芫花Daphne genkwa Sieb.et Zucc是瑞香科瑞香属植物，多分枝，花期3～5月，果期6～7月。生于海拔300～1 000 m，宜温暖的气候，性耐旱怕涝，广泛分布于河北、山西、陕西、甘肃、山东、江苏、安徽、浙江、江西、福建、台湾、河南、湖北、湖南、四川、贵州等省。其性温，味辛苦，有大毒。“芫花”之名来源于《神农本草经》，在其春季花尚未开时，采其花蕾干燥后入药，具有泻水逐饮、解毒杀虫等功效，常用于水肿胀满、痰饮急剧、气逆喘咳、二便不利等症。芫花的根皮也可药用，有消肿解毒、活血化瘀之效，可用于急性乳腺炎、痈疖肿毒、淋巴结结核、腹水、风湿痛、牙痛、跌打损伤等症。芫花具有广泛的生物活性，近年来关于其提取物如羟基芫花素、瑞香烷二萜等的研究屡见报道，芫花在抗病毒、抗肿瘤以及神经保护等方面有明显作用[1,2,3,4]。

目前对于芫花中活性成分的研究主要集中在花蕾和根部，为了进一步探明芫花枝叶的化学成分，开发枝叶的药用价值，本课题着重研究芫花枝叶部位三萜类化学成分及其α-葡萄糖苷酶抑制活性，共分离鉴定20个三萜类化合物，主要包括乌苏烷型三萜与齐墩果烷型三萜，包括山楂酸（maslinic acid,1）、常春藤皂苷元（hederagenin,2）、齐墩果酸（oleanolic acid,3）、3β,13β-dihydroxyolean-11-en-28-oic acid(4）、11-oxoerythrodiol(5）、3β-hydroxy-11-oxo-olean-12-en-28-oic acid(6）、坡模酸（pomolic acid,7）、3β-hydroxyolean-12-en-28-aldehyde(8）、3β-hydroxyolean-12-en-11-one(9）、3-羰基齐墩果酸（3-oxo-oleanolic acid,10）、古柯二醇（erythrodiol,11）、oleanolic acid 2-oxopropyl ester(12）、熊果酸（ursolic acid,13）、ilelatifol A(14）、3β-hydroxy-11-oxours-12-en-28-oic acid(15）、2α,3β-dihydroxyurs-12-en-28-oic acid(16）、3β-hydroxy-11-oxo-ursan-12-ene(17）、2-oxopropyl(3β)-3-hydroxyurs-12-en-28-oate(18）、熊果醛（ursolic aldehyde,19）和11α-methoxyurs-12-ene-3β,12-diol(20）。化合物6为首次从芫花中分离得到。化合物1～5、7～20为瑞香属首次分离得到。其中化合物1、3、4、13、15和16表现出高于阳性对照阿卡波糖的α-葡萄糖苷酶抑制活性。

1、仪器与材料

Bruker Avance DRX-600型核磁共振光谱仪（德国Bruker公司）；Agilent 6545 QTOF型质谱仪、Agilent 1260-6460型三重四极杆质谱仪（美国Agilent公司）；岛津LC-20A型型高效液相色谱仪（日本Shimadzu公司）；R-100型旋转蒸发仪（瑞士Buchi公司）；YMC-Pack ODS-A型色谱柱（250mm×10 mm,5μm，日本YMC株式会社）；D101-大孔吸附树脂（上海东鸿化工有限公司）；Sephadex LH-20（瑞典GE医疗生命科学公司）；薄层色谱硅胶板GF254及300～400目硅胶（青岛海洋化工有限公司）；用于柱色谱的所有溶剂均为分析级（天津富宇精细化工有限公司）；用于HPLC的溶剂为色谱级（瑞典欧森巴克化学公司）。阿卡波糖（acarbose，批号A3230505001，质量分数≥98%），北京索莱宝科技有限公司。

芫花于2019年6月采自山东省莱阳市，由山东中医药高等专科学校中药系魏国栋副教授鉴定为瑞香科瑞香属植物芫花D.genkwa Sieb.et Zucc的枝叶。样品标本（2019-06-DG）保藏于山东中医药高等专科学校中药系。

2、提取与分离

芫花干燥枝叶（5.0 kg），粉碎成粉末后用95%乙醇室温提取3次（每次用溶剂40 L,7 d/次）。提取液经减压蒸馏得到粗浸膏（500.2 g），粗浸膏加水混悬后依次用醋酸乙酯和正丁醇溶液萃取分别萃取3次，减压浓缩后得到醋酸乙酯浸膏（255.3 g）。将醋酸乙酯浸膏进行D-101大孔树脂柱色谱，依次用30%、50%、80%、95%的乙醇-水梯度洗脱。80%的馏分减压浓缩得到浸膏60.5 g，经过硅胶柱色谱粗分离（100～200目），以石油醚-醋酸乙酯（10∶1～0∶1）梯度洗脱，得到9个组分（A～I）。

E段（2.8 g）经过RP18反相柱色谱以甲醇-水体系（60%～100%）进行梯度洗脱，得到8个馏分E-F1～E-F8。E-F1(300.0 mg）通过半制备HPLC(85%乙腈-水）纯化得到化合物12(3.3 mg,t R=20 min）和18(5.2 mg,t R=21 min）。E-F2(800.0 mg）经过Sephadex LH-20凝胶（二氯甲烷-甲醇1∶1）色谱后通过半制备HPLC(85%乙腈-水）纯化得到化合物10(2.1 mg,t R=13 min）和11(15.3 mg,t R=17 min）。E-F3(500.0 mg）经过Sephadex LH-20凝胶（二氯甲烷-甲醇1∶1）色谱后通过半制备HPLC(75%乙腈-水）纯化得到化合物17(2.8 mg,t R=10min）和9(2.9 mg,t R=13 min）。E-F6(800.0 mg）经过Sephadex LH-20凝胶（二氯甲烷-甲醇1∶1）柱色谱后通过半制备HPLC(68%乙腈-水）纯化得到化合物8(10.1 mg,t R=20 min）、19(30.1 mg,t R=28 min）和20(4.4 mg,t R=40 min）。

