中草药  2016, Vol. 47 Issue (8): 1272-1277
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引用本文doi: 10.7501/j.issn.0253-2670.2016.08.004
夏文绮, 崔保松, 李帅. 四季青化学成分的研究[J]. 中草药, 2016, 47(8): 1272-1277.
XIA Wen-qi, CUI Bao-song, LI Shuai. Chemical constituents from leaves of Ilex chinensis[J]. Chinese Traditional and Herbal Drugs, 2016, 47(8): 1272-1277.
四季青化学成分的研究
夏文绮, 崔保松, 李帅     
中国医学科学院 北京协和医学院药物研究所 天然药物活性物质与功能国家重点实验室, 北京 100050
收稿日期: 2015-12-30;
基金项目: “十二五”重大专项“中药新药发现与评价技术平台”(2012ZX09301002-002)
作者简介: 夏文绮,女,硕士研究生,研究方向为中药有效成分及标准物质的研究。E-mail:xiawenqi@imm.ac.cn
通讯作者: 李帅 Tel:(010)63164628 E-mail:lishuai@imm.ac.cn
摘要: 目的 研究四季青(冬青Ilex chinensis叶)的化学成分。方法 应用多种色谱技术进行分离纯化,根据化合物的波谱数据进行结构鉴定。结果 从四季青70%乙醇提取物中分离得到15个三萜及其皂苷类化合物,分别鉴定为2α,3β-二羟基-23-去甲-4(24)-12(13)-二烯-齐墩果酸(1)、29-羟基常春藤皂苷元(2)、ilexosapogenin A(3)、救必应酸-3,23-缩丙酮(4)、2α,3β-二羟基-23-去甲-4(24)-12-二烯-乌索酸(5)、23-羟基乌索酸(6)、3β,23-二羟基-12,18-二烯-乌索酸(7)、积雪草酸(8)、泰国树脂酸-28-O-β-D-葡萄糖酯苷(9)、28-O-β-D-glucopyranoside spathodic acid(10)、坡模酸-28-O-β-D-葡萄糖酯苷(11)、rotungenoside(12)、3β,23-二羟基-12,18-二烯-乌索酸-28-O-β-D-葡萄糖酯苷(13)、3β,23-二羟基-12,19-二烯-乌索酸-28-O-β-D-葡萄糖酯苷(14)、3β,23-二羟基-12,19(29)-二烯-乌索酸-28-O-β-D-葡萄糖酯苷(15)。结论 化合物1、2为首次从冬青属植物中分离得到,所有化合物均为首次从该植物中分离得到。
关键词: 冬青     三萜     2α,3β-二羟基-23-去甲-4(24)-12(13)-二烯-齐墩果酸     29-羟基常春藤皂苷元     积雪草酸    
Chemical constituents from leaves of Ilex chinensis
XIA Wen-qi , CUI Bao-song, LI Shuai    
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Abstract: Objective To investigate the chemical constituents from the leaves of Ilex chinensis. Methods The constituents were isolated and purified by various chromatographic methods, and the structures were elucidated by spectroscopic analysis. Results Fifteen triterpenoid compounds were isolated from 70% ethanol extract in the leaves of I. chinensis and identified as 2α,3β-dihydroxy-23-norolean-4(24)-12(13)-dien-28-oic acid (1), 29-hydroxyhederagenin (2), ilexosapogenin A (3), rotundic acid isopropylidene (4), 2α,3β-dihydroxy-23-norurs-4(24)-12-dien-28-oic acid (5), 23-hydroxyursolic acid (6), 3β,23-dihydroxyursa-12,18-dien-28-oic acid (7), asiatic acid (8), 28-O-β-D-glucopyranoside siaresinolic acid (9), 28-O-β-D-glucopyranoside spathodic acid (10), 28-O-β-D-glucopyranoside pomolic acid (11), rotungenoside (12), 3β,23-dihydroxyurs-12,18-dien-28-O-β-D-glucopyranosyl ester (13), 3β,23-dihydroxyurs-12,19-dien-28-O-β-D-glucopyranosyl ester (14), and 3β,23-dihydroxyurs-12,19(29)-dien-28-O-β-D-glucopyranosyl ester (15). Conclusion Compounds 1 and 2 are isolated from the plants of Ilex L. for the first time. All compounds are isolated from this plant for the first time.
Key words: Ilex chinensis Sims     triterpenes     2α,3β-dihydroxy-23-norolean-4(24)-12(13)-dien-28-oic acid     29-hydroxyhederagenin     asiatic acid    

