• <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 ?

    大分子雜質(zhì)是雙黃連注射劑導(dǎo)致類過(guò)敏反應(yīng)的重要原因*

    2022-11-11 07:27:32萬(wàn)緒蓮韓依倫李律宇黎勇坤段為鋼
    關(guān)鍵詞:雙黃連大分子注射劑

    萬(wàn)緒蓮,夏 恒,韓依倫,李律宇,陳 歡,黎勇坤,段為鋼△

    (1.云南中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院,云南 昆明 650500;2.云南中醫(yī)藥大學(xué)中藥學(xué)院,云南 昆明 650500;3.云南中醫(yī)藥大學(xué)第三附屬醫(yī)院,云南 昆明 650500)

    Injection is a necessary dosage form used on emergent occasions or when drugs are with poor bioavailability.Drugs injected through subcutaneous tissue,musculartissue,orvein usually absorbed quickly and onset much faster than those via oral administration,though its delivery way is dangerous.Injections often cause more and/or heavier adverse reactions than other dosage forms,such as anaphylactoid reactions which mixed with anaphylactic reactions[1-2].Most anaphylactoid reactions usually happen less than 30 mins after administration,including itch,erythema,cough,hypotension,dyspnea,and shock;similar to those of anaphylactic reactions(allergy)[3].However,different from anaphylactic reaction,anaphylactoid reaction can be ignited by the first exposure to injections,while immunoglobin E(IgE)is not required to mediate such a reaction[3].Therefore,anaphylactoid reactions are called pseudo-allergic reactions[3-4].

    Although the mechanisms of anaphylactic reactions are largely illustrated,those of anaphylactoid reactions are still elusive.As for anaphylactoid reactions,the substances igniting,the targets mediating,and the pathways participating in the reaction are not fully clear.It has been reported that substances with small molecular weight such as compound 48/80(C48/80)can ignite the reaction,while the reaction is likely mediated by a Mas related G-protein coupled receptor(MRGPRX2)[3-5].The receptor proved to be an orphan receptor,is highly expressed in mast cells.The activation of the receptor triggers mast cell degranulation.The released substances,including histamine,will activate their downstream effectors and trigger the anaphylactoid reactions.Therefore,most of the studies on anaphylactoid reaction focused on small molecules[6].

    However,almost all injections can ignite anaphylactoid reactions as contrasted with their oral prepara tions.It seems that substances other than small molecules could also play a role in anaphylactoid reactions.For example,Shuanghuanglian injection(SHL),a traditional Chinese medicine injection made from the flower of Lonicera japonica,the root of Scutellaria baicalensis,and the fruit of Forsytnia suspensa,is widely used against virus infection and inflammation,without obvious anaphylactoid reactions[7-11].Three most frequent symptoms were in skin and mucosa,digestive system,and body temperature center[12].On the contrary,the similar reactions of its oral preparation with the same formula were seldom documented.The evidence suggests that the substances igniting anaphylactoid reactions can be kept out by walls of the gastrointestinal tract.Considering that macromolecular substances into blood flow can be prevented by the gastrointestinal tract[13],it can be suggested that macromo-lecular substances are at least partially responsible for the anaphylactoid reactions[14-15].

    As mentioned above,C48/80 is a widely used tool reagent in studying anaphylactoid reaction[5,16].The agent is produced from N-methyl-p-methoxy phenylethylamine and formaldehyde.According to its synthesis route,C48/80 is a mixture of polymers with a theoretical molecular weight of 629.1 Da,and the agent is possible to contain macromolecular substances.Unfortunately,the macromolecules in the substance have not been taken seriously.

    Though a study in 1976 reported that contaminated macromolecules may cause anaphylactoid reaction[17],the adverse reaction was yet ignored by pharmaceutical industries.Due to techniques of removing macromolecules are not widely introduced,all injections theoretically contain macromolecular impurities which may come from raw materials,contamination and excipients,as well as C48/80.Does macromolecular substances play an important part in anaphylactoid reactions?The main aim of the study is to illustrate that C48/80 and SHL really contain macromolecular impurities,and they play an important role in triggering anaphylactoid reactions.

    1 Materials and Methods

    1.1 Materials C48/80 was manufactured by Sigma-Aldrich.SHL was manufactured by Harbin Zhenbaodao Pharmaceutical Co.LTD(Harbin,China)and approved by China Food and Drug Administration(cFDA).Normal saline for injection(NS)was manufactured by Chenxin Pharmaceutical Co.LTD (Jining,China).Enzyme-linked immunosorbent assay(ELISA)kits for Guinea pig soluble complement terminal complex Sc5b-9 (Sc5b-9)(Lot.MM-8751501),complement factor B (CFb)(Lot.MM-8750901),mannose binding lectin(MBL)(Lot.MM-8751301),bradykinin(BK)(Lot.MM-8751801),β-hexosaminidase(β-HEX)(Lot.MM-196101),lysozyme (LZM)(Lot.MM-8752201),and histamine(HIS)(Lot.MM-033501)were manufactured by Jiangsu Enzyme industry Co.LTD(Yancheng,China).Cromolyn sodium (Lot.D16-J12G137501)and Evans Blue(Lot.L04D11 J133367)were manufactured by Shanghai Yuanye Biotechnology Co.LTD(Shanghai,China).Hematoxylin-eosin(HE)staining kits were manufactured by Boster Biological Technology Co.LTD(Wuhan,China).

