• <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 ?

    Three previously undescribed metabolites from Cordyceps cicadae JXCH-1,an entomopathogenic fungus

    2023-12-29 13:42:08JingFanPaiLiuKuanZhaoandHePingChen
    Natural Products and Bioprospecting 2023年6期

    Jing Fan,Pai Liu,Kuan Zhao and He-Ping Chen

    Abstract Three previously undescribed compounds,cordycicadione (1),cordycicadin F (2),and 7-hydroxybassiatin (3),were isolated from the cultures of Cordyceps cicadae JXCH1,an entomopathogenic fungus.Their structures and relative configurations were elucidated primarily by NMR spectroscopic analysis.The absolute configurations of 1 and 2 were determined by ECD calculations.Single-crystal X-ray diffraction method was adopted to determine the absolute configuration of 3.Compound 2 is a polycyclic polyketide with an unusual enol ether moiety and a spiro ring.The compounds obtained in this study were subjected to screening their inhibition against the proliferation of the human lung cancer cell line A549 and the production of nitric oxide in murine macrophages RAW264.7.

    Keywords Insect pathogenic fungus,Cordyceps cicadae,Steroid,Polyketide,Cyclic dipeptide

    1 Introduction

    The genusCordyceps,a group of insect-pathogenic fungi,includes many species with important medical applications in Chinese Medicine.The sensu stricto species used in Chinese Medicine areCordyceps sinensis,Cordyceps militaris,andCordyceps cicadae[1].Among the three species,C.sinensishas been used for treating diverse chronic diseases for more than 300 years,whileC.militarisis a species which has been widely used as a materia medica,but also used a food supplement.C.cicadaeis a parasitic fungus formed by cicada nymphs infected by the fungusC.cicadae(=Isaria cicadae) [2].Jin Chan Hua,a fungus-insect complex used in Chinese Medicine,is one of the earliest medicines recorded in classics of Traditional Chinese Medicine.Pharmacological experiments results suggested thatJin Chan Huais effective remedies for convulsions,chronic kidney diseases,measles,asthma,insomnia,and heart palpitations [3].However,although the medicinal values ofC.cicadaehave long been recognized,but unlike theC.militaris,theC.cicadaeare not cultivatable [4],and thereby,its shortage of supply has hampered the widely application of this species.

    Recently,there have been few studies on the chemical composition and pharmacological effects of theC.cicadaestrains,which have facilitated the alternative application of the crude fractionated products of this fungus.It is clear that the bioactive substances ofC.cicadaeinclude nucleosides,polysaccharides,sterols,cyclic dipeptides,amino acids,and other small organic compounds [5-8],which have extensive pharmacological effects including immunomodulatory,antitumor [9],neuroprotective [10],antibacterial [11],and anti-diabetic [12] activities.However,in terms of the exact chemical profiles of the natural products derived from this fungus,only a handful of compounds were reported.Cordycicadins A-D,purified from the fermentation broth of the fungusC.cicadaeJXCH1,are unprecedented C20polyketides with unusual exocyclic enol ether bridges [3].Biological evaluations of these polyketides indicate that they showed significant antifeedant activity against silkworm larvae,implying the potential of these compounds in green agrochemicals development.Therefore,the secondary metabolites ofC.cicadaeare indeed worth to be further investigated.Herein,we report the purification as well as structural elucidation of three metabolites from the cultures of the fungusC.cicadaeJXCH1.

    2 Results and discussion

    Cordycicadione (1,Fig.1) was obtained as a pale-yellow oil.It presented an ion peak with highest abundance atm/z497.28726 [M+Na]+in the (+)-HRESIMS analysis,which returned a molecular formula of C28H42O6(calcd for C28H42O6Na,497.28736),corresponding to eight degrees of unsaturation.The structural elucidation of1was accomplished by thorough analysis of the NMR spectra.The1H NMR spectroscopic data of1recorded in methanol-d4(Table 1) showed resonances for two CH3singlet protons atδH1.16 (Me-18),1.28 (Me-19),and four CH3doublets atδH0.74 (d,J=6.9 Hz,Me-28),0.76(d,J=6.9 Hz,Me-26),0.93 (d,J=7.0 Hz,Me-27),1.08 (d,J=6.9 Hz,Me-21),four oxygen-attached methine protons atδH3.30 (overlapped,H-23),3.76 (s,H-12),4.19(br.d,J=7.6 Hz,H-22),4.49 (m,H-16).The13C NMR as well as DEPT spectroscopic data of1recorded in methanol-d4(Table 2) displayed sharp carbon signals that were classified into six CH3,six CH2,nine CH [four oxygenated CH atδC74.8 (C-23),80.3 (C-12),83.5 (C-16) and 85.2 (C-22)],two double bond carbons,three quaternary carbons [sp3hybridized,one oxygenated,δC83.7 (C-14)],and two ketone carbons atδC201.0 (C-11) and 214.1(C-3).The 1D NMR spectroscopic data of1displayed similarity with the data of asperfloroid [13],an ergostane steroid purified from the fungusAspergillus flocculosus16D-1 which resides in sponge,except for the substitutions at C-3,C-5,C-6 and C-7.The C-5 and C-6 are double carbons of the reference compound asperfloroid,while C-5 of1is a methine,C-6 and C-7 are methylenes.These changes were substantiated by1H-1H COSY spectrum that showed correlations of H2-4/H-5/H2-6/H2-7(Fig.2).The HMBC correlations from H2-1,H2-2,and H2-4 to C-3 atδC214.1 suggested that C-3 was a ketone carbon in1,instead of being a hydroxymethine in asperfloroid (Fig.2).Analysis of the ROESY spectrum recorded in dimethyl sulfoxide-d6allowed to determine the relative configuration,which enabled the presence of some pivotal exchangeable protons.The ROESY correlations of H-12/Me-18/OH-14 indicated that OH-14 was β orientation,and OH-12 was α orientation.Moreover,the diagnostic ROESY correlations of Me-18/H-20,Me-18/H-15β(δH1.82),Me-18/H-22,and H-15α (δH2.20)/H-16/H-17 suggested theS*andR* configurations of C-16 and C-22,respectively (Fig.2).The relative stereochemistry of both C-23 and C-24 were assigned asR* by comparing the1H and13C NMR chemicals shifts with those of asperfloroid.Based on above-mentioned analysis,the ECD calculation method was adopted to assign the absolute configuration of1.As a result,the calculated ECD of 5S,10S,12R,13S,14S,16S,17R,20S,22R,23R,24Rshowed compatible curve with that of the experimental CD spectrum (Fig.3).Therefore,the evidence presents in above texts help to establish the structure of compound1(Fig.1).

