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暗紫贝母茎化学成分研究

李敏, 米雅慧, 蒯海敏, 胡晓龙, 汪豪

李敏,米雅慧,蒯海敏,等. 暗紫贝母茎化学成分研究[J]. 中国药科大学学报,2025,56(2):160 − 165. DOI: 10.11665/j.issn.1000-5048.2024040302
引用本文: 李敏,米雅慧,蒯海敏,等. 暗紫贝母茎化学成分研究[J]. 中国药科大学学报,2025,56(2):160 − 165. DOI: 10.11665/j.issn.1000-5048.2024040302
LI Min, MI Yahui, KUAI Haimin, et al. Chemical constituents from the stems of Fritillaria unibracteata[J]. J China Pharm Univ, 2025, 56(2): 160 − 165. DOI: 10.11665/j.issn.1000-5048.2024040302
Citation: LI Min, MI Yahui, KUAI Haimin, et al. Chemical constituents from the stems of Fritillaria unibracteata[J]. J China Pharm Univ, 2025, 56(2): 160 − 165. DOI: 10.11665/j.issn.1000-5048.2024040302

暗紫贝母茎化学成分研究

基金项目: 云南省重大科技专项(202402AA310001);云南省专家工作站项目 (202305AF150096);迪庆州-中国药科大学州校合作项目 (2021ZXYD01)
详细信息
    通讯作者:

    汪豪: Tel:025-83271328 E-mail:wanghao@cpu.edu.cn

  • 中图分类号: R284.1

Chemical constituents from the stems of Fritillaria unibracteata

Funds: This study was supported by the Major Science and Technology Special Projects of Yunnan Province (202402AA310001);the Expert Workstation Project of Yunnan Province (202305AF150096); Diqing State-China Pharmaceutical University State-School Cooperation Project (2021ZXYD01)
  • 摘要:

    采用硅胶及制备HPLC等柱色谱方法对暗紫贝母 (Fritillaria unibracteata P.K. Hsiao & K.C. Hsia) 茎乙醇提取物的化学成分进行分离纯化,通过NMR、ESI-MS等波谱技术鉴定化合物结构,分离鉴定了9个化合物,分别为:27-hydroxychlorogenone (1), sieboldogenin (2), (3β, 25S)-spirost-5-ene-3,17,27-triol (3), 拉肖皂苷元 (4), 替告皂苷元酮 (5), 啤酒甾醇 (6), 过氧化麦角甾醇 (7), 豆甾醇 (8), β-谷甾醇 (9)。其中化合物1为新化合物,化合物2~9均为首次从暗紫贝母茎中分离得到。体外活性初步评价了化合物1~9对A549细胞的细胞毒活性,结果显示,化合物6具有中等强度的细胞毒活性,IC50为(14.16±1.11)μmol/L。

    Abstract:

    Chemical investigation of the stems of Fritillaria unibracteata P.K. Hsiao & K.C. Hsia resulted in the isolation of nine compounds, by means of silica gel column chromatography, and preparative HPLC. Based on spectroscopic and chemical evidence, these compounds were identified as: 27-hydroxychlorogenone (1), sieboldogenin (2), (3β, 25S)-spirost-5-ene-3,17,27-triol (3), laxogenin (4), tigogenone (5), cerevisterol (6), ergosterol peroxide (7), stigmaterol (8), and β-sitosterol (9). Compound 1 was a new compound, and compounds 2-9 were isolated from the stems of Fritillaria unibracteata for the first time. The inhibitory effects of compounds 1−9 on A549 cells were determined using the MTT method. The results show that compound 6 exhibits moderate inhibitory activity with an IC50 value of (14.16 ± 1.11) μmol/L.

