• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    表面活性素單分子膜在空氣/水界面的遲滯現(xiàn)象

    2011-11-30 10:56:42宋昌盛葉汝強(qiáng)牟伯中
    物理化學(xué)學(xué)報(bào) 2011年9期
    關(guān)鍵詞:原子力華東理工大學(xué)聚集體

    楊 瑩 宋昌盛,2 葉汝強(qiáng) 牟伯中,*

    (1華東理工大學(xué)應(yīng)用化學(xué)研究所,上海200237;2太倉(cāng)出入境檢驗(yàn)檢疫局,江蘇太倉(cāng)215400)

    表面活性素單分子膜在空氣/水界面的遲滯現(xiàn)象

    楊 瑩1宋昌盛1,2葉汝強(qiáng)1牟伯中1,*

    (1華東理工大學(xué)應(yīng)用化學(xué)研究所,上海200237;2太倉(cāng)出入境檢驗(yàn)檢疫局,江蘇太倉(cāng)215400)

    表面活性素是一類具有較強(qiáng)表面活性的微生物脂肽類化合物,能在空氣/水界面形成不溶性單分子膜.利用Langmuir膜天平測(cè)定了表面活性素單分子膜的壓縮-擴(kuò)張循環(huán)曲線,發(fā)現(xiàn)單分子膜在經(jīng)歷了“平臺(tái)區(qū)”后出現(xiàn)較大的遲滯環(huán),遲滯環(huán)的形狀與亞相pH有關(guān).將“平臺(tái)區(qū)”的單分子膜轉(zhuǎn)移到云母表面后,用原子力顯微鏡(AFM)和掃描電子顯微鏡(SEM)均觀察到高度達(dá)幾十至數(shù)百納米的表面聚集體,說(shuō)明表面活性素在單分子膜的“平臺(tái)區(qū)”伴隨著自聚集.研究結(jié)果表明,表面活性素單分子膜在空氣/水界面的遲滯現(xiàn)象是分子浸入亞相和形成三維表面聚集體共同作用的結(jié)果.

    壓縮-擴(kuò)張循環(huán);遲滯環(huán);原子力顯微鏡;掃描電子顯微鏡;表面聚集體

    1 Introduction

    Surfactin is a fascinating group of lipopeptides produced by Bacillus subtilis.1Because of its exceptional surface activities,1-4and peculiar biological properties,5-7surfactin has a broad potential application in many fields,such as cosmetics,food additives,pharmacy,and oil recovery.8,9The structure of surfactin (Fig.1)contains a hydrophilic peptide loop of seven amino acids bonded to a linear hydrophobic fatty acid chain,10and consequently it exhibits amphiphilic behavior which is one of the main forces for self-assembly.11,12It is presumed that its biological activities,such as cytolytic property,5antiviral and hemolytic activities,6and the ability to inhibit formation of fibrin clots,1are a direct consequence of the interaction of surfactin with its target membrane.13To study the interactions between surfactin and biomembrane molecules,it seems important to study the structures and properties of surfactin and its analogues.An interest in the behavior of surfactin layers on water is due to the fact that the surface activity and biological property mostly occur at interfaces.14,15

    Several authors have experimentally investigated the interfacial behavior and molecular organization of surfactin at the air-water interface.4,16-18Maget-Dana and Ptak16firstly reported the interfacial behavior of surfactin.They found that there existed a“plateau region”in the compression isotherm during which the peptide loop of surfactin molecule would adopt a vertical orientation.Based on dark-field electron micrographs of single-layer Langmuir-Blodgett(LB)film of surfactin, Ishigami et al.4proposed that surfactin molecules would rather promote the formation of inhomogeneous multilayer films in three dimensions at high surface pressure.The interfacial behavior of surfactin analogues has been investigated by Eeman et al.17It was found that a more hydrophobic alkyl chain induced a higher surface pressure and the opening of the peptide ring reduced the maximal surface pressure.The plateau regions in isotherms were observed for all samples they used.On the basis of atomic force microscopy(AFM)observation for single-layer LB film,18it appears that surfactin film would transit from monolayer to multilayer during the“plateau region”.According to computer simulation of surfactin conformation at a hydrophobic-hydrophilic interface,Gallet et al.15suggested that some molecules would be organized into clusters or twodimensional micelles during the“plateau”transition.While the plateau is usually considered to be related with the conformational changes in the polar moiety,19,20the origin of the“plateau region”in surfactin monolayer is still unclear.

    Fig.1 Primary structure of surfactinApeptide loop containing seven amino acid residues bonds to a β-hydroxylfatty acid chain with 14 carbon atoms,denoted as SuC14in the context.

    In order to obtain information on molecular behavior of surfactin during the“plateau region”,hysteresis behavior was studied and surface morphologies of single-layer LB films were observed by AFM and scanning electron microscopy (SEM).The contribution of surface-to-bulk diffusion and formation of three-dimensional surface aggregates to the hysteresis loop were also discussed.

    2 Experimental

    2.1 Materials

    Surfactin samples used in the experiment were produced by Bacillus subtilis HSO121,21the isolation,purification,and structural analysis were conducted in our laboratory.22The surfactin with a β-hydroxyl fatty acid chain of 14 carbon atoms (denoted as SuC14)was collected by a semipreparative reversed-phase high performance liquid chromatography (HPLC),and its structure was determined by electrospray ionization quadruple-time-of-flight mass spectrometry(ESI QTOF MS),electroionization gas chromatography/mass spectrometry(EI GC/MS)combined with amino acid analysis and analytical HPLC.Hexane(≥99.0%)and chloroform(≥96.0%) were purchased from Shanghai Reagent Factory.