四季青为冬青科（Aquifoliaceae）冬青属Ilex L.植物冬青Ilex chinensis Sims的干燥叶，主要分布于秦岭南坡、长江流域及其以南广大地区[1]，秋、冬二季采收，晒干。四季青具有清热解毒、消肿祛瘀的功效，用于肺热咳嗽、咽喉肿痛、痢疾、胁痛、热淋；外治烧烫伤、皮肤溃疡[2]。现代药理实验也证明其具有抗菌[3]、抗炎[4, 5]、治疗烧烫伤[6]等作用。目前对四季青的研究报道多为粗提物的药理活性研究，对其传统药效物质基础的研究尚不充分，仅报道了13个三萜类化合物[7, 8, 9]，8个酚酸类化合物[10, 11, 12]，5个黄酮及3个甾体类化合物[13]。《中国药典》2015年版以其主要成分长梗冬青苷为定量测定依据，本研究在研制长梗冬青苷对照品的基础上，为进一步寻找和阐明其活性成分，对其化学成分进行了深入的研究，在已有化学成分研究报道的基础上，又从其70%乙醇提取物中分离得到8个三萜：2α,3β-二羟基-23-去甲-4(24)-12(13)-二烯-齐墩果酸 [2α,3β- dihydroxy-23-norolean-4(24)-12(13)-dien-28-oic acid，1]、29-羟基常春藤皂苷元（29-hydroxyhederagenin，2）、ilexosapogenin A（3）、救必应酸-3,23-缩丙酮（rotundic acid isopropylidene，4）、2α,3β-二羟基-23-去甲-4(24)-12-二烯-乌索酸 [2α,3β-dihydroxy-23- norurs-4(24)-12-dien-28-oic acid，5]、23-羟基乌索酸（23-hydroxyursolic acid，6）、3β,23-二羟基-12,18-二烯-乌索酸（3β,23-dihydroxyursa-12,18-dien-28-oic acid，7）、积雪草酸（asiatic acid，8）；7个三萜皂苷：泰国树脂酸-28-O-β-D-葡萄糖酯苷（28-O-β-D- glucopyranoside siaresinolic acid，9）、28-O-β-D- glucopyranoside spathodic acid（10）、坡模酸-28-O- β-D-葡萄糖酯苷（28-O-β-D-glucopyranoside pomolic acid，11）、rotungenoside（12）、3β,23-二羟基-12,18-二烯-乌索酸-28-O-β-D-葡萄糖酯苷（3β,23-dihydro- xyurs-12,18-dien-28-O-β-D-glucopyranosyl ester，13）、3β,23-二羟基-12,19-二烯-乌索酸-28-O-β-D-葡萄糖酯苷（3β,23-dihydroxyurs-12,19-dien-28-O-β-D-gluco- pyranosyl ester，14）、3β,23-二羟基-12,19(29)-二烯-乌索酸-28-O-β-D-葡萄糖酯苷 [3β,23-dihydroxyurs- 12,19(29)-dien-28-O-β-D-glucopyranosyl ester，15]。以上化合物均为首次从该植物中分离得到，化合物1、2为首次从冬青属植物中分离得到。

1 仪器与材料

Aglient 1000 series LC-MSD-trap-SL型ESI-MS质谱仪（美国Aglient公司），Mercury-400、Inova-500型核磁共振光谱仪（美国Varian公司），Shimadzu LC-6AD高效液相色谱泵（日本Shimadzu公司）配备SPD-20A双波长紫外吸收检测器，制备色谱柱为RP18（250 mm×10 mm，5 μm）半制备柱（日本YMC公司和Cosmosil公司）。葡聚糖凝胶Sephadex LH-20为瑞典GE Healthcare Bio-Science AB公司产品，硅胶预制板为烟台市化学工业研究所产品，色谱用硅胶为青岛海洋化工集团公司生产。所用试剂均购自北京化工厂，均为分析纯或色谱纯。

冬青叶于2010年3月采自江西省九江县，经江西省科学院生物资源研究所九江县森林植物研究所谭策铭研究员鉴定为冬青Ilex chinensis Sims的干燥叶。植物标本（ID-22233）保存于中国医学科学院药物研究所标本室。

