无柄灵芝子实体中的灵芝酸类化合物

刘文星 邱仕瑜 陈毅坚 董淼 周敏

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无柄灵芝子实体中的灵芝酸类化合物

    作者简介: 刘文星(1986−),男,云南人,硕士,讲师,研究方向:天然药物化学;E-mail:liuwenxing2017@163.com;
    通讯作者: 周敏, zhouminynun@163.com

Ganoderic acids from the fruiting bodies of Ganoderma resinaceum

    Corresponding author: ZHOU Min, zhouminynun@163.com
  • 摘要: 对无柄灵芝Ganoderma resinaceum子实体进行了化学成分研究,该大型真菌经过95%乙醇提取后,再使用正相柱色谱、反相柱色谱、MCI柱色谱以及半制备HPLC等技术手段分离得到了13个灵芝酸类化合物. 这些化合物的结构通过核磁共振技术(1H NMR、13C NMR)得到了鉴定,它们分别为resinacein D( 1 )、leucocontextin I( 2 )、ganolucidic acid γa( 3 )、resinacein R( 4 )、resinacein O( 5 )、甲基灵芝酸C( 6 )、灵芝酸AM17 )、灵芝酸H( 8 )、3β,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸( 9 )、3β,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸甲酯( 10 )、ganoderense A( 11 )、resinacein J( 12 )以及3β,7β,15β-三羟基-8,16-二烯-11,23-二羰基-羊毛甾烷-26-羧酸甲酯( 13 ),其中化合物2、3、6、8、9和化合物11为首次从该大型真菌中分离得到.
  • 图 1  无柄灵芝子实体中的灵芝酸类化合物

    Figure 1.  Ganoderic acids from the fruiting bodies of G. resinaceum

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出版历程
  • 收稿日期:  2019-09-25
  • 录用日期:  2020-03-30
  • 网络出版日期:  2020-04-11
  • 刊出日期:  2020-05-01

无柄灵芝子实体中的灵芝酸类化合物

    作者简介:刘文星(1986−),男,云南人,硕士,讲师,研究方向:天然药物化学;E-mail:liuwenxing2017@163.com
    通讯作者: 周敏, zhouminynun@163.com
  • 云南民族大学 化学与环境学院,云南 昆明 650500

摘要: 对无柄灵芝Ganoderma resinaceum子实体进行了化学成分研究,该大型真菌经过95%乙醇提取后,再使用正相柱色谱、反相柱色谱、MCI柱色谱以及半制备HPLC等技术手段分离得到了13个灵芝酸类化合物. 这些化合物的结构通过核磁共振技术(1H NMR、13C NMR)得到了鉴定,它们分别为resinacein D( 1 )、leucocontextin I( 2 )、ganolucidic acid γa( 3 )、resinacein R( 4 )、resinacein O( 5 )、甲基灵芝酸C( 6 )、灵芝酸AM17 )、灵芝酸H( 8 )、3β,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸( 9 )、3β,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸甲酯( 10 )、ganoderense A( 11 )、resinacein J( 12 )以及3β,7β,15β-三羟基-8,16-二烯-11,23-二羰基-羊毛甾烷-26-羧酸甲酯( 13 ),其中化合物2、3、6、8、9和化合物11为首次从该大型真菌中分离得到.

English Abstract

  • 无柄灵芝(Ganoderma resinaceum)属多孔菌科灵芝属,长期被用于预防和治疗各类疾病,具有抗癌、增强免疫力和保肝等多种药理作用[1],它的化学成分复杂,主要有三萜、杂萜、甾体、多糖、黄酮等成分[1-9]. 灵芝酸(ganoderic acid)是灵芝属真菌中含有的具有较高氧化程度的羊毛甾烷型三萜类化合物,具有广泛的药理活性,如抗肿瘤、保肝、镇痛、抗炎、抗衰老等[10]. 作者在无柄灵芝的初步研究中得到了1个新杂萜和少量灵芝酸类化合物[1],进一步的化学成分研究中得到了13个灵芝酸类化合物. 它们分别为:resinacein D(1)、leucocontextin I(2)、ganolucidic acid γa(3)、resinacein R(4)、resinacein O(5)、甲基灵芝酸C(6)、灵芝酸AM17)、灵芝酸H(8)、,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸(9)、3β,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸甲酯(10)、ganoderense A(11)、resinacein J(12)以及3β,7β,15β-三羟基-8,16-二烯-11,23-二羰基-羊毛甾烷-26-羧酸甲酯(13)(见图1),其中化合物2、3、6、8、9和化合物11为首次从无柄灵芝中分离得到.