    2.2 Surface pressure-molecular area(π-A) isotherm and preparation of LB films

    The π-A isotherms were recorded by a computer-controlled film balance(612D,Nima Technology,England).Surfactin was spread onto subphase from its hexane/chloroform(2:1,V/ V)solution with the concentration of 1 mmol·L-1.The subphase was water purified with a Hitech-Kflow system(resistivity 18 MΩ·cm,pH~6),its pH was adjusted with hydrochloric acid or sodium hydroxide and its temperature was controlled by a water bath.After 30 μL of the stock solution was spread, the monolayer film was maintained without compression for at least 15 min to ensure the complete evaporation of the solvents and then compressed by barriers with a speed of 0.1 nm2· min-1·molecule-1.For hysteresis measurements,a 10 s delay was used following each compression or expansion step,prior to the next compression or expansion.Each of the isotherms was performed three times and the isotherms were reproducible.

    Surfactin LB films were prepared by vertical dipping method.At the plateau region in the isotherm,where the change in area with change in surface pressure was large,the pressure control feedback was difficult to control.23Therefore,the films were transferred onto the freshly cleaved mica by the molecular area control.When molecular area reached 0.8 nm2·molecule-1,the LB film was deposited at a speed of 2 mm·min-1.

    2.3 AFM and SEM imaging

    Fig.2 Hysteresis cycles of SuC14monolayer on pure water(pH 6)at 25°C The solid and dashed lines,labeled with 1 and 2 respectively,are two consecutive hysteresis cycles at full expansion. The dotted lines are the hysteresis at 20 mN·m-1.

    Atomic force microscopy(AJ III,Aijian Nanotechnology, China)was used to observe the surface morphologies of LB films.All AFM measurements were performed at room temperature(20°C)in tapping mode,using silicon cantilever(Mikro-Masch Company,Estonia)with a resonance frequency in the range of 240-400 kHz,and a spring constant of 48 N·m-1.The AFM images were obtained with a maximum scan range of 18 μm×18 μm and the scan rate was 1-2 Hz.All images were gained from at least three macroscopically-separated areas on each sample,and at least two independent samples of each type were prepared and observed.

    The LB films were also observed by scanning electron microscopy(JSM-6390A,JEOL,Japan)after being gold-coated.

    3 Results and discussion

    3.1 Hysteresis loop

    Fig.2 shows the compression-expansion cycles of surfactin monolayer on pure water at 25°C.The dotted line exhibits the hysteresis at a lower surface pressure(20 mN·m-1).Two consecutive hysteresis cycles at full trough expansion are shown as the solid and dashed lines,labeled with 1 and 2,respectively.All cycles present the same feature:the expansion isotherm curves are all under the compression curves.However,there is a larger hysteresis at full trough expansion as compared to that expanded at the lower pressure.The 2nd cycle is shifted towards smaller molecular areas than the 1st cycle.

    Fig.3 Hysteresis cycles of SuC14monolayer on acidic subphase (pH 2)and alkaline subphase(pH 10)at 25°C

    We also studied the hysteresis cycles of surfactin monolayer on acidic subphase and alkaline subphase(shown in Fig.3).All these hysteresis cycles present similar characteristics:as the monolayers expanded,a sharp drop in the surface pressure is observed.With subphase pH decreased,hysteresis loop becomes smaller and the expansion isotherm curve undergoes a pseudo plateau.It is presumed that the difference results from the surface-to-bulk diffusion of molecules during the plateau region.At low pH,the peptide loop can be hardly ionized.As the pH increases,the peptide loop in surfactin tends to be ionized,and the increase of hydrophilicity leads some molecules to submerge into subphase.The solubilization and chain reorganization processes of molecules require time to return to their surface-adsorbed state at expanded areas.24The“l(fā)oss”of some molecules at interface at expansion areas induces lower surface pressure.

    3.2 Surface morphology studies

    To obtain information on the molecular behavior of surfactin in the plateau region,surfactin films were imaged by AFM after LB deposition.

    Fig.4 shows the representative AFM topographic images of SuC14LB films prepared at the subphase pH 2,6,and 10.The LB films were all transferred at the plateau region.At pH 2, regular spheres shown in Fig.4A are observed.The diameters of these spheres are in a scale of hundreds of nanometers,the heights range from tens to hundreds nanometers.At pH 6,the AFM image(Fig.4B)shows quasi-circular aggregates,and the size distribution is similar to that observed in Fig.4A.At pH 10,the number of aggregates is greatly decreased(Fig.4C). However,with the addition of calcium cations into the alkaline subphase,more aggregates which were bigger and higher were observed(Fig.4D).

    Fig.4 Top viewAFM images of single-layer SuC14LB film deposited at the plateau regions on water subphase(A)pH 2,(B)pH 6,(C)pH 10,(D)pH 10(containing calcium cation).All LB films were transferred at the pressure to which molecular area reaching at 0.8 nm2·molecule-1.

