• <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 ?

    Study of a water-soluble supramolecular complex of curcumin and β-cyclodextrin polymer with electrochemical property and potential anti-cancer activity

    2022-09-16 05:25:06WngZhngPingXioLiweiLinFngGuoQingyueWngYunzhePioGuowngDio
    Chinese Chemical Letters 2022年8期

    Wng Zhng, Ping Xio, Liwei Lin, Fng Guo, Qingyue Wng, Yunzhe Pio,Guowng Dio,?

    a School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China

    b Department of Applied Bioengineering, Graduate School of Convergence Science and Technology, Seoul National University, Suwon 443-270, Republic of Korea

    cShanghai Suni Biotechnology Development Co., Ltd., Shanghai 201300, China

    d Advanced Institutes of Convergence Technology, Suwon 443-270, Republic of Korea

    ABSTRACT As a member of the curcuminoid compound family, curcumin (Cur) has many interesting therapeutic properties.However, its low aqueous solubility and stability have resulted in poor bioavailability and restricted clinical efficacy.Based on size matching, β-cyclodextrin polymer (β-CDP), with its hydrophilic polymer chains and hydrophobic cavities, can form an inclusion complex with Cur.To improve the water solubility and stability of Cur, a simple and eco-friendly grinding method was designed to form β-CDP inclusion complexes.According to the Boltzmann–Hamel’s method and Job’s method, the molar ratio of the β-CD unit in β-CDP to Cur was determined to be 1:1.The diffusion coefficient and diffusion activation energy of Cur-β-CDP were calculated in an electrochemical study.This supramolecular complex worked well in vitro to inhibit the proliferation of hepatoma carcinoma cells HepG2.Remarkably, this method visibly reduced the undesirable side effects on normal cells, without weakening the anti-cancer activity of the drugs.We expect that the obtained host–guest complex will provide a new approach for delivering natural drug molecules, having low water solubility.

    Keywords:Cyclodextrin polymer Curcumin Solubility Anti-cancer activity Supramolecular complex

    Curcumin (Cur) is an orange polyphenol that is extracted from the rhizome of perennial plants in the Zingiberaceae family [1].The polyphenol structure, containing an unsaturated carbonyl scaffold, provides Cur with various therapeutic properties, such as being antioxidant, anti-inflammatory and antineoplastic.These properties have a wide range of applications in daily life, including use as anti-inflammatory drugs [2–4], antioxidants [5–7], food coloring agents [8], and so on [9–11].In addition, Cur has been shown to control the growth and propagation of many types of tumor cells [12–14].Mehrgan’s group synthetized Cur–silica nanoparticles as photosensitizers for the photodynamic treatment of human melanoma cancer cells, to improve their application in cancer treatment [15].Notably, Cur inhibits cell proliferation and promotes cell breakdown; it has been also shown to have effective therapeutic values [8].However, Cur has restricted applications owing to its low bioavailability, poor water solubility, and rapid metabolism [16].To solve these problems, we employed the supramolecular host compound,β-cyclodextrin polymer (β-CDP).

    Cyclodextrins (CDs), as 2nd-generation supramolecular host compounds, typically contain 6–12 D-glucosamine units [17].They possess a hollow circular truncated cone structure with a hydrophobic inner cavity and hydrophilic outer surface.CDs can accommodate various guest molecules in their cavities, to form stable host–guest inclusion complexes [18,19].Owing to its low cost,nontoxic nature, and pro-environment properties, CD is widely used in food [20,21], medicine [22,23], agriculture [24] and other fields [25,26].Recently,β-CDPs have received widespread attention as they show better water solubility and a higher inclusion efficiency than the parentβ-CD [27,28].Inclusion complexes withβ-CDP can be prepared via several techniques such as coprecipitation, kneading, slurry complexation, spray drying, and freeze drying techniques [29].Compared with CD, CDPs with distinct physical and chemical properties have been widely exploited for solubilization and pharmaceuticals [30–32].

    Fig.1.(A) The 1H NMR of Cur, Cur-β-CDP and β-CDP.(B) The potential solution structures of Cur.(C) UV–vis absorption spectra of Cur and β-CDP different molar ratios in water/ethanol mixed solution (Vwater:Vethanol = 1:1) with a constant concentration of [β-CDP]+[Cur] = 40 μmol/L: (a) [Cur] = 40 μmol/L; (b) β-CDP:[Cur] = 1:9; (c)β-CDP:[Cur] = 2:8; (d) [β-CDP]:[Cur] = 3:7; (e) [β-CDP]:[Cur] = 4:6; (f) [β-CDP]:[Cur] = 5:5; (g) [β-CDP]:[Cur] = 6:4; (h) [β-CDP]:[Cur] = 7:3; (i) [β-CDP]:[Cur] = 8:2;(j) [β-CDP]:[Cur] = 9:1; (k) [β-CDP] = 40 μmol/L.(D) Job plot showing the 1:1 binding stoichiometry of the complex between β-CDP and Cur by plotting the difference in absorption at 433 nm against the mole fraction of Cur at an invariant total concentration of Cur in solution.

    In this study, the Cur-β-CDP was prepared following a grinding method as shown in Fig.S1A (Supporting information), and the complex was evaluated using and the complex was evaluated using Fourier transform infrared (FT-IR), ultraviolet (UV), and nuclear magnetic resonance (NMR) spectroscopies.In Figs.S1B and C (Supporting information), Cur-β-CDP dispersed more easily in aqueous solution as compared to pure Cur.The aqueous inclusion complex solution was yellowish green, uniform, and stable, indicating thatβ-CDP could form water-soluble inclusion complexes with Cur.The improving water solubility of Cur-β-CDP allowed for the electrochemical analysis of Cur-β-CDP in the aqueous phase.In addition,we conducted anin vitrocytotoxicity study ofβ-CDP.The Cur-β-CDP in aqueous solutions was expected to enhance hydrophilicity and bioavailability of Cur.

