CN117815246A - 松杉灵芝菌丝体中三萜化合物的抗炎应用 - Google Patents
松杉灵芝菌丝体中三萜化合物的抗炎应用 Download PDFInfo
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Abstract
本发明提供了松杉灵芝菌丝体三萜化合物作为预防或治疗炎症的药物的应用；所述的松杉灵芝菌丝体三萜化合物为结构式4，5，7，8，9，11，12，13，14，15化合物中的一种或一种。结构式为4，5，7，8，9，11，12，13，14，15的化合物具有显著抑制TNF‑α、IL‑1β和IL‑6的活性，因而可作为预防和/或治疗炎症的药物或食品。
Description
技术领域
本发明涉及食用菌应用领域，具体的说涉及松杉灵芝菌丝体中三萜化合物的抗炎应用。
背景技术
松杉灵芝Ganoderma tsugae Murrill是担子菌门Basidiomycota、伞菌纲Agaricomycetes、多孔菌目Polyporales、多孔菌科Polyporaceae、灵芝属Ganoderma真菌，是主要分布于中国东北地区的灵芝物种。2001年，国家卫生部将其同灵芝、紫芝一同列入“可用于保健食品的真菌菌种名单”中，用于保健产品的开发。
炎症是一种较为常见的病理过程，机体受到创伤、感染等外界刺激后，巨噬细胞会产生大量肿瘤坏死因子(TNF-α)、白介素6(IL-6)、白介素1β(IL-1β)、白介素8(IL-8)等炎症因子。一般的，在关于药物抗炎活性研究中，选择NO、TNF-α、IL-6、IL-1β、PEG2的释放作为评价药物抗炎能力的指标。
真菌的不同生育阶段，会代谢产物不同的次生代谢产物。三萜是松杉灵芝主要的次级代谢产物，目前关于松杉灵芝菌丝体中的三萜化合物的生物活性研究，目前尚未有报道。
发明内容
本发明提供了松杉灵芝菌丝体三萜化合物作为预防或治疗炎症的药物的应用；其中所述的松杉灵芝菌丝体三萜化合物为结构式4，5，7，8，9，11，12，13，14，15化合物中的一种或一种以上：
其中所述的结构式4，5，7，8，9，11，12，13，14，15的化合物为：
上述结构式为4，5，7，8，9，11，12，13，14，15的化合物，其化学命名如下：
化合物4：15-OH-灵芝酸S
化合物5：灵芝酸Jb
化合物7：灵芝酸Ja
化合物8：灵芝酸X
化合物9：灵芝酸TQ
化合物11：灵芝酸Mf
化合物12：灵芝酸Y
化合物13：灵芝酸Z
化合物14：lanosta-7,9,(11),24-trien-3α-hydroxy-26-oic acid
化合物15：灵芝酸S1
本发明所提供的结构式4，5，7，8，9，11，12，13，14，15的化合物还可以作为预防和/或治疗炎症或在炎症治疗过程之中或之后起辅助作用的药物或食品中的用途。
本发明的创新点：
本发明首次从松杉灵芝发酵菌丝体中制备得到三萜化合物4，5，7，8，9，11，12，13，14，15，对分离制备的三萜化合物进行了抗炎活性验证，化合物4，5，7，8，9，11，12，13，14，15能有效抑制炎症因子TNF-α、IL-1β、IL-6的释放，可以用作预防或者治疗炎症的药物制剂或者食品。
附图说明
图1Fr1半制备液相色谱图
图2Fr2半制备液相色谱图
图3Fr3半制备液相色谱图
图4Fr4半制备液相色谱图
图5Fr5半制备液相色谱图
图6Fr6半制备液相色谱图
图7Fr7半制备液相色谱图
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：
松杉灵芝菌种Ganoderma tsugae Murrill，菌种保藏于中国林业大学生态与自然保护学院(菌种保藏号：Dai 20238)。
小鼠单核巨噬细胞RAW264.7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-5036。
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；
鼠源TNF-α、IL-6、IL-1β试剂盒：北京四正柏生物科技有限公司；
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、氘代氯仿：购自美国Sigma公司；
地塞米松：北京索莱宝科技有限公司；
其他试剂均为普通市售产品；
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；
Bruker AV 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
实施例1松杉灵芝菌丝体发酵
培养基配方：葡萄糖3％，酵母粉0.3％，MgSO4·7H2O 0.2％，KH2PO4 0.2％(上述均为重量百分比)，水余量，pH自然。
