Study On Chemical Constituents Of Nelumbinis Plumula With Inhibitory Activity Of The Pparγ Expression In 3T3-L1 Cells

Lotus plumule is the dried young cotyledon and radicle of the ripe seed of Nelumbo nucifera Gaertn.Currently,there are many studies on the quality control of Lotus plumule,most of which are based on the content of a single chemical marker.For example,the content of neferine was required to be more than 0.7%in Chinese Pharmacopoeia（2020）,and the content of liensinine or isoliensinine was also reported to control the quality of this herb.Lotus plumule contains various types of compounds such as alkaloids and flavonoids.Among these,further study is required to discover the one（s）to be used as quality markers that are closely realted to its clinic application.The clarification of the bioactive compoents of this herb will ensure the safety and effectiveness of Lotus plumule,and the development of the qulity control method.Therefore,in this thesis,the following studies were carried out,starting from discovety of the chemical constituents with inhibitory activity of the expression of PPARγin 3T3-L1 cells,in combination of ambient ionization mass spectrometry:1 Preliminary establishment of Cell-EFISI-MS methodIn order to quickly find and identify the material basis of Lotus plumule that enter the 3T3-L1-Lenti-PPARγ-Luc cells and exert its efficacy,a cell-EFISI-MS method was developed for the first time on the basis of the TLC-EFISI-MS approach.In this thesis,using the mouse 3T3-L1-Lenti-PPARγ-Luc cell as the model,we successfully established the online cell-EFISI-MS coupling method,by which the compounds entering the cell or binding to the membrane receptor can be directly analyzed from the 96-well plates.As a result,characteristic MS signal peaks with m/z625[M+H]⁺and 313[M+2H]²⁺,611[M+H]⁺and 306[M+2H]²⁺,597[M+H]⁺and299[M+2H]²⁺,were observed,which represented seven individual compounds with the same molecule weights based on the later phytochemical study of this herb.Through the bio-activity evaluation of the seven pure compounds on this cell model,it was found that all the seven compounds could enter the cell or bind to the membrane receptor,and six of them（1-seconeferine A,neferine,1’-epi-neferine,liensienine,isoliensienine,nelumboferine）showed significant inhibitory activity on the expression of PPARγ,with IC₅₀ values of 3.247μM,5.411μM,3.043μM,35.538μM,6.694μM and 18.626μM,respectively.In a word,we established a cell based EFISI-MS coupling method,and identified six biomarkers from the herb.Compared with the traditional cell analysis methods,our cell based EFISI-MS method can directly be carried out in situ and on-line analysis of the cell surface without complex pre-treatment such as scraping,crushing,centrifugation,extraction,enrichment and so on.2 Chemical constituents of Lotus plumule with inhibitory activity againstthe expression of PPARγin 3T3-L1 cellsTo verify the accurate structure of the components entering the cell or binding to the membrane receptor,the chemical constituents of Lotus plumule were isolated from its active CH₂Cl₂ and aqueous fractions.As a result,54 compounds were isolated and identified,including 49 alkaloids（mainly dibenzylisoquinoline,monobenzylisoquinoline and isoquinoline）.Among these,two new structure skeletons featured with benzo seven nitrogen heterocycles（azepinneferine A,1）,and bisbenzyl isoquinoline alkaloids with benzyl unit C-9 position or C-9’position replaced by terminal double bond（1S,1′R–neoneferine A,2;1R,1′S–neoneferine B,3;1R,1′R–neoneferine B,4;1R,1′S–neoisoliensinine A,5;and 1R,1′R–neoisoliensinine A,6）.The other 26 new compounds,including 14 split-ring dibenzylisoquinoline alkaloids（7-20）with cleavage of 1-or 1’-position C-N bond,8 six-membered nitrogen heterocycles substituted by aromatization of dibenzylisoquinoline alkaloids（21-28）,one common dibenzylisoquinoline alkaloid（29）and three simple isoquinoline alkaloids（30-32）were also iedntified from the CH₂Cl₂ fraction.A total of 38 compounds including 12 alkaloids,19 flavonoid glycosides,and 7 other compounds,were isolated and identified from the aqueous fraction.Among these,a new isoquinoline alkaloid（55）was included.The inhibitory activity of these new compounds on the expression of PPARγin the 3T3-L1-Lenti-PPARγ-Luc cells was determined.The results showed that compounds 1-17,19,23,26-27 and 29-30 had significant inhibitory activity on the expression of PPARγin3T3-L1-Lenti-PPARγ-Luc cells at the same dose as the positive drug T0070907（5μM）.Among them,the inhibitory effect of 1-2,4-7,9,10,12,14,17 and 27 was significantly higher than that of the positive control group.In summary,the chemical constituents of the active extracts of Nelumbinis plumula were systamatically studied,and a total of 85 compounds were isolated,purified and identified from the CH₂CL₂ and aqueous fractions with the inhibitory activity on the expression of PPARγin 3T3-L1 cells.In addiiton,the cell-EFISI-MS coupling method was preliminarily developed and applied to in situ characterized the active components entering cells by which six potential bio-markers of Lotus plumule with inhibing the expression of PPARγin 3T3-L1 cells were quickly located and identified as 1-seconeferine A,neferine,1’-epi-neferine,liensienine,isoliensienine,and nelumboferine.