    图  1  无柄灵芝子实体中的灵芝酸类化合物

    Figure 1.  Ganoderic acids from the fruiting bodies of G. resinaceum

    • DRX-400 型核磁共振仪(Bruker Corporation, United States);Agilent-1200型高效液相色谱仪(Agilent Corporation, United States);Venusil XBP-C18(Φ10 mm×250 mm,5 μm)半制备色谱柱(美国伯乐BIO-RAD公司);YMC-Pack ODS-A(Φ20 mm×250 mml.D.,S-5 μm)制备色谱柱(美国伯乐BIO-RAD公司);OSB-2100型旋转蒸发仪(EYELA);Bio-Rad SHZ-D(Ⅲ)型循环水式真空泵(予华仪器有限责任公司).

      0.130~0.216 mm、0.054~0.077 mm硅胶(青岛海洋化工有限公司);GF254(100 mm × 100 mm)薄层色谱板(青岛海洋化工有限公司);MCI(75~150 μm)(南京元宝峰医药科技有限公司);分析纯甲醇、乙腈(天津天泰精细化学品有限公司);工业级纯甲醇、乙醇、二氯甲烷、乙酸乙酯.

      本实验所用的无柄灵芝G. resinaceum购买于 2017 年 4 月,产地为云南省瑞丽市,样品经过云南民族大学杨青松副教授鉴定,保存于云南民族大学化学与环境学院(样品编号:201701G).

    • 45 kg 的无柄灵芝干燥后粉碎,用工业级乙醇加热回流提取 2 次(每次60 min),浓缩后用乙酸乙酯萃取,萃取液用旋转蒸发仪浓缩得1.0 kg浸膏. 浸膏首先经过正相硅胶柱色谱法分离,用二氯甲烷−甲醇为洗脱剂进行梯度洗脱(体积比分别为1∶0,140∶2,80∶2,8∶1,5∶1,0∶1),得到6个(A~F)组分.

      组分B(131.3 g)用石油醚−乙酸乙酯(体积比分别为20∶1,10∶1,8∶1,5∶1,2∶1)为洗脱剂进行硅胶柱色谱分离,得到5个组分(B-1~B-5),B-3(35.2 g)经过硅胶柱色谱和半制备液相色谱仪分离得到化合物1(59.6 mg).

      组分C(500.0 g)用正相硅胶柱色谱分离,用二氯甲烷−甲醇(体积比分别为1∶0,80∶2,40∶2,20∶2,10∶2,0∶1)梯度洗脱,得到6个(C-1~C-6)组分. C-2(51.2 g)以45%~95%甲醇水洗脱,用MCI中压柱层析分离,再用半制备液相色谱仪分离得到3个化合物,他们分别是化合物6(10.1 mg)、9(173.1 mg)和化合物10(47.8 mg);C-3(48.9 g)用MCI中压柱层析分离,分别用32%、45%、60%、75%、95%甲醇水洗脱,然后用半制备液相色谱仪分离得到5个化合物,它们分别是化合物2(17.2 mg)、3(23.2 mg)、8(31.8 mg)、11(25.0 mg)和化合物12(11.2 mg).

      组分D(300.0 g)用正相硅胶柱色谱分离,用二氯甲烷−甲醇(体积比分别为1∶0,140∶2,40∶2,20∶2,10∶2,4∶2,0∶1)梯度洗脱,得到7个组分(D-1~D-7). D-2(62.5 g)经过反相柱,用甲醇−水体系进行梯度洗脱,最后由半制备液相色谱仪进一步分离得到化合物4(69.7 mg)、5(48.2 mg)、7(164.6 mg)和化合物13(102.0 mg).

    • 化合物1 白色粉末,根据NMR数据推测其分子式为C30H45O5,不饱和度Ω = 8. 1H NMR (Pyridin-d5, 400 MHz) δ: 2.18 (1H, m, H-1a), 2.47~2.72 (1H, m, H-1b), 1.93~1.96 (2H, m, H-2), 3.47 (1H, dd-like, J = 9.6, 6.6 Hz, H-3), 2.00 (1H, m, H-5), 2.47~2.72 (2H, m, H-6), 4.79 (1H, dd, J = 4.7, 8.8 Hz, H-11), 2.47~2.72 (1H, m, H-12a), 2.31 (1H, m, H-12b), 2.00 (1H, m, H-15a), 2.47~2.72 (1H, m, H-15b), 1.36 (1H, m, H-16a), 2.00 (1H, m, H-16b), 1.70 (1H, m, H-17), 0.80 (3H, s, H-18), 1.33 (3H, s, H-19), 1.48 (1H, m, H-20), 1.04 (3H, d, J = 6.5 Hz, H-21), 1.63 (1H, m, H-22a), 1.22 (1H, m, H-22b), 2.18 (2H, m, H-23), 7.22 (1H, t, J = 6.8 Hz, H-24), 2.12 (3H, s, H-27), 1.13 (3H, s, H-28), 1.13 (3H, s, H-29), 1.55 (3H, s, H-30); 13C NMR (Pyridin-d5, 100 MHz) δ: 35.13 (C-1), 28.78 (C-2), 77.68 (C-3), 39.98 (C-4), 51.28 (C-5), 37.90 (C-6), 200.65 (C-7), 141.23 (C-8), 162.49 (C-9), 41.32 (C-10), 64.69 (C-11), 45.95 (C-12), 48.38 (C-13), 48.91 (C-14), 33.90 (C-15), 28.71 (C-16), 50.7 (C-17), 17.61 (C-18), 20.30 (C-19), 36.79 (C-20), 18.88 (C-21), 35.56 (C-22), 26.22 (C-23), 142.64 (C-24), 129.24 (C-25), 170.98 (C-26), 13.14 (C-27), 28.23 (C-28), 16.25 (C-29), 25.59 (C-30). 以上核磁数据与文献[4]报道一致,故鉴定化合物1为resinacein D.