    The formation of the Cur-β-CDP inclusion complex was determined using FT-IR spectroscopy.Fig.S2 (Supporting information)shows the FT-IR spectra ofβ-CDP, Cur and the inclusion complex.The peaks forβ-CDP were observed at 3394, 2929 and 1032 cm?1, which were assigned to the stretching vibration of –OH, the anisotropic stretching vibration of –CH2, and the stretching vibration of C–O–C, respectively [33].The FT-IR spectrum of Cur showed an absorption peak at 3505 cm?1, indicating the stretching vibration of the phenolic O–H [34].In addition, the red line shows the bending vibration of the olefinic C–H of Cur at 1429 cm?1and the aromatic C–O stretching vibration of Cur at 1278 cm?1.The characteristic peaks at 1598 (stretching vibration of the benzene ring of Cur) and 1510 cm?1(C–O and C–C vibrations of Cur)are shown in Fig.S2 (Supporting information).In the FT-IR spectrum of Cur-β-CDP, the characteristic peaks of Cur (as the guest molecule) nearly disappeared, while the peaks ofβ-CDP (as the host molecule) are retained owing to the host–guest interaction.Because of the shielding effect, only weak phenolic O–H and C–O vibrations at 1589 and 1517 cm?1were observed, suggested that Cur molecules entered the hydrophobic cavities ofβ-CDP.This resulted in a significant decrease in the infrared absorption peaks for Cur.Thus, the results indicated the formation of Cur-β-CDP.

    As shown in Fig.1A, the dimensional accommodations of Cur,Cur-β-CDP, andβ-CDP were studiedvia1H NMR spectroscopy.Cur usually exists in an enol form (Fig.1B) in DMSO–d6solvents[35,36].The1H NMR spectra of Cur andβ-CDP were consistent with those reported in the literatures [35,37].When host–guest interactions occur, the modification of the physical and chemical environment affects the atoms of Cur andβ-CDP, which should result in changes in their chemical shifts [38].The1H NMR spectra were obtained to confirm the formation of Cur-β-CDP.In the1H NMR spectra of Cur-β-CDP, allβ-CDP peaks were observed after the formation of the inclusion complex [39].However, their signals were slightly shifted owing to the host–guest interactions with Cur, as listed in Table S1 (Supporting information).In addition, the chemical shifts of protons Hcand Hgof Cur changed from 7.57–6.78 ppm to 7.56–6.77 ppm, respectively.According to reports by Wimmeret al.[40], Cur is an aliphatic guest complex, having a low association constant, that usually shows insignificant chemical shifts.This is similar to the above small chemical shifts seen in the CD complexes.Furthermore, the peaks for the Cur aromatic rings, ascribed from 6.40 ppm to 7.70 ppm after the inclusion complex formation,were significantly weakened compared with the1H NMR spectra of Cur in Fig.1A.This indicates that the aromatic rings of Cur were placed inside the CD cavities.Thus, the above results indicate that Cur andβ-CDP form inclusion complexes via supramolecular interactions.

    As shown in Fig.S3 (Supporting information), the Cur-β-CDP inclusion complex was also investigated using thermogravimetric analysis (TGA).From 139 °C to 400 °C, the mass loss of Cur approached nearly 60%, while the mass loss of Cur-β-CDP decreased between 299 °C to 398 °C.The decomposition temperature of Curβ-CDP was higher than that of Cur and close to that ofβ-CDP.This result was indicative of the formation of the Cur-β-CDP complex,which improves the thermal stability of Cur.The mass fraction of Cur in the inclusion complex was calculated to be approximately 20.2% based on the TGA results.

    Fig.2.(A) At 25 °C, the cyclic voltammograms of 1.0 mmol/L Cur-β-CDP in 0.1 mol/L PBS (pH 6.0) over fifteen consecutive cycles at a bare GCE at the scan rate of 50 mV/s at room temperature: (a) 1; (b) 2; (c) 3; (d) 4; (e) 5; (f) 6; (g) 7; (h) 8; (i) 9; (j) 10; (k) 11; (l) 12; (m) 13; (n) 14; (o) 15.(B) The electrochemical electrode reaction equation of Cur.(C) At 25 °C, the cyclic voltammograms of 1.0 mmol/L Cur-β-CDP in 0.1 mol/L PBS (pH 6.0) at different scan rate (mV/s): (a) 5; (b) 10; (c) 20; (d) 30; (e) 40; (f) 50;(g) 60; (h) 80; (i) 100; (j) 200; (k) 400; (1) 600; (m) 800; (n) 1000.The plot of (D) Ipa and (E) Ipc vs.v1/2.Data were taken from (C).

    To study the host–guest interaction between Cur andβ-CDP, a mixed solvent (ethanol and water,Vethanol:Vwater= 1:1) was chosen.Fig.S4A (Supporting information) shows the UV–vis spectra of a 1.0 × 10?5mol/L Cur solution with differing concentrations ofβ-CD unit inβ-CDP at 25 °C.It was found that the peak position of Cur at 433 nm did not change with the addition ofβ-CDP.However, the peak intensity increased with increasingβ-CDP content.If the mole ratio of Cur to the CD structural unit ofβ-CDP in the inclusion complex is assumed to be 1:1, the following equation can be used to calculate the dissociation constant of the inclusion complex [41]:

    whereHrepresents the host (CD unit inβ-CDP),Gis the guest(Cur), andHGis the inclusion complex (Cur-β-CDP).The initial concentrations ofHandGwere [H]0and [G]0, respectively, where[H]0?[G]0.ΔAis the change in absorbance, andΔεis the change in molar absorption coefficient.KDis the dissociation constant of the inclusion complex.