    Guinea pigs weighing 250~350 g and female Sprague-Dawley(SD)rats weighing 200 g were provided by Kunming Medical University.Animals were raised at 22℃temperature,at 45%~55% humiditycontrolled conditions,and under natural light.

    A multiple microplate reader of K6600A was manufactured by Beijing Kai’ao Technology Development Co.Ltd (Beijing,China).A lyophilizer of FDU-1100 was manufactured by EYELA(Tokyo,Japan).Ultra-filters of 10 kDa (1808013VS)were manufactured by Sartorius AG(Hamburg,Germany).Ultrapure water was obtained from a Milli-Q water purification system(EMD Millipore Group,Darmstadt,Germany).Other instruments or reagents used in the present study,if not mentioned,were made in China.

    1.2 Ethical approval and consent to participate Animal experiments were approved by the Animal Care and Use Committee of Jiangsu Provincial Institute of Materia Medica,Nanjing Tech University,Nanjing China(IACUC-20170830-02),and all the animals were treated under Guidelines for Ethical Review of Laboratory Animal Welfare of China(GB/T 35892-2018).Animal experiments were in accordance with ARRIVE guidelines(https://arriveguidelines.org).

    1.3 Macromolecular Substance Preparation The original C48/80 was dissolved in water for injection to obtain a 5%solution.The colorless solution was gradually added to the upper tube of the ultra-filter of 10 kDa.The ultra-filter was spun at 3 500 g and 4℃until more than a half volume was filtered through.Then,the solution passed through was collected as Component B containing substances with molecular weight below 10 kDa,and the solution in the upper tube was recovered by mixing with the water for injection and spun again.When the solution in the upper tube was diluted six times and ultrafiltered,the retained solution in the upper tube was collected as Component A containing substances with molecular weight above 10 kDa.

    The two components mentioned above were dried by the lyophilizer.Briefly,the solution was put in a plastic bottle of 250 mL,and frozen at-40 ℃overnight.Then,the frozen solution was put into the chamber of the lyophilizer and lyophilized for more than 48 h.Then,the bottle was weighed every 3 h,and when the total weight of the bottle did not decrease the lyophilization was ended.The net weight of the powder was calculated by subtracting the bottle weight from the total weight.The powder diluted with NS to an arranged concentration was used for subsequent studies.

    Similarly,components of the original SHL injection were prepared.Briefly,SHL was added to the upper tube of the ultra-filter of 10 kDa,and spun at 3 500 g and 4 ℃ until nine tenths of the volume was filtered through.The part retained in the upper tube was collected as component A of SHL and the part passed through was com ponent B.The obtained solutions,components A and B,were used directly without dilution or concentration[16].

    1.4 Systemic anaphylactoid reactions in guinea pigs evoked by macromolecules The original C48/80 and its components were dissolved in NS(0.75 mg/mL).Every 6 guinea pigs,including 3 male guinea pigs and 3 female guinea pigs (without pregnancy),were randomly arranged in a group to detect anaphylactoid reactions.Guinea pigs were intravenously administered with original C48/80 or its components (0.25 mg/kg).Animals administered with NS of the same volume were served as a negative control.According to Chi nese Pharmacopoeia (CP)of the 2015 edition(Book 4)[19],the symptoms appeared in 30 mins after administration were observed and their blood was immediately drawn without anticoagulation to obtain serum samples.The symptom scores were recorded according to Table 1.If an animal appeared two or more symptoms,only the higher or the highest score was used[13,16].

    Tab.1 Scores of symptoms

    Similarly,guinea pigs were intravenously administered with the original SHL injection and its components(3.4 mL/kg).Their symptoms appeared in 30 min after administration were also recorded according to Table 1 and their blood was collected 30 min after administration.

    The coagulated blood was spun at 3 000 g and 4℃for 5 min to obtain serum.SC5b-9,CFb,MBL,BK,β-HEX,LZM,and HIS in the serum were determined using ELISA kits according to protocols.

    1.5 Substances released from guinea pig leukocytes evoked by macromolecules Guinea pigs were anesthetized with urethane(1.0 g/kg)by intraperitoneal injection.Their abdomens were opened and their anticoagulated blood was drawn via the abdominal aortae.The blood was spun at 1 000 g and 4℃for 5 min to remove red blood cells,and the volume was recovered by adding NS.The suspension of 5 mL containing the whole white leukocytes was transferred to a six-well plate.When the original C48/80 or its components of 20 μg/mL were added in the suspension and mixed,the plate was kept at 37 ℃ for 30 min.The suspension of 1 mL was sampled and spun at 1 000 g and 4℃for 5 min to obtain its supernatant,and β-HEX,LZM and HIS in it were determined using ELISA kits[16].

