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作者简介:

叶洪波(1997-),硕士研究生,主要从事中药民族药药效物质基础及活性评价,(E-mail)yehongbo020060@163.com。

通讯作者:

周英,博士,教授,主要从事中药、民族药化学成分及中药新药研究,(E-mail)yingzhou71@126.com。

中图分类号:Q946

文献标识码:A

文章编号:1000-3142(2024)02-0373-09

DOI:10.11931/guihaia.gxzw202208026

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目录contents

    摘要

    红凉伞(Ardisia crenata var. bicolor)为贵州苗族常用药,具有清喉利咽、消肿止痛、祛风除湿等功效。为研究红凉伞根化学成分及体外抗炎活性,该文采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、ODS反相柱色谱及半制备HPLC等方法对红凉伞根70%乙醇提取物进行系统性研究,结合NMR、MS等现代波谱技术分析及文献对比进行化合物结构鉴定。采用脂多糖(LPS)诱导的小鼠RAW 264.7巨噬细胞的NO生成模型,评价化合物的抗炎活性。结果表明:(1)从红凉伞根70%乙醇提取物中分离得到10个化合物,分别鉴定为11-O-没食子酰岩白菜素(1)、11-O-(4-O-甲基没食子酰)岩白菜素(2)、11-O-香草酰岩白菜素(3)、6-O-(4-羟基苯甲酰基)岩白菜素(4)、11-丁香酰岩白菜素(5)、11-O-(3′,4′-二甲基没食子酸)-岩白菜素(6)、去甲氧基岩白菜素(7)、micractinin A(8)、monomethyl olivetol(9)、邻苯二甲酸二丁酯(10),其中化合物489为首次从紫金牛属中分离得到。(2)体外抗炎活性实验结果表明,化合物1-4对小鼠RAW 264.7细胞的NO生成具有明显的抑制作用( P<0.01),在20 μmol·mL-1浓度下,NO抑制率分别为67.09%、66.50%、59.83%、36.47%。该研究结果丰富了红凉伞根的化学成分,明确了其部分抗炎活性物质基础,为其药材资源的开发利用提供了理论依据。

    Abstract

    Ardisia crenata var. bicolor is a common medicine used by Miao Minority in Guizhou, which has the effects of clearing throat and benefiting pharynx, reducing swelling and relieving pain, dispelling wind and dehumidifying. In order to study the chemical constituents and anti-inflammatory activities of A. crenata var. bicolor roots, the 70% ethanol extact was separated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, ODS reverse column chromatography and semi-preparative HPLC. The structures of the compounds were identified by spectral data of NMR, MS and published literatures. Using lipopolysaccharide(LPS)-activated RAW 264.7 cell line model in vitro, compounds 1-4 were evaluated for the inhibition against nitric oxide(NO) production. The results were as follows: (1) Ten compounds were isolated from the 70% ethanol extract and identified as 11-O-galloylbergenin (1), 11-O-(4-O-methylgalloyl) bergenin (2), 11-O-vanilloylbergenin (3), 6-O-(4-hydroxy benzoyl) bergenin (4), 11-syringylbergenin (5), 11-O-(3′,4′-dimethylgalloyl)-bergenin (6), demethoxybergenin (7), micractinin A (8), monomethyl olivetol (9), and dibutyl phthalate (10). Among them, compounds 4, 8, 9 were obtained from Ardisia for the first time. (2) The results of anti-inflammatory activities in vitro showed that compounds 1-4 could significantly inhibit NO release in RAW 264.7 cells (P<0.01), and the inhibition rates of compounds at the concentration of 20 μmol·mL-1 were 67.09%, 66.50%, 59.83%, 36.47%, respectively. This study enriches the chemical constituents of A. crenata var. bicolor roots, clarifies the material basis of its anti-inflammatory activities, verifies the scientificity of its traditional usage, and provides new insight and scientific evidence for its rational development and utilization of the medicinal resources.

  • 红凉伞(Ardisia crenata var. bicolor)为紫金牛属(Ardisia)植物朱砂根(A. crenata)的变种,主要分布于我国华东、中南、西南等地,以根入药,具有清喉利咽、消肿止痛、祛风除湿等功效(中国科学院北京植物研究所,1974;国家药典委员会,2020)。红凉伞作为贵州特色苗药八爪金龙三大基原植物之一,收载于2003年版《贵州省中药材、民族药材质量标准》,在贵州苗族地区具有悠久的药用历史(贵州省药品监督管理局,2003),常用于咽炎、慢性支气管炎等疾病的治疗。此外,《本草纲目》中记载的“苗高尺许,叶似冬青叶,背甚赤”“主治咽喉肿痹,磨水或醋咽之,甚良。”揭示了红凉伞在咽炎治疗中具有良好药用价值(童家赟等,2017)。

  • 现有研究表明,红凉伞根中含有三萜皂苷类、香豆素类、糖苷类及甾体类化学成分(章为,2007;邹萍等,2009;王雪,2011),具有较强的抗氧化、抗肿瘤等药理活性(胡志霞等,2020;韩金芮,2021)。基于红凉伞在贵州苗族地区的广泛使用及其良好的抗炎生物活性,为进一步阐明红凉伞根中的抗炎活性物质基础,为后续的开发与利用提供理论依据,本文在前期研究基础上(叶洪波等,2022;Ye et al.,2022),对其70%乙醇提取物进行化学成分研究,采用柱色谱、制备型高效液相等分离技术,从水饱和正丁醇和乙酸乙酯萃取部位分离鉴定得到10个化合物(化合物1-10的结构如图1所示),并采用脂多糖(lipopolysaccharide,LPS)诱导的小鼠RAW 264.7巨噬细胞炎症模型评价化合物的抗炎活性,结果发现岩白菜素类成分具有较好抗炎活性。本研究结果丰富了红凉伞根的抗炎活性物质基础,可为红凉伞根资源的综合开发与利用提供参考。

  • 1 材料与仪器

  • 红凉伞于2019年10月采集于贵州省雷公山地区,经贵州中医药大学药学院魏升华教授鉴定为紫金牛科紫金牛属植物红凉伞(Ardisia crenata var. bicolor)。凭证标本(No.20190815)保存于贵州中医药大学中药民族药重点实验室。

