• <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 ?

    Cytotoxic effect of Rosa canina extract on human colon cancer cells through repression of telomerase expression

    2018-12-10 12:02:42IbrhimTurnSelimDemirKgnKilincSerpOzerYmnSemMisirHnieKrBernGencAhmetMenteseYukselAliyziciogluOrhnDeger
    Journal of Pharmaceutical Analysis 2018年6期

    Ibrhim Turn,Selim Demir,Kgn Kilinc,Serp Ozer Ymn,Sem Misir,e,Hnie Kr,Bern Genc,Ahmet Mentese,Yuksel Aliyzicioglu,Orhn Deger

    aDepartment of Genetic and Bioengineering,Faculty of Engineering and Natural Sciences,Gumushane University,29100 Gumushane,Turkey

    bMedicinal Plants,Traditional Medicine Practice and Research Center,Gumushane University,29100 Gumushane,Turkey

    cDepartment of Nutrition and Dietetics,Faculty of Health Sciences,Karadeniz Technical University,61080 Trabzon,Turkey

    dDepartment of Medical Biochemistry,Faculty of Medicine,Karadeniz Technical University,61080 Trabzon,Turkey

    eDepartment of Biochemistry,Faculty of Pharmacy,Cumhuriyet University,58140 Sivas,Turkey

    fProgram of Medical Laboratory Techniques,Vocational School of Health Sciences,Karadeniz Technical University,61080 Trabzon,Turkey

    Keywords:Apoptosis Colon cancer Cytotoxicity Rosa canina Telomerase

    A B S T R A C T Rosa canina is a member of the genus Rosa that has long been used for medical objectives.Several studies have reported cytotoxic effects of different Rosa species,but there has been only limited investigation of the cytotoxic effect of R.canina.The purpose of the current study was to examine the potential effect of R.canina extract on cell viability,the cell cycle,apoptosis,and the expression of telomerase in human colon cancer(WiDr)cells.The cytotoxic effect of the extract was determined using MTT assay.The mechanism involved in the cytotoxic effect of the extract was then evaluated in terms of apoptosis and the cell cycle using flow cytometry.Mitochondrial membrane potential(MMP)was investigated using the fluorometric method,and expression levels of telomerase were studied using RT-PCR.R.canina extract exhibited a selective cytotoxic effect on WiDr cells compared with normal colon cells.The extract induced cell cycle arrest at the S phase and apoptosis via reduced MMP in WiDr cells.R.canina extract significantly repressed telomerase expressions at treatment times of 48 and 72 h in WiDr cells.Our results suggest that R.canina may have considerable potential for development as a novel natural product-based anticancer agent.

    1.Introduction

    Rosa canina is a member of the genus Rosa and the family Rosaceae.The genus Rosa contains more than 100 species widely spread over Asia,Europe,the Middle East,and North America[1,2].

    Approximately 25%of all rose species are reported to grow in Turkey,especially in the regions of central and North-East Anatolia,and particularly in Gumushane and neighboring cities.R.canina contains many beneficial compounds,including vitamins,phenolic acids,proanthocyanidins,tannins, flavonoids,pentacyclic triterpenes,and minerals[1,3,4].It exhibits numerous biological properties,such as antioxidant,anti-inflammatory,anti-ulcerogenic,anti-obesity,antidiabetic,diuretic,antimutagenic,anticarcinogenic,anti-arthritic,neuroprotective,and antimicrobial effects[3–6].R.canina is one of the most commonly used medicinal plants in traditional medicine in both Western and Asian countries for the treatment of colds,asthma,hemorrhoids,infections,chronic pains,arthritis,and inflammatory diseases[1,2,4].The fruits of R.canina are consumed as a natural source of vitamin C in the forms of tea,snacks,jam,nectar,and dried pulp.There is also consider able interest in this speciesin various commercial spheres,especially the food,pharmaceutical,and cosmetic industries[2,3].

    Cancer is a pathological condition characterized by excessive cell growth and deriving from loss of control over the cell cycle and/or decreased apoptosis[7].Cancer cells exhibit characteristic features,such as avoidance of apoptosis,impaired cell cycle control,self-sufficiency in growth signaling,and telomerase activation[8,9].Telomerase is a ribonucleoprotein consisting of human telomerase reverse transcriptase(hTERT)and human telomerase RNA(hTR)[8].Its principal function is to synthesize telomeres using the RNA template instead of telomeric series lost during DNA replication in order to overcome the problem of end-replication[10].Telomerase reactivation in the cell is believed to be associated with carcinogenesis and is a significant step in tumor immortalization.Telomerase is reported to be active in more than 85%of all tumors but is present at low or undetectable levels in normal cells[8].Telomerase plays an important role in the unlimited growth of cancer cells,and inhibition of telomerase activity potentially represents a highly selective target for the treatment of cancer[11].

    Colon cancer is one of the most common types of cancers worldwide.While chemotherapy is one of the most widely used therapeutic strategies against colon cancer,it also has some limitations,such as normal cell toxicity and gradually increasing resistance in cancer cells.The discovery of new drugs for use in alternative strategies in cancer treatment is therefore highly desirable.Plants are regarded as very promising from this perspective,since they represent substantial sources of substances with various therapeutic uses.Most anticancer drugs are today produced from plants[12–14].