    A straight line was obtained by plotting([H]0[G]0)/ΔA versus[H]0, as shown in Fig.S4B (Supporting information).The good linear relationship (R2= 0.990) proved that the assumption of a 1:1 molar ratio of Cur andβ-CD units inβ-CDP was correct.According to the slope and intercept of the straight line, the dissociation constantKDof the inclusion complex was determined to be 6.4 ×10?3mol/L.Compared with the inclusion complex of Cur andβ-CD[42], the larger diffusion coefficient of Cur-β-CDP suggested thatβ-CDP could form a Cur inclusion complex more easily thanβ-CD.The result shows that the polymer chain has stronger intermolecular interactions thanβ-CD, inducing the formation of inclusion complexes more effectively [43].

    The stoichiometry of the inclusion complex was also confirmed using the continuous variation of Job’s method using UV–vis spectroscopy [44].In the UV–vis absorption spectra of Cur andβ-CDP in water/ethanol (Vwater:Vethanol= 1:1), the sum of the concentrations of both components remained constant.The Job’s plot of the complex formed between Cur and theβ-CD units inβ-CDP is shown in Figs.1C and D, confirming the formation of a 1:1 inclusion complex between Cur and theβ-CD unit inβ-CDP.The complexation betweenβ-CDP and Cur was also demonstrated by fluorescence spectrometer.Compared with the fluorescence spectra of Cur-β-CDP and Cur, the peak attributed to Cur in the inclusion complex showed a blue shift (Fig.S5 in Supporting information).It can be seen from the fluorescence spectra that the polarizability of Cur might be influenced when it was encapsulated in the hydrophobic cavity of CDP.

    Fig.2A displays the multi-cyclic voltammograms of 1.0 × 10?6mol/L Cur-β-CDP in 0.1 mol/L PBS (pH 6.0) at 25 °C with a scan rate of 50 mV/s over 15 consecutive cycles on a bare GCE.In the first cyclic scan, Cur-β-CDP showed an oxidation peak(P1) at 0.544 V and reduction peak (P2) at 0.251 V.In the second cycle, a new oxidation peak (P3) at 0.299 V formed in place of P1.However, virtually no change was observed in P2.After two cycles, only P2 and P3 were observed.This revealed that P1 was an irreversible oxidation peak, and that P2 and P3 were redox peaks, where the active group came from the product of the first irreversible cycle.This is similar to the electrochemical electrode reaction of Cur [45].The Cur structure contains two phenol rings that are substituted with two hydroxyl groups and two methoxy groups, which are involved in the electrochemical reactions of Curβ-CDP.The electrochemical electrode reaction is shown in Fig.2B.In the first cycle, the Cur in the CD cavity loses one proton and two electrons, resulting in a transition to an active intermediate state.In the second cycle, a reversible redox reaction of the active intermediate may involve the transfer of two electrons and two protons [46].The supramolecular interaction between Cur andβ-CDP has little effect on the good electrochemical redox capacity of Cur, while it is in the CD cavity.Moreover, the high solubility of Cur-β-CDP drives more Cur to participate in the electrochemical process.The good electrochemical activity of Cur-β-CDP was taken advantage of in a potential analytical application for determining Cur.

    The cyclic voltammograms of 1.0 mmol/L Cur-β-CDP in 0.1 mol/L PBS (pH 6.0) at 25 °C with various scan rates are shown in Fig.2C.The peak current (ip) was calculated according to the Randles–Sevick equation [31].wheren(n= 2) is the number of electrons transferred for the electrochemical reaction,Ais the geometric surface area of the electrode, andcis the total concentration of Cur in the inclusion complex.

    Fig.3.(A) The cell viability of different concentration of Cur, Cur unit in Cur-β-CDP and β-CD unit in β-CDP.(B) The effect of Cur, β-CDP and Cur-β-CDP on migration in HepG2 cells, the images of HepG2 cells treated with Cur, β-CDP and Cur-β-CDP for 0, 24, 48 h after scratching were captured, respectively.(C) Confocal images of Hepg2 cells incubated with Cur, β-CDP and Cur-β-CDP for 24 h.

    As shown in Figs.2D and E, the redox peak currents of Cur-β-CDP were directly proportional to the square root of the scan rate in the range of 0.005–1.000 V/s in PBS (pH 6.0).It was demonstrated that the linear relationship (R2= 0.990 andR2= 0.997)corresponds to the peak anode currentipa~and peak cathode currentipc~, respectively.This indicates that the electrochemical redox process of Cur-β-CDP is aquasi-reversible and diffusioncontrolled process under the experimental conditions.At most sweep speeds, the value ofipa/ipcwas close to 1 (theoreticallyipa/ipc= 1).The electron transfer of Cur on the electrode surface was prohibited because of the shielding effect ofβ-CDP, andipawas slightly less thanipc.For aquasi-reversible electrode process,the estimation of the diffusion coefficients of the oxidation state(DO) and reduction state (DR) could be determined from the slope of the Randles–Sevcik plot using Eq.2.DOandDRwere calculated as 7.94 × 10?8and 9.91 × 108cm2/s, respectively.

