山梗菜脂溶性化学成分的研究
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摘要
山梗菜是桔梗科(Campanulacea)半边莲属(Lobelia L.)植物山梗菜Lobeliasessilifolia Lamb.的根及或带根全草。产于我国云南西北部、广西北部、浙江、台湾、山东、河北、辽宁、吉林和黑龙江。目前对山梗菜有效成分的研究并不全面,药理学的研究也刚刚起步。为更加深入研究其化学成分,并为阐明其药效提供化学依据,有必要对其进行系统分离研究。
作者通过硅胶柱层析,凝胶柱层析等分离手段从山梗菜中分离出5个单体化合物,通过ESI-MS、~1HNMR、~(13)CNMR、HMBC、HMQC、DEPT、H~1-H~1COSY等光谱分析,确定它们的结构分别β-香树脂醇3-0-β-棕榈酸酯、马尼拉二醇3-0-β-棕榈酸酯、齐墩果酸、熊果酸、豆甾醇。其中豆甾醇在该属植物中首次发现。
五环三萜类化合物熊果酸和齐墩果酸有抗人肺癌细胞增殖、侵袭和诱导细胞凋亡的作用,并探讨其抗侵袭作用的机理。方法是药物处理后通过重建基底膜侵袭试验、对层粘连蛋白的粘附试验、趋化运动试验以及组织蛋白酶B活性测定观察细胞侵袭能力的变化。结果是熊果酸和齐墩果酸均可降低PGCL3细胞增殖能力(P＜0.001),ICm分别为44.73μmol/L和40.71μmol/L。30和40μmol/L熊果酸、和35和45μmol/L齐墩果酸均能抑制PGCl3细胞的侵袭能力(P＜0.001)且有剂量依赖性。抗侵袭作用机理的分析结果显示上述浓度的两种药物对细胞的运动、粘附能力及组织蛋白酶B分泌均有明显的抑制作用。
Lobelia sessilifolia Lamb is the root or the whole grass with root of Campanulacea.the study on active principles in the Lobelia sessilifolia Lamb aren't comprehensive and the study on pharmacology is just on the first step.Base of the previous research,I have got five compounds from Lobelia sessilifolia Lamb by using silica gel column chromatography, preparative thin-layer chromatography,reversed phase column chromatography,preparative HPLC.They are identified by EI-MS、~1HNMR、~(13)CNMR.The two compounds were determined asβ-amyrin 3-O-β-palmitate、maniladiol 3-O-β-palmitate、Oleanolic acid、ursolic acid、stigmasterol.
One of them was isolated from the genus Lobelia L.for the first time.
To study the anti—invasive effects and its mechanisim and apoptosis induction of pentacyclic triterpenoid including ursolic acid and oleanolic acid in highly potentially metastatic lung cancer cell line.The Methods are invasive a—bilit Y,the adhesive ability, the migration ability and the activity of cathepsin B(CB)of PGCI 3 cells treated with the two drugs were determined by the invasion test or reconstituted basement membranc,the laminin adhesion test,the chemotactic migration test and the enzymologicaI method of CB.The apoptosis of the cells was detected with acridine orange ethidium bromide fluorescent s.tain(A()/EB)and TU-NEL.The result is All of the two drugs can inhibit the proliferative and invasive ability,and induce apoptosis of the PGCL3 human lung cancer cell.The mechanism of anti-invasion may be to inhibit the adhesion,migration,and the CB secreation of the cell.