    Various studies have investigated the cytotoxic effects of different Rosa species.Olsson et al.[15]demonstrated that ethanolic extract of rose-hip has a cytotoxic effect on human colon(HT-29)and breast(MCF-7)cancer cells,while Fujii et al.[16]reported that ethanolic extract of R.canina exhibits a cytotoxic effect on mouse melanoma cells by inhibiting tyrosinase activity.Zamiri-Akhlaghi et al.[17]reported that ethanolic extract of R.damascena has a concentration-dependent cytotoxic effect against human cervix cancer(HeLa)cells.Recently,Jiménez et al.[18]demonstrated that R.canina extracts exhibit antiproliferative effects on human colon cancer(Caco-2)cells by increasing the number of apoptotic cells and cell cycle arrest at the S phase.However,no studies have examined the relationship between telomerase and R.canina extract in any cancer cell lines.The objective of this study was therefore to determine the cytotoxic effect of acidified dimethyl sulfoxide extract of R.canina on colon cancer(WiDr)cells,coupled with the mechanism of action.

    2.Experimental

    2.1.Chemicals

    Gentamicin and trypsin solutions were obtained from Biological Industries(Kibbutz Beit Haemek,Israel),Eagle's minimum essential medium(EMEM)from Lonza(Verviers,Belgium),and fetal bovine serum(FBS)from Biochrom(Berlin,Germany).All flow cytometry kits were purchased from Becton Dickinson(San Diego,CA,USA).All kits used for gene expression studies were purchased from Roche Diagnostics(Mannheim,Germany).The other principal chemicals used were obtained from Sigma(St.Louis,MO,USA).

    2.2.Sample collection and extract preparation

    Fully mature fruits of R.canina were harvested from Gumushane in the Eastern Anatolia region of Turkey.Samples were preserved in cool bags for transportation to the laboratory.These fruits were air-dried at 25°C and converted into a fine powder using a blender and milling procedures.The fruit powder(1 g)was extracted with 20 mL of dimethyl sulfoxide(DMSO)plus 0.5%(v/v)hydrogen chloride(HCl)in a mechanical shaker(Shell Lab,Cornelius,OR,USA)in the dark for 24 h at 45°C.The prepared 50 mg/mL stock acidified DMSO extract of R.canina was filtered with Whatman No.1 filter paper and a 0.2μm filter and then stored at-20°C until used in further experiments.

    2.3.Drug preparation and treatment

    Cisplatin was dissolved in DMSO and used as a reference compound in cytotoxicity experiments due to its use in colon cancer treatment[19].Final solvent concentrations of compounds were no higher than 0.5%in culture media in any experiment.That concentration was not sufficient to affect cell morphology or viability.

    2.4.Cell culture

    Human colon adenocarcinoma(WiDr,ATCC-CCL-218)cancer and human colon normal epithelial cell lines(CCD 841 CoN,ATCCCRL-1790)were supplied by the America Type Culture Collection(Manassas,VA,USA).Both cells were cultured in EMEM supplemented with 10%heat inactivated FBS and 1%antibiotic solution with a 5%CO2supply at 37°C.

    2.4.1.Cytotoxicity experiments

    MTT assay with a 72 h treatment time was employed to measure the cytotoxic effects of R.canina extract and cisplatin on colon cancer and normal cells[20].Brie fly,cells were seeded into a 96-well cell culture plate at 1×104cells per well.The cells were then treated with varying concentrations of R.canina extract(0–500 μg/mL)and cisplatin(0–10 μg/mL)for 72 h in a quadruplet manner.Subsequently,10 μL of MTT dye(0.25 mg/mL)was placed inside each well.The crystals that emerged were then dissolved in DMSO.Finally,absorbance was measured at 570 nm with the help of a microplate reader(Molecular Devices Versamax,California,USA).Optical densities were employed to calculate percentage viabilities in treated cells compared to untreated control cells.Logconcentrations versus%cell viabilities were plotted with a logarithmic graph,which was then used to determine the IC50values.

    2.4.2.Flow cytometry analysis for cell cycle distribution

    WiDr cells in the exponential growth phase were treated with 135,270,405 and 540 μg/mL concentrations of R.canina extract for 72 h,then harvested by trypsinization,and washed twice with buffer solution(containing sodium citrate,sucrose,and DMSO).After that procedure,250 μL of trypsin buffer was added to each tube and incubated for 10 min.Next,200 μL of trypsin inhibitor and RNase buffer was added to each tube and incubated for 10 min.Finally,200 μL of cold PI stain solution was added to each tube and incubated for 10 min in the dark on ice.Data from 30,000 cells per sample were collected and analyzed on a flow cytometer(BD Accuri C6,MI,USA).The percentage of cells in cycle phases was determined using MODFIT 3.0 verity software.The results were finally compared with the untreated control cells.

    2.4.3.Measurement of apoptosis using flow cytometry

    WiDr cells in the exponential growth phase were treated with 135,270,405 and 540 μg/mL concentrations of R.canina extract for 72 h,then harvested by trypsinization,and washed twice with icecold PBS.The cells were then resuspended with 100 μL of the binding buffer.In the next stage,5 μL of FITC Annexin V and 5 μL of propidium iodide(PI)were added to each tube and incubated for 10 min at room temperature in the dark.Finally,400 μL of the binding buffer was added to each tube and data from 10,000 cells per sample were collected and analyzed on a flow cytometer(BD Accuri C6,MI,USA)within 1 h.The results were compared with the untreated control cells.

    2.4.4.Determination of mitochondrial membrane potential(MMP)

    DiOC6(3),a lipophilic cationic dye,was used to determine the changes in MMP in this study.Normally,it tends to remain in the mitochondria.A decrease in dye intensity indicates disruption of MMP[21].Brie fly,cells were seeded into a 96-well black-walled plate at 1×103cells per well.The cells were then treated with different concentrations of R.canina extract(135–540 μg/mL)for 72 h.After treatment,the cells were washed twice with PBS and loaded with 10 nM DiOC6(3)for 30 min at 37°C in the dark.Finally,the fluorescence measurement was performed on a plate-reading fluorometer(Molecular Devices SpectraMax Paradigm Multi-Mode,Sunnyvale,CA,USA)with an excitation wavelength of 484 nm and an emission wavelength of 525 nm.Results were given as relative MMP compared to untreated control cells.

