Screening And Isolation Of Alkaloid Topoisomerase ? Inhibitors From Sophora Alopecuroides L

Sophora alopecuroides L,a perennial herbs,is widely distributed in the northwest of China,especially in Xing Jiang,Ning Xia,etc.Alkaloids are the main components of sophora alopecuroides L and have many pharmacological effects such as anti-tumor,anti-oxidation and neuroprotection,etc.DNA topoisomerase ?(Top ?)widely exists in prokaryotes and eukaryotes,is an important anti-tumor targets,then,tumor cell growth was inhibited by inhibiting enzyme activity.Based on the alkaloids of sophora alopecuroides L as the research object,the extraction and separation of alkaloids,Top ? targeted screening inhibitors and alkaloid pharmacological activities was studied,the main contents are as follows: 1.Isolation and purification of alkaloids from sophora alopecuroides L: The fruit parts of sophora alopecuroides L in Xingjiang were extracted,separated and purified.Various column analysis methods(silica gel column,Sephadex LH-20 gel column,C18 reverse silica gel column)and recrystallization were used.Physical and chemical identification and nuclear magnetic resonance spectroscopy(NMR)were used to identify five alkaloids isolated from matrine type,four of which were matrine,oxy-matrine,sophoridine,oxy-sophocarpine and one cytisine type alkaloid,cytisine.2.Top ? inhibitors targeting selection and determination of reaction conditions: Free Top ? enzyme as targets,the use of affinity ultrafiltration(UF)combined with ultrahigh performance liquid-mass spectrometry(UPLC-QTOF-MS)technology from the extract of sophora alopecuroides L screening identified six Top ? inhibitors,they are matrine,oxy-matrine,sophoridine,oxy-sophocarpine,sophoramine,and cytisine.Among them,it has been reported that sophoridine is a Top ? inhibitor.At the same time,examines the incubation time,temperature and concentration of organic reagents on the enzyme reaction of its influence,determine the final enzyme and the reaction conditions of alkaloid extract: 37 ?,incubation for 30 min,5% methanol soluble alkaloid extract,this condition has good adsorption effect.3.The pharmacological activity of the selected alkaloids was tested: In order to further confirm the anti-tumor activity of the screened alkaloids,matrine and cytisine were selected as the experimental group according to the absorption peak in the UPLC figure,and sophoridine as the control group,and the human breast cancer MDA-MB-468 cell line as the object for the in vitro tumor cell inhibition experiment.The results showed that all three alkaloids showed anti-MDA-MB-468 cell growth ability,and the inhibitory effect increased with the increase of the action time and concentration.The morphological changes of the cells were observed under an inverted microscope,including cell shrinkage,rupture,cell membrane blebbing and expanded intercellular spaces,indicating that the cells were in a state of apoptosis.