    Similarly,the suspension of 5 mL containing the whole leukocytes was transferred to a new six-well plate,and SHL or its components of 0.5 mL were added and mixed.The plate was also kept at 37℃.Thirty minutes later,β-HEX,LZM and HIS in the supernatant of the reaction suspension were determined using ELISA kits.

    1.6 Passive cutaneous anaphylactoid(PCA)reaction evoked by macromolecules in guinea pigs Every 4 guinea pigs,including 2 male guinea pigs and 2 female guinea pigs(without pregnancy)weighing about 300 g were randomly arranged in a group.They were pretreated with cromolyn sodium(50 mg/kg)or NS of the same volume by intraperitoneal injection for 5 days.The day before the macromolecular injection,their back fur was removed with a pair of scissors.On the next day,they were intravenously injected with 1 mL Evans Blue solution(0.5%,dissolved in NS).Fifteen minutes later,the original C48/80 or its components(0.75 mg/mL)of 0.1 mL were subcutaneously injected at one point.The administration of NS of the same volume was served as a negative control.Thirty minutes later,the animals were anesthetized with ure thane(1.0 g/kg),their back skin was cut,and the bluestaining areas were photographed.The Evans Blue in the skin(4.00 cm2)was extracted using 4 mL acetonenormal saline solution (acetone∶normal saline=7 ∶3)for 24 h.The extract was spun at 3 000 g for 5 min,and the Evans Blue in the supernatant was determined by the multiplate reader at 610 nm.Similarly,PCA reaction evoked by the original SHL and its components of 0.1 mL were tested[18].

    After Evans Blue was extracted,a piece of skin around the injection point was selected.The skin fixed by the acetone solution was routinely dehydrated with ethanol and embedded in paraffin.The skin was cut at 5 μm and stained with HE staining kits.The section was scanned using a fluorescence microscope in a light mode.

    1.7 Pulmonary vascular permeability affected by macromolecules Female rats were intraperitoneally treated with 10 mg cromolyn sodium or NS of the same volume.Thirty minutes later,they were anesthetized with urethane(1.0 g/kg).Their abdomens and chests were opened,and the superior and inferior venae cava were ligated with artery clamps.NS was perfused into the right ventricle under the pressure of 1 500 mmH2O.Their abdominal aortae were cut for discharging.When the pulmonary circulation was perfused for 5 min,the color of the lungs became light and the almost colorless perfusate was discharged from their aortae.Then,the original C48/80 or its components(62.5 μg/mL)of 1 mL were perfused into the system and kept for 10 min.After that,Evans Blue solution (50 mg in 1000 mL NS)of 1 mL was perfused into the system and kept for 10 min.Finally,the system was perfused with NS for 5 min,and the lungswereremoved from theirchestsand photographed.The lung stained in blue suggests the vascular permeability[18].

    Similarly,the effect of the original SHL and its components of the same volume(without dilution)on pulmonary vascular permeability were tested.

    1.8 Statistics Values were expressed as mean±standard deviation(SD)or median±inter-quartile range(IQR).If data exhibited the normal distribution,one-way analysis of variance(ANOVA)was performed to compare means between groups.If there was a significance,a post-hoc test(equal variance)or Tamhane’s test(unequal variances)was used to compare means between every two groups.If data did not exhibit the normal distribution,a non-parametric test of Mann-Whitney test was used to compare their median values between every two groups.Statistical significance was accepted at P<0.05.

    2 Results

    2.1 Preparation of components of C48/80 and SHL injection By ultrafiltration and lyophilization,the yield of component A(above 10 kD)of C48/80 was 12.5%,and that of component B(below 10 kD)was 87.5%;suggesting that there are adequate macromolecular substances in the original C48/80.The original SHL was a transparently yellow-brown solution.Interestingly,component A of SHL was much darker than the original,while component B was much lighter,suggesting that there are colored macromolecular substances in the original SHL.

    2.2 Systemic anaphylactoid reactionsevoked in guinea pigs by macromolecules The intravenously injected original C48/80(0.25 mg/kg)caused a moderate systemic anaphylactoid reaction.Its component A caused an even more serious reaction,while its component B caused a much weaker reaction(Fig.1A).Similar phenomena can be seen in SHL experiment because its component A also caused heaver anaphylactoid reactions(Fig.1B).The results suggested that macromolecular substances are one of the main factors responsible for the anaphylactoid reactions caused either by C48/80 or SHL.

    Fig.1 Systemic anaphylactoid reactions evoked by intravenously injected macromolecules of C48/80(A)and Shuanghuanglian injection(SHL)(B)in guinea pigs(n=6)

    To understand the mechanism of the systemic anaphylactoid reactions,some factors in their serum were detected.By comparing with the negative control(NS),the original C48/80 and its component A tended to increase all the factors involved in Fig.2,though they did not significantly affect the levels of SC5b-9(Fig.2A),MBL(Fig.2C),and LZM(Fig.2F).By comparing with the original C48/80,component A significantly increased the levels of CFb (Fig.2B),BK(Fig.2D),β-HEX(Fig.2E).However,SHL is different from C48/80.By comparing the negative control,the original SHL and its component A tended to decrease the factors mentioned above.By comparing with the original SHL,component A of SHL did not increase the levels of the above factors in serum (Fig.2),either.Sometimes,the component A significantly decreased SC5b-9(Fig.2A),CFb(Fig.2B),LZM(Fig.2F),and HIS(Fig.2G).