  • DRX-500 AVANCE III-600 MHz超导核磁共振仪、BRUKER AVANCE NEO-400 MHz超导核磁共振仪(德国Bruker公司);RID-20A示差折光检测器、Shimadzu LC-16D半制备型高效液相色谱(HPLC)仪(日本岛津公司);JEOL JMSHX110A型质谱(MS)仪(德国JEOL公司);AAPI 3200型质谱(MS)仪(美国Sciex公司);LCQ-DECA XP型LC-(ESI)MS质谱仪(美国Themo Finniga公司);RE-2000A旋转蒸发器(上海亚荣生化仪器厂);HERACELL150i/240i细胞培养箱(美国Themo Finniga公司)。C18反相柱填料ODS-A-HG(日本YMC公司);半制备柱( 250 mm ×10 mm, 5 μm,日本岛津公司);凝胶柱色谱填料(GE Healthcare Bio-Sciences,Uppsala,Sweden; LH-20);柱层析硅胶(80~100、200~300目,青岛海洋化工厂)。脂多糖(LPS,北京索莱宝科技有限公司);Cell Counting Kit-8(CCK8,中国biosharp公司);胎牛血清(fetal bovine serum,FBS)、 DEME 培养基(美国Gibco公司);NO检测试剂盒(南京建成生物工程研究所);地塞米松(dexamethasone,DEX,美国Sigma公司)。二氯甲烷、石油醚、乙酸乙酯、甲醇(均为天津市富宇精细化工有限公司生产的分析纯)。

  • 2 提取与分离

  • 取干燥红凉伞根15.8 kg,粉碎成粗粉,以液料比10∶1(V/V)的70%乙醇加热回流提取2次,每次2 h,合并过滤后的提取液,减压回收溶剂,得到红凉伞根浸膏5.1 kg。浸膏加适量蒸馏水混悬后依次用等体积石油醚、乙酸乙酯、水饱和正丁醇进行萃取,减压浓缩得到3个萃取部位。

  • 取350.0 g水饱和正丁醇萃取物,加入甲醇使其完全溶解,80目硅胶与样品溶液1∶1(m/m)拌匀,挥干溶剂备用。采用高于样品10倍量的200~300目硅胶,二氯甲烷湿法装柱,干法上样,以二氯甲烷-甲醇(1∶0~0∶1,V/V)进行梯度洗脱。减压回收溶剂,薄层硅胶板监视成分,合并相同组分,共得到8个流分(A-H)。流分D(15.4 g)经ODS柱色谱干法上样,以甲醇-水(1∶9~9∶1,V/V)进行梯度洗脱,正相薄层硅胶板监视,合并相同组分,得到9个流分(D1-D9)。流分D8经半制备型高效液相甲醇-水(40∶60,V/V)洗脱,得化合物1(20.0 mg)。

  • 取150.0 g乙酸乙酯层萃取物,加入乙酸乙酯使其完全溶解,80目硅胶与样品1∶1(m/m)混合均匀,挥干溶剂备用。200~300目硅胶与样品1∶10(m/m)湿法装柱,干法加入拌好样品的硅胶,以二氯甲烷-甲醇(1∶0~0∶1,V/V)梯度洗脱。回收溶剂,正相薄层硅胶板监视成分,合并相同的组分,得到7个流分(EA-EH)。流分EA(6.7 g)经ODS梯度洗脱后,经半制备型高效液相甲醇-水(79∶21,V/V)分离,得到化合物9(1.4 mg)。流分EB(1.7 g)经ODS梯度洗脱后,经半制备型高效液相甲醇-水(75∶25,V/V)分离,得到化合物8(0.6 mg)。流分ED(10.0 g)上清液经ODS反向柱层析梯度洗脱,回收流动相后正相薄层板监视成分,得到6个流分(ED1-ED6)。ED4经半制备型高效液相甲醇-水(42∶58,V/V)分离,得化合物2(2.4 mg);ED5经硅胶柱色谱二氯甲烷-甲醇(15∶1~5∶1,V/V)梯度洗脱,得到化合物3(1.5 mg)、4(1.2 mg);ED6经硅胶柱色谱二氯甲烷-甲醇(15∶1~5∶1,V/V)梯度洗脱,半制备型高效液相甲醇-水(56∶44,V/V)分离,得到化合物5(2.5 mg)和化合物6(4.5 mg)。流分EE(5.9 g)上清液经ODS反向色谱柱层析梯度洗脱,回收流动相后正相薄层板监视成分,合并相同的组分,得到6个流分(EE1-EE6)。EE2采用硅胶色谱柱和半制备型高效液相甲醇-水(40∶60,V/V)分离,得到化合物7(3.5 mg);EE6经半制备型高效液相甲醇-水(89∶11,V/V)分离,得到化合物10(5.0 mg)。

  • 3 结构鉴定

  • 化合物1  无色针晶。ESI-MS m/z: 481 [M+H]+,分子式为C21H20O131H-NMR(400 MHz,C5D5N)δ: 7.92(2H,s,H-2′,6′),7.79(1H,s,H-7),5.34(1H,dd,J = 12.1,2.0 Hz,H-10b),5.27(1H,d,J = 10.3 Hz,H-4a),4.81(1H,dd,J = 12.1,6.7 Hz,H-4),4.58(1H,dd, J = 10.3,9.3 Hz,H-11),4.49(1H,dd,J = 9.3,8.3 Hz,H-11),4.38(1H,ddd,J = 9.0,6.6,2.1 Hz,H-3),4.19(1H,dd,J = 9.9,8.3 Hz,H-2),3.97(3H,s,9-OCH3);13C-NMR(100 MHz,C5D5N)δ: 167.3(C-7′),164.4(C-6),152.8(C-8),149.4(C-10),147.7(C-3′,5′),142.1(C-9),141.4(C-4′),120.7(C-1′),119.5(C-6a),116.4(C-10a),111.3(C-7),110.4(C-2′,6′),81.1(C-4a),80.6(C-2),75.3(C-4),74.2(C-10b),71.6(C-3),64.5(C-11),60.3(9-OCH3)。以上数据与文献(叶海亚等,1996)报道基本一致,故鉴定化合物1为11-O-没食子酰岩白菜素(11-O-galloylbergenin)。

