47种植物提取物对脂肪酸合酶及乳腺癌细胞抑制作用

发布时间：2018-06-07 17:05

  本文选题：脂肪酸合酶 + 人乳腺癌细胞　； 参考：《中国林业科学研究院》2017年博士论文

【摘要】：脂肪酸合酶(E.C.2.1.3.85,FAS)是催化生物体内合成长链脂肪酸,参与能量代谢的一种重要的酶。研究表明,动物FAS是癌症治疗的潜在靶点。开发高活性、低毒的FAS抑制剂,对于癌症的防治具有重大意义。本文以FAS和人乳腺癌(MDA-MB-231)细胞为靶标,评价了47种植物醇提取物的抑制活性;以黄山栾树(Koelreuteria bipinnata Franch.var.integrifoliola(Merr.)T.Chen.)叶和竹叶为研究对象,采用生物活性追踪,利用植物化学手段,分离、鉴定活性成分,以期筛选出具有开发潜力的FAS抑制剂。主要研究结果如下:(1)评价了47种植物醇提取物对FAS和MDA-MB-231细胞的抑制活性,包括:构树Broussonetia papyifera(Linn.)L'Hert.ex Vent.、络石Trachelospermum jasminoides(Lindl.)Lem.、黄杜鹃Rhododendron molle(Blum)G.Don.、紫藤Sweet wisteria sinensis(Sims)Sweet.、白屈菜Chelidonium majus L.、苦豆子Sophora alopecuroides L.、夹竹桃Nerium indicum Mill.、糙叶树Aphananthe aspera(Thunb.)Planch.、大血藤Sargentodoxa cuneata(Oliv.)Rehd.、南五味子Kadsura longipedunculata Finet et Gagnep.、钩吻Gelsemium elegans(Gardn.Champ.)Benth.、黄山栾Koelreuteria bipinnata Franch.var.integrifoliola(Merr.)T.Chen、白背叶Mallotus apelta(Lour.)Muell.Arg.var.apelta、绿叶地锦Parthenocissus laetevirens Rehd、飞燕草Consolida ajacis(L.)Schur、石菖蒲Acorus tatarinowii Schot、芸实Caesalpinia decapetala(Roth)Alston、乌桕Sapium sebiferum(Linn.)Roxb.、杜英Elaeocarpus decipiens Hemsl.、美国凌霄Campsis radicans(L.)Seem.、水蓼Polygonum hydropiper L.、凬草Humulus scandens(Lour.)Merr.、七叶树Aesculus chinensis Bunge、山玉兰Magnolia delavayi Franch.、扶芳藤Euonymus fortunei(Turcz.)Hand.-Mazz.Pueraria lobata(Willd.)Ohwi、珍珠莲Ficus sarmentosa Buch.-Ham.ex J.E.Sm.var.henryi(King ex Oliv.)Corner、盐肤木Rhus chinensis Mil、华东野胡桃Jμglans cathayensis Dode var.formosana(Hayata)A.et R.H.Chang、青檀Pteroceltis tatarinowii Maxim.、鸡矢藤Paederia scandens(Lour.)Merr.、香花崖豆藤Millettia dielsiana Harms、粉叶爬山虎Parthenocissus thomsonii(Laws.)Planch.、山胡椒Lindera glauca(Sieb.et Zucc.)Bl.、狭叶山胡椒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)、阔叶箬竹Indocalamus latifolius(Keng)Meclure、毛竹Phyllostachys heterocycla(Carr.)Mitford cv.Pubescens、淡竹Phyllostachys glauca、甜龙竹Dendrocalamus giganteus Munro、水竹Phyllostachys heteroclada Oliver、刺竹B.blumeana Schult.f、佛肚竹Bambusa ventricosa Mc Clure、粉单竹Phyllostachys heteroclada Oliver、香竹Chimonocalamus delicatus Hsueh et Yi。