Inhibitory Effects Of 47plant Extracts On Fatty Acid Synthase And Breast Cancer Cell

Animal fatty acid synthase?E.C.2.3.1.85,FAS?is a key enzyme in energy metabolism,as it can catalyze the synthesis of long-chain fatty acids.Recently,FAS is reported to be a potential target for the treatment of various cancer.It is important to find FAS inhibitors with both potent activity and low toxicity for the development of new anti-tumor drugs.This study focused on the screening of novel FAS inhibitors with high bio-activity from plant resources.The inhibitory activity on FAS and breast cancer?MDA-MB-231?cells from 47 plant ethanol extracts were evaluated.Koelreuteria bipinnata Franch.var.integrifoliola for T.?Merr.?Chen.leaves and bamboo leaves were subjected toextraction,isolation,bio-activity evaluation and identification of active components,the main results are as follows:?1?Inhibitory activities on FAS and MDA-MB-231 of 47 kind of ethanol extracts were investigated including ethanol extracts of Broussonetia papyifera?Linn.?L'Hert.ex Vent.,Trachelospermum jasminoides?Lindl.?Lem.,Rhododendron molle?Blum?G.Don.,Sweet wisteria sinensis?Sims?Sweet.,Chelidonium majus L.,Sophora alopecuroides L.,Nerium indicum Mill.,Aphananthe aspera?Thunb.?Planch.,Sargentodoxa cuneata?Oliv.?Rehd.,Kadsura longipedunculata Finet et Gagnep.,Gelsemium elegans?Gardn.& Champ.?Benth.,Koelreuteria bipinnata Franch.var.integrifoliola?Merr.?T.Chen,Mallotus apelta?Lour.?Muell.Arg.var.apelta,Parthenocissus laetevirens Rehd,Consolida ajacis?L.?Schur,Acorus tatarinowii Schot,Caesalpinia decapetala?Roth?Alston,Sapium sebiferum?Linn.?Roxb.,Elaeocarpus decipiens Hemsl.,Campsis radicans?L.?Seem.,Polygonum hydropiper L.,Humulus scandens?Lour.?Merr.,Aesculus chinensis Bunge,Magnolia delavayi Franch.,Euonymus fortunei?Turcz.?Hand.-Mazz.Pueraria lobata?Willd.?Ohwi,Ficus sarmentosa Buch.-Ham.ex J.E.Sm.var.henryi?King ex Oliv.?Corner,Rhus chinensis Mil,J?glans cathayensis Dode var.formosana?Hayata?A.et R.H.Chang,Pteroceltis tatarinowii Maxim.,Paederia scandens?Lour.?Merr.,Millettia dielsiana Harms,Parthenocissus thomsonii?Laws.?Planch.,Lindera glauca?Sieb.et Zucc.?Bl.,Lindera angustifolia Cheng,Lindera praecox?Sieb.et Zucc.?Bl.,Ambrosia artemisiifolia L.;Pleioblastus amarus?Keng?,Indocalamus latifolius?Keng?Meclure,Phyllostachys heterocycla?Carr.?Mitford cv.Pubescens,Phyllostachys glauca,Dendrocalamus giganteus Munro,Phyllostachys heteroclada Oliver,B.blumeana Schult.f,Bambusa ventricosa McClure,Phyllostachys heteroclada Oliver and Chimonocalamus delicatus Hsueh et Yi.Some plant extracts presented significant proliferation inhibition activity,including Koelreuteria bipinnata Franch.var.integrifoliola?Merr.?T.Chen.,Kadsura longipedunculata Finet et Gagnep.,Aphananthe aspera?Thunb.?Planch.,Sargentodoxa cuneata?Oliv.?Rehd.and Rhododendron molle?Blum?G.Don..Further bio-assay guided screening showed that the n-butanol fraction of Koelreuteria bipinnata Franch.var.integrifoliola?Merr.?T.Chen.exhibited the most significant cytotoxicity,which an IC₅₀ value of 19.93 mg/L.The inhibitory activities of the different solvents extract on FAS were also measured by UV spectrophotometry.The results showed that the n-butanol fraction of Koelreuteria bipinnata Franch.var.integrifoliola?Merr.?T.Chen.had the highest inhibition activity,with the inhibition rate of 84.32% at a concentration of 100 mg/L.IC₅₀ value of 10 kinds of bamboo extracts were ranged from 512.45 mg/L to 647.09 mg/L on MDA-MB-231 cells,and 187.09 mg/L to 276.45mg/L on FAS.Pleioblastus amarus?Keng?and Phyllostachys heterocycla?Carr.?possessed high inhibitory activities.?2?According to the biological activity assay results,the n-butanol fraction of Koe lreuteria bipinnata Franch.var.integrifoliola?Merr.?T ethanol extracts further separated by HP-20 resin,and 60% ethanol sub fraction showed significant activity of FAS and MDA-MB-231 cell which IC₅₀ value were 19.45 mg/L,11.84 mg/L;this fraction was t hen separated by C18 into Fr1–Fr10,and Fr2 presented the strongest activity of which IC₅₀ value were 12.68 mg/L,5.26 mg/L.10 compounds were isolated from Fr2 by gel chromatography,and determined to be kaempferol-3-O-rutinoside,Rutin,Quercetin-3-O-?