  • 化合物2  白色固体粉末。ESI-MS m/z: 493 [M-H]-,分子式为C22H22O131H-NMR(400 MHz,C5D5N)δ: 7.79(2H,s,H-2′,6′),7.78(1H,s,H-7),5.34(1H,dd,J = 12.1,2.1 Hz,H-11),5.29(1H,d,J = 10.3 Hz,H-10b),4.84(1H,dd,J = 12.1,6.5 Hz,H-11),4.54~4.61(1H,m,H-4a),4.49(1H,t,J = 8.8 Hz,H-4),4.39(1H,ddd,J = 8.9,6.5,2.1 Hz,H-2),4.19(1H,dd,J = 10.0,8.3 Hz,H-3),4.03(3H,s,4′-OCH3),3.96(3H,s,9-OCH3); 13C-NMR(100 MHz,C5D5N)δ: 167.0(C-7′),164.4(C-6),152.9(C-8),152.5(C-3′,5′),149.4(C-10),142.1(C-9),141.8(C-4′),126.1(C-1′),119.6(C-6a),116.4(C-10a),111.4(C-7),110.3(C-2′,6′),81.2(C-4a),80.5(C-2),75.4(C-4),74.3(C-10b),71.6(C-3),64.8(C-11),60.3(4′-OCH3),60.2(9-OCH3)。以上数据与文献(左国营等,2007)报道基本一致,故鉴定化合物2为11-O-(4-O-甲基没食子酰)岩白菜素[11-O-(4-O-methylgalloyl)bergenin)]。

  • 化合物3 白色固体粉末。ESI-MS m/z: 477 [M-H]-,分子式为C22H22O121H-NMR(400 MHz,CD3OD)δ: 7.60(1H,dd,J = 8.5,2.1 Hz,H-6′),7.48(1H,d,J = 2.1 Hz,H-2′),7.08(1H,s,H-7),6.99(1H,d,J = 8.5 Hz,H-5′),5.03(1H,d,J = 10.4 Hz,H-10b),4.90(1H,m,H-11),4.38(1H,dd,J = 12.2,6.9 Hz,H-11),4.10(1H,dd,J = 10.5,9.4 Hz,H-4a),3.96(1H,ddd,J = 9.4,7.0,2.0 Hz,H-2),3.91(3H,s,3′-OCH3),3.88(3H,s,9-OCH3),3.84(1H,t,J = 9.0 Hz,H-4),3.54(1H,dd,J = 10.0,8.6 Hz,H-3); 13C-NMR(100 MHz,CD3OD)δ: 167.8(C-7′),165.7(C-6),153.7(C-4′),152.4(C-8),149.4(C-10),147.6(C-3′),142.3(C-9),123.7(C-6′),123.5(C-1′),119.4(C-6a),117.2(C-10a),117.0(C-5′),111.9(C-2′),111.2(C-7),81.3(C-4a),80.6(C-2),75.5(C-4),74.4(C-10b),71.9(C-3),64.9(C-11),60.9(9-OCH3),56.4(4′-OCH3)。以上数据与文献(Jia et al.,1995)报道基本一致,故鉴定化合物3为11-O-香草酰岩白菜素(11-O-vanilloylbergenin)。

  • 化合物4  黄色固体。ESI-MS m/z: 447 [M-H]-,分子式为C21H20O111H-NMR(400 MHz,C5D5N)δ: 8.27(2H,d,J = 8.5 Hz,H-2′,6′),7.77(1H,s,H-7),7.15(2H,d,J = 8.4 Hz,H-3′,5′),5.44(1H,dd,J = 12.0,2.1 Hz,H-11),5.34(1H,d,J = 10.4 Hz,H-10b),4.83(1H,dd,J = 12.0,7.0 Hz,H-11),4.68(1H,dd,J = 10.1,9.6 Hz,H-4a),4.52(1H,t,J = 8.8 Hz,H-4),4.45(1H,ddd,J = 9.4,6.9,2.1 Hz,H-2),4.22(1H,dd,J = 9.9,8.4 Hz,H-3),3.97(3H,s,9-OCH3); 13C-NMR(100 MHz,C5D5N)δ: 167.0(C-7′),164.7(C-6),164.2(C-4′),153.1(C-8),149.7(C-10),142.4(C-9),132.9(C-2′,6′),121.5(C-1′),119.9(C-6a),116.7(C-10a),116.6(C-3′,5′),111.6(C-7),81.5(C-4a),80.9(C-2),75.7(C-4),74.5(C-10b),72.0(C-3),64.9(C-11),60.6(9-OCH3)。以上数据与文献(Lin et al.,2012)报道基本一致,故鉴定化合物4为6-O-(4-羟基苯甲酰)岩白菜素[6-O-(4-hydroxy benzoyl)bergenin)]。

  • 化合物5  白色固体粉末。ESI-MS m/z: 531 [M+Na]+,分子式为C23H24O131H-NMR(400 MHz,C5D5N)δ: 7.79(2H,s,H-2′,6′),7.78(1H,s,H-7),5.33(1H,d,J = 10.4 Hz,H-10b),4.90(1H,m,H-11),4.70(1H,dd,J = 11.8,8.4 Hz,H-11),4.69(1H,dd,J = 10.4,9.3 Hz,H-4a),4.52(1H,t,J = 8.7 Hz,H-4),4.50(1H,ddd,J = 9.7,7.1,2.3 Hz,H-2),4.18(1H,t,J = 9.0 Hz,H-3),3.99(3H,s,9-OCH3),3.89(6H,s,3′,5′-OCH3);13C-NMR(100 MHz,C5D5N)δ: 167.0(C-7′),164.6(C-6),153.3(C-8),149.8(C-3′,5′),149.2(C-10),143.5(C-4′),142.4(C-9),120.1(C-1′),120.0(C-6a),116.6(C-10a),111.7(C-7),108.5(C-2′,6′),81.4(C-4a),80.7(C-2),75.7(C-4),74.4(C-10b),72.6(C-3),65.5(C-11),60.6(9-OCH3),56.7(3′,5′-OCH3)。以上数据与文献(张翠仙等,2010)报道基本一致,故鉴定化合物5为11-丁香酰岩白菜素(11-syringyl-bergenin)。

