• <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 ?

    Identification of Potential Flavonoid Inhibitors of the SARS-CoV-2 Main Protease 6YNQ:A Molecular Docking Study

    2020-12-31 08:13:04SUMITAroraGOVINDLohiyaKESHAVMoharirSAPANShahSUBHASHYene
    Digital Chinese Medicine 2020年4期

    SUMIT Arora,GOVIND Lohiya,KESHAV Moharir,SAPAN Shah,SUBHASH Yene

    a.Pharmacognosy and Phytochemistry Department,Gurunanak College of Pharmacy,Nagpur,Maharashtra 440026,India

    b.Pharmaceutics Department,Gurunanak College of Pharmacy,Nagpur,Maharashtra 440026,India

    c.Pharmaceutical Chemistry Department,Priyadarshini J.L.College of Pharmacy,Nagpur,Maharashtra 440016,India

    d.Pharmacology Department,Gurunanak College of Pharmacy,Nagpur,Maharashtra 440026,India

    ABSTRACT Objective Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent for coronavirus disease 2019(COVID-19),is responsible for the recent global pandemic.As there are no effective drugs or vaccines available for SARS-CoV-2,we investigated the potential of flavonoids against SARS-CoV-2 main protease 6YNQ.Methods In silico molecular simulation study against SARS-CoV-2 main protease 6YNQ.Results Among the 21 selected flavonoids,rutin demonstrated the highest binding energy (? 8.7 kcal/mol) and displayed perfect binding with the catalytic sites.Conclusions Our study demonstrates the inhibitory potential of flavonoids against SARS-CoV-2 main protease 6YNQ.These computational simulation studies support the hypothesis that flavonoids might be helpful for the treatment of COVID-19.

    Keywords COVID-19 SARS-CoV-2 Protease 6YNQ In silico Molecular simulation Virtual drug screening Flavonoids

    1 Introduction

    The unprecedented coronavirus disease 2019(COVID-19) outbreak has had a critical impact on countries across the globe and on people from every walk of life.As of the beginning of October 2020,the world has recorded 1 111 998 deaths due to COVID-19 and more than 39 944 882 confirmed cases[1].The causative agent of COVID-19,severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),belongs to theβ-coronavirus group.Antiviral drugs can target diverse phases of viral infection.In the case of SARSCoV-2,both structural and nonstructural proteins have been identified as potential drug targets.The main proteases (Mproor 3CLpro) of corona viruses tend to be highly conserved and are critical for viral replication.These proteins are responsible for the maturation of both nonstructural and structural viral proteins,making them a very attractive target for novel anti-coronavirus drugs.Thus,any inhibitor against these proteases (Mproor 3CLpro) that can block the replication of SARS-CoV-2 would be effective for the development of therapeutic agents or antiviral drugs against SARS-CoV-2[2].

    The main protease of SARS-CoV-2,6YNQ,is a homodimer bound to 2-methyl-1-tetralone,which has been expressed inEscherichia coliand has a known crystalline structure,comprising 306 amino acids[3].One important finding is that there have been no mutations in this protein to date.This viral protease is a multifunctional protein involved in the transcription and replication of various viral RNAs and is responsible for the cleavage of the functional replicase polyproteins at 11 different sites.Additionally,6YNQ[3]was shown to share 100% identity and similarity with 6LU7[4]using blast-2-seq,Smith-Waterman and Needleman-Wunsch sequence alignments.In addition,Protein Data Bank (PDB) structures of 6YNQ demonstrate a better resolution (1.8 ?),which ensures better protein structure evaluations.Furthermore,R and R free,who assess the similarity between the calculated values and the observed structural factor amplitudes,were lower for these structures than 6LU7.Although the major protease,spike protein,RdRp,and the papain-like protease have been evaluated as antiviral targets,6YNQ has not yet been evaluated for anti-COVID 19 activity.Co-crystallized ligand P6N (PDB ID 6YNQ) has been shown to interact with the binding site of Mpro,indicating its association with 6YNQ demonstrating the main interaction site for drug targeting[5].Similarly,6YNQ has been recently listed as a possible target for evaluating the efficacy of certain agents against COVID-19[6].Thus,6YNQ with its superior protein structure predictions is far more likely to yield reliable docking results,which is essential in the drug discovery process.

    Recently,the United States Food and Drug Administration approved the use of Gilead’s Remdesivir,marketed under the brand name Veklury,for treatment of COVID-19 for hospitalized patients.This is the first and only drug approved for adults and pediatric patients (age 12 years and older,weighing at least 40 kg) in the treatment of COVID-19 patients requiring hospitalization[7,8].Hydroxychloroquine was under early investigation for use in COVID-19 treatment;however,initial clinical trials were withdrawn amid safety concerns raised by the WHO[9].In order to identify any potential interactions with the SARSCoV-2 protease enzymes,many existing drugs such as Lopinavir,Oseltamivir,Ritonavir and Favipiravir using computational analysis have been evaluated[10,11].