保存在4℃冰箱中的菌种，经过活化，黑暗条件下在摇瓶中进行振荡培养7d(150r/min，26℃)，获得一级种子液。再将一级种子液按10％比例接入二级培养液(配方同上)中，150r/min、25℃避光培养21d，收集菌丝体，50℃下烘干备用。
实施例2松杉灵芝菌丝体的提取和萃取
将干燥的菌丝体用95％(重量百分比)乙醇水溶液常温下浸泡提取3次，每次24h，合并提取液，旋转蒸发浓缩得到松杉灵芝菌丝体醇提物。
进一步将松杉灵芝菌丝体醇提物用等体积的石油醚和乙酸乙酯依次萃取各三次，分别减压浓缩后得到石油醚萃取相和乙酸乙酯萃取相。
实施例3化合物的制备
对实施例2中得到的乙酸乙酯相通过中高压层析系统进行分离，首先选择色谱柱YMC C18手装柱(50μm,70×460mm)进行粗分离，流动相为乙腈(A)-0.01％冰醋酸水溶液(B)，流速20mL/min，洗脱程序为0-45min,A:55％；45-180min,A:60％；180-360min,A:70％；360-405min,A:75％；405-560min,A:100％，每400mL收集一个流份，经薄层色谱监测合并相似流份，最终得到7个组分：Fr 1(6.92g),Fr 2(2.87g),Fr 3(4.87g),Fr 4(2.64g),Fr 5(15.91g),Fr 6(2.35g),Fr 7(1.48g)。对7个组分通过中高压层析[色谱柱Agilent ZorbaxEclipse Plus-C18PrepH(5μm,4.6mm×250mm)，流动相为乙腈(A)-0.01％冰醋酸水溶液(B)，流速20mL/min，检测波长240nm]、制备薄层色谱层析和凝胶色谱层析(Sephadex LH-20)完成分离纯化。
Fr 1通过梯度洗脱(0-25min,A:45-50％,25-30min,A:50-100％)，得到化合物1(105.3mg,Rt:18.0min)。
Fr 2通过洗脱程序(0-30min,A:50-65％；30-40min,A:65-75％；40-45min,A:75-100％)，得到化合物2(24.7mg,Rt:27.2min)和化合物3(11.9mg,Rt:36.0min)。
Fr 3通过梯度洗脱(0-10min,A:60-80％；10-15min,A:80-85％；15-20min,A:85-100％)，得到化合物4(23.0mg,Rt:13.6min)。
Fr 4通过等度洗脱(0-50min,A:60％)，得到Fr 4-1、Fr 4-2，Fr 4-1为化合物5(6.8mg,Rt:17.0min)，Fr 4-2再通过凝胶色谱层析(Sephadex LH-20)，甲醇洗脱，得到化合物6(31.8mg,Rt:26.1min)。
Fr 5通过等度洗脱(0-50min,A:65％)，得到Fr 5-1、Fr 5-2、Fr 5-3，Fr 5-1通过制备薄层色谱层析，以氯仿：甲醇＝9:1展开，制备得到化合物7(6.0mg,Rt:20.1min)；Fr 5-2为化合物8(56.0mg,Rt:34.2min)；Fr 5-3通过制备薄层色谱层析，以氯仿：甲醇＝92:8展开，制备得到化合物9(23.3mg,Rt:40.8min)。
Fr 6通过等度洗脱(0-45min，A：70％)，得到化合物10(50.0mg,Rt:45.2min)，化合物11(20mg,Rt:26.2min)和化合物12(20.3mg,Rt:38.2min)。
Fr 7通过梯度洗脱(0-20min，A：80-86％；20-30min，A：86％)，得到化合物13(19.0mg,Rt:24.1min)，化合物14(5.4mg,Rt:25.8min)和化合物15(20.2mg,Rt:27.2min)。
实施例3化合物1-15的确认
利用质谱以及核磁检测等手段，对松杉灵芝菌丝体中得到15个化合物进行结构解析，确定其精准的化学结构，具体的15个化合物的确认信息如下：
化合物1[(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C30H44O5，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:483.32[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.81(1H,t,H-24),5.90(1H,d,J＝5.8Hz,H-7),5.36(1H,d,J＝7.7Hz,H-11),4.25(1H,m,H-15),3.73(1H,m,H-22),1.80(3H,s,H-27),1.18(3H,s,H-19),1.12(3H,s,H-29),1.07(3H,s,H-28),0.93(3H,s,H-30),0.89(1H,d,J＝6.6Hz,H-18),0.89(1H,d,H-21)。13C-NMR(CDCl3,125MHz)δ:217.0(C-3),173.6(C-26),144.9(C-9),141.1(C-8),140.0(C-24),130.5(C-25),121.2(C-7),117.0(C-11),74.5(C-15),72.8(C-22),52.1(C-14),50.8(C-5),47.6(C-4),45.3(C-17),44.3(C-13),41.6(C-20),38.9(C-12),38.7(C-16),37.4(C-10),36.8(C-1),35.1(C-23),34.9(C-2),25.5(C-28),23.8(C-6),22.6(C-29),22.3(C-19),17.5(C-30),16.1(C-18),13.1(C-27),12.1(C-21)。