  • 化合物6  白色固体粉末。ESI-MS m/z: 507 [M-H]-,分子式为C23H24O131H-NMR(400 MHz,C5D5N)δ: 7.90(1H,d,J = 2.0 Hz,H-6′),7.78(1H,s,H-7),7.55(1H,d,J = 2.0 Hz,H-2′),5.51(1H,dd,J = 12.0,2.2 Hz,H-11),5.33(1H,d,J = 10.4 Hz,H-10b),4.77(1H,dd,J = 12.0,7.6 Hz,H-11),4.66(1H,ddd,J = 9.8,7.6,2.2 Hz,H-4a),4.51(1H,t,J = 8.9 Hz,H-4),4.46(1H,ddd,J = 9.8,7.6,2.2 Hz,H-2),4.19(1H,dd,J = 9.9,8.4 Hz,H-3),3.99(3H,s,3′-OCH3),3.92(3H,s,4′-OCH3),3.86(3H,s,9-OCH3);13C-NMR(100 MHz,C5D5N)δ: 166.9(C-7′),164.7(C-6),154.3(C-4′),153.2(C-8),152.6(C-3′),149.7(C-10),142.5(C-5′),142.4(C-9),126.1(C-1′),119.9(C-6a),116.6(C-10a),112.9(C-2′),111.7(C-7),105.7(C-6′),81.4(C-4a),80.6(C-2),75.7(C-4),74.4(C-10b),72.2(C-3),65.4(C-11),60.7(4′-OCH3),60.6(9-OCH3),56.4(3′-OCH3)。以上数据与文献(Jia et al.,1995)报道基本一致,故鉴定化合物6为11-O-(3′,4′-二甲基没食子酸)-岩白菜素[11-O-(3′,4′-dimethylgalloyl)-bergenin)]。

  • 化合物7  白色固体粉末。HR-FAB-MS m/z: 299.075 1 [M+H]+(Calcd. for C13H15O8: 299.076 7),分子式为C13H14O81H-NMR(400 MHz,CD3OD)δ: 6.99(1H,d,J = 2.4 Hz,H-7),6.53(1H,d,J = 2.4 Hz,H-9),4.93(1H,d,J = 10.4 Hz,H-10b),4.05(1H,dd,J = 10.2,9.2 Hz,H-4a),4.01(1H,d,J = 10.0 Hz,H-11),3.81(1H,t,J = 9.0 Hz,H-4),3.69(1H,m,H-11),3.65(1H,m,H-2),3.42(1H,t,J = 9.1 Hz,H-3);13C-NMR(100 MHz,CD3OD)δ: 165.8(C-6),160.3(C-8),157.4(C-10),126.1(C-6a),115.7(C-10a),110.3(C-9),110.0(C-7),83.1(C-2),81.4(C-4a),75.6(C-4),74.3(C-10b),71.9(C-3),62.7(C-11)。以上数据与文献(Megumi et al.,2020)报道基本一致,故鉴定化合物7为去甲氧基岩白菜素(demethoxybergenin)。

  • 化合物8  白色固体粉末。HR-FAB-MS m/z: 649.341 4 [M+Na]+,(Calcd. for C29H54O14Na: 649.341 1),分子式为C29H54O141H-NMR(400 MHz,CD3OD)δ: 4.87(1H,m,H-1′),4.25(1H,d,J = 7.2 Hz,H-1″),4.16(1H,d,J = 1.8 Hz,H-1),4.14(1H,d,J = 2.8 Hz,H-1),3.99(1H,p,J = 5.1 Hz,H-2),3.89(3H,m,H-2′,3′,4″),3.87(1H,m,H-3),3.85(1H,d,J = 5.2 Hz,H-2″),3.78(1H,d,J = 3.5 Hz,H-4′),3.75(1H,d,J = 3.1 Hz,H-5′),3.72(1H,t,J = 2.4 Hz,H-5″),3.71(1H,d,J = 2.0 Hz,H-6″),3.68(1H,m,H-6′),3.67(1H,d,J = 4.6 Hz,H-3),3.52(1H,m,H-3″),2.36(2H,t,J = 7.5 Hz,H-2′′′),1.62(2H,t,J = 7.4 Hz,H-3′′′),1.26~1.31(16H,m,H-4′′′~13′′′),0.90(3H,t,J = 7.0 Hz,H-14′′′); 13C-NMR(100 MHz,CD3OD)δ: 175.5(C-1′′′),105.3(C-1″),100.5(C-1′),74.7(C-5″),74.6(C-3″),72.5(C-2″),72.1(C-3),71.5(C-4″),71.1(C-4′),70.2(C-5′),70.1(C-2),69.7(C-3′),67.8(C-6′),66.6(C-1),62.7(C-6″),34.9(C-2′′′),33.1,30.8,30.6,30.5,30.2,23.7(C-4′′′~13′′′),14.5(C-14′′′)。以上数据与文献(Tran et al.,2020)报道基本一致,故鉴定化合物8为micractinin A。

  • 化合物9  黄色油状物。1H-NMR(600 MHz,CD3OD)δ: 6.23(1H,dd,J = 2.2,1.4 Hz,H-6),6.22(1H,dd,J = 2.2,1.4 Hz,H-2),6.18(1H,t,J = 2.2 Hz,H-4),3.73(3H,s,3-OCH3),2.49(2H,t,J = 5.2 Hz,H-1′),1.59(2H,p,J = 7.6 Hz,H-2′),1.33(4H,m,H-3′,4′),0.91(3H,t,J = 7.1 Hz,H-5′); 13C-NMR(150 MHz,CD3OD)δ: 162.3(C-3),159.4(C-5),146.4(C-1),108.9(C-6),106.5(C-2),99.6(C-4),55.5(3-OCH3),37.1(C-1′),32.6(C-2′),32.2(C-3′),23.6(C-4′),14.4(C-5′)。以上数据与文献(Mcclanahan &Robertson,2004)报道基本一致,故鉴定化合物9为monomethyl olivetol。

