• <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.

    av在线播放精品| 99热这里只有是精品在线观看| 精品人妻一区二区三区麻豆| 伦理电影大哥的女人| 欧美精品一区二区大全| 国产av不卡久久| 亚洲高清免费不卡视频| 亚洲精品一区蜜桃| 久久久久久久久久人人人人人人| 日韩不卡一区二区三区视频在线| 69人妻影院| 成人av在线播放网站| 亚洲av在线观看美女高潮| 亚洲aⅴ乱码一区二区在线播放| 97精品久久久久久久久久精品| 成人美女网站在线观看视频| 亚洲经典国产精华液单| 亚洲美女视频黄频| 亚洲婷婷狠狠爱综合网| 国产乱人偷精品视频| 熟妇人妻久久中文字幕3abv| 亚洲激情五月婷婷啪啪| 中国国产av一级| 亚洲成人久久爱视频| 成人美女网站在线观看视频| 国产永久视频网站| 狂野欧美激情性xxxx在线观看| 国产片特级美女逼逼视频| 亚洲精品国产av蜜桃| 99re6热这里在线精品视频| 午夜日本视频在线| 听说在线观看完整版免费高清| 亚洲精品日本国产第一区| 国产亚洲av片在线观看秒播厂 | 国产黄a三级三级三级人| 美女cb高潮喷水在线观看| 别揉我奶头 嗯啊视频| 成人高潮视频无遮挡免费网站| 国产黄频视频在线观看| 校园人妻丝袜中文字幕| 18禁动态无遮挡网站| 欧美激情国产日韩精品一区| 亚洲欧美成人综合另类久久久| 日日干狠狠操夜夜爽| 国产精品久久久久久久久免| 岛国毛片在线播放| 欧美日韩精品成人综合77777| 久久综合国产亚洲精品| 麻豆av噜噜一区二区三区| 午夜免费男女啪啪视频观看| 久久99蜜桃精品久久| 国产亚洲最大av| 日韩 亚洲 欧美在线| 久久精品熟女亚洲av麻豆精品 | 毛片一级片免费看久久久久| 老女人水多毛片| 国产男女超爽视频在线观看| 黑人高潮一二区| 69av精品久久久久久| 欧美日韩亚洲高清精品| 大陆偷拍与自拍| 边亲边吃奶的免费视频| 精品一区在线观看国产| 91精品国产九色| 高清视频免费观看一区二区 | 婷婷色av中文字幕| 黄色一级大片看看| 99热网站在线观看| 一个人观看的视频www高清免费观看| 亚洲成人一二三区av| 日本熟妇午夜| 亚洲欧美成人综合另类久久久| 丝袜美腿在线中文| 国产日韩欧美在线精品| 两个人的视频大全免费| 国产综合精华液| 又爽又黄无遮挡网站| 国产伦一二天堂av在线观看| 久久精品夜夜夜夜夜久久蜜豆| 一二三四中文在线观看免费高清| 亚洲av电影不卡..在线观看| 精品不卡国产一区二区三区| 高清日韩中文字幕在线| 国产人妻一区二区三区在| 成人欧美大片| 深夜a级毛片| 啦啦啦中文免费视频观看日本| 一级av片app| 国产精品一区二区三区四区免费观看| 草草在线视频免费看| 午夜亚洲福利在线播放| 别揉我奶头 嗯啊视频| 美女脱内裤让男人舔精品视频| 美女国产视频在线观看| 老司机影院成人| 青春草视频在线免费观看| 国产成人精品久久久久久| 色哟哟·www| 少妇熟女欧美另类| 伊人久久精品亚洲午夜| 免费av毛片视频| 欧美成人一区二区免费高清观看| 大又大粗又爽又黄少妇毛片口| 午夜福利在线观看免费完整高清在| 亚洲精品色激情综合| 性色avwww在线观看| 亚洲精品一区蜜桃| 久久热精品热| 18禁在线播放成人免费| 内地一区二区视频在线| 三级男女做爰猛烈吃奶摸视频| 国产成人91sexporn| 最近手机中文字幕大全| 国产成人午夜福利电影在线观看| 热99在线观看视频| 能在线免费观看的黄片| 国产伦一二天堂av在线观看| 久久国内精品自在自线图片| 老师上课跳d突然被开到最大视频| 