    Interestingly,many bioactive compounds derived from plants that are known to exert some antiviral effects,have attracted researchers’ attention,with a large panel of these compounds under evaluation for SARS-CoV-2[12,13].Among them,flavonoids have been revealed as the most promising antiviral agents[14].Flavonoids are a large class of food additives that have a positive impact on health and which have been extensively evaluated against a wide range of DNA and RNA viruses.For example,the flavones apigenin,has been reported to exert an antiviral effect against picornavirus (RNA virus),while the flavonol quercetin-3-β-galactoside was found to competitively inhibit SARS-CoV 3CLproin anin vitroassay[15].Additionally,anin silicodocking simulation established that Biflavone adheres to the SARS-CoV 3CLprobinding pocket[16].The role of flavonoids and their interactions with diverse cellular targets and pathways involved in the viral life cycle have been widely demonstrated,and when these features are considered in conjunction with the structural diversity and degree of hydroxylation in flavonoids,it is obvious that these compounds could be used against SARS-CoV-2.

    In this study,we assessed the docking of 21 flavonoids and evaluated their potential as inhibitory compounds against the SARS-CoV-2 main protease 6YNQ using the AutoDock Vina software.Furthermore,we confirmed our initial findings and evaluated the structural flexibility of the docked poses for the best flavonoidsusing CABS-flex 2.0 software[17].

    2 Materials and Methods

    2.1 Platform for molecular docking

    The computational docking assessment of 21 flavonoid ligands for the SARS-CoV-2 main protease 6YNQ was performed using the AutoDock Vina software,and comparative docking was performed using Swiss dock (http://www.swissdock.ch/),an online server that uses EADock DSS software[18,19].

    2.2 Preparation of proteins and grids

    In silicoanalysis of 21 flavonoids was performed using a 1.80 ? crystal structure of 6YNQ from the SARSCoV-2 main protease in complex with an inhibitor 2-methyl-1-tetralone (PDB ID:6YNQ,with a resolution <2 ?,R-value free <0.25,R-value work <0.25),which was retrieved from the PDB (https://www.rcsb.org).The 6YNQ protein containsan a chain with(2~{S})-2-methyl-3,4-dihydro-2~{H}-naphthallen-1-one,which was used in the macromolecule preparation[4].The protein preparation parameters of AutoDock were then used to prepare the whole structure by deleting water molecules,adding hydrogen,and assigning partial charges using Kollman and Gasteiger,and the binding sites were identified after deleting the ligand.

    2.3 Ligand preparations

    For this investigation,the ligand (2~{S})-2-methyl-3,4-dihydro-2~{H}-naphthalen-1-one and the structures of each of the flavonoids under investigation were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and saved in SDF.Since the PDB,Partial Charge (Q) and Atom Type (T) (PDBQT)formats can all be used as input in the AutoDock Vina software,Open Babel (version 3.0.0) 21 was used to convert these SDF files to PDB[20].A total of 24 compounds were selected to target the main protease of SARS-CoV-2,including two approved anti-RdRp compounds (Remdesivir)[21]and a recently identified compound used to treat a mild to moderate cases of coronavirus (Favipiravir)[22].

    2.4 Determining compound active sites

    The active sites were defined as the coordinates of the ligand in the original target protein grids,and these active binding sites in the target protein were identified using the computed atlas for surface topography of proteins (CASTp)[23]and Biovia Discovery Studio 4.5[24].The amino acids in the active site were used to evaluate the Grid box and docking evaluation results.PyMol software (version 1.7.4)[25]and the Protein-Ligand Interaction Profiler (PLIP)web server[26]were used to profile the interactions between the ligand-protein complexes showing the lowest binding score and RMSD <2.0 ?.

    2.5 Protein ligand docking and visualization

    AutoDock Vina was used in all the docking experiments,using the optimized model as the docking target.Computational docking is executed to generate a population of promising orientations and conformations of the ligand within the binding site.The grid center for docking was set at X=3.789,Y=– 1.789,and Z=18.846,with the grid box set to 40 ?×40 ?×40 ?.Flavonoids were individually evaluated in the molecular docking,and prior to their first interactions,the classical MM2 force field was applied to optimize the structures of these small molecules,ensuring that their active sites were rigid.After validation of the docking protocol,virtual screening was accomplished using rigid molecular docking into the active site of the partner proteins.Throughout the virtual screening,both the macromolecule and the ligands were kept rigid.Finally,the binding energy limits were removed from the software,and the investigation of the 2D hydrogen-bond interactions could be completed using the Biovia Discovery Studio 4.5 program.This analysis produces a graphical output describing the hydrophobic bonds,hydrogen bonds,and their bond lengths in each docking pose.

    2.6 Physicochemical properties

    Lipinski’s rule was used to assess the physicochemical properties of all the selected flavonoids and predict their drug-like properties,and the Swiss ADME (http://www.swissadme.ch/) was used to compute the SMILES structures of each compound[27].

    2.7 Molecular dynamics simulations

    Molecular dynamics simulations were performed using the CABS Flex 2.0 server and were based on the coarse-grained simulations of protein motion[28]over 50 cycles and 50 trajectory frames of 10 ns each with some additional distance restraints including a global weight of 1.0 applied.These were built with Poisson-Boltzmann/Generalized Born (PB/GB) molecular mechanics,and the solvent probe radius was set to 1.4 ?,the minimum atomic radius was 1 ?,the salt radius was 2 ?,the ionic strength was 0.15,and the temperature of the simulation was 1.4.These restraints allowed us to analyze the conformational stability of the receptor-ligand complex system.