    2.4.5.Gene expression analysis

    Based on data from the cytotoxicity studies,the extract IC50value(270 μg/mL)was used to investigate gene expression.WiDr cells were treated with this concentration of extract for 24,48,and 72 h.After incubation,the cells were harvested for RNA isolation.

    2.4.6.RNA isolation and RT-PCR

    Total RNA isolation was performed using a highly pure RNA isolation kit in line with the manufacturer's recommmendations.RNA samples were converted to complementary DNA using a first strand cDNA synthesis kit as recommended by the manufacturer.A total of 100 ng/μL total RNA was employed for all reaction samples.Reaction compounds were incubated at 25 °C for 10 min,50 °C for 60 min,and 85°C for 5 min.

    The RT-PCR method using UPL probes was employed to measure hTERT expression.hTERT and GAPDH amplicon dimensions were 60 and 127 bp,respectively.Forward and reverse primers of hTERT and GAPDH were 5′-CCCCGGTTTCTATAAATTGAGC-3′,5′-CACCTTCCCCATGGTGTCT-3′, 5′-AGCCACGTCTCTACCTTGACA-3′,and 5′-CAGGTGAGCCACGAACTGT-3′,respectively.The reaction mixtures were incubated under the following conditions:pre-incubation 95 °C 10 min;amplification 95 °C 10 s 60 °C 30 s,72 °C 1 s,45 cycles and cooling 40°C 30 s,in a Roche Light Cycler 480-II device(Rotkreuz,Switzerland).Measurements were calculated using an advanced relative quantification module with mRNA levels being compared against the negative control samples(cells with no test compound)following normalization to GADPH mRNA levels.

    2.5.Statistical analysis

    All experiments were performed at least three times,the results being expressed as mean±standard deviation.Normal distribution was determined using the Kolmogorov-Smirnov test.One-way ANOVA was used to analyze intergroup differences.p<0.05 was regarded as significant.

    3.Results

    R.canina extract exhibited selective cytotoxicity in the colon cancer cell line compared to normal colon cells(Table 1 and Fig.1).

    The results of the cell cycle analysis are presented in Fig.2.All the concentrations of R.canina extract used(except for 135 μg/mL)significantly increased cell numbers at the S phase(p=0.0001).Additionally,these concentrations of R.canina extract significantly reduced cell numbers at the G0/G1phase compared to untreated cells(p=0.0001).

    The results of the Annexin V analysis are presented in Fig.3.R.canina extract significantly reduced the number of viable cells and increased the number of late and early apoptotic cells compared to untreated cells in a dose-dependent manner(p<0.05).

    Table 1 Cytotoxic effects of test compounds and their IC50values(μg/mL)(n=4).

    Fig.1.The anti-growth effect using MTT assay after treatment with R.canina extract for 72 h against WiDr cells and colon normal cells(n=4).

    Fig.2.Cell cycle analysis of WiDr cells treated for 72 h with R.canina extract at different concentrations(n=4).aRepresents significant results(p<0.05)compared with untreated WiDr cells.

    Fig.3.Apoptosis analysis of WiDr cells treated with different concentrations of R.canina extract for 72 h using Annexin-V FITC and propidium iodide staining(n=4).aRepresents significant results(p<0.05)compared with untreated WiDr cells.

    MMP analysis results are presented in Fig.4.All concentrations of R.canina extract significantly reduced the MMP in WiDr cells(p=0.001).The percentage reductions in MMP caused by R.canina extract were 12.7%,23.7%,57.9%,and 62.9%for concentrations of 135,270,405 and 540 μg/mL of R.canina extract,respectively.

    Fig.4.DiOC6staining for R.canina extract-induced dissipation of mitochondrial membrane potential in WiDr cells(n=4).aRepresents significant results(p<0.05)compared with untreated WiDr cells.

    Fig.5.Relative mRNA levels of hTERT after treatment with R.canina extract(270 μg/mL)at different treatment times(n=3).aRepresents significant results(p<0.05)compared with negative control cells.

    The IC50concentration(270 μg/mL)of R.canina extract significantly repressed hTERT expression at treatment times of 48 h and 72 h(p<0.05)in colon cancer cells(Fig.5).hTERT expression decreased by 36%and 63%after 48 and 72 h,respectively,compared to the negative control group.No statistically significant differences were regarded between the 48 and 72 h treatment groups(p>0.05).

    4.Discussion and conclusion

    The use of plants to treat diseases is known as herbal medicine and is regarded as a part of traditional treatment.Traditional medicine with plants is thought to go back to very early times in human history[22].Edible plants are rich in antioxidant molecules,such as vitamins,polyphenolic compounds and carotenoids.The fruits of R.canina have long been used for medical aims,and the biological functions of these fruits may be partly attributed to their phenolics and vitamin C contents[1,3].