国产老妇女一区| 我的女老师完整版在线观看| 精品人妻熟女av久视频| 国产伦一二天堂av在线观看| 七月丁香在线播放| 久久精品久久精品一区二区三区| 久久久久精品性色| 亚洲婷婷狠狠爱综合网| 国产黄色免费在线视频| 国产精品一区二区三区四区久久| 美女xxoo啪啪120秒动态图| 欧美xxxx性猛交bbbb| 国产69精品久久久久777片| 成人美女网站在线观看视频| 欧美最新免费一区二区三区| 日韩人妻高清精品专区| 精品一区二区免费观看| 嫩草影院精品99| 亚洲精品一二三| 精品不卡国产一区二区三区| 22中文网久久字幕| 搡女人真爽免费视频火全软件| 狂野欧美激情性xxxx在线观看| 国产精品熟女久久久久浪| 精品99又大又爽又粗少妇毛片| 亚洲人成网站在线观看播放| 日本免费在线观看一区| av免费观看日本| 亚洲内射少妇av| av卡一久久| 美女高潮的动态| 日韩视频在线欧美| 亚洲激情五月婷婷啪啪| 一个人免费在线观看电影| 成年av动漫网址| 永久网站在线| 一级二级三级毛片免费看| 欧美极品一区二区三区四区| 人妻夜夜爽99麻豆av| 国产亚洲最大av| 国产91av在线免费观看| 身体一侧抽搐| 国产精品爽爽va在线观看网站| 久热久热在线精品观看| 欧美日韩综合久久久久久| av免费在线看不卡| 在线免费观看不下载黄p国产| 黄色一级大片看看| 国产一区二区亚洲精品在线观看| 久久精品国产鲁丝片午夜精品| 免费观看av网站的网址| 男女国产视频网站| 成人亚洲精品av一区二区| 在线天堂最新版资源| 97在线视频观看| 亚洲精品国产av蜜桃| 身体一侧抽搐| 校园人妻丝袜中文字幕| 久久这里有精品视频免费| av国产免费在线观看| 最近手机中文字幕大全| 久久鲁丝午夜福利片| 亚洲综合色惰| 成年免费大片在线观看| 在线天堂最新版资源| 狂野欧美激情性xxxx在线观看| 99热这里只有精品一区| 三级国产精品欧美在线观看| 欧美日韩视频高清一区二区三区二| videos熟女内射| 国产极品天堂在线| 日韩av在线大香蕉| 啦啦啦韩国在线观看视频| 日本色播在线视频| 亚洲国产精品成人久久小说| 免费无遮挡裸体视频| 国产成人精品婷婷| 国产精品伦人一区二区| 国产一级毛片七仙女欲春2| 我要看日韩黄色一级片| 亚洲久久久久久中文字幕| 欧美区成人在线视频| 夫妻午夜视频| 日本一本二区三区精品| 日韩不卡一区二区三区视频在线| 国产乱来视频区| 老司机影院毛片| 国产一区二区亚洲精品在线观看| 精品人妻一区二区三区麻豆| 国产午夜精品久久久久久一区二区三区| 一区二区三区免费毛片| 亚洲精华国产精华液的使用体验| 七月丁香在线播放| 干丝袜人妻中文字幕| 在线天堂最新版资源| 乱人视频在线观看| 日韩视频在线欧美| 免费av毛片视频| 国产视频内射| av.在线天堂| 国产精品女同一区二区软件| 午夜精品一区二区三区免费看| 国产精品无大码| 99久久精品国产国产毛片| 亚洲,欧美,日韩| 久久久久久久久大av| 建设人人有责人人尽责人人享有的 | av在线蜜桃| 欧美日韩视频高清一区二区三区二| 国产一区有黄有色的免费视频 | 午夜爱爱视频在线播放| 大陆偷拍与自拍| 欧美日韩视频高清一区二区三区二| 免费少妇av软件| 日本色播在线视频| 午夜激情福利司机影院| 最近中文字幕2019免费版| 噜噜噜噜噜久久久久久91| 精品人妻一区二区三区麻豆| 欧美日韩精品成人综合77777| 国产人妻一区二区三区在| 三级经典国产精品| 91在线精品国自产拍蜜月| 99久久精品一区二区三区| 精品一区二区免费观看| 中文字幕免费在线视频6| 