      化合物2 白色粉末,根据NMR数据推测其分子式为C30H44O6,不饱和度Ω = 9. 1H NMR (CD3OD, 400 MHz) δ: 3.09 (1H, m, H-1a), 1.52 (1H, m, H-1b), 2.51 (1H, m, H-2a), 2.42 (1H, m, H-2b), 1.58 (1H, m, H-5), 1.55 (1H, m, H-6a), 1.88 (1H, m, H-6b), 2.51 (2H, m, H-7), 2.77 (1H, d, J = 17.0 Hz, H-12a), 2.36 (1H, d, J = 16.9 Hz, H-12b), 4.32 (1H, dd, J = 5.4, 9.0 Hz, H-15), 1.82~1.92 (2H, m, H-16), 1.82-1.92 (1H, m, H-17), 0.91 (3H, s, H-18), 1.21 (3H, s, H-19), 1.43 (1H, m, H-20), 1.11 (3H, d, J = 6.0 Hz, H-21), 1.39 (1H, m, H-22a), 1.50 (1H, m, H-22b), 2.08~2.27 (2H, m, H-23), 6.72 (1H, t, J = 7.1 Hz, H-24), 1.78 (3H, s, H-27), 1.17 (3H, s, H-28), 3.51 (1H, d, J = 11.5 Hz, H-29a), 3.97 (1H, d, J = 11.5 Hz, H-29b), 1.14 (3H, s, H-30); 13C NMR (CD3OD, 100 MHz) δ: 36.07 (C-1), 36.65 (C-2), 218.77 (C-3), 54.32 (C-4), 54.84 (C-5), 19.49 (C-6), 31.27 (C-7), 167.52 (C-8), 139.45 (C-9), 38.37 (C-10), 200.92 (C-11), 53.09 (C-12), 48.11 (C-13), 54.78 (C-14), 73.28 (C-15), 39.03 (C-16), 49.94 (C-17), 17.43 (C-18), 19.49 (C-19), 37.14 (C-20), 18.55 (C-21), 35.76 (C-22), 26.42 (C-23), 143.66 (C-24), 129.18 (C-25), 167.52 (C-26), 12.50 (C-27), 21.66 (C-28), 65.60 (C-29), 19.79 (C-30). 以上核磁数据与文献[11]报道一致,故鉴定化合物2为leucocontextin I.

      化合物3 白色粉末,根据NMR数据推测其分子式为C30H46O7,不饱和度Ω = 8. 1H NMR (CD3OD, 400 MHz) δ: 3.28 (1H, br.s, H-1a), 1.13 (1H, m, H-1b), 1.76 (2H, m, H-2), 3.48 (1H, dd-like, J = 12.0, 4.7 Hz, H-3), 1.32 (1H, m, H-5), 1.80 (1H, m, H-6a), 2.67 (1H, m, H-6b), 4.70 (1H, m, H-7), 2.37 (1H, d, J = 15.2 Hz, H-12a), 2.83 (1H, d, J = 15.2 Hz, H-12b), 4.50 (1H, m, H-15), 1.53~1.79 (1H, m, H-16a), 2.08 (1H, dd, J = 7.4, 12.6 Hz, H-16b), 1.52~1.80 (1H, m, H-17), 0.81 (3H, s, H-18), 0.95 (3H, s, H-19), 1.52~1.80 (1H, m, H-20), 1.21 (3H, d, J = 4.5 Hz, H-21), 1.52~1.80 (2H, m, H-22), 4.70 (1H, m, H-23), 6.55 (1H, d, J = 8.7 Hz, H-24), 1.84 (3H, s, H-27), 0.93 (3H, s, H-28), 0.89 (3H, s, H-29), 0.98 (3H, s, H-30); 13C NMR (CD3OD, 100 MHz) δ: 35.89 (C-1), 28.98 (C-2), 78.93 (C-3), 39.68 (C-4), 50.40 (C-5), 28.37 (C-6), 70.16 (C-7), 161.28 (C-8), 143.09 (C-9), 39.67 (C-10), 202.36 (C-11), 53.19 (C-12), 48.31 (C-13), 55.28 (C-14), 73.19 (C-15), 37.44 (C-16), 50.09 (C-17), 16.37 (C-18), 19.95 (C-19), 34.77 (C-20), 20.08 (C-21), 44.51 (C-22), 67.36 (C-23), 144.66 (C-24), 129.52 (C-25), 170.39 (C-26), 13.22 (C-27), 28.69 (C-28), 17.43 (C-29), 19.82 (C-30). 以上核磁数据与文献[12]报道基本一致,故鉴定化合物3为ganolucidic acid γa.