    Fig.2 Serum factors affected by C48/80 and Shuanghuanglian injection(SHL)in guinea pigs(mean±SD,n=6)

    To investigate whether the elevation of some factors mentioned above are derived from blood leukocytes,an in vitro experiment was performed.By comparing the original C48/80,its component A did not increase the release of β-HEX (Fig.3A)and LZM(Fig.3B),but significantly increased the release of HIS(Fig.3C);partly agreeing with the results in Fig.2 and suggesting that the anaphylactoid reactions caused by macromolecular substances in C48/80 is associated with the release of histamine from leukocytes.As for SHL,its component A did not increase the release of β-HEX(Fig.3A),LZM(Fig.3B)and HIS(Fig.3C);suggesting that the anaphylactoid reactions caused by macromolecular substances of SHL is less relevant to the blood leukocytes.

    Fig.3 Substances released from blood leukocytes(mean±SD,n=3)

    2.3 PCA reaction evoked by macromolecules in guinea pigs PCA reaction is believed to be a sensitive method to detect anaphylactoid reactions.Since anaphylactic or anaphylactoid reactions increase vascular permeability,the leached Evans Blue from vessels can be used to assess the intensity of the reaction.According to the results in Fig.4,normal saline for injection resulted almost no PCA reactions.However,the original C48/80 and its component A caused strong PCA reactions,and the reaction caused by its component B was much weaker(Fig.4A and 4C).It should be noted thatcomponentA caused the strongest PCA reaction among C48/80 groups.The reactions were partly prevented by cromolyn sodium(Fig.4B and 4C).

    Fig.4 Guinea pig’s back skin was stained with Evans Blue after passive cutaneous anaphylactoid(PCA)reaction (mean±SD,n=4)

    As for SHL,similar results were shown in Fig.4D,4E and 4D.The original SHL and its components caused even stronger PCA reactions than C48/80,respectively.Among the three SHL groups,component A caused the strongest PCA reaction,and component B caused the weakest.The reactions caused by SHL can also be partly antagonized by cromolyn sodium.

    Considering PCA reactions are acute injury on skin,regeneration rather than necrosis can be observed in the area near to the injection point.Local hydropic degeneration can be observed in the areas of skin with severe PCA reactions(Fig.5).The degree of hydropic degeneration was generally consistent with the results of PCA.

    Fig.5 Hematoxylin-eosin(HE)staining on guinea pig’s skin section.The fields were near to the injection sites

    2.4 Pulmonary vascular permeability affected by macromolecules To exclude the role of blood cells that may mediate vascular permeability,pulmonary perfusion experiment was carried out in female rats,and the results were showed in Fig.6.The lungs treated by NS was only slightly stained by Evans Blue(Fig.6A).The lungs treated by the original C48/80 and its component A was even heavily stained by the dye(Fig.6B and 6C).Unsurprisingly,the lungs treated by component B of C48/80 was almost negatively stained by the dye.The increased vascular permeability caused by macromolecular substances in C48/80 can almost be fully prevented by cromolyn sodium(Fig.6 E).

    Fig.6 Rat lungs stained with Evans Blue after perfusing with normal saline for injection(A),component A of C48/80(B),component B of C48/80(C),the original C48/80(D),cromolyn and component A of C48/80(E),cromolyn and component B of C48/80(F),component A of Shuanghuanglian injection(SHL)(G),component B of SHL(H),the original SHL(I),and cromolyn and component A of SHL(J).

    The lungs treated with the component A of SHL was also heavily stained by Evans Blue(Fig.6G),while those treated with the component B was only slightly stained(Fig.6H).The elevation in vascular permeability caused by the component A was almost fully prevented by cromolyn sodium(Fig.6J).

    3 Discussion

    The present study proved that C48/80 and SHL contain macromolecular impurities.Though C48/80 is a mixture of polymers,it was used to be believed that the agent only contains small molecules.However,more than ten percent of macromolecular substances were obtained by ultrafiltration.The role of the macromolecular substances in triggering anaphylactoid reaction was not noticed before.Similarly,like other herbal injections[13],macromolecular impurities were proved in SHL.The macromolecular substances in component A of SHL were not isolated in the present study,and the component would contain both the enriched macromolecular substances and other small molecules.Theoretically,component A of SHL should have a higher solids content than the original.In fact,our previous study[20]proved that the solids content of component A was only slightly and insignificantly higher than that of the original,while that of component B was slightly and insignificantly lower.Therefore,as for SHL,the solids dose of the original SHL and its components can be considered the same,and the difference in anaphylactoid reaction should be caused by the substances with different molecular weights.In addition,similar results were reported in an excipient(polysorbate 80)for injection[18].