化合物2[(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C32H46O6,白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:525.33[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.80(1H,t,J＝6.1Hz,H-24),5.93(1H,d,J＝6.7Hz,H-7),5.38(1H,d,J＝6.6Hz,H-11),5.05(1H,m,H-22),4.29(1H,m,H-15),2.07(3H,s,CH3COO-),1.87(3H,s,H-27),1.19(3H,s,H-29),1.13(3H,s,H-28),1.08(3H,s,H-30),0.97(1H,d,J＝6.8Hz,H-21),0.92(3H,s,H-19),0.63(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:217.1(C-3),172.5(CH3COO-),171.0(C-26),145.0(C-9),140.8(C-8),139.4(C-24),129.6(C-25),121.7(C-7),116.7(C-11),74.7(C-22),74.6(C-15),52.1(C-14),50.6(C-5),47.6(C-4),45.6(C-17),44.3(C-13),39.8(C-20),39.5(C-16),38.6(C-12),37.4(C-10),36.8(C-1),35.0(C-2),32.0(C-23),25.6(C-30),23.8(C-6),22.7(C-28),22.4(C-19),21.3(CH3COO-),17.3(C-29),16.0(C-18),13.0(C-21),12.5(C-27)。
化合物3[(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]
分子式为C32H48O7，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:567.34[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.78(1H,t,J＝7.5Hz,H-24),5.52(1H,d,J＝6.9Hz,H-7),5.39(1H,d,J＝6.6Hz,H-11),5.09(1H,m H-22),5.03(1H,t,J＝7.7Hz,H-15),2.09(3H,s,CH3COO-),2.06(3H,s,CH3COO-),1.86(3H,s,H-27),1.18(3H,s,H-29),1.12(3H,s,H-28),1.09(3H,s,H-30),0.98(3H,s,H-19),0.97(3H,d,J＝6.6Hz,H-21),0.67(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:216.9(C-3),172.3(CH3COO-),171.4(CH3COO-),170.9(C-26),144.8(C-9),140.3(C-8),139.2(C-24),129.6(C-25),121.5(C-7),116.8(C-11),77.4(C-15),74.6(C-22),51.5(C-14),50.5(C-5),47.6(C-4),45.6(C-17),44.1(C-13),39.7(C-20),38.1(C-12),37.4(C-10),36.8(C-16),36.8(C-1),35.0(C-2),32.1(C-23),25.6(C-30),23.8(C-6),22.7(C-28),22.4(C-19),21.6(CH3COO-),21.3(CH3COO-),18.5(C-29),16.0(C-18),12.9(C-21),12.5(C-27)。
化合物4[15-OH-灵芝酸S(15-hydroxy-ganoderic acid S)]