      化合物4 白色粉末,根据NMR数据推测其分子式为C31H48O8,不饱和度Ω = 8. 1H NMR (CD3OD, 400 MHz) δ: 2.69 (1H, dt, J = 13.5, 3.1 Hz, H-1a), 0.88 (1H, m, H-1b), 1.55~1.66 (2H, m, H-2), 3.14 (1H, dd, J = 11.5, 4.8 Hz, H-3), 0.87 (1H, d, J = 13.2 Hz, H-5), 2.02 (1H, m, H-6a), 1.55~1.66 (1H, m, H-6b), 4.51 (1H, dd, J = 10.0, 7.5 Hz, H-7), 2.23 (1H, d, J = 15.0 Hz, H-12a), 2.87 (1H, d, J = 15.5 Hz, H-12b), 4.75 (1H, dd, J = 9.2, 7.1 Hz, H-15), 1.73~1.83 (1H, m, H-16), 1.91 (1H, m, H-17), 0.92 (3H, s, H-18), 1.23 (3H, s, H-19), 1.98 (1H, m, H-20), 0.87 (3H, d, J = 6.3 Hz, H-21), 2.61 (1H, dd, J = 17.7, 2.2 Hz, H-22a), 2.48 (1H, dd, J = 17.7, 9.4 Hz, H-22b), 4.37 (1H, d, J = 5.0 Hz, H-24), 2.92 (1H, m, H-25), 1.00 (3H, d, J = 7.2 Hz, H-27), 0.95 (3H, s, H-28), 0.83 (3H, s, H-29), 1.24 (3H, s, H-30), 3.68 (3H, s, H-OCH3); 13C NMR (CD3OD, 100 MHz) δ: 35.87 (C-1), 28.34 (C-2), 78.88 (C-3), 39.65 (C-4), 50.37 (C-5), 28.97 (C-6), 70.13 (C-7), 161.21 (C-8), 143.02 (C-9), 39.65 (C-10), 202.24 (C-11), 53.08 (C-12), 48.32 (C-13), 55.29 (C-14), 73.18 (C-15), 36.99 (C-16), 49.30 (C-17), 17.57 (C-18), 19.85 (C-19), 33.27 (C-20), 20.21 (C-21), 46.60 (C-22), 212.82 (C-23), 78.68 (C-24), 43.37 (C-25), 176.00 (C-26), 11.43 (C-27), 28.72 (C-28), 16.40 (C-29), 20.03 (C-30), 52.46 (C-OCH3). 以上核磁数据与文献[4]报道一致,故鉴定化合物4为resinacein R.

      化合物5 白色粉末,根据NMR数据推测其分子式为C31H46O8,不饱和度Ω = 9. 1H NMR (Pyridin-d5, 400 MHz) δ: 3.21 (1H, dt-like, J = 13.8, 2.9 Hz, H-1a), 1.00 (1H, dd, J = 13.8, 4.2 Hz, H-1b), 1.89~1.93 (2H, m, H-2), 3.46 (1H, dd, J = 11.4, 4.6 Hz, H-3), 0.90 (1H, m, H-5), 2.22 (1H, m, H-6a), 1.90 (1H, m, H-6b), 5.14 (1H, m, H-7), 2.83 (1H, d-like, J = 16.8 Hz, H-12a), 2.98 (1H, d, J = 16.8 Hz, H-12b), 2.87 (1H, dd-like, J = 18.8, 8.4 Hz, H-16a), 2.29 (1H, dd, J = 18.8, 9.8 Hz, H-16b), 2.52 (1H, m, H-17), 1.25 (3H, s, H-18), 1.39 (3H, s, H-19), 2.28 (1H, m, H-20), 1.04 (3H, d, J = 6.6 Hz, H-21), 2.38 (1H, m, H-22a), 2.20 (1H, m, H-22b), 4.90 (1H, d, J = 4.6 Hz, H-24), 3.31 (1H, m, H-25), 1.04 (3H, d, J = 6.5 Hz, H-27), 1.14 (3H, s, H-28), 1.08 (3H, s, H-29), 1.42 (3H, s, H-30), 3.64 (3H, s, H-OCH3); 13C NMR (Pyridin-d5, 100 MHz) δ: 35.96 (C-1), 29.04 (C-2), 78.75 (C-3), 39.76 (C-4), 50.16 (C-5), 28.56 (C-6), 67.21 (C-7), 159.17 (C-8), 143.22 (C-9), 39.76 (C-10), 198.84 (C-11), 51.51 (C-12), 46.17 (C-13), 59.66 (C-14), 217.10 (C-15), 41.97 (C-16), 46.74 (C-17), 18.25 (C-18), 19.24 (C-19), 32.26 (C-20), 20.38 (C-21), 46.39 (C-22), 212.53 (C-23), 78.01 (C-24), 43.56 (C-25), 175.37 (C-26), 12.12 (C-27), 29.24 (C-28), 16.94 (C-29), 25.35 (C-30), 52.25 (C-OCH3). 以上核磁数据与文献[4]报道一致,故鉴定化合物5为resinacein O.