2 提取与分离

冬青干燥叶20 kg，用5倍量的70%乙醇加热回流提取3次，每次2 h，提取液减压回收。加适量水使浸膏分散溶解，用醋酸乙酯萃取得醋酸乙酯部位。醋酸乙酯部位浸膏460 g，经硅胶柱色谱，石油醚-丙酮梯度洗脱，得到12个组分A～L。组分D（12.5 g）经硅胶柱色谱，石油醚-丙酮（4︰1、3︰1、2︰1、1.5︰1、1︰1）梯度洗脱得到10个亚组分，其中亚组分DH（5.7 g）经反复硅胶、Sephadex LH-20柱色谱及HPLC制备分离得到化合物1（10 mg）、3（17 mg）、4（32 mg）、5（9 mg）、6（42 mg）、7（3 mg）。组分G（52.9 g）经硅胶柱色谱，氯仿-甲醇（50︰1→5︰1）梯度洗脱得到7个亚组分，其中亚组分GC（6.3 g）、GD（3.4 g）、GE（9.8 g）经反复硅胶、Sephadex LH-20柱色谱及HPLC制备分离得到化合物2（8 mg）、8（6 mg）、9（6 mg）、10（6 mg）、11（14 mg）、12（20 mg）、13（217 mg）、14（33 mg）、15（20 mg）。

3 结构鉴定

化合物1：白色粉末。ESI-MS m/z: 479 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 5.86 (1H,s,H-24a),5.50 (1H,t,J = 3.5 Hz,H-12),4.95 (1H,s,H-24b),4.36 (1H,d,J = 9.0 Hz,H-3),4.05 (1H,m,H-2),3.32 (1H,dd,J = 13.5,4.5 Hz,H-18),1.27 (3H,s,H-27),1.06 (3H,s,H-30),1.02 (3H,s,H-29),0.97 (3H,s,H-26),0.83 (3H,s,H-25)；13C-NMR (125 MHz,C5D5N) δ: 47.2 (C-1),73.9 (C-2),79.9 (C-3),150.7 (C-4),51.1 (C-5),26.5 (C-6),33.6 (C-7),40.3 (C-8),46.8 (C-9),39.3 (C-10),21.9 (C-11),123.0 (C-12),145.5 (C-13),42.8 (C-14),28.6 (C-15),24.2 (C-16),48.5 (C-17),42.6 (C-18),46.0 (C-19),32.1 (C-20),31.4 (C-21),34.7 (C-22),105.1 (C-24),15.6 (C-25),17.9 (C-26),25.2 (C-27),178.5 (C-28),33.7 (C-29),24.2 (C-30)。以上数据与文献报道一致[14]，故鉴定化合物1为2α,3β-二羟基-23-去甲-4(24)-12-二烯-齐墩果酸 。

化合物2：白色粉末。ESI-MS m/z: 511 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 5.56 (1H,t-like,H-12),4.20 (1H,d,J = 10.0 Hz,H-23a),4.21 (1H,m,H-3),3.73 (1H,d,J = 10.0 Hz,H-23b),3.60 (1H,d,J = 12.5 Hz,H-30a),3.45 (1H,d,J = 12.5 Hz,H-30b),1.28 (3H,s,H-27),1.24 (3H,s,H-29),1.08 (6H,s,H-24,H-25),1.00 (3H,s,H-26)；13C-NMR (125 MHz,C5D5N) δ: 39.2 (C-1),28.2 (C-2),74.3 (C-3),43.4 (C-4),49.1 (C-5),19.1 (C-6),33.4 (C-7),40.2 (C-8),48.6 (C-9),37.7 (C-10),24.3 (C-11),123.1 (C-12),145.5 (C-13),42.7 (C-14),28.9 (C-15),24.3 (C-16),47.6 (C-17),41.9 (C-18),41.7 (C-19),37.1 (C-20),29.6 (C-21),33.2 (C-22),68.5 (C-23),13.6 (C-24),16.4 (C-25),18.0 (C-26),26.6 (C-27),180.7 (C-28),73.9 (C-29),20.2 (C-30)。以上数据与文献报道一致[15]，故鉴定化合物2为29-羟基常春藤皂苷元。

