[0001] 本发明属于分析化学领域，涉及一种钩吻中生物碱的制备方法，具体地说是一种通过制备型三维高效液相色谱方法，实现钩吻中生物碱的高效分离纯化。

[0002] 天然化合物库是新药发现的重要来源。基于天然化合物库的组合化学和高通量筛选是现代新药发现最重要的手段(A.L.Harvey et al.,Natural products in drug discovery,Drug Discov Today,13,2008,894‑901)。药用植物是天然化合物的重要来源，富含生物碱的药用植物尤其受到关注，因为具有药理活性的生物碱占据活性天然化合物半数之多(G.A.Cordell et al.,The potential of alkaloids in drug discovery,
Phytotherapy Research,15,2001,183‑205)。生物碱的分离纯化对于天然化合物库扩建和新药发现具有重大意义。

[0003] 钩吻属植物钩吻富含生物碱，目前已分离得到120余种生物碱，因此新化合物发现较为困难。钩吻生物碱提取物具有抗癌、抗炎、镇痛、抗焦虑以及免疫调节等多种生理活性，但活性成分尚不明确(G.L.Jin et al.,Medicinal plants of the genus Gelsemium(Gelsemiaceae,Gentianales)‑‑a review of their phytochemistry,pharmacology,toxicology and traditional use,Journal of Ethnopharmacology,152,2014,33‑52)。
因此，开展钩吻中生物碱的分离纯化工作对全面认识钩吻药理活性具有非常重要的作用，对天然化合物库的发展具有重要意义。

[0004] 目前针对药用植物中的生物碱的分离纯化仍然面临非常大的挑战。生物碱的常规纯化方法包括硅胶柱色谱、pH区带高速逆流色谱以及制备型高效液相色谱(M.F.Bao et al.,Indole Alkaloids from Hunteria zeylanica,Journal of natural products,80,2017,790‑797；N.Du et al.,Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica,Scientific reports,7,2017,46067；L.Fang et al.,Large‑scale separation of alkaloids from Gelsemium elegans by pH‑zone‑refining counter‑current chromatography with a new solvent system screening method,Journal of chromatography A,1307,2013,80‑85；O.Sticher et al.,Natural product isolation,Naturalproduct Reports,25,2008,517‑554)。硅胶柱色谱存在生物碱死吸附、效率低及重复性差等问题；pH区带高效逆流色谱分辨率较低，无法实现微量成分的制备，也难以获得纯度较高的单体生物碱；制备型高效液相色谱具有高效、重复性好、可选择固定相多等优点，可用于钩吻生物碱分离纯化(O.Sticher et al.,Natural product isolation,Naturalproduct Reports,25,2008,517‑554)。然而，对于复杂程度较高的药用植物来说，常用的二维高效液相色谱的分离制备能力十分有限，无法适应复杂样品的纯化。
因此需要发展具有更强分离能力的三维高效液相色谱方法，实现复杂样品中的生物碱的分离纯化。

[0005] 针对上述问题，本发明的目的是发展一种三维高效液相色谱的方法，解决复杂样品中生物碱的分离纯化的问题。

[0006] 具体的技术方案为：

[0007] 本发明涉及一种钩吻中生物碱的制备的方法，具体是一种通过制备型三维高效液相色谱方法，流动相由水(A)、甲醇或乙腈(B)组成，采用甲酸、乙酸、氨水和三乙胺作为流动相添加剂，实现钩吻中生物碱的分离纯化。

[0008] 其中所述的第一维色谱柱为极性共聚的C18色谱柱XCharge C18；第二维色谱柱为可耐碱性的C8CE色谱柱；第三维色谱柱为可耐碱性的C18CE色谱柱。色谱操作参数如下：色谱柱内径为4.6‑100mm；样品浓度为1mg/mL‑1g/mL；进样量为1μL‑100mL；流速为0.7‑100mL/min；柱温为25‑40℃。

[0009] 操作步骤为：

[0010] 1)钩吻粗碱组分先经第一维高效液相色谱分离，色谱柱为极性共聚的碳十八柱，洗脱方式为线性梯度、台阶梯度，收集一维制备的馏分；

[0011] 2)将一维制备的馏分经过第二维分离，色谱柱为可耐碱性的反相柱，选择权利要求1所述的流动相对馏分进行第二维制备，获得二维馏分。

[0012] 3)将二维制备的馏分经过第三维分离，色谱柱为可耐碱性的反相柱，选择权利要求1所述的流动相对馏分进行第三维制备，获得生物简单体。

[0013] 所述钩吻粗碱的制备方法为：称取5公斤～200公斤的钩吻根茎粉末，用5～30倍量的50％～95％乙醇溶液在50‑100℃加热回流提取1‑5次，每次回流提取的时间为1‑3小时，合并提取液抽滤，用旋转蒸发仪40～80℃条件下减压回收乙醇，浓缩至20‑150L；加入5％～20％硫酸到pH＝3，采用乙酸乙酯等有机溶剂进行萃取，除去中性成分，收集水层。向水层加入10％～30％NaOH水溶液，使其pH＝10，再采用二氯甲烷、氯仿、正丁醇等有机溶剂对水层再次进行萃取，收集二氯甲烷层。20～40℃条件下减压浓缩得到粗碱。

[0014] 步骤1)中钩吻粗碱经第一维制备，梯度洗脱参数为：梯度时间范围为30‑150min，流动相如权利要求1中所述，采用线性梯度或台阶梯度进行洗脱，其中流动相(B)体积浓度变化为由0％～15％到60％～100％，添加剂甲酸的比例为0.05％～1％，流速为50‑100mL/min，检测器波长选择为190‑400nm；采用5％～30％甲酸溶液及甲醇或乙腈对钩吻粗碱进行溶解，得到浓度为50‑300mg/mL的样品，进样量为5‑100mL，根据色谱峰进行馏分收集，将每个馏分减压浓缩至干备用，以进行第二维分离。