    Fig.5 SEM images of single-layer SuC14LB film deposited at the plateau regions on water subphase(A)pH 2,(B)pH 6,(C)pH 10(containing calcium cation).All LB films were prepared at the conditions as same as forAFM observations.

    To reveal further details on the distribution of particles observed by AFM,all LB films,which were prepared at the conditions as same as for AFM observations,were characterized by SEM.The SEM micrographs are presented in Fig.5.At pH 2 and 6,homogeneous distributions of circular particles are observed,with the diameter of(393±52)and(371±101)nm,respectively.The particles in Fig.5C exhibit a much larger size but fewer amount compared with those in Fig.5(A,B).No particles are observed by SEM for LB film prepared on alkaline subphase(pH 10)(data not shown),which is probably due to the low contrast between the targets and background.

    The correspondence between the AFM images and SEM micrographs reveals that it would be a general phenomenon for surfactins that three-dimensional structures are formed in the plateau regions(for SuC13and SuC15,similar plateau regions and three-dimensional molecular particles are also observed, data not shown).For SuC14,the hydrophilic segment in molecule structure consists of a peptide loop of seven amino acids with two carboxyl residues(Glu1 and Asp5 in Fig.1).Several studies have proven that there exists hydrogen bond in surfactin molecules.4,15,26At low subphase pH,where the solubilization of surfactin molecule is low,the π-A isotherm shows a higher transition pressure reflecting the strong tendency of surfactin spreading on the subphase surface,and the protonation of the carboxylic moieties causes a reduction in both intra-and inter-molecular electrostatic repulsions,allowing a tighter closepacking of the alkyl chains at the interface,25and three hydrogen bonds in the most stable surfactin conformation assembled in monolayer15would be strengthened.Consequently molecules in Langmuir monolayer would pile up into supramolecular structures at high lateral pressure.When subphase is adjusted to pH 6 which is the pK(dissociation constant)of surfactin at interface,16the carboxyl groups are partially deprotonated, partial loss of surfactin molecules from the interface during the compression would occur besides the formation of supramolecular structures at high lateral pressure.At pH 10,the solubility of surfactin in water increase with ionization of the peptide loop,16which would induce molecules submerging into the subphase.On the other hand,the ionization of the peptide loop would greatly reduce the inter-and intra-molecular hydrogen bonds and hence surfactin molecules can hardly aggregate into supramolecular structure.However,the addition of calcium cations into the alkaline subphase would neutralize the two acidic amino residues and reduce electrostatic repulsions resulted from the ionization of the peptide loop.

    4 Conclusions

    The hysteresis behavior of surfactin monolayer on water subphase with different pH values and the LB film morphologies on mica substrates were investigated in this study.When compression cycle reached a plateau region at higher surface pressure,the expansion cycle showed a sharp drop in the surface pressure.With subphase pH decreased,hysteresis loop became smaller and the expansion isotherm underwent a pseudo plateau.AFM and SEM images of LB films at plateau region indicated that,when peptide loop in molecule structure was protonated or partially ionized,surfactin in monolayer could pile up into three-dimensional surface aggregates with the heights range from tens to hundreds nanometers.However,when peptide loop was fully ionized,surfactin in monolayer would prefer to submerge into subphase.Based on above findings,it is proposed that the formation of three-dimensional surface aggregates during the high-pressure transition induces the large hysteresis loop in surfactin monolayer,which is also attributed to the submergence of molecules into subphase when the peptide loop is ionized.The results presented here,concerning the hysteresis behavior in surfactin monolayer,provide new insights into the interfacial behavior of the amphiphilic lipopeptide.It is believed to be important to understand peculiar properties.

    However,when examining LB film by AFM and SEM,it is difficult to fully determine whether the observed structure is a characteristic of the original monolayer on water or a result of structural change upon the film transfer.As a result,in situ technologies,such as Brewster angle microscopy and in situ infrared spectroscopy,would be applied to acquire structural information at the molecular level in future.

    (1)Arima,K.;Kakinuma,A.;Tamura,G.Biochem.Biophys.Res. Commun.1968,31,488.

    (2) Deleu,M.;Razafindralambo,H.;Popineau,Y.;Jacques,P.; Thonart,P.;Paquot,M.Colloids Surf.A 1999,152,3.

    (3) Morikawa,M.;Hirata,Y.;Imanaka,T.Biochim.Biophys.Acta 2000,1488,211.

    (4) Ishigami,Y.;Osman,M.;Nakahara,H.;Sano,Y.;Ishiguro,R.; Matsumoto,M.Colloids Surf.B 1995,4,341.

    (5) Bernheimer,A.W.;Avigad,L.S.J.Gen.Microbiol.1970,61, 361.

    (6) Vollenbroich,D.;Pauli,G.;Ozel,M.;Vater,J.Appl.Environ. Microbiol.1997,63,44.

    (7) Kracht,M.;Rokos,H.;Ozel,M.;Kowall,M.;Pauli,G.; Vater,J.J.Antibiot.1999,52,613.

    (8) Desai,J.D.;Banat,I.M.Microbiol.Mol.Biol.Rev.1997, 61,47.

    (9) Schaller,K.D.;Fox,S.L.;Bruhn,D.F.;Noah,K.S.;Bala, G.A.Appl.Biochem.Biotechnol.2004,115,827.

    (10) Yang,S.Z.;Wei,D.Z.;Mu,B.Z.J.Biochem.Biophys. Methods 2006,68,69.