      化合物6 白色粉末,根据NMR数据推测其分子式为C31H48O7,不饱和度Ω = 8. 1H NMR (Pyridin-d5, 400 MHz) δ: 3.52 (1H, dd, J = 4.4, 11.3 Hz, H-3), 4.98~5.05 (1H, m, H-7), 4.98~5.05 (1H, m, H-15), 1.12 (3H, s, H-18), 1.54 (3H, s, H-19), 0.95 (3H, d, J = 6.3 Hz, H-21), 1.19 (3H, d, J = 7.2 Hz, H-27), 1.30 (3H, s, H-30), 1.12 (3H, s, H-31), 1.55 (3H, s, H-32), 3.64 (3H, s, H-OCH3); 13C NMR (Pyridin-d5, 100 MHz) δ: 35.82 (C-1), 29.42 (C-2), 77.91 (C-3), 39.69 (C-4), 50.24 (C-5), 29.14 (C-6), 69.78 (C-7), 160.80 (C-8), 142.23 (C-9), 39.43 (C-10), 200.29 (C-11), 53.08 (C-12), 47.87 (C-13), 55.08 (C-14), 72.66 (C-15), 37.36 (C-16), 49.00 (C-17), 17.80 (C-18), 20.05 (C-19), 33.52 (C-20), 20.62 (C-21), 50.14 (C-22), 209.03 (C-23), 47.14 (C-24), 35.40 (C-25), 176.60 (C-26), 17.51 (C-27), 29.14 (C-30), 17.02 (C-31), 20.23 (C-32), 52.01 (C-OCH3). 以上核磁数据与文献[13]对照一致,故鉴定化合物6为甲基灵芝酸C(methyl ganoderate C).

      化合物7 白色粉末,根据NMR数据推测其分子式为C30H42O7,不饱和度Ω = 10. 1H NMR (Pyridin-d5, 400 MHz) δ: 3. 45 (1H, dd, J = 5.1, 11.1 Hz, H-3), 1.06 (3H, s, H-18), 1.39 (3H, s, H-19), 1.05 (3H, d, J = 5.8 Hz, H-21), 1.38 (3H, d, J = 6.8 Hz, H-27), 1.15 (3H, s, H-28), 0.96 (3H, s, H-29), 1.75 (3H, s, H-30); 13C NMR (Pyridin-d5, 100 MHz) δ: 37.29 (C-1), 28.56 (C-2), 77.11 (C-3), 40.09 (C-4), 52.00 (C-5), 34.50 (C-6), 200.19 (C-7), 152.16 (C-8), 147.49 (C-9), 41.33 (C-10), 200.43 (C-11), 50.14 (C-12), 44.92 (C-13), 57.99 (C-14), 208.01 (C-15), 40.75 (C-16), 45.56 (C-17), 18.02 (C-18), 18.35 (C-19), 32.70 (C-20), 20.15 (C-21), 49.71 (C-22), 209.05 (C-23), 47.45 (C-24), 36.08 (C-25), 178.86 (C-26), 16.59 (C-27), 28.64 (C-28), 16.59 (C-29), 21.85 (C-30). 以上核磁数据与文献[14]对照一致,故鉴定化合物7为灵芝酸AM1 (ganoderic acid AM1).

