Studies On The Chemical Constituents And Bioactivities From The Seeds Of Orychophragmus Violaceus

Orychophragmus violaceus(L.)O.E.Schulz(Brassicaceae),introduced as an annual herb,mainly distributed in middle and east of Asia,was known as "Er-yue-lan" in China.Previous investigations on the chemical constituents of this plant resulted in isolation of flavonoids,phenolicacids and terpenoids.This plant has a long history as a traditional folk medicine for anti-tumor,anti-inflammatory,anti-bacterial and liver-protecting properties.Preliminary tests in vitro showed that the ethanol extract from the seeds of O.violaceus possessed cytotoxicity activities towards HepG2 cell lines.Based on the review of the latest five years on the chemical constituents and bioactivities of the Brassicaceae,chemical constituents of O.violaceus were studied systematically.Part of the isolated compounds were tested for their cytotoxicity activities,in addition,compounds with novel structures were tested for their radiation protection activities.The seeds of O.violaceus(40 kg)were extracted with 70%EtOH(240 kgx3)under reflux for 2 h each time.After evaporation of the solvent,the concentrated residue(4.0 kg)was suspended in water and partitioned with petroleum ether,EtOAc and n-BuOH successively.In this study,71 compounds were isolated from the 70%ethanol extract of the seeds of O.violaceus by different chromatographic methods such as silica gel CC,AB-8 macroporous resin column chromatography,Spehadex LH-20,ODS and preparative HPLC and 53 of them were identified on the basis of physical properties,MS,IR and NMR spectrum.10 of them were new compounds and they were identified as:orychophragine A(1),orychophragine B(2),orychophragine C(3),orychophramarin A(4),orychophramarin B(5),orychophramarin C(6),orychophramarin D(7),orychovioside A(8),orychoside A(9),orychoside B(10).Compounds 1-3 were the first examples of natural alkaloid products.Compounds 4-7 were four new isocoumarin derivatives.Compounds 8-10 were monoterpene glycoside,diterpene glycoside and triterpenoid glycoside,respectively.43 were known conpounds and they were identified as:rubusoside(11),mimengoside C(12),3-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1 ?3)-[?-D-glucopyranosyl-(1?2)]-?-D-fucopyranosyl-23,28-dihydroxy-oleane-11,13(18)-diene(13),3?-O-(?-D-xyl opyranosyl-(1?2)-[?-D-glucopy ranosyl-(1?4)-?-D-glucopy ranosyl-(1?3)]-?-D-fu copyranosyl-11,13(18)diene-olean-23a,28 diol(14),mimengoside D(15),mimengoside E(16),mimengoside F(17),buddlejasaponin ?(18),buddlejasaponin III(19),buddlejasaponin la(20),buddlejasaponin ?a(21),benzonic acid(22),2-hydroxy benzoic acid(23),4-hydroxy benzoic acid(24),4-hydroxy-3-methoxy-benzoic acid(25),3,4,5-trimethoxyb enzoic acid(26),7-hy droxy-6-methoxy coumarin(27),5,7-dihydroxy chromone(28),7,8-dimethylpyrazine(29),genkwanin(30),acacetin-7-O-neohesperidoside(31),isorhamnetin-3-O-?-D-glucopyranoside(32),apigenin-7-O-?-D-glucoside(33),chrysoeriol-7-O-?-D-glucoside(34),acacetin-7-O-?-D-glucoside(35),geristein(36),5,7,3',5 '-tetrahydroxyflavanone(37),5,7,3',5'-tetrahydroxy-flavanone-7-O-?-D-glucopyranoside(38),aromadendrin-7-O-?-D-glucopyranosi de(39),hesperetin-7-O-?-D-glucopy ranoside(40),1,3-dihydroxy anthraquinone(41),lariciresinol(42),lariciresinol-9-O-?-D-glucopyranoside(43),isolariciresinol-9-O-?-D-glucopyranoside(44),hexadecanoic acid(45),heneicosanoic acid(46),dioscin(47),gracillin(48),?-sitosterol(49),daucosterol(50),?-sitosteryl-3-O-?-D-glucopyranoside-20-O-palmitate(51),hypargyreusolide B(52),roseoside(53),respectively.All the compounds were isolated from the genus for the first time except compounds 22,37 and 50.A series of alkaloids with an unusual skeleton were isolated from the 70%ethanol extract of the seeds of O.violaceus,together with new isocoumarin derivatives,new triterpenoid saponins and flavonoids.These compounds enriched the chemical diversity of this genus.Part of the isolated compounds were tested for their cytotoxicity activities in vitro with the MTT assay.Orychophragines A-C(1-3)were evaluated for their cytotoxicity against HepG2,HCT-116,Hela and A549 cell lines.Compound 1 exhibited cytotoxicity with IC50 values of 7.73,10.79,11.91 and 9.93 ?M,respectively.Orychophramarins A-D(4-7)were assayed for their cytotoxicity against HCT-116 and Hela cell lines.Compound 4 showed significant cytotoxicity against HCT-116 and Hela cell lines compared with the positive control group(Cisplatin)with IC50 values of 5.10 and 8.91?M,respectively.Compounds 5 and 6 showed weak cytotoxicity against the tested tumor cell lines with IC50 values ranging from 10.12 to 28.30 ?M.Further mechanism study of 4 was carried out in HCT-116 cells.Results showed that 4 exhibited evident cell cycle retardation that arrested HCT-116 cells at G2 phase and induced apoptosis in HCT-116 cells.Compounds 9-21 were assayed for their cytotoxicity against HepG2 and A549 cell lines.Compounds 9,12,15,18 and 19 were found to possess obvious cytotoxicity.The new compound 9 exhibited potent cytotoxicity against the two cell lines with IC50 values of 7.13 and 8.77 ?M,respectively.In addition,Compounds 1-3 were evaluated for radiation protection against 60Co?irradiation in HUVEC cell line.Results showed that 3(at the concentration of 10 ?M)increased the survival rate of y irradiated HUVEC cells from(56.71 ± 0.92)%to(77.64± 1.19)%(P<0.01,GraphPad Prism 5).