    (11) Lang,S.Curr.Opin.Colloid Interface Sci.2002,7,12.

    (12) Terheiden,A.;Rellinghaus,B.;Stappert,S.;Acet,M.; Mayer,C.J.Chem.Phys.2004,121,510.

    (13) Grau,A.;Gomez-Fernandez,J.C.;Peypoux,F.;Ortiz,A. Biochim.Biophys.Acta 1999,1418,307.

    (14) Infante,M.R.;Moses,V.Int.J.Pept.Protein Res.1994,43, 173.

    (15) Gallet,X.;Deleu,M.;Razafindralambo,H.;Jacques,P.; Thonart,P.;Paquot,M.;Brasseur,R.Langmuir 1999,15, 2409.

    (16) Maget-Dana,R.;Ptak,M.J.Colloid Interface Sci.1992, 153,285.

    (17) Eeman,M.;Berquand,A.;Dufrene,Y.F.;Paquot,M.; Dufour,S.;Deleu,M.Langmuir 2006,22,11337.

    (18) Song,C.S.;Ye,R.Q.;Mu,B.Z.Colloids Surf.A 2007,302, 82.

    (19) Youm,S.G.;Paeng,K.;Choi,Y.W.;Park,S.;Sohn,D.;Seo, Y.S.;Satija,S.K.;Kim,B.G.;Kim,S.;Park,S.Y. Langmuir 2005,21,5647.

    (20) Islam,M.N.;Kato,T.J.Chem.Phys.2004,121,10217.

    (21) Lü,Y.N.;Yang,S.Z.;Mu,B.Z.Microbiology 2005,32,67. [呂應(yīng)年,楊世忠,牟伯中.微生物學(xué)通報(bào),2005,32,67.]

    (22) Liu,X.;Haddad,N.I.A.;Yang,S.;Mu,B.Protein Pept. Lett.2007,14,766.

    (23) Alonso,C.;Alig,T.;Yoon,J.;Bringezu,F.;Warriner,H.; Zasadzinski,J.A.Biophys.J.2004,87,4188.

    (24) Zhu,J.;Eisenberg,A.;Lennox,R.B.Macromolecules 1992, 25,6547.

    (25) Petriat,F.;Giasson,S.Langmuir 2005,21,7326.

    (26) Song,C.S.;Ye,R.Q.;Mu,B.Z.Colloids Surf.A 2008,330,49.

    May 17,2011;Revised:June 29,2011;Published on Web:July 11,2011.

    Hysteresis Behavior of Surfactin Monolayer at the Air/Water Interface

    YANG Ying1SONG Chang-Sheng1,2YE Ru-Qiang1MU Bo-Zhong1,*
    (1Institute of Applied Chemistry,East China University of Science and Technology,Shanghai 200237,P.R.China;
    2Taicang Entry-Exit Inspection and Quarantine Bureau,Taicang 215400,Jiangsu Province,P.R.China)

    Surfactin,one of the most surface-active microbial lipopeptides,can readily form an insoluble monolayer at the air/water interface.Consecutive compression-expansion cycles of surfactin with a β-hydroxyl fatty acid chain consisting of 14 carbon atoms were studied by a Langmuir film balance.A larger hysteresis loop was observed when the compression isotherm reached a plateau compared with that expanded at a lower surface pressure(20 mN·m-1).The 2nd cycle was shifted towards smaller molecular areas compared with the 1st cycle.We also studied the hysteresis cycles of the surfactin monolayer on subphase of different pH values.With a decrease in the subphase pH the hysteresis loop became smaller and the expansion isotherm curve underwent a longer pseudo plateau.Furthermore,the morphologies of the surfactin monolayers in the plateau region,which were transferred onto a mica surface,were characterized by atomic force microscopy(AFM)and scanning electron microscopy(SEM).Both AFM and SEM images gave three-dimensional surface aggregates with heights ranging from tens to hundreds of nanometers.The above results suggest that the formation of three-dimensional surface aggregates at the plateau region induces a large hysteresis loop in the surfactin monolayer,which can also be attributed to the submergence of molecules into the subphase when the peptide loop in the surfactin molecule is ionized.

    Compression-expansion cycle;Hysteresis loop;AFM;SEM;Surface aggregate

    O647

    ?Corresponding author.Email:bzmu@ecust.edu.cn;Tel:+86-21-64252063;Fax:+86-21-64252458.

    The project was supported by the National High Technology Research and Development Program of China(863)(2009AA063503).