    Components A of C48/80 caused more serous anaphylactoid reactions than the original(Fig.1),suggesting that macromolecular substances may play an important role in triggering the reactions.The reactions might asso ciate with the activation of complement system because component A of C48/80 tended to elevate the level of SC5b-9,CFB,and MBL,though some of which did not reach a significant high level(Fig.2).Considering there was an elevation of BK in serum,the macromolecular substances of C48/80 activate bradykinin system.The anaphylactoid reaction might associate with the activation of mast cells,too,because component A of C48/80 elevated the level of β-HEX,LZM,and HIS both in vivo(Fig.2)and in vitro(Fig.3),which are most likely to be released from mast cells[21].

    Different from C48/80,the macromolecular substances in SHL caused even more serous anaphylactoid reactions than that in C48/80,but rarely elevated the levels of the serum factors(Fig.2).Further,SHL groups tended to inhibit β-HEX,LZM,and HIS in vitro(Fig.3).The possible explanation is that SHL has pharmacological effects against virus and inflammation[22],and the serum factors that can be elevated by C48/80 were inhibited by SHL.The results suggested that the mechanism of SHL could be different from that of C48/80 in triggering anaphylactoid reactions.

    Compared with anaphylactoid reaction ignited by intravenous administration,PCA reaction is a more sensitive test with visual results.PCA reaction seldomly causes animal death,which were supported by the present study.The advantage of PCA reaction is that the degree of anaphylactoid reaction can be quantitatively evaluated according to the exudation of Evans Blue.This means that the principle of the test is to evaluate vascular permeability.Due to the limited understandingofanaphylactoid reaction,intravenous investigation is still necessary for intravenous injections,since PCA reaction cannot find a lethal reaction of an intravenous injection.

    The macromolecular substances from C48/80 and SHL enhanced the vascular permeability in the pulmonary perfusion tests(Fig.6).Considering blood cells including leukocytes,were removed by perfusate,and large molecules are unlikely to cross the vascular wall into the interstitial space to activate mast cells where they reside,it can be deduced that the increase of vascular permeability is likely to be resulted from a direct effect on epithelia.

    Cromolyn sodium is a mast cell stabilizer with anti-inflammatory activity.The mechanism may be linked tointerferences with the antigen-stimulated calcium transport across the mast cell membrane,thereby inhibiting mast cell release of histamine,leukotrienes,and other substances that cause anaphylactoid reactions[23].Considering cromolyn sodium also reduces vessel permeability[24],it is understandable that the PCA reactions and the increase of vascular permeability caused by macromolecular substances from C48/80 and SHL were prevented by cromolyn sodium.

    4 Conclusion

    All the results suggested that macromolecular substances are one of the main factors responsible for anaphylactoid reactions caused both by C48/80 and SHL.The direct increase of vascular permeability participates the anaphylactoid reactions ignited by C48/80 and SHL,and mast cell degranulation is also involved in the reaction of C48/80.

