本文選題：脂肪酸合酶 + 人乳腺癌細胞��； 參考：《中國林業(yè)科學研究院》2017年博士論文

【摘要】：脂肪酸合酶(E.C.2.1.3.85,FAS)是催化生物體內合成長鏈脂肪酸,參與能量代謝的一種重要的酶。研究表明,動物FAS是癌癥治療的潛在靶點。開發(fā)高活性、低毒的FAS抑制劑,對于癌癥的防治具有重大意義。本文以FAS和人乳腺癌(MDA-MB-231)細胞為靶標,評價了47種植物醇提取物的抑制活性;以黃山欒樹(Koelreuteria bipinnata Franch.var.integrifoliola(Merr.)T.Chen.)葉和竹葉為研究對象,采用生物活性追蹤,利用植物化學手段,分離、鑒定活性成分,以期篩選出具有開發(fā)潛力的FAS抑制劑。主要研究結果如下:(1)評價了47種植物醇提取物對FAS和MDA-MB-231細胞的抑制活性,包括:構樹Broussonetia papyifera(Linn.)L'Hert.ex Vent.、絡石Trachelospermum jasminoides(Lindl.)Lem.、黃杜鵑Rhododendron molle(Blum)G.Don.、紫藤Sweet wisteria sinensis(Sims)Sweet.、白屈菜Chelidonium majus L.、苦豆子Sophora alopecuroides L.、夾竹桃Nerium indicum Mill.、糙葉樹Aphananthe aspera(Thunb.)Planch.、大血藤Sargentodoxa cuneata(Oliv.)Rehd.、南五味子Kadsura longipedunculata Finet et Gagnep.、鉤吻Gelsemium elegans(Gardn.Champ.)Benth.、黃山欒Koelreuteria bipinnata Franch.var.integrifoliola(Merr.)T.Chen、白背葉Mallotus apelta(Lour.)Muell.Arg.var.apelta、綠葉地錦Parthenocissus laetevirens Rehd、飛燕草Consolida ajacis(L.)Schur、石菖蒲Acorus tatarinowii Schot、蕓實Caesalpinia decapetala(Roth)Alston、烏桕Sapium sebiferum(Linn.)Roxb.、杜英Elaeocarpus decipiens Hemsl.、美國凌霄Campsis radicans(L.)Seem.、水蓼Polygonum hydropiper L.、凬草Humulus scandens(Lour.)Merr.、七葉樹Aesculus chinensis Bunge、山玉蘭Magnolia delavayi Franch.、扶芳藤Euonymus fortunei(Turcz.)Hand.-Mazz.Pueraria lobata(Willd.)Ohwi、珍珠蓮Ficus sarmentosa Buch.-Ham.ex J.E.Sm.var.henryi(King ex Oliv.)Corner、鹽膚木Rhus chinensis Mil、華東野胡桃Jμglans cathayensis Dode var.formosana(Hayata)A.et R.H.Chang、青檀Pteroceltis tatarinowii Maxim.、雞矢藤Paederia scandens(Lour.)Merr.、香花崖豆藤Millettia dielsiana Harms、粉葉爬山虎Parthenocissus thomsonii(Laws.)Planch.、山胡椒Lindera glauca(Sieb.et Zucc.)Bl.、狹葉山胡椒Lindera angustifolia Cheng、大果山胡椒Lindera praecox(Sieb.et Zucc.)Bl.、豚草Ambrosia artemisiifolia L.和苦竹Pleioblastus amarus(Keng)、闊葉箬竹Indocalamus latifolius(Keng)Meclure、毛竹Phyllostachys heterocycla(Carr.)Mitford cv.Pubescens、淡竹Phyllostachys glauca、甜龍竹Dendrocalamus giganteus Munro、水竹Phyllostachys heteroclada Oliver、刺竹B.blumeana Schult.f、佛肚竹Bambusa ventricosa Mc Clure、粉單竹Phyllostachys heteroclada Oliver、香竹Chimonocalamus delicatus Hsueh et Yi。結果表明:黃山欒、南五味子、糙葉樹、大血藤和黃杜鵑提取物對MDA-MB-231細胞具有顯著抑制活性,供試濃度為100 mg/L時,抑制率分別76.68±0.16%、71.02±0.12%、62.70±1.26%、61.54±0.35%和55.79±1.71%,對照藥劑白藜蘆醇的抑制率為75.20±0.77%。黃山欒正丁醇相對MDA-MB-231細胞的IC_(50)值為19.93 mg/L;在100 mg/L時,對FAS的抑制率為84.32%。10種竹葉提取物對MDA-MB-231細胞IC_(50)值為512.45mg/L~647.09mg/L,對FAS的IC_(50)值為187.09~276.45mg/L,苦竹和毛竹具有較強的抑制活性。(2)通過活性追蹤,研究了黃山欒樹葉中抑制FAS和MDA-MB-231細胞的活性成分。黃山欒樹葉乙醇提取物經液液分配,其正丁醇萃取相活性最高,進一步經大孔樹脂分離,60%乙醇相活性最強,對FAS和MDA-MB-231細胞的IC_(50)值分別為19.45 mg/L、11.84mg/L。中壓分離60%乙醇相,活性檢測顯示Fr2為主要活性流分,IC_(50)值分別為12.68 mg/L、5.26 mg/L。利用凝膠色譜和制備色譜對Fr2進行反復分離,得到10個單體,分別為山奈酚-3-O-蕓香糖苷,蘆丁,槲皮素-3-O-β-D-半乳糖苷,山奈酚-3-O-2′′-O-沒食子�；|香糖苷,山柰酚-3-O-α-L-鼠李糖基-(1-6)-β-D-半乳糖苷,槲皮苷,槲皮素-3-O-β-D-吡喃阿拉伯糖苷,紫云英苷-6′′-O-沒食子酸酯,山柰酚-3-O-β-刺槐雙糖苷,山柰酚-3-O-葡萄糖苷�；钚詼y定表明,在10 mg/L和100 mg/L時,10種單體化合物均對FAS表現一定的抑制作用;在50 mg/L時,對MDA-MB-231細胞的抑制作用不顯著。