[0015] 步骤2)中选取一维制备馏分中的代表性馏分，采用C8CE色谱柱进行第二维制备；洗脱参数为：梯度时间范围为20‑80min，流动相为权利要求1中所述流动相，流动相(B)体积浓度变化为由10％～60％到40％～100％，添加剂氨水的比例为0.05％～5％，流速为10‑
100mL/min，检测波长为190‑400nm。根据色谱分离情况进行馏分收集，将每个馏分减压浓缩至干备用，以进行第三维分离。

[0016] 步骤3)中选取二维制备馏分中的代表性馏分，采用C18CE色谱柱进行第三维制备；洗脱参数为：梯度时间范围为20‑80min，流动相为权利要求1中所述流动相，流动相(B)体积浓度变化为由10％～60％到40％～100％，添加剂乙酸的比例为0.05％～5％、三乙胺的比例为0.05％～5％，流速为10‑100mL/min，检测波长为190‑400nm。分别收集每个馏分中的色谱峰，并进行浓缩，经核磁实验确定，获得生物碱单体P1‑P24。

[0017] 所述化合物P1为4R‑Gelsemine N‑oxide，分子式为C20H22N2O3，分子量为338；所述化合物P2为4S‑Gelsemine N‑oxide，分子式为C20H22N2O3，分子量为338；所述化合物P3为11‑hydroxykoumine，分子式为C20H22N2O2，分子量为322；所述化合物P4为11‑hydroxygelsenicine，分子式为C19H22N2O4，分子量为342；所述化合物P5为18,19‑(S)‑hydroxydihydrogelsevirine，分子式为C21H26N2O5，分子量为386；所述化合物P6为11‑Methoxyl‑14‑hydroxygelseniceine，分子式为C20H24N2O5，分子量为372；所述化合物P7为19(R)‑hydroxygelselegine，分子式为C20H26N2O5，分子量为374；所述化合物P8为
gelsemoxonine A，分子式为C20H26N2O6，分子量为390；所述化合物P9为gelsemoxonine，分子式为C19H22N2O5，分子量为358；所述化合物P10为19‑(R)‑Hydroxydihydrogelsevirine，分子式为C21H26N2O4，分子量为370；所述化合物P11为15‑hydroxyhumantenine，分子式为C21H26N2O4，分子量为370；所述化合物P12为16‑epi‑voacarpine，分子式为C21H24N2O4，分子量为368；所述化合物P13为koumidine，分子式为C19H22N2O，分子量为294；所述化合物P14为Nb‑demethylgelsevirine，分子式为C20H22N2O3，分子量为338；所述化合物P15为normacusine B，分子式为C19H22N2O，分子量为294；所述化合物P16为19‑(Z)‑akuammidine，分子式为C21H24N2O3，分子量为352；所述化合物P17为khasuanine A，分子式为C21H24N2O3，分子量为
352；所述化合物P18为19‑(E)‑akuammidine，分子式为C21H24N2O3，分子量为352；所述化合物P19为koumine，分子式为C20H22N2O，分子量为306；所述化合物P20为gelsenicine，分子式为C19H22N2O3，分子量为326；所述化合物P21为dihydrokoumine，分子式为C20H22N2O，分子量为
308；所述化合物P22为gelsevirine，分子式为C21H24N2O3，分子量为352；所述化合物P23为(4R)‑Gelsevirine N4‑oxide，分子式为C21H24N2O4，分子量为368；所述化合物P24为11‑methoxy‑19‑hydroxygelselegine，分子式为C21H28N2O6，分子量为404。四个新化合物的结构信息如下：

[0018]

[0019] 本发明的有益效果

[0020] 该方法解决钩吻中生物碱的分离纯化的问题，为其他中药、生物样品中生物碱的分离纯化提供了良好的技术方案。附图说明

[0021] 图1为钩吻总碱到化合物的制备流程图，(a)钩吻粗碱在XCharge C18色谱柱上的第一维制备结果；(b)钩吻馏分3在C8CE色谱柱上的第二维制备结果；(c)钩吻馏分3‑6在C18CE色谱柱上的第三维制备结果。

[0022] 图2为实施例4钩吻馏分(a)3‑2、(b)3‑4、(c)3‑9和(d)3‑10在C18CE色谱柱上的第三维制备结果。

[0023] 下面通过具体实施例对本发明进行说明，但本发明并不局限于此。

[0024] 下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。

[0025] 实施例1：钩吻生物碱富集

[0026] 称取198公斤的钩吻根茎粉末，用10倍质量的70％乙醇溶液在70℃加热回流提取3次，每次回流提取的时间为2小时，合并提取液抽滤，用旋转蒸发仪55℃条件下减压回收乙醇，浓缩至85L；加入10％硫酸到pH＝3，采用乙酸乙酯进行萃取，除去中性成分，收集水层。向水层加入20％NaOH水溶液，使其pH＝10，再采用二氯甲烷对水层再次进行萃取，收集二氯甲烷层。30℃条件下减压浓缩得到粗碱粉末。

[0027] 实施例2：钩吻粗碱一维制备

[0028] 钩吻粗碱先经过第一维制备，色谱条件：色谱柱为XCharge C18色谱柱；流动相组成为体积浓度0.1％甲酸‑水(v/v)(A)和体积浓度0.1％甲酸‑甲醇(B)；洗脱梯度为0‑14min，体积浓度0％B；14‑28min，体积浓度6％B；28‑42min，体积浓度12％B；42‑56min，体积浓度18％B；56‑70min，体积浓度24％B；84‑98min，体积浓度30％B；流速为300mL/min；检测波长为254nm；样品溶液浓度为188mg/mL，进样体积为80mL；按色谱峰进行馏分接取，共收集
8个馏分(分别为馏分1至馏分8)，每个馏分50℃减压浓缩至干后备用。

[0029] 实施例3：钩吻馏分二维制备

[0030] 选取一维制备馏分中的代表性馏分3，采用C8CE色谱柱进行第二维制备。色谱条件：色谱柱为C8CE色谱柱；流动相组成为0.125％氨水‑水(v/v)(A)和甲醇(B)；洗脱梯度为0‑10min，体积浓度45‑50％B；10‑30min，体积浓度50‑55％B；30‑40min，体积浓度55‑60％B；
40‑45min，体积浓度60‑100％B；45‑55min，体积浓度100％B；流速为300mL/min；检测波长为
254nm；样品溶液浓度为553mg/mL，进样体积为17mL；按色谱峰进行馏分接取，共收集10个馏分(分别为馏分3‑1、3‑2、3‑3、3‑4、3‑5、3‑6、3‑7、3‑8、3‑9至3‑10)，每个馏分50℃减压浓缩至干后备用。

