• <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 ?

    Insight into the interaction of inhaled corticosteroids with human serum albumin:A spectroscopic-based study

    2018-03-06 01:51:06CrlottPontremoliNdiBreroGuidoViscrdiSonjVisentin
    Journal of Pharmaceutical Analysis 2018年1期

    Crlott Pontremoli,Ndi Brero,Guido Viscrdi,Sonj Visentin

    aDepartment of Applied Science and Technology(DISAT),Politecnico of Torino,Corso Duca degli Abruzzi 24,Torino 10129,Italy

    bDepartment of Chemistry and NIS Interdepartmental Centre,University of Torino,via Pietro Giuria 7,10125 Torino,Italy

    cMolecular Biotechnology and Health Sciences Department,University of Torino,via Quarello 15,10135 Torino,Italy

    1.Introduction

    The study of protein–drug interactions plays an important role in pharmacokinetics and pharmacodynamics of drugs.They influence the distribution and elimination speed;only non-binding drug can spread and reach the target producing a biological response.One of the most important factors affecting the distribution and the free,active concentration of many administered drugs is binding affinity for human serum albumin(HSA).Drug binding to HSA increases drug half-life and lowers the free drug concentration in blood,which makes it extremely important for clinical care.In early drug discovery,the plasma protein binding is important in order to evaluate drug dosing needs and clearance from the body.

    HSA is the most abundant drug carrier protein,with a well known primary structure.Its tertiary structure has been determined by X-ray crystallography[1].It has an important role in maintaining the colloidal osmotic pressure in blood and in the transport of exogenous and endogenous substances,including fatty acids,amino acids,steroids,bilirubin and drugs[2,3].Backbone of protein consists of a single polypeptide chain of 585 amino acid residues that form three homologous domains(I,II,and III),stabilized by 17 disulfide bridges due to the 34 cysteines present in the molecule;each domain contains two subdomains(A and B),respectively constituted by 6 and 4 α-helices[4–6].Crystallographic studies have revealed that HSA possesses binding sites for aromatic and heterocyclic ligands within two hydrophobic pockets:in subdomains IIA(Sudlow's site I:warfarin-binding site)and IIIA(Sudlow's site II:indole/benzodiazepine site).Both hydrophobic and electrostatic interactions play a major role in controlling the affinity towards drug binding for sites I and II.For site I,mainly hydrophobic interactions are dominant,while for site II,a combination of hydrophobic,hydrogen bonding and electrostatic interactions plays a crucial role.The tryptophan residue(Trp 214)of HSA is in subdomain IIA(site I)and plays a crucial role in spectrophotometric studies[2,3].When a ligand binds to one domain,it can induce distinct conformational changes on the other domain,as both sub domains share a common interface.For this reason,the binding of a drug to serum albumin may change considerably the binding abilities of HSA towards other molecules[7].

    Fig.1.The interaction of ICSs with albumin plays an important role in governing systemic side effects.

    From a pharmaceutical point of view,the interaction between inhaled corticosteroids(ICSs)and HSA is very interesting.Corticosteroids are the most potent and effective anti-inflammatory agents in many respiratory chronic diseases.In this case,the preferred way of administration of a corticosteroid is inhalation;this way permits to deliver the drug directly to the lung,where it acts locally in order to minimise the systemic side effects,compared to oral or parenteral administration.Clinical studies have shown that ICSs significantly reduce airway hy-perresponsiveness,effectively prevent acute exacerbations,improve lung function and decrease symptoms severity[8].Corticosteroids are involved in different physiological processes;in particular,they alter the production of inflammatory mediators in the airways,such as macrophages,eosinophils,lymphocytes,mast cells and dendritic cells[9].

    It is well known that the safety and efficacy profile of an ICS is influenced by the pharmacokinetic properties and associated pharmacodynamic effects of the drug[10,11].If ICSs are not bound,they can circulate freely and cause systemic adverse effects.In fact,the freely circulating ICSs can bind to nonpulmonary glucocorticoids receptors and cause adverse effects such as a reduction in the function of the hypothalamic-pituitary-adrenal(HPA)axis and growth impairment[12].Extensive protein binding can,therefore,be viewed as a way to temporarily remove an ICS that is available to the tissues from the systemic circulation,thereby reducing the potential for development of adverse effects[12–14].Among other pharmacological properties,high plasma protein binding is a desirable property for any ICS,since this reduces the potential for systemic side effects[15].Therefore,adetailed investigation of drug–protein interaction assumes significance for thorough understanding of the pharmacokinetic behaviour of corticosteroids and for the design of analogues with effective pharmacological properties(Fig.1).

    The purpose of this study was to evaluate the extent of protein binding of different ICSs,like betamethasone(A), flunisolide(B),prednisolone(C)and triamcinolone(D)(Fig.2)in order to develop a rapid spectroscopic method to study the interaction and eventually compare the affinity of ICSs with HSA.To our knowledge,the interaction between ICS and HSA has never been investigated by spectroscopic techniques.

    In this study,UV–Vis and fluorescence spectroscopy were used to elucidate the mode of binding and probable structural alterations of HSA upon drug binding.The binding constant and the nature of binding forces were determined.Lastly,the thermodynamic and F?rster's parameters associated with the binding process were also calculated.All the data obtained could clarify the type of interaction that can occur between ICS and HSA and could be fundamental to understand if and how the structural features of the drugs could modulate this interaction.Moreover,it may pave the comprehension of the bioavailability of corticosteroids,justifying the major use as inhaled administration,and facilitate the interpretation of absorption and distribution process of corticosteroids.

    2.Materials and methods

    2.1.Materials

    Albumin from human serum(HSA)lyophilized powder,≥97%(agarose gel electrophoresis),was purchased from Sigma Aldrich(Italy).To prepare the stock solution(100μM),HSA was dissolved in 2 mM phosphate buffer solution(PBS,pH 7.4).

    Betamethasone(≥98%), flunisolide(≥97%),prednisolone(≥99%)and triamcinolone were all purchased from Sigma Aldrich(Italy);the stock solutions(3 mM)were prepared by dissolving drugs in a solution of 96%ethanol and PBS(1:1,v/v).

    2.2.Apparatus

    All fluorescence spectra were recorded with a Horiba Jobin Yvon Fluorolog3 TCSPC spectrofluorophotometer(Bernsheim,Germany)with 1.0 cm quartz cells.UV–Vis spectra were recorded on a UH5300 Hitachi spectrophotometer(Hitachi Europe,Milan,Italy).The pH measurements were made with a Eutech Instruments pH2700(Landsmeer,The Netherlands).

