香鳞毛蕨中生物活性物质的筛选
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摘要
香鳞毛蕨Dryopteris fragrans(L.)Schott为鳞毛蕨科鳞毛蕨属植物,落叶多年生草本,生于高寒地区的滑石坡、森林中的碎石坡上和火山周围的岩浆缝隙中。该植物在黑龙江、辽宁、吉林、内蒙古和河北地区均有分布,在我省主要分布于五大连池、塔河县白卡兽山、呼中大白山的高山地带及小兴安岭北部地区。香鳞毛蕨药用历史悠久,生理活性较强,民间应用广泛。为了从香鳞毛蕨中寻找新的药用活性先导化合物,本文对其进行了系统研究。
将香鳞毛蕨乙醇提取物经过AB-8大孔吸附树脂吸附,得到水洗脱组分、30%乙醇洗脱组分、70%乙醇洗脱组分和90%乙醇洗脱组分。通过对2种肿瘤细胞的抗肿瘤活性筛选,对T淋巴细胞进行的免疫调节活性筛选,确定30%乙醇洗脱组分为具有抗肿瘤及免疫调节作用的有效部位。
在前期有效部位确定的基础上,通过硅胶、ODS柱色谱和HPLC等现代提取分离方法和手段,对30%乙醇洗脱组分进行分离,在分离过程中始终贯穿活性跟踪,分别对Bel-7402、A549肿瘤细胞株进行了抗肿瘤活性筛选,对T淋巴细胞进行了免疫调节活性筛选。其中得到具有抗肿瘤作用的活性组分2个,具有免疫调节作用的活性组分2个,对活性组分进一步分离得到10个化合物,通过~1H-NMR、~(13)C-NMR、HMBC和DEPT等现代波谱学测试技术,并结合ESI-MS质谱,以及与文献数据对比对其进行了结构鉴定。当化合物的浓度在5μg/mL时,化合物3对2种肿瘤细胞株均具有极强抑制作用,化合物10、5协同ConA对T淋巴细胞具有显著的促进作用,说明具有非常好的免疫调节活性。
本论文对香鳞毛蕨进行了化学成分的活性跟踪分离,最终得到了3个有活性的先导化合物,并且此3个化合物的生物活性为首次确定,为进一步研发新药奠定了基础。
Dryopteris fragrans(L.)Scott, a deciduous perennial herb in Dryopteris family of Dryopteris genus, grows in sloping land with stones of high cold area and forest or in the cracks of magma near the volcano, and distributes in Heilongjiang, Jilin, and Hebei province. In Heilongjiang, it mainly distributes in Wu-da-lian-chi, Bai-ka-shou mountain of Ta-he town, alpine region of Da-bai Mountain and the north area of Xiao -xing-an-ling Mountain. Dryopteris fragrans(L.)Scott, with strong biological activity, have a long history and has been widely used. To find the new active compound, this paper carried out a series of systematic researches.
After the ethanol extracts of Dryopteris fragrans(L.)Scott were absorbed in macroporous absorption resin AB-8, 30% ethanol eluate, 70% ethanol eluate and 90% ethanol eluate were gathered. Through screening the anti-tumor activities of two tumors cells and the immunoregulation activities of T lymphatic cells, 30% ethanol eluate was determined as the analgestic effective parts.
On the basis of former effective parts determined, 30% ethanol elute was isolated by Silica gel, ODS chromatography and HPLC. Active track was fully used during the isolating process, which performed the anti-tumor active screening to Be1-7402,A549 and the immunoregulation active screening to T lymphatic cells respectively. During the research, two anti-tumor active eluates and two immunoregulation active eluates were found. Moreover, ten compounds were collected by means of further isolation to eluate. Besides, structure identification of the eluates was done through H-NMR, C-NMR, HMBC and DEPT combing ESI-MS as well as the comparison based on the related literature datas. When the compound was at 5μg/mL, compound number three had fairly strong inhibited effective parts to two kinds of tumor cells, while compoud number ten and five along with ConA had positive effects on T lymphatic cells, which indicated that they had fine immunoregulation effective parts.
As a whole, this paper researched on activity-guided isolation of chemical components from Dryopteris fragrans(L.)Scott and acquired three effective leading compounds. The bioactivities of the three compounds were confirmed for the first time, which made a foundation to develop new medicines in future.