    The cyclic voltammograms of Cur-β-CDP in 0.1 mol/L PBS (pH 6.0) at different temperatures are shown in Fig.S6A (Supporting information).It clearly shows that the peak currentipand peak potentialEpwere affected by the temperature.When the temperature increased,Epmoved in a negative direction, andipincreased.By using Eq.3, the diffusion coefficient of Cur-β-CDP at a series of temperatures can be obtained.The linear fitting relationship between the diffusion coefficientDand temperature was obtained as follows [31].

    whereEdis the diffusion activation energy of the inclusion compound, andD0is the diffusion coefficient of the inclusion compound at infinite temperature.A linear fit of logDto the reciprocal of temperature produced a straight line (R2= 0.9940).The straight line is shown in Fig.S6B (Supporting information); moreover,Edwas calculated as 37.36 kJ/mol.

    Cur toxicity affects unhealthy cells through membranemediated mechanisms, and usually exhibits lower cytotoxicity against normal cells than traditional chemotherapeutic drugs [47].Despite these attractive properties, the use of pure Cur in cancer therapy is still restricted owing to its low bioavailability and solubility under physiological conditions.In the following study, the host–guest strategy was selected to embed Cur in a water-soluble polymeric matrix, which could efficiently control the release of Cur and increase its bioavailability.

    To determine the cytotoxic potential of Cur-β-CDP, HepG2 cells were incubated with various concentrations of Cur,β-CDP, and Cur-β-CDP for 24 h, which was followed by evaluation of cell viabilityviaMTT assay.The results of the cytotoxic effects against HepG2 are shown in Fig.3A.Overall,β-CDP was almost nontoxic to HepG2 cells at concentrations of 5–80 μmol/mL, indicating thatβ-CDP could be a potential pharmaceutical carrier [48].Both pure Cur and Cur-β-CDP showed cytotoxic effects on HepG2 cells, thereby verifying the anti-cancer activity of Cur-β-CDP resulting from released Cur.The viability of HepG2 cells decreased from 94.04% to 51.98% with increasing concentrations of Cur ranging from 5 μmol/mL to 80 μmol/mL.In parallel, the viability of HepG2 cells decreased from 91.94% to 44.81% with increasing concentrations of Cur-β-CDP.The results indicate that the Cur inclusion complex exhibited stronger inhibitory effects on HepG2 cells than Cur.In addition, the IC50values of Cur and the Cur-β-CDP complex were measured as 32 and 28 mg/L, respectively.These results demonstrated that host-guest complexation withβ-CDP improved the bioavailability of Cur.And it indicated that Cur-β-CDP exhibited stronger inhibitory effects on HepG2 cells than Cur and Cur-pillar[5]arene [49].

    The cellular morphological photos of the HepG2 cells with different concentrations of the inclusion complex (1–80 μmol/L) for 24 h were microscopically observed, as shown in Fig.S7 (Supporting information).With an increasing concentration of Cur-β-CDP,the number of cells decreased gradually, and the morphology of the cells became round compared to the control cells.This alteration was consistent with the detection of cell viability.It was clear that, for the inclusion complex,β-CDP served as a complexation host to enhance the water solubility of Cur, and improved the anticancer activity of Cur.

    The synergistic effect of the host–guest complexation betweenβ-CDP and Cur prevented the proliferation of the liver cells.The formation of the inclusion complex could protect Cur against biological degradation, which was a relevant factor to ensure the cytotoxic effect of Cur on the cancer cells [50].

    To further investigate thein vitrocell efficacy of Cur-β-CDP, Cur,β-CDP, and Cur-β-CDP were chosen to study cellular uptakeviaconfocal fluorescence microscopy, wherein blue spots indicate the nuclei of HepG2 cells.Cur and Cur-β-CDP are shown in green, owing to the fluorescence of Cur.Moreover,β-CDP has no fluorescence.The HepG2 cells incubated with Cur-β-CDP displayed strong fluorescence, which was distributed over a large area of the cell,except for the nucleus, whereas the HepG2 cells incubated with Cur exhibited a slightly weaker fluorescence that was distributed in a smaller area of the cell, again except for the nucleus (Fig.3B).This suggests both Cur and Cur-β-CDP are mainly distributed in the cytoplasm.Therefore, compared to Cur, the HepG2 cells absorbed more Cur-β-CDP.The phagocytic effect of Cur-β-CDP was slightly better than that of Cur, indicating the high-water solubility and drug absorption of Cur-β-CDP.

    Cell migration contributes to the metastasis of malignant tumors, leading to a poor prognosis for cancer patients.We performed anin vitrowound healing assay to study cancer cell migration in response to mechanical scratch wounds.The wound healing process was followed by taking time-lapse images at 0, 24 and 48 h, as shown in Fig.3C.The wound was 34.3% closed at 24 h and 63.8% closed at 48 h for untreated cells.The results show the effect of Cur,β-CDP, and Cur-β-CDP (percentage of healing rate after 24 h and 48 h respectively.The inhibitory effects of Cur and Cur-β-CDP on cell migration were almost the same.After 48 h,compared with Cur andβ-CDP, Cur-β-CDP inhibited cell migration more strongly in Fig.S8 (Supporting information).This significantly inhibited the healing rate within 48 h.Thus, Cur-β-CDP can release Cur sustainably for long-term treatment of malignant tumors.These results show that Cur can be released from the Curβ-CDP complex and inhibit tumor cell migration.The influence of Cur-β-CDP was even stronger within 48 h compared with Cur, indicating that Cur-β-CDP was stable and that Cur could be released sustainably.