[0031] 一维制备馏分中的其它馏分同样按上述条件和过程进行二维制备；

[0032] 实施例4：钩吻馏分三维制备

[0033] 选取二维制备馏分中的馏分3‑2、3‑4、3‑6、3‑9和3‑10进行第三维制备。色谱条件：色谱柱为C18CE色谱柱；流动相组成为：0.1％乙酸三乙胺‑水(v/v)(A)和0.1％乙酸三乙胺‑乙腈(v/v)(B)；流速为3mL/min；检测波长为254nm；柱温为30℃；其他条件如下：3‑2，洗脱梯度为0‑30min，体积浓度30‑50％B，样品溶液浓度为120mg/mL，进样体积为90μL；3‑4，洗脱梯度为0‑20min，体积浓度22‑25％B；20‑30min，体积浓度25‑100％B，样品溶液浓度为95mg/mL，进样体积为90μL；3‑6，洗脱梯度为0‑40min，体积浓度23‑30％B，样品溶液浓度为107mg/mL，进样体积为90μL；3‑9，洗脱梯度为0‑30min，体积浓度30‑50％B，样品溶液浓度为95mg/mL，进样体积为90μL；3‑10，洗脱梯度为0‑30min，体积浓度30‑50％B；30‑40min，体积浓度
50％B，样品溶液浓度为115mg/mL，进样体积为90μL。收集色谱峰，50℃减压浓缩至干，得到化合物P1～P24。高效液相色谱检测纯度大于95％。

[0034] 二维制备馏分中的其它馏分同样按上述条件和过程进行三维制备；

[0035] 实施例4：化合物鉴定数据

[0036] 化合物P1～P24经理化测定，数据如下：

[0037] P1(4R‑Gelsemine N‑oxide)，HR‑ESI‑MS(m/z):339.1707[M+H]+；1H NMR(400MHz,MeOD)δ7.43(d,J＝7.7Hz,1H,H‑9),7.22(td,J＝7.7,1.1Hz,1H,H‑10),7.00(td,J＝7.7,1.1Hz,1H,H‑11),6.87(d,J＝7.7Hz,1H,H‑12),6.25(dd,J＝17.9,11.0Hz,1H,H‑19),5.16(dd,J＝11.0,0.8Hz,1H,H‑18b),5.06(dd,J＝17.8,0.9Hz,1H,H‑18a),4.14(overlapped,
2H,H‑17α,H‑5),4.09(dd,J＝11.3,2.0Hz,1H,H‑17β),3.76(m,1H,H‑3),3.50(d,J＝
12.3Hz,1H,H‑21α),3.37(d,J＝12.2Hz,1H,H‑21β),3.32(s,3H,N‑Me),3.25(m,1H,H‑6),
2.88(dd,J＝14.5,2.9Hz,1H,H‑14α),2.76(dd,J＝8.4,2.1Hz,1H,H‑16),2.56(m,1H,H‑
13
15),2.09(ddd,J＝14.5,5.8,2.8Hz,1H,H‑14β). C NMR(151MHz,MeOD)δ179.68(C‑2),
142.67(C‑13),137.00(C‑19),132.08(C‑8),129.67(C‑9),129.61(C‑11),122.90(C‑10),
115.15(C‑18),110.52(C‑12),87.09(C‑5),80.88(C‑21),70.39(C‑3),61.37(C‑17),54.91(C‑20),53.65(C‑7),53.55(C‑6),50.95(N‑Me),38.85(C‑16),36.02(C‑15),23.27(C‑14)；

[0038] P2(4S‑Gelsemine N‑oxide),HR‑ESI‑MS(m/z):339.1707[M+H]+；1H NMR(400MHz,MeOD)δ8.38(s,1H,N‑H),7.43(d,J＝7.6Hz,1H,H‑9),7.25(td,J＝7.7,1.1Hz,1H,H‑10),7.03(td,J＝7.7,1.1Hz,1H,H‑11),6.88(d,J＝7.7Hz,1H,H‑12),6.19(dd,J＝17.9,
11.1Hz,1H,H‑19),5.14(m,1H,H‑18a),5.05(dd,J＝17.9,0.8Hz,1H,H‑18b),4.22(dd,J＝
11.4,2.5Hz,1H,H‑17α),4.07(s,1H,H‑5),4.02(dd,J＝11.4,2.0Hz,1H,H‑17α),3.86(d,J＝8.2Hz,1H,H‑16),3.74(t,J＝2.6Hz,1H,H‑3),3.63(d,J＝12.3Hz,1H,H‑21α),3.19(s,
3H,N‑Me),3.15(d,J＝12.4Hz,1H,H‑21β),2.86(dd,J＝14.5,2.9Hz,1H,H‑14α),2.65(m,
13
1H,H‑15),2.60(s,1H,H‑6),2.10(ddd,J＝14.5,5.8,2.8Hz,1H,H‑14β). C NMR(151MHz,MeOD)δ179.24(C‑2),142.77(C‑13),136.46(C‑19),131.70(C‑8),129.79(C‑9),129.43(C‑
11),122.94(C‑10),115.06(C‑18a),110.64(C‑12),86.36(C‑5),80.06(C‑21),70.65(C‑
3),61.84(C‑17α),59.35(C‑6),55.32(C‑7),51.39(N‑Me),35.90(C‑16),35.39(C‑15),
23.32(C‑14)；

[0039] P3(11‑hydroxykoumine),HR‑ESI‑MS(m/z):323.1745[M+H]+；

[0040]

[0041]

