Chemical Constituents From Blossoms Of Citrus Aurantium L.var.amara Engl And Their Bioactivities

Citrus aurantium L.var.amara Engl.(CAVA),belonging to Citrus genus,is consumed as an edible and medicinal resource in China.However,little information is available about its pharmacological activities.In the present study,bioactivity-guided isolation of CAVA was performed and their bioactivities were systematically investigated and compared using various models both in vitro and in vivo(1)Total flavonoids CAVAF,alkaloids CAVAA,polysaccharides CAVAP,coumarins CAVAC,essential oil CAVAO,chloroform fractions CHL,ethyl acetate fractions EA and n-butanol fractions n-Bu were extracted from blossoms of CAVA.Their chemical compositions and contents were determined.Bioactivity screening assays demonstrated that different constituents varied significantly toward different sources of free radicals and other oxidants.CAVAP exhibited greater immune-enhancement capacity,while CAVAO,CHL and EA showed great anti-inflammation potential.As for inhibitory effects on macrophage-deprived foam cells and differentiation of 3T3-L1 preadipocytes,CHL and EA presented greater potency.(2)CAVAP could significantly stimulated production of IL-6 and TNF-α,up-regulated expression levels of iNOS,IL-6,TNF-α and IL-1β mRNA,and promoted phosphorylation of ERK,JNK,P38 and P65 in macrophages.Box-Behnken design(BBD)was performed to optimize the extraction process of CAVAP and the yield of CAVAP was significantly increased Further isolation and purification of CAVAP was performed,yielding two novel polysaccharides CAVAP-I and CAVAP-II.CAVAP-I and CAVAP-II differed from each other in molecular weights,monosaccharide compositions and glycosidic bonds.(1→3,6)-β-D-Gal and(1→5)-α-L-Ara composed the backbone of CAVAP-I and CAVAP-II,respectively.Similar to CAVAP,CAVAP-II might also act via modulation of MAPK and NF-κB pathways,and CAVAP-II showed greater potency than CAVAP-I(3)CAVAO could significantly inhibit secretion of IL-6,TNF-α,and IL-1β as well as their gene expression levels in LPS-induced macrophages.CAVAO also markedly decreased COX-2 expression,NF-κB nuclear translocation,IκBα degradation and phosphorylation,IκKα/β,JNK and P38 phosphorylation.GC-MS assay indicated that linalool,α-terpineol,(R)-limonene,and linalyl acetate might be the compounds responsible for the anti-inflammatory activity of CAVAO.It was possible that the anti-inflammation activity of CAVAO was a result of a complex interaction among its various chemical compounds,which might produce synergistic effects(4)Nobiletin(NOB),caffeine(CAF),homoeriodictyol(HE),choerospondin(CHO),hesperetin-7-O-β-D-glucopyranoside(HG),bergaptol(BER),naringin(NARG)and 7-methylxanthine(MET)were identified from blossoms of CAVA based on bioactivity-guided isolation.HE and BER exhibited greater efficacy in inhibiting LPS-induced NO accumulation HG,the structural analog of HE,also markedly inhibited NO secretion.HE,HG and BER might exert anti-inflammatory effects through regulating MAPK and NF-κB pathways.The number and position of hydroxyl groups and the sugar substitution,might partially explain the bioactivity discrepancy of HE and HG(5)Oil Red O staining showed that HE,CHO,HG and BER might exhibit greater inhibition on ox-LDL-induced lipid accumulation.BODIPY staining,Dil-ox-LDL uptake,cholesterol influx and efflux measurements indicated that HE,CHO,HG and BER could significantly inhibit formation of macrophage-deprived foam cells.RT-qPCR and western blot assays suggested that different compounds mainly acted via differential regulation of ABC A1,ABCG1,SRB1,SRA1 and CD36 expression(6)EA extracts could significantly inhibit differentiation of 3T3-L1 preadipocytes,attenuate antioxidant responses and affect expression of genes involved in lipid and glucose metabolism in C.elegans.TG quantification in various mutants suggested that EA extracts might inhibit lipid accumulation in C.elegans dependent on mdt-15.EA extracts could significantly decrease HFD-induced body weight gain,affect plasma biochemical parameters,prevent epididymal adipose hypertrophy,liver oxidative injuries and steatosis,and reverse gut dysbiosis in mice Further investigation showed that NOB,HE,HG and NARG might inhibit differentiation of 3T3-L1 preadipocytes through modulating expression of C/EBPα,FAS,Leptin and PPARy mRNAIn conclusion,these results showed that CAVA had various pharmacological activities,which could be developed into functional food and bring lots of economic and social benefits.