  • 暗紫贝母 (Fritillaria unibracteata P.K. Hsiao & K.C. Hsia) 为百合科 (Liliaceae) 贝母属植物,主产于四川西北部及青海东南部,生于海拔3200~4500米的草地上[1]。暗紫贝母是《中华人民共和国药典》(2020版)收载中药川贝母的基源植物之一,通常以鳞茎入药,具有清热润肺、化痰止咳、散结消痈的功效[2]。现代研究表明,贝母属植物还含有生物碱、萜类、甾体、多糖等多种化学成分,并且具有广泛的药理作用,如Shen等[3]从伊贝母中分离得到的甾体化合物对大鼠脑胶质瘤C6细胞和人宫颈癌HeLa细胞具有明显的抗增殖作用;从暗紫贝母中分离得到的甾体生物碱具有较弱的抗炎活性[4];从川贝母、暗紫贝母等多个贝母品种中提取得到的总皂苷部分均具有明显的抗炎活性[5]。目前对暗紫贝母化学成分的研究主要集中于鳞茎部位[4, 68],非药用部位的化学成分未见报道。川贝母价格昂贵,人工种植及采集过程中,大量地上部位作为废料丢弃。为了揭示暗紫贝母地上部位药效物质基础及为暗紫贝母地上部分的综合开发利用提供参考依据,本研究对暗紫贝母茎进行了较为系统的化学成分研究,分离鉴定了9个化合物,分别为:27-hydroxychlorogenone (1), sieboldogenin (2), (3β, 25S)-spirost-5-ene-3,17,27-triol (3), 拉肖皂苷元 (4), 替告皂苷元酮 (5), 啤酒甾醇 (6), 过氧化麦角甾醇 (7), 豆甾醇 (8), β-谷甾醇 (9)。其中化合物1为新化合物,化合物2~9均为首次从暗紫贝母茎中分离得到。采用MTT法测定化合物1~9对A549细胞的抑制作用,结果显示化合物6具有中等强度的细胞毒活性,IC50为(14.16 ± 1.11) μmol/L。

    Avance-300, 500及600型核磁共振仪 (德国布鲁克公司);LC-20AT型高效液相色谱仪,LC-8A型制备液相色谱仪,LCMS-2020四极杆液质联用仪 (日本岛津公司);Agilent 6500 UPLC-Q-TOF质谱仪 (美国安捷伦公司);JASCO P-1020型旋光仪 (日本佳司科公司);柱色谱硅胶 (青岛海洋化工公司);GF254薄层色谱硅胶板 (烟台化工研究所);柱色谱RP-C18 (50 μm, 日本YMC公司);Sephadex LH-20 (美国GE Healthcare公司);MCI gel (CHP20P, 75~150 μm, 日本三菱化学公司);岛津Shim-Pack VP-ODS分析色谱柱 (4.6 mm×250 mm, 5 μm);岛津Shim-Pack Prep-ODS制备色谱柱 (20 mm×250 mm, 15 μm);人非小细胞肺癌细胞株A549、胎牛血清、DMEM培养液 (江苏凯基生物技术股份有限公司);Infinite 200 Pro全波长酶标 (瑞士帝肯公司);其他试剂均为市售分析纯。

    暗紫贝母茎由香格里拉市天泉川贝科技有限公司提供,经中国药科大学中药学院汪豪教授鉴定为Fritillaria unibracteata P.K. Hsiao & K.C. Hsia的干燥茎干,植物标本存放于中国药科大学天然药物化学实验室,标本号为AZ20221116。