[0042] P4(11‑hydroxygelsenicine),HR‑ESI‑MS(m/z):343.1706[M+H]+；1H NMR(600MHz,DMSO)δ9.59(s,1H,H‑11),7.24(d,J＝8.2Hz,1H,H‑9),6.42(dd,J＝8.1,2.3Hz,1H,H‑10),6.32(d,J＝2.2Hz,1H,H‑12),4.20(m,1H,H‑5),4.18(dd,J＝10.7,0.8Hz,1H,H‑17α),4.11(dd,J＝10.7,3.2Hz,1H,H‑17β),3.80(s,3H,N‑OMe),3.52(dd,J＝4.4,1.8Hz,1H,H‑3),
2.77(t,J＝9.2Hz,1H,H‑15),2.45(m,2H,H‑16),2.26(m,2H,H‑19β),2.09(dd,J＝14.6,
1.6Hz,1H,H‑14α),1.97(ddd,J＝15.0,10.2,4.9Hz,1H,H‑14β),1.93(dd,J＝15.3,1.9Hz,
13
1H,H‑6β),1.14(t,J＝7.3Hz,3H,H‑18). C NMR(151MHz,DMSO)δ181.74(C‑20),170.99(C‑
2),157.61(C‑11),138.65(C‑13),125.52(C‑9),122.21(C‑8),108.97(C‑10),94.44(C‑
12),74.64(C‑3),71.72(C‑5),62.91(N‑OMe),61.32(C‑17α),54.95(C‑7),41.77(C‑15),
40.06(C‑16),37.44(C‑6α),26.63(C‑14α),24.86(C‑19α),9.88(C‑18).

[0043] P5(18,19‑(S)‑hydroxydihydrogelsevirine),HR‑ESI‑MS(m/z):387.1991[M+H+]；

[0044]

[0045]

[0046] P6(11‑Methoxyl‑14‑hydroxygelseniceine),HR‑ESI‑MS(m/z):373.1802[M+H]+；1
H NMR(400MHz,MeOD)δ7.44(d,J＝8.3Hz,1H,H‑9),6.63(dd,J＝8.4,2.4Hz,1H,H‑10),
6.54(d,J＝2.4Hz,1H,H‑12),4.42(dd,J＝10.8,3.4Hz,1H,H‑17β),4.31(overlapped,3H,H‑5,H‑14,H‑17α),3.91(s,3H,N‑OMe),3.81(s,3H,Ar‑OMe),3.58(m,1H,H‑3),2.88(dd,J＝
8.6,1.0Hz,1H,H‑15),2.69(dt,J＝22.6,7.5Hz,1H,H‑19α),2.59(overlapped,2H,H‑16,H‑
19β),2.49(dd,J＝15.6,4.7Hz,1H,H‑6α),2.08(dd,J＝15.6,2.2Hz,1H,H‑6β),1.28(t,J＝
13
7.4Hz,3H,H‑18). C NMR(151MHz,MeOD)δ184.24(C‑20),173.41(C‑2),161.94(C‑11),
140.13(C‑13),126.78(C‑9),124.84(C‑8),109.24(C‑10),95.15(C‑12),80.82(C‑3),
72.73(C‑5),66.83(C‑14),63.98(N‑OMe),62.07(C‑17α),56.04(Ar‑OMe),55.07(C‑7),
53.11(C‑15),39.71(C‑16),38.51(C‑6α),26.56(C‑19α),10.17(C‑18).

[0047] P7(19(R)‑hydroxygelselegine),HR‑ESI‑MS(m/z):375.2026[M+H]+；

[0048]

[0049]

[0050] P8(gelsemoxonine A),HR‑ESI‑MS(m/z):391.1894[M+H]+；

[0051]

[0052]

[0053] P9(gelsemoxonine),HR‑ESI‑MS(m/z):359.1666[M+H]+；1H NMR(400MHz,Methanol‑d4)δ7.57(dd,J＝7.7,1.1Hz,1H,H‑9),7.37(td,J＝7.7,1.2Hz,1H,H‑11),7.17(td,J＝7.7,1.1Hz,1H,H‑10),7.08(dd,J＝7.8,0.9Hz,1H,H‑12),4.51(d,J＝2.4Hz,1H,H‑
14α),4.21(m,2H,H‑17α),4.03(s,3H,N‑OMe),3.83(ddd,J＝8.6,4.8,1.6Hz,1H,H‑5),3.73(d,J＝2.7Hz,1H,H‑3),3.33(m,1H,H‑16),2.69(dq,J＝18.3,7.2Hz,1H,H‑19α),2.53(overlapped,2H,H‑6α,19β),2.24(dd,J＝16.0,4.8Hz,1H,H‑6β),1.08(t,J＝7.2Hz,3H,H‑
13
18). C NMR(151MHz,MeOD)δ211.58(C‑20),175.42(C‑2),139.27(C‑13),132.15(C‑8),
129.81(C‑11),126.64(C‑9),125.12(C‑10),108.53(C‑12),80.04(C‑3),69.29(C‑14),
67.89(C‑15),64.18(N‑OMe),62.18(C‑17),56.75(C‑5),55.84(C‑7),35.32(C‑16),34.97(C‑6),30.03(C‑19),7.38(C‑18).

[0054] P10(19‑(R)‑Hydroxydihydrogelsevirine),HR‑ESI‑MS(m/z):371.2053[M+H]+；1H NMR(400MHz,MeOD)δ7.53(d,J＝7.6Hz,1H,H‑9),7.35(td,J＝7.7,0.9Hz,1H,H‑11),7.10(td,J＝7.7,1.0Hz,1H,H‑10),7.04(d,J＝7.7Hz,1H,H‑12),5.16(q,J＝6.6Hz,1H,H‑19),4.12(dd,J＝11.2,2.1Hz,1H,H‑17α),3.99(overlapped,4H,H‑17β,N‑OMe),3.84(s,1H,H‑
3),3.82(s,1H,H‑5),3.29(d,J＝10.8Hz,1H,H‑21),2.73(d,J＝10.9Hz,1H,H‑21β),2.65(dd,J＝14.8,2.9Hz,1H,H‑14α),2.60(d,J＝8.9Hz,1H,H‑16),2.51(s,3H,N‑Me),2.28(overlapped,2H,H‑6,H‑15),2.13(ddd,J＝14.8,5.6,2.6Hz,1H,H‑14α),1.09(d,J＝
13
6.6Hz,3H,H‑18). C NMR(151MHz,MeOD)δ175.32(C‑2),140.94(C‑13),129.83(C‑9),
129.53(C‑11),128.96(C‑8),124.04(C‑10),108.59(C‑12),72.97(C‑5),70.44(C‑3),
64.02(N‑OMe),61.58(C‑17),58.94(C‑21),57.79(C‑20),53.67(C‑7),48.69(C‑6),40.22(N‑Me),39.60(C‑16),36.57(C‑15),23.12(C‑14),19.81(C‑18).