    2.3.Experimental conditions

    UV–Vis measurements were carried out in the range of 200–400 nm.UV–Vis absorption spectra were recorded at room temperature,by using different concentrations of drugs(betametha-sone,prednisolone,and triamcinolone=2.0,4.0,6.0,8.0,10.0μM;flunisolide=5.0,10.0,15.0,20.0,25.0μM).

    Fluorescence quenching spectra were measured in the range of 300–500 nm upon excitation at 280 nm.The excitation and emission slits were 6 nm and 10 nm,respectively.The fluorescence spectra were performed at three different temperatures(296 K,303 K,and 310 K).5μM HSA was titrated by successive additions of drug solutions at different concentrations.To reach protein saturation,it is necessary to use a range from 50μM to 500μM for betamethasone and from 50μM to 700μM for flunisolide,prednisolone and triamcinolone.

    Fluorescence resonance energy transfer(FRET)measurements were performed at room temperature(296 K).The overlaps were obtained by using the emission spectrum of 5 μM HSA(λexcitationat 280 nm;the excitation and emission slits were 6 nm and 10 nm,respectively)and the absorption spectra of drugs(betamethasone,prednisolone,and triamcinolone=10.0μM; flunisolide=25.0μM).

    3.Results and discussion

    3.1.UV–Vis spectroscopy

    Absorption spectroscopy is one of the techniques used to explore the structural changes of protein and to investigate proteinligand complex formation[16].HSA has two main absorption bands,and one of them is located at 280 nm,which is the absorption band of the tryptophan(Trp 214)[17,18].

    The absorption spectra of the protein at room temperature in absence and in presence of different concentrations of drugs(betamethasone,prednisolone,and triamcinolone=2.0,4.0,6.0,8.0,10.0μM; flunisolide=5.0,10.0,15.0,20.0,25.0μM)were recorded and are shown in Fig.S1.As can be seen,for every sample,the absorption intensity of HSA at around 280 nm increased with the addition of increased concentrations of drugs.Moreover,the absorption spectrum of protein-drug complex was different from that of albumin and drugs alone.The maximum peak position of HSA-drug complex was slightly shifted towards lower wavelength region.These results confirmed that every studied drug can bind the protein.

    3.2.Fluorescence quenching mechanism

    The fluorescence spectrum of albumin was recorded in absence and in presence of drugs at different concentrations.Fig.3 shows spectra at room temperature.HSA shows a typical strong fluorescence emission peak at 350 nm,which does not shift in the presence of the drugs.Every analysed molecule causes a concentration-dependent quenching of the intrinsic fluorescence of protein that decreases gradually with the increase of drug concentration.The quenching of protein fluorescence by drugs was due to the formation of a protein-drug complex.This means that the microenvironment of HSA was changed during the binding interaction.In order to obtain thermodynamic parameters,binding studies were performed at three different temperatures(296 K,303 K and 310 K)and the obtained steady-state maximum fluorescence intensity was recorded.

    Fluorescence quenching data were treated by different methods,as reported in the following paragraphs,to evaluate the equilibrium association(KA)and dissociation(KD)constants.

    3.2.1.Stern-Volmer equation

    First of all, fluorescence quenching of albumin was analysed by Stern-Volmer Eq.(1)[19]:

    whereF0is the fluorescence intensities in the absence of quencher,Fis the steady-state fluorescence intensity in the presence of the quencher,and[Q]is the concentration of the quencher.KSVis the Stern-Volmer quenching constant and describes a collisional quenching of fluorescence.Quenching data are presented as plots ofF0/Fvs.[Q],yielding an intercept of one on the y-axis and a slope equal toKSV.Fig.4 shows Stern-Volmer plots of the fluorescence quenching of HSA by drugs at different temperatures.

    A linear Stern-Volmer plot,however,does not define the quenching mechanism.In order to distinguish dynamic from static quenching,the dependence of the interaction of a drug,described byKSV,on temperature has been proposed.TheKSVvalues decrease with an increase in temperature for static quenching and the reverse effect can be observed for dynamic quenching[19].

    As shown in Table 1,theKSVof the protein-drug complexes A,B,and C decreases with increased temperature.This indicates that a possible static quenching interaction between protein and drug occurs[20].TheKSVof protein-triamcinolone complex is similar to negligible variations by changing the temperature,but it is possible to observe a slight increase also in this case,so probably a static quenching interaction between protein and drug may also occur.

    Fig.3.Fluorescence spectra of HSA–drugs interaction(T=296 K).(A)HSA–betamethasone;(B)HSA– flunisolide;(C)HSA–prednisolone and(D)HSA–triamcininolone.λex=280 nm.

    3.2.2.Non-linear least squares

    Non-linear least squares fit procedure is a simple method to analyse fluorescence data at different temperatures[21]based on Eq.(2):

    where[Q]is the drug concentration,yis the specific binding derived by measuring fluorescence intensity,Bmaxis the maximum amount of the protein-drug complex formed at saturation,andKDis the equilibrium dissociation constant.Fig.5 shows the binding curves obtained;the percentage of bound HSA,i.e.y,derived from the fluorescence intensity emission maximum,is plotted against the drug concentration.

    The correspondingKDandKA(which are reciprocals of each other)at different temperatures are shown in Table 2.The binding constant calculated for HSA-triamcinolone complex suggests the lower affinity of this drug for the protein than the other tested drugs,as reported in literature[15].TheKAof protein-triamcinolone complex is similar to negligible variations by changing the temperature,but it is also possible to observe a slight increase.In this case,affinity seems to be higher for flunisolide.By increasing temperature,for A,B and C HSA-drug complexes,the value of association constant decreases.

    3.3.Binding parameters

    By double logarithm regression curve(shown in Eq.(3))[22],it is possible to obtain the number of binding sites(n).Eq.(3)describes the relationship between fluorescence intensity and the quencher concentration:

    whereF0is the fluorescence intensity of the protein alone,Fis the fluorescence intensity after the addition of the quencher,and[Q]is the quencher/drug concentration.The plots obtained using Eq.(3)are shown in Fig.S2.The slope of the line is the n value.If the value of n is equal to 1,it means that a strong binding exists between the protein and the drug[22].

