药学学报  2016, Vol. 51 Issue (7): 1110-1116   PDF    
引用本文
YU Jin-qian, WANG Zhao-ping, ZHU Heng, LI Gang, WANG Xiao. Chemical constituents of Lonicera japonica roots and their anti-inflammatory effects[J]. Acta Pharm Sin, 2016, 51(7): 1110-1116.  
于金倩, 王召平, 朱姮, 李岗, 王晓. 忍冬根的化学成分及其抗炎作用[J]. 药学学报, 2016, 51(7): 1110-1116.  
忍冬根的化学成分及其抗炎作用
于金倩1, 王召平2, 朱姮2, 李岗2, 王晓1,2     
1. 山东省中药质量控制技术重点实验室, 山东省分析测试中心, 山东 济南 250014;
2. 山东中医药大学药学院, 山东 济南 250014
收稿日期: 2016-03-14; 修回日期: 2016-04-07.
基金项目: 国家自然科学基金面上资助项目（81473298）；山东省科技发展计划项目（2014GSF119031，2014GZX219003）.
* 通讯作者: Tel: 86-531-82605319, Fax: 86-531-82964889, E-mail: wangx@sdas.org
摘要: 为了研究忍冬（Lonicera japonica Thunb.）根的化学成分，采用硅胶、Sephadex LH-20等柱色谱和制备液相色谱等对其95%乙醇提取物进行分离纯化，共得到17个化合物，并通过质谱、核磁共振波谱等分别鉴定为1-oxo-（1H）-cyclopenta[b]benzofuran-7-carbaldehyde（1）、4-羟基桂皮酸（2）、绿原酸（3）、loganin aglycone（4）、咖啡酸（5）、裂环马钱素二甲基乙缩醛（6）、korolkoside（7）、松柏苷（8）、獐牙菜苷（9）、断氧化马钱子苷（10）、5-O-咖啡酰奎宁酸（11）、3-O-咖啡酰奎宁酸甲酯（12）、3-O-咖啡酰奎宁酸乙酯（13）、3,5-O-二咖啡酰奎宁酸（14）、4,5-O-二咖啡酰奎宁酸（15）、grandifloroside（16）和3,4-O-二咖啡酰奎宁酸（17）。其中化合物1为新化合物，化合物8为首次从该植物中分离得到，其他成分均为首次从忍冬根中分离得到。运用斑马鱼抗炎模型，测得化合物1、3、14~17具有抗炎活性。
关键词: 忍冬根     化学成分     结构鉴定     斑马鱼     抗炎    
Chemical constituents of Lonicera japonica roots and their anti-inflammatory effects
YU Jin-qian1, WANG Zhao-ping2, ZHU Heng2, LI Gang2, WANG Xiao1,2     
1. Shandong Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Jinan 250014, China;
2. College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250014, China
Abstract: To study the chemical composition and their anti-inflammatory activities of honeysuckle (Lonicera japonica Thunb.) roots, seventeen compounds were isolated from the roots of L. japonica Thunb. by various chromatography, including silica gel, Sephadex LH-20 and preparative HPLC. Their structures were identified by MS, IR, and nuclear magnetic resonance spectra, as 1-oxo-(1H)-cyclopenta[b]benzofuran-7-carbaldehyde (1), 4-hydroxycinnamic acid (2), chlorogenic acid (3), loganin aglycone (4), caffeic acid (5), secologanin dimethyl acetal (6), korolkoside (7), coniferin (8), sweroside (9), secoxyloganin (10), 5-O-caffeoylquinic acid (11), chlorogenic acid methyl ester (12), chlorogenic acid ethyl ester (13), 3, 5-O-dicaffeoylquinic acid (14), 4, 5-O-dicaffeoylquinic acid (15), grandifloroside (16), and 4, 5-O-dicaffeoylquinic acid (17). Among those, compound 1 is a new compound, and compound 8 is found in L. japonica for the first time. Compounds 1, 3, 14-17 showed significant anti-inflammatory activities against macrophage in zebrafish.
Key words: roots of Lonicera japonica     chemical constituent     identification     zebrafish     anti-inflammatory    

忍冬根为忍冬科忍冬属植物忍冬 (Lonicera japonica Thunb.) 的干燥根。金银花与忍冬藤是忍冬的常用入药部位,具有抗菌消炎[1, 2]、清热解毒、疏散风热的功效,临床上常用于治疗痈疮、喉痹、热血毒痢、风热感冒、温病发热等疾病[3]。金银花与忍冬藤作为常用中药材,在化学成分与药理方面已进行了系统性研究,但是忍冬根却缺少系统性研究,为了进一步开发利用忍冬的植物资源,扩大药用部位,故对忍冬根进行了系统的化学成分研究。