    Colon cancer is one of the leading causes of death in the USA and the second most common form of fatal cancer worldwide.Almost half of all patients diagnosed with colon cancer are reported to die.Fewer than 10%of patients of with metastatic colon cancer survive for more than 5 years.Surgical treatment,chemotherapy,radiotherapy or combinations of these are generally applied in cancers of the digestive system[13].Chemotherapy is an effective means of treating several types of cancer,including colon cancer,but the cost represents an important public health problem in developing countries.In addition,gradual resistance developed in cancer cells to chemotherapeutic drugs and healthy cells being affected by them reduce the success levels of chemotherapy[14].New strategies are therefore required in order to solve these problems.More than 60%of anticancer drugs in use today are isolated from natural products of plant origin[23].Natural product research for cancer treatment is therefore one of these new strategies due to its effectiveness against cancer cells and the fact that it is harmless to normal cells[12].Although experimental studies have shown that phenolic compounds in particular exhibit anticancer properties,the entire mechanism involved is as yet unclear.The mechanisms that have been proposed in the context of the anticancer effect of phenolic compounds include their ability to scavenge free radicals,their ability to induce xenobiotic metabolism,their ability to regulate gene expression,and their ability to modulate DNA repair,apoptosis and cell signaling,such as cell replication and invasion[14].However,the number of cytotoxicity studies performed with Rosa species is very limited[15–18].The purpose of this study was therefore to determine the cytotoxic effect of R.canina extract in colon cancer cell lines,one of the most common forms of cancer worldwide,and to identify the probable mechanisms of action.In vitro analyses are first performed in determining the potential biological bene fits of any compound.If positive results are obtained from that analysis,then in vivo studies are performed in the second stage[7].The WiDr cell line is derived from a human colon carcinoma and is commonly used in cancer research involving in vitro colon cancer models[24,25].This study was therefore planned on a colon cancer cell line(WiDr)under in vitro conditions.An effective and acceptable anticancer compound has to meet various criteria,including exhibiting no harmful effects on healthy cells[7].Cytotoxicity experiments were therefore conducted in WiDr cells together with normal colon cells.In order to investigate the antiproliferative activity of R.canina extract,WiDr and colon normal cells were treated with different concentrations of extract for 72 h.R.canina extract exhibited reasonable selective cytotoxic effects against WiDr cells compared with normal colon cells.In terms of cytotoxic activity,Lee et al.[26]demonstrated that methanolic extracts of R.rugosa stem act as anti-prostate cancer agents,while Olsson et al.[15]demonstrated that ethanolic extract of rose hip has a cytotoxic effect on HT-29 and MCF-7 cells.Tumbas et al.[27]reported that quercetin,ellagic acid and vitamin C are the most abundant antioxidant compounds in R.canina.However,only vitamin C-free extract,which is rich in polyphenols,exhibits cytotoxic activity in three human cancer cell lines(HeLa,MCF-7 and HT-29 with IC50values of approximately 81,248 and 364 μg/mL,respectively).They concluded that vitamin C and flavonoids are responsible for the antioxidant activity of R.canina,while only polyphenols contribute to its cytotoxic activity.Olech et al.[28]reported that teas and tinctures prepared from R.rugosa exhibit cytotoxic effects against human ovarian,lung,cervix,and breast cancer cells without harming human skin fibroblast cells.In another study,Guimar?es et al.[29]showed that methanolic extract of R.canina from Portugal exhibits cytotoxic effect against human lung,colon,cervix and liver cancer cells,while no effect was observed on breast cancer cells.Artun et al.[30]recently reported that methanolic extract of R.damascena exhibits selective cytotoxic effects on human cervix cancer(HeLa)cells compared to normal kidney epithelial(Vero)cells.Additionally,not only the extracts of Rosa species but also various compounds isolated from Rosa species(such as bee pollen polysaccharides,phenylethanoids,and essential oils)have been reported to exhibit cytotoxic effects in colon,leukemia,liver,breast and oropharyngeal epidermoid carcinoma cell lines[13,31,32].

    Loss of control of the cell cycle is reported to play a role in the development of cancer.Therefore,halting the cycle of cancer cells at any stage is one of the basic aims in the approach to treating cancer[9].We performed cell cycle analysis to assess population cell death and to determine whether R.canina extract can induce cell cycle arrest.As shown in Fig.2,R.canina extract induced significant dose-dependent accumulation of cells at the S phase.Consistent with our results,Jiménez et al.[18]reported that R.canina extracts arrested the cell cycle of Caco-2 cells at the S phase.Cagle et al.[33]demonstrated that an 80%methanolic extract of R.canina exhibited antiproliferative effects on human glioblastoma cells by increasing cell cycle arrest at the G2/M phase and blocking both the MAPK and AKT signaling mechanisms.No findings were determined concerning apoptosis in that study.

    Attenuate or extinct apoptosis capacity has been identified in a range of cancer types.Raising apoptosis levels through the use of different agents is therefore another approved target mechanism component of anticancer strategies[7].We therefore examined the capability of varying concentrations of R.canina extract to reduce the growth of WiDr cells by increasing levels of apoptosis using the Annexin V/propidium iodide double-staining assay.Our results showed that R.canina extract significantly induced cell apoptosis in a concentration-dependent manner(Fig.3).Mitochondrial depolarization is a major component of apoptosis induction[9].We therefore investigated alterations in MMP following R.canina extract treatment.As shown in Fig.4,R.canina extract significantly induced loss of MMP in a concentration-dependent manner.These results suggest that the type of cell death induced by R.canina extract is mitochondria-dependent apoptosis.Several studies have investigated the apoptotic properties of Rosa species in many different cancer cells.Khatib et al.[34]reported that essential oil from R.damascena exhibits cytotoxic effects against a gastric cancer cell line by inducing apoptosis.Erguven et al.[35]demonstrated that ethanolic extract of R.agrestis exhibits concentration-dependent cytotoxic effects on human endometrial adenocarcinoma cellsby inducing apoptoticcell death.Jiménez et al.[18]reported that R.canina extracts have cytotoxic effects on Caco-2 cells by increasing the number of apoptotic cells.