[0055] P11(15‑hydroxyhumantenine),HR‑ESI‑MS(m/z):371.2012[M+H]+；1H NMR(400MHz,DMSO)δ7.46(d,J＝7.5Hz,1H,H‑9),7.34(t,J＝7.7Hz,1H,H‑11),7.11(t,J＝
7.5Hz,1H,H‑10),7.04(d,J＝7.7Hz,1H,H‑12),5.78(q,J＝6.8Hz,1H,H‑19),4.25(dd,J＝
10.4,5.1Hz,1H,H‑17α),3.96(d,J＝10.4Hz,1H,H‑17α),3.89(s,3H,N‑OMe),3.54(d,J＝
7.7Hz,1H,H‑14α),3.37(overlapped,3H,H‑14α,H‑21),2.50(d,J＝15.3Hz,1H,H‑14α),
2.26(overlapped,4H,H‑14α,N‑Me),2.15(m,1H,H‑14α),1.90(dd,J＝15.3,7.8Hz,1H,H‑14
13
β),1.63(d,J＝7.0Hz,3H,H‑18),1.58(m,1H,H‑6α). C NMR(151MHz,DMSO)δ173.99(C‑2),
143.05(C‑20),139.01(C‑13),129.51(C‑8),128.41(C‑11),126.82(C‑9),123.14(C‑10),
116.52(C‑19),107.42(C‑12),71.25(C‑3),66.94(C‑15),63.73(N‑OMe),62.37(C‑17),
60.63(C‑5),54.18(C‑7),47.68(N‑Me),43.88(C‑16),42.71(C‑21),37.02(C‑14),26.08(C‑6),13.09(C‑18).

[0056] P12(16‑epi‑voacarpine),HR‑ESI‑MS(m/z):369.1789[M+H]+；1H NMR(600MHz,MeOD)δ8.45(s,1H,N‑H),7.46(d,J＝7.9Hz,1H,H‑9),7.35(d,J＝8.2Hz,1H,H‑12),7.11(ddd,J＝8.1,7.2,1.0Hz,1H,H‑11),7.01(m,1H,H‑10),5.34(m,1H,H‑19),4.59(d,J＝5.8Hz,1H,H‑5),4.25(m,1H,H‑21),3.70(s,3H,COOCH3),3.59(d,J＝10.6Hz,1H,H‑17α),
3.52(d,J＝10.6Hz,1H,H‑17β),3.41(dd,J＝10.0,8.6Hz,1H,H‑21β),3.31(m,36H,H‑6β),
3.28(dd,J＝3.4,2.6Hz,1H,H‑15),3.18(dd,J＝16.4,6.2Hz,1H,H‑6α),2.32(dd,J＝14.4,
3.8Hz,1H,H‑14β),1.91(dd,J＝14.4,2.2Hz,1H,H‑14α),1.65(dt,J＝6.9,2.0Hz,3H,H‑
13
18). C NMR(151MHz,MeOD)δ176.43(COOCH3),138.68(C‑2),137.53(C‑13),127.09(C‑8),
123.08(C‑11),120.15(C‑10),119.64(C‑9,C‑20),117.95(C‑19),112.47(C‑12),107.85(C‑7),82.80(C‑3),64.29(C‑17),59.80(C‑5),58.33(C‑16),54.93(COOCH3),52.65(C‑
21),38.04(C‑15),35.47(C‑14),22.43(C‑6),13.11(C‑18).

[0057] P13(koumidine),HR‑ESI‑MS(m/z):295.1820[M+H]+；1H NMR(400MHz,MeOD)δ7.41(d,J＝7.7Hz,1H,H‑9),7.28(d,J＝8.0Hz,1H,H‑12),7.06(m,1H,H‑11),6.98(m,1H,H‑10),5.39(m,1H,H‑19),4.17(dd,J＝10.2,3.1Hz,1H,H‑3),3.80(d,J＝17.2Hz,1H,H‑21α),3.67(d,J＝18.0Hz,1H,H‑21β),3.62(m,1H,H‑5),3.52(dd,J＝10.8,6.5Hz,1H,H‑17α),3.16(dd,J＝10.8,8.9Hz,1H,H‑17β),3.04(d,J＝15.6Hz,1H,H‑6β),2.94(dd,J＝16.2,5.8Hz,
1H,H‑6α),2.46(dd,J＝5.6,2.9Hz,1H,H‑15),2.27(m,1H,H‑16),1.94(m,1H,H‑14α),1.86
13
(dt,J＝13.0,3.3Hz,1H,H‑14β),1.61(d,J＝6.8Hz,3H,H‑18). C NMR(151MHz,MeOD)δ
141.50(C‑20),138.20(C‑2),137.90(C‑13),127.43(C‑8),122.20(C‑11),119.85(C‑10),
118.76(C‑9),115.68(C‑19),111.97(C‑12),105.83(C‑7),61.12(C‑17),54.55(C‑21),
54.24(C‑5),51.13(C‑3),44.10(C‑16),35.00(C‑15),29.23(C‑14),23.27(C‑6),12.55(C‑
18).