    國(guó)家高技術(shù)研究發(fā)展計(jì)劃項(xiàng)目(863)(2009AA063503)資助

    猜你喜歡
    原子力華東理工大學(xué)聚集體
    銅納米簇聚集體的合成、發(fā)光與胞內(nèi)溫度傳感
    一種新型聚集誘導(dǎo)發(fā)光的片狀銀納米簇聚集體的合成
    原子力顯微鏡(AFM)用于瀝青老化行為微觀表征研究綜述
    石油瀝青(2022年3期)2022-08-26 09:13:44
    類胡蘿卜素聚集體的研究進(jìn)展
    華東理工大學(xué)藝術(shù)設(shè)計(jì)與傳媒學(xué)院設(shè)計(jì)作品選登
    單浩作品選登
    原子力顯微鏡—熒光顯微鏡聯(lián)用技術(shù)在活細(xì)胞單分子檢測(cè)中的應(yīng)用
    The Immoral Duchess
    原子力顯微鏡在材料成像中的應(yīng)用
    化工管理(2015年8期)2015-12-21 08:37:22
    水合物聚集體受力分析及臨界聚集體粒徑的計(jì)算
    石油化工(2014年1期)2014-06-07 05:57:08
    国产日韩一区二区三区精品不卡| 亚洲欧美一区二区三区国产| 免费少妇av软件| 岛国毛片在线播放| 亚洲欧洲国产日韩| 美女福利国产在线| 自线自在国产av| 99国产精品免费福利视频| 午夜福利网站1000一区二区三区| 一边亲一边摸免费视频| 国产老妇伦熟女老妇高清| 免费黄网站久久成人精品| 91精品三级在线观看| 男女边摸边吃奶| 国产成人精品婷婷| 99久久精品国产国产毛片| 丁香六月天网| 99视频精品全部免费 在线| 国产极品粉嫩免费观看在线| 精品国产国语对白av| 亚洲av综合色区一区| 考比视频在线观看| 大香蕉久久成人网| 午夜免费鲁丝| 久久人人97超碰香蕉20202| 精品少妇黑人巨大在线播放| 日韩中字成人| av国产精品久久久久影院| 亚洲成人一二三区av| 亚洲国产精品999| 婷婷色麻豆天堂久久| 日本猛色少妇xxxxx猛交久久| 热99国产精品久久久久久7| 日本91视频免费播放| 欧美最新免费一区二区三区| 亚洲成国产人片在线观看| 最后的刺客免费高清国语| av线在线观看网站| 国产有黄有色有爽视频| 久久久国产一区二区| 亚洲av免费高清在线观看| 精品亚洲成国产av| 国产熟女欧美一区二区| 18禁裸乳无遮挡动漫免费视频| 咕卡用的链子| 国产免费又黄又爽又色| 日韩av在线免费看完整版不卡| 九色成人免费人妻av| 五月天丁香电影| 国产又爽黄色视频| 国产女主播在线喷水免费视频网站| 日韩中文字幕视频在线看片| 下体分泌物呈黄色| 欧美老熟妇乱子伦牲交| 亚洲天堂av无毛| 看免费av毛片| 精品亚洲乱码少妇综合久久| 在线看a的网站| 777米奇影视久久| 高清欧美精品videossex| 91成人精品电影| 国产av码专区亚洲av| 亚洲欧美成人综合另类久久久| 久久青草综合色| 精品卡一卡二卡四卡免费| 国产一级毛片在线| 久久毛片免费看一区二区三区| 老司机影院毛片| 国产一区二区在线观看av| 王馨瑶露胸无遮挡在线观看| 国产成人精品一,二区| av.在线天堂| 少妇的丰满在线观看| av不卡在线播放| 国产精品麻豆人妻色哟哟久久| 中文字幕人妻丝袜制服| 国国产精品蜜臀av免费| 国产精品久久久久久精品古装| 亚洲,欧美,日韩| 哪个播放器可以免费观看大片| 看免费av毛片| 国产亚洲av片在线观看秒播厂| 精品国产露脸久久av麻豆| 我的女老师完整版在线观看| 久久国内精品自在自线图片| 久久国产精品男人的天堂亚洲 | 下体分泌物呈黄色| 亚洲成av片中文字幕在线观看 | 最近最新中文字幕免费大全7| 在线观看免费高清a一片| av线在线观看网站| 国产女主播在线喷水免费视频网站| 国产亚洲午夜精品一区二区久久| 成人二区视频| www.av在线官网国产| 精品福利永久在线观看| 日本午夜av视频| 狂野欧美激情性xxxx在线观看| a级毛色黄片| 欧美激情国产日韩精品一区| 26uuu在线亚洲综合色| 国产免费视频播放在线视频| 亚洲欧美色中文字幕在线| 成人毛片60女人毛片免费| 夫妻午夜视频| 欧美 日韩 精品 国产| videosex国产| 各种免费的搞黄视频| 老司机亚洲免费影院| 青春草视频在线免费观看| 国产精品 国内视频| 伦理电影免费视频| 欧美国产精品va在线观看不卡| 日本欧美视频一区| 最近中文字幕2019免费版| 日韩av不卡免费在线播放| 日本91视频免费播放| 国产午夜精品一二区理论片| 在线天堂中文资源库| 亚洲欧美精品自产自拍| 国产国语露脸激情在线看| 