F段（2.0 g）以二氯甲烷-甲醇（1∶0～100∶1）梯度洗脱得到5个馏份F-S1～F-S5。F-S4(300.0mg）经过Sephadex LH-20凝胶（二氯甲烷-甲醇1∶1）色谱后通过半制备HPLC(55%乙腈-水）纯化得到化合物2(3.0 mg,t R=20 min）。F-S5(200.0 mg）经过Sephadex LH-20凝胶（二氯甲烷-甲醇1∶1）色谱后通过半制备HPLC(60%乙腈-水）纯化得到化合物1(3.0 mg,t R=34min）和16(5.1 mg,t R=42min）。

G段（7.0 g）进行RP18反相柱色谱，以60%～100%的甲醇-水进行梯度洗脱得到10个部分，G-F1～F10。G-F10(800.0 mg）经过Sephadex LH-20凝胶（100%甲醇）色谱后通过半制备液相HPLC(50%乙腈-水）纯化得到化合物4(30.0 mg,t R=25min）。G-F9(500.0 mg）经过Sephadex LH-20凝胶（100%甲醇）色谱后通过半制备液相HPLC(69%乙腈-水）纯化得到化合物6(2.0 mg,t R=18 min）、15(2.3 mg,t R=20 min）和7(2 mg,t R=23 min）。G-F7(500.0 mg）经过Sephadex LH-20凝胶（100%甲醇）色谱后通过半制备液相HPLC(70%乙腈-水）纯化得到化合物5(7.6 mg,t R=39 min）和14(4.7mg,t R=36 min）。

H段（5.0 g）以二氯甲烷-甲醇（1∶0～100∶1）梯度洗脱进行硅胶柱色谱分离，得到3个部分H-S1～H-S3。H-S2(3.0 g）经过反复重结晶得到化合物13(2.0 g）。H-S2(500.0 mg）经过Sephadex LH-20凝胶色谱柱色谱（二氯甲烷-甲醇1∶1）后经过半制备HPLC(88%乙腈-水）纯化得到化合物3(5.6mg,t R=26.5 min）。

3、结构鉴定

化合物1：白色粉末，ESI-MS m/z:473.4[M+H]+，推测该化合物分子式为C30H48O4,1H-NMR(600 MHz,C5D5N) δ:5.49 (1H,t,J=3.8 Hz,H-12),4.12 (1H,m,H-2),3.42 (1H,d,J=9.3 Hz,H-3),3.32(1H,dd,J=14.0,4.6 Hz,H-18),2.27 (1H,dd,J=12.4,4.5 Hz,H-1a),2.10 (2H,m,H2-11),2.08 (1H,m,H-22a),2.02 (2H,m,H-16),1.86 (1H,m,H-22b),1.86(1H,m,H-9),1.84 (1H,m,H-19a),1.57 (1H,m,H-6a),1.56 (1H,m,H-7a),1.49 (1H,m,H-21a),1.45 (1H,m,H-6b),1.36 (1H,m,H-7b),1.34 (2H,m,H-1b,H-19b),1.30 (3H,s,Me-23),1.29 (3H,s,Me-27),1.23 (2H,q,J=3.3 Hz,H2-15),1.21 (1H,d,J=3.5 Hz,H-21b),1.10 (3H,s,Me-24),1.06 (1H,m,H-5),1.05 (3H,s,Me-30),1.02 (3H,s,Me-25),1.01 (3H,s,Me-26),0.96(3H,s,Me-29);13C-NMR (150 MHz,C5D5N) δ:180.6(C-28),145.3 (C-13),122.9 (C-12),84.2 (C-3),69.0 (C-2),56.3 (C-5),48.6 (C-9),48.2 (C-1),47.1 (C-17),46.8(C-19),42.6 (C-14),42.4 (C-18),40.3 (C-4),40.2 (C-8),38.9 (C-10),34.6 (C-21),33.7 (C-29),33.6 (C-7),33.6 (C-22),31.4 (C-20),29.7 (C-23),28.7 (C-15),26.6(C-27),24.3 (C-16),24.2 (C-30),24.1 (C-11),19.3 (C-6),18.1 (C-24),17.9 (C-26),17.3 (C-25)。以上波谱数据与文献报道一致[5]，故鉴定化合物1为山楂酸。