    猜你喜歡
    雙黃連大分子注射劑
    國(guó)家藥監(jiān)局關(guān)于修訂輔酶Q10注射劑說(shuō)明書的公告(2022年第11號(hào))
    中老年保健(2022年4期)2022-08-22 02:58:30
    還原真實(shí)的雙黃連
    不一樣的“雙黃連”
    注射用丹參多酚酸與12種常用注射劑的配伍穩(wěn)定性
    中成藥(2018年2期)2018-05-09 07:20:11
    雙黃連系列制劑制備工藝再評(píng)價(jià)
    中成藥(2017年5期)2017-06-13 13:01:12
    半柔性大分子鏈穿越微孔行為的研究
    中藥注射劑樹(shù)脂檢查方法的改進(jìn)
    中成藥(2016年8期)2016-05-17 06:08:44
    微流控超快混合器及生物大分子折疊動(dòng)力學(xué)應(yīng)用研究進(jìn)展
    無(wú)敵上上簽
    意林(2014年10期)2014-07-28 18:17:07
    3種常用抗病毒中藥注射劑的配伍觀察
    九色亚洲精品在线播放| 赤兔流量卡办理| 黄色怎么调成土黄色| 日本一区二区免费在线视频| 美女主播在线视频| 久9热在线精品视频| 午夜av观看不卡| 国产成人精品久久二区二区91| 一二三四社区在线视频社区8| 在线观看免费视频网站a站| 精品一品国产午夜福利视频| 国产真人三级小视频在线观看| 十八禁高潮呻吟视频| 亚洲欧美激情在线| 亚洲中文字幕日韩| 国产欧美日韩一区二区三区在线| 亚洲 国产 在线| 涩涩av久久男人的天堂| 侵犯人妻中文字幕一二三四区| 精品一区在线观看国产| 桃花免费在线播放| 丰满迷人的少妇在线观看| 亚洲精品日韩在线中文字幕| 亚洲九九香蕉| 久久久久精品人妻al黑| 视频区欧美日本亚洲| 岛国毛片在线播放| 夫妻性生交免费视频一级片| 啦啦啦 在线观看视频| 五月开心婷婷网| 国产一卡二卡三卡精品| 国产精品麻豆人妻色哟哟久久| 久久久久久久国产电影| 妹子高潮喷水视频| 久久久久网色| 最近手机中文字幕大全| 午夜激情av网站| 国产亚洲av高清不卡| 97人妻天天添夜夜摸| 五月天丁香电影| 九色亚洲精品在线播放| 久久国产精品影院| 国产精品国产三级国产专区5o| 亚洲精品一卡2卡三卡4卡5卡 | 国产精品av久久久久免费| 一区二区日韩欧美中文字幕| 国产日韩欧美视频二区| 99九九在线精品视频| 99久久99久久久精品蜜桃| 在线观看免费高清a一片| 王馨瑶露胸无遮挡在线观看| 免费一级毛片在线播放高清视频 | xxxhd国产人妻xxx| 人人妻人人添人人爽欧美一区卜| xxxhd国产人妻xxx| 黄色毛片三级朝国网站| 99国产精品99久久久久| 久久国产亚洲av麻豆专区| av国产精品久久久久影院| 国产精品欧美亚洲77777| 欧美日韩视频高清一区二区三区二| 亚洲国产精品一区三区| av国产精品久久久久影院| 99九九在线精品视频| 精品少妇内射三级| av不卡在线播放| 老司机深夜福利视频在线观看 | 久久精品久久精品一区二区三区| 久久 成人 亚洲| 美国免费a级毛片| 曰老女人黄片| 亚洲专区国产一区二区| 99国产精品一区二区三区| 亚洲av片天天在线观看| 亚洲午夜精品一区,二区,三区| 久久久久久久国产电影| 热99久久久久精品小说推荐| 国产精品久久久av美女十八| 中文乱码字字幕精品一区二区三区| 天天躁日日躁夜夜躁夜夜| 美女国产高潮福利片在线看| 在线观看www视频免费| 亚洲欧美激情在线| 精品亚洲乱码少妇综合久久| 巨乳人妻的诱惑在线观看| 无遮挡黄片免费观看| 午夜福利乱码中文字幕| 国产精品久久久人人做人人爽| a级片在线免费高清观看视频| 免费在线观看完整版高清| 熟女av电影| 国产一区二区三区综合在线观看| 久久精品国产亚洲av涩爱| 性高湖久久久久久久久免费观看| 成年美女黄网站色视频大全免费| 久久精品国产亚洲av高清一级| 婷婷色麻豆天堂久久| 久久免费观看电影| 国产黄色免费在线视频| 十八禁人妻一区二区| 欧美日韩一级在线毛片| 最新在线观看一区二区三区 | 一级片'在线观看视频| 爱豆传媒免费全集在线观看| 国产成人欧美在线观看 | 国产一区二区在线观看av| 久久热在线av| 一本久久精品| 国产精品国产三级国产专区5o| 久久久久久久大尺度免费视频| 日本91视频免费播放| 国产在线视频一区二区| 亚洲七黄色美女视频| 日韩一卡2卡3卡4卡2021年| av一本久久久久| 青青草视频在线视频观看| 一本色道久久久久久精品综合| av网站在线播放免费| 91老司机精品| 日日摸夜夜添夜夜爱| 老司机在亚洲福利影院| 久久久久久久大尺度免费视频| 日本欧美视频一区| 国产淫语在线视频| 免费在线观看完整版高清| 永久免费av网站大全| 国产女主播在线喷水免费视频网站| 中文欧美无线码| 亚洲激情五月婷婷啪啪| 菩萨蛮人人尽说江南好唐韦庄| 中文字幕最新亚洲高清| av在线播放精品| av电影中文网址| 在线观看www视频免费| 国产精品99久久99久久久不卡| 99国产精品一区二区三区| 99热全是精品| 亚洲欧美精品综合一区二区三区| 色播在线永久视频| 久久午夜综合久久蜜桃| av天堂久久9| 亚洲综合色网址| 久久人人爽av亚洲精品天堂| 9色porny在线观看| 99九九在线精品视频| 国产黄色视频一区二区在线观看| www.