    The number of binding sites is easily calculated:for HSA–betamethasone complex is 1.50(296 K),1.30(303 K),and 1.50(310 K),for HSA– flunisolide is 1.30(296 K),0.92(303 K),and 1.10(310 K),for HSA–prednisolone complex is 1.30(296 K),1.40(303 K),and 1.30(310 K)and for HSA–triamcinolone is 1.30(296 K),1.40(303 K),and 1.30(310 K).Almost all values are approximately equal to 1,indicating that there is one independent binding site on HSA for each analysed drug[23].

    3.4.Site marker competitive binding experiments

    Fig.4.The Stern-Volmer plots of the fluorescence quenching of HSA by drugs at different temperatures.(A)betamethasone;(B) flunisolide;(C)prednisolone;and(D)triamcinolone.

    Table 1The quenching constants(KSVin M-1)of HSA and drugs at different temperatures.

    In order to further investigate drug binding site and to precisely determine the location of corticosteroids on HSA,competitive binding tests were carried out.Warfarin and ibuprofen are two specific markers for HSA binding sites I and II,respectively[24].In the site marker competitive experiment,warfarin or ibuprofen was gradually added to the solution of HSA-corticosteroids complex and then fluorescence intensity of the system was recorded.As shown in Fig.S3,with the addition of warfarin in the HSA-drug solution,the fluorescence intensity was slightly higher than that without warfarin,including a red shift.Then,when increasing warfarin concentration into the solution of HSA-corticosteroids complex,the fluorescence intensity of HSA solution decreased gradually,reaching saturation at 1 mM of site marker with an effective displacement of about 98%–99%for all the tested drugs,indicating that the binding of the corticosteroids to HSA was affected by warfarin addition.On the contrary,the addition of ibuprofen to the HSA-drug complex only promoted a slight enhancement of the fluorescence intensity(Fig.S4),indicating that site II marker did not prevent the binding of corticosteroids in its usual binding location.These results suggest that corticosteroids compete with warfarin for binding to HSA to site I[24].

    3.5.Thermodynamic parameters

    The interaction forces between small molecules and macromolecules include four binding modes:H-bonding,Van der Waals,electrostatics and hydrophobic interactions[25].The model of interaction between drug and the protein could be obtained,according to the data of enthalpy(ΔH)and entropy change(ΔS)[26]:(1)ΔH>0 andΔS>0,hydrophobic forces;(2)ΔH<0 and ΔS<0,van der Waals interactions and hydrogen bonds;(3)ΔH<0 andΔS>0,electrostatic interactions.The thermodynamic parameters,enthalpy and entropy of the HSA-drugs complex reaction are important to confirm binding modes.The temperature-dependence of the binding constant was analysed at 296 K,303 K,and 310 K and thermodynamic parameters were calculated from the following Van’t Hoff equations[19]:

    whereKAis the binding constant,R is the gas constant andTis the experimental temperature.The values ofΔHandΔSobtained for the binding sites are shown in Table S1.The negative sign forΔG means that the binding process is spontaneous for every studied interaction[23].

    Fig.5.The binding curves of HSA–drugs complex at different temperatures.(A)betamethasone;(B) flunisolide;(C)prednisolone and(D)triamcinolone.

    From Table S1 it can be seen that for HSA-betamethasone,HSA-flunisolide and HSA-prednisolone complexes,bothΔHandΔShave negative values.This indicates that van der Waals interactions and hydrogen bonds may play a major role in the binding.Conversely,in the formation of HSA-triamcinolone complex,an exothermic reaction occurs,characterized by a negativeΔHvalue and a positiveΔSvalue.From the point of view of water structure,a positiveΔSvalue is frequently taken as a typical evidence for hydrophobic interaction.Furthermore,specific electrostatic interactions between ionic species in aqueous solution are characterized by a positiveΔSvalue and a negativeΔHvalue[27].In order to evaluate the thermodynamic parameters of HSA-triamcinolone complex,it is not possible to take account of a single intermolecular force model.As described in literature for another complex(HSA-dexamethasone)[28],the binding,in this case,might involve hydrophobic interaction strongly,as evidenced by the positive values of ΔS,but electrostatic interaction can not be excluded either.

    3.6.Energy transfer

    FRET is a simple method to measure the distance between the acceptor(ligand)and the donor(tryptophan residues in the protein)[29].According to F?rster's non-radiative energy transfer theory,energy efficiency(E),critical energy-transfer distance(R0,E=50%),the energy donor and the energy acceptor distance(r)and the overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor(J)can be calculated by the following equations[30]:

    Table 2Values of the equilibrium dissociation and association constants of HSA-drug complexes at different temperatures obtained by a non linear fit equation.

    Table 3Parameters of J,E,R0and r of HSA-drug complexes at 296 K.

    wherek2is the orientation factor,φis the fluorescence quantum yield of the donor,nis the refractive index of the medium,F(λ)is the fluorescence intensity of the donor at wavelength λ and ε(λ)is the molar absorption coefficient of the acceptor at wavelength λ.In this case,k2=2/3,n=1.336 andφ=0.118[16].

    The overlaps of the emission spectra of the protein and the absorption spectra of drugs at room temperature were obtained(Fig.S5).Using these equations,it is possible to calculate J,E,R0and r for every interaction.Data are reported in Table 3.

    The distance r<7 nm indicates that the energy transfer between protein and drugs occurred with a high possibility[16,23].This is in agreement with conditions of F?ster's non-radiative energy transfer theory[31],indicating again the static quenching interaction between protein and drugs.According to Stern-Volmer plots,data obtained with different methods are comparable with each other.

    3.7.Conformation investigation

    Synchronous fluorescence spectroscopy introduced by Lloyd[32]has been used to investigate the conformational change of proteins.The synchronous fluorescence spectrum could be obtained by synchronously scanning the excitation and emission monochromators with a wavelength difference between excitation and emission as a constant.The intrinsic fluorescence of HSA is manifested by emission of Trp and Tyr residues present in the protein[33].

    The synchronous fluorescence spectra obtained with Δλ=60 nm exclusively characterize the fluorescence of tryptophan residue.After complex formation,the local environment could change and induce a red or blue shift of the tryptophan emission spectra.The shift in the position of fluorescence emission maximum corresponds to changes of the polarity around the chromophore molecule.A blue shift of λmaxmeans that the aminoacid residues are located in a more hydrophobic environment and are less exposed to the solvent,while a red shift of λmaximplies that the amino acid residues are in a polar environment and are more exposed to the solvent[34].By synchronous fluorescence spectral changes of aminoacid residues,the conformational changes of protein can be predicted.We can also obtain the information about the location of corticosteroids binding site from the synchronous fluorescence data.