    Fig. 1 The chemical structures of compounds 1-3

    Table 1 1H NMR Spectroscopic data of compounds 1-3 (600 MHz)

    Table 2 13C NMR spectroscopic data of compounds 1-3(150 MHz)

    Cordycicadin F (2,Fig.1),a pale-yellow oil,possesses the molecular formula of C19H26O5from the ion peak atm/z335.18518 [M+H]+(calcd for C19H27O5,335.18530)in (+)-HRESIMS analysis.The1H NMR spectroscopic data of2(Table 1) showed two doublet methyl signals atδH1.14 (d,J=6.2 Hz,Me-19),1.40 (d,J=6.2 Hz,Me-1),five oxygen-connected methine protons atδH3.41 (m,H-18),3.89 (m,H-15),3.99 (dd,J=4.9,2.1 Hz,H-14),4.62 (overlapped,H-9),4.68 (m,H-2).The13C NMR along with the DEPT spectroscopic data of2(Table 2)indicated resonances for 19 carbons attributable to two CH3carbon atδC20.6 (C-1) and 22.2 (C-19),seven CH2carbons atδC26.1 (C-16),28.3 (C-17),31.6 (C-3),31.8(C-4),45.6 (C-11),45.8 (C-13),46.3 (C-8),six methines[five oxygenated ones atδC73.1 (C-18),75.8 (C-15),78.0(C-9),78.3 (C-14),and 83.0 (C-2)],one pair of double bonds atδC111.4 (C-6) and 174.1 (C-5),one ketal carbon and one ketone carbon atδC117.1 (C-12) andδC206.6 (C-7),respectively.These data of2(Tables 1 and 2)resembled those of the known compound opaliferin [14],a polyketide isolated from the entomopathogenic fungusCordycepssp.NBRC 106954.The difference between compound2and opaliferin is that the C-14 and C-18 of opaliferin are each substituted with a hydroxy group,while the C-14 and C-18 of2are attached by an ether bond.This change was reinforced by the HMBC correlations from H-18 to C-14 (Fig.2).The tetrahydrofuran moiety (C-1-C-5) was established by HMBC correlations from H-2,H2-3,and H2-4 to C-5.The presence of cyclopentanone moiety (C-6-C-10) was confirmed by the HMBC correlations of H-8,H-9 to C-6,C-7,and of H-10,H-11 to C-6.The HMBC correlation from H2-4 to C-6 and chemical shifts of C-5 (δC174.1),C-6 (δC111.4)in13C NMR spectrum suggested the existence of an enol moiety.The HMBC correlations from H2-11 to C-12,H2-13 to C-11,H2-13 to C-12,H-14 to C-12,H-15 and H-9 to C-12,together with the chemical shift of C-12 (δC117.1) indicated that C-12 was an acetal carbon which connected to C-9 and C-15 by two epoxy bonds.All of these data were highly similar to opaliferin.Furthermore,the configurations of the chiral carbons of2were consistent with those of opaliferin,which were assigned as 2R,5E,9S,10R,12R,14S,15S,18Rby biosynthetic considerations,comparison with the NMR data,and the ROESY correlations of H-14/H-15/H-18,and H-9/H-10 (Fig.2),and confirmed by ECD calculation (Fig.4).Therefore,compound2was established and was trivially named cordycicadin F.