    干燥的暗紫贝母茎5.0 kg,粉碎成粗粉,95%乙醇室温浸渍提取4次,每次7 d,合并醇提液,减压浓缩得稠浸膏,加水混悬后依次用石油醚、乙酸乙酯和水饱和正丁醇萃取。取石油醚部位 (83.5 g),经硅胶柱色谱 (石油醚-乙酸乙酯, 100∶1~0∶1) 洗脱,TLC检识,合并得到流份Frs. 1~10。Fr. 5经Sephadex LH-20柱色谱 (氯仿-甲醇, 1∶1) 分离,得到流份Frs. 5-1~5-7。Fr. 5-4在甲醇中重结晶,沉淀部分经硅胶柱色谱 (石油醚-乙酸乙酯, 15∶1) 纯化,得到化合物5 (8.2 mg);Fr. 5-4母液部分依次经硅胶柱色谱 (石油醚-乙酸乙酯, 30∶1~0∶1) 和制备HPLC (甲醇-水, 90∶10) 分离,得到化合物7 (18.6 mg)。Fr. 10依次经硅胶柱色谱 (石油醚-乙酸乙酯, 5∶1~3∶1),和制备HPLC (甲醇-水,80∶20) 分离,得到4 (43.5 mg)。取乙酸乙酯部位 (23.5 g),经MCI柱色谱 (乙醇-水, 40∶60~100∶0) 洗脱,TLC检识,合并得到流份Frs. A~G。Fr. D经Sephadex LH-20柱色谱 (氯仿-甲醇, 1∶1) 洗脱,得到流份Frs. D-1~D-5。Fr. D-3经C18柱色谱 (甲醇-水, 50∶50~90∶10) 洗脱,得到流份Frs. D-3-1~D-3-15。Fr. D-3-8经硅胶柱色谱 (石油醚-丙酮, 5∶1~3∶1) 洗脱,再分别经硅胶柱色谱 (石油醚-丙酮, 1∶1) 及制备HPLC (乙腈-水, 30∶70) 纯化,得到化合物1 (1.7 mg), 2 (46.1 mg)。Fr. D-3-9经硅胶柱色谱反复分离纯化,依次以石油醚-丙酮 (3∶1),石油醚-乙酸乙酯 (1∶1) 洗脱,得到化合物3 (3.7 mg)。Fr. E经C18柱色谱 (甲醇-水, 60∶40~100∶ 0) 分离,得到流分Frs. E-1~E-13。Fr. E-11经硅胶柱色谱 (石油醚-丙酮, 3∶1) 分离,得到化合物6 (1.9 mg)。Fr. G依次经Sephadex LH-20 (甲醇), 硅胶柱色谱 (石油醚-丙酮, 20∶1) 分离纯化,得到化合物8 (8.5 mg), 9 (21.1 mg)。化合物1~9的结构式见图1

    Figure  1.  Chemical structures of compounds 19

    A549细胞以每毫升5×104个细胞密度接种至96孔板中,每孔100 µL,置37 ℃、5% CO2培养箱中培养24 h,弃去孔内上清液,空白组加入含10% FBS的DMEM培养基200 µL,实验组中加入相应浓度的含化合物1~9或卡铂培养基200 µL,每组设置3个复孔,继续培养24 h。随后,每孔加入MTT溶液 (5 mg/mL) 20 µL,放入培养箱中避光孵育4 h。最后,弃去孔内上清液,每孔加入DMSO 150 µL于摇床上振荡10 min,用酶标仪490 nm波长处测定吸收度,并用GraphPad Prism 8.0计算IC50