两个人看的免费小视频| 中文字幕精品免费在线观看视频 | 亚洲第一区二区三区不卡| 日本午夜av视频| 伦理电影免费视频| 女性被躁到高潮视频| 久久精品国产鲁丝片午夜精品| 精品少妇黑人巨大在线播放| 一级,二级,三级黄色视频| 中国三级夫妇交换| 久久97久久精品| 久久精品久久久久久久性| 日韩精品免费视频一区二区三区 | 国产亚洲欧美精品永久| 婷婷色av中文字幕| 制服丝袜香蕉在线| 久久青草综合色| av黄色大香蕉| 免费高清在线观看视频在线观看| 一级毛片我不卡| 精品一品国产午夜福利视频| 欧美日韩精品成人综合77777| 咕卡用的链子| 人人妻人人爽人人添夜夜欢视频| 中国三级夫妇交换| 蜜桃国产av成人99| 男女下面插进去视频免费观看 | 成人亚洲精品一区在线观看| 多毛熟女@视频| 亚洲精品中文字幕在线视频| 亚洲国产精品一区二区三区在线| 91在线精品国自产拍蜜月| av在线app专区| 欧美精品人与动牲交sv欧美| 久久韩国三级中文字幕| 亚洲人成网站在线观看播放| 亚洲国产av新网站| 秋霞在线观看毛片| 国产有黄有色有爽视频| 视频中文字幕在线观看| av在线app专区| 一级,二级,三级黄色视频| av国产精品久久久久影院| av在线老鸭窝| 免费人妻精品一区二区三区视频| 中文字幕精品免费在线观看视频 | 亚洲成人手机| 热99久久久久精品小说推荐| 国产成人免费观看mmmm| 性色av一级| 久久久久久久久久人人人人人人| 中国国产av一级| 久久精品国产a三级三级三级| 永久网站在线| 国精品久久久久久国模美| 国产成人精品一,二区| av在线观看视频网站免费| 美女福利国产在线| a级片在线免费高清观看视频| 亚洲欧美成人综合另类久久久| 精品一区二区三区视频在线| 看十八女毛片水多多多| 亚洲欧洲精品一区二区精品久久久 | 亚洲精品,欧美精品| 18禁在线无遮挡免费观看视频| 欧美97在线视频| 黄片无遮挡物在线观看| 多毛熟女@视频| 亚洲欧美日韩卡通动漫| 日本wwww免费看| 成人影院久久| 女性被躁到高潮视频| 国产日韩欧美在线精品| 99国产综合亚洲精品| 免费高清在线观看视频在线观看| 国产亚洲午夜精品一区二区久久| 成年人午夜在线观看视频| 午夜福利视频在线观看免费| 国产白丝娇喘喷水9色精品| 观看av在线不卡| 综合色丁香网| 国产一级毛片在线| 成年人免费黄色播放视频| 日本av免费视频播放| 天堂8中文在线网| 久久精品久久久久久久性| 人人妻人人爽人人添夜夜欢视频| 国产激情久久老熟女| 欧美亚洲日本最大视频资源| 天天躁夜夜躁狠狠久久av| 日日摸夜夜添夜夜爱| 夜夜爽夜夜爽视频| 日韩精品有码人妻一区| 国产精品一区www在线观看| 伦理电影大哥的女人| 97在线视频观看| 成年av动漫网址| 久久久久久久久久久久大奶| 一本大道久久a久久精品| 高清视频免费观看一区二区| 少妇的逼好多水| 亚洲精品国产av蜜桃| 精品福利永久在线观看| 午夜福利影视在线免费观看| 国产一区二区激情短视频 | 国产免费一区二区三区四区乱码| 久久精品国产亚洲av涩爱| 国产欧美另类精品又又久久亚洲欧美| 国产深夜福利视频在线观看| 国产极品天堂在线| 久久久久网色| 国产精品久久久久久久电影| 黄色毛片三级朝国网站| 亚洲人成网站在线观看播放| 国产亚洲欧美精品永久| 久久久国产欧美日韩av| 草草在线视频免费看| 侵犯人妻中文字幕一二三四区| 国产男女超爽视频在线观看| 91在线精品国自产拍蜜月| 午夜免费观看性视频| 日韩精品有码人妻一区| 国产亚洲一区二区精品| 亚洲精品视频女| 人妻人人澡人人爽人人| 亚洲成色77777| 精品第一国产精品| 午夜免费男女啪啪视频观看| 自拍欧美九色日韩亚洲蝌蚪91| 黄色 视频免费看| 成人毛片a级毛片在线播放| 久久久久久久精品精品| 久热这里只有精品99| 久久精品国产a三级三级三级| 2022亚洲国产成人精品| 午夜激情av网站| 99精国产麻豆久久婷婷| 嫩草影院入口| 成人免费观看视频高清| 视频中文字幕在线观看| 日韩成人伦理影院| 免费av中文字幕在线| 久久精品国产鲁丝片午夜精品| 国产精品成人在线| av女优亚洲男人天堂| 夫妻性生交免费视频一级片| 亚洲精品av麻豆狂野| 国产精品国产av在线观看| 十分钟在线观看高清视频www| 晚上一个人看的免费电影| 国产精品 国内视频| 人妻人人澡人人爽人人| www.av在线官网国产| 久久午夜综合久久蜜桃| 另类亚洲欧美激情| 有码 亚洲区| 免费黄频网站在线观看国产| 国产成人免费观看mmmm| 久久精品国产鲁丝片午夜精品| 男人操女人黄网站| 久久久精品区二区三区| 国产精品国产三级专区第一集| 免费日韩欧美在线观看| 在线天堂最新版资源| 高清不卡的av网站| 