    The molecular formula of the colorless crystals 7-hydroxybassiatin (3,Fig.1) was determined as C15H19NO4by the protonated ion peak atm/z278.13851[M+H]+(calcd for C15H20NO4,278.13868) in the HRESIMS analysis,requiring seven degrees of double bond equivalence.The proton NMR data of3(Table 1)showed three singlet methyls [two methyl protons atδH1.02 (Me-9) and 1.07 (Me-8),oneN-methyl protons atδH3.05 (Me-17)],two methine protons atδH3.10 (s,H-6) and 4.67 (t,J=4.8 Hz,H-3),one pair of methylene protons atδH3.28 (dd,J=14.2,4.8 Hz,H-10),3.37 (dd,J=14.2,4.8 Hz,H-10),one exchangeable proton atδH4.80 (s,OH-7),and five aromatic protons.The13C NMR and DEPT spectroscopic data of2(Table 2) presented carbon signals for three methyl carbons atδC24.0 (C-8),27.0 (C-9) and 32.3 (C-17),one methylene carbon atδC37.4 (C-10),seven methines [one oxygenated atδC80.4(C-6),the others atδC63.1 (C-3),128.7 (C-14),129.8(C-13),129.8 (C-15),130.7 (C-12),130.7 (C-16)],two quaternary carbons atδC72.2 (C-7) and 136.1 (C-11),and two ketone carbons atδC166.9 (C-2) and 167.5(C-5).The aforementioned data exhibited high similarity to bassiatin,which was isolated from the cultured broth of the fungusBeauνeria bassianaK-717 [15].The 2D NMR spectroscopic analysis revealed that the major structural discrepancy between3and bassiatin was that the substituted pattern of C-7.The C-7 of bassiatin is a methine while C-7 of3is attached by a hydroxy group.The difference was fully corroborated by the HMBC correlations from OH-7 to C-6,C-7 and C-9.Therefore,the absolute configuration of3was assigned as 3S,6Rby single-crystal X-ray diffraction analysis with the Flack parameter of 0.01(4) (Fig.5).Hence,the structure of compound3was established (Fig.1),and trivially named 7-hydroxybassiatin.

    Fig. 2 Key 2D NMR correlations of compounds 1-3

    Fig. 3 Experimental CD and calculated ECD of 1

    Fig. 4 Experimental CD and calculated ECD of 2

    The compounds isolated were tested for their cytotoxicity toward A549 cell line (human lung cancer),and the anti-inflammatory activity in murine RAW264.7 macrophages by evaluating the production of nitric oxide(NO).However,the results suggested that they were devoid of any activity.

    3 Conclusion

    Fig. 5 X-ray ORTEP drawing of compound 3

    Three new compounds isolated from the cultures of the entomopathogenic fungusC.cicadaeJXCH1.The isolates in this study were subjected to screening their inhibition against the proliferation of the A549,a human lung cancer cell line,and the production of nitric oxide in murine macrophages RAW264.7.Although they were devoid of any activity in the biological activity screening model used in this study,but these compounds may play important or untapped roles during the process of fungal invasion into the insect hosts.Besides,this study extends the diversity of secondary metabolites in the fungusC.cicadae,which also promotes the understanding of chemical basis of traditional Chinese medicine.In subsequent experiments,we also hope to find molecules with novel skeleton or better activity fromC.cicadaeto provide more lead compounds for drug research and development.

    4 Experimental section

    4.1 General experimental procedures

    The laboratory instruments used in this study are as same as those reported in the literature [16-18].

    4.2 Fungal material

    A flower-like cicadae larva-fungi complex was collected in Dajueshan Natural Reserve,Fuzhou,Jiangxi Province,China,in June 2018.The “flower” part of the complex was wash by disinfectant distilled water for three times.The inner part of the “flower” which contained powder-like spores was transferred onto the potatodextrose-agar (PDA) plate supplemented with ampicillin and streptomycin.The fungus was purified by several times of efforts,and it was identified by sequencing the internal transcribed spacer region,which was amplified from the genomic DNA of this fungus with the primers ITS1 and ITS4.As a result,the sequencing data (Gen-Bank Accession No.OP591391) suggest that the fungus wasC.cicadae.A voucher specimen of the cicadae larvafungi complex and the fungus (JXCH1) was kept by the Mushroom Bioactive Natural Products Research Group of South-Central Minzu University.

    The fresh strain ofC.cicadaeJXCH1 was grew on PDA medium for seven days (25 ℃).Then the plate agar was cut into small cubes to inoculate 200 Erlenmeyer flasks (500 mL),each containing 250 mL of culture medium which consists of 5 g glucose,1.5 g pork peptone,5 g yeast extract,0.5 g KH2PO4,0.5 g MgSO4per liter.The flasks were cultured on dark shakers at 25 °C and 160 rpm for 25 days.

    4.3 Extraction and isolation

    Based on previous experience on handling the cultures of this fungus [3],50 L of methanol was added into the cultures and soaked for more than 3 days in room temperature,followed by centrifugation to break apart the mycelia and broth.The liquid phase was evaporated in vacuo to 3 L,and it was further partitioned with ethyl acetate (EtOAc) (3 × 3 L).The EtOAc layer was gathered and concentrated to give the extract A (20.2 g).The mycelia part was soaked in 5 L MeOH (3 × 3 days).The MeOH extract was combined and removed in reduced pressure,then dissolved in water and partitioned against EtOAc to give the extract B (33.3 g).The total crude extract (A+B,53.5 g) was fractionated by MPLC with a stepwise gradient of MeOH-H2O (20:80-100:0)to afford fifteen fractions (A-O).

    Fraction I was subjected to a size-exclusion CC(Sephadex LH-20) eluting with acetone and yielded ten subfractions (I1-I10).Subfraction I2 further separated by prep-HPLC (H2O-MeCN: 75:25-50:50,25 min,4 mL min-1) to gain six subfractions (I2a-I2f).Subfraction I2b was separated into three subfractions (I2b1-I2b3)by prep-HPLC (H2O-MeOH: 60:40-40:60,30 min,3 mL min-1).Subfraction I2b1 was purified on prep-HPLC (H2O-MeCN: 69:31-57:43,15 min,4 mL min-1)to yield compounds3(tR=14.5 min,3.9 mg).

