Study On Isolation And Identification Of Active Ingredients From Citrus Aurantium L.var.amara Engl And Their Bioactivities

Citrus aurantium L.var.amara Engl(CAVA),a subgenus of genus Citrus(Rutaceae),is the variant of Citrus aurantium L mainly distributed in Fujian,Guangdong,Sichuan,Zhejiang in China.Blossoms of CAVA were used as edible and medicinal resource nowadys in china.Blossoms of CAVA were employed to trate swelling pain in the chest and abdomen,depression of QI,nausea and vomiting,phlegm and retained fluid,gastroptosis,archoptosis and descensus uteri in folk prescription.Its flower is rich in volatile oil,flavonoids and alkaloids.In addition,phytochemical studies have revealed the presence of cardiac glycosides,vitamins,essential amino acids,fibers,coumarins and minerals.Recent pharmacological researches revealed that CAVA exhibits various biological activities such as anti-inflammatory,anti-oxidant,anti-bacterial,anti-virus,anti-tumor,anti-lipidemia,immunomodulatory and gastrointestinal propulsive motility capacities.With the help of the traditional natural products analysis technologies combined with modern research means for active substances,the present study made a thorough inquiry on active fractions of CAVA from three aspects of separation and purification,structural identification,pharmacological action and its mechanism evaluation,main contributions of this work are as follows:(1)A comprehensive evalution of antioxidant and antitumor activities of ethanol extract of CAVA and its five polar fractions was performed via three kinds of tumor cells(HepG2,MCF-7,H1299)and four kinds of antioxidant assays(Total reducing power,DPPH,ABTS,FRAP).The results showed that after fractionated extraction on CAVA ethanol extract,the active substances with significant antioxidant and antitumor abilities were mainly concentrated in petroleum ether fraction,ethyl acetate fraction and n-Butanol fraction.(2)In virtue of traditional column chromatography(silical gel column,sephadex LH-20 column,polyamide column)and semi-preparative/analytical HPLC,active fractions of CAVA were separated and purified.The structure of compound was charactered via mass spectrometry,NMR spectrum and their physicochemical properties.Ten compounds were obtained,and they were nobiletin,caffeine,naringenin,homoeriodictyol,hesperetin-7-O-?-D-glucoside,bergaptol,choerospodin,7-methylxanthine,naringin and neohesperidin,respectively.Among them,homoeriodictyol,hesperetin-7-O-?-D-glucoside,bergaptol,choerospodin,7-methylxanthine and caffeine were isolated from blossoms of CAVA for the first time.(3)Systematical research on antioxidant and antitumor activities of ten compounds was undertaken using the same assays as crude extracts.Hesperetin-7-O-?-D-glucoside exhibited the strongest inhibitory effect on proliferation of HepG2 cell lines,followed by naringenin.Their inhibitory effect was significantly better than that of the control group at the maximum safety concentration.Meanwhile,inhibitory effect of bergaptol and choerospodin is equivalent to that of 5-F.The most obvious effect on inhibiting MCF-7 cell lines proliferation was naringenin,then hesperetin-7-O-?-D-glucoside.Antiproliferative effect of the former is better than that of 5-F,while the latter is equivalent to that of 5-F.A significant reduction on cell viability of H1299 cells intervened by neohesperidin was observed using MTT assay and hesperetin-7-O-?-D-glucoside came second.Their suppressive effect was inferior to control group.In deterdmination of antioxidant capacity of ten compounds,bergaptol exhibited the most visible total reducing power,whereas bergaptol and neohesperidin presented the most remarkable DPPH radical scavenging capacity.Forthermore,bergaptol and homoeriodictyol provided a significant scavenging ability agains ABTS radical.The highest FRAP value came from hesperetin-7-O-?-D-glucoside,and it means that hesperetin-7-O-?-D-glucoside had the most notable ferric ion reducing antioxidant power.The experimental results suggested that homoeriodictyol,hesperetin-7-O-?-D-glucoside,bergaptol,naringenin and neohesperidin may be responsible for the significant antioxidant and antitumor properties of CAVA.(4)RAW264.7 cell was used to determine the toxicity of ten compounds.Meanwhile homoeriodictyol displayed the most remarkable anti-inflammatory capacity according to the release of NO of LPS-stimulated macrophage RAW264.7 cells.ELLISA,RT-PCR and Western Blot were employed to make a thorough inquiry on anti-inflammatory mechanism of homoeriodictyol.The results showed that homoeriodictyol ccan significantly inhibited the release of IL-6 and TNF-?,and mRNA expression of iNOS,IL-6,TNF-?,IL-? and COX-2.Moreover,homoeriodictyol suppressed the phosphorylation of MAPKs and the phosphorylation NF-?B through suppressing nuclear transcription of P65,degradation and phosphorylation of I?B-?,phosphorylation of I?K?/?,p-JNK and p-ERK,and activation of COX-2,thus performing the anti-inflammatory capacity.