    3 Results

    3.1 Molecular docking

    The results of the molecular docking study using AutoDock Vina revealed the binding energies of the selected compounds,inhibitors and reference molecules (Table1).Additionally,these results were compared with SwissDock.The algorithm consisted of several steps,including the generation of as many binding modes in the local docking as possible,estimating CHARMM energies on the grid,the evaluation of the most favored binding modes using FACTS,and then clustering evaluation.Cluster “0”has the best full fitness (FF) score.We submitted both our protein and phytochemicals one by one.After each docking run,the output clusters were identified and the individual conformer from each cluster with the most favorable binding mode and most negative FF score were chosen for further evaluation as these had the best fit.

    Table1 Ligands with their binding energy and Lipinski rule parameters from the PDB for 6YNQ from SARS-CoV-2

    The inhibitor 2-methyl-1-tetralone binds to the active site of 6YNQ with the lowest binding energy(– 5.6 kcal/mol),where CYS A:145 and HIS A:163 represent the catalytic residues (Figure1A).Favipiravir (binding energy was – 5.5 kcal/mol) docked with a confirmation that forms conventional hydrogen bonds with HIS A:163,GLU A:166,MET A:165 and ASN A:142 along with pi donor hydrogen bonds with CYS A:145 and LEU A:141 (Figure1B),suggesting its interaction with the catalytic residues.In contrast,Remdesivir had the lowest binding energy of– 8.6 kcal/mol,more than the inhibitor and Favipiravir,but failed to make hydrogen bonds with the catalytic residues and rather formed bonds with GLU A:166,HIS A:41,and THR A:25 along with pialkyl interactions with PRO A:168 (Figure1C).

    Among the 21 flavonoids,rutin was shown to bind with the lowest binding energy,– 8.7 kcal/mol,which was close to the Remdesivir binding energy,forming conventional hydrogen bonds with the catalytic active site residues CYS A:145,HIS A:163,SER A:46,THR A:45,HIS A:41,GLU A:166,and GLN A:189 and additional Van der Waals interactions with ASN A:142.In addition,it produced a pialkyl interaction with MET A:49,re-enforcing its binding (Figure2).However,the other flavonoid baicalein demonstrated a binding energy of – 7.9 kcal/mol and formed hydrogen bonds with the active site HIS A:163 and GLU A:166,a pi alkyl interaction with the other catalytic residue CYS A:145,various Van der Waals interactions with LEU A:141,and a pi bond with MET A:49 and HIS A:41 (Figure3).Fisetin presented with a binding energy of – 7.3 kcal/mol and was shown to interact with various amino acid residues producing hydrogen bonds with active site HIS A:163,LEU A:141,GLU A:166,ARG A:188,GLN A:192,THR A:190,PRO A:168 and GLN A:189,and pi alkyl interactions with the other catalytic residues CYS A:145 and PRO A:168(Figure4).The other flavonoids presented with binding energy values ranging from – 8.4 kcal/mol to– 5.6 kcal/mol,although there were no hydrogen bonds with the catalytic site.

    3.2 Physicochemical characterization

    Furthermore,the physicochemical properties of the compounds were studied to predict the pharmacokinetics of the drugs,using Lipinski’s rule.Lipinski’s rules describe orally active drug compounds as having a molecular weight (MW) of <500 Da,an octanol-water partition coefficient (LogP) of <5,a polar surface area (PSA) of <150 ?,number of hydrogen bond donors (HBDs) <5,number of hydrogen bond acceptors (HBAs) <10,and number of rotatable bonds (RBs) <10[29].The Lipinski values for each of the selected compounds are listed in Table1.

    3.3 Molecular dynamics

    The structural flexibility of the best three phytoconstituents in complex with 6YNQ was evaluated using CABS-flex 2.0.To validate the docking results,the structural PDB file was provided to the server with default parameters to obtain the maximum simulation output[30].The root mean square fluctuation (RMSF) values (Figure5) explain the fluctuation of each amino acid residue in the best docked ligand in order to validate the conformational stability of the protein-ligand docked complexes(Figure6).

    4 Discussion

    Molecular docking studies of flavonoids with SARSCoV-2 main protease 6YNQ exhibited promising results based on their binding energies,as determined by AutoDock Vina.In this study,some known antiviral and other flavonoids were selected for targeting SARS-CoV-2 main protease 6YNQ,and molecular docking studies were carried out to assess their potential antiviral effect.To evaluate the binding between the flavonoids and the targets,we selected 21 flavonoids against 6YNQ,along with their known inhibitor 2-methyl-1-tetralone,and reference compounds Remdesivir and Favipiravir.Our results suggest that most of the ligands present with nearly the same score in either docking method,with a corresponding correlation coefficient of 0.752 7 between docking scores obtained using AutoDock vina and Delta G by SwissDock,supporting the accuracy of the AutoDock vina predictions.Based on these results,three flavonoids,rutin,baicalein and fisetin,should be considered potential inhibitors of SARS-CoV-2 main protease 6YNQ acting via Mpro6YNQ inhibition.