自偷自拍.com| 国产av一区二区精品久久| 国产精品二区激情视频| 久久精品国产亚洲av高清一级| 国产精品一国产av| 国产极品粉嫩免费观看在线| 99久久综合免费| 国产精品免费视频内射| 亚洲成av片中文字幕在线观看| 久久精品人人爽人人爽视色| 99久久人妻综合| 天天躁狠狠躁夜夜躁狠狠躁| 欧美老熟妇乱子伦牲交| 久久国产精品大桥未久av| 国产熟女午夜一区二区三区| 国产高清国产精品国产三级| 夜夜骑夜夜射夜夜干| 超碰97精品在线观看| 巨乳人妻的诱惑在线观看| 黄色一级大片看看| 51午夜福利影视在线观看| 亚洲黑人精品在线| 两性夫妻黄色片| 国产精品免费大片| 国产成人精品久久二区二区免费| 欧美国产精品va在线观看不卡| 亚洲五月色婷婷综合| 欧美在线黄色| 国产欧美亚洲国产| 国产高清不卡午夜福利| 国产免费福利视频在线观看| 少妇人妻 视频| av天堂久久9| 国产精品久久久久成人av| 精品少妇一区二区三区视频日本电影| 亚洲av成人不卡在线观看播放网 | 久久久精品免费免费高清| 女性生殖器流出的白浆| 国产精品一国产av| 国产精品免费大片| 热re99久久国产66热| 色婷婷久久久亚洲欧美| 国产视频一区二区在线看| 国产在线一区二区三区精| 久久久精品免费免费高清| 国产精品一区二区在线观看99| 一区二区三区精品91| e午夜精品久久久久久久| 啦啦啦中文免费视频观看日本| a级毛片黄视频| 国产日韩欧美亚洲二区| 国产在线免费精品| 免费高清在线观看视频在线观看| 在线观看免费午夜福利视频| 国产极品粉嫩免费观看在线| 成人18禁高潮啪啪吃奶动态图| 老司机亚洲免费影院| 国产亚洲一区二区精品| 韩国高清视频一区二区三区| 韩国高清视频一区二区三区| 亚洲国产看品久久| 亚洲九九香蕉| 制服诱惑二区| 免费久久久久久久精品成人欧美视频| 国产免费一区二区三区四区乱码| 欧美成人精品欧美一级黄| 欧美黑人精品巨大| 捣出白浆h1v1| 丝袜人妻中文字幕| 国产视频首页在线观看| 亚洲国产欧美网| 激情视频va一区二区三区| videos熟女内射| 成人亚洲精品一区在线观看| 久9热在线精品视频| 黄色a级毛片大全视频| 亚洲五月色婷婷综合| 久久狼人影院| 香蕉丝袜av| 女人精品久久久久毛片| 国产成人a∨麻豆精品| 少妇 在线观看| 另类亚洲欧美激情| 视频区欧美日本亚洲| 嫁个100分男人电影在线观看 | 无遮挡黄片免费观看| 久久女婷五月综合色啪小说| 国产免费福利视频在线观看| 日本色播在线视频| 久久综合国产亚洲精品| 高清欧美精品videossex| 精品人妻1区二区| videosex国产| 国产色视频综合| 日韩一卡2卡3卡4卡2021年| 亚洲中文日韩欧美视频| 飞空精品影院首页| 国产精品二区激情视频| 汤姆久久久久久久影院中文字幕| 国产精品一区二区在线不卡| 热99国产精品久久久久久7| 又紧又爽又黄一区二区| 97在线人人人人妻| 黄色视频不卡| 国产精品秋霞免费鲁丝片| 丝袜美足系列| 伊人久久大香线蕉亚洲五| 国产精品九九99| 欧美日韩一级在线毛片| 亚洲中文日韩欧美视频| 乱人伦中国视频| 国产av精品麻豆| 日本wwww免费看| 王馨瑶露胸无遮挡在线观看| 精品人妻一区二区三区麻豆| 99国产综合亚洲精品| 性少妇av在线| 国产在视频线精品| av在线老鸭窝| 大话2 男鬼变身卡| 色综合欧美亚洲国产小说| 一二三四在线观看免费中文在| 一区二区日韩欧美中文字幕| 免费高清在线观看视频在线观看| 午夜免费鲁丝| 亚洲精品中文字幕在线视频| 国产精品国产av在线观看| 欧美精品一区二区大全| 男女国产视频网站| 日韩一区二区三区影片| 蜜桃在线观看..| 又大又爽又粗| 人人妻人人添人人爽欧美一区卜| 日韩一区二区三区影片| 免费在线观看完整版高清| 婷婷色综合大香蕉| 免费一级毛片在线播放高清视频 | 国产淫语在线视频| 国产福利在线免费观看视频| 亚洲精品乱久久久久久| 不卡av一区二区三区| 精品视频人人做人人爽| 国产精品国产三级专区第一集| 黄片播放在线免费| 黄色 视频免费看| 国产精品.