结果与讨论

本研究从忍冬根分离得到17个化合物,分别为1-oxo-(1H)-cyclopenta [b] benzofuran-7-carbaldehyde (1)、4-羟基桂皮酸 (4-hydroxycinnamic acid,2)、绿 原酸 (chlorogenic acid,3)、loganin aglycone (4)、咖啡酸 (caffeic acid,5)、裂环马钱素二甲基乙缩醛 (secologanin dimethyl acetal,6)、korolkoside (7)、 松 柏苷 (coniferin,8)、獐牙菜苷 (sweroside,9)、断氧 化马钱子苷 (secoxyloganin,10)、5-O-咖啡酰奎宁酸 (5-O-caffeoylquinic acid,11)、3-O-咖啡酰奎宁酸甲酯 (chlorogenic acid methyl ester,12)、3-O-咖啡酰奎宁 酸乙酯 (chlorogenic acid ethyl ester,13)、3,5-O-二咖啡酰奎宁酸 (3,5-O-dicaffeoylquinic acid,14)、4,5-O-二咖啡酰奎宁酸 (4,5-O-dicaffeoylquinic acid,15)、grandifloroside (16) 和3,4-O-二咖啡酰奎宁酸 (3,4- O-dicaffeoylquinic acid,17)。其中化合物1为新化合物 (图 1),化合物8为首次从该植物中分离得到,其他成分均为首次从忍冬根中分离得到。

Figure 1 Structure and key HMBC correlations (→) of compound 1

化合物1 棕色油状物,ESI-MS m/z 198.0,(-) HR-ESI-MS给出准分子离子峰197.030 5 [M-H]- (C12H6O3,计算值198.031 7),确定分子式为C12H6O3,不饱和度为10。红外光谱中1 605、1 516和1 447 cm-1说明化合物结构中含有苯环,1 681 cm-1说明化合物结构中含有羰基。

1H NMR (400 MHz,DMSO-d6) (表 1) 数据显示该化合物在低场区有1个醛基氢信号δH 9.69 (1H,s)、

Table 1 1H NMR (400 MHz) and 13C NMR (100 MHz) data of compound 1 (in DMSO-d6,J in Hz)

一组ABX自旋体系芳基质子信号δH 7.26 (dd,8.4,1.2)、7.23 (d,1.2) 和6.90 (d,8.4) 及2个邻位顺式偶合的烯烃质子信号δH 7.78 (d,8.4)、6.81 (d,8.4)。13C NMR结合HSQC图谱 显示12个碳信号: 1个醛基碳 (δC 191.3)、1个与双键偶合的羰基碳 (δC 168.1) 和10个芳香碳 (δC 161.8,153.2,146.5,131.9,129.1,124.9,122.6,116.0,115.5,114.8)。上述官能团中不饱和度为7,表明化合物1为三环结构。通过HSQC谱指认了所有与氢相连的碳信号。在HMBC谱中(图 1),醛基氢质子δH 9.69与δC 146.5 (C-7)、124.9 (C-6)、114.8 (C-8) 存在相关,推测存在苯甲醛的结构; δH 7.78 (H-3) 与δC 168.1 (C-1)、161.8 (C-3a)、122.6 (C-1a)、115.5 (C-2) 存在相关,δH 6.81 (H-2) 与δC 168.1 (C-1)、161.8 (C-3a)、122.6 (C-1a) 存在相关,推测存在环戊二烯 酮的结构; δH 7.26 (H-6) 与δC 191.3 (CHO)、153.2 (C-4a)、146.5 (C-7)、114.8 (C-8) 存在相关,δH 7.23 (H-8) 与δC 191.3 (CHO)、153.2 (C-4a)、146.5 (C-7)、124.9 (C-6)、122.6 (C-1a) 存在相关,δH 6.90 (H-5) 与δC 153.2 (C-4a)、146.5 (C-7)、129.1 (C-8a) 存在相关,确证苯甲醛与环戊二烯酮通过骈合的呋喃环连在一起。因此,确定化合物1结构式如图 1所示,命名为1-oxo-(1H)-cyclopenta[b]benzofuran-7-carbaldehyde。

采用斑马鱼抗炎模型测定了化合物1～5、9、10、12、14～17的抗炎活性。荧光显微镜下观察巨噬细胞炎症反应,计算炎症细胞数量,统计样品对炎症反应的影响,并观察胚胎死亡或畸形情况。结果表明,化合物1、3、16、17在100 μg·mL&# 8722;1浓度下表现出抗炎活性,化合物14和15在3个浓度下均表现出抗炎活性。实验结果见表 2。

Table 2 Effect of compounds against macrophage in zebrafish. Cell count of macrophage in the blank control: 3.43 ± 0.79; cell count of macrophage in the CuSO4 control: 17 ± 5.35; positive control: when the concentration of aspirin is 3.6 μg·mL-1,cell count of macrophage is 7.52 ± 1.88; the results are expressed in average value ± standard deviation,n = 2. P < 0.05 vs CuSO4 control
实验部分