    Fractions K-M were combined and subjected to a size-exclusion CC (Sephadex LH-20) eluting with acetone and yielded fifteen subfractions (K1-K15).Compounds1(tR=23.26 min,2.4 mg) and2(tR=19.55 min,3.0 mg) were isolated from subfraction K6 by prep-HPLC (H2O-MeCN: 70:30-46:54,30 min,4 mL min-1).

    4.4 Characterization data

    4.4.1 Cordycicadione (1)

    Pale-yellow oil;[α]23.0D +46.9 (c0.05,MeOH);UV(MeOH) λmax(logε) 245.0 (3.50) nm;1H NMR(600 MHz,CD3OD and DMSO-d6) data,see Table 1,13C NMR (150 MHz,CD3OD and DMSO-d6) data,see Table 2;HRESIMSm/z497.28726 [M+Na]+(calcd for C28H42O6Na,497.28736).

    4.4.2 Cordycicadin F (2)

    4.4.3 7-Hydroxybassiatin (3)

    4.5 Computational details

    The Gaussian 16 package was used as the software for all the calculation jobs [19].Firstly,conformational analyses of the candidate molecules were searched at MMFF94s force field.The returned conformers which occupy more than 1% proportion were optimized by the density functional theory (DFT) method at the B3LYP/6-31G(d) level.The optimized conformers were subjected to electronic CD calculations.The method used in ECD calculation was time-dependent DFT (TD-DFT) at B3LYP/6-31G(d,p) level,the solvent model was IEFPCM model in MeOH (Additional file 1).The ECD calculation results were processed by the SpecDis software (v1.71)[20],the calculated ECD and experimental CD curves were plotted in the Microsoft Office Excel program.

    4.6 Single-crystal X-ray diffraction data of 3

    A colorless block-like sample with the molecular formula of C15H19NO4,M=277.31,approximate dimensions 0.228 mm × 0.247 mm × 0.322 mm,was used for the X-ray crystallographic analysis on the BRUKER D8 QUEST diffractometer.The integration of the data using an orthorhombic unit cell yielded a total of 20,646 reflections to a maximumθangle of 79.51° (0.78 ? resolution),of which 3218 were independent (average redundancy 6.416,completeness=99.4%,Rint=3.45%,Rsig=2.93%) and 3160 (98.20%) were greater than 2σ(F2).The final cell constants ofa=6.7753(4) ?,b=12.8266(8)?,c=17.1279(11) ?,α=90.00°,β=90.00°,γ=90.00°,volume=1488.48(16) ?3,T=273(2) K.Data were corrected for absorption effects using the Multi-Scan method (SADABS).The structure was solved and refined using the Bruker SHELXTL Software Package,using the space groupP212121,with Z=4,μ(CuKα)=1.54178.The final anisotropic full-matrix least-squares refinement onF2with 189 variables converged at R1=2.87%,for the observed data andwR2=7.66% for all data.The goodness-of-fit was 1.053.The absolute configuration was determined by the Flack parameter=0.01(4),which was determined using 1303 quotients [(I+)-(I-)]/[(I+)+(I-)][21].The crystallographic data have been deposited to the Cambridge Crystallographic Data Centre (CCDC 2250285).

    4.7 Biological activity assays

    4.7.1 Anti-proliferative assay

    The anti-proliferative of the isolated compounds in this study against the five cancer cell lines were conducted by the same protocol in Ref.[22].

    4.7.2 Anti-inflammatory activity assay

    The anti-inflammatory of the isolated compounds against the murine RAW264.7 macrophages were conducted by the same protocol in Ref.[22].

    Supplementary Information

    The online version contains supplementary material available at https:// doi.org/ 10.1007/ s13659-023-00410-2.

    Additional file 1: Figure S1.1H NMR spectrum of compound1(600 MHz,CD3OD).Figure S2.13C and DEPT NMR spectra of compound1(150 MHz,CD3OD).Figure S3.HSQC spectrum of compound1(CD3OD).Figure S4.1H-1H COSY spectrum of compound1(CD3OD).Figure S5.HMBC spectrum of1(CD3OD).Figure S6.HRESIMS report of compound1.Figure S7.1H NMR spectrum of compound1(600 MHz,DMSO-d6).Figure S8.13C and DEPT NMR spectra of compound1(150 MHz,DMSO-d6).Figure S9.HSQC spectrum of compound1(DMSO-d6).Figure S10.1H-1H COSY spectrum of compound1(DMSO-d6).Figure S11.HMBC spectrum of compound1(DMSO-d6).Figure S12.ROESY spectrum of compound1(DMSO-d6).Figure S13.1H NMR spectrum of compound2(600 MHz,CD3OD).Figure S14.13C and DEPT NMR spectra of compound2(150 MHz,CD3OD).Figure S15.HSQC spectrum of compound2(CD3OD).Figure S16.1H-1H COSY spectrum of compound2(CD3OD).Figure S17.HMBC spectrum of compound2(CD3OD).Figure S18.ROESY spectrum of compound2(CD3OD).Figure S19.HRESIMS report of compound2.Figure S20.1H NMR spectrum of compound3(600 MHz,CD3COCD3).Figure S21.13C and DEPT NMR spectra of compound3(150 MHz,CD3COCD3).Figure S22.HSQC spectrum of compound3(CD3COCD3).Figure S23.1H-1H COSY spectrum of compound3(CD3COCD3).Figure S24.HMBC spectrum of compound3(CD3COCD3).Figure S25.ROESY spectrum of compound3(CD3COCD3).Figure S26.HRESIMS report of compound3.Figure S27.CD spectrum of1.Figure S28.CD spectrum of compound2.Figure S29.CD spectrum of compound3.ECD calculation details of compounds1and2.