    Telomerase activation is one of the characteristic features of cancer cells[8,11].Telomerase is reported to be active in more than 85%of all tumors,but it is present at low or undetectable levels in normal cells[8].Telomerase plays an important role in the unlimited proliferation of cancer cells,so inhibition of telomerase activity is regarded as a highly selective target for cancer treatment[11].Moreover,increased telomerase expression has been reported in several types of epithelial cancer,including colon cancer[36].To the best of our knowledge,no previous studies have investigated the effect of R.canina extract on telomerase expression in any human cancer cells.Our findings show that the IC50(270 μg/mL)concentration of R.canina extract significantly repressed hTERT expression at treatment times of 48 and 72 h.Inhibition of telomerase in cancer cells is known to result in induction of cell death.However,there is increasing evidence that telomerase plays a key role in apoptosis regulation by changing MMP or metal homeostasis in mitochondria in a manner unrelated to its classic role[37].Additionally,global expression profiling studies have stated that hTERT expression affects 284 genes,the function of which involves a wide range of cellular processes,including cell cycle regulation,metabolism,differentiation,apoptosis,and cell signaling[38].Suppression of hTERT expression also causes elevated p53 and p21 transcription,and p21 mediates p53-dependent growth arrest in cancer cells[37].Polyphenols are also capable of downregulating hTERT expression in various cancer cell lines.This con firms previous reports showing such downregulation with some polyphenols(resveratrol,apigenin,baicalein, chrysin,galangin,and kaempferol)in colon and esophageal squamous cancer cells[11,39].We therefore think that downregulation of telomerase by R.canina extract(rich in polyphenols)may contribute to its apoptotic and antiproliferative activity in WiDr cells.

    One limitation of this research is that in vitro studies cannot be extrapolated to potential activity in vivo.Further studies are now necessary to understand the exact interaction of the involved signaling pathways.

    Conflicts of interest

    The authors declare that there are no conflicts of interest.

    Acknowledgments

    The authors would like to thank the Foundation of Scientific Research of Gumushane University for financially supporting this research under Project No:13.F5119.02.1.The authors also wish to thank Professor Ersan Kalay from the Medical Biology Department,Karadeniz Technical University for professional assistance with the flow cytometry studies.