薄层硅胶GF254、柱色谱硅胶 (200～300目,青岛海洋化工厂); 分析型色谱柱: YMC-PEAK ODS-A column (250 mm × 4.6 mm,i.d. 5 μm); 旋转蒸发器R-3 (瑞士BUCHI公司); 普源L-3000系列 (北京普源精电科技有限公司) 高效液相色谱仪; Waters 600型 (美国Waters公司) 高效液相色谱仪; Bruker-400核磁共振波谱仪 (瑞士布鲁克公司); Agilent 1200RRLC- 6410 QQQ-MS/MS质谱仪; Nicolet710傅里叶变换红外光谱仪; 斑马鱼养殖饲养设备 (北京爱生科技公司); Forma 3111型水套式CO2培养箱 (美国Forma公司); SZX16型荧光显微镜及DP2-BSW图像采集系统 (日本Olympus公司)。HPLC用甲醇、乙腈为色谱纯 (美国Tedia公司); HPLC用水为娃哈哈纯净水; CuSO4 (Sigma公司); JS7系巨噬细胞荧光转基因斑马鱼(山东省科学院生物研究所药物筛选研究室); 提取分离用试剂 (乙醇、石油醚、乙酸乙酯、甲醇、二氯甲烷等) 均为分析纯。

实验用忍冬根药材采购于山东省平邑县,经山东中医药大学李佳教授鉴定为忍冬科忍冬属植物忍冬 (Lonicera japonica Thunb.) 的干燥根,标本 (No. 20130910) 收藏于山东省分析测试中心标本室。

1 提取与分离

干燥忍冬根18 kg,粉碎,95% 乙醇回流提取3次,分别提取2、2和1 h,提取液合并、浓缩,得乙醇提取物; 依次用等体积石油醚、乙酸乙酯、正丁醇萃取4次,合并萃取液,分别合并每部分萃取液,减压浓缩干燥得乙酸乙酯浸膏 (460 g)。乙酸乙酯浸膏通过硅胶柱色谱(CH2Cl2-CH3OH,100∶0→0∶100) 分为B1-B13段。B4部分 (CH2Cl2-CH3OH,50∶1) 经逆流色谱,逆流体系为石油醚-乙酸乙酯-甲醇-水 (6∶4∶1∶9),得到化合物1 (10 mg) 和2 (9 mg),经硅胶柱色谱得化合物3 (30 mg) 和4 (9 mg)。B5部分 (CH2Cl2-CH3OH,50∶1) 经硅胶柱色谱、Sephadex LH-20得化合物5 (10 mg)。B6部分 (CH2Cl2-CH3OH,25∶1) 经中压高效制备液相得到化合物6 (13 mg)。B7部分 (CH2Cl2-CH3OH,15∶1) 经硅胶柱色谱、Sephadex LH-20得化合物7 (10 mg)。B8部分 (CH2Cl2- CH3OH,10∶1) 经硅胶柱色谱、Sephadex LH-20、高效制备液相色谱得化合物 8 (4 mg)、化合物9 (30 mg) 和化合物10 (47 mg)。B9部分 (CH2Cl2-CH3OH,10∶1) 经硅胶柱色谱、Sephadex LH-20、高效制备液相色谱得化合物11 (8 mg)、化合物12 (13 mg)、化合物13 (19 mg)、化合物14 (12 mg)、化合物15 (7 mg)、化合物16 (52 mg) 和化合物17 (4 mg)。

2 结构鉴定

化合物1,棕色油状物。(-) HR-ESI-MS给出准分子离子峰m/z 197.030 5 [M-H]- (C12H6O3计算值198.031 7)。红外光谱 (IR): 3 176、1 681、1 605、1 516和1 447 cm-1。NMR数据见表 1。

化合物2,淡黄色粉末,ESI-MS: m/z 163.8 [M- H]- (C9H8O3); 1H NMR (400 MHz,DMSO-d6) δ:7.52 (1H,d,J = 16.0 Hz,H-7),7.46 (2H,m,H-2,6),6.80 (2H,d,J = 8.0 Hz,H-3,5),6.31 (1H,d,J = 16.0 Hz,H-8); 13C NMR (100 MHz,DMSO-d6) δ: 125.8 (C-1),130.5 (C-2),166.2 (C-3),160.0 (C-4),116.1 (C-5),132.6 (C-6),144.4 (C-7),115.3 (C-8),168.6 (C-9)。该化合物波谱数据与文献[4]报道的4-羟基桂皮酸数据基本一致,故鉴定化合物为4-羟基桂皮酸。