国产成人一区二区在线| 久久午夜综合久久蜜桃| 18+在线观看网站| 亚洲综合色网址| 国产深夜福利视频在线观看| 亚洲欧洲国产日韩| 久久人人97超碰香蕉20202| 国产精品成人在线| 性色av一级| 男女边摸边吃奶| 老司机影院成人| 大陆偷拍与自拍| 一二三四中文在线观看免费高清| 精品少妇黑人巨大在线播放| 在线观看美女被高潮喷水网站| 欧美变态另类bdsm刘玥| 国产精品欧美亚洲77777| 国产成人精品一,二区| 亚洲国产精品专区欧美| 黑人巨大精品欧美一区二区蜜桃 | 午夜福利视频在线观看免费| 一级毛片 在线播放| 日韩制服丝袜自拍偷拍| 亚洲av成人精品一二三区| 香蕉精品网在线| 国产精品成人在线| 婷婷色av中文字幕| 亚洲天堂av无毛| 国产精品一二三区在线看| 伊人亚洲综合成人网| 99久久精品国产国产毛片| 成人亚洲欧美一区二区av| av国产精品久久久久影院| 深夜精品福利| 色视频在线一区二区三区| 日韩成人伦理影院| 人人澡人人妻人| 国产又色又爽无遮挡免| 久久ye,这里只有精品| 免费人成在线观看视频色| 亚洲精品乱久久久久久| 免费少妇av软件| 一本—道久久a久久精品蜜桃钙片| 99久久人妻综合| 日韩精品有码人妻一区| 久久精品国产亚洲av天美| 国产一区二区在线观看日韩| 久久久久久久大尺度免费视频| 大香蕉久久成人网| 成人二区视频| 看非洲黑人一级黄片| 成人二区视频| 久久精品国产自在天天线| 婷婷成人精品国产| 日本午夜av视频| 国产精品久久久久久精品古装| av在线观看视频网站免费| 99热全是精品| 亚洲精品乱码久久久久久按摩| 久久久久网色| 久久久亚洲精品成人影院| 男人爽女人下面视频在线观看| 欧美日韩一区二区视频在线观看视频在线| 亚洲综合精品二区| 最近最新中文字幕大全免费视频 | 日本av免费视频播放| 韩国高清视频一区二区三区| 成人手机av| 一级黄片播放器| 日韩一本色道免费dvd| 9热在线视频观看99| 天天操日日干夜夜撸| 国产片特级美女逼逼视频| 欧美xxⅹ黑人| 国产片特级美女逼逼视频| 国产福利在线免费观看视频| 人妻少妇偷人精品九色| 国产黄色免费在线视频| 另类精品久久| 欧美人与性动交α欧美精品济南到 | 午夜福利影视在线免费观看| 亚洲欧美精品自产自拍| 9热在线视频观看99| 美女大奶头黄色视频| 王馨瑶露胸无遮挡在线观看| 日韩在线高清观看一区二区三区| 欧美另类一区| 两个人免费观看高清视频| 99热国产这里只有精品6| 久久人人爽人人片av| 国产黄色免费在线视频| 人妻少妇偷人精品九色| 国产有黄有色有爽视频| 久久久久视频综合| 在线观看免费日韩欧美大片| 欧美国产精品一级二级三级| 天天躁夜夜躁狠狠躁躁| 永久免费av网站大全| 91精品伊人久久大香线蕉| 久久久久久人妻| 亚洲色图 男人天堂 中文字幕 | 亚洲人与动物交配视频| 香蕉丝袜av| 国产亚洲最大av| av黄色大香蕉| 天堂中文最新版在线下载| 观看美女的网站| 国产成人精品一,二区| 最黄视频免费看| 美国免费a级毛片| 国产精品麻豆人妻色哟哟久久| 91精品三级在线观看| 人人澡人人妻人| 亚洲精品一二三| 亚洲国产av新网站| 人妻系列 视频| 天堂中文最新版在线下载| 国精品久久久久久国模美| 国产成人一区二区在线| 精品久久蜜臀av无| 国产欧美日韩一区二区三区在线| 欧美激情 高清一区二区三区| 国产亚洲最大av| 一区二区日韩欧美中文字幕 | 毛片一级片免费看久久久久| 亚洲,欧美精品.| www.色视频.com| 久久精品久久久久久久性| 伊人亚洲综合成人网| 丰满少妇做爰视频| 欧美激情极品国产一区二区三区 | 不卡视频在线观看欧美| 黑人欧美特级aaaaaa片| 秋霞伦理黄片| 国产一区二区在线观看日韩| 欧美成人午夜精品| 制服丝袜香蕉在线| 欧美人与性动交α欧美软件 | 亚洲五月色婷婷综合| 女人久久www免费人成看片| 久久国产亚洲av麻豆专区| 成人午夜精彩视频在线观看| 少妇被粗大猛烈的视频| 一边摸一边做爽爽视频免费| 久久人妻熟女aⅴ| 国产免费一区二区三区四区乱码| 免费在线观看完整版高清| 一本色道久久久久久精品综合| 十八禁高潮呻吟视频| 少妇人妻久久综合中文| 欧美成人午夜免费资源| 香蕉国产在线看| 国产精品99久久99久久久不卡 | 国产亚洲精品第一综合不卡 | 免费看av在线观看网站| 母亲3免费完整高清在线观看 | 精品第一国产精品| 久热这里只有精品99| 亚洲精品色激情综合| 老熟女久久久| 日产精品乱码卡一卡2卡三| 青春草视频在线免费观看| 男女免费视频国产| 久久综合国产亚洲精品| 久久精品熟女亚洲av麻豆精品| 亚洲成av片中文字幕在线观看 | 18在线观看网站| 