  • 化合物10  黄色油状物。ESI-MS m/z: 279 [M+H]+,分子式为C16H22O41H-NMR(600 MHz,CD3OD)δ: 7.73(2H,dd,J = 5.7,3.3 Hz,H-3,6),7.62(2H,dd,J = 5.7,3.3 Hz,H-4,5),4.30(4H,t,J = 6.7 Hz,H-1′,1″),1.73(4H,m,H-2′,2″),1.47(4H,m,H-3′,3″),0.99(6H,t,J = 7.4 Hz,H-4′,4″); 13C-NMR(150 MHz,CD3OD)δ: 169.3(C-7,8),133.6(C-1,2),132.3(C-4,5),129.9(C-3,6),66.7(C-1′,1″),31.7(C-2′,2″),20.3(C-3′,3″),14.0(C-4′,4″)。以上数据与文献(赵邯涛等,2022)报道基本一致,故鉴定化合物10为邻苯二甲酸二丁酯(dibutyl phthalate)。

  • 图1 化合物1-10的结构

  • Fig.1 Structures of compounds 1-10

  • 4 抗炎活性测定

  • 采用Cell Counting Kit-8(CCK8)试剂盒检测药物对小鼠巨噬细胞RAW 264.7的细胞毒作用,采用Griess法检测细胞NO的释放量(于玲玲等,2021;王研等,2022)。

  • 4.1 CCK8试验

  • 取对数生长期的RAW 264.7细胞,调整细胞密度至每毫升1×105个,接种于96孔板中(每孔100 μL),设置给药组和空白对照组。在37℃、5% CO2的恒温细胞培养箱中培养24 h,按照CCK8试剂盒说明书进行后续实验操作,于450 nm处测定光密度(optical density,OD)。细胞活力评价公式:细胞存活率(%)=(OD给药组/OD空白对照组)×100。用GraphPad Prism 8软件处理实验数据,结果如图2所示。由图2可知,在20 μmol·mL-1浓度下,化合物1-4的细胞存活率均在90%以上。因此,化合物对小鼠RAW 264.7细胞无细胞毒作用。

  • 4.2 NO释放量试验

  • 取对数生长期的RAW 264.7细胞,接种于96孔板中,加入脂多糖(LPS)诱导巨噬细胞形成炎症模型,同时加入20 μmol·mL-1的待测化合物,试验还设置空白对照组、模型组及阳性对照组(地塞米松),每组3个复孔。在37℃、5% CO2恒温培养箱中培养24 h。按照NO试剂盒说明书操作,于540 nm处测定OD值。NO抑制率计算公式:RI(%)=[(CLPS组-C给药组)/(CLPS组-C空白对照组)]×100(其中C表示NO含量)。

  • 图2 化合物1-4对小鼠RAW 264.7细胞存活率的影响(n=3)

  • Fig.2 Effects of compounds 1-4 on cell viability of mouse RAW 264.7 cells (n=3)

  • 数据采用GraphPad Prism 8统计分析软件进行单因素方差分析(one-way ANOVA)和方差齐性检验进行组间比较,数据以均值±标准差(x-±s)表示,P<0.01表示有显著性,P<0.05表示有统计学意义,结果如表1和图3所示。

  • 与空白对照组相比,模型组细胞NO含量显著增加(P<0.01),表明造模成功。与模型组相比,在20 μmol·mL-1浓度下,给药组所有化合物对RAW 264.7小鼠巨噬细胞NO释放均具有一定的抑制作用,其中化合物4抑制率最小,为36.47%,化合物1抑制率最大,为67.09%。

  • 5 讨论与结论

  • 本研究从红凉伞根70%乙醇提取部位分离鉴定10个单体化合物,包括7个岩白菜素类衍生物(化合物1-7)和3个其他化合物(化合物8-10),其中化合物489为首次从紫金牛属中分离得到。

  • 表1 化合物1-4对NO释放抑制率的影响(n=3)

  • Table1 Effects of compounds 1-4 on inhibition ratio of NO production (n=3)

  • 注:所有的化合物均在20 μmol·mL-1浓度下进行试验; DEX. 地塞米松。

  • Note: All compounds are tested at the concentration of 20 μmol·mL-1; DEX. Dexamethasone.

  • 图3 化合物1-4对NO释放抑制率的影响 (n=3)

  • Fig.3 Effects of compounds 1-4 on inhibition ratio of NO production (n=3)

  • 炎症是机体在受到机体内外物理、化学等刺激时作出的生理反应,通常表现为红、肿、热、痛等症状。适当的炎症反应有助于机体抵御损伤,促进伤口愈合,但过度炎症反应会对人体正常组织器官造成损伤且可能促进某些疾病的发生与发展(李丽等,2023;刘雅清等,2023)。NO在炎症级联反应,特别是炎症反应的发生和信号传导方面起到关键调节作用(羊波等,2016)。有研究发现,岩白菜素通过影响细胞炎症因子的释放、生物酶活性及相关炎症通路蛋白的表达来起到良好的抗炎作用(de Oliveira et al.,2019;郑胜眉等,2020;蓝颖等,2021)。

  • 本研究采用LPS诱导RAW 264.7细胞产生NO为评价模型,筛选了化合物1-4对NO生成的抑制作用。本研究结果显示,化合物1-4对LPS诱导RAW 264.7细胞产生NO均具有不同程度的抑制作用,其中化合物1-3抑制作用较强,20 μmol·mL-1浓度下NO抑制率分别为69.81%、67.09%和66.50%,与40 μg·mL-1浓度下DEX抑制率相当。结合构效关系分析,岩白菜素类化合物苯环上3′,4′,5′位羟基或甲氧基的取代可能是其发挥抗炎作用的关键官能团。岩白菜素及其衍生物已被报道具有较强的抗炎、抗艾滋病毒、抗糖尿病等生物活性(Piacente et al.,1996;Zhang &Lee,2003;Li et al.,2005;张东东等,2020;周金娜等,2020),其中良好的抗炎活性,在本研究中得到进一步证实,其抗炎机制可能与减少相关炎症因子的释放、降低炎症通路蛋白的表达有关。综上所述,红凉伞根化学成分的良好抗炎活性为其作为咽喉疾病用药提供科学依据。本研究结果丰富了红凉伞的化学成分,明确了其具有抗炎效果的物质基础,验证了其在贵州苗族地区用法的科学性,为药材资源的进一步开发与利用提供了重要依据。

  • 参考文献

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    • JIA ZH, MITSUNAGA K, KOIKE K, et al. , 1995. New bergenin derivatives from Ardisia crenata [J]. Nat Med, 49(2): 187-189.