    The synchronous fluorescence spectra of HSA in presence of betamethasone, flunisolide,prednisolone and triamcinolone were recorded and are shown in Fig.6.For every analysed complex,the emission maximum of tryptophan residues showed significant red shift of tryptophan residue fluorescence,confirming that every analysed drugs reached subdomain IIA,where only one Trp residue(Trp 214)is located.High concentration of drugs makes protein molecules extend,thus reducing energy transfer between aminoacids and reducing the fluorescence intensity[35].Since Trp 214 is at site I,these results indicated that corticosteroids can bind to HSA in the hydrophobic cavity on subdomain IIA,which is in full agreement with quenching and competitive binding experiments.

    Fig.6.Synchronous fluorescence spectra of HSA-drugs at 296 K,Δλ=60 nm.[HSA]:black line;[Drugs]:50.0–800.0 μM.(A)betamethasone;(B) flunisolide;(C)prednisolone;and(D)triamcinolone.

    4.Conclusions

    Drug binding to HSA is a major problem in pharmaceutical research because the binding to albumin influences the effective drug concentration that can reach the target site.In this work,the interaction of albumin with four different corticosteroids was investigated at different temperatures by different spectroscopic approaches.UV–Vis spectroscopy confirmed that all the investigated drugs can bind to HSA to form a protein–drug complex.Quenching fluorescence data revealed that the protein can be bound by the studied corticosteroids,around the Trp 214(as confirmed by competitive binding experiments and synchronous fluorescence)and that the quenching is governed by a static quenching(data are comparable with results obtained by FRET).According to thermodynamic parameters(negativeΔHandΔSvalues),the hydrogen bonds and van der Waals forces play a major role in the binding process between albumin and betamethasone, flunisolide and prednisolone,while hydrophobic forces may play a major role in stabilizing albumin-triamcinolone complex.The evaluation of the equilibrium association(KA)and dissociation(KD)constants was obtained by a non-linear method at different temperatures.The data showed that temperature does not influence the formation of HSA-triamcinolone complex,while it can influence the interaction between albumin and betamethasone, flunisolide and prednisolone.This means that the drug structure may play a crucial role in the binding with the protein.Usually,drugs bind to HSA high-affinity sites with typical association constants in the range of 104–106M-1[36].As reported in literature[8],serum analysis revealed that the present corticosteroids can bind the HSA in a range between 70%and 80%thus with a low affinity.These data are in agreement with the equilibrium association constants obtained in this study.Usually a binder with high affinity,such as warfarin,showsKAaround 105M-1[37]while the studied ICSs haveKAin the order of 103M-1.

    The present spectroscopic approach offers a fast screening method to eventually investigate the structure-activity relationship(SAR)of new therapeutic molecules since it can discriminate the binding affinity with simple and reliable experiments.

    Conflicts of interest

    The authors declare that there are no Conflicts of interest.

    This work was supported by a grant from the University of Torino(Ricerca Locale ex-60%,Bando 2015).

    Appendix A.Supplementary material

    Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jpha.2017.07.003.

    [1]X.M.He,D.C.Carter,Atomic structure and chemistry of human serum albumin,Nature 358(1992)209–215.

    [2]T.K.Maiti,K.S.Ghosh,J.Debnath,et al.,Binding of all-trans retinoic acid to human serum albumin: fluorescence,FT-IR and circular dichroism studies,Int.J.Biol.Macromol.38(2006)197–202.

    [3]M.X.Xie,M.Long,Y.Liu,et al.,Characterization of the interaction between human serum albumin and morin,Biochim.Biophys.Acta 2006(1760)1184–1191.

    [4]Y.Wang,H.Yu,X.Shi,et al.,Structural mechanism of ring opening reaction of glucose by human serum albumin,J.Biol.Chem.288(2013)15980–15987.

    [5]F.Keshavarz,M.M.Alavianmehr,R.Youse fi,Molecular interaction of benzalkoniumibuprofenate and its discrete ingredients with human serum albumin,Phys.Chem.Res.2(2013)111–116.

    [6]C.D.Kanakis,P.A.Tarantilis,M.G.Polissiou,et al.,Antioxidant flavonoids bind human serum albumin,J.Mol.Struct.798(2006)69–74.

    [7]L.Trynda-Lemiesz,Paclitaxel–HSA interaction.Binding sites on HSA molecule,Bioorg.Med.Chem.12(2004)3269–3275.

    [8]J.W.Georgitis,The 1997 asthma management guidelines and therapeutic issues relating to the treatment of asthma,Chest 155(1999)210–217.

    [9]P.J.Barnes,Effect of corticosteroids on airway hyperresponsiveness,Am.Rev.Respir.Dis.141(1990)70–76.

    [10]C.Crim,L.N.Pierre,P.T.Daley-Yates,A review of the pharmacology and pharmacokinetics of inhaled fluticasone propionate and mometasone furoate,Clin.Ther.231(2001)1339–1354.

    [11]H.Derendorf,Pharmacokinetic and pharmacodynamics properties of inhaled corticosteroids in relation to efficacy and safety,Respir.Med.91(1997)22–28.

    [12]O.D.Wolthers,J.W.Honour,Measures of hypothalamic-pituitary-adrenal function in patients with asthma treated with inhaled glucocorticoids:clinical and research implications,J.Asthma 36(1999)477–486.

    [13]S.Rohatagi,S.Appajosyula,H.Derendorf,et al.,Risk-benefit value of inhaled glucocorticoids:a pharmacokinetic/pharmacodynamic perspective,J.Clin.Pharmacol.44(2004)37–47.

    [14]H.Derendorf,G.Hochhaus,B.Meibohm,et al.,Pharmacokinetics and pharmacodynamics of inhaled corticosteroids,J.Allergy Clin.Immunol.101(1998)440–446.

    [15]H.Derendorf,R.Nave,A.Drollmann,et al.,Relevance of pharmacokinetics and pharmacodynamics of inhaled corticosteroids to asthma,Eur.Respir.J.28(2006)1042–1050.