    Rutin demonstrated strong inhibition of 6YNQ,forming conventional hydrogen bonds with the catalytic active site residue CysA 145 and having the lowest binding energy (? 8.7 kcal/mol) of any of the compounds.Taken together,this suggests that it exhibits the strongest and most stable binding.This result is in agreement with previously published data that suggest that rutin (docking score:? 9.16 kcal/mol) is the most potent inhibitor for 6LU7[31].Other researchers have also reported that rutin is an effective inhibitor of various targets of the SARS-CoV-2 proteases[32].These studies have confirmed that CysA 145 is a critical residue within the binding pocket of these proteases falling within a 6 ? radius around the catalytic center of these proteins[33,34],and support the application of rutin as a competitive SARS-CoV 3CLproinhibitor that interacts via hydrogen bonding with the catalytically active residue CysA 145.

    One of the most studied flavonoids,baicalein,also forms hydrogen bonds with these proteins,targeting the other catalytic residue,i.e.,histidine.Numerous studies have reported that baicalein and its analogs are strong inhibitors of SARS-CoV-2 3CLproand helicase,suggesting that baicalein is a potential candidate for combating coronavirus disease[35,36].In addition,a traditional Chinese medicine formulation containing baicalein was evaluated in a neutralization study using a fRhK4 cell line infected with 10 strains of SARS-CoV-2 from 10 different patients and shown to effectively neutralize these viruses,supporting the potential clinical application of this product[37].The flavonol fisetin also produced both hydrogen and pi alkyl bonds with the catalytic center of 6YNQ,although its binding energy was somewhat lower than rutin and baicalein.Other studies have reported binding of fisetin with 6LU7[38].Given these results,we propose that a combination of rutin,baicalein and fisetin may produce a synergistic inhibition of both catalytically active residues in 6YNQ,improving its overall inhibition.

    Lipinski’s rule is a major deciding factor when evaluating the potential of drug candidates and is often used to determine whether a compound with particular pharmacological or biological actions possesses the necessary physical and chemical properties for administration in humans.Evaluation of the molecular properties of the compounds based on the computed partition coefficient (LogP) demonstrated that these compounds have relatively good lipophilicity,as the LogPvalues were less than 5[39,40].These results also demonstrated that both baicalein and fisetin strictly followed Lipinski’s rule with zero violations,indicating that both compounds are likely to possess active drug characteristics

    Low RMSF values imply limited motion within a system,while high values in the molecular dynamics simulations reflect more flexibility[41].The results of the molecular dynamics simulations show that there are appropriate secondary structure residues with theα-helix andβ-sheet of the protein that present with minimal fluctuation when evaluated using efficient constraints in the all-atom molecular dynamics algorithm,a classical simulation approach for proteins.Rutin,baicalein and fisetin were shown to maintain their molecular interactions with the target protein under all of these conditions,confirming their likely interaction.

    Interestingly,these three flavonoids are nutraceuticals and act as vital nutritional component of various fruits and vegetables.Thus,we anticipate that this nutraceutical has the potential to enhance immunity and inhibit COVID-19 infections in the population[42].Furthermore,combination therapy of synthetic drugs with flavonoids often results in superior outcomes for antiviral treatments[43,44].Most flavonoid evaluations for COVID-19 have focused on 3CL as the main viral protease[45];however,our study demonstrates the potential for flavonoid treatments to affect other targets including 6YNQ of SARS-CoV-2.

    Conclusions

    Human health and safety is intrinsically linked with the need to find and test novel interventions for COVID-19 (SARS-CoV-2),making any study related to these endeavors critical to global concerns.Here,we have used computational docking studies of various flavonoids against the SARS-CoV-2 main protease 6YNQ to help identify novel therapeutic effectors.We evaluated a library of 21 flavonoids and revealed that rutin,baicalein and fisetin bind the target efficiently and may have value as potential inhibitors.Thus,we conclude that these phytochemicals can be used as potential antiviral candidates and suggest that furtherin vitroorin vivoexperiments may provide better insight into the optimal flavonoid structure for preventing and treating COVID-19.

    Acknowledgements

    The authors are thankful to the Principal,Gurunanak College of Pharmacy,Principal,Priyadarshini J.L.College of Pharmacy and management of the Sikh Education Society for extending facilities.

    Competing Interests

    The authors declare no conflict of interest.