美女xxoo啪啪120秒动态图| 人妻 亚洲 视频| 国产精品免费大片| 乱码一卡2卡4卡精品| 国产成人免费观看mmmm| 新久久久久国产一级毛片| 精品一区二区免费观看| 国产爽快片一区二区三区| 亚洲天堂av无毛| 欧美成人精品欧美一级黄| 一区二区三区精品91| 少妇熟女欧美另类| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 一个人免费看片子| 亚洲av成人精品一二三区| 精品久久久精品久久久| 亚洲国产精品成人久久小说| 精品国产一区二区三区久久久樱花| 国产高清国产精品国产三级| 国产在线一区二区三区精| 国产 一区精品| 最近的中文字幕免费完整| 在线看a的网站| 最后的刺客免费高清国语| 亚洲成色77777| 男男h啪啪无遮挡| 久久久欧美国产精品| 大陆偷拍与自拍| 午夜av观看不卡| 三上悠亚av全集在线观看| 婷婷色综合大香蕉| 热re99久久精品国产66热6| 全区人妻精品视频| 免费日韩欧美在线观看| 欧美精品一区二区大全| 九色亚洲精品在线播放| 日韩av在线免费看完整版不卡| 欧美xxxx性猛交bbbb| 精品人妻偷拍中文字幕| 国产 精品1| 岛国毛片在线播放| 91精品伊人久久大香线蕉| 国产在线视频一区二区| 丰满迷人的少妇在线观看| 国产av一区二区精品久久| √禁漫天堂资源中文www| 精品一品国产午夜福利视频| 久久久国产一区二区| 精品视频人人做人人爽| 九色成人免费人妻av| 久久婷婷青草| 久久久精品94久久精品| 亚洲人成网站在线观看播放| 狂野欧美激情性xxxx在线观看| 亚洲图色成人| 1024视频免费在线观看| 春色校园在线视频观看| 91精品伊人久久大香线蕉| 国产欧美日韩一区二区三区在线| 国产欧美日韩综合在线一区二区| 三上悠亚av全集在线观看| 观看av在线不卡| 中文字幕免费在线视频6| 99热网站在线观看| 久久久a久久爽久久v久久| 桃花免费在线播放| 亚洲伊人色综图| 十分钟在线观看高清视频www| 免费黄频网站在线观看国产| 亚洲一级一片aⅴ在线观看| 天天影视国产精品| 在线观看三级黄色| 成年女人在线观看亚洲视频| 免费av中文字幕在线| 人妻一区二区av| 亚洲欧美日韩卡通动漫| 久久精品人人爽人人爽视色| 高清不卡的av网站| 国产成人精品在线电影| 美女福利国产在线| 五月开心婷婷网| av国产久精品久网站免费入址| 欧美日韩综合久久久久久| 又黄又粗又硬又大视频| 一级毛片黄色毛片免费观看视频| 国产亚洲精品久久久com| 纵有疾风起免费观看全集完整版| 国产精品99久久99久久久不卡 | 亚洲精品日本国产第一区| 男的添女的下面高潮视频| 久久久久国产精品人妻一区二区| 男女边摸边吃奶| 韩国高清视频一区二区三区| 成人黄色视频免费在线看| 九九爱精品视频在线观看| 精品国产一区二区久久| 亚洲欧美色中文字幕在线| 午夜久久久在线观看| 熟女av电影| 久久97久久精品| 免费高清在线观看日韩| 国产亚洲精品久久久com| 午夜福利网站1000一区二区三区| 一二三四在线观看免费中文在 | 亚洲 欧美一区二区三区| 日韩熟女老妇一区二区性免费视频| 成人无遮挡网站| 国产福利在线免费观看视频| 狂野欧美激情性bbbbbb| 91国产中文字幕| 免费黄网站久久成人精品| 女人精品久久久久毛片| 亚洲国产看品久久| 亚洲欧洲精品一区二区精品久久久 | 精品国产乱码久久久久久小说| 日韩电影二区| av卡一久久| 亚洲精品日韩在线中文字幕| 老女人水多毛片| 天堂8中文在线网| 国产欧美亚洲国产| 久久久久国产精品人妻一区二区| 七月丁香在线播放| 亚洲丝袜综合中文字幕| 制服人妻中文乱码| 国产日韩欧美亚洲二区| 99国产综合亚洲精品| 精品国产国语对白av| 大话2 男鬼变身卡| 日韩 亚洲 欧美在线| 只有这里有精品99| 女的被弄到高潮叫床怎么办| 亚洲av成人精品一二三区| 久久久a久久爽久久v久久| 国产精品三级大全| 精品少妇黑人巨大在线播放| 亚洲高清免费不卡视频| 日本黄大片高清| 人妻系列 视频| 国产欧美日韩综合在线一区二区| 久久久久久久亚洲中文字幕| 在线观看国产h片| 国产白丝娇喘喷水9色精品| 老司机亚洲免费影院| 只有这里有精品99| 久久精品国产亚洲av天美| 午夜激情久久久久久久| 人妻一区二区av| 99九九在线精品视频| 午夜福利网站1000一区二区三区| 丰满饥渴人妻一区二区三| 国产一区二区在线观看av| 精品一品国产午夜福利视频| 天美传媒精品一区二区| 久久女婷五月综合色啪小说| 曰老女人黄片| 亚洲色图 男人天堂 中文字幕 | 免费看光身美女| 一区二区三区乱码不卡18| 久久免费观看电影| 99热这里只有是精品在线观看| 国产一区亚洲一区在线观看|