[0058] P14(Nb‑demethylgelsevirine),HR‑ESI‑MS(m/z):339.1708[M+H]+；1H NMR(600MHz,MeOD)δ7.58(d,J＝7.6Hz,1H,H‑9),7.39(td,J＝7.7,1.1Hz,1H,H‑11),7.14(td,J＝7.7,1.1Hz,1H,H‑10),7.07(dd,J＝7.8,0.5Hz,1H,H‑12),6.23(dd,J＝17.8,11.1Hz,1H,H‑19),5.28(dd,J＝11.1,0.5Hz,1H,H‑18b),5.19(dd,J＝17.8,0.6Hz,1H,H‑18a),4.40(d,J＝0.9Hz,1H,H‑5),4.10(dd,J＝11.4,2.0Hz,1H,H‑17β),4.04(dd,J＝11.3,2.2Hz,1H,H‑17α),3.97(s,3H,N‑OMe),3.82(m,1H,H‑3),3.20(d,J＝12.0Hz,1H,H‑21α),3.13(d,J＝
11.9Hz,1H,H‑21β),2.87(dd,J＝14.7,2.9Hz,1H,H‑14α),2.69(m,1H,H‑15),2.52(d,J＝
13
7.9Hz,1H,H‑16),2.16(ddd,J＝14.7,5.8,2.8Hz,1H,H‑14β),2.08(m,1H,H‑6). C NMR(151MHz,MeOD)δ173.09(C‑2),140.74(C‑13),135.90(C‑19),130.23(C‑11),129.44(C‑9),
127.90(C‑8),124.35(C‑10),116.58(C‑18),108.76(C‑12),70.50(C‑3),66.17(C‑5),
64.01(N‑OMe),60.86(C‑17),54.14(C‑21),53.06(C‑6),52.97(C‑7),52.28(C‑20),41.10(C‑16),36.72(C‑15),23.39(C‑14).

[0059] P15(normacusine B),HR‑ESI‑MS(m/z):295.1775[M+H]+；1H NMR(600MHz,MeOD)δ8.46(s,1H,N‑H),7.46(m,1H,H‑9),7.34(dt,J＝8.2,0.8Hz,1H,H‑12),7.13(ddd,J＝8.2,
7.1,1.1Hz,1H,H‑11),7.04(ddd,J＝7.9,7.2,0.9Hz,1H,H‑10),5.53(m,1H,H‑19),4.70(d,J＝9.2Hz,1H,H‑3),4.09(d,J＝16.4Hz,1H,H‑21α),4.00(d,J＝16.5Hz,1H,H‑21β),3.53(d,J＝1.5Hz,1H,H‑17α),3.35(m,1H,H‑17β),3.25(dd,J＝16.1,5.1Hz,1H,H‑6α),2.95(d,J＝16.1Hz,1H,H‑6β),2.54(m,1H,H‑15),2.36(ddd,J＝12.6,10.4,2.1Hz,1H,H‑5),1.95
13
(m,2H,H‑14),1.66(dt,J＝6.9,1.4Hz,3H,H‑18). C NMR(151MHz,MeOD)δ138.54(C‑2),
134.97(C‑13),130.63(C‑20),127.99(C‑8),123.21(C‑9),121.79(C‑18),120.48(C‑10),
118.99(C‑19),112.36(C‑12),103.42(C‑7),64.47(C‑17),58.41(C‑21),53.38(C‑5),
52.66(C‑3),44.16(C‑16),34.64(C‑14),34.50(C‑15),26.86(C‑6),12.79(C‑18).[0060] P16(19‑(Z)‑akuammidine),HR‑ESI‑MS(m/z):353.1918[M+H]+；1H NMR(400MHz,DMSO)δ10.66(s,1H,N‑H),7.31(d,J＝7.6Hz,1H,H‑9),7.23(d,J＝8.0Hz,1H,H‑12),6.97(m,1H,H‑11),6.90(m,1H,H‑10),5.32(qd,J＝6.6,4.4Hz,1H,H‑19),4.67(t,J＝5.2Hz,1H,OH),4.06(d,J＝8.7Hz,1H,H‑3),3.53(m,2H,H‑17),3.47(s,2H,H‑21),3.23(m,1H,H‑6β),
2.87(s,3H,COOCH3),2.65(m,3H,H‑14β,H‑15),2.45(m,1H,H‑6α),1.75(m,1H,H‑14α),1.54
13
(d,J＝6.7Hz,3H,H‑18). C NMR(151MHz,DMSO)δ172.75(COOCH3),139.80(C‑20),138.83(C‑2),136.25(C‑13),126.45(C‑8),120.09(C‑11),118.05(C‑9),117.21(C‑10),115.06(C‑19),110.89(C‑12),103.97(C‑7),67.52(C‑17),57.38(C‑5),52.76(C‑21),51.32(C‑
16),50.32(C‑3),49.55(COOCH3),35.49(C‑15),30.13(C‑14),24.05(C‑6),12.29(C‑13).[0061] P17(khasuanine A),HR‑ESI‑MS(m/z):353.1972[M+H]+；1H NMR(400MHz,MeOD)δ
7.37(d,J＝7.7Hz,1H,H‑9),7.26(d,J＝8.1Hz,1H,H‑12),7.04(m,1H,H‑11),6.96(m,1H,H‑
10),5.45(m,1H,H‑19),4.23(d,J＝9.5Hz,1H,H‑16),3.78(d,J＝9.7Hz,1H,H‑15α),3.68(d,J＝8.6Hz,1H,H‑15β),3.63(m,1H,H‑5),3.52(m,1H,H‑22α),3.41(dd,J＝16.6,3.1Hz,
1H,H‑22β),3.25(dd,J＝4.1,1.3Hz,1H,H‑21),2.94(s,3H,H‑23),2.84(m,2H,H‑6β),2.70(ddd,J＝13.1,4.2,1.9Hz,1H,H‑17α),1.90(m,1H,H‑17β),1.68(dt,J＝6.8,1.9Hz,2H,H‑
13
18). C NMR(151MHz,MeOD)δ174.69(C‑3),138.48(C‑2),138.45(C‑13),137.76(C‑20),
127.92(C‑8),122.10(C‑11),119.72(C‑10),118.61(C‑9),111.97(C‑12),106.10(C‑7),
68.93(C‑15),59.04(C‑5),56.09(C‑22),52.65(C‑15),51.98(C‑23),51.61(C‑16),30.29(C‑21),30.07(C‑17),25.01(C‑6),13.30(C‑18).