    亚洲国产精品专区欧美| 国产 一区精品| av一本久久久久| 成人毛片60女人毛片免费| 高清毛片免费看| 久久久久精品性色| 精品一区在线观看国产| 国产av不卡久久| av免费观看日本| 亚洲精品亚洲一区二区| 免费高清在线观看视频在线观看| 日韩人妻高清精品专区| 欧美xxⅹ黑人| 永久网站在线| 午夜福利高清视频| 91久久精品电影网| 纵有疾风起免费观看全集完整版| 熟女电影av网| 制服丝袜香蕉在线| 久久久久久九九精品二区国产| 国产高潮美女av| 免费看光身美女| 麻豆国产97在线/欧美| 18禁裸乳无遮挡动漫免费视频 | 久久国内精品自在自线图片| 简卡轻食公司| 超碰97精品在线观看| 在线观看人妻少妇| 免费看不卡的av| 国产老妇女一区| 免费看a级黄色片| 一级毛片 在线播放| av在线天堂中文字幕| 亚洲av免费高清在线观看| 久久久久久久国产电影| 国产淫语在线视频| 一级爰片在线观看| 少妇的逼水好多| av天堂中文字幕网| 一区二区三区精品91| 国产成人精品婷婷| 69人妻影院| 男人爽女人下面视频在线观看| 18禁动态无遮挡网站| 在线观看人妻少妇| 麻豆成人av视频| 国产伦理片在线播放av一区| 国产伦精品一区二区三区四那| 黄色欧美视频在线观看| av专区在线播放| 亚洲欧美成人精品一区二区| 亚洲国产精品999| 亚洲国产日韩一区二区| 在线精品无人区一区二区三 | 国产毛片在线视频| www.av在线官网国产| 人体艺术视频欧美日本| 99精国产麻豆久久婷婷| 亚洲激情五月婷婷啪啪| 日韩av免费高清视频| 国产亚洲av嫩草精品影院| 如何舔出高潮| 大香蕉久久网| 最近最新中文字幕免费大全7| 欧美xxxx黑人xx丫x性爽| 亚洲图色成人| 全区人妻精品视频| 啦啦啦在线观看免费高清www| 国产亚洲精品久久久com| 真实男女啪啪啪动态图| 边亲边吃奶的免费视频| 国产精品精品国产色婷婷| 三级国产精品片| 欧美老熟妇乱子伦牲交| 免费高清在线观看视频在线观看| 一个人观看的视频www高清免费观看| 欧美极品一区二区三区四区| 最近中文字幕2019免费版| 亚洲一级一片aⅴ在线观看| 久久国内精品自在自线图片| 91精品一卡2卡3卡4卡| 蜜臀久久99精品久久宅男| 国产黄a三级三级三级人| 国产精品久久久久久精品电影小说 | 老司机影院毛片| 大香蕉久久网| 人人妻人人看人人澡| 岛国毛片在线播放| 国产欧美亚洲国产| 午夜福利网站1000一区二区三区| 日韩一本色道免费dvd| 3wmmmm亚洲av在线观看| 国产精品人妻久久久影院| 美女xxoo啪啪120秒动态图| 美女内射精品一级片tv| 少妇 在线观看| 久久精品国产亚洲av天美| 秋霞伦理黄片| 欧美最新免费一区二区三区| 亚洲自拍偷在线| 国产精品人妻久久久影院| 亚洲美女视频黄频| 色视频在线一区二区三区| 在线免费观看不下载黄p国产| 人人妻人人看人人澡| 免费高清在线观看视频在线观看| 免费观看无遮挡的男女| 久久精品国产亚洲av天美| 久久久久久久久久人人人人人人| 久久久久久伊人网av| 久久韩国三级中文字幕| 亚洲在线观看片| av专区在线播放| 少妇猛男粗大的猛烈进出视频 | 日本三级黄在线观看| 狠狠精品人妻久久久久久综合| 欧美3d第一页| 男人狂女人下面高潮的视频| tube8黄色片| 日韩成人伦理影院| 肉色欧美久久久久久久蜜桃 | 啦啦啦啦在线视频资源| 国产亚洲av片在线观看秒播厂| 国产精品不卡视频一区二区| 国产精品一区二区在线观看99| 国产精品国产三级专区第一集| 九色成人免费人妻av| 亚洲四区av| av专区在线播放| 久久99蜜桃精品久久| 免费观看性生交大片5| 高清毛片免费看| 日本免费在线观看一区| 狂野欧美激情性xxxx在线观看| 欧美3d第一页| 身体一侧抽搐| 在线观看美女被高潮喷水网站| 中文字幕制服av| 免费大片黄手机在线观看| 久久99精品国语久久久| av在线观看视频网站免费| 最近的中文字幕免费完整| 韩国av在线不卡| 国产在线一区二区三区精| 欧美老熟妇乱子伦牲交| 人妻夜夜爽99麻豆av| 搡女人真爽免费视频火全软件| 久久久久久久午夜电影| 欧美日韩国产mv在线观看视频 | 久久人人爽人人片av| 99久久人妻综合| 欧美老熟妇乱子伦牲交| 久久热精品热| 啦啦啦在线观看免费高清www| 日本一二三区视频观看| 波多野结衣巨乳人妻| 