化合物3,白色粉末,ESI-MS: m/z 353.3 [M-H]- (C16H18O9); 1H NMR (400 MHz,DMSO-d6) δ: 7.44 (1H,d,J = 15.0 Hz,H-7'),7.04 (1H,brs,H-2'),6.99 (1H,d,J = 7.8 Hz,H-6'),6.78 (1H,d,J = 8.4 Hz,H-5'),6.17 (1H,d,J = 15.0 Hz,H-8'),5.07 (1H,m,H-3),3.93 (1H,m,H-5),3.41 (1H,m,H-4),1.80～2.02 (4H,m,H-2,6); 13C NMR (100 MHz,DMSO-d6) δ: 73.5 (C-1),37.2 (C-2),68.0 (C-3),70.3 (C-4),70.3 (C-5),36.1 (C-6),174.5 (C-7),125.6 (C-1'),114.8 (C-2'),145.5 (C-3'),148.4 (C-4'),115.8 (C-5'),121.4 (C-6'),145.0 (C-7'),114.3 (C-8'),165.7 (C-9')。该化合物波谱数据与文献[5]报道的绿原酸数据基本一致,故鉴定化合物为绿原酸。

化合物4,白色粉末,ESI-MS: m/z 229.1 [M+H]+ (C11H16O5); 1H NMR (400 MHz,DMSO-d6) δ: 7.39 (1H,s,H-3),4.77 (1H,s,H-1),3.88 (1H,s,H-8a),2.96 (1H,d,J = 8.0 Hz,H-5),2.09 (1H,d,J = 8.0 Hz,H-7),1.71 (1H,s,H-8b),1.65 (1H,d,J = 8.0 Hz,H-9),1.34 (1H,s,H-6),1.01 (3H,d,J = 8.0 Hz,H-CH3); 13C NMR (100 MHz,DMSO-d6) δ: 96.6 (C-1),152.5 (C-3),111.2 (C-4),32.2 (C-5),42.5 (C-6),72.4 (C-7),41.7 (C-8),46.8 (C-9),167.7 (10-CO),51.3 (11-OCH3),14.5 (12-CH3)。该化合物波谱数据与文献[6]报道的loganin aglycone数据基本一致,故鉴定化合物为loganin aglycone。

化合物5,淡黄色粉末,ESI-MS: m/z 178.9 [M- H]- (C9H8O4); 1H NMR (400 MHz,DMSO-d6) δ: 7.44 (1H,d,J = 16.0 Hz,H-7),7.05 (1H,s,H-2),6.97 (1H,d,J = 8.0 Hz,H-6),6.79 (1H,d,J = 8.0 Hz,H-5),6.22 (1H,d,J = 16.0 Hz,H-8); 13C NMR (100 MHz,DMSO- d6)δ: 126.2 (C-1),115.1 (C-2),146.1 (C-3),148.6 (C-4),115.7 (C-5),121.6 (C-6),144.9 (C-7),116.3 (C-8),168.5 (C-9)。该化合物波谱数据与文献[7]报道的咖啡酸数据基本一致,故鉴定化合物为咖啡酸。

化合物6,白色无定形粉末,ESI-MS: m/z 457.3 [M+Na]+ (C19H30O11); 1H NMR (400 MHz,DMSO-d6) δ: 7.41 (1H,s,H-3),5.65 (1H,m,H-8),5.43 (1H,d,J = 4.0 Hz,H-1),5.35 (1H,d,J = 16.0 Hz,H-10b),5.26 (1H,d,J = 8.0 Hz,H-10a),4.53 (1H,d,J = 8.0 Hz,Glu-1),4.42 (1H,s,H-7),3.63 (3H,s,7-OCH3),3.46 (3H,s,7-OCH3),3.00 (1H,m,H-5),2.78 (1H,m,H-9),2. 01 (1H,m,H-6e),1.50 (1H,m,H-6a); 13C NMR (100 MHz,DMSO-d6) δ: 96.2 (C-1),151.9 (C-3),110.3 (C-4),28.2 (C-5),32.1 (C-6),102.6 (C-7),52.2 (7- OCH3),52.3 (7-OCH3),134.9 (C-8),43.6 (C-9),119.7 (C-10),167.1 (C-11),51.4 (11-OCH3),99.2 (Glu-1),73.5 (Glu-2),77.2 (Glu-3),70.5 (Glu-4),77.7 (Glu-5),61.6 (Glu-6)。该化合物波谱数据与文献[8]报道的裂环马钱素二甲基乙缩醛数据基本一致,故鉴定该化合物为裂环马钱素二甲基乙缩醛。