化合物3：白色粉末。ESI-MS m/z: 511 [M＋Na]+。1H-NMR (500 MHz,CD3OD) δ: 5.25 (1H,t,J = 3.5 Hz,H-12),3.55 (1H,dd,J = 10.5,5.0 Hz,H-3),3.47 (1H,d,J = 11.0 Hz,H-23a),3.28 (1H,d,J = 11.0 Hz,H-23b),3.19 (1H,d,J = 3.5 Hz,H-19),3.00 (1H,brs,H-18),1.24 (3H,s,H-27),0.91 (6H,s,H-29,30),0.88 (3H,s,H-25),0.71 (3H,s,H-26),0.65 (3H,s,H-24)；13C-NMR (125 MHz,CD3OD) δ: 39.3 (C-1),27.4 (C-2),74.1 (C-3),43.3 (C-4),48.6 (C-5),19.3 (C-6),33.5 (C-7),40.7 (C-8),48.4 (C-9),38.1 (C-10),24.8 (C-11),124.9 (C-12),144.6 (C-13),42.6 (C-14),28.7 (C-15),29.5 (C-16),46.8 (C-17),45.2 (C-18),82.5 (C-19),36.0 (C-20),29.5 (C-21),34.1 (C-22),67.5 (C-23),12.7 (C-24),16.1 (C-25),17.8 (C-26),25.1 (C-27),181.0 (C-28),29.5 (C-29),25.2 (C-30)。以上数据与文献报道一致[16]，故鉴定化合物3为ilexosapogenin A。

化合物4：白色粉末。ESI-MS m/z: 551 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 5.59 (1H,brs,H-12),3.64 (1H,dd,J = 11.5,3.5 Hz,H-3),3.61 (1H,d,J = 11.0 Hz,H-23a),3.54 (1H,d,J = 11.0 Hz,H-23b),1.72 (3H,s,H-27),1.52 (3H,s,H-29),1.47 (3H,s,H-33),1.45 (3H,s,H-26),1.14 (3H,s,H-31),1.12 (3H,d,J = 7.0 Hz,H-30),1.07 (3H,s,H-25),0.86 (3H,s,H-24)；13C-NMR (125 MHz,C5D5N) δ: 39.1 (C-1),24.9 (C-2),78.0 (C-3),42.3 (C-4),54.8 (C-5),18.2 (C-6),33.2 (C-7),37.6 (C-8),47.9 (C-9),37.2 (C-10),24.0 (C-11),128.0 (C-12),140.1 (C-13),40.7 (C-14),29.5 (C-15),27.1 (C-16),48.5 (C-17),51.8 (C-18),72.9 (C-19),42.6 (C-20),24.2 (C-21),38.7 (C-22),72.8 (C-23),17.3 (C-24),13.1 (C-25),17.0 (C-26),26.6 (C-27),180.8 (C-28),27.3 (C-29),16.8 (C-30),19.8 (C-31),99.2 (C-32),30.5 (C-33)。以上数据与文献报道一致[17]，故鉴定化合物4为救必应酸-3,23-缩丙酮。

化合物5：白色粉末。ESI-MS m/z: 479 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 5.86 (1H,s,H-24a),5.50 (1H,brs,H-12),4.95 (1H,s,H-24b),4.37 (1H,d,J = 9.0 Hz,H-3),4.05 (1H,m,H-2),2.65 (1H,d,J = 11.5 Hz,H-18),1.22 (3H,s,H-27),1.09 (3H,s,H-26),1.00 (3H,d,J = 6.5 Hz,H-29),0.96 (3H,d,J = 6.0 Hz,H-30),0.82 (3H,s,H-25)；13C-NMR (125 MHz,C5D5N) δ: 48.5 (C-1),73.9 (C-2),80.0 (C-3),150.7 (C-4),51.1 (C-5),21.9 (C-6),32.4 (C-7),40.0 (C-8),45.9 (C-9),39.1 (C-10),24.3 (C-11),124.3 (C-12),140.0 (C-13),43.2 (C-14),29.0 (C-15),25.1 (C-16),48.7 (C-17),54.1 (C-18),39.9 (C-19),40.5 (C-20),31.5 (C-21),25.3 (C-22),105.1 (C-24),15.7 (C-25),17.9 (C-26),24.3 (C-27),180.4 (C-28),18.0 (C-29),21.9 (C-30)。以上数据与文献报道一致[18]，故鉴定化合物5为2α,3β-二羟基-23-去甲-4(24)-12(13)-二烯-乌索酸。