精品久久久久久久久亚洲| 一区二区三区免费毛片| 18禁动态无遮挡网站| 亚洲精品日本国产第一区| 69人妻影院| 免费黄色在线免费观看| 99热这里只有精品一区| 国产成人freesex在线| 男女下面进入的视频免费午夜| 国产高清有码在线观看视频| 水蜜桃什么品种好| 赤兔流量卡办理| 男女边摸边吃奶| 激情五月婷婷亚洲| 亚洲欧美日韩另类电影网站 | 有码 亚洲区| av国产久精品久网站免费入址| 久久人人爽人人片av| 精品久久久久久久人妻蜜臀av| 国产av码专区亚洲av| 少妇丰满av| 听说在线观看完整版免费高清| 好男人视频免费观看在线| 超碰97精品在线观看| 涩涩av久久男人的天堂| 91久久精品国产一区二区成人| videossex国产| 国产精品嫩草影院av在线观看| 亚洲精品456在线播放app| 中国美白少妇内射xxxbb| 身体一侧抽搐| 精品午夜福利在线看| 最近中文字幕高清免费大全6| 99热全是精品| av专区在线播放| 你懂的网址亚洲精品在线观看| 日本色播在线视频| 国产一区二区亚洲精品在线观看| 亚洲婷婷狠狠爱综合网| 超碰97精品在线观看| 在线播放无遮挡| 两个人的视频大全免费| 日韩不卡一区二区三区视频在线| 最新中文字幕久久久久| 久久精品久久久久久噜噜老黄| 久久久久精品久久久久真实原创| av一本久久久久| 一本久久精品| 极品教师在线视频| 国产 一区 欧美 日韩| 亚洲av成人精品一区久久| 波多野结衣巨乳人妻| 国产av码专区亚洲av| 精品国产乱码久久久久久小说| 久久久精品欧美日韩精品| 性插视频无遮挡在线免费观看| 国产黄色视频一区二区在线观看| 国产精品麻豆人妻色哟哟久久| 免费av不卡在线播放| 黄片wwwwww| 色综合色国产| 日日啪夜夜撸| 一边亲一边摸免费视频| 在线 av 中文字幕| 黄色配什么色好看| 欧美性感艳星| 成人一区二区视频在线观看| 亚洲成人中文字幕在线播放| 五月天丁香电影| 亚洲,欧美,日韩| 成人国产av品久久久| 美女被艹到高潮喷水动态| 亚洲精品日韩av片在线观看| 秋霞在线观看毛片| 国产高潮美女av| 午夜福利视频精品| 国产伦理片在线播放av一区| 国产亚洲午夜精品一区二区久久 | 国产成人精品婷婷| 中文字幕人妻熟人妻熟丝袜美| 国产黄频视频在线观看| 国产精品久久久久久精品电影小说 | 18禁在线播放成人免费| 国产免费一区二区三区四区乱码| 嘟嘟电影网在线观看| h日本视频在线播放| 亚洲成人精品中文字幕电影| 大片电影免费在线观看免费| 一级毛片久久久久久久久女| 精品一区二区三区视频在线| 国产成人精品福利久久| 欧美日韩视频高清一区二区三区二| 丝袜喷水一区| 国产成人精品久久久久久| 精品人妻偷拍中文字幕| 夜夜爽夜夜爽视频| av在线亚洲专区| 我的老师免费观看完整版| 舔av片在线| 中文字幕人妻熟人妻熟丝袜美| 国产精品无大码| 久久久久精品性色| 日本黄大片高清| 国精品久久久久久国模美| 成人欧美大片| 国产一区亚洲一区在线观看| 日本三级黄在线观看| 中文欧美无线码| 国产精品久久久久久精品电影小说 | 免费av观看视频| 中国三级夫妇交换| 日日啪夜夜爽| 国产伦精品一区二区三区视频9| 99热6这里只有精品| 国产高清不卡午夜福利| 性插视频无遮挡在线免费观看| 亚洲性久久影院| 国产精品三级大全| 国产成人精品一,二区| 看非洲黑人一级黄片| 亚洲人与动物交配视频| 三级国产精品欧美在线观看| 亚洲va在线va天堂va国产| 一个人看视频在线观看www免费| 日韩av免费高清视频| av黄色大香蕉| 亚洲av成人精品一二三区| 麻豆精品久久久久久蜜桃| 午夜福利在线观看免费完整高清在| av国产精品久久久久影院| 国产成人午夜福利电影在线观看| 一边亲一边摸免费视频| 国产一区有黄有色的免费视频| 久久久亚洲精品成人影院| 一级片'在线观看视频| 欧美亚洲 丝袜 人妻 在线| 菩萨蛮人人尽说江南好唐韦庄| 国产精品久久久久久av不卡| 日日啪夜夜爽| 深夜a级毛片| 一级毛片我不卡| 欧美高清成人免费视频www| 国产精品秋霞免费鲁丝片| 免费黄色在线免费观看| 夜夜看夜夜爽夜夜摸| 免费观看性生交大片5| 国产色爽女视频免费观看| 久久精品人妻少妇| 一区二区三区四区激情视频| 国产免费又黄又爽又色| 中文欧美无线码| 国产在视频线精品| 熟女av电影| 久久久久久久久大av| 国产视频内射| 