化合物7,淡黄色粉末,ESI-MS: m/z 827.3 [M+ Na]+ (C36H52O20)。1H NMR (400 MHz,DMSO-d6) δ: 7.47 (1H,s,H-3b),7.39 (1H,s,H-3a),5.60 (1H,m,H-8a),5.60 (1H,m,H-8b),5.44 (1H,d,J = 4.8 Hz,H- 1b),5.33～5.22 (2H,m,H-10a),5.28 (1H,d,J = 10.4 Hz,H-1a),4.66 (1H,d,J = 7.6 Hz,H-1'),4.60 (1H,m,H-7b),4.51 (1H,d,J = 7.6 Hz,H-1"),4.38 (1H,m,H-7a),4.06 (1H,m,H-6'),3.68 (1H,m,H-6"),3.64 (3H,s,H-12a),3.60 (3H,s,H-12b),3.43 (1H,m,H-6"),3.41 (1H,m,H-6'),3.36 (1H,m,H-2"),3.26 (1H,m,H-5'),3.18 (6H,s,H-13a,H-14a),3.16 (1H,m,H-4'),3.14 (2H,m,H-5",H-3"),3.04 (1H,m,H-3'),3.03 (1H,m,H-4"),2.95 (1H,m,H-2'),2.87 (1H,d,J = 4.8 Hz,H-6b),2.76 (1H,d,J = 6.0 Hz,H-5a),2.59 (2H,s,H-9),1.97 (1H,m,H-6a),1.94 (1H,s,H-6b); 13C NMR (100 MHz,DMSO-d6) δ: 167.1 (C-11a),167.0 (C-11b),152.5 (C-3b),151.9 (C-3a),134.9 (C-8a),134.8 (C-8b),119.9 (C-10a),119.8 (C-10b),110.3 (C-4a),110.1 (C-4b),102.5 (C-7a),100.8 (C-7b),100.0 (C-1'),99.1 (C-1"),96.8 (C-1a),96.1 (C-1b),80.3 (C-4'),77.8 (C-5"),77.2 (C-3"),74.3 (C-3'),73.5 (C-2'),73.4 (C-2"),70.5 (C-4"),67.9 (C-6'),66.6 (C-5'),61.6 (C-6"),53.3 (C-13a),52.2 (C-14a),51.6 (C-12a),51.5 (C-12b),43.6 (C-9a),43.6 (C-9b),34.3 (C-6b),32.2 (C-6a),28.4 (C-5b),28.1 (C-5a)。该化合物波谱数据与文献[9]报道的korolkoside数据基本一致,故鉴定该化合物为korolkoside。

化合物8,白色粉末,ESI-MS: m/z 341.2 [M-H]- (C16H22O8); 1H NMR (400 MHz,DMSO-d6) δ: 7.06 (1H,s,H-2),7.02 (1H,d,J = 5.6 Hz,H-5),6.88 (1H,d,J = 5.6 Hz,H-6),6.48 (1H,d,J = 10.4 Hz,H-7),6.28 (1H,m,H-8),4.88 (1H,d,J = 7.2 Hz,H-1'),3.78 (3H,s,10-OCH3),3.66 (1H,m,H-6'α),3.45 (1H,m,H-6'β),3.24 (2H,m,H-2',3'),3.16 (1H,m,H-4'); 13C NMR (100 MHz,DMSO-d6) δ: 130.8 (C-1),109.6 (C-2),148.9 (C-3),145.8 (C-4),115.0 (C-5),118.9 (C-6),128.3 (C-7),128.8 (C-8),61.5 (C-9),55.5 (10-OCH3),99.8 (C-1'),73.2 (C-2'),76.7 (C-3'),69.5 (C-4'),76.9 (C-5'),61.5 (C-6')。该化合物波谱数据与文献[10]报道的松柏苷数据基本一致,故鉴定化合物为松柏苷。

化合物9,淡黄色粉末,ESI-MS: m/z 357.3 [M- H]- (C16H22O9); 1H NMR (400 MHz,DMSO-d6) δ: 7.49 (1H,s,H-3),5.50 (1H,m,H-8),5.44 (1H,s,H-1),5.34 (1H,brs,H-10b),5.24 (1H,dd,J = 10.0,11.2 Hz,H- 10a),4.52 (1H,d,J = 7.2 Hz,H-1'),4.36 (1H,m,H-7b),4.28 (1H,m,H-7a),3.00 (1H,m,H-5),2.66 (1H,brs,H-9),1.74 (1H,d,J = 12.0 Hz,H-6b),1.50 (1H,d,J = 12.0 Hz,H-6a); 13C NMR (100 MHz,DMSO-d6) δ: 96.0 (C-1),151.9 (C-3),106.3 (C-4),27.2 (C-5),24.7 (C-6),68.1 (C-7),132.9 (C-8),41.9 (C-9),120.7 (C-10),165.1 (C-11),98.5 (C-1'),73.6 (C-2'),77.7 (C-3'),70.5 (C-4'),76.8 (C-5'),61.5 (C-6')。该化合物波谱数据与文献[11]报道的獐芽菜苷数据基本一致,故鉴定化合物为獐芽菜苷。