分子式为C30H44O4，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:467.32[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.87(1H,t,J＝7.6Hz,H-24),5.88(1H,d,J＝6.9Hz,H-7),5.37(1H,d,J＝9.0Hz,H-11),4.29(1H,dd,J＝9.7Hz,H-15),1.83(3H,s,H-27),1.19(3H,s,H-29),1.12(3H,s,H-19),1.07(3H,s,H-28),0.93(3H,s,H-30),0.91(1H,d,J＝6.6Hz,H-21),0.63(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:217.0(C-3),173.1(C-26),145.3(C-24),144.8(C-9),141.0(C-8),127.0(C-25),121.2(C-7),117.0(C-11),74.6(C-15),52.0(C-14),50.6(C-5),48.9(C-17),47.5(C-4),44.4(C-13),40.0(C-16),38.5(C-12),37.3(C-10),36.7(C-1),36.0(C-20),34.9(C-2),34.8(C-22),25.9(C-23),25.5(C-28),23.7(C-6),22.5(C-29),22.3(C-19),18.3(C-21),17.1(C-30),16.1(C-18),12.1(C-27)。
化合物5[灵芝酸Jb(ganodermic acid Jb)]
分子式为C30H46O4，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:469.34[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.79(1H,t,J＝7.5Hz,H-24),5.87(1H,d,J＝6.8Hz,H-7),5.32(1H,d,J＝6.4Hz,H-11),1.82(3H,s,H-27),0.99(3H,s,H-19),0.98(3H,s,H-29),0.94(3H,s,H-28),0.92(1H,d,J＝6.5Hz,H-21),0.87(3H,s,H-30),0.62(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:170.7(C-26),146.2(C-9),143.6(C-24),140.6(C-8),27.3(C-25),121.3(C-7),115.8(C-11),78.6(C-3),74.2(C-15),51.9(C-14),48.7(C-17),48.5(C-5),44.3(C-13),39.4(C-16),38.6(C-4),38.4(C-12),37.4(C-10),35.9(C-20),35.8(C-1),34.8(C-22),28.0(C-29),27.3(C-2),25.7(C-23),22.9(C-6),22.7(C-19),18.2(C-21),17.1(C-28),15.8(C-18),15.7(C-30),12.0(C-27)。
化合物6[灵芝酸P(ganoderic acid P)]
分子式为C34H50O7，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:569.36[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.88(1H,td,J＝11.2Hz,J＝1.6Hz,H-24),5.50(1H,d,J＝4.8Hz,H-7),5.35(1H,d,J＝6.5Hz,H-11),5.09(1H,m,H-15),3.74(1H,m,H-3),2.09(3H,s,CH3COO-),2.11(3H,s,CH3COO-),1.85(3H,s,H-27),1.08(3H,s,H-30),1.01(3H,s,H-28),0.99(3H,s,H-19),0.94(1H,d,J＝6.8Hz,H-21),0.99(3H,s,H-29),0.68(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:171.9(C-26),171.4(CH3COO-),170.9(CH3COO-),146.2(C-9),140.2(C-8),139.3(C-24),129.4(C-25),121.7(C-7),115.5(C-11),76.3(C-15),76.2(C-3),74.9(C-22),51.6(C-14),45.6(C-17),44.1(C-13),43.1(C-5),39.8(C-20),38.1(C-12),37.5(C-10),37.5(C-16),36.9(C-4),32.1(C-23),30.1(C-1),28.4(C-28),25.7(C-2),23.2(C-6),23.0(C-29),22.9(C-19),21.7(CH3COO-),21.3(CH3COO-),18.8(C-30),16.0(C-18),12.9(C-21),12.5(C-27)。