    In conclusion, we characterized the host–guest interactions between Cur andβ-CDP using various methods.The dissociation constant of the Cur ?CD unit inβ-CDP was calculated to be 6.4 × 10?3mol/L and the binding stoichiometry was confirmed to be 1:1.The water solubility of the inclusion complex was much higher than that of pure Cur.The host–guest interaction of Curβ-CDP in water was also studiedviaCV.The diffusion coefficients of the oxidized and reduced states were calculated as 7.94 × 10?8and 9.91 × 10?8cm2/s, respectively.The diffusion activation energy was calculated as 37.36 kJ/mol.Notably, Cur could serve as an included guest molecule and as an anti-cancer agent, which could strengthen biomedical applications.Cur-β-CDP effectively inhibited the growth of HepG2 cells, while having little effect on non-tumor cells.Moreover, this study demonstrated a promising approach for the improvement of Cur drugs with low water solubility, by enhancing the solubility and improving their anti-cancer performance.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (Nos.21703200 and 21773203), the Chey Institute for Advanced Studies International Scholar Exchange Fellowship for the Academic Year of 2021–2022, and China Scholarship Council Program (No.201908320084).

    Supplementary materials

    Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.12.037.

    国产亚洲欧美精品永久| svipshipincom国产片| 色婷婷av一区二区三区视频| 一区二区三区精品91| 亚洲成av片中文字幕在线观看| 9热在线视频观看99| 亚洲色图综合在线观看| 国产精品免费大片| 操美女的视频在线观看| 日韩一卡2卡3卡4卡2021年| 日本av手机在线免费观看| 秋霞在线观看毛片| 青春草视频在线免费观看| 亚洲欧美色中文字幕在线| 少妇 在线观看| 久久久久久亚洲精品国产蜜桃av| 亚洲av日韩在线播放| 国产有黄有色有爽视频| 大香蕉久久网| 日韩三级视频一区二区三区| 如日韩欧美国产精品一区二区三区| av又黄又爽大尺度在线免费看| 中国国产av一级| 欧美日韩福利视频一区二区| 亚洲国产日韩一区二区| 老熟女久久久| 亚洲精品国产av成人精品| 丰满迷人的少妇在线观看| 搡老乐熟女国产| 国产一区二区 视频在线| 99久久99久久久精品蜜桃| 久久 成人 亚洲| 欧美日韩福利视频一区二区| 久久女婷五月综合色啪小说| 日本精品一区二区三区蜜桃| 在线观看一区二区三区激情| 国产精品麻豆人妻色哟哟久久| 黄色视频不卡| 嫁个100分男人电影在线观看| 久久久国产成人免费| 91成年电影在线观看| 精品亚洲成a人片在线观看| 动漫黄色视频在线观看| 亚洲欧美精品综合一区二区三区| 曰老女人黄片| 日韩制服丝袜自拍偷拍| 免费黄频网站在线观看国产| 久热爱精品视频在线9| 久久午夜综合久久蜜桃| 亚洲av男天堂| 如日韩欧美国产精品一区二区三区| av福利片在线| 国产日韩一区二区三区精品不卡| 在线观看免费高清a一片| 国产成人啪精品午夜网站| 亚洲精品日韩在线中文字幕| 精品人妻熟女毛片av久久网站| 久久精品aⅴ一区二区三区四区| 久久精品国产亚洲av香蕉五月 | 老鸭窝网址在线观看| 久久久精品免费免费高清| 天天躁夜夜躁狠狠躁躁| 侵犯人妻中文字幕一二三四区| a 毛片基地| 久久久久久人人人人人| 考比视频在线观看| avwww免费| 午夜两性在线视频| 在线观看免费高清a一片| 大片电影免费在线观看免费| av又黄又爽大尺度在线免费看| 