[0062] P18(19‑(E)‑akuammidine),HR‑ESI‑MS(m/z):353.1915[M+H]+；1H NMR(400MHz,MeOD)δ7.44(d,J＝7.7Hz,1H,H‑9),7.29(d,J＝8.0Hz,1H,H‑12),7.07(m,1H,H‑11),6.99(m,1H,H‑10),5.32(m,1H,H‑19),4.29(d,J＝6.4Hz,1H,H‑5),4.14(dd,J＝10.4,3.3Hz,1H,H‑3),3.69(s,3H,N‑Me),3.64(overlapped,4H,H‑17,H‑21),3.28(d,J＝16.9Hz,1H,H‑6α),3.20(t,J＝2.9Hz,1H,H‑15),3.08(dd,J＝16.3,6.5Hz,1H,H‑6β),1.98(ddd,J＝13.1,
10.5,2.4Hz,1H,H‑14α),1.85(dt,J＝13.5,3.5Hz,1H,H‑14β),1.64(dt,J＝6.9,2.0Hz,3H,
13
H‑18). C NMR(151MHz,MeOD)δ138.12(C‑20),137.54(C‑2),136.49(C‑13),127.52(C‑8),
122.33(C‑11),119.91(C‑9),119.06(C‑10),117.71(C‑19),112.01(C‑12),105.76(C‑7),
64.42(C‑17),56.14(C‑21),55.43(C‑5),55.35(C‑16),52.48(N‑Me),50.21(C‑3),32.32(C‑15),29.88(C‑14),23.15(C‑6),13.07(C‑18).

[0063] P19(koumine),HR‑ESI‑MS(m/z):307.1747[M+H]+；1H NMR(400MHz,Methanol‑d4)δ7.63(d,J＝7.2Hz,1H,H‑12),7.53(d,J＝7.5Hz,1H,H‑9),7.38(td,J＝7.6,1.3Hz,1H,H‑
11),7.31(td,J＝7.5,1.2Hz,1H,H‑10),4.91(ddd,J＝3.6,2.3,1.1Hz,1H,H‑3),4.86(dd,J＝17.7,1.2Hz,1H,H‑18a),4.80(dd,J＝11.3,1.1Hz,1H,H‑18b),4.67(dd,J＝17.6,
11.2Hz,1H,H‑19),4.30(dd,J＝12.0,4.6Hz,1H,H‑17α),3.62(d,J＝12.0Hz,1H,H‑17β),
3.25(d,J＝11.8Hz,1H,H‑21α),2.99(d,J＝11.9Hz,1H,H‑21β),2.91(td,J＝3.7,2.1Hz,
1H,H‑16),2.86(m,1H,H‑5),2.72(dt,J＝14.9,3.8Hz,1H,H‑3α),2.63(s,3H,N‑H),2.40(t,J＝1.9Hz,1H,H‑6α),2.39(d,J＝3.5Hz,1H,H‑15),2.35(m,1H,H‑6β),1.85(dt,J＝14.8,
13
2.3Hz,1H,H‑6β). C NMR(151MHz,MeOD)δ187.96(C‑2),155.00(C‑13),144.65(C‑8),
138.05(C‑19),129.50(C‑11),127.72(C‑10),124.61(C‑9),121.44(C‑12),116.86(C‑18),
71.90(C‑3),61.96(C‑17),59.04(C‑7),58.18(C‑21),57.73(C‑5),46.63(C‑20),42.87(N‑Me),38.63(C‑16),34.03(C‑15),30.48(C‑6),25.96(C‑14).

[0064] P20(gelsenicine),HR‑ESI‑MS(m/z):327.1668[M+H]+；1H NMR(400MHz,Methanol‑d4)δ7.54(d,J＝7.5Hz,1H,H‑9),7.29(td,J＝7.8,0.8Hz,1H,H‑11),7.09(td,J＝7.6,0.7Hz,1H,H‑10),6.94(d,J＝7.7Hz,1H,H‑12),4.31(m,1H,H‑5),4.27(dd,J＝11.1,3.1Hz,
1H,H‑17β),3.91(s,3H,N‑OMe),3.67(dd,J＝4.3,1.9Hz,1H,H‑3),2.99(t,J＝9.2Hz,1H,H‑
15),2.66(dd,J＝16.7,7.6Hz,1H,H‑19α),2.59(m,1H,H‑16),2.48(dd,J＝15.5,4.6Hz,2H,H‑6α),2.30(dd,J＝15.0,2.2Hz,1H,H‑14α),2.18(ddd,J＝14.9,10.0,4.6Hz,1H,H‑14β),
13
2.09(dd,J＝15.5,2.3Hz,1H,H‑6β),1.26(t,J＝7.5Hz,3H,H‑18). C NMR(151MHz,MeOD)δ
187.55(C‑20),173.06(C‑2),139.11(C‑13),133.50(C‑8),129.34(C‑11),125.92(C‑9),
124.62(C‑10),107.78(C‑12),76.25(C‑3),73.27(C‑5),63.88(N‑OMe),62.62(C‑17),
57.42(C‑7),43.63(C‑15),40.93(C‑16),38.61(C‑6),28.01(C‑14),27.99(C‑19),10.24(C‑18).

[0065] P21(dihydrokoumine),HR‑ESI‑MS(m/z):309.1982[M+H]+；1H NMR(400MHz,MeOD)δ7.21(d,J＝7.4Hz,1H,H‑9),7.03(td,J＝7.7,1.1Hz,1H,H‑11),6.75(td,J＝7.4,0.8Hz,
1H,H‑10),6.66(d,J＝7.7Hz,1H,H‑12),5.57(dd,J＝18.3,11.0Hz,1H,H‑19),4.63(dd,J＝
5.2,1.0Hz,1H,3),4.24(m,1H,H‑18),4.20(dd,J＝11.9,5.0Hz,1H,H‑18β),3.62(d,J＝
11.9Hz,1H,H‑17),3.42(d,J＝2.3Hz,1H,H‑2),2.75(overlapped,2H,H‑5,H‑21),2.60(overlapped,2H,H‑6β,H‑16),2.50(s,3H,N‑Me),2.46(d,J＝11.7Hz,1H,H‑21β),2.21
13
(overlapped,3H,H‑14,H‑15),2.06(dd,J＝14.6,3.9Hz,1H,H‑6α). C NMR(151MHz,MeOD)δ
153.54(C‑13),142.59(C‑19),135.12(C‑8),128.88(C‑11),124.25(C‑9),119.84(C‑10),
113.64(C‑18),111.46(C‑12),76.94(C‑2),70.47(C‑3),62.43(C‑17),59.10(C‑5),58.88(C‑21),47.24(C‑7),42.83(C‑20),42.45(N‑Me),38.53(C‑16),33.04(C‑6),30.98(C‑15),
20.87(C‑14).