国产永久视频网站| 日韩亚洲欧美综合| 精品国产一区二区三区久久久樱花 | 久久久久免费精品人妻一区二区| 一边亲一边摸免费视频| 免费黄频网站在线观看国产| 免费观看a级毛片全部| 熟女人妻精品中文字幕| 国产一级毛片七仙女欲春2| 久久6这里有精品| 天堂av国产一区二区熟女人妻| 草草在线视频免费看| 韩国高清视频一区二区三区| av网站免费在线观看视频 | 麻豆成人av视频| 国产精品一区二区三区四区久久| 国产大屁股一区二区在线视频| 日本色播在线视频| 国产麻豆成人av免费视频| 国产熟女欧美一区二区| 欧美xxxx黑人xx丫x性爽| 中文资源天堂在线| 久久久久久国产a免费观看| 色尼玛亚洲综合影院| 免费高清在线观看视频在线观看| 能在线免费观看的黄片| 夫妻性生交免费视频一级片| 男女视频在线观看网站免费| 国产成人freesex在线| 精品久久久久久久久av| 国产精品麻豆人妻色哟哟久久 | 午夜精品一区二区三区免费看| 最新中文字幕久久久久| 婷婷色av中文字幕| 国产黄频视频在线观看| 国产视频内射| 欧美精品一区二区大全| 成年人午夜在线观看视频 | 国产av国产精品国产| 国产成人午夜福利电影在线观看| 国产精品日韩av在线免费观看| 午夜爱爱视频在线播放| 99视频精品全部免费 在线| 一区二区三区四区激情视频| 97超碰精品成人国产| 国产精品人妻久久久影院| 精品一区二区三区视频在线| 伦精品一区二区三区| 国产一区二区亚洲精品在线观看| 日本黄色片子视频| 在线a可以看的网站| 天美传媒精品一区二区| 中国美白少妇内射xxxbb| 亚洲av成人av| 亚洲av成人精品一区久久| 最新中文字幕久久久久| 中文字幕免费在线视频6| 亚洲欧美精品专区久久| 久99久视频精品免费| 午夜福利在线观看免费完整高清在| 大片免费播放器 马上看| 亚洲,欧美,日韩| 淫秽高清视频在线观看| 日韩欧美一区视频在线观看 | 欧美+日韩+精品| 国产一区二区亚洲精品在线观看| 国产欧美日韩精品一区二区| 亚洲精品乱久久久久久| av黄色大香蕉| 菩萨蛮人人尽说江南好唐韦庄| 免费看日本二区| 免费高清在线观看视频在线观看| 韩国av在线不卡| 久久热精品热| 97精品久久久久久久久久精品| 国产免费一级a男人的天堂| 亚洲精品国产av成人精品| 国产精品1区2区在线观看.| 啦啦啦中文免费视频观看日本| 久久久久免费精品人妻一区二区| 久久久精品欧美日韩精品| 国产淫语在线视频| 一级片'在线观看视频| 日韩欧美国产在线观看| www.色视频.com| 中文字幕免费在线视频6| 哪个播放器可以免费观看大片| 建设人人有责人人尽责人人享有的 | 国产单亲对白刺激| 日本与韩国留学比较| 久久人人爽人人片av| 日韩av不卡免费在线播放| 成人性生交大片免费视频hd| 国产女主播在线喷水免费视频网站 | 看免费成人av毛片| 亚洲aⅴ乱码一区二区在线播放| 不卡视频在线观看欧美| 日本av手机在线免费观看| 91精品一卡2卡3卡4卡| 亚洲欧洲日产国产| 日韩视频在线欧美| 一级a做视频免费观看| 联通29元200g的流量卡| 国产国拍精品亚洲av在线观看| 亚洲在线自拍视频| 亚洲av免费在线观看| 精品久久久久久久久久久久久| 男人舔女人下体高潮全视频| 精品99又大又爽又粗少妇毛片| 3wmmmm亚洲av在线观看| 亚洲美女搞黄在线观看| 国产精品av视频在线免费观看| 特大巨黑吊av在线直播| 久久精品国产自在天天线| 一级av片app| 精品人妻视频免费看| 欧美3d第一页| 自拍偷自拍亚洲精品老妇| 亚洲精品第二区| 在线观看av片永久免费下载| 