作者通过硅胶柱层析,凝胶柱层析等分离手段从山梗菜中分离出5个单体化合物,通过ESI-MS、~1HNMR、~(13)CNMR、HMBC、HMQC、DEPT、H~1-H~1COSY等光谱分析,确定它们的结构分别β-香树脂醇3-0-β-棕榈酸酯、马尼拉二醇3-0-β-棕榈酸酯、齐墩果酸、熊果酸、豆甾醇。其中豆甾醇在该属植物中首次发现。
五环三萜类化合物熊果酸和齐墩果酸有抗人肺癌细胞增殖、侵袭和诱导细胞凋亡的作用,并探讨其抗侵袭作用的机理。方法是药物处理后通过重建基底膜侵袭试验、对层粘连蛋白的粘附试验、趋化运动试验以及组织蛋白酶B活性测定观察细胞侵袭能力的变化。结果是熊果酸和齐墩果酸均可降低PGCL3细胞增殖能力(P＜0.001),ICm分别为44.73μmol/L和40.71μmol/L。30和40μmol/L熊果酸、和35和45μmol/L齐墩果酸均能抑制PGCl3细胞的侵袭能力(P＜0.001)且有剂量依赖性。抗侵袭作用机理的分析结果显示上述浓度的两种药物对细胞的运动、粘附能力及组织蛋白酶B分泌均有明显的抑制作用。
Lobelia sessilifolia Lamb is the root or the whole grass with root of Campanulacea.the study on active principles in the Lobelia sessilifolia Lamb aren't comprehensive and the study on pharmacology is just on the first step.Base of the previous research,I have got five compounds from Lobelia sessilifolia Lamb by using silica gel column chromatography, preparative thin-layer chromatography,reversed phase column chromatography,preparative HPLC.They are identified by EI-MS、~1HNMR、~(13)CNMR.The two compounds were determined asβ-amyrin 3-O-β-palmitate、maniladiol 3-O-β-palmitate、Oleanolic acid、ursolic acid、stigmasterol.
One of them was isolated from the genus Lobelia L.for the first time.
To study the anti—invasive effects and its mechanisim and apoptosis induction of pentacyclic triterpenoid including ursolic acid and oleanolic acid in highly potentially metastatic lung cancer cell line.The Methods are invasive a—bilit Y,the adhesive ability, the migration ability and the activity of cathepsin B(CB)of PGCI 3 cells treated with the two drugs were determined by the invasion test or reconstituted basement membranc,the laminin adhesion test,the chemotactic migration test and the enzymologicaI method of CB.The apoptosis of the cells was detected with acridine orange ethidium bromide fluorescent s.tain(A()/EB)and TU-NEL.The result is All of the two drugs can inhibit the proliferative and invasive ability,and induce apoptosis of the PGCL3 human lung cancer cell.The mechanism of anti-invasion may be to inhibit the adhesion,migration,and the CB secreation of the cell.
引文
[1]中国科学院昆明植物研究所.云南植物志,第二卷.科学出扳社1979,521-534
[2]Kaji ishimaru,Himshi Yonemitsu.Koichiro Shimomura.Lobetyolin andlobetyol from hairy root culture of Lobelia inflate,Phytochemistry,1991.30(7):2255-2257
[3]张明哲、曹立伟.紫燕草化学成分的研究,北京大学学报(自然科学扳).1991,27(2):205-9
[4]张明哲、王景朝.江南山梗菜化学成分的研究,植物学报,1992,34(1):58-61
[5]Kanji ishimaru,ShinjiSadoshima,SumanaNeera,et al.A polyacetylenegentiobio side from hairy roots of Lobeila inflate.,Phytochemistry,1992,31(5):1577-1579
[6]Yoshitama,K.An acylated delphinidin 3-rutinoside-5.3',5'-trlglucoside from Lobelia erinus Phytochemistry,1977,16:1857
[7]Taro Kondo,Junko Yamashiki,kiyoko,kawahori,et al.Structureof Lobelinin A and B.noval anthocyanins acylated with three and four different organic acids,espectively,Tetrahedron Letters,1989,30(44):6055-6058
[8]Norio Saito,Kenjiro Toki,Harumitsu Kuwano Acylated cyaniding-3-rutinoside -5.3'-djglucoside from the purple-red flower of Lobelia erinus.Phytochemistry,1995.39(2),423-6.