[0066] P22(gelsevirine),HR‑ESI‑MS(m/z):353.1857[M+H]+；1H NMR(400MHz,Methanol‑d4)δ7.54(d,J＝7.6Hz,1H,H‑9),7.34(td,J＝7.7,1.0Hz,1H,H‑11),7.09(td,J＝7.7,1.0Hz,1H,H‑10),7.03(d,J＝7.7Hz,1H,H‑12),6.21(dd,J＝17.8,11.0Hz,1H,H‑19),5.09(dd,J＝11.0,1.2Hz,1H,H‑18b),5.01(dd,J＝17.8,1.2Hz,1H,H‑18a),4.11(dd,J＝11.0,
2.2Hz,1H,H‑17α),3.96(overlapped,3H,N‑OMe),3.93(overlapped,1H,H‑17β),3.72(tt,J＝2.9,1.1Hz,1H,H‑3),3.52(d,J＝1.4Hz,1H,H‑5),2.81(dd,J＝14.5,3.0Hz,1H,H‑14α),
2.76(d,J＝10.5Hz,1H,H‑21α),2.48(d,J＝8.3Hz,1H,H‑16),2.37(d,J＝6.0Hz,1H,H‑15),
2.32(d,J＝10.6Hz,1H,H‑21β),2.25(s,3H,N‑Me),2.04(ddd,J＝14.5,5.7,2.8Hz,1H,H‑14
13
β),1.95(m,1H,H‑6). C NMR(151MHz,MeOD)δ174.59(C‑2),140.68(C‑13),139.26(C‑19),
129.59(C‑9,C‑11),129.21(C‑8),124.03(C‑10),113.83(C‑18),108.40(C‑12),73.33(C‑
5),70.73(C‑3),66.60(C‑21),63.96(N‑OMe),62.17(C‑17),55.17(C‑20),53.65(C‑17),
52.22(C‑6),40.36(N‑Me),38.88(C‑16),37.11(C‑15),23.91(C‑14).

[0067] P23((4R)‑Gelsevirine N4‑oxide),HR‑ESI‑MS(m/z):369.1771[M+H]+；1H NMR(400MHz,MeOD)δ7.66(dd,J＝7.4,0.4Hz,1H,H‑12),7.58(d,J＝7.6Hz,1H,H‑9),7.44(td,J＝7.6,1.2Hz,1H,H‑11),7.36(td,J＝7.5,1.1Hz,1H,H‑10),5.00(d,J＝17.9Hz,1H,H‑18a),4.95(d,J＝11.4Hz,2H,H‑18b),4.64(dd,J＝17.9,11.4Hz,1H,H‑19),4.38(dd,J＝
12.5,5.0Hz,1H,H‑17α),4.09(d,J＝13.9Hz,1H,H‑21α),4.00(d,J＝13.9Hz,1H,H‑21β),
3.73(overlapped,2H,H‑5,H‑17β),3.56(s,3H,N‑OMe),3.45(m,1H,H‑6α),3.14(m,1H,H‑
16),2.79(dt,J＝15.1,3.8Hz,1H,H‑14α),2.58(dd,J＝15.4,3.7Hz,1H,H‑6β),2.48(m,1H,
13
H‑15),1.91(dt,J＝15.1,2.0Hz,1H,H‑14β). C NMR(151MHz,MeOD)δ186.14(C‑2),155.18(C‑13),143.35(C‑8),134.97(C‑19),130.14(C‑11),128.19(C‑10),124.72(C‑9),121.91(C‑12),119.14(C‑18a),74.11(C‑5),73.02(C‑21),71.52(C‑3),60.77(C‑17),59.66(C‑
7),56.62(N‑OMe),47.28(C‑20),35.86(C‑16),31.54(C‑15),27.49(C‑6),25.23(C‑14).[0068] P24(11‑methoxy‑19‑hydroxygelselegine),HR‑ESI‑MS(m/z):405.2030[M+H]+；1H NMR(400MHz,DMSO)δ7.29(d,J＝8.3Hz,1H,H‑9),6.64(dd,J＝8.3,2.4Hz,1H,H‑10),6.60(d,J＝2.3Hz,1H,H‑12),4.32(q,J＝6.1Hz,1H,H‑19),4.23(d,J＝10.8Hz,1H,H‑17α),4.13(dd,J＝10.9,3.9Hz,1H,H‑17β),3.91(s,3H,N‑OMe),3.78(s,3H,O‑Me),3.47(overlapped,
2H,H‑5,H‑21α),3.39(d,J＝6.1Hz,1H,H‑3),3.30(d,J＝10.9Hz,1H,H‑21β),2.67(m,1H,H‑
16),2.31(ddd,J＝10.3,5.8,1.5Hz,1H,H‑15),2.20(d,J＝15.9Hz,1H,H‑14α),2.05(dd,J＝15.8,3.7Hz,1H,H‑6β),1.92(m,1H,H‑14β),1.81(dd,J＝15.8,2.4Hz,1H,H‑6α),1.20(d,
13
J＝6.3Hz,3H,H‑18). C NMR(151MHz,DMSO)δ174.78(C‑2),159.71(C‑11),138.85(C‑13),
126.31(C‑9),123.37(C‑8),107.97(C‑10),94.06(C‑12),74.69(C‑3),68.61(C‑20),67.18(C‑19),63.46(C‑17),62.82(C‑21),62.03(N‑OMe),59.08(C‑5),56.48(C‑7),55.51(OMe),
38.56(C‑16),35.41(C‑15),33.32(C‑6),22.72(C‑14),19.26(C‑18).