化合物2：白色粉末，ESI-MS m/z:473.4[M+H]+，推测该化合物分子式为C30H48O4。1H-NMR(600 MHz,C5D5N) δ:5.48 (1H,t,J=3.7 Hz,H-12),4.19 (1H,m,H-3),4.17 (1H,d,J=10.0 Hz,H-23a),3.70 (1H,d,J=10.4 Hz,H-23b),3.28 (1H,dd,J=14.0,4.6 Hz,H-18),2.15 (1H,m,H-15a),2.07 (1H,m,H-16a),2.00 (1H,m,H-22a),1.96 (1H,m,H-16b),1.92(1H,m,H-11a),1.86 (1H,m,H-2a),1.78 (1H,m,H-19a),1.77 (1H,m,H-22b),1.76 (1H,m,H-9),1.66 (1H,m,H-6a),1.60 (1H,m,H-7a),1.54 (1H,m,H-1a),1.52(1H,m,H-5),1.43 (1H,m,H-6b),1.41 (1H,m,H-21a),1.27 (1H,m,H-19b),1.26 (1H,m,H-7b),1.22 (3H,s,Me-27),1.18 (1H,m,H-21b),1.14 (1H,m,H-15b),1.08 (1H,dt,J=7.6,3.8 Hz,H-1b),1.04 (3H,s,Me-24),1.03 (3H,s,Me-26),0.98 (3H,s,Me-30),0.95 (3H,s,Me-25),0.91 (3H,s,Me-29),0.98 (3H,s,Me-30);13C-NMR (150 MHz,C5D5N) δ:180.5 (C-28),145.2(C-13),123.0 (C-12),73.7 (C-3),68.2 (C-23),49.0 (C-5),48.5 (C-9),47.0 (C-17),46.8 (C-19),43.3 (C-4),42.6 (C-14),42.4 (C-18),40.1 (C-8),39.2 (C-1),37.6(C-10),34.6 (C-21),33.6 (C-22),33.6 (C-29),33.4 (C-7),31.3 (C-20),28.7 (C-15),28.1 (C-2),26.5 (C-27),24.2 (C-30),24.1 (C-11),24.1 (C-16),19.0 (C-6),17.9 (C-26),16.4 (C-25),13.5 (C-24)。以上波谱数据与文献报道一致[6,7]，故鉴定化合物2为常春藤皂苷元。

化合物3：白色粉末，ESI-MS m/z:455.8[M-H]–，推测该化合物分子式为C30H48O3。1H-NMR (600MHz,CD3OD) δ:5.24 (1H,t,J=3.8 Hz,H-12),3.15(1H,dd,J=11.6,4.6 Hz,H-3),2.85 (1H,dd,J=4.6,13.7 Hz,H-18),2.01 (1H,td,J=13.4,4.0 Hz,H-16a),1.16 (3H,s,Me-27),0.97 (3H,s,Me-23),0.94 (3H,s,Me-25),0.94 (3H,s,Me-30),0.91 (3H,s,Me-29),0.82(3H,s,Me-26),0.78 (3H,s,Me-24),0.75 (1H,m,H-5);13C-NMR (150 MHz,CD3OD) δ:182.1 (C-28),145.3 (C-13),123.6 (C-12),79.7 (C-3),56.8 (C-5),49.8(C-29),47.7 (C-17),47.3 (C-19),42.9 (C-14),42.8 (C-18),40.6 (C-8),39.8 (C-1),39.8 (C-4),38.2 (C-10),34.9 (C-21),34.0 (C-22),33.9 (C-7),33.6 (C-29),31.6(C-20),28.8 (C-15),28.7 (C-23),27.9 (C-2),26.4 (C-27),24.5 (C-11),24.1 (C-16),24.0 (C-30),19.5 (C-6),17.7 (C-26),16.3 (C-24),15.9 (C-25)。以上波谱数据与文献报道一致[8]，故鉴定化合物3为齐墩果酸。

化合物4：白色粉末，ESI-MS m/z:471.4[M-H]–，推测该化合物分子式为C30H48O4。1H-NMR (600MHz,CD3OD) δ:6.04 (1H,dd,J=10.4,1.7 Hz,H-12),5.35 (1H,dd,J=10.3,3.2 Hz,H-11),3.06 (1H,dd,J=11.6,4.8 Hz,H-3),2.17 (1H,td,J=13.3,5.8 Hz,H-16a),2.02 (1H,dd,J=13.8,3.4 Hz,H-18),1.02 (3H,s,Me-27),0.95 (3H,s,Me-26),0.89 (3H,s,Me-23),0.88 (3H,s,Me-29),0.84 (3H,s,Me-30),0.81 (3H,s,Me-25),0.69 (3H,s,Me-24);13C-NMR (150 MHz,CD3OD) δ:182.7 (C-28),137.4 (C-11),127.8 (C-12),92.1 (C-13),79.5 (C-3),56.0 (C-18),54.5 (C-5),51.8(C-9),45.6 (C-17),42.9 (C-14),42.6 (C-8),40.0 (C-4),39.4 (C-1),38.2 (C-19),37.5 (C-10),35.2 (C-22),33.5(C-29),32.3 (C-21),32.3 (C-7),28.3 (C-23),28.3 (C-20),27.7 (C-15),26.5 (C-2),23.9 (C-16),22.4 (C-30),19.6 (C-26),18.8 (C-27),18.7 (C-6),18.5 (C-25),15.7(C-24)。以上波谱数据与文献报道一致[9]，故鉴定化合物4为3β,13β-dihydroxyolean-11-en-28-oic acid。