      化合物8 黄色油状,根据NMR数据推测其分子式为C30H40O7,不饱和度Ω = 11. 1H NMR (Pyridin-d5, 400 MHz) δ: 3.47 (1H, dd, J =5.4, 10. 6 Hz, H-3), 0.85 (3H, s, H-18), 1.33 (3H, s, H-19), 2.28 (3H, s, H-21), 6.40 (1H, s, H-22), 1.42 (3H, d, J = 7.0 Hz, H-27), 1.16 (3H, s, H-28), 1.07 (3H, s, H-29), 1.78 (3H, s, H-30); 13C NMR (Pyridin-d5, 100 MHz) δ: 34.45 (C-1), 28.52 (C-2), 77.05 (C-3), 41.30 (C-4), 49.41 (C-5), 37.00 (C-6), 199.97 (C-7), 147.81 (C-8), 152.15 (C-9), 39.97 (C-10), 199.65 (C-11), 49.12 (C-12), 46.12 (C-13), 57.25 (C-14), 207.55 (C-15), 37.47 (C-16), 51.49 (C-17), 18.13 (C-18), 18.20 (C-19), 153.97 (C-20), 21.52 (C-21), 125.73 (C-22), 199.36 (C-23), 48.70 (C-24), 36.21 (C-25), 178.85 (C-26), 18.06 (C-27), 28.46 (C-28), 16.45 (C-29), 22.50 (C-30). 以上核磁数据与文献[15]对照一致,故鉴定化合物8为灵芝酸H(ganoderenic acid H).

      化合物9 黄色针状结晶,根据NMR数据推测其分子式为C30H42O7,不饱和度Ω = 10. 1H NMR (CD3OD, 400 MHz) δ: 1.12 (1H, dd, J = 6.9, 10.7 Hz, H-1a), 2.83~2.90 (1H, m, H-1b), 1.64 (2H, m, H-2), 3.13 (1H, dd, J = 5.1, 11.8 Hz, H-3), 0.95 (1H, m, H-5), 2.12 (1H, dd, J = 7.8, 12.8 Hz, H-6a), 1.60 (1H, m, H-6b), 4.80 (1H, m, H-7), 2.55 (1H, d, J = 16.0 Hz, H-12a), 2.83~2.90 (1H, m, H-12b), 5.73 (1H, s, H-16), 1.19 (3H, s, H-18), 1.18 (3H, s, H-19), 3.00 (1H, dd, J = 6.9, 13.6 Hz, H-20), 1.10 (3H, d, J = 6.9 Hz, H-21), 2.83~2.90 (2H, m, H-22), 2.74 (1H, dd, J = 5.9, 17.5 Hz, H-24a), 2.83~2.90 (1H, m, H-24b), 2.83~2.90 (1H, m, H-25), 1.18 (3H, d, J = 3.0 Hz, H-27), 1.00 (3H, s, H-28), 0.81 (3H, s, H-29), 1.52 (3H, s, H-30); 13C NMR (CD3OD, 100 MHz) δ: 35.90 (C-1), 28.28 (C-2), 78.84 (C-3), 39.73 (C-4), 50.47 (C-5), 27.37 (C-6), 68.31 (C-7), 159.30 (C-8), 143.66 (C-9), 40.26 (C-10), 199.48 (C-11), 45.43 (C-12), 52.73 (C-13), 59.54 (C-14), 212.11 (C-15), 123.45 (C-16), 189.49 (C-17), 31.54 (C-18), 19.06 (C-19), 29.95 (C-20), 19.82 (C-21), 48.33 (C-22), 208.74 (C-23), 47.11 (C-24), 35.86 (C-25), 179.41 (C-26), 17.52 (C-27), 28.75 (C-28), 16.33 (C-29), 33.68 (C-30). 以上核磁数据与文献[16]报道一致,故鉴定化合物9为,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸.

      化合物10 白色粉末,根据NMR数据推测其分子式为C31H44O7,不饱和度Ω = 10. 1H NMR (Pyridin-d5, 400 MHz) δ: 1.21 (1H, m, H-1a), 2.92~3.28 (1H, m, H-1b), 1.93~1.96 (2H, m, H-2), 3.51 (1H, dd, J = 4.5, 11.6 Hz, H-3), 1.14 (1H, m, H-5), 1.93~1.96 (2H, m, H-6), 5.15 (1H, dd, J = 7.9, 9.5 Hz, H-7), 2.77 (1H, d, J = 16.9, H-12a), 2.92~3.28 (1H, m, H-12b), 5.95 (1H, s, H-16), 1.47 (3H, s, H-18), 1.42 (3H, s, H-19), 2.92~3.28 (1H, m, H-20), 1.17 (3H, d, J = 7.1 Hz, H-21), 2.92~3.28 (1H, m, H-22a), 2.59 (1H, dd, J = 17.5, 4.5 Hz, H-22b), 2.92~3.28 (2H, m, H-24), 2.92~3.28 (1H, m, H-25), 1.10 (3H, d, J = 6.9 Hz, H-27), 1.29 (3H, s, H-28), 1.11 (3H, s, H-29), 1.59 (3H, s, H-30), 3.62 (3H, s, H-31); 13C NMR (Pyridin-d5, 100 MHz) δ: 35.78 (C-1), 28.98 (C-2), 77.83 (C-3), 39.69 (C-4), 50.17 (C-5), 27.80 (C-6), 67.66 (C-7), 159.52 (C-8), 142.82 (C-9), 39.95 (C-10), 198.20 (C-11), 45.19 (C-12), 52.15 (C-13), 58.72 (C-14), 210.73 (C-15), 123.18 (C-16), 187.90 (C-17), 31.51 (C-18), 19.18 (C-19), 29.36 (C-20), 19.72 (C-21), 47.95 (C-22), 207.10 (C-23), 46.68 (C-24), 35.26 (C-25), 176.47 (C-26), 17.45 (C-27), 29.12 (C-28), 16.68 (C-29), 33.67 (C-30), 52.02 (C-31). 以上核磁数据与文献[16]报道一致,故鉴定化合物10为3β,7β-二羟基-11,15,23-三羰基-羊毛甾烷-8,16-二烯-26-羧酸甲酯.