[9]Michiko Yamanaka,Koichiro Shimomura,Kuniroshi Yoshihira.Glucosylation of phenolics by hairy root cultures of Lobelia sessilifolia.Phytochemistry 1995,40(4):1149-50
[10]李琦,师彦平,OE.Edwards.红山梗菜化学成分研究,高等学校化学学报.1993,14(10):1391-1393
[11]扬靖华,李子燕,汪云松,等.长萼野烟化学成分的研究(Ⅱ),中草药,2001,32(3):207
[12]国家医药管理局中草药情报中心站编,植物药有技成分手册,人民卫生出版社,673-4,1986
[13]顾学裘.彭海卿.药剂专利手册.天律:天律科学技术出版社,1987.238
[14]王婧婧,范秀珍,陈融,等.不同浓度半边莲生物总碱对血管紧张素Ⅱ诱导的人脐动脉平滑肌细胞增殖的影响,滨州医学院学报,2004,27(4):320
[15]江苏新医学院:中药大辞典.1977,1187
[16]Ostlund R E.Phytosterols in human nutrition[J].An—nual Review of Nutrition,2002,22:533-549.
[17]David K,Shirley C C.Phytosterols—health benefits andpotential concerns:a review[J]Nutrition Research.2005,25:413-428.
[18]IFST updated information statement on phytosterol esters.[EB/01].http～//fatsandoilsrefining.productfinder.corn/pages-213988.html,2005.
[19]Bergmann K,Sudhop T,LutjOhann D.Cholesterol and plantsterolabsorption:recent insights[J].The Ameri—can Journal of Cardiology.2005.96:10-14.
[20]Thompson G R,Grundy S M.History and development of plant sterol and Cardiology,2005,96:3-9.
[21]McCann S E,Freudenheim J L,Marshall J R,et al.Risk of human ovarian cancer is related to dietary intakeof selected nutrients,phytochemicals and food groups[J].Journal of Nutrition,2003,133:1937-1942.
[22]De S E,Bofetta P,Ronco A L,et al.Plant sternls andrisk of stomach cancer:a case-control study in uruguay[J].Nutrition and Cancer,2000,37:140-144.
[23]Lj J H,Awad A B,Fink GoS,et al.Measurement varia—bility of plasma sitosterol and campesterol,two new biomarkers for cancer prevention [J].European Journalof Can cer Prevention.2001.10:245-249.
[24]Awad A B,Gan Y,Fink C S.Efect of JB—sitosterol,aplant sterolon growth,protein phosphatase 2A andphospholipid D in LNCaP cells[J].Nutrition and Canc—er.2000,36:74-78
[25]Awad A B,Downie A,Fink C S,et al.Dietary phytos—terol Inhibits the growth and metastasis of DA—MB—231 human breast cancer cells grown in SCID mice[J].Antican cer Research.2000,20:821-824.
[26]Awad A B,Williams H,Fink C S,et al.In vitro and invlvo(SCID mice)efect of phytosterols on the growthand dissemination of human prostate cancer PC —3 cells[J].European Journal of Cancer Prevention,2001,10:507-513.
[27]Awad A B,Chan K C,Downie A C,et al.Peanuts as asource of 3—sitosterol,a sterol with anticancer proper—ties[J].Nutrition and Cancer,2000,36:238-241.
[28]Tapiero H,Townsend D M,Tew K D.Phytosterols inthe prevention of human pathologies[J].Biomedicine& Pharmacotherapy.2003.57:321-325.
[29]Ganry O.Phytocstrogens and prostate cancer risk[J].Preventive Medicine,2005,41:1-6.
[30]Awad A B,Williams H,Fink C S.Efect of phytoster-ols oncholesterolmetabolism and MAP kinase in MDA—MB—231humanbreastcancercells[J].Journal of Nutrition Biochemistry,2003,14:111-119.
[31]Choi Y H,Kong K R,Kim Y A,et al.Induction of Bax and activation of caspases during 8—sitosterol me diated apoptosis in human colon cancer cells[J].Inter—national Journal of Oncology,2003,23:1657-1662.
