以生物活性为导向的荜茇抗乳腺癌的物质基础及其作用机制研究

The nearly mature or mature fruit of the Piper longum Linn. is widely used in traditional ethnic medicine, and is a main component of the anticancer compound recipe for the treatment of gynecologic tumors clinically used in Uyghur medicine. The previous scientific research proved that piperlongumine and piperine, the amide alkaloids in Piper longum Linn., showed remarkable bioactivity against breast cancer both in vitro and in vivo. Since the Piper longum Linn. contains more compounds with the same type of amide alkaloid structure as piperlongumine and piperine, are there any other more active amide alkaloids besides piperlongumine and piperine? Are the amide alkaloids the bioactive constituents of the Piper longum Linn.? So, firstly, the more active and chemically more diverse fruit of the Piper longum Linn. will be screened as the research object on the basis of the comparisons of the bioactivity against breast cancer and the diversity of the chemical constituents between the nealy mature and mature fruits, then the active compounds will be quickly identified and purified by the method of anti-breast cancer tracking in vitro with the combination of the technique of HPLC-DAD-MS/MS, and the database of UV spectroscopy and LC-MS spectroscopy of the active fractions will be established after the structures of these components were determined. The anti-breast cancer activities of all isolated compounds will be evaluated. In addition, the underlying mechanism of the activity for the chosen compounds will be investigated. This research can not only provide the scientific evidences for the way of the usage of the Piper longum Linn. in the traditional medicine, but also make a contribution to the rational exploration of the Piper longum Linn. by the confirmation of its bioactive constituents aginst breast cancer and their mechanisms.

荜茇以近成熟或者成熟果穗在民族药中广泛应用，并作为维吾尔医药抗妇科肿瘤有效复方的主要组方药材应用于现代临床。研究表明荜茇中的酰胺类生物碱荜茇酰胺和胡椒碱具有显著抗乳腺癌作用。荜茇含有丰富的酰胺类生物碱，除了荜茇酰胺和胡椒碱，还含有非常多的同类型化合物，是否存在活性更佳的酰胺类生物碱？胺类生物碱是否是荜茇抗癌功效的物质基础？本项目拟以荜茇近成熟果穗和成熟果穗的抗乳腺癌活性和化学成分多样性的对比研究为前期，筛选出抗癌活性较优、化学成分多样性较为丰富的果穗作为研究对象，采用活性跟踪方法，结合HPLC-DAD-MS/MS快速化学检识技术，对荜茇中的抗乳腺癌活性成分进行快速分离纯化并进行结构鉴定，建立活性组分的图谱数据库。对得到的所有化合物进行抗乳腺癌活性评价，选取活性强的新颖先导化合物进行作用机制研究。本研究通过对荜茇抗乳腺癌的活性物质基础和作用机制的确定，为荜茇药用价值的合理开发做出贡献。

荜茇作为中药、维药、藏药、蒙药及印度阿育吠陀医学中的常用药材具有广泛的生理活性，对其开展活性及其物质基础研究对荜茇药用价值的开发和活性先导化合物的发现均具有重要意义。本项目对比了荜茇近成熟果穗和成熟果穗的化学成分和它们对MCF-7、Hela和A54 9的抑制活性，发现二者在化学成分和体外抗肿瘤方面无显著区别，为荜茇在民族药中的用法提供了理论依据。发现荜茇的乙酸乙酯和石油醚部位具有一定的抑制人乳腺癌细胞株MCF-7的作用。对乙酸乙酯部位开展了系统的化学成分分离纯化和结构鉴定，鉴定出32个化合物，其中21个为酰胺类生物碱，5个酰胺类生物碱为荜茇中首次得到。并总结了荜茇中酰胺类生物碱的紫外吸收光谱与其结构之间的关系，建立了乙酸乙酯活性部位中酰胺类生物碱的紫外全波长扫描图谱数据库。利用UHPLC-Q-Orbitrap HRMS/MS进一步从乙酸乙酯活性部位中鉴定出61个酰胺类生物碱，建立了乙酸乙酯活性组分的液-质联用图谱数据库。利用气质联用（GC-MS）从石油醚活性部位中鉴定出48个化合物，其中23个为酰胺类生物碱，占该部位总含量的74.43%。分析可知，酰胺类生物碱可能是活性部位中的主要活性成分。对所得的酰胺类生物碱单体化合物进行了针对MCF-7的细胞毒测试，发现其对MCF-7只有弱的细胞毒或无明显细胞毒活性，分析部位对MCF-7的抑制活性可能是通过单体化合物之间的协同作用而产生。此外，对所得酰胺类生物碱单体化合物进行了体外逆转肿瘤多药耐药（MDR）、降糖和促黑色素生成以及酪氨酸酶活性筛选。所测试的11个单体化合物均未表现出对MCF-7/ADR细胞的多药耐药逆转活性；发现4个化合物对蛋白酪氨酸磷酸酶1B（PTP1B）有显著抑制作用，其中2个还对α-葡萄糖苷酶有明显抑制活性，对构效关系进行了分析；发现乙醇提取物和5个化合物对B16细胞有显著促进黑色素生成作用，并有一定的增强酪氨酸酶活性作用，表明其可能是通过增强B16细胞中酪氨酸酶活性而起到促进黑色素生成的作用。本研究为荜茇药用价值的进一步开发提供了理论依据，并为先导化合物的发现奠定了物质基础。