    Acknowledgements

    This work was financially supported by the National Natural Science Foundation of China (grant number 81903512) and the Fundamental Research Funds for the Central Universities,South-Central Minzu University (Grant Number CPT22033).The authors thank the Analytical & Measuring Center,School of Pharmaceutical Sciences,South-Central Minzu University for the spectra test.

    Author contributions

    KZ and HPC designed the experiment;JF and PL performed the isolation and identification of all the compounds;PL measured the NMR spectra;JF wrote the original manuscript;PL,KZ,and HPC revised the manuscript.

    Availability of data and materials

    All data generated and analyzed during this study are included in this published article and its Additional file 1.

    Declarations

    Competing interests

    The authors declare that there is no competing interests or personal relationships that could affect the work reported in this article.

    Received:29 August 2023

    Accepted:11 October 2023

    成人无遮挡网站| 日韩亚洲欧美综合| 超碰av人人做人人爽久久| xxx大片免费视频| 日本色播在线视频| 亚洲av日韩在线播放| 欧美不卡视频在线免费观看| 亚洲精品一二三| 亚洲av成人精品一区久久| 欧美成人午夜免费资源| 亚洲欧美一区二区三区国产| 午夜精品在线福利| 在线观看免费高清a一片| 超碰av人人做人人爽久久| 国产亚洲最大av| 免费电影在线观看免费观看| 日本色播在线视频| av在线蜜桃| 国产老妇女一区| 亚洲经典国产精华液单| 欧美日本视频| 久久这里有精品视频免费| 黄色欧美视频在线观看| 亚洲国产精品国产精品| 婷婷色av中文字幕| 免费在线观看成人毛片| 熟女人妻精品中文字幕| 久久午夜福利片| 亚洲aⅴ乱码一区二区在线播放| 狂野欧美激情性xxxx在线观看| 精品久久国产蜜桃| 亚洲欧洲日产国产| 亚洲精品国产成人久久av| 久久久精品94久久精品| 国产男人的电影天堂91| 少妇丰满av| 亚洲国产精品专区欧美| 18禁在线播放成人免费| 婷婷六月久久综合丁香| 五月伊人婷婷丁香| 日本与韩国留学比较| 男女啪啪激烈高潮av片| av在线蜜桃| av网站免费在线观看视频 | 80岁老熟妇乱子伦牲交| 国产免费又黄又爽又色| 老女人水多毛片| 久久精品国产亚洲av天美| 精品99又大又爽又粗少妇毛片| 国产精品久久久久久久久免| 成人鲁丝片一二三区免费| 秋霞在线观看毛片| 欧美xxxx性猛交bbbb| 麻豆成人午夜福利视频| 日本一二三区视频观看| 亚洲aⅴ乱码一区二区在线播放| 啦啦啦中文免费视频观看日本| 舔av片在线| 777米奇影视久久| 最近最新中文字幕免费大全7| 男人舔女人下体高潮全视频| 亚洲精品日韩av片在线观看| av免费观看日本| 亚洲欧美日韩无卡精品| 亚洲精品视频女| 日日啪夜夜撸| ponron亚洲| 成人毛片a级毛片在线播放| 人妻夜夜爽99麻豆av| 人体艺术视频欧美日本| 高清午夜精品一区二区三区| 欧美不卡视频在线免费观看| 国产精品.