    [16]H.M.Ma,X.Chen,N.Zhang,et al.,Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins,Spectrochim.Acta Part A:Mol.Biomol.Spectrosc.72(2009)465–469.

    [17]J.Liu,J.N.Tian,J.Zhang,et al.,Interaction of magnolol with bovine serum albumin:a fluorescence-quenching study,Anal.Bioanal.Chem.376(2003)864–867.

    [18]Y.Yue,X.Chen,J.Qin,et al.,Characterization of the mangiferin-human serum albumin complex by spectroscopic and molecular modeling approaches,J.Pharm.Biomed.49(2009)753–759.

    [19]J.R.Lakowicz,Principles of Fluorescence Spectroscopy,Springer-Verlag,New York,2006.

    [20]Y.J.Hu,Y.Liu,W.Jiang,et al.,Fluorometric investigation of the interaction of bovine serum albumin with surfactants and 6-mercaptopurine,J.Photochem.Photobiol.B 80(2005)235–242.

    [21]N.Barbero,E.Barni,C.Barolo,et al.,A study of the interaction between fluorescein sodium salt and bovine serum albumin by steady-state fluorescence,Dyes Pigments 80(2009)307–313.

    [22]K.H.Ulrich,Molecular aspects of ligand binding to serum albumin,Pharmacol.Rev.33(1981)17–53.

    [23]B.Valeur,J.C.Brochon,New Trends in Fluorescence Spectroscopy,Springer Press,Berlin,1999.

    [24]F.Ding,N.Li,B.Han,et al.,The binding of C.I.Acid Red 2 to human serum albumin:determination of binding mechanism and binding site using fluorescence spectroscopy,Dyes Pigments 83(2009)249–257.

    [25]J.B.Madsen,K.I.Pakkanen,S.Lee,Investigation of the thermostability of Bovine Submaxillary Mucin(BSM)and its impact on lubrication,APCBEE Proc.7(2013)21–26.

    [26]Y.N.Ni,G.L.Liu,S.Kokot,Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2-isopropylphenate with bovine serum albumin,Talanta 76(2008)513–521.

    [27]D.P.Ross,S.Subramanian,Thermodynamics of protein association reactions:forces contributing to stability,Biochemistry 20(1981)3096–3102.

    [28]P.N.Naik,S.A.Chimatadar,S.T.Nandibewoor,Interaction between a potent corticosteroid drug–dexamethasone with bovine serum albumin and human serum albumin:a fluorescence quenching and fourier transformation infrared spectroscopy study,J.Photochem.Photobiol.B 100(2010)147–159.

    [29]T.F?rster,Transfer mechanisms of electronic excitation,Discuss.Faraday Soc.27(1959)7–17.

    [30]C.Pontremoli,N.Barbero,G.Viscardi,S.Visentin,Mucin–drugs interaction:the case of theophylline,prednisolone and cephalexin,Bioorg.Med.Chem.23(2015)6581–6586.

    [31]F.L.Cui,J.Fan,J.P.Li,et al.,Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin:investigation by fluorescence spectroscopy,Bioorg.Med.Chem.12(2004)151–157.

    [32]P.Qu,H.Lu,X.Y.Ding,et al.,Study on the interaction of 6-thioguanine with bovine serum albumin by spectroscopic techniques,J.Mol.Struct.920(2009)172–177.

    [33]B.Tang,M.Du,Z.Z.Chen,et al.,Studies on luminescence of Trp and Tyr residues in protein denaturation by three-dimensional-synchronous-polarized spectrofluorimetry,Acta Chim.Sin.62(2004)1153–1157.

    [34]T.C.O’Haver,A.F.Fell,G.Smith,Derivative spectroscopy and its applications in analysis,Anal.Proc.19(1982)22–46.

    [35]X.H.Wu,J.H.Zhou,X.T.Gu,et al.,Study on interaction between hypocrellin A and hemoglobin or myoglobin using synchronous fluorescence spectra,Spectrosc.Spect.Anal.26(2006)2287–2290.

    [36]L.Trynda-Lemiesz,K.Wiglusz,Interactions of human serum albumin with meloxicam.Characterization of binding site,J.Pharm.Biomed.52(2010)300–304.

    [37]S.Baroni,M.Mattu,A.Vannini,R,et al.,Effect of ibuprofen and warfarin on the allosteric properties of haem–human serum albumin.A spectroscopic study,Eur.J.Biochem.268(2001)6214–6220.