日韩,欧美,国产一区二区三区| 大码成人一级视频| 国产欧美日韩一区二区三区在线 | 国产精品一区二区在线观看99| 看非洲黑人一级黄片| 日韩免费高清中文字幕av| 精品久久久噜噜| 久久99精品国语久久久| 大片免费播放器 马上看| 大又大粗又爽又黄少妇毛片口| 日产精品乱码卡一卡2卡三| 亚洲欧美日韩卡通动漫| 国产成人精品一,二区| av天堂中文字幕网| 亚洲在久久综合| 男女下面进入的视频免费午夜| 大片免费播放器 马上看| 国产在线一区二区三区精| 日韩人妻高清精品专区| 久久久久久久精品精品| 日韩一区二区三区影片| 日韩成人伦理影院| 日韩不卡一区二区三区视频在线| 国内精品宾馆在线| 日日摸夜夜添夜夜添av毛片| 久久精品久久久久久噜噜老黄| 好男人在线观看高清免费视频| 成人一区二区视频在线观看| 97人妻精品一区二区三区麻豆| 亚洲精品成人av观看孕妇| 久久久久久九九精品二区国产| 在线 av 中文字幕| 午夜福利高清视频| 亚洲欧美日韩另类电影网站 | 欧美三级亚洲精品| 美女cb高潮喷水在线观看| 国产伦在线观看视频一区| 亚洲美女视频黄频| 国产黄片美女视频| 一级毛片黄色毛片免费观看视频| 日本午夜av视频| 日日摸夜夜添夜夜爱| 欧美丝袜亚洲另类| 黄色欧美视频在线观看| av.在线天堂| 18禁在线播放成人免费| 国产日韩欧美亚洲二区| 午夜福利在线在线| 欧美成人一区二区免费高清观看| 在线观看一区二区三区激情| 成人特级av手机在线观看| freevideosex欧美| 丝袜美腿在线中文| 亚洲三级黄色毛片| 婷婷色麻豆天堂久久| 一区二区三区四区激情视频| 亚洲精品成人av观看孕妇| 在线精品无人区一区二区三 | 免费高清在线观看视频在线观看| 国模一区二区三区四区视频| 日韩成人伦理影院| 一级毛片 在线播放| 国产乱来视频区| av在线亚洲专区| 狂野欧美激情性xxxx在线观看| 国产精品国产av在线观看| 97在线视频观看| 国产伦精品一区二区三区四那| 男女边吃奶边做爰视频| 少妇的逼水好多| 噜噜噜噜噜久久久久久91| 赤兔流量卡办理| 日韩强制内射视频| 日韩在线高清观看一区二区三区| 国产综合精华液| 高清视频免费观看一区二区| 日本av手机在线免费观看| 国产成人午夜福利电影在线观看| 在线观看美女被高潮喷水网站| 国产成人freesex在线| 久久97久久精品| 黄色配什么色好看| 最后的刺客免费高清国语| 精品国产一区二区三区久久久樱花 | 国产精品三级大全| 另类亚洲欧美激情| 黄色怎么调成土黄色| 岛国毛片在线播放| 最后的刺客免费高清国语| 免费观看在线日韩| 亚洲国产精品成人久久小说| 国产亚洲5aaaaa淫片| 国产精品久久久久久精品古装| 少妇人妻一区二区三区视频| 午夜精品国产一区二区电影 | 秋霞在线观看毛片| 成人黄色视频免费在线看| 亚洲人成网站在线播| 亚洲欧美成人综合另类久久久| 又爽又黄a免费视频| 高清日韩中文字幕在线| 一级毛片aaaaaa免费看小| 国产一区二区亚洲精品在线观看| 在线播放无遮挡| 日本av手机在线免费观看| 国产精品一区二区三区四区免费观看| 国语对白做爰xxxⅹ性视频网站| 插逼视频在线观看| 精品一区二区三区视频在线| 午夜日本视频在线| 国产精品蜜桃在线观看| 我的女老师完整版在线观看| 狠狠精品人妻久久久久久综合| 国产亚洲5aaaaa淫片| 欧美日韩国产mv在线观看视频 | 精品人妻视频免费看| 午夜激情福利司机影院| 久久精品国产亚洲av天美| 久久久亚洲精品成人影院| 卡戴珊不雅视频在线播放| 亚洲国产精品999| 中文天堂在线官网| 男人舔奶头视频| 国产精品99久久99久久久不卡 | 亚洲在线观看片| 日韩一本色道免费dvd| av国产久精品久网站免费入址| www.av在线官网国产| av一本久久久久| 高清在线视频一区二区三区| 亚洲精品自拍成人| 在现免费观看毛片| .