化合物6：白色粉末。ESI-MS m/z: 495 [M＋Na]+。1H-NMR (500 MHz,CD3OD) δ: 5.25 (1H,t,J = 3.5 Hz,H-12),3.63 (1H,dd,J = 11.0,4.5 Hz,H-3),3.55 (1H,d,J = 11.0 Hz,H-23a),3.31 (1H,d,J = 11.0 Hz,H-23b),2.22 (1H,d,J = 6.5 Hz,H-18),1.15 (3H,s,H-27),1.01 (3H,s,H-25),0.99 (3H,d,J = 6.5 Hz,H-29),0.91 (3H,d,J = 6.5 Hz,H-30),0.87 (3H,s,H-26),0.73 (3H,s,H-24)；13C-NMR (125 MHz,CD3OD) δ: 39.7 (C-1),27.5 (C-2),73.9 (C-3),43.3 (C-4),48.0 (C-5),19.1 (C-6),33.8 (C-7),40.4 (C-8),48.0 (C-9),37.9 (C-10),24.1 (C-11),126.9 (C-12),139.7 (C-13),43.3 (C-14),29.2 (C-15),25.3 (C-16),48.6 (C-17),54.4 (C-18),40.8 (C-19),40.4 (C-20),31.8 (C-21),38.1 (C-22),67.4 (C-23),12.8 (C-24),16.4 (C-25),17.7 (C-26),24.4 (C-27),181.6 (C-28),17.8 (C-29),21.6 (C-30)。以上数据与文献报道一致[19]，故鉴定化合物6为23-羟基乌索酸。

化合物7：淡黄色粉末。ESI-MS m/z: 493 [M＋Na]+。1H-NMR (400 MHz,CD3OD) δ: 5.33 (1H,t,J = 4.0 Hz,H-12),3.57 (1H,dd,J = 11.6,5.2 Hz,H-3),3.47 (1H,d,J = 11.2 Hz,H-23a),3.23 (1H,d,J = 11.2 Hz,H-23b),1.68 (3H,s,H-29),1.04 (3H,d,J = 8.5 Hz,H-30),0.97 (3H,s,H-25),0.94 (3H,s,H-27),0.89 (3H,s,H-26),0.65 (3H,s,H-24)；13C-NMR (125 MHz,CD3OD) δ: 40.1 (C-1),27.6 (C-2),73.8 (C-3),43.3 (C-4),49.1 (C-5),19.1 (C-6),35.3 (C-7),40.1 (C-8),49.3 (C-9),37.8 (C-10),24.1 (C-11),127.1 (C-12),140.0 (C-13),45.8 (C-14),29.3 (C-15),29.8 (C-16),49.6 (C-17),132.7 (C-18),136.8 (C-19),35.7 (C-20),27.6 (C-21),32.5 (C-22),67.2 (C-23),12.9 (C-24),17.0 (C-25),18.7 (C-26),22.3 (C-27),180.6 (C-28),19.8 (C-29),19.0 (C-30)。以上数据与文献报道一致[20, 21]，故鉴定化合物7为3β,23-二羟基-12,18-二烯-乌索酸。

化合物8：白色粉末。ESI-MS m/z: 487 [M－H]−。1H-NMR (500 MHz,C5D5N) δ: 5.48 (1H,t-like,H-12),4.23～4.30 (3H,m,H-2,3,23a),3.75 (1H,d,J = 10.5 Hz,H-23b),2.64 (1H,d,J = 11.5 Hz,H-18),1.15 (3H,s,H-24),1.09 (9H,s,H-25～27),0.98 (3H,d,J = 6.5 Hz,H-29),0.93 (3H,d,J = 6.0 Hz,H-30)；13C-NMR (125 MHz,C5D5N) δ: 48.3 (C-1),66.9 (C-2),78.6 (C-3),44.1 (C-4),48.4 (C-5),19.0 (C-6),33.6 (C-7),40.5 (C-8),48.5 (C-9),38.8 (C-10),24.2 (C-11),126.0 (C-12),139.9 (C-13),43.0 (C-14),29.1 (C-15),25.4 (C-16),48.6 (C-17),54.0 (C-18),39.9 (C-19),39.9 (C-20),31.6 (C-21),38.0 (C-22),69.4 (C-23),15.0 (C-24),18.0 (C-25),18.0 (C-26),24.3 (C-27),180.5 (C-28),18.1 (C-29),21.9 (C-30)。以上数据与文献报道一致[22]，故鉴定化合物8为积雪草酸。