化合物10,淡黄色粉末,ESI-MS: m/z 405.1 [M+ H]+ (C17H24O11); 1H NMR (400 MHz,DMSO-d6) δ:7.44 (1H,s,H-3),5.46 (1H,m,H-8),5.39 (1H,brs,H-1),5.25 (1H,m,H-10a),5.20 (1H,m,H-10b),4.51 (1H,d,J = 8.0 Hz,H-1'),3.61 (3H,s,12-OCH3),3.04 (1H,d,J = 8.0 Hz,H-5),2.76 (1H,d,J = 16.4 Hz,H- 6a),2.72 (1H,m,H-9),2.12 (1H,m,H-6b); 13C NMR (100 MHz,DMSO-d6) δ: 95.9 (C-1),152.3 (C-3),108.8 (C-4),27.4 (C-5),34.3 (C-6),173.7 (C-7),133.8 (C-8),43.6 (C-9),120.3 (C-10),166.9 (C-11),51.4 (12-OCH3),99.1 (C-1'),73.5 (C-2'),77.2 (C-3'),70.5 (C-4'),77.8 (C-5'),61.5 (C-6')。该化合物数据与文献[12]报道的断氧化马钱子苷数据基本一致,故鉴定化合物为断氧化马钱子苷。

化合物11,白色粉末,ESI-MS: m/z 353.2 [M-H]- (C16H18O9); 1H NMR (400 MHz,DMSO-d6) δ: 7.45 (1H,d,J = 16.0 Hz,H-7'),7.06 (1H,brs,H-2'),6.98 (1H,brs,H-6'),6.78 (1H,brs,H-5'),6.19 (1H,d,J = 16.0 Hz,H-8'),5.09 (1H,m,H-3),3.93 (1H,m,H-5),3.55 (1H,brs,H-4),1.77～2.00 (4H,m,H-2,6); 13C NMR (100 MHz,DMSO-d6) δ:74.4 (C-1),37.9 (C-2),69.3 (C-3),71.5 (C-4),67.7 (C-5),37.5 (C-6),175.8 (C-7),126.1 (C-1'),115.3 (C-2'),146.1 (C-3'),148.7 (C-4'),116.3 (C-5'),121.8 (C-6'),145.4 (C-7'),114.8 (C-8'),166.4 (C-9')。该化合物波变数据与文献[13]报道的5-O-咖啡酰奎宁酸数据基本一致,故鉴定化合物为5-O-咖啡酰奎宁酸。

化合物12,淡黄色粉末,ESI-MS: m/z 367.0 [M- H]- (C17H20O9); 1H NMR (400 MHz,DMSO-d6) δ: 7.40 (1H,d,J = 16.0 Hz,H-7'),7.03 (1H,s,H-2'),6.98 (1H,d,J = 7.6 Hz,H-6'),6.78 (1H,d,J = 7.6 Hz,H-5'),6.12 (1H,d,J = 15.6 Hz,H-8'),5.01 (1H,m,H-3),3.89 (1H,d,J = 7.2 Hz,H-5),3.56 (4H,brs,H-4,H-8),2.11 (H,m,H-6a),2.09 (1H,brs,H-2a),1.94 (1H,m,H-6b),1.76 (1H,t,J = 11.2 Hz,H-2b); 13C NMR (100 MHz,DMSO-d6) δ: 73.5 (C-1),35.6 (C-2),71.5 (C-3),69.8 (C-4),67.3 (C-5),37.7 (C-6),174.1 (C-7),52.2 (C-8),125.7 (C-1'),115.0 (C-2'),146.1 (C-3'),149.1 (C-4'),116.3 (C-5'),121.8 (C-6'),145.6 (C-7'),114.2 (C-8'),165.9 (C-9')。该化合物波谱数据与文献[14]报道的3-O-咖啡酰奎宁酸甲酯数据基本一致,故鉴定化合物为3-O-咖啡酰奎宁酸甲酯。