[32]Bouic P J D.Sterols and sterolins:new drugs for theimmune system [J].Therapeutic Focus,2002,7:775-778.
[33]Vivancos M,Moreno[J]—Sitosterol modulates an—tioxidant enzyme response in RAW 264.7 macrophages[J].Free Radical Biology & Medicine,2005,39:91-97.
[34]Laura C B,Joan Caries E G,Vicent R.Changes in in—testinal and liver global gene expression in response to aphytosterol—enriched diet[J].Atherosclerosis,2005,181:75-85.
[35]Ju Y H,Clausen h M,Alfred K F,et al itosterol,Sitosterol Glucoside,and a Mixture of JB—Sitos—terol and 8—Sitosterol glucoside modulate the growth ofestrogen—responsive breast cancer cells in vitro and inovariectomized athymic mice[J].Journal of Nutrition,2004.134:1145-1151.Phytosterols and its anticancer efectZHANG Qiang.
[36]龚慕辛,贾富霞,王敏,等.不同提取方法对女贞子中齐墩果酸提出量的影响(J3.北京中医,1999,10(3):53.
[37]崔星明,王勇为,陈光宇.超临界流体萃取芦笋中熊果酸的研究F.J3.上海农业学 报,2004,21(1):123-123.
[38]雒廷亮,张景伟,曹文豪,等。山茱萸中熊果酸提取工艺研究进展(J3.河南化工,2005,22(2):6-8.
[39]黄书铭,杨新林,张自强,等.超声循环提取灵芝中三萜类化合物的研究F.J3.中草药,2004,35(5):508-510.
[40]仲兆金,刘浚.衍生法分离茯苓三萜(J3.中药材,2002,25(4):247
[41]易中宏,郑一敏,胥秀英,等.分光光度法测定茯苓中总三萜类成分CJ3.药,2005,16(4):847-848.
[42]黄书铭,杨新林,黄健,等。紫外分光光度法快速测定灵芝样品中三萜类化合物的含量(J3.北京理工人学学报,2004,24(6):555-558.
[43]张雁冰,王克让,刘宏民.马桑叶中总三萜酸的含量测定(J3.时珍国医国药,2006,17(4):529-530.
[44]Corthout J,Naessens T,Apers S,et al.Short communicationquantitative determination of ginsenosides from Panax ginsengroots and ginse ng preparations by thin layer chromatography—densitometrCJ7.J Pharm Biomed Aanl,1991,21:187-192.
[45]Kuanzinger A.Baumelster A,Cuda K,et al.Determ inationofll—ket0—bo swellic acid in human plasm(J3.J Pharm Biomed Anal,2002,28:729-739.
[46]Andrisano V,Bonazzi D,Carrini V.HPLC analysis ofliquo ricetriterpeno ids application to the quality control of pharmaceuticals(J3.J Pharm Biomed Anal.1995,13(4:5):597-605.
[47]8iiehele B.Simmet T,Analysis.of 12 different pentacyclic trierpenic acids from frank incense in,human plasma by high—performanceliquid chromatography and photodiode array detection(J].J Chromatography B.2003,795:355-462.
[48]Wang X M,Sakuma T,A safu—A djaye E,et al.Determination of ginsenosides in plant extracts from Panax ginseng and Panaxqu inquefolius L.by LC/MS MS[J]·Anal Chem,1999,71:1579-1584.