化合物9：白色粉末。ESI-MS m/z: 657 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.39 (1H,d,J = 8.0 Hz,H-1′),5.53 (1H,t,J = 3.0 Hz,H-12),3.58～3.60 (2H,m,H-18,H-19),3.42 (1H,dd,J = 11.5,4.0 Hz,H-3),1.62 (3H,s,H-27),1.22 (3H,s,H-23),1.18 (3H,s,H-29),1.14 (3H,s,H-30),1.04 (3H,s,H-26),0.98 (3H,s,H-24),0.95 (3H,s,H-25)；13C-NMR (125 MHz,C5D5N) δ: 39.3 (C-1),28.6 (C-2),78.6 (C-3),39.9 (C-4),56.4 (C-5),19.5 (C-6),33.7 (C-7),40.7 (C-8),48.9 (C-9),38.0 (C-10),24.7 (C-11),123.5 (C-12),144.8 (C-13),42.6 (C-14),29.6 (C-15),28.5 (C-16),47.0 (C-17),45.1 (C-18),81.6 (C-19),36.0 (C-20),29.4 (C-21),33.5 (C-22),29.3 (C-23),17.0 (C-24),16.0 (C-25),18.1 (C-26),25.1 (C-27),177.7 (C-28),29.2 (C-29),25.4 (C-30),96.4 (C-1′),74.7 (C-2′),79.8 (C-3′),71.6 (C-4′),79.5 (C-5′),62.6 (C-6′)。以上数据与文献报道一致[23]，故鉴定化合物9为泰国树脂酸-28-O-β-D-葡萄糖酯苷。

化合物10：白色粉末。ESI-MS m/z: 673 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.41 (1H,d,J = 8.0 Hz,H-1′),5.54 (1H,t,J = 3.0 Hz,H-12),4.53 (1H,d,J = 10.5 Hz,H-24a),4.32 (1H,t,J = 9.0 Hz,H-3′),3.71 (1H,d,J = 10.5 Hz,H-24b),3.62 (2H,m,H-3,19),3.56 (1H,s,H-18),1.64 (3H,s,H-27),1.56 (3H,s,H-23),1.16 (6H,s,H-26,29),1.01 (3H,s,H-30),0.95 (3H,s,H-25)；13C-NMR (125 MHz,C5D5N) δ: 38.4 (C-1),28.2 (C-2),80.0 (C-3),42.9 (C-4),56.3 (C-5),19.1 (C-6),33.3 (C-7),40.0 (C-8),48.2 (C-9),37.1 (C-10),24.4 (C-11),122.9 (C-12),144.1 (C-13),41.8 (C-14),28.8 (C-15),27.8 (C-16),46.3 (C-17),44.4 (C-18),80.9 (C-19),35.4 (C-20),28.7 (C-21),32.8 (C-22),23.4 (C-23),64.3 (C-24),15.8 (C-25),17.3 (C-26),24.2 (C-27),177.0 (C-28),28.5 (C-29),24.7 (C-30),95.7 (C-1′),74.0 (C-2′),78.8 (C-3′),70.9 (C-4′),79.2 (C-5′),61.9 (C-6′)。以上数据与文献报道一致[24]，故鉴定化合物10为28-O-β-D-glucopyranoside spathodic acid。

化合物11：白色粉末。ESI-MS m/z: 657 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.34 (1H,d,J = 8.5 Hz,H-1′),5.60 (1H,t-like,H-12),3.45 (1H,dd,J = 12.0,4.5 Hz,H-3),2.97 (1H,s,H-18),1.72 (3H,s,H-27),1.44 (3H,s,H-29),1.25 (6H,s,H-23,H-26),1.09 (3H,d,J = 6.5 Hz,H-30),1.07 (3H,s,H-24),0.99 (3H,s,H-25)；13C-NMR (125 MHz,C5D5N) δ: 39.6 (C-1),28.6 (C-2),78.7 (C-3),39.9 (C-4),56.4 (C-5),19.5 (C-6),34.1 (C-7),41.1 (C-8),48.3 (C-9),38.2 (C-10),24.6 (C-11),128.9 (C-12),139.8 (C-13),42.6 (C-14),29.8 (C-15),26.6 (C-16),49.1 (C-17),55.0 (C-18),73.1 (C-19),42.2 (C-20),27.2 (C-21),37.9 (C-22),29.3 (C-23),17.0 (C-24),16.2 (C-25),17.9 (C-26),25.1 (C-27),177.4 (C-28),27.5 (C-29),17.2 (C-30),96.3 (C-1′),74.6 (C-2′),79.8 (C-3′),71.7 (C-4′),79.5 (C-5′),62.8 (C-6′)。以上数据与文献报道一致[16]，故鉴定化合物11为坡模酸-28-O-β-D-葡萄糖酯苷。