    化合物1  白色粉末 (CHCl3),茴香醛-浓硫酸反应显蓝色。$ {\text{[}\text{α}\text{]}}_{\text{D}}^{\text{20}}\text{:} $–28.0° (c 0.05, CHCl3); HR-ESI-MS m/z: 445.2952 (Calcd. for [C27H40O5+H]+: 445.2948),结合1H, 13C NMR信息,推测化合物1分子式为C27H40O51H NMR谱图中显示化合物含有3个甲基质子信号 [δH 0.99 (3H, d, J = 7.0 Hz, H-21), 0.98 (3H, s, H-19), 0.80 (3H, s, H-18)],2组氧取代亚甲基质子信号 [δH 3.72 (1H, dd, J = 11.7, 2.9 Hz, H-26a), 3.53 (1H, m, H-26b); 3.48 (2H, m, H-27)],及1个氧取代次甲基质子信号 [δH 4.43 (1H, td, J = 7.9, 5.8 Hz, H-16)]。13C NMR和DEPT谱图显示化合物含有27个碳原子,包括2个羰基碳信号 [δC 211.0 (C-3), 208.7 (C-6)],2个氧取代亚甲基碳信号 [δC 65.0 (C-27), 63.1 (C-26)],1个氧取代次甲基碳信号δC 80.4 (C-16),以及一个连氧的季碳信号δC 109.5 (C-22)。化合物1与已知化合物chlorogenone的核磁数据对比发现,二者A-E环的1H, 13C NMR数据基本一致[9],提示化合物1为螺甾烷醇类化合物,具有A/B trans, B/C trans, C/D trans, D/E cis, 且H-5, Me-21为α构型。与chlorogenone相比,化合物1多出1组氧取代亚甲基信号 [δH 3.65 (H-27), δC 65.0 (C-27)],缺少27-CH3的信号,并且C-25向低场位移 +7.8×10−6,C-24, 26分别向高场位移 −5.6×10−6, −3.8×10−6,提示化合物1的C-27可能存在氧取代官能团。在1H-1H COSY谱图中,可见以下一组质子自旋偶合体系:δH 1.95, 1.68 (H-23a, 23b) ↔ 1.69, 1.52 (H-24a, 24b) ↔ 1.81 (H-25) ↔ 3.72, 3.53 (H-26a, 26b)[3.48 (H-27)]。在HMBC谱图中,可观察到δH 3.65 (H-27) 与δC 23.1 (C-24), 38.0 (C-25), 63.1 (C-26) 的远程相关信号,因此确定C-27存在羟基取代。将化合物1与分离得到的已知化合物2的F环13C NMR数据进行比较,发现二者F环碳原子化学位移基本一致,提示C-25位上的羟甲基为α取向。在NOESY谱图中,可见Me-18/H-20, H-20/H-23b, H-23b/H-25的NOE相关信号,表明H-25为β取向,进一步证实化合物1中C-25位上的羟甲基为α构型。化合物1的NMR波谱数据归属见表11H-1H COSY, HMBC和NOESY的关键相关信号见图2图3。综上分析,化合物1的结构鉴定为 (20S, 25S)-5α-spirostane-27-ol-3,6-dione,命名为27-hydroxychlorogenone,为一新化合物。

    Table  1.  NMR data of compound 1a
    PositonδH bδC cHMBC
    11.62, 2.13, m38.1C-3, 5
    22.04, 2.35, m37.0C-1, 3, 5, 10
    3211.0
    42.07, 2.43, m37.5C-2, 3, 5, 6
    52.59, m57.5C-3, 6, 10, 19
    6208.7
    72.03, m
    2.41, dd (16.0, 6.1)
    46.6C-6, 9
    81.65, m37.3
    91.37, m53.4
    1041.2
    111.46, 1.67, m21.5
    121.26, 1.83, m39.4C-9, 11, 18
    1341.0
    141.38, m56.3C-8
    151.30, 1.36, m31.6C-14, 16
    164.43, 1H, td (7.9, 5.8)80.4C-13
    171.84, m62.0C-12, 14, 16, 18, 20, 21
    180.80, 3H, s16.4C-12, 14, 17
    190.98, 3H, s12.6C-1, 5, 9, 10
    201.91, m41.7C-17
    210.99, 3H, d (7.0)14.4C-17, 20, 22
    22109.5
    231.68, 1.95, m30.7C-25
    241.52, 1.69, m23.1C-22, 26
    251.81, m38.0C-27
    263.53, m
    3.72, dd (11.7, 2.9)
    63.1C-22, 24, 25,
    273.48, 2H, m65.0C-24, 25, 26
    a Measured in CDCl3. Assignments were established by DEPT, 1H-1H COSY, HMQC and HMBC experiments. J values (in Hz) are in parentheses;b Measured at 600 MHz; c Measured at 150 MHz
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    化合物2  白色粉末 (CHCl3),ESI-MS m/z: 447 [M+H]+,结合1H, 13C NMR信息,确定其分子式为C27H42O51H NMR (CDCl3, 300 MHz) δ: 4.42 (1H, td, J = 7.9, 6.0 Hz, H-16), 3.68 (1H, dd, J = 10.2, 3.4 Hz, H-26a), 3.48 (2H, m, H-27), 3.43~3.36 (2H, m, H-3, 26b), 0.98 (3H, d, J = 6.8 Hz, H-21), 0.81 (3H, s, H-19), 0.77 (3H, s, H-18)。13C NMR (CDCl3, 125 MHz) δ: 210.5 (C-6), 109.6 (C-22), 80.7 (C-16), 70.8 (C-3), 65.2 (C-27), 63.3 (C-26), 62.2 (C-17), 57.0 (C-5), 56.7 (C-14), 54.1 (C-9), 46.9 (C-7), 41.8 (C-20), 41.1 (C-10), 41.1 (C-13), 39.7 (C-12), 38.3 (C-25), 37.5 (C-8), 36.8 (C-1), 31.7 (C-15), 30.9 (C-23), 30.9 (C-2), 30.2 (C-4), 23.3 (C-24), 21.5 (C-11), 16.6 (C-18), 14.6 (C-21), 13.4 (C-19)。与文献对照[10],鉴定化合物2为sieboldogenin。