久久人人爽av亚洲精品天堂| 午夜福利视频精品| 久久久久久久久免费视频了| 国产成人一区二区三区免费视频网站| 久久免费观看电影| 中文字幕另类日韩欧美亚洲嫩草| 91av网站免费观看| 最新的欧美精品一区二区| 日韩有码中文字幕| 亚洲天堂av无毛| 高清黄色对白视频在线免费看| 国产一区二区三区综合在线观看| 精品一区二区三卡| 亚洲精品一二三| 在线看a的网站| 一区二区av电影网| 免费人妻精品一区二区三区视频| 美女高潮喷水抽搐中文字幕| 丝瓜视频免费看黄片| 女人被躁到高潮嗷嗷叫费观| 99热全是精品| 黑人巨大精品欧美一区二区mp4| 亚洲专区国产一区二区| 国产亚洲精品一区二区www | 热re99久久国产66热| 精品一区在线观看国产| 国产精品一区二区在线不卡| a级毛片黄视频| 黄网站色视频无遮挡免费观看| 国产老妇伦熟女老妇高清| 亚洲av电影在线观看一区二区三区| 操出白浆在线播放| 18在线观看网站| 免费高清在线观看视频在线观看| 国产高清videossex| 9191精品国产免费久久| 国产精品一区二区免费欧美 | svipshipincom国产片| 亚洲欧美成人综合另类久久久| 后天国语完整版免费观看| 亚洲熟女精品中文字幕| av又黄又爽大尺度在线免费看| 狂野欧美激情性xxxx| 中文字幕人妻丝袜制服| 精品一区二区三卡| 男女午夜视频在线观看| 午夜福利视频在线观看免费| 成人免费观看视频高清| 国产精品亚洲av一区麻豆| 高潮久久久久久久久久久不卡| 91九色精品人成在线观看| 天天躁日日躁夜夜躁夜夜| 国产精品一区二区精品视频观看| 美女国产高潮福利片在线看| 黑人操中国人逼视频| 99九九在线精品视频| 我要看黄色一级片免费的| 欧美日韩av久久| 精品国产超薄肉色丝袜足j| 亚洲专区中文字幕在线| 日韩电影二区| 中文字幕人妻丝袜一区二区| 久久久久精品人妻al黑| 久久毛片免费看一区二区三区| 国产精品久久久人人做人人爽| 热99久久久久精品小说推荐| 欧美日韩中文字幕国产精品一区二区三区 | 国产一卡二卡三卡精品| av线在线观看网站| 午夜免费成人在线视频| 日本91视频免费播放| 久久久久国产一级毛片高清牌| 男女国产视频网站| 女警被强在线播放| 一进一出抽搐动态| 国产精品秋霞免费鲁丝片| 欧美日韩福利视频一区二区| 99久久国产精品久久久| 人妻久久中文字幕网| 国产精品欧美亚洲77777| 国产高清videossex| 韩国高清视频一区二区三区| 国产精品av久久久久免费| 成年女人毛片免费观看观看9 | 91精品伊人久久大香线蕉| av天堂久久9| 亚洲avbb在线观看| 亚洲综合色网址| 久久综合国产亚洲精品| 一本大道久久a久久精品| 亚洲精品久久成人aⅴ小说| 免费高清在线观看视频在线观看| 国产激情久久老熟女| 精品卡一卡二卡四卡免费| 美女福利国产在线| 中文字幕av电影在线播放| 黑人欧美特级aaaaaa片| 欧美性长视频在线观看| 婷婷成人精品国产| 国产一区有黄有色的免费视频| 女性生殖器流出的白浆| 2018国产大陆天天弄谢| 国产一级毛片在线| 国产成人精品无人区| 日韩中文字幕视频在线看片| av国产精品久久久久影院| 亚洲 欧美一区二区三区| 国产成人av激情在线播放| 成人18禁高潮啪啪吃奶动态图| 国产亚洲午夜精品一区二区久久| 久久ye,这里只有精品| 黑人操中国人逼视频| 好男人电影高清在线观看| 18禁国产床啪视频网站| 国产成人精品在线电影| kizo精华| 久久中文看片网| 美女高潮到喷水免费观看| 永久免费av网站大全| 欧美精品一区二区免费开放| 啦啦啦在线免费观看视频4| 动漫黄色视频在线观看| 欧美午夜高清在线| 黄片小视频在线播放| 99re6热这里在线精品视频| 色94色欧美一区二区| 国产精品 国内视频| 国产成人av教育| 99国产精品99久久久久| 午夜福利视频在线观看免费| 免费观看av网站的网址| 久久九九热精品免费| 精品熟女少妇八av免费久了| 日韩一卡2卡3卡4卡2021年| 免费久久久久久久精品成人欧美视频| 黄色怎么调成土黄色| 下体分泌物呈黄色| 成年人免费黄色播放视频| 亚洲色图 男人天堂 中文字幕| 免费久久久久久久精品成人欧美视频| 免费在线观看日本一区| 女人爽到高潮嗷嗷叫在线视频| 欧美人与性动交α欧美软件| 成人av一区二区三区在线看 | 免费看十八禁软件| 天堂8中文在线网| 亚洲精品国产区一区二| 欧美av亚洲av综合av国产av| 国产主播在线观看一区二区| 人人妻人人爽人人添夜夜欢视频| 中文字幕另类日韩欧美亚洲嫩草| 国产97色在线日韩免费| av在线播放精品| 亚洲精品在线美女| av视频免费观看在线观看| 纵有疾风起免费观看全集完整版| 久久综合国产亚洲精品| 美女中出高潮动态图| 在线观看免费午夜福利视频| 丝袜脚勾引网站| 国产三级黄色录像| 淫妇啪啪啪对白视频 | 最近中文字幕2019免费版| 日韩一卡2卡3卡4卡2021年| 成人18禁高潮啪啪吃奶动态图| 一级毛片精品| 亚洲一区中文字幕在线| 女人被躁到高潮嗷嗷叫费观| 99精品欧美一区二区三区四区| 母亲3免费完整高清在线观看| 极品人妻少妇av视频| 久久久久久久精品精品| 国产又爽黄色视频| 亚洲欧美成人综合另类久久久| 亚洲精品第二区| 日韩熟女老妇一区二区性免费视频| 亚洲国产av影院在线观看| 精品国产国语对白av| 12—13女人毛片做爰片一| 日本五十路高清| 欧美午夜高清在线| 日韩电影二区| 大陆偷拍与自拍| 国产亚洲精品久久久久5区| 青草久久国产| 亚洲午夜精品一区,二区,三区| 久久精品国产亚洲av香蕉五月 | 2018国产大陆天天弄谢| 亚洲情色 制服丝袜| 亚洲 欧美一区二区三区| 美女国产高潮福利片在线看| videos熟女内射| 黄频高清免费视频| 熟女少妇亚洲综合色aaa.