萜化合物。其名称和结构式如下所示：
化合物1，3β,15α,22β-trihydroxy-lanosta-7,9(11),24-trien-26-oic acid。
化合物2，(22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oicacid，为新化合物。
化合物3，(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid。
化合物4，(22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid，为新化合物。
化合物5，(22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid，为新化合物。
化合物6，(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid。
化合物7，(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid。
化合物8，(22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid，新化合物。
化合物9，lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid。
化合物10，(22S,24E)-22β-hydroxy-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid，新化合物。
化合物11，(24Z)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid，新化合物。
化合物12，(22S,24Z)-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oicacid，新化合物。
实施例4实施例3制备的结构式1～12的化合物的抗肿瘤活性
测试用肿瘤细胞：人白血病细胞为K562细胞用含体积百分含量10％的胎牛血清的DMEM培养液培养，人肺癌细胞为A549细胞、人肝癌细胞为HepG2细胞用含体积百分含量10％的胎牛血清的RPMI 1640培养液培养。上述细胞的培养条件均为37℃、5％CO2培养箱中常规培养，隔天传代。
被测试样品溶液，化合物样品配制：精确称量化合物，无菌操作条件下，用DMSO分别配制成20mg/mL的溶液，依次将结构式1～12的化合物逐级稀释至浓度为10mg/mL、4mg/mL、2mg/mL。
细胞增殖测定：细胞生长至对数期时，将2×104个/mL混悬液铺到96孔板中，每孔加入199μL。并加入1μL不同浓度的化合物，每个样品3个重复，置于培养箱中，待培养72小时后，用酶标仪测定96孔板570nm和600nm下的吸光值，分别记为A1和A2。每孔加入30μL的0.1mg/mL的Alamar blue检测试剂，再放入培养箱培养3-5h。观察到颜色变化后，重复测定570nm和600nm下的吸光值，分别记为A3和A4。测定样品对细胞的增殖抑制率按公式1计算。
化合物抑制细胞增殖的结果如表5所示。
表5化合物1-12对几种肿瘤细胞的IC50值
本发明的12个三萜类化合物据具有不同程度的抑制多种肿瘤细胞(人白血病细胞为K562细胞、人肺癌细胞为A549细胞、人肝癌细胞为HepG2)增殖作用，特别是化合物7、8、9抑制肿瘤细胞的增殖作用具有突出的效果。
Claims (3)
1.一种紫芝发酵菌丝体中三萜化合物，其结构式为如下结构式2、4、5、8、10、11和12的化合物或其药用盐、晶体或水合物：
2.一种用于预防或治疗肿瘤的制剂，其含有结构式1-12所示的一种或一种以上化合物或其药用盐以及医学上可接受的载体；
其中所述的结构式1-12的化合物为：
3.紫芝发酵菌丝体中三萜化合物作为预防或治疗肿瘤的药物的应用；其中所述的紫芝发酵菌丝体中三萜化合物为结构式1-12的化合物中的一种或一种以上，
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069116A (zh) * | 2013-03-29 | 2014-10-01 | 双鹤生物科技股份有限公司 | 赤灵酸s用于制造癌症治疗药物的用途 |
| US20140294753A1 (en) * | 2013-03-29 | 2014-10-02 | Double Crane Biotechnology Co. LTD | Novel use of ganodermic acids for treating cancer |
| CN115894591A (zh) * | 2022-10-20 | 2023-04-04 | 上海市农业科学院 | 一种赤芝菌丝体中三萜化合物及其应用 |
| CN115894590A (zh) * | 2022-10-20 | 2023-04-04 | 上海市农业科学院 | 一种紫芝发酵菌丝体中三萜化合物及其抗炎应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069116A (zh) * | 2013-03-29 | 2014-10-01 | 双鹤生物科技股份有限公司 | 赤灵酸s用于制造癌症治疗药物的用途 |
| US20140294753A1 (en) * | 2013-03-29 | 2014-10-02 | Double Crane Biotechnology Co. LTD | Novel use of ganodermic acids for treating cancer |
| CN115894591A (zh) * | 2022-10-20 | 2023-04-04 | 上海市农业科学院 | 一种赤芝菌丝体中三萜化合物及其应用 |
| CN115894590A (zh) * | 2022-10-20 | 2023-04-04 | 上海市农业科学院 | 一种紫芝发酵菌丝体中三萜化合物及其抗炎应用 |
Non-Patent Citations (3)
| Title |
|---|
| ZHOU YAN等: "Fast analysis of triterpenoids in Ganoderma lucidum spores by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry", 《BIOMEDICAL CHROMATOGRAPHY》, vol. 27, no. 11, pages 1561 * |
| 冯娜等: "灵芝菌丝体三萜及其药理活性的研究进展", 《菌物学报》, vol. 41, no. 9, pages 1346 - 1347 * |
| 刘如明: "灵芝酸的结构修饰及其新型衍生物的抗肿瘤机制研究", 《华东理工大学博士学位论文》, pages 61 - 75 * |
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