化合物7[灵芝酸Ja(ganodermic acid Ja)]
分子式为C30H46O4，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:469.34[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.77(1H,m,H-24),5.58(1H,d,J＝6.2Hz,H-7),5.33(1H,d,J＝5.7Hz,H-11),5.09(1H,m,H-22),4.27(1H,t,J＝8.3Hz,H-15),1.80(3H,s,H-27),0.99(3H,s,H-19),0.98(3H,s,H-29),0.94(3H,s,H-30),0.93(3H,s,H-28),0.91(1H,d,J＝6.4Hz,H-21),0.61(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:173.3(C-26),146.4(C-9),143.9(C-24),140.9(C-8),128.1(C-25),121.5(C-7),115.8(C-11),76.4(C-3),74.8(C-15),52.3(C-14),48.9(C-17),44.5(C-13),43.2(C-5),40.1(C-16),38.6(C-12),37.5(C-4),37.5(C-10),36.1(C-20),35.1(C-22),30.1(C-1),28.4(C-28),25.8(C-23),25.7(C-2),23.1(C-6),23.0(C-29),22.9(C-19),18.5(C-21),17.6(C-30),16.1(C-18),12.5(C-27)。
化合物8[灵芝酸X(ganoderic acid X)]
分子式为C32H48O5，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:511.35[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.86(1H,t,J＝7.7Hz,H-24),5.48(1H,d,J＝4.8Hz,H-7),5.35(1H,d,J＝4.8Hz,H-11),5.07(1H,dd,J＝9.5Hz,H-15),3.47(1H,t,J＝3.0Hz,H-3),2.09(3H,s,CH3COO-),1.83(3H,s,H-27),1.02(3H,s,H-28),0.99(3H,s,H-29),0.99(3H,s,H-30),0.93(3H,s,H-19),0.92(1H,d,J＝6.5Hz,H-21),0.66(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:172.6(C-26),171.5(CH3COO-),146.1(C-9),145.3(C-24),140.4(C-8),126.9(C-25),121.5(C-7),115.8(C-11),77.6(C-15),76.3(C-3),51.6(C-14),49.0(C-17),44.3(C-13),43.1(C-5),38.2(C-12),37.5(C-10),37.5(C-4),37.2(C-16),36.2(C-20),34.9(C-22),30.1(C-1),28.4(C-28),26.2(C-23),25.7(C-2),23.2(C-6),23.0(C-19),22.9(C-29),21.7(CH3COO-),18.7(C-30),18.4(C-21),16.2(C-18),12.3(C-27)。
化合物9[灵芝酸TQ(ganodermic acid TQ)]
分子式为C32H46O5，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:509.33[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.83(1H,m,H-24),5.52(1H,d,J＝6.9Hz,H-7),5.40(1H,d,J＝6.5Hz,H-11),5.09(1H,dd,J＝8.3Hz,H-15),2.09(3H,s,CH3COO-),1.82(3H,s,H-27),1.18(3H,s,H-19),1.12(3H,s,H-29),1.10(3H,s,H-28),1.00(3H,s,H-30),0.92(1H,d,J＝6.4Hz,H-21),0.68(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:217.0(C-3),173.3(C-26),171.5(CH3COO-),144.7(C-9),144.6(C-24),140.6(C-8),127.6(C-25),121.2(C-7),117.1(C-11),77.4(C-15),51.5(C-14),50.6(C-5),49.0(C-17),47.6(C-4),44.2(C-13),38.2(C-12),37.4(C-10),37.2(C-2),36.8(C-1),36.1(C-20),35.0(C-16),34.9(C-22),26.1(C-23),25.6(C-28),23.8(C-6),22.7(C-29),22.4(C-19),21.6(CH3COO-),18.5(C-30),18.4(C-21),16.2(C-18),12.3(C-27)。