| 日韩免费高清中文字幕av| 丝袜在线中文字幕| 一区福利在线观看| 老汉色av国产亚洲站长工具| 国产精品久久久久久人妻精品电影 | 欧美精品高潮呻吟av久久| 亚洲av日韩精品久久久久久密| 国产黄色免费在线视频| 老司机午夜十八禁免费视频| 亚洲综合色网址| 一本综合久久免费| 涩涩av久久男人的天堂| 欧美在线一区亚洲| av不卡在线播放| 国产一卡二卡三卡精品| 最新的欧美精品一区二区| 久久久久精品国产欧美久久久 | 两性午夜刺激爽爽歪歪视频在线观看 | 如日韩欧美国产精品一区二区三区| 欧美精品啪啪一区二区三区 | 亚洲天堂av无毛| 50天的宝宝边吃奶边哭怎么回事| 一边摸一边抽搐一进一出视频| 亚洲精品中文字幕在线视频| 亚洲欧美色中文字幕在线| 成年美女黄网站色视频大全免费| 午夜成年电影在线免费观看| 亚洲一区中文字幕在线| 国产精品国产av在线观看| 欧美人与性动交α欧美精品济南到| 国产精品熟女久久久久浪| 一区福利在线观看| 超色免费av| 搡老乐熟女国产| 我要看黄色一级片免费的| 亚洲精品国产色婷婷电影| 亚洲av成人一区二区三| 国产一级毛片在线| 18禁国产床啪视频网站| 亚洲熟女精品中文字幕| 咕卡用的链子| 久久女婷五月综合色啪小说| 少妇的丰满在线观看| 97精品久久久久久久久久精品| 成年动漫av网址| 亚洲国产欧美一区二区综合| 中文字幕色久视频| 丰满人妻熟妇乱又伦精品不卡| 91九色精品人成在线观看| 韩国精品一区二区三区| 国产欧美日韩精品亚洲av| 免费在线观看黄色视频的| 老司机影院成人| 日韩三级视频一区二区三区| 国产精品亚洲av一区麻豆| 男人爽女人下面视频在线观看| av线在线观看网站| 国产伦理片在线播放av一区| 18在线观看网站| 丰满迷人的少妇在线观看| 狠狠精品人妻久久久久久综合| 啦啦啦 在线观看视频| av片东京热男人的天堂| 国产精品一区二区精品视频观看| 久久精品人人爽人人爽视色| 一级片'在线观看视频| 亚洲三区欧美一区| 深夜精品福利| 叶爱在线成人免费视频播放| 国产色视频综合| 欧美少妇被猛烈插入视频| 一级a爱视频在线免费观看| 午夜免费观看性视频| 日韩有码中文字幕| 国产精品熟女久久久久浪| 国产伦理片在线播放av一区| 国产色视频综合| 欧美久久黑人一区二区| 手机成人av网站| 韩国精品一区二区三区| 成年美女黄网站色视频大全免费| 久久国产精品人妻蜜桃| 老熟女久久久| 男女免费视频国产| 亚洲欧美成人综合另类久久久| 精品卡一卡二卡四卡免费| 免费黄频网站在线观看国产| cao死你这个sao货| 可以免费在线观看a视频的电影网站| 国产高清国产精品国产三级| a 毛片基地| 男女床上黄色一级片免费看| 久久九九热精品免费| netflix在线观看网站| 人妻 亚洲 视频| 欧美一级毛片孕妇| 亚洲精品中文字幕在线视频| 亚洲国产精品一区二区三区在线| 亚洲av电影在线观看一区二区三区| 欧美亚洲日本最大视频资源| 亚洲男人天堂网一区| 亚洲人成电影观看| 啦啦啦中文免费视频观看日本| 日本五十路高清| 亚洲av欧美aⅴ国产| 一区二区三区四区激情视频| 国产又色又爽无遮挡免| 少妇人妻久久综合中文| 狂野欧美激情性bbbbbb| 在线天堂中文资源库| 久久九九热精品免费| netflix在线观看网站| 99国产极品粉嫩在线观看| 女性生殖器流出的白浆| 欧美一级毛片孕妇| 亚洲精品中文字幕在线视频| 超色免费av| 国产精品久久久久久精品电影小说| 视频区欧美日本亚洲| www.精华液| 国产一级毛片在线| av有码第一页| 免费不卡黄色视频| 在线观看舔阴道视频| 国产在线观看jvid| 一级,二级,三级黄色视频| av在线播放精品| 少妇被粗大的猛进出69影院| 两人在一起打扑克的视频| 亚洲欧美激情在线| 国产三级黄色录像| 两人在一起打扑克的视频| 美国免费a级毛片| 国产在线观看jvid| 亚洲av国产av综合av卡| 一级毛片女人18水好多| 日本91视频免费播放| 大香蕉久久成人网| 亚洲自偷自拍图片 自拍| 老汉色av国产亚洲站长工具| 久久久久久亚洲精品国产蜜桃av| 久久久久国产精品人妻一区二区| 日本欧美视频一区| 99国产精品一区二区蜜桃av | 成人国产一区最新在线观看| 少妇裸体淫交视频免费看高清 | 日韩欧美国产一区二区入口| 老司机影院毛片| 一级毛片电影观看| 妹子高潮喷水视频| 五月开心婷婷网| 亚洲色图综合在线观看| 精品国产一区二区三区久久久樱花| 我要看黄色一级片免费的| 99国产精品一区二区蜜桃av | 大片电影免费在线观看免费| 老熟妇仑乱视频hdxx| 午夜福利免费观看在线| 大片电影免费在线观看免费| 狂野欧美激情性bbbbbb| 国产欧美日韩一区二区三 | 免费久久久久久久精品成人欧美视频| 