(3)苦竹、毛竹、闊葉箬竹、淡竹竹葉提取物中黃酮含量與其對FAS和MDA-MB-231細胞抑制活性正相關。利用大孔樹脂分離,追蹤活性流分。結果表明,竹葉提取物50%乙醇相為主要抑制FAS活性部位;95%乙醇相為主要抑制MDA-MB-231細胞活性部位流式細胞術檢測苦竹大孔流分誘導MDA-MB-231細胞凋亡作用,發(fā)現70%、95%乙醇相均具有誘導癌細胞凋亡作用,在100、200、400 mg/L濃度下,70%和95%乙醇相對MDA-MB-231細胞的凋亡率分別為11.3%、23.3%、36.7%和11.1%、23.1%、38.7%。(4)建立了同時檢測竹葉中10種黃酮類化合物的UPLC-MS/MS分析方法,明確了竹葉黃酮在不同流分中的含量及分布規(guī)律。竹葉黃酮碳苷主要分布在50%乙醇相;黃酮苷元主要分布在70%乙醇相,結合活性測定結果,推測黃酮碳苷可能是抑制FAS的主要活性成分,且異葒草苷和異牡荊苷可能是主要活性單體;黃酮苷元可能是是抑制MDA-MB-231細胞作用的主要成分。(5)測定了16種竹葉黃酮對FAS的抑制作用,結果表明,異牡荊苷活性最為突出,IC_(50)為4.56mg/L,明顯優(yōu)于陽性對照白藜蘆醇。木犀草素、槲皮素、異葒草苷、芹菜素、山奈酚、苜蓿素均具有顯著抑制活性,IC_(50)值依次為:12.05mg/L、21.71 mg/L、27.67 mg/L、42.81 mg/L、56.60 mg/L、89.70 mg/L。構效關系研究表明,黃酮B環(huán)6號位置的取代基能提高其抑制FAS的活性,C環(huán)上3號位置-OH取代對抑制FAS的活性是不利的,而B環(huán)上-OH越多活性越強,且3′位置的-OH的活性要強于5′位置。木犀草素、槲皮素、芹菜素、山奈酚、苜蓿素、異牡荊苷對MDA-MB-231細胞均具有顯著的抑制活性,IC_(50)值依次為41.10mg/L、51.17mg/L、57.04mg/L、64.44mg/L、145.66mg/L、181.36mg/L,異葒草苷未對MDA-MB-231細胞表現出增殖抑制活性。竹葉黃酮對細胞內FAS抑制活性測定結果表明,在50mg/L時,芹菜素、木犀草素、槲皮素、山奈酚、異牡荊苷和苜蓿素處理MDA-MB-231細胞24 h,胞內FAS剩余酶活為60.74%、58.69%、77.62%、61.12%、89.76%、86.75%,異牡荊苷未對胞內FAS表現出強抑制活性,可能與化合物進入細胞的方式和效率有關。綜上所述,本文評價了47種植物醇提取物對FAS和人乳腺癌(MDA-MB-231)細胞的抑制活性,篩選出黃山欒樹等5種具有開發(fā)FAS抑制劑潛力的植物資源;明確了竹葉黃酮對FAS的抑制作用,發(fā)現異牡荊苷對FAS具有極強的體外抑制活性。研究結果對于黃山欒樹等植物資源的開發(fā)利用以及利用竹葉黃酮開發(fā)FAS抑制劑具有重要參考價值。
[Abstract]:E.C.2.1.3.85 (FAS) is an important enzyme that catalyzes the endogenous growth chain fatty acids and participates in energy metabolism. The study shows that animal FAS is a potential target for cancer treatment. The development of high active and low toxic FAS inhibitors is of great significance for the prevention and control of cancer. This article is targeted at FAS and human breast cancer (MDA-MB-231) cells as the target. The inhibitory activity of 47 kinds of plant alcohol extracts was evaluated. The leaves and bamboo leaves of the Koelreuteria bipinnata Franch.var.integrifoliola (Merr.) T.Chen. (Merr. T.Chen.) were used as the research object. The bioactive tracking was used, and the chemical methods were used to isolate and identify the active components, so as to screen out the potential FAS inhibitors. The results are as follows: (1) the inhibitory activities of 47 kinds of plant alcohol extracts on FAS and MDA-MB-231 cells were evaluated, including Broussonetia papyifera (Linn.) L'Hert.ex Vent., Trachelospermum jasminoides (Lindl.) Lem., Rhododendron Rhododendron. L., Sophora alopecuroides L., Nerium indicum Mill., Aphananthe aspera (Thunb.) Planch.. Foliola (Merr.) T.Chen, Mallotus apelta (Lour.) Muell.Arg.var.apelta, green leaf Kam Parthenocissus laetevirens Rehd. Campsis radicans (L.) Seem., Polygonum hydropiper L. of water Polygonum, Humulus scandens (Lour.) Merr., seven leaf tree Aesculus. Yi (King ex Oliv.) Corner, salt skin Rhus chinensis Mil, East Huadong wild walnut J micron glans cathayensis Dode. Lindera glauca (Sieb.et Zucc.) Bl., narrow leaf pepper