久久久| 精品酒店卫生间| 精品国内亚洲2022精品成人| 联通29元200g的流量卡| 80岁老熟妇乱子伦牲交| 日韩在线高清观看一区二区三区| 亚洲精华国产精华液的使用体验| 国产欧美日韩精品一区二区| 99久久精品一区二区三区| 国产在线男女| 大香蕉久久网| 不卡视频在线观看欧美| 人妻制服诱惑在线中文字幕| av在线亚洲专区| 91狼人影院| 久久亚洲国产成人精品v| 国产大屁股一区二区在线视频| 国产大屁股一区二区在线视频| 免费少妇av软件| 久久精品熟女亚洲av麻豆精品 | 黄色配什么色好看| 亚洲av中文字字幕乱码综合| 久久久欧美国产精品| 在线播放无遮挡| 日日摸夜夜添夜夜添av毛片| 国产精品99久久久久久久久| 欧美高清成人免费视频www| 午夜免费观看性视频| 少妇的逼水好多| 国产成人午夜福利电影在线观看| 极品少妇高潮喷水抽搐| 精品久久久久久成人av| 国产乱人偷精品视频| 中文字幕制服av| 亚洲av在线观看美女高潮| 亚洲不卡免费看| 亚洲av不卡在线观看| 熟妇人妻不卡中文字幕| 免费看日本二区| 18禁动态无遮挡网站| 高清欧美精品videossex| 99久国产av精品国产电影| 亚洲欧美中文字幕日韩二区| 少妇人妻精品综合一区二区| 少妇熟女欧美另类| 青春草国产在线视频| 成年免费大片在线观看| 蜜桃亚洲精品一区二区三区| 国产亚洲av片在线观看秒播厂 | 日本三级黄在线观看| 国产 一区 欧美 日韩| 一级爰片在线观看| 欧美变态另类bdsm刘玥| 国产淫语在线视频| 亚洲精品乱码久久久v下载方式| 韩国高清视频一区二区三区| 亚洲国产色片| 国产亚洲一区二区精品| 亚洲精品456在线播放app| 免费黄色在线免费观看| 亚洲成人一二三区av| 久久精品国产鲁丝片午夜精品| 国产高清三级在线| 国产精品不卡视频一区二区| 99久久九九国产精品国产免费| 在线a可以看的网站| 边亲边吃奶的免费视频| 91av网一区二区| xxx大片免费视频| 日韩强制内射视频| 欧美成人一区二区免费高清观看| 精品人妻熟女av久视频| 亚洲av不卡在线观看| 免费观看精品视频网站| 中文资源天堂在线| 九九在线视频观看精品| 天天躁夜夜躁狠狠久久av| 久久精品夜夜夜夜夜久久蜜豆| 久久国产乱子免费精品| 丰满人妻一区二区三区视频av| 久久久国产一区二区| 国产综合精华液| videossex国产| 少妇熟女欧美另类| 色尼玛亚洲综合影院| 国产综合懂色| 色5月婷婷丁香| 最近中文字幕2019免费版| 2022亚洲国产成人精品| 99久久中文字幕三级久久日本| 久久久国产一区二区| 综合色丁香网| 18禁裸乳无遮挡免费网站照片| 少妇熟女欧美另类| 中文在线观看免费www的网站| 国产亚洲av片在线观看秒播厂 | 波野结衣二区三区在线| 亚洲av中文av极速乱| www.色视频.com| 国产精品爽爽va在线观看网站| 高清av免费在线| 日韩av在线大香蕉| 日韩 亚洲 欧美在线| 欧美高清性xxxxhd video| 可以在线观看毛片的网站| 女人久久www免费人成看片| 黄片无遮挡物在线观看| 亚洲最大成人av| 亚洲人与动物交配视频| 别揉我奶头 嗯啊视频| 久久久a久久爽久久v久久| 亚洲最大成人中文| 午夜福利网站1000一区二区三区| 免费观看a级毛片全部| 久久人人爽人人爽人人片va| 美女内射精品一级片tv| 久久精品国产亚洲网站| 精品久久久噜噜| 不卡视频在线观看欧美| 日韩人妻高清精品专区| 日韩不卡一区二区三区视频在线| 高清av免费在线| 毛片一级片免费看久久久久| 午夜激情福利司机影院| 老女人水多毛片| 免费看光身美女| 国产高潮美女av| 日产精品乱码卡一卡2卡三| 99久久精品热视频| 乱人视频在线观看| 一区二区三区乱码不卡18| 狂野欧美白嫩少妇大欣赏| 久久久久久久亚洲中文字幕| 国产午夜精品论理片| 欧美区成人在线视频| 少妇被粗大猛烈的视频| 综合色av麻豆| 大片免费播放器 马上看| 韩国av在线不卡| 老女人水多毛片| 免费看光身美女| 蜜臀久久99精品久久宅男| 亚洲自偷自拍三级| 欧美成人a在线观看| 国产亚洲一区二区精品| 国产成人a区在线观看| 女人久久www免费人成看片| 免费高清在线观看视频在线观看| 99热网站在线观看| 有码 亚洲区| 欧美日韩国产mv在线观看视频 | 麻豆久久精品国产亚洲av| 日韩中字成人| 国产视频首页在线观看| 日韩欧美精品免费久久| 国产v大片淫在线免费观看| a级一级毛片免费在线观看| 精品国产一区二区三区久久久樱花 | 成年女人在线观看亚洲视频 | 日韩一区二区视频免费看| 国产黄片美女视频| 国产 一区精品| 综合色av麻豆| 成年人午夜在线观看视频 | 国产成人午夜福利电影在线观看| 亚洲欧美精品自产自拍| 亚洲av日韩在线播放| 日韩精品有码人妻一区| 中国国产av一级| 国产伦精品一区二区三区视频9| 91久久精品国产一区二区三区| 一区二区三区乱码不卡18| 亚洲av不卡在线观看| kizo精华| 成人亚洲欧美一区二区av| 又大又黄又爽视频免费| 久久6这里有精品| 男人狂女人下面高潮的视频| 日韩在线高清观看一区二区三区| 男女边摸边吃奶| 亚洲成人精品中文字幕电影| 欧美精品国产亚洲| 嫩草影院精品99| 乱系列少妇在线播放| 青春草视频在线免费观看| 亚洲欧美精品自产自拍| 精品久久久久久电影网| 亚洲欧美日韩卡通动漫| 蜜桃亚洲精品一区二区三区| 99re6热这里在线精品视频| 国产熟女欧美一区二区| 亚洲国产精品sss在线观看| 