化合物10[灵芝酸Me(ganoderic acid Me)]
分子式为C34H50O6，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:553.36[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.75(1H,t,J＝7.6Hz,H-24),5.49(1H,d,J＝5.4Hz,H-7),5.35(1H,d,J＝5.9Hz,H-11),5.08(1H,m,H-15),4.47(1H,m,H-3),2.09(3H,s,CH3COO-),2.06(3H,s,CH3COO-),1.79(3H,s,H-27),1.07(3H,s,H-30),1.00(3H,s,H-19),0.99(3H,s,H-29),0.93(1H,d,J＝6.5Hz,H-21),0.89(3H,s,H-28),0.67(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:171.5(C-26),171.5(CH3COO-),170.7(CH3COO-),145.8(C-9),143.1(C-24),140.2(C-8),128.0(C-25),121.1(C-7),115.7(C-11),78.3(C-3),77.6(C-15),51.4(C-14),48.9(C-17),44.1(C-13),43.9(C-5),38.0(C-12),37.3(C-10),37.0(C-16),36.5(C-4),36.0(C-20),34.8(C-22),30.6(C-1),27.8(C-28),25.8(C-23),23.1(C-2),22.8(C-6),22.7(C-19),22.4(C-29),21.4(CH3COO-),21.3(CH3COO-),18.4(C-30),18.2(C-21),15.9(C-18),12.4(C-27)。
化合物11[灵芝酸Mf(ganoderic acid Mf)]
分子式为C32H48O5，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:511.35[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.89(1H,t,J＝7.6Hz,H-24),5.85(1H,d,J＝6.7Hz,H-7),5.33(1H,d,J＝6.8Hz,H-11),4.67(1H,t,J＝3.1Hz,H-3),4.29(1H,m,H-15),2.06(3H,s,CH3COO-),1.84(3H,s,H-27),1.01(3H,s,H-19),0.99(3H,s,H-29),0.99(3H,s,H-30),0.92(1H,d,J＝6.5Hz,H-21),0.88(3H,s,H-28),0.63(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:173.1(C-26),171.1(CH3COO-),146.3(C-9),145.6(C-24),140.9(C-8),127.0(C-25),121.4(C-7),115.8(C-11),78.3(C-3),74.9(C-15),52.2(C-14),49.0(C-17),44.6(C-13),44.2(C-5),40.2(C-16),38.6(C-12),37.5(C-10),36.7(C-4),36.1(C-20),34.9(C-22),30.8(C-1),28.0(C-28),26.1(C-23),23.3(C-2),23.0(C-6),22.9(C-19),22.7(C-29),21.6(CH3COO-),18.5(C-21),17.4(C-30),16.1(C-18),12.1(C-27)。
化合物12[灵芝酸Y(ganoderic acid Y)]