久久精品夜色国产| 国产精品福利在线免费观看| 女人被狂操c到高潮| 只有这里有精品99| 干丝袜人妻中文字幕| 亚洲欧美日韩无卡精品| 国产91av在线免费观看| 免费av毛片视频| 中文字幕人妻熟人妻熟丝袜美| 国产精品一及| 中文字幕久久专区| 国产精品三级大全| 欧美日本视频| 日本免费a在线| 高清视频免费观看一区二区 | 肉色欧美久久久久久久蜜桃 | 欧美一级a爱片免费观看看| 五月玫瑰六月丁香| 久久这里有精品视频免费| a级毛片免费高清观看在线播放| 日韩一区二区视频免费看| 国产精品日韩av在线免费观看| 小蜜桃在线观看免费完整版高清| 久久综合国产亚洲精品| 国产毛片a区久久久久| 久久韩国三级中文字幕| 少妇高潮的动态图| 欧美xxxx黑人xx丫x性爽| 熟女电影av网| 成人av在线播放网站| 日韩人妻高清精品专区| 色视频www国产| 亚洲精品日韩av片在线观看| 久久精品国产亚洲av天美| 国产伦精品一区二区三区视频9| 亚洲精品乱码久久久v下载方式| 成人国产麻豆网| 精品久久久久久久久久久久久| 国产 亚洲一区二区三区 | 中文在线观看免费www的网站| 最近最新中文字幕免费大全7| 18禁动态无遮挡网站| 人妻夜夜爽99麻豆av| 一个人观看的视频www高清免费观看| 国产激情偷乱视频一区二区| 五月玫瑰六月丁香| 亚洲精品一区蜜桃| 又粗又硬又长又爽又黄的视频| 青春草视频在线免费观看| 97在线视频观看| 国产精品不卡视频一区二区| 亚洲欧美日韩卡通动漫| 九九在线视频观看精品| 成人av在线播放网站| 亚洲av国产av综合av卡| 干丝袜人妻中文字幕| 26uuu在线亚洲综合色| 高清在线视频一区二区三区| 久久久国产一区二区| 亚洲人成网站在线观看播放| 日本爱情动作片www.在线观看| 成年版毛片免费区| 婷婷色综合www| 91久久精品电影网| 成人鲁丝片一二三区免费| 97人妻精品一区二区三区麻豆| 九九爱精品视频在线观看| 国产 亚洲一区二区三区 | 久久久久久久亚洲中文字幕| 国产黄a三级三级三级人| 亚洲精品一二三| 久久精品久久精品一区二区三区| 99热这里只有是精品在线观看| 蜜臀久久99精品久久宅男| 亚洲成人一二三区av| 亚洲av成人精品一二三区| 真实男女啪啪啪动态图| 色哟哟·www| 国产伦在线观看视频一区| 免费观看性生交大片5| 亚洲欧美日韩东京热| 视频中文字幕在线观看| 中文资源天堂在线| 亚洲av男天堂| 欧美性感艳星| 午夜精品一区二区三区免费看| 国产精品av视频在线免费观看| 久久久久久久久久久丰满| 1000部很黄的大片| 日本一二三区视频观看| 欧美日韩综合久久久久久| 欧美激情在线99| 亚洲av成人精品一区久久| 国内精品一区二区在线观看| 亚洲精品日韩av片在线观看| 亚洲av电影不卡..在线观看| 亚洲欧洲国产日韩| 国产91av在线免费观看| 五月伊人婷婷丁香| 韩国av在线不卡| 欧美三级亚洲精品| 婷婷色综合大香蕉| 久久精品久久久久久久性| 国产永久视频网站| 成人av在线播放网站| 1000部很黄的大片| 亚洲av免费高清在线观看| 人人妻人人澡人人爽人人夜夜 | 一个人免费在线观看电影| 久久久a久久爽久久v久久| 久久精品人妻少妇| 国产精品久久久久久精品电影小说 | 国产高清国产精品国产三级 | 能在线免费观看的黄片| 成人毛片60女人毛片免费| 国产精品熟女久久久久浪| 欧美三级亚洲精品| 禁无遮挡网站| 欧美三级亚洲精品| 国产亚洲av嫩草精品影院| 免费av不卡在线播放| 日日摸夜夜添夜夜爱| 大香蕉久久网| 国产免费视频播放在线视频 | 亚洲婷婷狠狠爱综合网| 三级经典国产精品| 国产成人a∨麻豆精品| 看十八女毛片水多多多| 嫩草影院精品99| 国产伦理片在线播放av一区| 我要看日韩黄色一级片| 亚洲在久久综合| 久久韩国三级中文字幕| 在线a可以看的网站| 亚洲国产最新在线播放| .