精品久久国产蜜桃| 午夜精品在线福利| 男女那种视频在线观看| 欧美日韩综合久久久久久| 国产精品av视频在线免费观看| 日本免费a在线| 韩国高清视频一区二区三区| 国产成人一区二区在线| 边亲边吃奶的免费视频| 免费不卡的大黄色大毛片视频在线观看 | 一个人观看的视频www高清免费观看| 一级黄片播放器| 尾随美女入室| 国产乱人视频| av一本久久久久| 简卡轻食公司| 亚洲av成人精品一区久久| 天堂中文最新版在线下载 | 精品熟女少妇av免费看| 免费不卡的大黄色大毛片视频在线观看 | 天堂俺去俺来也www色官网 | 女人久久www免费人成看片| 国产黄色免费在线视频| 日韩av免费高清视频| 久久久色成人| 一级爰片在线观看| 日本免费在线观看一区| 亚洲av.av天堂| 久久97久久精品| 简卡轻食公司| 亚洲在线观看片| 国产成人精品久久久久久| 亚洲精品乱久久久久久| 亚洲最大成人手机在线| 天堂网av新在线| ponron亚洲| 男女边吃奶边做爰视频| 三级毛片av免费| 国产亚洲精品av在线| 午夜福利在线在线| 国产精品女同一区二区软件| 建设人人有责人人尽责人人享有的 | 日韩欧美一区视频在线观看 | 男女边摸边吃奶| 97超视频在线观看视频| 久久午夜福利片| 午夜久久久久精精品| 国产午夜福利久久久久久| 美女黄网站色视频| 亚洲最大成人手机在线| 真实男女啪啪啪动态图| 精品不卡国产一区二区三区| 亚洲怡红院男人天堂| 天美传媒精品一区二区| 久久精品久久久久久久性| 人妻一区二区av| 久久国产乱子免费精品| videossex国产| 久久久久久久亚洲中文字幕| 欧美日韩一区二区视频在线观看视频在线 | 久热久热在线精品观看| 日本熟妇午夜| 在线观看一区二区三区| 18禁在线无遮挡免费观看视频| 国产高清三级在线| 成年女人看的毛片在线观看| 国产精品一及| 欧美日韩国产mv在线观看视频 | 日产精品乱码卡一卡2卡三| 亚洲国产欧美人成| 少妇人妻精品综合一区二区| 80岁老熟妇乱子伦牲交| 亚洲自拍偷在线| 91精品伊人久久大香线蕉| 伊人久久国产一区二区| 高清午夜精品一区二区三区| 97超视频在线观看视频| 亚洲成人久久爱视频| 麻豆成人av视频| 男人舔女人下体高潮全视频| 国语对白做爰xxxⅹ性视频网站| 亚洲三级黄色毛片| 日韩一本色道免费dvd| 激情五月婷婷亚洲| 亚洲精品成人av观看孕妇| 免费av观看视频| 91aial.com中文字幕在线观看| 女人十人毛片免费观看3o分钟| 亚洲av在线观看美女高潮| 亚洲国产高清在线一区二区三| 男女边吃奶边做爰视频| 非洲黑人性xxxx精品又粗又长| 内射极品少妇av片p| 精品国产三级普通话版| 国产亚洲91精品色在线| 嫩草影院新地址| 男人狂女人下面高潮的视频| 国产毛片a区久久久久| 国产精品av视频在线免费观看| 在现免费观看毛片| 久久久欧美国产精品| 26uuu在线亚洲综合色| 亚洲精品国产av成人精品| 国产成人a区在线观看| 亚洲精品自拍成人| 免费观看性生交大片5| 日日啪夜夜爽| 久久热精品热| 一个人免费在线观看电影| 九九爱精品视频在线观看| 大陆偷拍与自拍| 一边亲一边摸免费视频| 青春草国产在线视频| 伦理电影大哥的女人| 国产成人91sexporn| 国产黄色视频一区二区在线观看| 国产精品99久久久久久久久| 少妇丰满av| 午夜福利视频1000在线观看| 欧美高清成人免费视频www| 深夜a级毛片| 午夜老司机福利剧场| 日韩一区二区视频免费看| 欧美日韩在线观看h| 色播亚洲综合网| 一级爰片在线观看| 午夜福利在线观看免费完整高清在| 狂野欧美激情性xxxx在线观看| 99热这里只有精品一区| 欧美日韩一区二区视频在线观看视频在线 | 少妇熟女欧美另类| 人妻制服诱惑在线中文字幕| 亚洲伊人久久精品综合| 精品一区二区三区视频在线| 国产精品不卡视频一区二区| 国产精品久久久久久精品电影| 亚洲精品日本国产第一区| 亚洲国产日韩欧美精品在线观看| 亚洲综合色惰| 日韩一区二区三区影片| 网址你懂的国产日韩在线| 亚洲av福利一区| 成人鲁丝片一二三区免费| 人体艺术视频欧美日本| 夫妻午夜视频| 午夜福利成人在线免费观看| 免费观看在线日韩| 日本免费a在线| videos熟女内射| 国内少妇人妻偷人精品xxx网站| 校园人妻丝袜中文字幕| 久久久a久久爽久久v久久| 自拍偷自拍亚洲精品老妇| 两个人视频免费观看高清| 国产在视频线在精品| 国产极品天堂在线| 性色avwww在线观看| 国产一区有黄有色的免费视频 | 亚洲伊人久久精品综合| 亚洲国产色片| 黄色配什么色好看| 国产 亚洲一区二区三区 | 18禁裸乳无遮挡免费网站照片| 少妇的逼水好多| 草草在线视频免费看| 简卡轻食公司| 成人亚洲精品一区在线观看 | av女优亚洲男人天堂| 一个人看视频在线观看www免费| av免费在线看不卡| 麻豆乱淫一区二区| h日本视频在线播放| 久久久精品欧美日韩精品| 