    Figure  2.  Key 1H-1H COSY and HMBC (H→C) correlations of compound 1
    Figure  3.  Key NOESY correlations of compound 1

    化合物3  白色粉末 (CHCl3),ESI-MS m/z: 447 [M+H]+,结合1H, 13C NMR信息,确定其分子式为C27H42O51H NMR (CDCl3, 300 MHz) δ: 5.34 (1H, d, J = 5.1 Hz, H-6), 3.98 (1H, t, J = 7.6 Hz, H-16), 3.75 (1H, dd, J = 11.4, 3.3 Hz, H-26a), 3.54 (2H, m, H-27), 3.50-3.41 (2H, m, H-3, 26b), 1.02 (3H, s, H-19), 0.91 (3H, d, J = 7.2 Hz, H-21), 0.82 (3H, s, H-18)。13C NMR (CDCl3, 125 MHz) δ: 141.0 (C-5), 121.5 (C-6), 110.5 (C-22), 91.2 (C-16), 90.3 (C-17), 71.9 (C-3), 65.1 (C-27), 63.3 (C-26), 53.0 (C-14), 49.8 (C-9), 44.8 (C-13), 44.0 (C-20), 42.4 (C-4), 38.1 (C-25), 37.4 (C-1), 36.8 (C-10), 32.2 (C-2), 31.8 (C-8), 31.8 (C-12), 31.8 (C-23), 31.4 (C-7), 30.4 (C-15), 22.7 (C-24), 20.8 (C-11), 19.6 (C-19), 17.2 (C-18), 8.2 (C-21)。与文献对照[1112],鉴定化合物3为 (3β, 25S)-spirost-5-ene-3,17,27-triol。

    化合物4  白色粉末 (CHCl3),ESI-MS m/z: 431 [M+H]+,结合1H, 13C NMR信息,确定其分子式为C27H42O41H NMR (CDCl3, 300 MHz) δ: 4.37 (1H, td, J = 7.9, 4.3 Hz, H-16), 3.53 (1H, m, H-26a), 3.42 (1H, m, H-3), 3.32 (1H, t, J = 10.8 Hz, H-26b), 0.94 (3H, d, J = 6.7 Hz, H-21), 0.76 (3H, d, J = 6.7 Hz, H-27), 0.74 (6H, s, H-18, 19)。13C NMR (CDCl3, 125 MHz) δ: 210.7 (C-6), 109.4 (C-22), 80.5 (C-16), 70.6 (C-3), 67.0 (C-26), 62.1 (C-17), 56.9 (C-5), 56.6 (C-14), 54.0 (C-9), 46.8 (C-7), 41.7 (C-20), 41.0 (C-10), 41.0 (C-13), 39.6 (C-12), 37.5 (C-8), 36.7 (C-1), 31.7 (C-15), 31.4 (C-23), 30.7 (C-2), 30.3 (C-25), 30.1 (C-4), 28.8 (C-24), 21.5 (C-11), 17.2 (C-27), 16.5 (C-18), 14.5 (C-21), 13.3 (C-19)。与文献对照[10],鉴定化合物4为拉肖皂苷元 (laxogenin)。