化合物5：白色粉末，ESI-MS m/z:457.3[M+H]+，推测该化合物分子式为C30H48O3。1H-NMR(600 MHz,CDCl3) δ:5.56 (1H,s,H-12),3.46 (1H,d,J=10.9 Hz,H-28a),3.22 (1H,m,H-3),3.21 (1H,m,H-28b),2.77 (1H,dt,J=13.6,3.7 Hz,H-1a),2.33 (1H,s,H-9),2.15 (1H,dd,J=13.6,4.6 Hz,H-18),1.75 (1H,m,H-15a),1.75 (1H,m,H-19a),1.65 (2H,m,H2-2),1.38 (3H,s,Me-27),1.35 (1H,m,H-16a),1.33 (1H,m,H-21a),1.18 (1H,m,H-15b),1.15 (1H,m,H-19b),1.12(3H,s,Me-25),1.10 (3H,s,Me-26),1.00 (3H,s,Me-26),0.98 (1H,m,H-1b),0.91 (3H,s,Me-30),0.89 (3H,s,Me-29),0.80 (3H,s,Me-24),0.69 (1H,dd,J=11.7,1.9 Hz,H-5);13C-NMR (150 MHz,CDCl3) δ:200.3(C-11),169.6 (C-13),128.4 (C-12),78.9 (C-3),69.8 (C-28),61.9 (C-9),55.1 (C-5),45.5 (C-8),45.0 (C-19),43.6 (C-14),42.8 (C-18),39.3 (C-1),39.3 (C-4),37.2(C-17),37.1 (C-10),34.0 (C-21),33.1 (C-29),32.8 (C-7),31.2 (C-20),30.8 (C-22),28.2 (C-24),27.4 (C-2),26.0 (C-15),23.5 (C-27),23.5 (C-30),21.6 (C-16),18.7(C-26),17.6 (C-6),16.5 (C-25),15.7 (C-23)。以上波谱数据与文献报道一致[10]，故鉴定化合物5为11-oxoerythrodiol。

化合物6：白色粉末，ESI-MS m/z:469.4[M-H]–，推测该化合物分子式为C30H46O4。1H-NMR (600MHz,CDCl3) δ:5.63 (1H,s,H-12),3.22 (1H,dd,J=10.9,5.3 Hz,H-3),2.96 (1H,dd,J=14.0,4.7 Hz,H-18),1.09 (3H,s,Me-23),0.98 (3H,s,Me-30),0.94(3H,s,Me-29),0.93 (3H,s,Me-26),0.90 (3H,s,Me-25),0.77 (3H,s,Me-24);13C-NMR (150 MHz,CDCl3) δ:200.5 (C-11),182.9 (C-28),168.5 (C-13),128.1 (C-12),78.8 (C-3),61.8 (C-9),55.0 (C-5),46.0 (C-17),45.0 (C-14),44.1 (C-19),43.4 (C-8),41.3 (C-18),39.1 (C-4),39.1 (C-1),37.3 (C-10),33.6 (C-21),32.9 (C-29),32.8(C-7),31.6 (C-22),30.7 (C-2),28.1 (C-23),27.8 (C-15),27.2 (C-2),23.6 (C-27),23.4 (C-30),22.6 (C-16),19.3 (C-26),17.3 (C-6),16.3 (C-25),15.6 (C-24)。以上波谱数据与文献报道一致[11]，故鉴定化合物6为3β-hydroxy-11-oxo-olean-12-en-28-oic acid。

化合物7：白色粉末，ESI-MS m/z:471.4[M-H]–，推测该化合物分子式为C30H48O4。1H-NMR (600MHz,CD3OD) δ:5.29 (1H,s,H-12),3.17 (1H,m,H-3),2.51 (1H,s,H-18),2.50 (1H,m,H-2a),2.07～1.91(2H,m,H-11),1.93～1.79 (1H,m,H-9),1.79～1.70(3H,m,H-15a,21a,22a),1.70～1.62 (2H,m,H-2b,6),1.61～1.50 (5H,m,H-1a,1b,16,21b),1.47～1.30 (2H,m,H-1b,7b),1.34 (3H,s,Me-27),0.99 (3H,s,Me-23),0.95 (3H,s,Me-25),0.94 (3H,d,J=6.6 Hz,Me-30),0.90 (1H,m,H-5),0.81 (3H,s,Me-24),0.79 (3H,s,Me-26);13C-NMR (150 MHz,CD3OD) δ:140.1 (C-13),129.4 (C-12),79.8 (C-3),73.6 (C-19),56.8 (C-5),55.1 (C-18),43.1 (C-20),42.6 (C-14),41.1 (C-8),39.9(C-4),39.8 (C-10),39.1 (C-22),34.2 (C-7),29.6 (C-15),28.7 (C-23),27.9 (C-21),27.3 (C-16),27.1 (C-29),26.7 (C-2),24.8 (C-27),24.7 (C-11),19.6 (C-1),17.5(C-24),16.6 (C-26),16.3 (C-30),15.9 (C-25)。以上波谱数据与文献报道一致[12]，故鉴定化合物7为pomolic acid。

化合物8：白色粉末，ESI-MS m/z:463.4[M+Na]+，推测该化合物分子式为C30H48O2。1H-NMR(600 MHz,CDCl3) δ:9.39 (1H,s,H-28),5.34 (1H,t,J=3.7 Hz,H-12),3.20 (1H,dd,J=11.4,4.4 Hz,H-3),2.62 (1H,m,H-18),1.97 (1H,m,H-11a),1.88 (1H,m,H-11b),1.13 (3H,s,Me-27),0.98 (3H,s,Me-23),0.91(3H,s,Me-29),0.91 (3H,s,Me-24),0.90 (3H,s,Me-30),0.77 (3H,s,Me-25),0.73 (3H,s,Me-26);13C-NMR (150 MHz,CDCl3) δ:207.7 (C-28),143.1 (C-13),123.4 (C-12),79.1 (C-3),55.3 (C-5),49.2 (C-9),47.7(C-17),45.7 (C-19),41.8 (C-14),40.5 (C-18),39.7 (C-8),38.9 (C-4),38.6 (C-1),37.1 (C-10),33.3 (C-21),33.2 (C-7),32.9 (C-22),30.8 (C-29),28.2 (C-20),27.9(C-2),27.3 (C-23),26.9 (C-15),25.7 (C-27),23.6 (C-16),23.6 (C-30),22.2 (C-11),18.4 (C-6),17.2 (C-26),15.7 (C-24),15.5 (C-15)。以上波谱数据与文献报道一致[13]，故鉴定化合物8为3β-hydroxyolean-12-en-28-aldehyde。