极品少妇高潮喷水抽搐| 亚洲av国产av综合av卡| 69人妻影院| 日韩欧美一区视频在线观看 | 3wmmmm亚洲av在线观看| 男插女下体视频免费在线播放| 日韩电影二区| 久久久久精品久久久久真实原创| 在线 av 中文字幕| 久久久久国产网址| or卡值多少钱| 国产色爽女视频免费观看| 亚洲自拍偷在线| 亚洲四区av| 黄色一级大片看看| 精品久久国产蜜桃| av线在线观看网站| 久久久午夜欧美精品| 亚洲综合精品二区| 99re6热这里在线精品视频| 亚洲精品乱久久久久久| 晚上一个人看的免费电影| 国产精品综合久久久久久久免费| 六月丁香七月| 日日摸夜夜添夜夜添av毛片| 七月丁香在线播放| 美女高潮的动态| 中文天堂在线官网| 亚洲婷婷狠狠爱综合网| 久久久久久久久中文| 美女黄网站色视频| 99久国产av精品| 亚洲第一区二区三区不卡| 深爱激情五月婷婷| 久99久视频精品免费| 麻豆成人av视频| 菩萨蛮人人尽说江南好唐韦庄| 好男人在线观看高清免费视频| 亚洲国产日韩欧美精品在线观看| 久久精品国产自在天天线| 午夜福利视频精品| 久久亚洲国产成人精品v| 国产成人91sexporn| 三级毛片av免费| 国产精品一区二区在线观看99 | 国产黄a三级三级三级人| 自拍偷自拍亚洲精品老妇| 亚洲内射少妇av| 2021少妇久久久久久久久久久| 人人妻人人看人人澡| av在线观看视频网站免费| 久久久久久国产a免费观看| 99热全是精品| 日本-黄色视频高清免费观看| 男女边吃奶边做爰视频| 色尼玛亚洲综合影院| 国产永久视频网站| 日韩av在线大香蕉| 日韩视频在线欧美| 日韩欧美精品v在线| 一区二区三区乱码不卡18| 18+在线观看网站| 男女啪啪激烈高潮av片| 精品久久久精品久久久| 亚洲国产日韩欧美精品在线观看| 免费播放大片免费观看视频在线观看| 久久久色成人| 国产毛片a区久久久久| 色综合站精品国产| 久久久久久久久久久丰满| 午夜福利高清视频| 久久6这里有精品| 久久精品熟女亚洲av麻豆精品 | 神马国产精品三级电影在线观看| 两个人视频免费观看高清| 天堂网av新在线| 又大又黄又爽视频免费| 在线免费观看不下载黄p国产| 亚洲成人av在线免费| 欧美性感艳星| 亚洲国产精品国产精品| 男人舔奶头视频| 天天躁日日操中文字幕| 波多野结衣巨乳人妻| 日韩人妻高清精品专区| 69av精品久久久久久| 可以在线观看毛片的网站| 精品久久久久久电影网| 日韩一区二区三区影片| 午夜福利网站1000一区二区三区| 免费观看无遮挡的男女| 91精品一卡2卡3卡4卡| 亚洲精品色激情综合| 亚洲美女搞黄在线观看| 亚洲精品视频女| 欧美成人一区二区免费高清观看| 亚洲人成网站在线观看播放| 婷婷六月久久综合丁香| 乱系列少妇在线播放| 亚洲成色77777| 美女xxoo啪啪120秒动态图| 最近最新中文字幕大全电影3| 97人妻精品一区二区三区麻豆| 老司机影院成人| 黄色日韩在线| 一二三四中文在线观看免费高清| 亚洲精品一二三| 亚洲av电影在线观看一区二区三区 | 久久97久久精品| 亚洲无线观看免费| 中国国产av一级| 亚洲欧美成人精品一区二区| 国产成人福利小说| 一级毛片aaaaaa免费看小| 美女黄网站色视频| 99久久人妻综合| 五月伊人婷婷丁香| 女人被狂操c到高潮| 日韩av不卡免费在线播放| 在线免费十八禁| 三级国产精品欧美在线观看| 午夜福利视频1000在线观看| 伦理电影大哥的女人| 亚洲欧美日韩卡通动漫| 国产成人freesex在线| 国产黄片视频在线免费观看| 欧美日本视频| 最近视频中文字幕2019在线8| kizo精华| 欧美性感艳星| 尾随美女入室| 舔av片在线| 亚洲在久久综合| 国产综合精华液| 夫妻午夜视频| 青春草视频在线免费观看| 国产亚洲午夜精品一区二区久久 | videos熟女内射| 一级毛片 在线播放| 久久久a久久爽久久v久久| 日韩大片免费观看网站| 国产高清三级在线| 亚洲av成人av| 啦啦啦韩国在线观看视频| 亚洲国产欧美人成| 国产午夜精品一二区理论片| 亚洲精品国产成人久久av| 亚洲精品一二三| 一夜夜www| 99久久九九国产精品国产免费| 一区二区三区免费毛片| 三级男女做爰猛烈吃奶摸视频| 女人十人毛片免费观看3o分钟| 大香蕉久久网| 国产高清有码在线观看视频| 国产精品99久久久久久久久| 两个人视频免费观看高清| 欧美不卡视频在线免费观看| 久久午夜福利片| 国产熟女欧美一区二区| 久久久色成人| 中文字幕av成人在线电影| 久久人人爽人人片av| 深夜a级毛片| 中文字幕免费在线视频6| 国产一区二区在线观看日韩| 九九爱精品视频在线观看| 国产成人免费观看mmmm| 黄色欧美视频在线观看| 男女边吃奶边做爰视频| 91狼人影院| 国产av国产精品国产| 在线a可以看的网站| 国产亚洲精品久久久com| 国产一级毛片七仙女欲春2| 日韩 亚洲 欧美在线| 久久99精品国语久久久| 国产精品久久久久久久久免|