    化合物5  白色粉末 (CHCl3),ESI-MS m/z: 437 [M+Na]+,结合1H, 13C NMR信息,确定其分子式为C27H42O31H NMR (CDCl3, 300 MHz) δ: 4.39 (1H, m, H-16), 3.47 (1H, d, J = 4.6 Hz, H-26a), 3.36 (1H, t, J = 10.8 Hz, H-26b), 1.02 (3H, s, H-19), 0.96 (3H, d, J = 6.9 Hz, H-21), 0.79 (3H, s, H-18), 0.78 (3H, d, J = 6.3 Hz, H-27)。13C NMR (CDCl3, 125 MHz) δ: 212.1 (C-3), 109.4 (C-22), 80.9 (C-16), 67.0 (C-26), 62.4 (C-17), 56.2 (C-14), 54.0 (C-9), 46.8 (C-5), 44.8 (C-4), 41.8 (C-20), 40.7 (C-13), 40.1 (C-12), 38.7 (C-1), 38.3 (C-2), 35.9 (C-10), 35.2 (C-8), 32.0 (C-7), 31.9 (C-15), 31.5 (C-23), 30.4 (C-25), 29.0 (C-6), 29.0 (C-24), 21.4 (C-11), 17.3 (C-27), 16.6 (C-18), 14.6 (C-21), 11.7 (C-19)。与文献对照[13],鉴定化合物5为替告皂苷元酮 (tigogenone)。

    化合物6  白色粉末 (CHCl3),ESI-MS m/z: 453 [M+Na]+,结合1H NMR, 13C NMR信息,确定其分子式为C28H46O31H NMR (CDCl3, 300 MHz) δ: 5.35 (1H, d, J = 4.9 Hz, H-7), 5.23 (1H, dd, J = 15.0, 6.9 Hz, H-23), 5.17 (1H, dd, J = 15.3, 7.5 Hz, H-22), 4.07 (1H, m, H-3), 3.62 (1H, d, J = 4.9 Hz, H-6), 1.08 (3H, s, H-19), 1.02 (3H, d, J = 6.6Hz, H-21), 0.91 (3H, d, J = 6.8 Hz, H-28), 0.83 (3H, d, J = 6.7 Hz, H-26), 0.82 (3H, d, J = 6.8 Hz, H-27), 0.60 (3H, s, H-18)。13C NMR (CDCl3, 125 MHz) δ: 144.2 (C-8), 135.5 (C-22), 132.4 (C-23), 117.7 (C-7), 76.1 (C-5), 73.9 (C-6), 67.9 (C-3), 56.2 (C-17), 54.9 (C-14), 43.9 (C-13), 43.7 (C-9), 43.0 (C-24), 40.5 (C-20), 39.7 (C-4), 39.4 (C-12), 37.3 (C-10), 33.2 (C-25), 33.1 (C-1), 31.0 (C-2), 28.1 (C-16), 23.1 (C-15), 22.2 (C-11), 21.3 (C-21), 20.1 (C-26), 19.8 (C-27), 19.0 (C-19), 17.7 (C-28), 12.5 (C-18)。与文献对照[14],鉴定化合物6为啤酒甾醇 (cerevisterol)。