化合物9：白色粉末，ESI-MS m/z:441.6[M+H]+，推测该化合物分子式为C30H48O2。1H-NMR(600 MHz,CDCl3) δ:5.58 (1H,s,H-12),3.23 (1H,dd,J=11.1,5.3 Hz,H-3),2.79 (1H,dt,J=13.5,3.6 Hz,H-9),1.36 (3H,s,Me-27),1.13 (3H,s,Me-24),1.13(3H,s,Me-23),1.00 (3H,s,Me-26),0.90 (3H,s,Me-29),0.88 (3H,s,Me-30),0.86 (3H,s,Me-25),0.80 (3H,s,Me-28);13C-NMR (150 MHz,CDCl3) δ:200.5 (C-11),170.8 (C-13),128.3 (C-12),78.9 (C-3),61.9 (C-9),55.1 (C-5),47.7 (C-18),45.6 (C-14),45.3 (C-19),43.5(C-8),39.3 (C-1),37.2 (C-4),36.7 (C-22),34.6 (C-21),33.2 (C-29),32.9 (C-7),32.5 (C-17),31.2 (C-6),28.9(C-28) 28.2 (C-23),27.5 (C-2),26.6 (C-16),26.5 (C-15),23.6 (C-30),23.6 (C-27),18.9 (C-6),17.6 (C-26),16.5 (C-24),15.7 (C-25)。以上波谱数据与文献报道一致[14]，故鉴定化合物9为3β-hydroxyolean-12-en-11-one。

化合物10：白色粉末，ESI-MS m/z:477.8[M+Na]+，推测该化合物分子式为C30H46O3。1H-NMR(600 MHz,CDCl3) δ:5.29 (1H,t,J=3.7 Hz,H-12),2.83 (1H,dd,J=13.8,4.7 Hz,H-18),2.54 (1H,ddd,J=15.8,11.1,7.3 Hz,H-2a),2.36 (1H,ddd,J=15.9,6.8,3.6 Hz,H-2b),1.14 (3H,s,Me-27),1.08 (3H,s,Me-23),1.04 (3H,s,Me-24),1.02 (3H,s,Me-25),0.92(3H,s,Me-29),0.90 (3H,s,Me-30),0.80 (3H,s,Me-26);13C-NMR (150 MHz,CDCl3) δ:217.9 (C-3),184.4(C-28),143.8 (C-13),122.5 (C-12),55.4 (C-5),47.6 (C-4),47.0 (C-9),46.7 (C-17),45.9 (C-19),41.8 (C-14),41.1 (C-18),39.4 (C-1),39.2 (C-8),36.9 (C-10),34.3(C-2),33.9 (C-21),33.2 (C-29),32.5 (C-22),32.3 (C-7),30.8 (C-20),27.8 (C-15),26.6 (C-24),26.0 (C-27),23.7 (C-30),23.6 (C-11),23.0 (C-16),21.6 (C-23),19.7(C-6),17.1 (C-26),15.1 (C-25)。以上波谱数据与文献报道一致[15]，故鉴定化合物10为3-羰基齐墩果酸。

化合物11：白色粉末，ESI-MS m/z:465.4[M+Na]+，推测该化合物分子式为C30H50O2。1H-NMR(600 MHz,CDCl3) δ:5.19 (1H,t,J=3.7 Hz,H-12),3.55 (1H,d,J=11.0 Hz,H-28a),3.22 (1H,m,H-3),3.21 (1H,d,J=11.3 Hz,H-28b),1.98 (1H,dd,J=13.5,4.7 Hz,H-18),1.92～1.83 (3H,m,H2-11,16α),1.75～1.68 (2H,m,H-19α,15β),1.17 (3H,s,Me-27),1.00(3H,s,Me-23),0.94 (3H,s,Me-26),0.93 (3H,s,Me-25),0.89 (3H,s,Me-29),0.87 (3H,s,Me-30),0.79 (3H,s,Me-24);13C-NMR (150 MHz,CDCl3) δ:144.3 (C-13),122.5 (C-12),79.2 (C-3),69.9 (C-28),55.3 (C-5),47.7 (C-9),46.6 (C-19),42.5 (C-18),41.9 (C-14),39.9(C-8),38.9 (C-4),38.7 (C-1),37.1 (C-10),37.1 (C-17),34.2 (C-21),33.3 (C-29),32.7 (C-7),31.2 (C-22),31.1(C-20),28.2 (C-23),27.4 (C-2),26.1 (C-27),25.7 (C-15),23.73 (C-30),23.67 (C-11),22.1 (C-16),18.5 (C-6),16.9 (C-26),15.7 (C-24),15.7 (C-25)。以上波谱数据与文献报道一致[16]，故鉴定化合物11为古柯二醇。