分子式为C30H46O3，白色无定型粉末，易溶于甲醇、丙酮。负离子ESI-MS m/z:453.34[M-H]-。1H-NMR(500MHz,CD3OD)δ:6.80(1H,t,J＝7.4Hz,H-24),5.50(1H,d,J＝6.7Hz,H-7),5.34(1H,d,J＝6.5Hz,H-11),3.34(1H,m,H-3),1.83(3H,s,H-27),1.00(3H,s,H-19),0.99(3H,s,H-29),0.96(1H,d,J＝6.5Hz,H-21),0.90(3H,s,H-30),0.87(3H,s,H-28),0.60(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:171.5(C-26),146.8(C-9),144.3(C-24),143.3(C-8),128.0(C-25),121.1(C-7),116.9(C-11),79.2(C-3),51.7(C-17),51.0(C-14),50.1(C-5),44.5(C-13),39.4(C-4),38.6(C-12),38.1(C-10),36.9(C-20),36.6(C-1),35.6(C-22),32.2(C-15),28.8(C-28),28.6(C-2),28.0(C-16),26.3(C-23),26.1(C-30),23.7(C-6),23.1(C-19),18.7(C-21),16.4(C-29),16.1(C-18),12.4(C-27)。
化合物13[灵芝酸Z(ganoderic acid Z)]
分子式为C30H48O3，白色无定型粉末，易溶于甲醇、丙酮。负离子ESI-MS m/z:455.36[M-H]-。1H-NMR(500MHz,CD3OD)δ:6.92(1H,t,J＝7.6Hz,H-24),3.26(1H,m,H-3),1.86(3H,s,H-27),1.02(3H,s,H-19),1.00(3H,s,H-29),0.96(1H,d,J＝6.3Hz,H-21),0.90(3H,s,H-30),0.83(3H,s,H-28),0.71(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:171.5(C-26),146.8(C-9),144.3(C-24),143.3(C-8),128.0(C-25),121.1(C-7),116.9(C-11),79.2(C-3),51.7(C-17),51.0(C-14),50.1(C-5),44.5(C-13),39.4(C-4),38.6(C-12),38.1(C-10),36.9(C-20),36.6(C-1),35.6(C-22),32.2(C-15),28.8(C-28),28.6(C-2),28.0(C-16),26.3(C-23),26.1(C-30),23.7(C-6),23.1(C-19),18.7(C-21),16.4(C-29),16.1(C-18),12.4(C-27)。
化合物14[lanosta-7,9,(11),24-trien-3α-hydroxy-26-oic acid]
分子式为C30H46O3，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:453.34[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.90(1H,td,J＝7.7Hz,J＝1.5Hz,H-24),5.48(1H,t,J＝4.5Hz,H-7),5.35(1H,d,J＝6.4Hz,H-11),3.46(1H,m,H-3),1.84(3H,s,H-27),1.00(3H,s,H-19),0.98(3H,s,H-30),0.94(3H,s,H-29),0.93(3H,s,H-28),0.89(1H,d,J＝1.0Hz,H-21),0.58(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:172.8(C-26),146.1(C-9),145.9(C-24),142.8(C-8),126.7(C-25),120.4(C-7),116.1(C-11),76.4(C-3),51.0(C-17),50.6(C-14),44.0(C-13),43.4(C-5),37.9(C-12),37.5(C-4),37.4(C-10),36.3(C-20),34.9(C-22),31.6(C-16),30.1(C-1),28.4(C-28),28.1(C-15),26.1(C-23),25.9(C-30),25.7(C-2),23.2(C-6),23.0(C-29),22.8(C-19),18.5(C-21),15.8(C-18),12.2(C-27)。
化合物15[灵芝酸S1(ganoderic acid S1)]