    化合物7  无色结晶 (CHCl3),ESI-MS m/z: 451 [M+Na]+,结合1H, 13C NMR信息,确定其分子式为C28H44O31H NMR (CDCl3, 300 MHz) δ: 6.50 (1H, d, J = 8.5 Hz, H-7), 6.24 (1H, d, J = 8.5 Hz, H-6), 5.22 (1H, dd, J = 15.2, 7.1 Hz, H-23), 5.13 (1H, dd, J = 15.2, 7.8 Hz, H-22), 3.97 (1H, m, H-3), 0.99 (3H, d, J = 6.6 Hz, H-21), 0.90 (3H, d, J = 6.8Hz, H-28), 0.88 (3H, s, H-19), 0.83 (3H, d, J = 6.8 Hz, H-27), 0.81 (3H, s, H-18), 0.81 (3H, d, J = 6.8 Hz, H-26)。13C NMR (CDCl3, 125 MHz) δ: 135.4 (C-6), 135.4 (C-7), 132.5 (C-23), 130.9 (C-22), 81.7 (C-5), 79.0 (C-8), 66.6 (C-3), 56.4 (C-17), 51.9 (C-14), 51.3 (C-9), 44.7 (C-13), 42.9 (C-24), 39.9 (C-20), 39.5 (C-12), 37.1 (C-4), 37.1 (C-10), 34.9 (C-1), 33.2 (C-25), 30.3 (C-2), 28.8 (C-16), 23.6 (C-11), 21.0 (C-21), 20.8 (C-15), 20.1 (C-26), 19.8 (C-27), 18.3 (C-19), 17.7 (C-28), 13.0 (C-18)。与文献对照[15],鉴定化合物7为过氧麦角甾醇 (ergosterol peroxide)。

    化合物8  无色晶体 (CHCl3),分子式C29H48O。1H NMR (CDCl3, 300 MHz) δ: 5.35 (1H, d, J = 4.8 Hz, H-6), 5.15 (1H, dd, J = 15.2, 8.5 Hz, H-22), 5.01 (1H, dd, J = 15.2, 8.5 Hz, H-23), 3.53 (1H, m, H-3), 1.02 (3H, d, J = 6.7 Hz, H-21), 1.01 (3H, s, H-19), 0.84 (3H, d, J = 6.4 Hz, H-26), 0.80 (3H, t, J = 6.0 Hz, H-29), 0.79 (3H, d, J = 6.5 Hz, H-27), 0.69 (3H, s, H-18)。13C NMR (CDCl3, 125 MHz) δ: 140.9 (C-5), 138.5 (C-22), 129.5 (C-23), 121.9 (C-6), 72.0 (C-3), 57.1 (C-14), 56.2 (C-17), 51.4 (C-24), 50.3 (C-9), 42.5 (C-4), 42.4 (C-13), 40.6 (C-20), 39.9 (C-12), 37.4 (C-1), 36.7 (C-10), 32.1 (C-25), 32.1 (C-8), 32.0 (C-7), 31.9 (C-2), 29.1 (C-16), 25.6 (C-28), 24.5 (C-15), 21.4 (C-21), 21.3 (C-26), 21.2 (C-11), 19.6 (C-29), 19.1 (C-27), 12.4 (C-29), 12.2 (C-18)。与文献对照[16],鉴定化合物8为豆甾醇 (stigmasterol)。

    化合物9  白色结晶状粉末 (CHCl3),分子式C29H50O。1H NMR (CDCl3, 300 MHz) δ: 5.35 (1H, d, J = 5.4 Hz, H-6), 3.51 (1H, m, H-3), 1.00 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.84 (3H, t, J = 6.5 Hz, H-29), 0.83 (3H, d, J = 6.5 Hz, H-27), 0.81 (3H, d, J = 6.7 Hz, H-26), 0.67 (3H, s, H-18)。13C NMR (CDCl3, 125 MHz) δ: 140.9 (C-5), 121.9 (C-6), 72.0 (C-3), 56.9 (C-14), 56.2 (C-17), 50.3 (C-9), 46.0 (C-24), 42.5 (C-4), 42.5 (C-13), 40.0 (C-12), 37.4 (C-1), 36.7 (C-10), 36.3 (C-20), 34.1 (C-22), 32.1 (C-7), 32.1 (C-8), 31.8 (C-2), 29.4 (C-25), 28.4 (C-16), 26.3 (C-23), 24.5 (C-15), 23.3 (C-28), 21.3 (C-11), 20.0 (C-21), 19.5 (C-19), 19.2 (C-26), 18.9 (C-27), 12.1 (C-29), 12.0 (C-18)。与文献对照[17],鉴定化合物9β-谷甾醇 (β-sitosterol)。