化合物12：白色粉末，ESI-MS m/z:535.4[M+Na]+，推测该化合物分子式为C33H52O4。1H-NMR(600 MHz,CDCl3) δ:5.29 (1H,t,J=3.7 Hz,H-12),4.59 (1H,d,J=16.8 Hz,H-1'a),4.54 (1H,d,J=16.7,H-1'b),3.21 (1H,dd,J=11.3,4.3 Hz,H-3),2.87 (1H,dd,J=13.8,4.7 Hz,H-18),2.16 (3H,s,3'-Me),1.14(3H,s,27-Me),0.99 (3H,s,23-Me),0.93 (3H,s,29-Me),0.91 (3H,s,Me-24),0.90 (3H,s,Me-30),0.78(3H,s,Me-25),0.72 (3H,s,Me-26);13C-NMR (150MHz,CDCl3) δ:202.5 (C-2'),177.2 (C-28),143.7 (C-13),122.7 (C-12),79.2 (C-3),68.2 (C-1'),55.4 (C-5),47.8 (C-9),47.0 (C-17),46.1 (C-19),41.9 (C-14),41.4(C-18),39.5 (C-8),38.9 (C-4),38.6 (C-1),37.2 (C-10),34.0 (C-21),33.2 (C-30),32.8 (C-22),32.5 (C-7),30.8(C-20),28.2 (C-23),27.8 (C-15),27.3 (C-2),26.5 (C-3'),26.0 (C-27),23.8 (C-29),23.6 (C-16),23.3 (C-11),18.5 (C-6),17.1 (C-26),15.7 (C-25),15.5 (C-24)。以上波谱数据与文献报道一致[17]，故鉴定化合物12为oleanolic acid 2-oxopropyl ester。

化合物13：白色粉末，ESI-MS m/z:457.4[M+H]+，推测该化合物分子式为C30H48O3。1H NMR (600MHz,CD3OD) δ:5.23 (1H,t,J=3.7 Hz,H-12),3.15(1H,dd,J=11.7,4.6 Hz,H-3),2.20 (1H,dd,J=11.4,1.8 Hz,H-18),1.12 (3H,s,Me-23),0.98 (3H,s,Me-27),0.96 (3H,d,J=5.0 Hz,Me-29),0.96 (3H,s,Me-26),0.89 (3H,d,J=6.5 Hz,Me-30),0.85 (3H,s,Me-24),0.78 (3H,s,Me-25);13C-NMR (150 MHz,CD3OD) δ:181.7 (C-28),139.6 (C-13),126.9 (C-12),79.7 (C-3),56.7 (C-5),54.4 (C-18),49.0 (C-9),43.2 (C-14),40.8 (C-8),40.4 (C-19),40.0 (C-1),39.8 (C-4),38.1 (C-10),38.1 (C-22),34.3 (C-7),31.8 (C-21),29.2(C-15),28.8 (C-23),27.9 (C-2),25.3 (C-16),24.4 (C-11),24.1 (C-27),21.6 (C-30),19.5 (C-6),17.8 (C-29),17.7 (C-26),16.4 (C-24),16.0 (C-25)。以上波谱数据与文献报道一致[18,19]，故鉴定化合物13为熊果酸。

化合物14：白色粉末，ESI-MS m/z:457.3[M+H]+，推测该化合物分子式为C30H48O3。1H-NMR(600 MHz,CDCl3) δ:5.51 (1H,s,H-12),3.46 (1H,d,J=10.9 Hz,H-28a),3.22 (1H,dd,J=11.4,4.9 Hz,H-3),3.16 (1H,d,J=11.0 Hz,H-28b),2.32 (1H,s,H-9),1.32 (3H,s,Me-27),1.16 (3H,s,Me-23),1.15 (3H,d,J=6.9 Hz,Me-30),0.96 (3H,s,Me-24),0.82 (3H,d,J=6.5 Hz,Me-29),0.81 (3H,s,Me-25);13C-NMR(150 MHz,CDCl3) δ:199.8 (C-11),163.8 (C-13),130.7(C-12),78.9 (C-3),69.9 (C-28),61.7 (C-9),55.0 (C-5),54.1 (C-18),45.2 (C-8),43.8 (C-14),39.4 (C-20),39.4(C-1),39.3 (C-4),39.1 (C-19),38.5 (C-17),37.1 (C-10),35.0 (C-22),32.9 (C-7),30.4 (C-21),28.2 (C-23),27.4 (C-2),26.7 (C-15),22.8 (C-16),21.3 (C-30),20.7(C-27),18.5 (C-26),17.7 (C-6),17.5 (C-29),16.7 (C-25),15.7 (C-24)。以上波谱数据与文献报道一致[20,21]，故鉴定化合物14为ilelatifol A。

化合物15：白色粉末，ESI-MS m/z:505.4[M+Cl]–，推测该化合物分子式为C30H46O4。1H-NMR(600 MHz,CDCl3) δ:5.56 (1H,s,H-12),1.31 (3H,s,Me-27),1.12 (3H,s,Me-25),0.99 (3H,s,Me-24),0.97(3H,s,Me-20),0.97 (3H,d,J=6.2,Hz,Me-30),0.87(3H,d,J=6.4 Hz,Me-29),0.79 (3H,s,Me-5);13C-NMR (150 MHz,CDCl3) δ:200.5 (C-11),179.7 (C-28),163.7 (C-13),130.6 (C-12),78.7 (C-3),61.5 (C-9),55.0(C-5),52.9 (C-18),47.4 (C-17),44.7 (C-8),43.9 (C-14),39.2 (C-1),39.1 (C-4),38.7 (C-20),38.6 (C-19),37.2 (C-10),36.1 (C-22),33.1 (C-7),30.4 (C-21),28.5(C-15),28.1 (C-23),27.1 (C-2),23.9 (C-16),21.0 (C-27),21.0 (C-30),18.9 (C-26),17.5 (C-6),17.1 (C-29),16.3 (C-25),15.6 (C-24)。以上波谱数据与文献报道一致[22,23]，故鉴定化合物15为3β-hydroxy-11-oxours-12-en-28-oic acid。