    国产亚洲欧美精品永久| 少妇熟女欧美另类| 久久久a久久爽久久v久久| 国产在线免费精品| 在线观看一区二区三区| 观看美女的网站| 亚洲精品第二区| 国产色爽女视频免费观看| 人人妻人人澡人人爽人人夜夜| 成人黄色视频免费在线看| 男女边吃奶边做爰视频| 麻豆成人午夜福利视频| 国产伦精品一区二区三区四那| 精品国产露脸久久av麻豆| av国产免费在线观看| 亚洲av电影在线观看一区二区三区| 亚洲四区av| 乱码一卡2卡4卡精品| 精品酒店卫生间| 哪个播放器可以免费观看大片| a级毛片免费高清观看在线播放| 婷婷色综合www| 18禁动态无遮挡网站| 国产毛片在线视频| 日本午夜av视频| 黑人猛操日本美女一级片| 99久久精品国产国产毛片| 精品人妻一区二区三区麻豆| 美女脱内裤让男人舔精品视频| 日本wwww免费看| 亚洲欧美日韩卡通动漫| 免费看av在线观看网站| 久久久a久久爽久久v久久| 亚洲欧美日韩东京热| 伦理电影大哥的女人| 亚洲精品久久久久久婷婷小说| 国国产精品蜜臀av免费| 久久久久国产精品人妻一区二区| 欧美高清成人免费视频www| 欧美精品人与动牲交sv欧美| 多毛熟女@视频| 久久久久精品性色| 中文资源天堂在线| 亚洲精品,欧美精品| 日韩强制内射视频| 欧美3d第一页| 伦理电影大哥的女人| 久久精品国产自在天天线| 偷拍熟女少妇极品色| 日韩一区二区三区影片| 国产伦精品一区二区三区四那| 男的添女的下面高潮视频| 国产亚洲91精品色在线| a 毛片基地| 精品一品国产午夜福利视频| 纵有疾风起免费观看全集完整版| 国产高清国产精品国产三级 | 成人亚洲精品一区在线观看 | 免费在线观看成人毛片| 日日摸夜夜添夜夜添av毛片| 婷婷色综合大香蕉| 成人无遮挡网站| 亚洲精品一区蜜桃| 日本黄大片高清| 五月玫瑰六月丁香| 新久久久久国产一级毛片| 亚洲经典国产精华液单| 十分钟在线观看高清视频www | 国产成人aa在线观看| 亚洲丝袜综合中文字幕| 亚洲精品乱码久久久久久按摩| 午夜激情久久久久久久| 乱系列少妇在线播放| av视频免费观看在线观看| 亚洲精品乱码久久久久久按摩| 一级毛片电影观看| 晚上一个人看的免费电影| 国产在线视频一区二区| 亚洲综合色惰| 国产精品久久久久久精品电影小说 | 汤姆久久久久久久影院中文字幕| 91在线精品国自产拍蜜月| 晚上一个人看的免费电影| 女性生殖器流出的白浆| 国产欧美另类精品又又久久亚洲欧美| 简卡轻食公司| 91狼人影院| 深夜a级毛片| 嫩草影院新地址| 美女视频免费永久观看网站| 最近中文字幕2019免费版| 七月丁香在线播放| 狂野欧美激情性bbbbbb| 国产一区二区三区综合在线观看 | 观看免费一级毛片| 亚洲一区二区三区欧美精品| 久久青草综合色| 欧美日韩亚洲高清精品| 色网站视频免费| 国产成人免费观看mmmm| 亚洲国产色片| 亚洲,欧美,日韩| 亚洲成人中文字幕在线播放| 夜夜骑夜夜射夜夜干| 国产日韩欧美在线精品| 午夜日本视频在线| 91久久精品国产一区二区成人| 九九爱精品视频在线观看| 国产av精品麻豆| 免费不卡的大黄色大毛片视频在线观看| 热99国产精品久久久久久7| 精品视频人人做人人爽| 街头女战士在线观看网站| 视频中文字幕在线观看| 一个人免费看片子| 亚洲真实伦在线观看| 视频中文字幕在线观看| 免费黄频网站在线观看国产| 国产黄色免费在线视频| 啦啦啦视频在线资源免费观看| 亚洲精品乱码久久久v下载方式| 成人亚洲精品一区在线观看 | 啦啦啦啦在线视频资源| 99视频精品全部免费 在线| 亚洲自偷自拍三级| 乱码一卡2卡4卡精品| 日本黄色日本黄色录像| 日日撸夜夜添| a级毛色黄片| 久久鲁丝午夜福利片| 国产精品秋霞免费鲁丝片| 免费观看的影片在线观看| 日韩电影二区| 久久99精品国语久久久| 久久韩国三级中文字幕| 午夜免费观看性视频| 免费高清在线观看视频在线观看| 亚洲高清免费不卡视频| 一级毛片aaaaaa免费看小| 2022亚洲国产成人精品| 毛片一级片免费看久久久久| h视频一区二区三区| av在线观看视频网站免费| 久久久久久久精品精品| 久久国产精品男人的天堂亚洲 | 狠狠精品人妻久久久久久综合| 伦理电影大哥的女人| 国产欧美另类精品又又久久亚洲欧美| 超碰97精品在线观看| 网址你懂的国产日韩在线| 黑人高潮一二区| 亚洲人与动物交配视频| 看十八女毛片水多多多| 欧美国产精品一级二级三级 | 国产精品一区二区三区四区免费观看| 色婷婷av一区二区三区视频| 一级黄片播放器| 国产成人精品福利久久| 欧美少妇被猛烈插入视频| 国产一级毛片在线| 免费观看av网站的网址| 国产高清国产精品国产三级 | 日韩av免费高清视频| 久久99热6这里只有精品| 国产精品.久久久| 黑人高潮一二区| 麻豆乱淫一区二区| 熟女电影av网| 青青草视频在线视频观看| 国产久久久一区二区三区| 日本黄大片高清| 一级毛片久久久久久久久女| 又黄又爽又刺激的免费视频.| 日韩av在线免费看完整版不卡| 国产高清有码在线观看视频| 国产欧美日韩精品一区二区| 亚洲天堂av无毛| 午夜老司机福利剧场| 久久久精品免费免费高清| 亚洲成人中文字幕在线播放| 欧美精品亚洲一区二区| 黑丝袜美女国产一区| 亚洲内射少妇av| 成人二区视频| 久久久亚洲精品成人影院| 女人十人毛片免费观看3o分钟| 欧美高清性xxxxhd video| 久久国产精品大桥未久av | 蜜桃亚洲精品一区二区三区| 夜夜爽夜夜爽视频| 亚洲色图av天堂| 亚洲av国产av综合av卡| 精品熟女少妇av免费看| 高清日韩中文字幕在线| 国产亚洲精品久久久com| 国产成人午夜福利电影在线观看| 肉色欧美久久久久久久蜜桃| 国产成人免费观看mmmm| 能在线免费看毛片的网站| 精品亚洲乱码少妇综合久久| 精品酒店卫生间| av国产免费在线观看| av在线播放精品| 如何舔出高潮| 