化合物13,淡黄色粉末,ESI-MS: m/z 381.1 [M- H]- (C18H22O9); 1H NMR (400 MHz,DMSO-d6) δ: 7.02 (1H,s,H-2'),6.95 (1H,d,J = 7.6 Hz,H-6'),6.78 (1H,d,J = 7.6 Hz,H-5'),6.13 (1H,d,J = 15.6 Hz,H-8'),5.02 (1H,m,H-3),4.05 (2H,m,H-8),3.89 (1H,m,H-5),3.58 (1H,brs,H-4),2.11 (1H,m,H-6a),2.08 (1H,brs,H-2a),1.95 (1H,m,H-6b),1.77 (1H,t,J = 10.0 Hz ,H-2b),1.14 (3H,t,J = 6.8 Hz,H-9); 13C NMR (100 MHz,DMSO-d6) δ: 73.5 (C-1),35.7 (C-2),71.5 (C-3),70.0 (C-4),67.5 (C-5),37.7 (C-6),173.5 (C-7),60.8 (C-8),14.2 (C-9),125.8 (C-1'),115.0 (C-2'),146.1 ( C-3'),149.1 (C-4'),116.3 (C-5'),121.7 (C-6'),145.5 (C-7'),114.2 (C-8'),165.9 (C-9')。该化合物波谱数据与文献[15]报道的3-O-咖啡酰奎宁酸乙酯数据基本一致,故鉴定化合物为3-O-咖啡酰奎宁酸乙酯。

化合物14,白色粉末,ESI-MS: m/z 515.4 [M-H]- (C25H24012); 1H NMR (400 MHz,DMSO-d6) δ: 7.51 (1H,d,J = 14.8 Hz,H-7'),7.43 (1H,d,J = 14.8 Hz,H-7''),7.06 (2H,d,J = 5.6 Hz,H-2',2''),6.97 (2H,d,J = 6.4 Hz,H-6',6''),6.78 (2H,brs,H-5',5''),6.23 (1H,d,J = 15.8 Hz,H-8'),6.19 (1H,d,J = 15.8 Hz,H-8''),5.33 (1H,m,H-5),5.13 (1H,m,H-3),3.84 (1H,s,H-4),2.14 (1H,m,H-6α),1.99 (2H,m,H-2),1.91 (1H,m,H-6β); 13C NMR (100 MHz,DMSO-d6) δ: 72.9 (C-1),36.2 (C-2),71.1 (C-3),68.0 (C-4),71.4 (C-5),35.2 (C-6),175.8 (C-7),126.1 (C-1'),126.0 (C-1''),115.2 (C-2'),114.6 (C-2''),145.2 (C-3',3''),148.9 (C-4'),148.7 (C-4''),116.3 (C-5',5''),166.1 (C-9'),166.6 (C-9''),121.7 (C-6',6''),146.1 (C-7'),145.6 (C-7''),116.2 (C-8'),115.3 (C-8'')。该化合物波谱数据与文献[5]报道的3,5- O-二咖啡酰奎宁酸数据基本一致,故鉴定化合物为3,5-O-二咖啡酰奎宁酸。

化合物15,淡黄色粉末,ESI-MS: m/z 539.3 [M+ Na]+ (C25H24O12); 1H NMR (400 MHz,DMSO-d6) δ: 7.51 (1H,d,J = 16.0 Hz,H-7"),7.46 (1H,d,J = 16.0 Hz,H-7'),7.08 (2H,brs,H-2",2'),7.01 (2H,brs,H-6',6"),6.80 (2H,brs,H-5',5"),6.27 (1H,d,J = 16.0,H-8'),6.18 (1H,d,J = 16.0 Hz,H-8"),5.43 (1H,s,H-5),5.02 (1H,s,H-4),4.23 (1H,brs,H-3),1.94～2.19 (4H,m,H-2,6); 13C NMR (100 MHz,DMSO-d6) δ: 74.2 (C-1),38.0 (C-2),68.2 (C-3),73.9 (C-4),66.9 (C-5),37.8 (C-6),175.4 (C-7),125.9 (C-1',1"),115.3 (C-2",2'),146.0 (C-3',3"),148.9 (C-4',4"),116.3 (C-5'),116.3 (C-5"),121.8 (C-6'),121.9 (C-6"),146.1 (C-7',7"),165.1 (C-9'),166.5 (C-9"),114.1 (C-8'),114.4 (C-8")。该化合物波谱数据与文献[16]报道的4,5-O-二咖啡酰奎宁酸数据基本一致,故鉴定化合物为4,5-O-二咖啡酰奎宁酸。