-D-galactopyranoside,kaempferol-3-O-[2??-O-galloylrutinoside],kaempferol-3-O-[?-L-rhamn opyranosyl-?1-6?-?-D-galac-topyranoside],Quercitrin,Quercetin-3-O-?-D-arabinopyranoside,astragalin6??-O-gallate,kaempferol-3-O-?-robinobinoside,and kaempferol-3-O-gluside.Th e activity assay showed that 10 compounds inhibited the activity of FAS at concentration 10 mg/L and 100 mg/L,but at concentration of 50 mg/L,the inhibitory effect on MDA-MB-231 cells was not significant.?3?The inhibitory effects of bamboo leaves flavonoids from the species of Pleioblastus amarus?Indocalamus latifolius?Phyllostachys heterocycla cv.pubescens and Phyllostachys glauca on the FAS and human breast cancer cell MDA-MB-231 in vitro were positively associated with the content of flavonoids.After HP-20 resin purification,it showed that 50% ethanol fraction had the strongest inhibitory activities on FAS and 95% ethanol fraction possessed the strongest inhibitory activities on MDA-MB-231 cells.Flow cytometric were employed to detect the inducing apoptosis activities of Pleioblastus amarus?Keng?macroporous on MDA-MB-231,and found that 70% as well as 95% ethanol fraction were both capable of inducing apoptosis of cancer cells.The apoptosis rate in the concentration of 100,200,400mg/L of 70% and 95% were 11.3%,23.3%,36.7% and 11.1%,23.1%,38.7%,respectively.?4?A UPLC-MS/MS analysis method was established for simultaneous determination of 10 flavonoids in bamboo leaves.The content and distribution of flavonoids in different flow fractions were determined.Bamboo leaf flavone C-glycosides mainly distributed in 50% ethanol fraction;flavonoid aglycones are mainly distributed in 70% ethanol fraction.According to the inhibitory assay,C-glycosylflavones might be the main active ingredient for the inhibition of FAS,isoorientin and isovitexin might be the main active monomer;flavonoid glycosides were considered to be the major components for the inhibition of MDA-MB-231 cells.?5?Inhibitory effects of 16 bamboo leaves flavonoid monomers on FAS were determined.The IC₅₀ value of isovitexin was 4.56mg/L,which was higher than the positive control of resveratrol.Luteolin,quercetin,isoorientin,apigenin,kaempferol,tricin also presented some inhibition activity,and the IC₅₀ values were: 12.05mg/L,21.71 mg/L,27.67 mg/L,42.81 mg/L,56.60 mg/L,89.70 mg/L respectively.The structure-activity relationship showed that the substitution position of flavonoid B ring 6 could increase the inhibitory activity on FAS,-OH substitution at position 3 on the C ring is detrimental to the inhibition of FAS activity,and more-OH on the B ring,the active stronger.Luteolin,quercetin,apigenin,kaempferol,tricin,isovitexin showed significant inhibitory activity on MDA-MB-231 cell,IC₅₀ value were 41.10mg/L,51.17mg/L,57.04mg/L,64.44mg/L,145.66mg/L,181.36mg/L,respectively.For the etermination of the inhibitory activity of FAS in MDA-MB-231 cell,MDA-MB-231 cells were treated with apigenin,luteolin,quercetin,kaempferol,isovitexin and tricin in 24 h at the concentration of 50mg/L,intracellular FAS residual activity were 60.74%,58.69%,77.62%,61.12%,89.76%,86.75%.The weak inhibitory activity showed by isovitexin might be related to the way and efficiency of compounds entering cell.In summary,this paper evaluated inhibitory activities of 47 species of plant ethanol extracts on FAS and human breast cancer cells?MDA-MB-231?,showed evidence of Koelreuteria bipinnata Franch.var.integrifoliola?Merr.?T.Chen.and other 4 plants had the potential to be developed to FAS inhibitors and even anti-tumor drug precursors.The inhibitory effect of flavonoids from bamboo leaves on FAS was confirmed and very strong inhibitory activity of isovitexin on FAS in vitro was found.The results has important reference value for the utilization of Koelreuteria bipinnata Franch.var.integrifoliola?Merr.?T.Chen.resources and use of bamboo leaf flavonoids in FAS inhibitor development.