    18禁动态无遮挡网站| 天天躁夜夜躁狠狠久久av| 一区在线观看完整版| 少妇的逼好多水| 久久6这里有精品| 下体分泌物呈黄色| 91久久精品国产一区二区三区| 亚洲成人手机| 欧美丝袜亚洲另类| 国产又色又爽无遮挡免| 亚洲精品自拍成人| 麻豆国产97在线/欧美| 性色avwww在线观看| 亚洲精品中文字幕在线视频 | 亚洲成人一二三区av| 青春草视频在线免费观看| 久久女婷五月综合色啪小说| 国内揄拍国产精品人妻在线| 大片免费播放器 马上看| 欧美 日韩 精品 国产| 久久久久性生活片| 国产精品麻豆人妻色哟哟久久| 女性生殖器流出的白浆| 男女下面进入的视频免费午夜| 国产精品女同一区二区软件| 超碰97精品在线观看| 小蜜桃在线观看免费完整版高清| 日韩一本色道免费dvd| 国产欧美另类精品又又久久亚洲欧美| 91精品伊人久久大香线蕉| 最近的中文字幕免费完整| 国产免费又黄又爽又色| 亚洲第一区二区三区不卡| 国产一区二区在线观看日韩| 国产高清有码在线观看视频| 亚洲精品456在线播放app| 另类亚洲欧美激情| 久久久午夜欧美精品| 日韩三级伦理在线观看| 久久国内精品自在自线图片| 国产亚洲av片在线观看秒播厂| 亚洲中文av在线| 只有这里有精品99| 国产亚洲最大av| 中文乱码字字幕精品一区二区三区| 极品少妇高潮喷水抽搐| 人体艺术视频欧美日本| 高清日韩中文字幕在线| 日日摸夜夜添夜夜爱| 美女高潮的动态| 人妻 亚洲 视频| 天堂中文最新版在线下载| 日本wwww免费看| 日本av手机在线免费观看| 久久精品夜色国产| 亚洲精华国产精华液的使用体验| 国产黄频视频在线观看| 综合色丁香网| 免费看日本二区| 人人妻人人爽人人添夜夜欢视频 | 国产精品无大码| 黑人猛操日本美女一级片| 毛片女人毛片| 欧美一级a爱片免费观看看| 久久国产精品大桥未久av | 天美传媒精品一区二区| 久热久热在线精品观看| 五月开心婷婷网| 国产真实伦视频高清在线观看| 亚洲精品国产成人久久av| 亚洲欧美清纯卡通| 青春草视频在线免费观看| 日本-黄色视频高清免费观看| 99热网站在线观看| 18禁裸乳无遮挡免费网站照片| 亚洲人成网站在线播| 99re6热这里在线精品视频| 久久久久久九九精品二区国产| 自拍欧美九色日韩亚洲蝌蚪91 | 欧美一级a爱片免费观看看| 在线天堂最新版资源| 纯流量卡能插随身wifi吗| 18+在线观看网站| 22中文网久久字幕| 国产精品免费大片| 在现免费观看毛片| 午夜免费观看性视频| 国产免费一级a男人的天堂| 亚洲精品乱久久久久久| 亚洲欧美中文字幕日韩二区| 男女无遮挡免费网站观看| 黄色配什么色好看| 麻豆成人av视频| 最近手机中文字幕大全| 久久精品夜色国产| 国产91av在线免费观看| 免费少妇av软件| 人人妻人人看人人澡| 国产成人精品福利久久| 成人毛片60女人毛片免费| 日本午夜av视频| 啦啦啦在线观看免费高清www| 91在线精品国自产拍蜜月| 成年人午夜在线观看视频| 干丝袜人妻中文字幕| 亚洲国产毛片av蜜桃av| 久久久精品免费免费高清| 成人亚洲精品一区在线观看 | 国产精品av视频在线免费观看| 国产精品人妻久久久影院| 久久青草综合色| 国产乱来视频区| 观看免费一级毛片| 纵有疾风起免费观看全集完整版| 黄色怎么调成土黄色| 18禁动态无遮挡网站| 一级a做视频免费观看| 国产精品一区www在线观看| 亚洲三级黄色毛片| 欧美精品一区二区免费开放| 亚洲精品国产av成人精品| 欧美97在线视频| 日韩av不卡免费在线播放| 亚洲av电影在线观看一区二区三区| 日韩一区二区三区影片| 亚洲经典国产精华液单| 亚洲内射少妇av| 国产中年淑女户外野战色| 97超碰精品成人国产| 国产精品成人在线| 午夜免费鲁丝| 又爽又黄a免费视频| 成人毛片60女人毛片免费| av一本久久久久| 美女高潮的动态| 国产在线免费精品| 亚洲精品色激情综合| 久久久久国产精品人妻一区二区| 麻豆成人av视频| 日韩精品有码人妻一区| 2021少妇久久久久久久久久久| 久久婷婷青草| 26uuu在线亚洲综合色| 97热精品久久久久久| 观看免费一级毛片| av在线观看视频网站免费| 日本爱情动作片www.在线观看| 日日啪夜夜爽| 国产黄色免费在线视频| 久久韩国三级中文字幕| 亚洲丝袜综合中文字幕| 亚洲美女视频黄频| 亚洲欧美中文字幕日韩二区| 欧美+日韩+精品| 久久久久精品久久久久真实原创| 男人爽女人下面视频在线观看| 久久久欧美国产精品| 哪个播放器可以免费观看大片| 91久久精品国产一区二区成人| 亚洲精品一区蜜桃| 日韩av不卡免费在线播放| 国产黄片视频在线免费观看| tube8黄色片| 边亲边吃奶的免费视频| 国产精品精品国产色婷婷| 亚洲不卡免费看| av.在线天堂| 夫妻午夜视频| 精品99又大又爽又粗少妇毛片| 你懂的网址亚洲精品在线观看| 亚洲内射少妇av| 国产亚洲一区二区精品| 三级经典国产精品| 国产亚洲午夜精品一区二区久久| 亚洲美女搞黄在线观看| 久久久久久久久久久免费av| 国产亚洲欧美精品永久| 99热这里只有精品一区| 日韩人妻高清精品专区| 国产精品女同一区二区软件| 久久精品国产鲁丝片午夜精品| 在线看a的网站| 亚洲自偷自拍三级| 中文字幕制服av| 亚洲电影在线观看av| 又粗又硬又长又爽又黄的视频| 最近手机中文字幕大全| 日日啪夜夜爽| 亚洲欧美精品自产自拍| 午夜免费鲁丝| 成人一区二区视频在线观看| 亚洲欧美成人综合另类久久久| 男人和女人高潮做爰伦理| 中国美白少妇内射xxxbb| 男女下面进入的视频免费午夜| 国产亚洲精品久久久com| 小蜜桃在线观看免费完整版高清| 18禁裸乳无遮挡动漫免费视频| 99热6这里只有精品| 亚洲最大成人中文| 777米奇影视久久| 性色av一级| 国产精品一区二区在线观看99| 免费久久久久久久精品成人欧美视频 | 