Isolation Of Active Phthalides Regulating Nuclear Receptor TR3 From Angelicae Sinensis Radix,Chuanxiong Rhizoma And Ligustici Rhizoma Et Radix

Orphan nuclear receptor TR3 is one of the ideal targets regulating cell proliferation and apoptosis by participating in a variety of physiological activities in different periods of various cells,which can promote proliferation,but also can inhibit proliferation and promote apoptosis.Previous experiments and literature studies have shown that some phthalides can target nuclear receptor TR3 to play anti-tumor,anti-inflammatory and other biological activities.In this study,several Chinese medicines:Angelicae Sinensis Radix,Chuanxiong Rhizoma and Ligustici Rhizoma et Radix containing phthalides were selected to isolate and study these phthalides and the activity of regulation of nuclear receptor TR3.In this study,the conventional silica gel column chromatography,C18 column chromatography,medium pressure preparation and high performance liquid phase preparation were used,and 26 phthalides were identified by one-dimensional and two-dimensional NMR spectroscopy,high-resolution mass spectrometry,ultraviolet and other spectroscopic data analysis.Among these,there are:10 mono phthalides:Senkyunolide I(15),Z-6-hydroxy-7-methoxydihydroligustilide(16),Senkyunolide A(17),Neocnidilide(18),11-Angeloysenkyunolide F(19),Senkyunolide B(20),Senkyunolide C(21),Z-ligustilide(24),E-ligustilide(25),Butylphthalide(26);16 dimer phthalates:Angeolide(1),Tokinolide B methyl ester(2),Angeolide methyl ester(3),Z,Z'-6.6',7.3'a-diligustilide(4),Gelispirolide(5),Z-6.8',7.3 '-diligustilide(6),3E,3'E-6.6,,7.3'a-diligustilide(7),3Z-6R,7S,3'R,8'R-6.8',7.3,-diligustilide(8),3Z,3'E-6.6,,7.3'a-diligustilide(9),3Z-6S,7R,3'S,8,R-6.8',7.3,-diligustilide(10),3E-6R,7S,3'S,8,R-6.8.,7,3'-diligustilide(11),Chuanxiongdiolide A(12),3,3'Z-6.T,7.6'-diligustilide(13),Tokinolide A(14),Senkyunolide P(22),Z'-3,8-dihydro-6,6',7,3'a-diligustilide(23).And phthalides 7-11 are new compounds.In order to detect whether these phthalidess have the activity of regulating the orphan nuclear receptor TR3,a number of phthalids(C8-C29)extracted before and the phthalides isolated from Angelicae sinensis Radix,Chuanxiong Rhizoma and Ligustici Rhizoma et Radix are selected in this study.The effects of compounds on TR3 transcription activation were detected by double reporting gene analysis method,and it was found that compounds 1,C4,C8,C11,C12 and C20 had obvious transcription activation effect on nuclear receptor TR3,especially on C12,while compounds 4,6 and 24 had obvious transcription inhibitory effect on nuclear receptor TR3.This study also used the report gene analysis to detect the effect of phthalides on NF-?B transcription activation,found that compound 2,5 can significantly inhibit NF-?B transcription activation,compounds 13,14,20,24 and so on also show a certain degree of inhibition effect.In order to observe the cytotoxicity of these phthalides,the cytotoxicity was detected in human lung cancer cells H460 by MTT method,and the experimental results showed that the compound C28 could strongly inhibit the proliferation of H460 cells at concentration 20?M,compounds 1,14,C8,C11,C12 could significantly inhibit cell proliferation.The above preliminary bioactivity results provide some reference for the follow-up study on the function and mechanism of TR3 cell signal regulation related diseases.