分子式为C30H44O3，白色无定型粉末，易溶于氯仿、甲醇、丙酮。负离子ESI-MS m/z:451.33[M-H]-。1H-NMR(500MHz,CDCl3)δ:6.90(1H,t,J＝6.3Hz,H-24),5.51(1H,d,J＝6.2Hz,H-7),5.39(1H,d,J＝5.5Hz,H-11),1.85(3H,s,H-27),0.88(3H,s,H-30),1.20(3H,s,H-19),1.13(3H,s,H-28),1.09(3H,s,H-29),0.94(1H,d,J＝5.4Hz,H-21),0.60(3H,s,H-18)。13C-NMR(CDCl3,125MHz)δ:217.1(C-3),173.0(C-26),145.9(C-24),144.8(C-9),143.0(C-8),126.8(C-25),120.2(C-7),117.4(C-11),51.1(C-17),50.9(C-5),50.5(C-14),47.7(C-4),44.0(C-13),38.0(C-12),37.4(C-10),36.8(C-1),36.4(C-20),35.1(C-2),34.9(C-22),31.7(C-15),28.1(C-16),26.1(C-23),25.6(C-28),25.6(C-30),23.9(C-6),22.7(C-29),22.3(C-19),18.5(C-21),15.9(C-18),12.2(C-27)。
实施实例4化合物的抗炎活性检测
本实施例采用LPS刺激诱导的RAW264.7细胞模型，通过Griess试剂法检测细胞外NO含量，测定上述化合物4，5，7，8，9，11，12，13，14，15对NO释放的抑制作用。
小鼠巨噬细胞RAW264.7细胞的培养与处理
使用含10％胎牛血清、100U/mL青霉素和100μg/mL链霉素混合液的DMEM高糖酚红培养液，在37℃、5％CO2的恒温培养箱中培养RAW264.7细胞。
(1)化合物4，5，7，8，9，11，12，13，14，15对LPS刺激的RAW264.7细胞释放NO的影响
取对数时期的RAW264.7细胞，用无色RPMI1640培养液稀释成5×105个/mL的单细胞悬液，接种于96孔板中(每孔194μL)，每组设三个平行孔。CO2培养箱中培养24h后分别加入1μL浓度为2μmol/mL的化合物4，5，7，8，9，11，12，13，14，15和阳性对照地塞米松(样品终浓度均为10nmol/mL)，继续孵育1h后加入5μL浓度为100μg/mL的LPS，同时设定LPS组(加入1μL PBS+5μL LPS)和空白对照组(加入1μL PBS+5μL PBS)。培养48h后，吸取100μL培养上清于酶标板，各孔加入50μL Griess试剂，室温避光孵育10min后，用酶标仪在543nm处测定OD值。根据NaNO2标准曲线计算各组细胞培养上清液中NO的浓度以及对NO释放的抑制率，测定样品对细胞抑制率计算按下列公示计算。
实验结果如下表1所示：
表1化合物对NO释放的抑制率比较
实验结果表明，化合物8、9均具有较强的抑制NO产生的活性。
(2)化合物对LPS诱导的RAW264.7细胞炎性因子表达的影响
取对数时期的RAW264.7巨噬细胞，将其接种于96孔培养板中，控制细胞数量约为5×105个/mL。之后置于37℃，5％CO2细胞培养箱中培养24h。CO2培养箱中培养24h后分别加入1μL浓度为2μmol/mL的化合物4，5，7，8，9，11，12，13，14，15和阳性对照地塞米松(样品终浓度均为10nmol/mL)，继续孵育1h后加入5μL浓度为100μg/mL的LPS，同时设定LPS组(加入1μLPBS+5μL LPS)和空白对照组(加入1μL PBS+5μL PBS)。培养48h后，收集细胞培养上清，用ELISA法检测TNF-α、IL-1β和IL-6含量。
实验结果分别如表2、3、4所示。
表2化合物对LPS诱导的RAW264.7细胞TNF-α释放的影响
不同字母表示差异显著(P＜0.05)
表3化合物对LPS诱导的RAW264.7细胞IL-1β释放的影响
不同字母表示差异显著(P＜0.05)
表4化合物对LPS诱导的RAW264.7细胞IL-6释放的影响
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不同字母表示差异显著(P＜0.05)
实验结果(表2、3、4)表明：模型组中炎症因子TNF-α、IL-1β、IL-6显著升高，发现化合物4，5，7，8，9，11，12，13，14，15均能抑制TNF-α、IL-1β和IL-6释放。化合物5、7、8、9、13、14、15具有显著抑制炎症因子TNF-α的释放；化合物4，5，7，8，9，11，12，13，14，15具有显著抑制IL-1β炎症因子释放的活性；化合物4、5、7、8、9、11则具有显著抑制IL-6炎症因子释放的活性。
Claims (2)
1.松杉灵芝菌丝体三萜化合物用于制备预防或治疗炎症的药物的应用；其中所述的松杉灵芝菌丝体三萜化合物为结构式4，5，7，8，9，11，12，13，14，15化合物中的一种或一种以上：
其中所述结构式4，5，7，8，9，11，12，13，14，15的化合物为：
2.结构式4，5，7，8，9，11，12，13，14，15的化合物用于制备预防和/或治疗炎症或在炎症治疗过程之中或之后起辅助作用的药物或食品中的用途。
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