      化合物11 黄色针状结晶,根据NMR数据推测其分子式为C30H40O7,不饱和度Ω = 11. 1H NMR (Pyridin-d5, 400 MHz) δ: 3.02~3.26 (1H, m, H-1a), 1.55 (1H, m, H1b), 1.88~2.00 (2H, m, H-2), 3.51 (1H, t, J = 8.0 Hz, H-3), 1.88~2.00 (1H, m, H-5), 2.63~2.81 (2H, m, H-6), 3.24 (1H, d, J = 16.7 Hz, H-12a), 2.73 (1H, d, J = 16.7 Hz, H-12b), 6.00 (1H, s, H-16), 1.32 (3H, s, H-18), 1.20 (3H, s, H-19), 3.02~3.19 (1H, m, H-20), 1.09 (3H, d, J = 8.2 Hz, H-21), 2.98 (1H, d, J = 7.4 Hz, H-22a), 2.86 (1H, d, J = 6.3 Hz, H-22b), 3.02~3.19 (1H, m, H-24a), 2.58 (1H, dd, J = 4.7, 17.4 Hz, H-24b), 3.28 (1H, m, H-25), 1.36 (3H, d, J = 7.2 Hz, H-27), 1.14 (3H, s, H-28), 1.08 (3H, s, H-29), 1.62 (3H, s, H-30); 13C NMR (Pyridin-d5, 100 MHz) δ: 34.76 (C-1), 28.47 (C-2), 77.13 (C-3), 39.75 (C-4), 50.00 (C-5), 36.12 (C-6), 198.43 (C-7), 151.08 (C-8), 152.56 (C-9), 41.30 (C-10), 200.22 (C-11), 45.19 (C-12), 52.04 (C-13), 55.95 (C-14), 203.33 (C-15), 124.03 (C-16), 183.50 (C-17), 30.26 (C-18), 17.74 (C-19), 29.24 (C-20), 19.91 (C-21), 48.23 (C-22), 207.57 (C-23), 47.05 (C-24), 35.87 (C-25), 178.60 (C-26), 17.93 (C-27), 28.32 (C-28), 16.13 (C-29), 33.03 (C-30). 以上核磁数据与文献[17]对照一致,故鉴定化合物11为ganoderense A.

      化合物12 白色粉末,根据NMR数据推测其分子式为C30H44O8,不饱和度Ω = 9. 1H NMR (CD3OD, 400 MHz) δ: 2.81 (1H, m, H-1a), 1.00 (1H, m, H-1b), 1.57~1.73 (2H, m, H-2), 3.17 (1H, m, H-3), 0.97 (1H, d, J = 12.6 Hz, H-5), 2.13 (1H, dd, J = 7.4, 12.6 Hz, H-6a), 1.57~1.73 (1H, m, H-6b), 4.49 (1H, dd, J = 7.5, 9.8 Hz, H-7), 3.14 (1H, m, H-12a), 2.67 (1H, d, J = 15.6 Hz, H-12b), 5.46 (1H, s, H-15), 5.39 (1H, s, H-16), 1.18 (3H, s, H-18), 1.24 (3H, s, H-19), 1.35 (3H, s, H-21), 3.02 (1H, d, J = 16.2 Hz, H-22a), 2.67 (1H, d, J = 16.2 Hz, H-22b), 2.83 (1H, m, H-24a), 2.59 (1H, m, H-24b), 2.81 (1H, m, H-25), 1.16 (3H, d, J = 6.9 Hz, H-27), 1.02 (3H, s, H-28), 0.85 (3H, s, H-29), 1.30 (3H, s, H-30); 13C NMR (CD3OD, 100 MHz) δ: 35.89 (C-1), 29.05 (C-2), 78.95 (C-3), 39.74 (C-4), 50.67 (C-5), 28.39 (C-6), 70.13 (C-7), 161.95 (C-8), 142.86 (C-9), 39.92 (C-10), 202.36 (C-11), 50.12 (C-12), 53.05 (C-13), 58.87 (C-14), 77.68 (C-15), 127.74 (C-16), 156.11 (C-17), 23.36 (C-18), 19.60 (C-19), 73.60 (C-20), 28.89 (C-21), 53.50 (C-22), 212.06 (C-23), 49.70 (C-24), 36.34 (C-25), 181.25 (C-26), 16.40 (C-27), 28.74 (C-28), 17.67 (C-29), 24.04 (C-30). 以上核磁数据与文献[4]对照一致,故鉴定化合物12为resinacein J.