[2]Kaji ishimaru,Himshi Yonemitsu.Koichiro Shimomura.Lobetyolin andlobetyol from hairy root culture of Lobelia inflate,Phytochemistry,1991.30(7):2255-2257
[3]张明哲、曹立伟.紫燕草化学成分的研究,北京大学学报(自然科学扳).1991,27(2):205-9
[4]张明哲、王景朝.江南山梗菜化学成分的研究,植物学报,1992,34(1):58-61
[5]Kanji ishimaru,ShinjiSadoshima,SumanaNeera,et al.A polyacetylenegentiobio side from hairy roots of Lobeila inflate.,Phytochemistry,1992,31(5):1577-1579
[6]Yoshitama,K.An acylated delphinidin 3-rutinoside-5.3',5'-trlglucoside from Lobelia erinus Phytochemistry,1977,16:1857
[7]Taro Kondo,Junko Yamashiki,kiyoko,kawahori,et al.Structureof Lobelinin A and B.noval anthocyanins acylated with three and four different organic acids,espectively,Tetrahedron Letters,1989,30(44):6055-6058
[8]Norio Saito,Kenjiro Toki,Harumitsu Kuwano Acylated cyaniding-3-rutinoside -5.3'-djglucoside from the purple-red flower of Lobelia erinus.Phytochemistry,1995.39(2),423-6.
[9]Michiko Yamanaka,Koichiro Shimomura,Kuniroshi Yoshihira.Glucosylation of phenolics by hairy root cultures of Lobelia sessilifolia.Phytochemistry 1995,40(4):1149-50
[10]李琦,师彦平,OE.Edwards.红山梗菜化学成分研究,高等学校化学学报.1993,14(10):1391-1393
[11]扬靖华,李子燕,汪云松,等.长萼野烟化学成分的研究(Ⅱ),中草药,2001,32(3):207
[12]国家医药管理局中草药情报中心站编,植物药有技成分手册,人民卫生出版社,673-4,1986
[13]顾学裘.彭海卿.药剂专利手册.天律:天律科学技术出版社,1987.238
[14]王婧婧,范秀珍,陈融,等.不同浓度半边莲生物总碱对血管紧张素Ⅱ诱导的人脐动脉平滑肌细胞增殖的影响,滨州医学院学报,2004,27(4):320
[15]江苏新医学院:中药大辞典.1977,1187
[16]Ostlund R E.Phytosterols in human nutrition[J].An—nual Review of Nutrition,2002,22:533-549.
[17]David K,Shirley C C.Phytosterols—health benefits andpotential concerns:a review[J]Nutrition Research.2005,25:413-428.
[18]IFST updated information statement on phytosterol esters.[EB/01].http～//fatsandoilsrefining.productfinder.corn/pages-213988.html,2005.
[19]Bergmann K,Sudhop T,LutjOhann D.Cholesterol and plantsterolabsorption:recent insights[J].The Ameri—can Journal of Cardiology.2005.96:10-14.
[20]Thompson G R,Grundy S M.History and development of plant sterol and Cardiology,2005,96:3-9.
[21]McCann S E,Freudenheim J L,Marshall J R,et al.Risk of human ovarian cancer is related to dietary intakeof selected nutrients,phytochemicals and food groups[J].Journal of Nutrition,2003,133:1937-1942.
[22]De S E,Bofetta P,Ronco A L,et al.Plant sternls andrisk of stomach cancer:a case-control study in uruguay[J].Nutrition and Cancer,2000,37:140-144.
[23]Lj J H,Awad A B,Fink GoS,et al.Measurement varia—bility of plasma sitosterol and campesterol,two new biomarkers for cancer prevention [J].European Journalof Can cer Prevention.2001.10:245-249.
[24]Awad A B,Gan Y,Fink C S.Efect of JB—sitosterol,aplant sterolon growth,protein phosphatase 2A andphospholipid D in LNCaP cells[J].Nutrition and Canc—er.2000,36:74-78
[25]Awad A B,Downie A,Fink C S,et al.Dietary phytos—terol Inhibits the growth and metastasis of DA—MB—231 human breast cancer cells grown in SCID mice[J].Antican cer Research.2000,20:821-824.
[26]Awad A B,Williams H,Fink C S,et al.In vitro and invlvo(SCID mice)efect of phytosterols on the growthand dissemination of human prostate cancer PC —3 cells[J].European Journal of Cancer Prevention,2001,10:507-513.
[27]Awad A B,Chan K C,Downie A C,et al.Peanuts as asource of 3—sitosterol,a sterol with anticancer proper—ties[J].Nutrition and Cancer,2000,36:238-241.