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    • LIU YQ, LIU B, MA XR, et al. , 2023. Anti-inflammatory effect of Huangqintang on LPS-induced RAW264. 7 inflammatory cells [J]. Chin J Exp Trad Med Form, 29(7): 20-28. [刘雅清, 刘滨, 马旭冉, 等, 2023. 黄芩汤对LPS诱导的RAW264. 7炎症细胞的抗炎作用 [J]. 中国实验方剂学杂志, 29(7): 20-28. ]

    • LI YF, HU LH, LOU FC, et al. , 2005. PTP1B inhibitors from Ardisia japonica [J]. J Asian Nat Prod Res, 7(1): 13-18.

    • MCCLANAHAN RH, ROBERTSON LW, 2004. Microbial transformation of olivetol by Fusarium roseum [J]. J Nat Prod, 48(4): 660-663.

    • MEGUMI S, TOSHIKAZU S, NIJSIRI R, et al. , 2002. Ardisiphenols and other antioxidant principles from the fruits of Ardisia colorata [J]. Chem Pharm Bull, 50(11): 1484-1487.

    • PIACENTE S, PIZZA C, DETOMMASI N, et al. , 1996. Constituents of Ardisia japonica and their in vitro anti-HIV activity [J]. J Nat Prod, 59(6): 565-569.

    • TONG JY, LIANG ZT, ZHAO ZZ, et al. , 2017. Investigation of genus Ardisia in bencao literature [J]. Chin J Chin Mat Med, 42(2): 396-404. [童家赟, 梁之桃, 赵中振, 等, 2017. 本草著作中紫金牛属药物基原考 [J]. 中国中药杂志, 42(2): 396-404. ]

    • TRAN H, YOUN UJ, KIM JA, et al. , 2020. Glycerols and fatty acids isolated from Micractinium sp. KSF0031 [J]. Biochem Syst Ecol, 89(1): 1-4.

    • WANG X, 2011. Study on the chemical constituents of antitumor metastasis from Ardisia crenata [D]. Tianjin: Tianjin Medical University: 6-31. [王雪, 2011. 红凉伞抗肿瘤转移化学成分研究 [D]. 天津: 天津医科大学: 6-31. ]

    • WANG Y, LIANG XB, ZHAO ZZ, 2022. Diterpenoids from whole herb of Euphorbia helioscopia [J]. Chin Tradit Herb Drugs, 53(15): 4625-4633. [王妍, 梁旭博, 赵珍珠, 2022. 泽漆全草中二萜类成分研究 [J]. 中草药, 53(15): 4625-4633. ]

    • YANG B, YING Y, CHEN LL, et al. , 2016. Advance in studies on anti-inflammatory mechanism of flavonoids [J]. Chin Pharm, 19(7): 1369-1373. [羊波, 应茵, 陈苓丽, 等, 2016. 黄酮类化合物抗炎作用机制研究进展 [J]. 中国药师, 19(7): 1369-1373. ]

    • YE HB, WEI X, YIN X, et al. , 2022. Two new phenol compounds from roots of Ardisia crenata [J]. Nat Prod Res, 15: 1-4.

    • YE HB, ZHOU YQ, LIAO ZR, et al. , 2022. Isolation and identification of triterpenoids from the roots of Ardisia crenata var. bicolor [J]. J Chin Med Mat, 45(2): 346-350. [叶洪波, 周永强, 廖张蓉, 等, 2022. 红凉伞根三萜类化学成分分离与鉴定 [J]. 中药材, 45(2): 346-350. ]

    • YE HY, CHEN CX, HAO XJ, 1996. The chemical constituents from Ostryopsis nobilis [J]. Acta Bot Yunnan, 18(4): 473-475. [叶海亚, 陈昌祥, 郝小江, 1996. 滇虎榛中的化学成分 [J]. 云南植物研究, 18(4): 473-475. ]

    • YU LL, NING DS, FU YX, et al. , 2021. Chemical constituents and their anti-inflammatory activities of Callicarpa longissim [J]. Guihaia, 41(11): 1875-1881. [于玲玲, 宁德生, 符毓夏, 等, 2021. 尖尾枫化学成分及其抗炎活性研究 [J]. 广西植物, 41(11): 1875-1881. ]

    • ZHANG CX, PENG GT, HE XX, et al. , 2010. Chemical constituents of Psychotria sp. (Ⅰ) [J]. Acta Sci Nat Univ Sunyatseni, 49(4): 147-149. [张翠仙, 彭光天, 何细新, 等, 2010. 九节属药用植物Psychotria sp. 化学成分分析(Ⅰ) [J]. 中山大学学报(自然科学版), 49(4): 147-149. ]

    • ZHANG DD, LI XY, LIU P, et al. , 2020. Chemical constituents and cytotoxic activities of Cissus javana from Myanmar [J]. Nat Prod Res Dev, 32(4): 612-621. [张东东, 李兴玉, 刘珮, 等, 2020. 缅甸产青紫葛的化学成分及细胞毒活性研究 [J]. 天然产物研究与开发, 32(4): 612-621. ]

    • ZHANG W, 2007. Study and development of antitumor active constituents from Ardisia bicolor Walker [D]. Changsha: Central South University: 6-46. [章为, 2007. 两色紫金牛抗肿瘤活性成分研究及开发 [D]. 长沙: 中南大学: 6-46. ]

    • ZHANG ZY, LEE SY, 2003. PTP1B inhibitors as potential therapeutics in the treatment of type 2 diabetes and obesity [J]. Expert Opin Investig Drugs, 12(2): 223-233.