化合物16：白色粉末，ESI-MS m/z:473.8[M+H]+，推测该化合物分子式为C30H48O4。1H-NMR(600 MHz,C5D5N) δ:5.46 (1H,t,H-12),4.08 (1H,m,H-2),3.39 (1H,d,J=9.5 Hz,H-3),2.62 (1H,d,J=11.5 Hz,H-18),1.25 (3H,s,Me-23),1.18 (3H,s,Me-27),1.06 (3H,s,Me-26),1.05 (3H,s,Me-24),0.98 (3H,d,J=6.5 Hz,Me-30),0.97 (3H,s,Me-25),0.93 (3H,d,J=6.5 Hz,Me-29);13C-NMR (150 MHz,C5D5N)δ:180.3 (C-28),139.7 (C-13),125.9 (C-12),84.2 (C-3),68.9 (C-2),56.3 (C-5),53.9 (C-18),48.4 (C-1),48.4(C-17),48.4 (C-9),42.9 (C-14),40.4 (C-4),40.2 (C-8),39.8 (C-19),39.8 (C-20),38.8 (C-10),37.8 (C-22),33.9(C-7),31.4 (C-16),29.7 (C-21),29.0 (C-23),25.3 (C-15),24.3 (C-27),24.1 (C-11),21.8 (C-29),19.2 (C-6),18.1 (C-24),17.9 (C-30),17.8 (C-26),17.3 (C-25)。以上波谱数据与文献报道一致[24]，故鉴定化合物16为2α,3β-dihydroxyurs-12-en-28-oic acid。

化合物17：白色粉末，ESI-MS m/z:441.4[M+H]+，推测该化合物分子式为C30H48O2。1H-NMR(600 MHz,CDCl3) δ:5.54 (1H,s,H-12),3.23 (1H,dd,J=11.4,5.0 Hz,H-3),2.32 (1H,s,H-9),1.67 (1H,m,H-18),1.29 (3H,s,Me-27),1.17 (3H,s,Me-26),1.00(3H,s,Me-25),0.94 (3H,s,Me-24),0.81 (3H,s,Me-23),0.81 (3H,d,J=3.9 Hz,Me-30),0.80 (3H,d,J=6.5 Hz,Me-29);13C-NMR (150 MHz,CDCl3) δ:200.0(C-1),165.1 (C-13),130.6 (C-12),79.0 (C-3),61.7 (C-9),59.1 (C-18),55.1 (C-5),45.3 (C-14),43.8 (C-8),41.1 (C-22),39.4 (C-20),39.4 (C-1),39.4 (C-19),39.3(C-4),37.1 (C-10),34.1 (C-17),33.0 (C-7),31.0 (C-21),29.0 (C-28),28.2 (C-23),27.7 (C-16),27.5 (C-2),27.4 (C-15),21.3 (C-30),20.7 (C-27),18.7 (C-26),17.7(C-6),17.6 (C-29),16.7 (C-25),15.7 (C-24)。以上波谱数据与文献报道一致[25]，故鉴定化合物17为3β-hydroxy-11-oxo-ursan-12-ene。

化合物18：白色粉末，ESI-MS m/z:513.8[M+H]+，推测该化合物分子式为C33H52O4。1H-NMR(600 MHz,CDCl3) δ:5.26 (1H,d,J=3.1 Hz,H-12),4.54 (2H,m,H2-1'),3.22 (1H,dd,J=11.4,4.7 Hz,H-3),2.15 (3H,s,Me-3'),1.09 (3H,s,Me-27),0.99 (3H,s,Me-23),0.95 (3H,d,J=6.4 Hz,Me-30),0.92 (3H,s,Me-25),0.86 (3H,d,J=6.7 Hz,Me-29),0.78 (3H,s,Me-24),0.74 (3H,s,Me-26);13C-NMR (150 MHz,CDCl3) δ:202.6 (C-2'),177.0 (C-28),138.1 (C-13),125.9 (C-12),79.2 (C-3),68.2 (C-1'),55.4 (C-5),53.0(C-18),48.4 (C-17),47.7 (C-9),42.2 (C-14),39.7 (C-8),39.2 (C-19),39.0 (C-20),38.9 (C-1),38.8 (C-4),37.1 (C-10),36.8 (C-22),33.1 (C-7),30.8 (C-21),28.3(C-23),28.1 (C-15),27.4 (C-2),26.5 (C-3'),24.4 (C-16),23.7 (C-27),23.4 (C-11),21.3 (C-30),18.4 (C-6),17.2 (C-29),15.8 (C-24),15.6 (C-25)。以上波谱数据与文献报道一致[26]，故鉴定化合物18为2-oxopropyl(3β)-3-hydroxyurs-12-en-28-oate。