    采用MTT法评价已分离鉴定的9个化合物的体外抗肿瘤活性,结果见表2。结果显示,化合物6对A549细胞具有中等强度的增殖抑制作用,其IC50为(14.16 ± 1.11) μmol/L。

    Table  2.  Antiproliferative activities of compounds 19 in A549 cells
    Compd. IC50 /(μmol/L) Compd. IC50/(μmol/L)
    1 >50 6 14.16 ± 1.11
    2 >50 7 >50
    3 >50 8 >50
    4 >50 9 >50
    5 >50 Carboplatin 8.34 ± 0.95
    下载: 导出CSV 
    | 显示表格

    暗紫贝母通常以鳞茎入药,其他非药用部分往往被作为废物丢弃。本研究对暗紫贝母茎乙醇提取物的化学成分进行初步研究,分离并鉴定了9个化合物,均为甾体化合物,化合物1为新化合物,化合物2~9为首次从暗紫贝母茎中分离得到。同时,通过MTT法对分离鉴定得到的9个化合物进行了体外抗肿瘤初步活性测试,结果表明,化合物6对A549细胞具有中等强度的细胞毒活性,其他化合物对A549细胞的IC50均大于50 μmol/L,提示暗紫贝母中此类螺甾烷醇类化合物可能不具有明显的抗肿瘤作用,少数C21甾醇具有中等抗肿瘤活性。基于此,后续将继续对分离得到的化合物进行抗炎及免疫调节的体外活性测试。

  • Figure  1.   Chemical structures of compounds 19

    Figure  2.   Key 1H-1H COSY and HMBC (H→C) correlations of compound 1

    Figure  3.   Key NOESY correlations of compound 1

    Table  1   NMR data of compound 1a

    PositonδH bδC cHMBC
    11.62, 2.13, m38.1C-3, 5
    22.04, 2.35, m37.0C-1, 3, 5, 10
    3211.0
    42.07, 2.43, m37.5C-2, 3, 5, 6
    52.59, m57.5C-3, 6, 10, 19
    6208.7
    72.03, m
    2.41, dd (16.0, 6.1)
    46.6C-6, 9
    81.65, m37.3
    91.37, m53.4
    1041.2
    111.46, 1.67, m21.5
    121.26, 1.83, m39.4C-9, 11, 18
    1341.0
    141.38, m56.3C-8
    151.30, 1.36, m31.6C-14, 16
    164.43, 1H, td (7.9, 5.8)80.4C-13
    171.84, m62.0C-12, 14, 16, 18, 20, 21
    180.80, 3H, s16.4C-12, 14, 17
    190.98, 3H, s12.6C-1, 5, 9, 10
    201.91, m41.7C-17
    210.99, 3H, d (7.0)14.4C-17, 20, 22
    22109.5
    231.68, 1.95, m30.7C-25
    241.52, 1.69, m23.1C-22, 26
    251.81, m38.0C-27
    263.53, m
    3.72, dd (11.7, 2.9)
    63.1C-22, 24, 25,
    273.48, 2H, m65.0C-24, 25, 26
    a Measured in CDCl3. Assignments were established by DEPT, 1H-1H COSY, HMQC and HMBC experiments. J values (in Hz) are in parentheses;b Measured at 600 MHz; c Measured at 150 MHz
    下载: 导出CSV

    Table  2   Antiproliferative activities of compounds 19 in A549 cells

    Compd. IC50 /(μmol/L) Compd. IC50/(μmol/L)
    1 >50 6 14.16 ± 1.11
    2 >50 7 >50
    3 >50 8 >50
    4 >50 9 >50
    5 >50 Carboplatin 8.34 ± 0.95
    下载: 导出CSV
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  • 收稿日期:  2024-04-02
  • 刊出日期:  2025-04-24

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