      化合物13 黄色油状物,根据NMR数据推测其分子式为C31H46O7,不饱和度Ω = 9. 1H NMR (CD3OD, 400 MHz) δ: 2.87 (1H, m, H-1a), 1.59 (1H, m, H-1b), 1.65 (2H, m, H-2), 2.77 (1H, dd, J = 6.9, 16.9 Hz, H-3), 0.95 (1H, m, H-5), 2.15 (1H, dd, J = 7.3, 12.4 Hz, H-6), 4.51 (1H, dd, J = 7.3, 10.1 Hz, H-7), 3.03 (1H, d, J = 15.3 Hz, H-12), 2.91 (1H, m, H-12), 5.47 (1H, s, H-15), 5.29 (1H, s, H-16), 1.31 (3H, s, H-18), 1.26 (3H, s, H-19), 2.66 (1H, m, H-20), 1.16 (3H, d, J = 6.9 Hz, H-21), 2.56 (1H, dd, J = 6.8, 16.1 Hz, H-22), 2.62 (1H, m, H-24), 2.67 (1H, m, H-25), 1.07 (3H, d, J = 6.7 Hz, H-27), 1.04 (3H, s, H-28), 0.87 (3H, s, H-29), 1.04 (3H, s, H-30), 3.66 (3H, s, H-OCH3); 13C NMR (CD3OD, 100 MHz) δ: 35.76 (C-1), 28.35 (C-2), 77.95 (C-3), 39.90 (C-4), 50.62 (C-5), 28.93 (C-6), 70.11 (C-7), 161.96 (C-8), 142.68 (C-9), 39.67 (C-10), 201.67 (C-11), 47.23 (C-12), 52.89 (C-13), 57.44 (C-14), 78.81 (C-15), 125.68 (C-16), 155.71 (C-17), 22.75 (C-18), 19.67 (C-19), 28.60 (C-20), 20.73 (C-21), 49.01 (C-22), 209.57 (C-23), 49.32 (C-24), 35.79 (C-25), 177.82 (C-26), 17.33 (C-27), 28.77 (C-28), 16.44 (C-29), 23.33 (C-30), 52.30 (C-OCH3). 以上核磁数据与文献[18]报道一致,故鉴定化合物13为3β,7β,15β-三羟基-8,16-二烯-11,23-二羰基-羊毛甾烷-26-羧酸甲酯.

    • 本实验对无柄灵芝G. resinaceum子实体的95%乙醇提取物通过正相柱色谱、反相柱色谱、MCI柱色谱以及半制备HPLC等技术手段进行了化学成分研究,得到了13个灵芝酸类化合物,它们的结构通过核磁共振技术(1H NMR、13C NMR)进行了鉴定,其中化合物2、3、6、8、9和化合物11为首次从该大型真菌中分离得到,所有化合物均已在灵芝属真菌中分离得到.

      根据文献报道,灵芝酸类化合物C-24/C-25双键的存在是抑制α葡萄糖苷酶的关键因素,而C-11位和C-23位羟基的存在会降低对α葡萄糖苷酶的抑制率,C-15位羟基和C-26位羧酸的存在则增强抑制率,故化合物1在质量浓度为2.0 mmol/L时对α葡萄糖苷酶的抑制率为30.3%,而化合物4、5和12则没有明显的抑制α葡萄糖苷酶活性[4]. 从文献报道的其他活性测试结果来看,这些化合物的活性研究还不够广泛和深入. 如化合物2对K562、SMMC-7721和MCF-7细胞没有明显的细胞毒活性[11];化合物3没有引发hPXR促使CYP3A4表达的能力,也没有对HL-60、SW480、A549、SMMC-7721和MCF-7明显的细胞毒活性[12];化合物11没有明显的抑制硫氧还蛋白氧化还原酶活性[17],等. 根据灵芝酸类化合物的药理活性报道,结合本实验提供的物质基础,可以进一步探索这些化合物未知的活性,或对这些化合物进行结构修饰,从而增强其抑制α葡萄糖苷酶的活性,对研究具有抗糖尿病活性药物的先导化合物具有重要的研究价值和广阔的运用前景.

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