化合物19：白色粉末，ESI-MS m/z:441.7[M+H]+，推测该化合物分子式为C30H48O2。1H-NMR(600 MHz,CDCl3) δ:9.32 (1H,s,H-28),5.31 (1H,t,J=3.7 Hz,H-12),3.21 (1H,dd,J=11.3,4.7 Hz,H-3),1.09 (3H,s,Me-27),0.99 (3H,s,Me-23),0.96 (3H,d,J=6.3 Hz,Me-30),0.92 (3H,s,Me-24),0.88 (3H,d,J=6.5 Hz,Me-29),0.78 (3H,s,Me-25),0.77 (3H,s,Me-26);13C-NMR (150 MHz,CDCl3) δ:207.6 (C-28),137.9 (C-13),126.3 (C-12),79.1 (C-3),55.3 (C-5),52.7(C-18),50.3 (C-17),47.7 (C-9),42.3 (C-14),39.9 (C-8),39.1 (C-19),38.9 (C-20),38.9 (C-4),38.8 (C-1),37.0 (C-10),33.2 (C-22),32.0 (C-7),30.3 (C-21),28.3(C-15),27.3 (C-2),27.0 (C-23),23.4 (C-16),23.3 (C-11),23.3 (C-27),21.2 (C-30),18.4 (C-6),17.3 (C-26),16.8 (C-29),15.8 (C-24),15.6 (C-25)。以上波谱数据与文献报道一致[27]，故鉴定化合物19为熊果醛。

化合物20：白色粉末，ESI-MS m/z:507.2[M+Cl]–，推测该化合物分子式为C31H52O3。1H-NMR(600 MHz,CDCl3) δ:4.53 (1H,s,12-OH),4.25 (1H,d,J=10.3 Hz,H-11),3.24 (1H,q,J=8.1,5.6 Hz,H-3),3.17 (3H,s,11-OMe),2.23 (1H,m,H-18),1.87 (1H,d,J=10.4 Hz,H-9),1.79 (1H,td,J=13.5,5.0 Hz,H-15a),1.20 (3H,s,Me-27),1.11 (3H,s,Me-25),1.11(3H,s,Me-26),1.02 (3H,s,Me-23),0.93 (3H,d,J=6.4 Hz,Me-30),0.92 (3H,d,J=6.4 Hz,Me-29),0.82(3H,s,Me-24),0.81 (3H,s,Me-28);13C-NMR (150MHz,CDCl3) δ:142.2 (C-12),118.4 (C-13),78.8 (C-3),76.8 (C-11),55.6 (C-5),51.5 (11-OMe),47.8 (C-18),46.4 (C-9),43.0 (C-14),41.8 (C-22),40.9 (C-19),40.7 (C-8),39.6 (C-20),39.3 (C-4),39.1 (C-1),38.5 (C-10),34.4 (C-7),33.5 (C-17),31.4 (C-21),28.7 (C-28),28.5 (C-23),27.7 (C-16),27.6 (C-2),27.3 (C-15),24.0(C-27),21.4 (C-30),18.4 (C-6),18.2 (C-26),17.1 (C-29),16.3 (C-25),15.9 (C-24)。以上波谱数据与文献报道一致[28]，故鉴定化合物20为11α-methoxyurs-12-ene-3β,12-diol。

4、活性测试

采用Omar等[29]的方法测试化合物1～20对α-葡萄糖苷酶的抑制活性。测试化合物在100μmol/L时对α-葡萄糖苷酶活性的抑制率，对抑制率>50%的化合物进行半数抑制浓度（median inhibition concentration,IC50）值测定。其中化合物1、3、4、13、15和16对α-葡萄糖苷酶的抑制率>50%。化合物1、3、4、13、15和16测试IC50值：用倍比稀释法设置浓度范围为100、50、25、12.5、6.25、3.13、1.56μmol/L，通过SPSS 18.0软件中的probit回归法计算得到IC50值分别为（15.75±0.40）、（34.18±2.26）、（20.55±1.35）、（24.76±0.21）、（34.74±0.54）、（35.60±1.66）μmol/L，抑制活性均高于阳性对照阿卡波糖[IC50为（536.9±24.0）μmol/L]。其中，化合物1、3、4为齐墩果烷型三萜，化合物13、15、16为乌苏烷型三萜，化合物1的IC50值最小，对α-葡萄糖苷酶的抑制活性最为显著。可以看出，在同个骨架中，化合物1相比其他2个化合物，其C-2上多连了1个α-羟基，而化合物4的羟基在C-13上，说明2位上的羟基也许由于化学位阻相较于13位羟基的更小，所以能够与α-葡萄糖苷酶的结合更为紧密，从而更好地抑制起发挥作用。同时，20位的偕二甲基对于抑制活性也有发挥一定作用的可能性。

5、讨论

本课题对芫花枝叶部分的三萜类化学成分及α-葡萄糖苷酶抑制活性进行了研究，从芫花干燥枝叶的95%乙醇提取物的醋酸乙酯部位中分离鉴定了20个三萜类化合物。芫花的天然产物中，有从花蕾中分离得到的具有抗肿瘤活性显著的瑞香烷型二萜，也有从根部分离得到的具有神经保护活性的愈创木烷型倍半萜，而鲜少对其枝叶进行系统的化学成分研究。本课题则对芫花的枝叶部分进行了系统的三萜类成分的分离鉴定，并得到了具有抑制α-葡萄糖苷酶的活性化合物。但本研究仍不够深入，后续应进一步分离得到更多此结构类型的成分以及活性化合物，即可进一步探讨其构效关系；同时也可能挖掘到结构新颖的化合物从而扩大天然产物库，后期将对其他极性部位和药理作用展开进一步研究，以便为芫花的综合利用提供科研数据和理论基础。

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基金资助:国家自然科学基金资助项目(22177016);山东省中医药科技发展计划项目(2020M037);2019年医疗服务与保障能力提升补助资金“全国中药资源普查项目”(财社[2019]39号);

文章来源:薛俊娟,叶国华,魏国栋,等.芫花枝叶三萜类化学成分及其α-葡萄糖苷酶抑制活性研究[J].中草药,2024,55(08):2524-2532.