蜜桃久久精品国产亚洲av| 亚洲第一区二区三区不卡| 搡老乐熟女国产| 免费黄网站久久成人精品| 永久免费av网站大全| 日本av手机在线免费观看| 国产高清三级在线| 日韩伦理黄色片| 色婷婷久久久亚洲欧美| 亚洲中文av在线| 国产男女内射视频| 国产乱人偷精品视频| 亚洲激情五月婷婷啪啪| 午夜老司机福利剧场| 国产av一区二区精品久久 | 久久精品国产亚洲网站| 在线天堂最新版资源| 亚洲av电影在线观看一区二区三区| 直男gayav资源| 久久久久久九九精品二区国产| 久久国产亚洲av麻豆专区| 一级爰片在线观看| 99热国产这里只有精品6| 在线免费十八禁| 不卡视频在线观看欧美| 久久99热这里只有精品18| 王馨瑶露胸无遮挡在线观看| 国产 精品1| 高清毛片免费看| 亚洲中文av在线| 免费高清在线观看视频在线观看| 搡老乐熟女国产| 欧美高清性xxxxhd video| 在线观看av片永久免费下载| 亚洲四区av| 国产探花极品一区二区| 亚洲自偷自拍三级| av线在线观看网站| 亚洲精品日韩av片在线观看| 国产成人aa在线观看| 波野结衣二区三区在线| 国产精品一区二区在线不卡| 在线观看人妻少妇| 精品久久久久久久久亚洲| 亚洲四区av| 成人高潮视频无遮挡免费网站| 最近最新中文字幕免费大全7| 久久精品久久久久久久性| 国产高清不卡午夜福利| 噜噜噜噜噜久久久久久91| 精品人妻一区二区三区麻豆| 亚洲天堂av无毛| 欧美精品亚洲一区二区| 人人妻人人澡人人爽人人夜夜| 成人特级av手机在线观看| 精品亚洲成国产av| 久久精品国产自在天天线| 国产成人aa在线观看| 久久国产精品大桥未久av | 色视频www国产| 大陆偷拍与自拍| 高清av免费在线| 亚洲国产精品国产精品| 少妇被粗大猛烈的视频| 妹子高潮喷水视频| 精品人妻偷拍中文字幕| 美女cb高潮喷水在线观看| 久久精品国产亚洲av涩爱| 黑人高潮一二区| 欧美 日韩 精品 国产| 在线观看av片永久免费下载| 亚洲国产色片| 国产精品久久久久久久电影| 国产免费视频播放在线视频| 97超碰精品成人国产| 黑人高潮一二区| av线在线观看网站| 看非洲黑人一级黄片| 国产精品一区www在线观看| av福利片在线观看| 天天躁日日操中文字幕| 九草在线视频观看| 久久97久久精品| 久久亚洲国产成人精品v| 在线观看三级黄色| videos熟女内射| 看非洲黑人一级黄片| 久久久久性生活片| 久久精品人妻少妇| 夜夜骑夜夜射夜夜干| 一个人看视频在线观看www免费| 高清av免费在线| 高清在线视频一区二区三区| 亚洲欧美成人综合另类久久久| 国产视频内射| 男女边摸边吃奶| 日韩亚洲欧美综合| 美女脱内裤让男人舔精品视频| 最近中文字幕高清免费大全6| 另类亚洲欧美激情| 亚洲欧洲日产国产| 亚洲三级黄色毛片| 在线观看三级黄色| 亚洲av成人精品一区久久| 久久精品久久久久久噜噜老黄| 干丝袜人妻中文字幕| 国产永久视频网站| 女性被躁到高潮视频| 波野结衣二区三区在线| 91精品伊人久久大香线蕉| 欧美+日韩+精品| 久久久精品94久久精品| 国产免费又黄又爽又色| 日韩中文字幕视频在线看片 | 97在线视频观看| videos熟女内射| 日韩伦理黄色片| 啦啦啦啦在线视频资源| 国产一区二区三区av在线| 51国产日韩欧美| 午夜福利在线观看免费完整高清在| 女人十人毛片免费观看3o分钟| 国产精品不卡视频一区二区| 搡老乐熟女国产| 大又大粗又爽又黄少妇毛片口| 久久久久久久久大av| 成年av动漫网址| 国产一区二区三区av在线| 一级黄片播放器| 国内少妇人妻偷人精品xxx网站| 精品久久久精品久久久| .国产精品久久| 啦啦啦中文免费视频观看日本| 日日摸夜夜添夜夜添av毛片| 丰满乱子伦码专区| 少妇的逼水好多| 亚洲国产欧美在线一区| 在线观看一区二区三区激情| 亚洲一级一片aⅴ在线观看| 久久精品国产亚洲av天美| 中文欧美无线码| 丝袜脚勾引网站| 欧美日韩一区二区视频在线观看视频在线| 久久午夜福利片| 精品酒店卫生间| 黄色怎么调成土黄色| 亚洲欧美成人精品一区二区| 欧美国产精品一级二级三级 | 成人毛片60女人毛片免费| 日本一二三区视频观看| 亚洲国产高清在线一区二区三| 毛片一级片免费看久久久久| 亚洲av二区三区四区| 免费观看无遮挡的男女| 少妇高潮的动态图| 91精品伊人久久大香线蕉| 日韩,欧美,国产一区二区三区| 超碰av人人做人人爽久久| 国产成人午夜福利电影在线观看| 狂野欧美激情性xxxx在线观看| 久久这里有精品视频免费| 国产国拍精品亚洲av在线观看| 一级爰片在线观看| av又黄又爽大尺度在线免费看| 在线观看人妻少妇| 日韩中文字幕视频在线看片 | 亚洲国产精品一区三区| 国产精品不卡视频一区二区| 日韩成人av中文字幕在线观看| 日韩成人伦理影院| 成人漫画全彩无遮挡| 久久久欧美国产精品| 一级毛片电影观看| 在线播放无遮挡| 国产高清国产精品国产三级 | 91精品国产国语对白视频| 亚洲国产日韩一区二区| 亚洲,一卡二卡三卡| 岛国毛片在线播放| 毛片一级片免费看久久久久| 99精国产麻豆久久婷婷| 久久国内精品自在自线图片| 人妻夜夜爽99麻豆av| 精品一区二区三区视频在线| 国产永久视频网站| 免费在线观看成人毛片| 欧美日韩在线观看h| 少妇熟女欧美另类| 国产真实伦视频高清在线观看| 欧美3d第一页| 最黄视频免费看| 成年人午夜在线观看视频| 久久鲁丝午夜福利片| 一区二区三区四区激情视频| 尾随美女入室| 国产精品欧美亚洲77777| 日日啪夜夜爽| 午夜激情久久久久久久| 人人妻人人看人人澡| 国产亚洲最大av| 国产伦在线观看视频一区| 日本av手机在线免费观看| www.av在线官网国产| 高清不卡的av网站| 久久久久久久久久成人| 成人特级av手机在线观看| 国产在线一区二区三区精| 欧美日韩一区二区视频在线观看视频在线| 国产精品女同一区二区软件| 97在线人人人人妻| 国产精品偷伦视频观看了| 51国产日韩欧美| 亚洲精品,欧美精品| 蜜桃亚洲精品一区二区三区| 国产伦精品一区二区三区视频9| 中文在线观看免费www的网站| 国产精品人妻久久久影院| 久久婷婷青草| 超碰av人人做人人爽久久| 免费大片黄手机在线观看| 一区二区三区免费毛片| 寂寞人妻少妇视频99o| av女优亚洲男人天堂| 国产黄色免费在线视频| 特大巨黑吊av在线直播| 最近最新中文字幕免费大全7| 中文字幕久久专区| 91久久精品国产一区二区三区| 伦精品一区二区三区| 亚洲欧美日韩卡通动漫| av.