国产在线视频一区二区| 观看av在线不卡| 女人十人毛片免费观看3o分钟| 亚洲国产成人一精品久久久| 岛国毛片在线播放| 男女下面进入的视频免费午夜| 啦啦啦中文免费视频观看日本| 男人狂女人下面高潮的视频| 欧美激情国产日韩精品一区| 亚洲美女黄色视频免费看| 国产综合精华液| 国产无遮挡羞羞视频在线观看| 欧美+日韩+精品| 日本av免费视频播放| 日韩成人av中文字幕在线观看| 韩国av在线不卡| 国产毛片在线视频| 国产极品天堂在线| 91久久精品国产一区二区三区| 中文乱码字字幕精品一区二区三区| 一级毛片aaaaaa免费看小| 亚洲精品视频女| 丰满乱子伦码专区| 日韩欧美精品免费久久| 亚洲人成网站在线播| 人妻一区二区av| 精品人妻偷拍中文字幕| 在线观看三级黄色| 婷婷色综合www| 国产大屁股一区二区在线视频| 国产伦理片在线播放av一区| 国产乱人视频| 麻豆国产97在线/欧美| 国产片特级美女逼逼视频| av视频免费观看在线观看| 久久99精品国语久久久| 欧美另类一区| 日韩亚洲欧美综合| 在线观看免费视频网站a站| 亚洲精品日韩在线中文字幕| 欧美一区二区亚洲| 中文字幕制服av| 99视频精品全部免费 在线| 精品久久国产蜜桃| 国产精品99久久99久久久不卡 | 国产黄片美女视频| 久久久久精品久久久久真实原创| 丰满迷人的少妇在线观看| 久久久久性生活片| 免费人成在线观看视频色| 国产av码专区亚洲av| 亚洲一级一片aⅴ在线观看| 一区二区三区免费毛片| tube8黄色片| 国产极品天堂在线| 最近2019中文字幕mv第一页| 人妻夜夜爽99麻豆av| 一二三四中文在线观看免费高清| 人妻一区二区av| 又粗又硬又长又爽又黄的视频| 亚洲人成网站在线播| 日韩av在线免费看完整版不卡| 一本久久精品| 色网站视频免费| 久久青草综合色| 欧美变态另类bdsm刘玥| 精品午夜福利在线看| 纯流量卡能插随身wifi吗| 午夜日本视频在线| 亚洲人与动物交配视频| 91久久精品电影网| 亚洲四区av| 亚洲成色77777| 成人漫画全彩无遮挡| 联通29元200g的流量卡| 日韩制服骚丝袜av| 内地一区二区视频在线| 最黄视频免费看| 国产视频内射| 久久久亚洲精品成人影院| 日本一二三区视频观看| 大香蕉久久网| 免费观看无遮挡的男女| 国产探花极品一区二区| 99久久中文字幕三级久久日本| 精品少妇久久久久久888优播| 久久久久久人妻| 日韩欧美 国产精品| 一本久久精品| 亚洲成色77777| 一边亲一边摸免费视频| 美女高潮的动态| 两个人的视频大全免费| 99久国产av精品国产电影| 成人亚洲精品一区在线观看 | 另类亚洲欧美激情| 日本猛色少妇xxxxx猛交久久| 久久精品国产鲁丝片午夜精品| 亚洲天堂av无毛| 日韩一区二区三区影片| 欧美亚洲 丝袜 人妻 在线| 在现免费观看毛片| 高清黄色对白视频在线免费看 | 久久久久久久亚洲中文字幕| 亚洲av.av天堂| 欧美高清性xxxxhd video| 黄片无遮挡物在线观看| 日韩 亚洲 欧美在线| 身体一侧抽搐| 精品一区二区三区视频在线| 亚洲精品aⅴ在线观看| 美女内射精品一级片tv| 久久精品国产亚洲av涩爱| 在线观看国产h片| 男女无遮挡免费网站观看| 最近最新中文字幕免费大全7| 九九爱精品视频在线观看| 亚洲欧美精品专区久久| 亚洲av综合色区一区| 亚洲精品视频女| 人妻少妇偷人精品九色| 日韩av不卡免费在线播放| 国产亚洲精品久久久com| 成人亚洲欧美一区二区av| av国产精品久久久久影院| 高清视频免费观看一区二区| 久久久久久久精品精品| 精品少妇黑人巨大在线播放| 日韩成人av中文字幕在线观看| 久久亚洲国产成人精品v| 欧美少妇被猛烈插入视频| 久久精品熟女亚洲av麻豆精品| 国产精品人妻久久久影院| 精品亚洲成国产av| av免费在线看不卡| 最近最新中文字幕大全电影3| www.色视频.com| 国产久久久一区二区三区| 久久精品久久久久久噜噜老黄| 嘟嘟电影网在线观看| 亚洲精品,欧美精品| 高清不卡的av网站| 国产探花极品一区二区| 女人十人毛片免费观看3o分钟| 十八禁网站网址无遮挡 | 国产精品一二三区在线看| 亚洲精品乱码久久久久久按摩| 水蜜桃什么品种好| 久久久午夜欧美精品| 国产黄色视频一区二区在线观看| 日韩一本色道免费dvd| 在线 av 中文字幕| 亚洲精品aⅴ在线观看| 国产老妇伦熟女老妇高清| 在线看a的网站| 夜夜骑夜夜射夜夜干| 欧美日韩一区二区视频在线观看视频在线| 国产精品熟女久久久久浪| av.在线天堂| 毛片女人毛片| 三级国产精品欧美在线观看| 久久久久精品性色| 这个男人来自地球电影免费观看 | 美女cb高潮喷水在线观看| .国产精品久久| 好男人视频免费观看在线| 波野结衣二区三区在线| 亚洲欧美清纯卡通| 寂寞人妻少妇视频99o| 国内精品宾馆在线| 久久热精品热| 韩国高清视频一区二区三区| 观看av在线不卡| 欧美极品一区二区三区四区| 免费播放大片免费观看视频在线观看| 尤物成人国产欧美一区二区三区| 亚洲av男天堂| 久久国产精品男人的天堂亚洲 | 九草在线视频观看| 一区二区三区免费毛片| 欧美xxⅹ黑人| 欧美xxxx性猛交bbbb| 久久久久久九九精品二区国产| 亚洲欧美一区二区三区国产| 久久久午夜欧美精品| 青春草国产在线视频| 久久99蜜桃精品久久| 国产69精品久久久久777片| 亚洲精品日韩av片在线观看| 高清毛片免费看| 国产精品av视频在线免费观看| 国产乱人偷精品视频| 亚洲国产精品专区欧美| 少妇精品久久久久久久| 国产亚洲午夜精品一区二区久久| 日韩成人伦理影院| 亚洲,一卡二卡三卡| 国产黄片美女视频| 国产亚洲精品久久久com| 大话2 男鬼变身卡| 国产成人aa在线观看| 亚洲精品久久久久久婷婷小说| 欧美丝袜亚洲另类| 男的添女的下面高潮视频| 