Lindera angustifolia Cheng, big fruit mountain pepper Lindera praecox (Sieb.et Zucc.). Ubescens, bamboo Phyllostachys glauca, sweet dragon bamboo Dendrocalamus giganteus Munro, bamboo Phyllostachys heteroclada Oliver, bamboo B.blumeana Schult.f, the bamboo B.blumeana Schult.f. The MDA-MB-231 cells have significant inhibitory activity to MDA-MB-231 cells. When the test concentration is 100 mg/L, the inhibition rates are 76.68 + 0.16%, 71.02 + 0.12%, 62.70 + 1.26%, 61.54 + 0.35% and 55.79 +, respectively. The inhibition rate of resveratrol is 75.20 + 0.77%. IC_ (50) values of MDA-MB-231 cells in Mount Huangshan Luan Luan. For 19.93 mg/L, at 100 mg/L, the inhibition rate to FAS was 84.32%.10 species of bamboo leaf extract, IC_ (50) was 512.45mg/L~647.09mg/L, IC_ (50) value of FAS was 187.09~276.45mg/L, bitter bamboo and Phyllostachys pubescens had strong inhibitory activity. (2) through activity tracing, the inhibition of FAS and MDA-MB-231 cells in the leaves of Mount Huangshan Luan was studied. The ethanol extracts from the leaves of Mount Huangshan tree leaves are the most active in the extraction phase of n-butanol. The activity of the extraction phase of n-butanol is the highest. The activity of the 60% ethanol phase is the strongest, the IC_ (50) value of the FAS and MDA-MB-231 cells is 19.45 mg/L, the 11.84mg/L. medium pressure separation 60% ethanol phase, the activity detection shows that the Fr2 is the main active fraction, and the IC_ (50) value is respectively. 12.68 mg/L and 5.26 mg/L. were repeatedly separated by gel chromatography and preparative chromatography, and 10 monomers were obtained, namely, acetonide -3-O- rutin, rutin, quercetin -3-O- beta -D- galactoside, kaempferol -3-O-2 '-O- gallooside, kaempferol -3-O- alpha -L- mouse rhinosinyl - (1-6) - beta -D- galactoside, quercetin and quercetin -3-O- beta -D- Piran glycoside, -3-O- '-O-' -O- gallate, kaempferol -3-O- beta locust biglycoside, kaempferol -3-O- glucoside. The activity determination showed that the 10 monomers were inhibited at FAS at 10 mg/L and 100 mg/L, and the inhibitory effect on MDA-MB-231 cells was not significant in 50 mg /L. (3) bitter. Bamboo, Phyllostachys pubescens, broadleaf Lo bamboos, the flavonoid content in the extracts from the leaves of light bamboo leaves are positively related to the inhibitory activity of FAS and MDA-MB-231 cells. The results show that the 50% ethanol phase of the bamboo leaf extract mainly inhibits the active part of the FAS, and the 95% ethanol phase is the main inhibition of the flow cytometry of the active part of the MDA-MB-231 cells. The apoptosis of MDA-MB-231 cells was induced by the large pore flow of bitter bamboo. It was found that the 70%, 95% ethanol phase could induce apoptosis of cancer cells. Under the concentration of 100200400 mg/L, the apoptosis rate of 70% and 95% ethanol relative