化合物12：白色粉末。ESI-MS m/z: 673 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.33 (1H,d,J = 8.5 Hz,H-1′),5.58 (1H,t-like,H-12),4.53 (1H,d,J = 10.0 Hz,H-24a),3.70 (1H,d,J = 10.0 Hz,H-24b),3.64 (1H,m,H-3),2.96 (1H,s,H-18),1.70 (3H,s,H-27),1.55 (3H,s,H-23),1.43 (3H,s,H-29),1.20 (3H,s,H-26),1.09 (3H,d,J = 6.5 Hz,H-30),0.96 (3H,s,H-25)；13C-NMR (125 MHz,C5D5N) δ: 39.3 (C-1),28.9 (C-2),80.8 (C-3),43.6 (C-4),56.9 (C-5),19.8 (C-6),34.3 (C-7),41.0 (C-8),48.4 (C-9),37.6 (C-10),24.8 (C-11),128.8 (C-12),139.8 (C-13),42.6 (C-14),29.7 (C-15),26.6 (C-16),49.1 (C-17),54.9 (C-18),73.1 (C-19),42.5 (C-20),27.2 (C-21),38.2 (C-22),24.1 (C-23),65.1 (C-24),16.6 (C-25),17.8 (C-26),25.0 (C-27),177.4 (C-28),27.5 (C-29),17.2 (C-30),96.3 (C-1′),74.6 (C-2′),79.8 (C-3′),71.7 (C-4′),79.5 (C-5′),62.8 (C-6′)。以上数据与文献报道一致[25]，故鉴定化合物12为rotungenoside。

化合物13：白色粉末。ESI-MS m/z: 655 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.36 (1H,d,J = 8.5 Hz,H-1′),5.78 (1H,t-like,H-12),4.20 (1H,m,H-3),4.01 (1H,d,J = 9.5 Hz,H-23a),3.74 (1H,d,J = 9.5 Hz,H-23b),1.83 (3H,s,H-29),1.22 (3H,s,H-25),1.08 (3H,s,H-27),1.07 (3H,s,H-26),1.05 (3H,s,H-24),1.04 (3H,d,J = 6.5 Hz,H-30)；13C-NMR (125 MHz,C5D5N) δ: 39.8 (C-1),27.2 (C-2),73.6 (C-3),43.4 (C-4),48.8 (C-5),19.2 (C-6),35.1 (C-7),39.9 (C-8),49.1 (C-9),37.5 (C-10),24.0 (C-11),127.2 (C-12),139.2 (C-13),45.4 (C-14),31.4 (C-15),29.6 (C-16),50.4 (C-17),134.4 (C-18),136.4 (C-19),35.8 (C-20),28.3 (C-21),35.3 (C-22),68.1 (C-23),13.8 (C-24),17.3 (C-25),18.9 (C-26),22.6 (C-27),175.2 (C-28),20.0 (C-29),19.2 (C-30),96.3 (C-1′),74.6 (C-2′),79.4 (C-3′),71.6 (C-4′),79.7 (C-5′),62.7 (C-6′)。以上数据与文献报道一致[21]，故鉴定化合物13为3β,23-二羟基-12,18-二烯-乌索酸-28-O-β-D-葡萄糖酯苷。