[28]Tapiero H,Townsend D M,Tew K D.Phytosterols inthe prevention of human pathologies[J].Biomedicine& Pharmacotherapy.2003.57:321-325.
[29]Ganry O.Phytocstrogens and prostate cancer risk[J].Preventive Medicine,2005,41:1-6.
[30]Awad A B,Williams H,Fink C S.Efect of phytoster-ols oncholesterolmetabolism and MAP kinase in MDA—MB—231humanbreastcancercells[J].Journal of Nutrition Biochemistry,2003,14:111-119.
[31]Choi Y H,Kong K R,Kim Y A,et al.Induction of Bax and activation of caspases during 8—sitosterol me diated apoptosis in human colon cancer cells[J].Inter—national Journal of Oncology,2003,23:1657-1662.
[32]Bouic P J D.Sterols and sterolins:new drugs for theimmune system [J].Therapeutic Focus,2002,7:775-778.
[33]Vivancos M,Moreno[J]—Sitosterol modulates an—tioxidant enzyme response in RAW 264.7 macrophages[J].Free Radical Biology & Medicine,2005,39:91-97.
[34]Laura C B,Joan Caries E G,Vicent R.Changes in in—testinal and liver global gene expression in response to aphytosterol—enriched diet[J].Atherosclerosis,2005,181:75-85.
[35]Ju Y H,Clausen h M,Alfred K F,et al itosterol,Sitosterol Glucoside,and a Mixture of JB—Sitos—terol and 8—Sitosterol glucoside modulate the growth ofestrogen—responsive breast cancer cells in vitro and inovariectomized athymic mice[J].Journal of Nutrition,2004.134:1145-1151.Phytosterols and its anticancer efectZHANG Qiang.
[36]龚慕辛,贾富霞,王敏,等.不同提取方法对女贞子中齐墩果酸提出量的影响(J3.北京中医,1999,10(3):53.
[37]崔星明,王勇为,陈光宇.超临界流体萃取芦笋中熊果酸的研究F.J3.上海农业学 报,2004,21(1):123-123.
[38]雒廷亮,张景伟,曹文豪,等。山茱萸中熊果酸提取工艺研究进展(J3.河南化工,2005,22(2):6-8.
[39]黄书铭,杨新林,张自强,等.超声循环提取灵芝中三萜类化合物的研究F.J3.中草药,2004,35(5):508-510.
[40]仲兆金,刘浚.衍生法分离茯苓三萜(J3.中药材,2002,25(4):247
[41]易中宏,郑一敏,胥秀英,等.分光光度法测定茯苓中总三萜类成分CJ3.药,2005,16(4):847-848.
[42]黄书铭,杨新林,黄健,等。紫外分光光度法快速测定灵芝样品中三萜类化合物的含量(J3.北京理工人学学报,2004,24(6):555-558.
[43]张雁冰,王克让,刘宏民.马桑叶中总三萜酸的含量测定(J3.时珍国医国药,2006,17(4):529-530.
[44]Corthout J,Naessens T,Apers S,et al.Short communicationquantitative determination of ginsenosides from Panax ginsengroots and ginse ng preparations by thin layer chromatography—densitometrCJ7.J Pharm Biomed Aanl,1991,21:187-192.
[45]Kuanzinger A.Baumelster A,Cuda K,et al.Determ inationofll—ket0—bo swellic acid in human plasm(J3.J Pharm Biomed Anal,2002,28:729-739.
[46]Andrisano V,Bonazzi D,Carrini V.HPLC analysis ofliquo ricetriterpeno ids application to the quality control of pharmaceuticals(J3.J Pharm Biomed Anal.1995,13(4:5):597-605.
[47]8iiehele B.Simmet T,Analysis.of 12 different pentacyclic trierpenic acids from frank incense in,human plasma by high—performanceliquid chromatography and photodiode array detection(J].J Chromatography B.2003,795:355-462.
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