Chemical Constituents And Bioactivities Of Orychophragmus Violaceus

Orychophragmus violaceus is distributed in most areas of China.It is known as "Er-yue-lan"and Cui zi hua"in China.As a traditional herb in China,it is used for the treatment of heat toxic dieases and jaundice dieases.Previous research on the chemical constituents of this plant resulted in isolation of flavonoids,alkaloids,isocoumarins,phenolic acids,saponins and other main components exhibit various bioactivities.We have investigated the chemical constituents of the seed and herb of Orychophragmus violaceus,and evaluated the anti-radiation,antiinflammatory,anti-tumor,anti-oxidant activities of them systematically.We have isolated four new alkaloids(orychophragines A-C)from the seeds of O.violaceus.And they were tested for cytotoxicity activities,results showed that orychophragine A significantly inhibit the HepG2,A549,HCT-116 cell lines.In addition,orychophragine C was evaluated for radiation protection against 60Co γ irradiation(dose of 8.0 Gy,dose rate of 0.8 Gy/min)in HUVEC cell line.Results showed that orychophragine C at a concentration of 25 μM increased the cell viability of γ irradiated HUVECs from 56.7±0.92%to 77.6±1.19%.The above finding provided a scientific basis for the further investigation of O.violaceus,and there are few reports on the chemical composition and bioactivities of O.violaceus.Therefore we focused on the research of the seeds and herb of O.violaceus.As continuing the study of previous work,the 25%ethanol part of the n-butanol extraction part of O.violaceus seeds were isolated by reversedphase silica gel column chromatography,phase silica gel column chromatography,Sephadex LH-20 gel column,recrystallization and HPLC.Six compounds were obtained from this part and structures of these compounds were identified by means of modern spectroscopy techniques such as nuclear magnetic resonance spectroscopy(NMR),mass spectrum(MS),infrared absorption spectrum(IR).Two pairs of new pentacyclic spermidine alkaloid enantiomers:(+)-orychovioline A(1),(-)orychovioline A(2),(+)-orychovioline B(3),(-)-orychovioline B(4),along with two known compounds,spicatolignan B(5),(2S)-2-benzoyloxy-3-butenyl glucosinolate(6)were identified.Among them,compounds 1～4 are new compounds,5 was isolated from the genus for the first time.The 70%ethanol extract of the herb of O.violaceus were systematically separated by above medthods.In this study,forty-five compounds were isolated and thirty-seven were identified by the above separation means.They were identified as 4-hydroxy-5-methoxy-3quinolinecarbonitrile(7),β-aromatic alcohol(8),2-methyl-1,3,6trihydroxy-9,10-anthraquinone(9),carotene(10),lauric acid monoglyceride(11),adenine(12),isovanillic acid(13),3,4’,6,8-tetrahydroxyflavone-7-C-glucoside(14),cappariloside A(15),kaempferol-3-Oβ-D-galactose-7-O-α-L-rhamnoside(16),1-methyl malate(17),phydroxyben-zoic acid(18),2,3-dihydroxybenzoic acid(19),phenylacetamide(20),syringic acid(21),vanillic acid(22),uracil(23),kaempferol-3-O-β-D-glucopyranosyl-7-O-α-L-rhamnoside(24),uridine(25),kaempferol-3-O-α-L-arabinosyl-7-O-α-L-rhamnoside(26),liriodendrin(27),isoflavin(28),1-ribosealcohol-2,3-diketone-1,2,3,4tetrahydro-6,7-dimethyl-quinoxaline(29),3,6-dibenzyl-2,5dioxopiperazine(30),perlolyrine(31),cyclo-Leu-Ile(32),quercetin-3-O-β-D-glucosyl-7-O-α-L-rhamnoside(33),kaempferol-7-O-α-Lrhamnoside(34),quercetin(35),Indole-3-acetonitrile-4-methoxy-2-S-β-D-glucopyranoside(36),trans-[3-(4’-hydroxyphenyl)-2-propenoic acid](37),4-oxo-1,4-dihydroquinoline-3-carbonitrile(38),trans-ferulic acid(39),kaempferol-3-O-β-D-glucopyranoside(40),quercetin-7-O-α-Lrhamnopyranoside(41),3,4-dimethoxy cinnamic acid(42),orychophragine E(43).Compound 43 is a new compound.Compounds 7 and 38 were isolated from nature for the first time.All compounds were isolated from the genus for the first time except compounds 10,13,21-23,35.(±)-orychovioline A,(+)-orychovioline A(1),(-)-orychovioline A(2),(±)-orychovioline B,(+)-orychovioline B(3)and(-)-orychovioline B(4)were evaluated for their radiation protection against 60Co γ irradiation in HUVEC cell line.Results showed that(-)-orychovioline A(1)and(±)orychovioline A at concentrations of 25 μM increased the cell viability ofγ irradiated HUVECs from 52.4±2.3%to 66.4±2.8%and 62.2±2.6%,respectively.Single-cell electrophoresis showed that(-)-orychovioline A(1)reduced radiation damage to HUVEC cellular DNA.(±)-orychovioline A,(+)-orychovioline A(1),(-)-orychovioline A(2),(±)-orychovioline B,(+)-orychovioline B(3)and(-)-orychovioline B(4)were assessed the anti-inflammatory effects in terms of their abilities to inhibit the production of NO by LPS-stimulated the murinemacrophage cell line RAW264.7 with Griess method.Results showed compound 2 exhibited obvious anti-inflammatory activities,with the IC50 value of 20.3± 1.58 μM,which was equivalent to Dexamethasone.All compounds isolated from the herb of O.violaceus were evaluated for their radiation protection against X-ray irradiation in HUVEC cell line.Results showed that compunds 7,15,18,20,25,28,29,36,38,43 exhibited anti-radiation activities,especially compound 20 at a concentration of 25 μM increased the cell viability of y irradiated HUVEC cells from 38.7±1.26%to 56.2±2.66%.Moreover,the compounds isolated from the herb of O.violaceus were also tested the anti-inflammatory effects with Griess method.Compounds 9 and 31 exhibited moderate anti-inflammatory activities,and the IC50 values were 31.4±1.54 and 27.3±1.29 μM,respectively.All compounds isolated from the herb of O.violaceus were tested for their cytotoxicity activities against Hela and SH-SY5Y cell lines with the Counting Kit-8(CCK-8)assay.Compound 31 only showed obvious cytotoxicity against SH-SY5Y cell line with IC50 value of 23.4±2.88 μM.Part of compounds were tested the anti-oxidant properties by the ABTS and FRAP assays.From the results,compounds 14,19,33,34 and 35 were exhibit antioxidant activities.