化合物16,白色粉末,ESI-MS: m/z 537.4 [M- H]- (C25H30O13); 1H NMR (400 MHz,DMSO-d6) δ: 7.47 (1H,d,J = 16.0 Hz,H-β),7.43 (1H,s,H-3),7.04 (1H,s,H-2"),6.98 (1H,d,J = 6.4 Hz,H-6"),6.76 (1H,d,J = 8.0 Hz,H-5"),6.20 (1H,d,J = 16.0 Hz,H-α),5.72 ( 1H,m,H-8 ),5.45 (1H,d,J = 4.8 Hz,H-1),5.31 (1H,d,J = 17.6 Hz,H-10),5.23 (1H,d,J = 10.0 Hz,H-10),4.53 (1H,d,J = 8.0 Hz,H-1'),4.13 (1H,s,H-7),4.09 ( 1H,s H-7),3.81( 1H,s,H-6'),3.66 (1H,d,J = 12.0 Hz,H-6'),3.15～3.44 (H-2'-H-5'),2.76 (1H,d,J = 3.6 Hz,H-5),2.58 (1H,s,H-9),1.97 ( 1H,s,H-6),1.76 (1H,brs,H-6); 13C NMR (100 MHz,DMSO-d6) δ: 99.2 (C-1),151.8 (C-3),110.8 (C-4),30.5 (C-5),29.5 (C-6),61.6 (C-7),135.3 (C-8),43.7 (C-9),119.3 (C-10),166.9 (C-11),95.9 (C-1'),73.5 (C-2'),77.2 (C-3'),70.5 (C-4'),77.8 (C-5'),62.9 (C-6'),125.9 (C-1"),114.4 (C-2"),146.1 (C-3"),148.9 (C-4"),116.3 (C-5"),121.8 (C-6"),115.2 (C-α),145.5 (C-β),168.4 (C-CO)。该化合物波谱数据与文献[17]报道的grandifloroside数据基本一致,故鉴定为grandifloroside。

化合物17,白色粉末,ESI-MS: m/z 539.0 [M+ Na]+ (C25H24O12); 1H NMR (400 MHz,DMSO-d6) δ: 7.54 (1H,d,J = 15.2 Hz,H-7'),7.44 (1H,d,J = 15.2 Hz,H-7''),7.05 (2H,m,H-2',2''),7.00 (2H,m,H-6',6''),6.77 (2H,brs,H-5',5''),6.29 (1H,d,J = 15.6 Hz,H-8'),6.16 (1H,d,J = 15.6 Hz,H-8''),5.28 (1H,brs,H-3),4.98 (1H,brs,H-4),4.15 (1H,brs,H-5),2.24 (2H,m,H-6),1.90～2.02 (2H,m,H-2); 13C NMR (100 MHz,DMSO-d6) δ: 73.6 (C-1),38.2 (C-2),68.3 (C-3),72.5 (C-4),65.8 (C-5),36.2 (C-6),173.9 (C-7),125.9 (C-1'),125.8 (C-1''),115.4 (C-2'),115.2 (C-2''),146.0 (C-3'),146.1 (C-3''),149.1 (C-4'),149.2 (C-4''),116.3 (C-5',5''),121.9 (C-6'),121.8 (C-6''),146.1 (C-7'),146.2 (C-7''),114.3 (C-8''),113.7 (C-8'),165.7 (C-9'),166.4 (C-9'')。该化合物波谱数据与文献[18]报道的3,4-O-二咖啡酰奎宁酸数据基本一致,故鉴定化合物为3,4-O-二咖啡酰奎宁酸。

3 抗炎活性测定 3.1 斑马鱼抗炎模型的培养

JS7系巨噬细胞荧光转基因斑马鱼,雌雄斑马鱼分开喂养,照明14 h,黑暗10 h交替进行,以人工颗粒状饵料和刚孵出的卤虫无节幼体定时喂养。取健康性成熟的斑马鱼按雌雄比例1∶1放入交配缸内,次日9～10 h收集受精卵,取卵后对受精卵进行消毒和洗涤,最终将受精卵移至斑马鱼胚胎培养用水 (含5.0 mmol·L-1 NaCl、0.17 mmol·L-1 KCl、0.4 mmol·L-1 CaCl2、0.16 mmol·L-1 MgSO4) 中,28 ℃下控光培养。

3.2 不同化合物对转基因斑马鱼抗炎活性研究

受精卵发育3 dpf (days post fertilization) 时,在体视 显微镜下挑选正常的斑马鱼胚胎,移入6孔培养板 中,每孔20枚,每次每组两个重复孔,实验重复两次。分别加入不同浓度 (1、10和100 μg·mL-1) 的12个化合物溶液,加培养水至6.0 mL; 阴性对照组加入0.1% DMSO,加盖并分别将受试组与阴性对照组斑马鱼置于光照培养箱 (28 ℃) 让胚胎继续发育。CuSO4避光处理斑马鱼1 h后,在室温条件下利用4% 多聚甲醛 (PFA) 将各组斑马鱼固定。固定1 h后,清除4% PFA并使用磷酸盐缓冲液 (PBST) 清洗斑马鱼。另设空白对照组斑马鱼,此组斑马鱼发育3 dpf时,使用0.1% DMSO处理,随后置于光照培养箱 (28 ℃) 让胚胎继续发育。1 h后此组斑马鱼不添加20 μmol·L-1 CuSO4溶液,继续在28 ℃条件下避光培养。1 h后,利用4% PFA在室温条件下将其固定。固定1 h后去除PFA,并使用PBST清洗空白对照组斑马鱼。

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