日韩中文字幕欧美一区二区| 最近最新免费中文字幕在线| 国产精品一区二区在线观看99| 国产亚洲午夜精品一区二区久久| 乱人伦中国视频| 又大又爽又粗| 精品久久久精品久久久| 国产91精品成人一区二区三区 | 国产一区二区三区av在线| 真人做人爱边吃奶动态| 欧美老熟妇乱子伦牲交| av片东京热男人的天堂| 又紧又爽又黄一区二区| 最近中文字幕2019免费版| 丝袜美足系列| 亚洲av日韩在线播放| 亚洲专区国产一区二区| 69精品国产乱码久久久| 在线观看人妻少妇| 中文字幕色久视频| 老熟女久久久| 国产熟女午夜一区二区三区| 777久久人妻少妇嫩草av网站| 国产精品九九99| 亚洲精品国产一区二区精华液| 天天躁夜夜躁狠狠躁躁| 亚洲黑人精品在线| 色播在线永久视频| 十八禁网站网址无遮挡| av天堂久久9| 女人高潮潮喷娇喘18禁视频| 国产精品成人在线| 精品少妇一区二区三区视频日本电影| 亚洲天堂av无毛| 日本五十路高清| 欧美另类亚洲清纯唯美| 男女床上黄色一级片免费看| 91九色精品人成在线观看| 欧美在线一区亚洲| 婷婷成人精品国产| 亚洲国产av影院在线观看| 亚洲国产成人一精品久久久| 中文欧美无线码| 国产精品99久久99久久久不卡| 老司机影院毛片| 搡老乐熟女国产| 国产成人系列免费观看| 男人操女人黄网站| 亚洲一区中文字幕在线| 99国产综合亚洲精品| av天堂久久9| 久久精品aⅴ一区二区三区四区| 欧美一级毛片孕妇| 久久天堂一区二区三区四区| 中文字幕高清在线视频| 国产在线免费精品| 免费在线观看日本一区| 亚洲成人国产一区在线观看| 2018国产大陆天天弄谢| 新久久久久国产一级毛片| 精品国产乱码久久久久久小说| 亚洲精品成人av观看孕妇| 天天操日日干夜夜撸| 午夜日韩欧美国产| 日韩三级视频一区二区三区| 久久午夜综合久久蜜桃| 精品一区二区三卡| 久久久久国产一级毛片高清牌| 91成人精品电影| 热99re8久久精品国产| 999久久久精品免费观看国产| a在线观看视频网站| 少妇 在线观看| 99香蕉大伊视频| 国产日韩欧美在线精品| 黄色a级毛片大全视频| 亚洲av电影在线进入| 99久久99久久久精品蜜桃| 午夜福利在线免费观看网站| 国产1区2区3区精品| 亚洲精品一二三| 多毛熟女@视频| 97在线人人人人妻| 久久久国产成人免费| 久久国产精品影院| 狠狠狠狠99中文字幕| 欧美 亚洲 国产 日韩一| 国产精品av久久久久免费| 男男h啪啪无遮挡| 日本黄色日本黄色录像| 91老司机精品| 日本黄色日本黄色录像| 亚洲成人手机| 亚洲精品自拍成人| 99久久人妻综合| 欧美另类一区| 亚洲人成77777在线视频| 女性被躁到高潮视频| 电影成人av| 黑人巨大精品欧美一区二区mp4| 亚洲av国产av综合av卡| 精品福利观看| 欧美精品一区二区大全| 美国免费a级毛片| 久久久国产精品麻豆| 精品国产乱子伦一区二区三区 | 黄色片一级片一级黄色片| 老汉色∧v一级毛片| 国产熟女午夜一区二区三区| 午夜视频精品福利| 后天国语完整版免费观看| 每晚都被弄得嗷嗷叫到高潮| 波多野结衣一区麻豆| 不卡一级毛片| 黄片大片在线免费观看| 精品福利观看| 人成视频在线观看免费观看| 欧美 亚洲 国产 日韩一| 俄罗斯特黄特色一大片| 一级毛片精品| 性高湖久久久久久久久免费观看| 啦啦啦在线免费观看视频4| 深夜精品福利| 色视频在线一区二区三区| 日本精品一区二区三区蜜桃| 久久 成人 亚洲| 久久久久久久久久久久大奶| 久久精品国产a三级三级三级| 国产高清videossex| 精品国产一区二区三区久久久樱花| 两性午夜刺激爽爽歪歪视频在线观看 | 欧美日韩亚洲国产一区二区在线观看 | 免费少妇av软件| 久久久久国产一级毛片高清牌| 国产真人三级小视频在线观看| 一级片'在线观看视频| 国产淫语在线视频| 成人三级做爰电影| 天天躁狠狠躁夜夜躁狠狠躁| 亚洲免费av在线视频| videos熟女内射| 性少妇av在线| 天天躁狠狠躁夜夜躁狠狠躁| 91麻豆av在线| 两个人免费观看高清视频| 亚洲精品国产一区二区精华液| 啦啦啦免费观看视频1| 大片电影免费在线观看免费| 亚洲精品美女久久久久99蜜臀| 丰满少妇做爰视频| 久久精品国产亚洲av高清一级| av免费在线观看网站| 国产精品 国内视频| 黑人巨大精品欧美一区二区mp4| 一级,二级,三级黄色视频| av免费在线观看网站| 这个男人来自地球电影免费观看| 一边摸一边做爽爽视频免费| 18在线观看网站| 亚洲成人免费电影在线观看| 久9热在线精品视频| 亚洲精品一区蜜桃| 国产精品亚洲av一区麻豆| 在线观看一区二区三区激情| 亚洲avbb在线观看| 久久av网站| 女性被躁到高潮视频| 三级毛片av免费| 免费在线观看完整版高清| 岛国在线观看网站| 一区二区三区精品91| 好男人电影高清在线观看| 十分钟在线观看高清视频www| 久久狼人影院| 久久ye,这里只有精品| 99精品欧美一区二区三区四区| 看免费av毛片| av有码第一页| 午夜日韩欧美国产| 免费观看人在逋| 黄色视频在线播放观看不卡| 丝袜人妻中文字幕| 超碰97精品在线观看| 黄色片一级片一级黄色片| av线在线观看网站| 熟女少妇亚洲综合色aaa.| 久久国产精品大桥未久av| 亚洲精品第二区|