插阴视频在线观看视频| 国产在线视频一区二区| 91久久精品国产一区二区三区| a 毛片基地| 波野结衣二区三区在线| 色视频在线一区二区三区| 成人综合一区亚洲| 精品国产三级普通话版| 中文字幕精品免费在线观看视频 | 亚洲一区二区三区欧美精品| 国产高清有码在线观看视频| 中文字幕久久专区| 精品国产三级普通话版| 99热这里只有是精品在线观看| 欧美三级亚洲精品| 免费观看无遮挡的男女| 中文资源天堂在线| 久久久国产一区二区| 精品亚洲乱码少妇综合久久| 欧美精品一区二区免费开放| 夜夜骑夜夜射夜夜干| 大香蕉久久网| 久久人人爽av亚洲精品天堂 | 国产精品三级大全| 涩涩av久久男人的天堂| 在线观看一区二区三区| 人妻系列 视频| 欧美精品一区二区大全| 性色av一级| 3wmmmm亚洲av在线观看| 一个人免费看片子| 国产精品爽爽va在线观看网站| 亚洲久久久国产精品| 欧美zozozo另类| 久久国产精品大桥未久av | 黄色配什么色好看| 校园人妻丝袜中文字幕| 精品国产一区二区三区久久久樱花 | 高清在线视频一区二区三区| 啦啦啦在线观看免费高清www| 亚洲丝袜综合中文字幕| 视频中文字幕在线观看| 精品国产一区二区三区久久久樱花 | 日本欧美视频一区| 成人漫画全彩无遮挡| 国产精品一区二区三区四区免费观看| 久久国内精品自在自线图片| 2022亚洲国产成人精品| 内地一区二区视频在线| 久久久国产一区二区| 尾随美女入室| 亚洲成人手机| av网站免费在线观看视频| 韩国高清视频一区二区三区| 精品一区二区免费观看| 精品视频人人做人人爽| 日韩成人av中文字幕在线观看| 久久女婷五月综合色啪小说| 国产人妻一区二区三区在| 久久99蜜桃精品久久| 亚洲国产成人一精品久久久| 五月玫瑰六月丁香| 国产成人一区二区在线| 王馨瑶露胸无遮挡在线观看| 在线观看av片永久免费下载| 亚洲国产成人一精品久久久| 777米奇影视久久| 午夜免费男女啪啪视频观看| 亚洲色图综合在线观看| 九九在线视频观看精品| 国产精品99久久99久久久不卡 | 一区二区三区精品91| 免费黄网站久久成人精品| 日韩三级伦理在线观看| 国产成人一区二区在线| 最近手机中文字幕大全| 亚洲精品日韩在线中文字幕| 国产久久久一区二区三区| 国产亚洲91精品色在线| 国产探花极品一区二区| 国产亚洲精品久久久com| 国产精品国产三级专区第一集| 亚洲av不卡在线观看| 日韩大片免费观看网站| 欧美老熟妇乱子伦牲交| 成人无遮挡网站| 久久精品人妻少妇| 国产爽快片一区二区三区| 啦啦啦在线观看免费高清www| 伦精品一区二区三区| 18禁裸乳无遮挡免费网站照片| 日韩av不卡免费在线播放| 亚洲国产成人一精品久久久| 国产亚洲91精品色在线| 伊人久久精品亚洲午夜| 男人和女人高潮做爰伦理| 欧美亚洲 丝袜 人妻 在线| 直男gayav资源| 一级毛片电影观看| 黄色日韩在线| 日韩 亚洲 欧美在线| 在线观看一区二区三区| av专区在线播放| 久久6这里有精品| 三级国产精品欧美在线观看| 如何舔出高潮| 高清av免费在线| 一个人看视频在线观看www免费| 一级毛片我不卡| 国产极品天堂在线| 又爽又黄a免费视频| 日韩强制内射视频| 国产av精品麻豆| 国产乱来视频区| 国产成人免费观看mmmm| 欧美老熟妇乱子伦牲交| 婷婷色综合www| 国产精品成人在线| 国产 一区 欧美 日韩| 久久ye,这里只有精品| 成人亚洲欧美一区二区av| 一级黄片播放器| 亚洲综合色惰| 亚洲国产精品999| 国产一区二区三区综合在线观看 | 自拍偷自拍亚洲精品老妇| 亚洲精品一区蜜桃| 中国国产av一级| 国产av码专区亚洲av| 国产亚洲一区二区精品| 亚洲精品色激情综合| 91狼人影院| 99久久精品国产国产毛片| 欧美日韩在线观看h| 日本欧美视频一区| 久久97久久精品| 人妻夜夜爽99麻豆av| 久久久久久久大尺度免费视频| 777米奇影视久久| 亚洲国产日韩一区二区| 熟女人妻精品中文字幕| 精品一区二区免费观看| 中国美白少妇内射xxxbb| 春色校园在线视频观看| 亚洲天堂av无毛| 精品视频人人做人人爽| 天天躁日日操中文字幕| 2022亚洲国产成人精品| 丝袜喷水一区| 久久精品久久精品一区二区三区| 日韩欧美 国产精品| 亚洲不卡免费看| 亚洲av福利一区| 亚洲最大成人中文| 水蜜桃什么品种好| 亚洲综合色惰| 欧美+日韩+精品| 老司机影院成人| 国产真实伦视频高清在线观看| 男人添女人高潮全过程视频| 啦啦啦视频在线资源免费观看| 亚洲激情五月婷婷啪啪| 老师上课跳d突然被开到最大视频| 国产综合精华液| 国产免费一级a男人的天堂| 狠狠精品人妻久久久久久综合| a级毛片免费高清观看在线播放| 最近中文字幕2019免费版| 亚洲成人一二三区av| 日韩大片免费观看网站| 在线精品无人区一区二区三 | 国产 一区精品| 成年女人在线观看亚洲视频| 好男人视频免费观看在线| 自拍偷自拍亚洲精品老妇| 日韩视频在线欧美| 在线观看免费高清a一片| 汤姆久久久久久久影院中文字幕| 亚洲人成网站高清观看| 国产av国产精品国产| 日韩人妻高清精品专区| 伊人久久国产一区二区| 日本av手机在线免费观看| 在线观看av片永久免费下载| 又爽又黄a免费视频| 久久久久久久精品精品| 男女啪啪激烈高潮av片| 国产色爽女视频免费观看| 久久6这里有精品| 午夜福利在线在线| 国产色爽女视频免费观看| 女人十人毛片免费观看3o分钟| 婷婷色麻豆天堂久久| 亚洲第一av免费看| 男男h啪啪无遮挡| 亚洲精品久久久久久婷婷小说| 日本欧美视频一区| 美女内射精品一级片tv| 深夜a级毛片| 小蜜桃在线观看免费完整版高清| 中文字幕精品免费在线观看视频 | 成人一区二区视频在线观看| 欧美xxxx黑人xx丫x性爽| 中文欧美无线码| 成人午夜精彩视频在线观看| 一级毛片我不卡| 日韩伦理黄色片|