国产精品久久| 亚洲aⅴ乱码一区二区在线播放| 91狼人影院| 亚洲国产欧美人成| 国产 一区 欧美 日韩| 国产精品av视频在线免费观看| 久久久久性生活片| 身体一侧抽搐| 午夜老司机福利剧场| 国产黄片美女视频| 欧美zozozo另类| 日韩国内少妇激情av| 欧美一区二区亚洲| 亚洲精品一二三| 一级a做视频免费观看| 九九爱精品视频在线观看| av在线观看视频网站免费| 51国产日韩欧美| 麻豆久久精品国产亚洲av| videossex国产| 网址你懂的国产日韩在线| 国产黄片视频在线免费观看| 国产成人一区二区在线| av免费观看日本| 精品人妻视频免费看| 最近的中文字幕免费完整| 人体艺术视频欧美日本| 精品久久久久久久末码| 色综合色国产| 一区二区三区四区激情视频| 色5月婷婷丁香| 国国产精品蜜臀av免费| 国产免费又黄又爽又色| 丰满人妻一区二区三区视频av| 欧美成人精品欧美一级黄| 国产真实伦视频高清在线观看| 免费黄色在线免费观看| 亚洲一区二区三区欧美精品 | 国产免费一级a男人的天堂| 亚洲人与动物交配视频| 99热国产这里只有精品6| 国产日韩欧美在线精品| 久久综合国产亚洲精品| 九九在线视频观看精品| 欧美xxxx性猛交bbbb| 国产一区二区在线观看日韩| 国产免费又黄又爽又色| 午夜免费鲁丝| av福利片在线观看| 少妇人妻精品综合一区二区| 精品国产三级普通话版| 日韩一区二区视频免费看| 免费观看性生交大片5| 欧美日韩一区二区视频在线观看视频在线 | 直男gayav资源| 一级a做视频免费观看| 国产成人freesex在线| 美女cb高潮喷水在线观看| 久久久午夜欧美精品| 国产欧美日韩一区二区三区在线 | 搞女人的毛片| 一级毛片我不卡| 亚洲av中文av极速乱| 欧美高清性xxxxhd video| 少妇人妻久久综合中文| 新久久久久国产一级毛片| 中国三级夫妇交换| 国产高清三级在线| 亚州av有码| 久久久久久久亚洲中文字幕| 免费av毛片视频| 欧美丝袜亚洲另类| 中文字幕av成人在线电影| 熟女人妻精品中文字幕| 汤姆久久久久久久影院中文字幕| 色视频www国产| 少妇丰满av| 久久久久久久国产电影| 亚洲熟女精品中文字幕| 狠狠精品人妻久久久久久综合| 亚洲精品成人久久久久久| 蜜桃亚洲精品一区二区三区| 亚洲成人中文字幕在线播放| 中文字幕制服av| 日本wwww免费看| 久久久久久久久久久丰满| 国产伦精品一区二区三区视频9| 伊人久久国产一区二区| 七月丁香在线播放| 国产精品麻豆人妻色哟哟久久| 精品久久国产蜜桃| 三级经典国产精品| 精品人妻熟女av久视频| 国产一区二区在线观看日韩| 久久这里有精品视频免费| 久久韩国三级中文字幕| 亚洲精品亚洲一区二区| 日本三级黄在线观看| av一本久久久久| 久久久久久久久久人人人人人人| 三级国产精品片| 99热网站在线观看| 国产毛片在线视频| 国产精品熟女久久久久浪| 国产精品无大码| 免费少妇av软件| av国产精品久久久久影院| 免费观看在线日韩| 欧美 日韩 精品 国产| xxx大片免费视频| 精品少妇黑人巨大在线播放| 2021少妇久久久久久久久久久| 亚洲熟女精品中文字幕| 国产淫语在线视频| 国产高清有码在线观看视频| 国内少妇人妻偷人精品xxx网站| 亚洲美女搞黄在线观看| 麻豆国产97在线/欧美| 亚洲色图av天堂| 国产中年淑女户外野战色| 精品久久久久久电影网| 人妻一区二区av| 国产精品熟女久久久久浪| 美女主播在线视频| 亚洲丝袜综合中文字幕| 少妇猛男粗大的猛烈进出视频 | 欧美3d第一页| 少妇的逼好多水| 人人妻人人澡人人爽人人夜夜| 最近2019中文字幕mv第一页| 国产伦精品一区二区三区视频9| 91精品一卡2卡3卡4卡| 久久国内精品自在自线图片| 3wmmmm亚洲av在线观看| 国产午夜精品一二区理论片| 一本久久精品| 五月伊人婷婷丁香| 美女被艹到高潮喷水动态| 日日摸夜夜添夜夜爱| 亚洲一区二区三区欧美精品 | 丰满乱子伦码专区| 高清午夜精品一区二区三区| 极品教师在线视频| 一级a做视频免费观看| 亚洲成人av在线免费| 成年人午夜在线观看视频| 日韩伦理黄色片| 成人午夜精彩视频在线观看| 18禁裸乳无遮挡免费网站照片| 国产 一区精品| 联通29元200g的流量卡| 一区二区三区免费毛片| 精品人妻视频免费看| 午夜免费观看性视频| 最后的刺客免费高清国语| 日本色播在线视频| 少妇的逼水好多| 国产亚洲精品久久久com| 最新中文字幕久久久久| 国产精品不卡视频一区二区| 国产精品嫩草影院av在线观看| 亚洲欧美成人综合另类久久久| 中文资源天堂在线| av女优亚洲男人天堂| 国产成人91sexporn| 国产毛片在线视频| 国产人妻一区二区三区在| 99久久中文字幕三级久久日本| 久久鲁丝午夜福利片| 国产精品嫩草影院av在线观看| 国产精品福利在线免费观看| 色视频www国产|