to MDA-MB-231 cells was 11.3%, 23.3%, 36.7% and 11.1%, 23.1%, 38.7% respectively. (4) was established to detect the flavonoids in bamboo leaves at the same time. The content and distribution of flavonoids in bamboo leaves were determined by UPLC-MS/MS analysis. The flavonoid glycosides of bamboo leaves were mainly distributed in the 50% ethanol phase. The flavonoid glycosides were mainly distributed in the 70% ethanol phase. Combined with the results of the activity determination, it was suggested that the flavonoid glycosides may be the main active components to inhibit the FAS, and the isovitex and the isovitex glucoside may be Main active monomer; flavonoid glycosides may be the main components to inhibit the action of MDA-MB-231 cells. (5) the inhibitory effects of 16 kinds of bamboo leaf flavonoids on FAS were measured. The results showed that the activity of isomonidin was the most prominent, IC_ (50) was 4.56mg/L, which was obviously better than the positive control resveratrol, luteolin, quercetin, isovitrine, apigenin, anononol, alalum, and alaline The IC_ (50) value in turn is 12.05mg/L, 21.71 mg/L, 27.67 mg/L, 42.81 mg/L, 56.60 mg/L, and 89.70 mg/L. structure-activity relationship studies show that the substituent of the flavonoid B ring 6 position can improve its activity to inhibit FAS. The -OH substitution on 3 position on the C ring is unfavorable to the inhibition of FAS activity, and the more activity on the ring is stronger, The activity of -OH in the 3 'position was stronger than the 5' position. Luteolin, quercetin, apigenin, alfalfa, alfalfa and alfalfa had significant inhibitory activity to MDA-MB-231 cells, and IC_ (50) values were 41.10mg/L, 51.17mg/L, 57.04mg/L, 64.44mg/L, 145.66mg/ L, 181.36mg/L, and isovitoside did not inhibit the proliferation of MDA-MB-231 cells. Activity. The results of intracellular FAS inhibitory activity of bamboo leaf flavonoids showed that at 50mg/L, apigenin, luteolin, quercetin, Alanin, alfalfa and alfalfa treated MDA-MB-231 cells 24 h, and the intracellular FAS residual enzyme activities were 60.74%, 58.69%, 77.62%, 61.12%, 89.76%, 86.75%. In this paper, the inhibitory activity of 47 kinds of plant alcohol extracts on FAS and human breast cancer (MDA-MB-231) cells was evaluated, and 5 plant resources with the potential of developing FAS inhibitors were screened out in Mount Huangshan Luan tree, and the inhibitory effect of yellow ketone on FAS was clarified, and it was found that the ISO Vitex was in the presence of FAS. The results are of great reference value for the development and utilization of plant resources such as Mount Huangshan Luan tree and the utilization of bamboo leaf flavonoids for the development of FAS inhibitors.
【學位授予單位】：中國林業(yè)科學研究院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R91

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