    • ZHAO HT, LIU WY, LI XG, et al. , 2022. Chemical constituents from the fibrous roots of Coptis chinensis [J]. Chin Tradit Patent Med, 44(1): 122-126. [赵邯涛, 刘武阳, 李学刚, 等, 2022. 黄连须根化学成分的研究 [J]. 中成药, 44(1): 122-126. ]

    • ZHENG SM, ZHOU X, HUANG WT, et al. , 2020. Effect of bergenin on LPS induced inflammatory factor production and cell morphological changes in RAW264. 7 cells [J]. J Chin Med Mat, 43(1): 206-210. [郑胜眉, 周兴, 黄文涛, 等, 2020. 岩白菜素对LPS诱导RAW264. 7细胞炎性因子产生及细胞形态变化的影响 [J]. 中药材, 43(1): 206-210. ]

    • ZHOU JN, ZHANG RP, ZOU C, et al. , 2020. Synthesis of novel derivatives of bergenin and its antitussive and antispasmodic activity [J]. J Kunming Med Univ, 41(8): 14-17. [周金娜, 张荣平, 邹澄, 等, 2020. 岩白菜素新颖衍生物合成及其镇咳祛痰活性 [J]. 昆明医科大学学报, 41(8): 14-17. ]

    • ZUO GY, LI ZQ, CHEN LR, et al. , 2007. Gallicacid esters of bergenin from Saxifraga melanocentra (Saxifragaceae) and their inhibition against HCV NS3 protease [J]. Acta Bot Yunnan, 29(4): 486-488. [左国营, 李正全, 陈丽蓉, 等, 2007. 黑蕊虎耳草中岩白菜素没食子酸酯类及其对丙型肝炎丝氨酸蛋白酶的抑制作用 [J]. 云南植物研究, 29(4): 486-488. ]

    • ZOU P, HUANG J, GUO HC, et al. , 2009. Saponins from the roots of Ardisia crenata f. hortensis [J]. Nat Prod Res Dev, 21(2): 249-251. [邹萍, 黄静, 郭弘川, 等, 2009. 红凉伞根茎皂苷化学成分研究 [J]. 天然产物研究与开发, 21(2): 249-251. ]

  • 参考文献

    • Beijing Institute of Botany, Chinese Academy of Sciences, 1974. Atlas of higher plants in China : Vol. 3 [M]. Beijing: Science Press: 221-224. [中国科学院北京植物研究所, 1974. 中国高等植物图鉴: 第3册 [M]. 北京: 科学出版社: 221-224. ]

    • Chinese Pharmacopoeia Committee, 2020. Chinese pharma-copoeia [M]. Beijing: China Medical Science Press: 143. [国家药典委员会, 2020.中华人民共和国药典 [M]. 北京: 中国医药科技出版社: 143. ]

    • DE OLIVEIRA GA, ALARCÓN DE LA LASTRA C, ROSILLO MÁ, et al. , 2019. Preventive effect of bergenin against the development of TNBS-induced acute colitis in rats is associated with inflammatory mediators and NLRP3/ASC inflammasome signaling path ways [J]. Chem-Biol Inter, 297: 25-33.

    • Guizhou Drug Administration, 2003. Quality standards of traditional Chinese medicine and ethnic medicine in Guizhou Province [M]. Guiyang: Guizhou Science and Technology Press: 118. [贵州省药品监督管理局, 2003. 贵州省中药材、民族药材质量标准 [M]. 贵阳: 贵州科技出版社: 118. ]

    • HAN JR, 2021. Comparative study on antioxidant and anti-tumor activities of Radix Ardisiae from different sources [D]. Guiyang: Guizhou University: 4-5. [韩金芮, 2021. 不同基原八爪金龙抗氧化及抗肿瘤活性对比研究 [D]. 贵阳: 贵州大学: 4-5. ]

    • HU ZX, WANG WY, WANG Y, et al. , 2020. Effect of extract of Ardisia crenata var. bicolor regulating MNX1-AS1 on proliferation and apoptosis of breast cancer cells [J]. New Chin Med, 52(22): 10-14. [胡志霞, 王文元, 王岩, 等, 2020. 红凉伞提取物调控MNX1-AS1对乳腺癌细胞增殖和凋亡的影响 [J]. 新中医, 52(22): 10-14. ]

    • JIA ZH, MITSUNAGA K, KOIKE K, et al. , 1995. New bergenin derivatives from Ardisia crenata [J]. Nat Med, 49(2): 187-189.

    • LAN Y, ZHANG KF, LIAN YY, et al. , 2021. Study on the protective effect of bergenin on acute liver injury in rats based on IL-6/JAK2/STAT3 signaling pathway [J]. Nat Prod Res Dev, 33(9): 1445-1451. [蓝颖, 张可锋, 连苑宇, 等, 2021. 基于IL-6/JAK2/STAT3信号通路研究岩白菜素对急性肝损伤大鼠的保护作用 [J]. 天然产物研究与开发, 33(9): 1445-1451. ]

    • LI L, LEI Y, WANG Y, et al. , 2023. Chemical constituents of Pimpinella candolleana and their anti-inflammatory activities [J]. Guihaia, 43(6): 1114-1123. [李丽, 雷艳, 汪洋, 等, 2023. 杏叶防风的化学成分及抗炎活性研究 [J]. 广西植物, 43(6): 1114-1123. ]

    • LIN CF, HWANG TL, LIN SY, et al. , 2012. Bioactive isocoumarins from Cissus pteroclada [J]. J Tradit Chin Med, 23(1): 41-49.

    • LIU YQ, LIU B, MA XR, et al. , 2023. Anti-inflammatory effect of Huangqintang on LPS-induced RAW264. 7 inflammatory cells [J]. Chin J Exp Trad Med Form, 29(7): 20-28. [刘雅清, 刘滨, 马旭冉, 等, 2023. 黄芩汤对LPS诱导的RAW264. 7炎症细胞的抗炎作用 [J]. 中国实验方剂学杂志, 29(7): 20-28. ]

    • LI YF, HU LH, LOU FC, et al. , 2005. PTP1B inhibitors from Ardisia japonica [J]. J Asian Nat Prod Res, 7(1): 13-18.

    • MCCLANAHAN RH, ROBERTSON LW, 2004. Microbial transformation of olivetol by Fusarium roseum [J]. J Nat Prod, 48(4): 660-663.

    • MEGUMI S, TOSHIKAZU S, NIJSIRI R, et al. , 2002. Ardisiphenols and other antioxidant principles from the fruits of Ardisia colorata [J]. Chem Pharm Bull, 50(11): 1484-1487.