将香鳞毛蕨乙醇提取物经过AB-8大孔吸附树脂吸附,得到水洗脱组分、30%乙醇洗脱组分、70%乙醇洗脱组分和90%乙醇洗脱组分。通过对2种肿瘤细胞的抗肿瘤活性筛选,对T淋巴细胞进行的免疫调节活性筛选,确定30%乙醇洗脱组分为具有抗肿瘤及免疫调节作用的有效部位。
在前期有效部位确定的基础上,通过硅胶、ODS柱色谱和HPLC等现代提取分离方法和手段,对30%乙醇洗脱组分进行分离,在分离过程中始终贯穿活性跟踪,分别对Bel-7402、A549肿瘤细胞株进行了抗肿瘤活性筛选,对T淋巴细胞进行了免疫调节活性筛选。其中得到具有抗肿瘤作用的活性组分2个,具有免疫调节作用的活性组分2个,对活性组分进一步分离得到10个化合物,通过~1H-NMR、~(13)C-NMR、HMBC和DEPT等现代波谱学测试技术,并结合ESI-MS质谱,以及与文献数据对比对其进行了结构鉴定。当化合物的浓度在5μg/mL时,化合物3对2种肿瘤细胞株均具有极强抑制作用,化合物10、5协同ConA对T淋巴细胞具有显著的促进作用,说明具有非常好的免疫调节活性。
本论文对香鳞毛蕨进行了化学成分的活性跟踪分离,最终得到了3个有活性的先导化合物,并且此3个化合物的生物活性为首次确定,为进一步研发新药奠定了基础。
Dryopteris fragrans(L.)Scott, a deciduous perennial herb in Dryopteris family of Dryopteris genus, grows in sloping land with stones of high cold area and forest or in the cracks of magma near the volcano, and distributes in Heilongjiang, Jilin, and Hebei province. In Heilongjiang, it mainly distributes in Wu-da-lian-chi, Bai-ka-shou mountain of Ta-he town, alpine region of Da-bai Mountain and the north area of Xiao -xing-an-ling Mountain. Dryopteris fragrans(L.)Scott, with strong biological activity, have a long history and has been widely used. To find the new active compound, this paper carried out a series of systematic researches.
After the ethanol extracts of Dryopteris fragrans(L.)Scott were absorbed in macroporous absorption resin AB-8, 30% ethanol eluate, 70% ethanol eluate and 90% ethanol eluate were gathered. Through screening the anti-tumor activities of two tumors cells and the immunoregulation activities of T lymphatic cells, 30% ethanol eluate was determined as the analgestic effective parts.
On the basis of former effective parts determined, 30% ethanol elute was isolated by Silica gel, ODS chromatography and HPLC. Active track was fully used during the isolating process, which performed the anti-tumor active screening to Be1-7402,A549 and the immunoregulation active screening to T lymphatic cells respectively. During the research, two anti-tumor active eluates and two immunoregulation active eluates were found. Moreover, ten compounds were collected by means of further isolation to eluate. Besides, structure identification of the eluates was done through H-NMR, C-NMR, HMBC and DEPT combing ESI-MS as well as the comparison based on the related literature datas. When the compound was at 5μg/mL, compound number three had fairly strong inhibited effective parts to two kinds of tumor cells, while compoud number ten and five along with ConA had positive effects on T lymphatic cells, which indicated that they had fine immunoregulation effective parts.
As a whole, this paper researched on activity-guided isolation of chemical components from Dryopteris fragrans(L.)Scott and acquired three effective leading compounds. The bioactivities of the three compounds were confirmed for the first time, which made a foundation to develop new medicines in future.
引文
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[4]沈志滨,金哲雄,张德连.香鳞毛蕨的生药学研究[J].中草药,2002,33(7):661-663.
[5]傅沛云.东北植物检索表(第二版).科学出版社,1995.
[6]王全喜,邵成文,曹建国等.北蕨类植物配子体发育的研究(Ⅺ鳞毛蕨科)[J].哈尔滨师范大学自然科学学报,1995,4(11):83-88.
[7]张彦龙,匡宏枫等.香鳞毛蕨挥发油成分的研究.中草药,2006,(7):32-33.
[8] Hideyuki Ito, Takashi Muranaka, Kazuko Mori.Ichthyotoxic Phloroglucinol Derivatives from Dryopteris fragrans and Their Anti-tumor Promoting Activity[J].Chem Pharm.bull, 2000, 48(8):1190-1195.
[9]沈志滨,罗文英,严优芍.香鳞毛蕨中间苯三酚衍生物的研究[J].中草药,2005,29(6):559-561.
[10]沈志滨,张维库,马英丽.香鳞毛蕨化学成分的研究(Ⅲ)[J].中草药,2006,37(7):995-997.
[11]张彦龙.香鳞毛蕨治疗类风湿性关节炎有效部位的药理作用和化学成分的研究[D].黑龙江:黑龙江中医药大学,2003.
[12]沈志滨,金哲雄,张德连.香鳞毛蕨治疗银屑病的药理作用研究[J].中草药,2002,33(5):844-845.
[13]沈志滨,马英丽,江蔚新.香鳞毛蕨对真菌的抑制作用[J].中草药,2005,36(5):735-736.
[14]罗容,金哲雄,张延萍.香鳞毛蕨擦剂的药效学研究[J].哈尔滨商业大学学报(自然科学版),2003,19(4):388-340.
[15]李红枝,沈志滨,赵瑛.香鳞毛蕨软膏对大鼠实验性体癣的治疗作用[J].中药材,2005,28(10):491-492.
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[18] Wu ZH .The Catalogue of Fern on Family and Genus in China (中国蕨类植物科属志) [M]. Beijing: Science Press,1991.
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