国产精品久久| 亚洲婷婷狠狠爱综合网| 在线a可以看的网站| 一级毛片我不卡| 在线天堂最新版资源| 禁无遮挡网站| 1000部很黄的大片| 亚洲成人久久爱视频| 22中文网久久字幕| 51国产日韩欧美| 国产精品国产三级专区第一集| 亚洲欧美一区二区三区国产| 22中文网久久字幕| 97精品久久久久久久久久精品| 亚洲精品乱码久久久久久按摩| 久久久久九九精品影院| 欧美区成人在线视频| 亚洲四区av| 亚洲欧美日韩东京热| 免费观看无遮挡的男女| 黄色配什么色好看| 一区二区三区免费毛片| 午夜福利在线观看免费完整高清在| 国产精品一区www在线观看| 男女啪啪激烈高潮av片| 日韩一区二区三区影片| 午夜福利在线在线| 青春草国产在线视频| 2022亚洲国产成人精品| 国产精品国产三级国产专区5o| 国产精品国产三级专区第一集| 日韩一区二区三区影片| 日产精品乱码卡一卡2卡三| 韩国高清视频一区二区三区| 中文欧美无线码| 欧美 日韩 精品 国产| 美女主播在线视频| 别揉我奶头 嗯啊视频| 亚洲av男天堂| 亚洲在线自拍视频| 麻豆国产97在线/欧美| kizo精华| 一级毛片久久久久久久久女| 亚洲欧美日韩东京热| 久久韩国三级中文字幕| 精品不卡国产一区二区三区| 毛片女人毛片| 三级国产精品欧美在线观看| 成人性生交大片免费视频hd| freevideosex欧美| 国产精品一二三区在线看| 少妇人妻一区二区三区视频| 狠狠精品人妻久久久久久综合| 亚洲怡红院男人天堂| 亚洲精华国产精华液的使用体验| 久久久久九九精品影院| 精品人妻视频免费看| 免费观看精品视频网站| 国产av国产精品国产| 在线观看av片永久免费下载| 久久精品国产鲁丝片午夜精品| or卡值多少钱| 欧美三级亚洲精品| 欧美日韩亚洲高清精品| 97在线视频观看| 欧美日韩综合久久久久久| 欧美丝袜亚洲另类| 免费电影在线观看免费观看| 国产伦在线观看视频一区| 精品欧美国产一区二区三| 少妇丰满av| 国产成人午夜福利电影在线观看| 久久久久九九精品影院| av免费观看日本| 国产伦精品一区二区三区四那| 欧美xxxx黑人xx丫x性爽| 亚洲乱码一区二区免费版| 欧美97在线视频| 免费观看无遮挡的男女| 亚洲精品自拍成人| 中文字幕人妻熟人妻熟丝袜美| 欧美日韩视频高清一区二区三区二| 人妻夜夜爽99麻豆av| 日本三级黄在线观看| 美女xxoo啪啪120秒动态图| 国产精品一区二区性色av| 午夜福利成人在线免费观看| 国产精品爽爽va在线观看网站| 欧美日韩一区二区视频在线观看视频在线 | 大片免费播放器 马上看| av在线观看视频网站免费| 欧美一级a爱片免费观看看| 毛片一级片免费看久久久久| 自拍偷自拍亚洲精品老妇| 中文字幕久久专区| 18禁在线无遮挡免费观看视频| 街头女战士在线观看网站| 欧美潮喷喷水| 一区二区三区乱码不卡18| 国产一区二区三区av在线| 久久久精品免费免费高清| 欧美日韩国产mv在线观看视频 | 久久久午夜欧美精品| 真实男女啪啪啪动态图| 丝袜美腿在线中文| 人人妻人人澡欧美一区二区| 亚洲av成人精品一区久久| 国产成人a∨麻豆精品| 亚洲欧美精品专区久久| av专区在线播放| 国产在视频线精品| 97人妻精品一区二区三区麻豆| 精品久久久久久久人妻蜜臀av| 日韩欧美精品免费久久| 亚洲精品日韩av片在线观看|