结果表明:黄山栾、南五味子、糙叶树、大血藤和黄杜鹃提取物对MDA-MB-231细胞具有显著抑制活性,供试浓度为100 mg/L时,抑制率分别76.68±0.16%、71.02±0.12%、62.70±1.26%、61.54±0.35%和55.79±1.71%,对照药剂白藜芦醇的抑制率为75.20±0.77%。黄山栾正丁醇相对MDA-MB-231细胞的IC_(50)值为19.93 mg/L;在100 mg/L时,对FAS的抑制率为84.32%。10种竹叶提取物对MDA-MB-231细胞IC_(50)值为512.45mg/L~647.09mg/L,对FAS的IC_(50)值为187.09~276.45mg/L,苦竹和毛竹具有较强的抑制活性。(2)通过活性追踪,研究了黄山栾树叶中抑制FAS和MDA-MB-231细胞的活性成分。黄山栾树叶乙醇提取物经液液分配,其正丁醇萃取相活性最高,进一步经大孔树脂分离,60%乙醇相活性最强,对FAS和MDA-MB-231细胞的IC_(50)值分别为19.45 mg/L、11.84mg/L。中压分离60%乙醇相,活性检测显示Fr2为主要活性流分,IC_(50)值分别为12.68 mg/L、5.26 mg/L。利用凝胶色谱和制备色谱对Fr2进行反复分离,得到10个单体,分别为山奈酚-3-O-芸香糖苷,芦丁,槲皮素-3-O-β-D-半乳糖苷,山奈酚-3-O-2′′-O-没食子酰基芸香糖苷,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-没食子酸酯,山柰酚-3-O-β-刺槐双糖苷,山柰酚-3-O-葡萄糖苷。活性测定表明,在10 mg/L和100 mg/L时,10种单体化合物均对FAS表现一定的抑制作用;在50 mg/L时,对MDA-MB-231细胞的抑制作用不显著。(3)苦竹、毛竹、阔叶箬竹、淡竹竹叶提取物中黄酮含量与其对FAS和MDA-MB-231细胞抑制活性正相关。利用大孔树脂分离,追踪活性流分。结果表明,竹叶提取物50%乙醇相为主要抑制FAS活性部位;95%乙醇相为主要抑制MDA-MB-231细胞活性部位流式细胞术检测苦竹大孔流分诱导MDA-MB-231细胞凋亡作用,发现70%、95%乙醇相均具有诱导癌细胞凋亡作用,在100、200、400 mg/L浓度下,70%和95%乙醇相对MDA-MB-231细胞的凋亡率分别为11.3%、23.3%、36.7%和11.1%、23.1%、38.7%。(4)建立了同时检测竹叶中10种黄酮类化合物的UPLC-MS/MS分析方法,明确了竹叶黄酮在不同流分中的含量及分布规律。竹叶黄酮碳苷主要分布在50%乙醇相;黄酮苷元主要分布在70%乙醇相,结合活性测定结果,推测黄酮碳苷可能是抑制FAS的主要活性成分,且异荭草苷和异牡荆苷可能是主要活性单体;黄酮苷元可能是是抑制MDA-MB-231细胞作用的主要成分。(5)测定了16种竹叶黄酮对FAS的抑制作用,结果表明,异牡荆苷活性最为突出,IC_(50)为4.56mg/L,明显优于阳性对照白藜芦醇。木犀草素、槲皮素、异荭草苷、芹菜素、山奈酚、苜蓿素均具有显著抑制活性,IC_(50)值依次为:12.05mg/L、21.71 mg/L、27.67 mg/L、42.81 mg/L、56.60 mg/L、89.70 mg/L。构效关系研究表明,黄酮B环6号位置的取代基能提高其抑制FAS的活性,C环上3号位置-OH取代对抑制FAS的活性是不利的,而B环上-OH越多活性越强,且3′位置的-OH的活性要强于5′位置。木犀草素、槲皮素、芹菜素、山奈酚、苜蓿素、异牡荆苷对MDA-MB-231细胞均具有显著的抑制活性,IC_(50)值依次为41.10mg/L、51.17mg/L、57.04mg/L、64.44mg/L、145.66mg/L、181.36mg/L,异荭草苷未对MDA-MB-231细胞表现出增殖抑制活性。竹叶黄酮对细胞内FAS抑制活性测定结果表明,在50mg/L时,芹菜素、木犀草素、槲皮素、山奈酚、异牡荆苷和苜蓿素处理MDA-MB-231细胞24 h,胞内FAS剩余酶活为60.74%、58.69%、77.62%、61.12%、89.76%、86.75%,异牡荆苷未对胞内FAS表现出强抑制活性,可能与化合物进入细胞的方式和效率有关。综上所述,本文评价了47种植物醇提取物对FAS和人乳腺癌(MDA-MB-231)细胞的抑制活性,筛选出黄山栾树等5种具有开发FAS抑制剂潜力的植物资源;明确了竹叶黄酮对FAS的抑制作用,发现异牡荆苷对FAS具有极强的体外抑制活性。研究结果对于黄山栾树等植物资源的开发利用以及利用竹叶黄酮开发FAS抑制剂具有重要参考价值。
[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.
【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R91

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