在线天堂| 亚洲,一卡二卡三卡| 成人免费观看视频高清| 日本一二三区视频观看| 日韩av在线免费看完整版不卡| 欧美丝袜亚洲另类| 26uuu在线亚洲综合色| 又爽又黄a免费视频| 国产亚洲91精品色在线| 国产伦理片在线播放av一区| 亚洲精品久久午夜乱码| .国产精品久久| 热99国产精品久久久久久7| 22中文网久久字幕| 久久毛片免费看一区二区三区| 久久久久人妻精品一区果冻| 国产精品一区二区三区四区免费观看| 国产精品爽爽va在线观看网站| 80岁老熟妇乱子伦牲交| 深爱激情五月婷婷| 国产一区二区三区综合在线观看 | 国产高潮美女av| 欧美精品亚洲一区二区| av免费观看日本| 精品人妻一区二区三区麻豆| 男女边吃奶边做爰视频| 国产探花极品一区二区| 日韩免费高清中文字幕av| 国产永久视频网站| 晚上一个人看的免费电影| 久久久久久久久大av| 久久精品熟女亚洲av麻豆精品| 99九九线精品视频在线观看视频| 网址你懂的国产日韩在线| 日本一二三区视频观看| 亚洲精品久久久久久婷婷小说| 国产精品久久久久久久久免| 中国三级夫妇交换| 免费看不卡的av| 女人十人毛片免费观看3o分钟| 国产有黄有色有爽视频| 久久精品夜色国产| 久久久久精品久久久久真实原创| 夜夜爽夜夜爽视频| 美女xxoo啪啪120秒动态图| 亚洲中文av在线| 亚洲av免费高清在线观看| 久久久午夜欧美精品| 精品99又大又爽又粗少妇毛片| 日韩中文字幕视频在线看片 | 久久午夜福利片| 永久免费av网站大全| 超碰97精品在线观看| 亚洲av.av天堂| 久久精品熟女亚洲av麻豆精品| 国产综合精华液| 国产高清不卡午夜福利| 少妇 在线观看| 99热国产这里只有精品6| 精品久久久噜噜| 久久人人爽av亚洲精品天堂 | 国产淫片久久久久久久久| freevideosex欧美| 亚洲av在线观看美女高潮| 一级a做视频免费观看| 亚洲欧美日韩东京热| 99久久精品国产国产毛片| 午夜福利在线在线| 精品亚洲乱码少妇综合久久| 有码 亚洲区| 麻豆成人av视频| 好男人视频免费观看在线| 两个人的视频大全免费| 久热久热在线精品观看| 亚洲国产欧美在线一区| 99re6热这里在线精品视频| 亚洲综合精品二区| 免费高清在线观看视频在线观看| 97在线人人人人妻| 国产精品一区二区在线不卡| 色网站视频免费| 亚洲精品一二三| 美女主播在线视频| 少妇精品久久久久久久| 91久久精品国产一区二区三区| 国产欧美亚洲国产| 精品一区二区三区视频在线| 亚洲成人中文字幕在线播放| 国产美女午夜福利| 亚洲真实伦在线观看| 高清在线视频一区二区三区| 搡老乐熟女国产| 一个人看的www免费观看视频| 中文字幕精品免费在线观看视频 | 欧美成人a在线观看| 纵有疾风起免费观看全集完整版| 亚洲av不卡在线观看| 久久久久精品久久久久真实原创| a级一级毛片免费在线观看| 少妇猛男粗大的猛烈进出视频| 男的添女的下面高潮视频| 最近手机中文字幕大全| 91aial.com中文字幕在线观看| 新久久久久国产一级毛片| 伦理电影免费视频| 尾随美女入室| 久久国内精品自在自线图片| 成人黄色视频免费在线看| 一级爰片在线观看| 一级片'在线观看视频| 91狼人影院| 夜夜骑夜夜射夜夜干| 欧美精品一区二区免费开放| 一级爰片在线观看| 一级毛片电影观看| 大陆偷拍与自拍| 天堂俺去俺来也www色官网| 亚洲av男天堂| 日本欧美视频一区| 日本av手机在线免费观看| 在线看a的网站| 在线亚洲精品国产二区图片欧美 | 免费人妻精品一区二区三区视频| 久久国内精品自在自线图片| 日本午夜av视频| 久久婷婷青草| 国产精品精品国产色婷婷| 亚洲国产高清在线一区二区三| 亚洲av综合色区一区| 少妇被粗大猛烈的视频| 赤兔流量卡办理| 欧美成人一区二区免费高清观看| 日韩一本色道免费dvd| 欧美 日韩 精品 国产| 一级a做视频免费观看| .国产精品久久| 免费观看的影片在线观看| 亚洲精品日韩av片在线观看| 久久婷婷青草| 一区二区三区免费毛片| 制服丝袜香蕉在线| 午夜精品国产一区二区电影| 国产亚洲最大av| 你懂的网址亚洲精品在线观看| 简卡轻食公司| 国产成人91sexporn| 伦精品一区二区三区| 成年人午夜在线观看视频| 蜜臀久久99精品久久宅男| 少妇精品久久久久久久| 国精品久久久久久国模美| 亚洲激情五月婷婷啪啪| 日本黄色片子视频| 菩萨蛮人人尽说江南好唐韦庄| 国产伦在线观看视频一区| 男人爽女人下面视频在线观看| 草草在线视频免费看| 国产成人精品一,二区| 久久97久久精品| 亚洲精品亚洲一区二区| 国产色爽女视频免费观看| 国产一区亚洲一区在线观看| 精品酒店卫生间| 久久久久性生活片| 自拍欧美九色日韩亚洲蝌蚪91 | 妹子高潮喷水视频| 亚洲欧美一区二区三区黑人 | 亚洲成人中文字幕在线播放| 亚洲丝袜综合中文字幕| 日韩欧美精品免费久久| 亚洲欧美日韩无卡精品| 极品少妇高潮喷水抽搐| 日本爱情动作片www.在线观看| 日韩av不卡免费在线播放| 国产一级毛片在线| 国产精品欧美亚洲77777| 日本免费在线观看一区| 午夜精品国产一区二区电影| 国产男人的电影天堂91| 人人妻人人爽人人添夜夜欢视频 | 丰满人妻一区二区三区视频av|