化合物14：白色粉末。ESI-MS m/z: 655 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.37 (1H,d,J = 8.5 Hz,H-1′),5.69 (1H,t-like,H-12),4.20 (1H,m,H-3),4.04 (1H,d,J = 9.5 Hz,H-23a),3.72 (1H,d,J = 9.5 Hz,H-23b),3.55 (1H,s,H-18),1.66 (3H,s,H-30),1.59 (3H,s,H-29),1.18 (3H,s,H-25),1.07 (3H,s,H-27),1.04 (3H,s,H-26),1.02 (3H,s,H-24)；13C-NMR (125 MHz,C5D5N) δ: 39.7 (C-1),28.3 (C-2),73.8 (C-3),43.4 (C-4),49.1 (C-5),19.0 (C-6),34.4 (C-7),40.3 (C-8),48.8 (C-9),37.6 (C-10),24.2 (C-11),128.2 (C-12),138.3 (C-13),44.2 (C-14),24.1 (C-15),29.1 (C-16),47.8 (C-17),50.9 (C-18),129.2 (C-19),124.3 (C-20),29.0 (C-21),33.4 (C-22),68.3 (C-23),13.7 (C-24),17.1 (C-25),18.7 (C-26),22.7 (C-27),176.7 (C-28),17.8 (C-29),20.8 (C-30),96.3 (C-1′),74.7 (C-2′),79.4 (C-3′),71.6 (C-4′),79.8 (C-5′),62.7 (C-6′)。以上数据与文献报道一致[21]，故鉴定化合物14为3β,23-二羟基-12,19-二烯-乌索酸- 28-O-β-D-葡萄糖酯苷。

化合物15：白色粉末。ESI-MS m/z: 655 [M＋Na]+。1H-NMR (500 MHz,C5D5N) δ: 6.35 (1H,d,J = 8.5 Hz,H-1′),5.58 (1H,t-like,H-12),5.21 (1H,brs,H-29a),5.06 (1H,brs,H-29b),4.20 (1H,m,H-3),3.82 (1H,brs,H-18),4.07 (1H,d,J = 10.0 Hz,H-23a),3.73 (1H,d,J = 10.0 Hz,H-23b),1.21 (3H,s,H-27),1.20 (3H,s,H-25),1.08 (3H,s,H-26),1.07 (3H,d,J = 6.5 Hz,H-30),1.03 (3H,s,H-24)；13C-NMR (125 MHz,C5D5N) δ: 39.4 (C-1),26.3 (C-2),73.9 (C-3),43.3 (C-4),49.2 (C-5),19.1 (C-6),33.7 (C-7),40.3 (C-8),48.6 (C-9),37.8 (C-10),24.4 (C-11),129.1 (C-12),138.0 (C-13),43.4 (C-14),28.2 (C-15),29.6 (C-16),50.3 (C-17),52.8 (C-18),153.9 (C-19),38.0 (C-20),31.1 (C-21),37.6 (C-22),68.3 (C-23),13.6 (C-24),16.7 (C-25),17.9 (C-26),26.7 (C-27),176.5 (C-28),110.9 (C-29),19.9 (C-30),96.4 (C-1′),74.6 (C-2′),79.4 (C-3′),71.6 (C-4′),79.9 (C-5′),62.7 (C-6′)。以上数据与文献报道一致[21]，故鉴定化合物15为3β,23-二羟基- 12,19(29)-二烯-乌索酸-28-O-β-D-葡萄糖酯苷。

4 讨论

本研究从四季青中分离得到15个化合物，其中包括8个三萜苷元和7个三萜皂苷类化合物，其中，化合物1～3为齐墩果酸型三萜；4～8为乌苏酸型三萜；9～10为齐墩果酸的三萜皂苷；11～15为乌苏酸的三萜皂苷，三萜皂苷均为28位羧基与葡萄糖结合成的酯苷。化合物1和5为23位降碳，24位成环外双键的三萜；化合物4为3和23位羟基与丙酮形成缩酮结构；化合物7、13～15为含两个双键的乌苏烷型三萜，其中化合物7、13为共轭双键，化合物15为29位成环外双键。

据文献报道化合物5对炎症因子的产生具有明显的抑制作用[26]；化合物6、8可以较好地消除巴豆油导致的小鼠耳肿胀[27]。此外，四季青中已报道的成分救必应酸[9]具有明显的抗菌和抗炎活性[28, 29]，原儿茶酸也具有较好的抗炎作用[30, 31]。因此，四季青的清热解毒、消肿祛瘀的传统功效可能与其所含的三萜和酚酸类活性成分有关，两类成分之间是否存在协同作用值得进一步深入研究。

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