    • PIACENTE S, PIZZA C, DETOMMASI N, et al. , 1996. Constituents of Ardisia japonica and their in vitro anti-HIV activity [J]. J Nat Prod, 59(6): 565-569.

    • TONG JY, LIANG ZT, ZHAO ZZ, et al. , 2017. Investigation of genus Ardisia in bencao literature [J]. Chin J Chin Mat Med, 42(2): 396-404. [童家赟, 梁之桃, 赵中振, 等, 2017. 本草著作中紫金牛属药物基原考 [J]. 中国中药杂志, 42(2): 396-404. ]

    • TRAN H, YOUN UJ, KIM JA, et al. , 2020. Glycerols and fatty acids isolated from Micractinium sp. KSF0031 [J]. Biochem Syst Ecol, 89(1): 1-4.

    • WANG X, 2011. Study on the chemical constituents of antitumor metastasis from Ardisia crenata [D]. Tianjin: Tianjin Medical University: 6-31. [王雪, 2011. 红凉伞抗肿瘤转移化学成分研究 [D]. 天津: 天津医科大学: 6-31. ]

    • WANG Y, LIANG XB, ZHAO ZZ, 2022. Diterpenoids from whole herb of Euphorbia helioscopia [J]. Chin Tradit Herb Drugs, 53(15): 4625-4633. [王妍, 梁旭博, 赵珍珠, 2022. 泽漆全草中二萜类成分研究 [J]. 中草药, 53(15): 4625-4633. ]

    • YANG B, YING Y, CHEN LL, et al. , 2016. Advance in studies on anti-inflammatory mechanism of flavonoids [J]. Chin Pharm, 19(7): 1369-1373. [羊波, 应茵, 陈苓丽, 等, 2016. 黄酮类化合物抗炎作用机制研究进展 [J]. 中国药师, 19(7): 1369-1373. ]

    • YE HB, WEI X, YIN X, et al. , 2022. Two new phenol compounds from roots of Ardisia crenata [J]. Nat Prod Res, 15: 1-4.

    • YE HB, ZHOU YQ, LIAO ZR, et al. , 2022. Isolation and identification of triterpenoids from the roots of Ardisia crenata var. bicolor [J]. J Chin Med Mat, 45(2): 346-350. [叶洪波, 周永强, 廖张蓉, 等, 2022. 红凉伞根三萜类化学成分分离与鉴定 [J]. 中药材, 45(2): 346-350. ]

    • YE HY, CHEN CX, HAO XJ, 1996. The chemical constituents from Ostryopsis nobilis [J]. Acta Bot Yunnan, 18(4): 473-475. [叶海亚, 陈昌祥, 郝小江, 1996. 滇虎榛中的化学成分 [J]. 云南植物研究, 18(4): 473-475. ]

    • YU LL, NING DS, FU YX, et al. , 2021. Chemical constituents and their anti-inflammatory activities of Callicarpa longissim [J]. Guihaia, 41(11): 1875-1881. [于玲玲, 宁德生, 符毓夏, 等, 2021. 尖尾枫化学成分及其抗炎活性研究 [J]. 广西植物, 41(11): 1875-1881. ]

    • ZHANG CX, PENG GT, HE XX, et al. , 2010. Chemical constituents of Psychotria sp. (Ⅰ) [J]. Acta Sci Nat Univ Sunyatseni, 49(4): 147-149. [张翠仙, 彭光天, 何细新, 等, 2010. 九节属药用植物Psychotria sp. 化学成分分析(Ⅰ) [J]. 中山大学学报(自然科学版), 49(4): 147-149. ]

    • ZHANG DD, LI XY, LIU P, et al. , 2020. Chemical constituents and cytotoxic activities of Cissus javana from Myanmar [J]. Nat Prod Res Dev, 32(4): 612-621. [张东东, 李兴玉, 刘珮, 等, 2020. 缅甸产青紫葛的化学成分及细胞毒活性研究 [J]. 天然产物研究与开发, 32(4): 612-621. ]

    • ZHANG W, 2007. Study and development of antitumor active constituents from Ardisia bicolor Walker [D]. Changsha: Central South University: 6-46. [章为, 2007. 两色紫金牛抗肿瘤活性成分研究及开发 [D]. 长沙: 中南大学: 6-46. ]

    • ZHANG ZY, LEE SY, 2003. PTP1B inhibitors as potential therapeutics in the treatment of type 2 diabetes and obesity [J]. Expert Opin Investig Drugs, 12(2): 223-233.

    • ZHAO HT, LIU WY, LI XG, et al. , 2022. Chemical constituents from the fibrous roots of Coptis chinensis [J]. Chin Tradit Patent Med, 44(1): 122-126. [赵邯涛, 刘武阳, 李学刚, 等, 2022. 黄连须根化学成分的研究 [J]. 中成药, 44(1): 122-126. ]

    • ZHENG SM, ZHOU X, HUANG WT, et al. , 2020. Effect of bergenin on LPS induced inflammatory factor production and cell morphological changes in RAW264. 7 cells [J]. J Chin Med Mat, 43(1): 206-210. [郑胜眉, 周兴, 黄文涛, 等, 2020. 岩白菜素对LPS诱导RAW264. 7细胞炎性因子产生及细胞形态变化的影响 [J]. 中药材, 43(1): 206-210. ]

    • ZHOU JN, ZHANG RP, ZOU C, et al. , 2020. Synthesis of novel derivatives of bergenin and its antitussive and antispasmodic activity [J]. J Kunming Med Univ, 41(8): 14-17. [周金娜, 张荣平, 邹澄, 等, 2020. 岩白菜素新颖衍生物合成及其镇咳祛痰活性 [J]. 昆明医科大学学报, 41(8): 14-17. ]

    • ZUO GY, LI ZQ, CHEN LR, et al. , 2007. Gallicacid esters of bergenin from Saxifraga melanocentra (Saxifragaceae) and their inhibition against HCV NS3 protease [J]. Acta Bot Yunnan, 29(4): 486-488. [左国营, 李正全, 陈丽蓉, 等, 2007. 黑蕊虎耳草中岩白菜素没食子酸酯类及其对丙型肝炎丝氨酸蛋白酶的抑制作用 [J]. 云南植物研究, 29(4): 486-488. ]

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