久久久| av片东京热男人的天堂| 我要看黄色一级片免费的| 考比视频在线观看| 免费观看a级毛片全部| netflix在线观看网站| 午夜免费观看性视频| 又大又黄又爽视频免费| 日本av手机在线免费观看| 18禁观看日本| 午夜福利免费观看在线| 一本—道久久a久久精品蜜桃钙片| 老司机亚洲免费影院| 免费少妇av软件| 纵有疾风起免费观看全集完整版| 国产高清videossex| 亚洲欧美激情在线| 日本黄色日本黄色录像| 菩萨蛮人人尽说江南好唐韦庄| 亚洲欧美清纯卡通| 久久精品熟女亚洲av麻豆精品| 只有这里有精品99| 99国产精品99久久久久| 少妇精品久久久久久久| 欧美在线一区亚洲| 黑人欧美特级aaaaaa片| 每晚都被弄得嗷嗷叫到高潮| 亚洲人成77777在线视频| 激情五月婷婷亚洲| 黄色 视频免费看| 精品国产一区二区三区久久久樱花| 国产激情久久老熟女| 999精品在线视频| 最近中文字幕2019免费版| 免费久久久久久久精品成人欧美视频| 亚洲精品成人av观看孕妇| 亚洲精品美女久久av网站| xxx大片免费视频| 久热爱精品视频在线9| 亚洲成色77777| 又粗又硬又长又爽又黄的视频| 一区二区三区激情视频| 黑丝袜美女国产一区| 国产男女内射视频| 女人被躁到高潮嗷嗷叫费观| 欧美激情极品国产一区二区三区| 久久久久久亚洲精品国产蜜桃av| 欧美日韩精品网址| 亚洲久久久国产精品| 日本wwww免费看| 国产精品久久久久久精品电影小说| 久久久久精品人妻al黑| 成人国产av品久久久| www.av在线官网国产| 亚洲欧美日韩另类电影网站| 搡老岳熟女国产| 日韩大码丰满熟妇| 久久青草综合色| 国产亚洲av片在线观看秒播厂| 久久天躁狠狠躁夜夜2o2o | a级片在线免费高清观看视频| 少妇人妻久久综合中文| 亚洲欧美激情在线| 99精国产麻豆久久婷婷| 久久ye,这里只有精品| 无遮挡黄片免费观看| 汤姆久久久久久久影院中文字幕| 亚洲精品国产av成人精品| 99国产精品免费福利视频| 桃花免费在线播放| 一边摸一边做爽爽视频免费| 精品一品国产午夜福利视频| 大话2 男鬼变身卡| 免费在线观看日本一区| 国产无遮挡羞羞视频在线观看| 久久久欧美国产精品| 99久久精品国产亚洲精品| 久久人人爽人人片av| 90打野战视频偷拍视频| 美女扒开内裤让男人捅视频| 纯流量卡能插随身wifi吗| 大型av网站在线播放| 久久精品久久精品一区二区三区| 国产一区有黄有色的免费视频| 如日韩欧美国产精品一区二区三区| 免费一级毛片在线播放高清视频 | 久久女婷五月综合色啪小说| 久久ye,这里只有精品| 男人舔女人的私密视频| 人成视频在线观看免费观看| 国产一区二区 视频在线| 国产野战对白在线观看| 亚洲精品日韩在线中文字幕| 亚洲国产欧美网| 热99久久久久精品小说推荐| 日本wwww免费看| 国产日韩欧美视频二区| 成在线人永久免费视频| av一本久久久久| 精品人妻一区二区三区麻豆| 欧美亚洲 丝袜 人妻 在线| 午夜福利视频精品| 国产精品国产av在线观看| 少妇人妻久久综合中文| 亚洲成人免费电影在线观看 | 色播在线永久视频| 久久久精品免费免费高清| 桃花免费在线播放| 国产一级毛片在线| 成年美女黄网站色视频大全免费| 国产一区二区 视频在线| 国产又爽黄色视频| 欧美日韩视频精品一区| 亚洲欧美中文字幕日韩二区| 久久鲁丝午夜福利片| 成人三级做爰电影| 欧美成人精品欧美一级黄| 久久久久久久久久久久大奶| 99久久综合免费| 国产午夜精品一二区理论片| 日本一区二区免费在线视频| 亚洲国产精品成人久久小说| 成年人免费黄色播放视频| av一本久久久久| 人人妻,人人澡人人爽秒播 | 亚洲欧洲日产国产| 成年av动漫网址| 欧美亚洲 丝袜 人妻 在线| 女人爽到高潮嗷嗷叫在线视频| 亚洲第一青青草原| 777米奇影视久久| 三上悠亚av全集在线观看| 国产精品 国内视频| 咕卡用的链子| 亚洲,欧美,日韩| 国产精品免费大片| 亚洲av在线观看美女高潮| 久久精品亚洲av国产电影网| 一边摸一边抽搐一进一出视频| 午夜福利在线免费观看网站| 久久久久国产一级毛片高清牌| 亚洲专区中文字幕在线| 久久精品成人免费网站| 高清欧美精品videossex| 色94色欧美一区二区| 久久人人爽av亚洲精品天堂| 一区二区日韩欧美中文字幕| 色综合欧美亚洲国产小说| 97在线人人人人妻| 亚洲人成电影免费在线| 美女视频免费永久观看网站| 电影成人av| 高清欧美精品videossex| 成人亚洲精品一区在线观看| 50天的宝宝边吃奶边哭怎么回事| 成人午夜精彩视频在线观看| 精品熟女少妇八av免费久了| 久久国产精品人妻蜜桃| 国产无遮挡羞羞视频在线观看| 51午夜福利影视在线观看| 国产av国产精品国产| 亚洲av美国av| svipshipincom国产片| 久久久国产欧美日韩av| 国产国语露脸激情在线看| 国产高清视频在线播放一区 | 日本色播在线视频| 久久人人爽人人片av| 精品福利永久在线观看| 久久精品国产综合久久久| 岛国毛片在线播放| 黄片播放在线免费| kizo精华| 精品欧美一区二区三区在线| 亚洲图色成人| 日本色播在线视频| 激情视频va一区二区三区| a级毛片在线看网站| 在现免费观看毛片| 国产有黄有色有爽视频| 91精品伊人久久大香线蕉| 亚洲久久久国产精品| www.熟女人妻精品国产| 午夜福利乱码中文字幕| 自拍欧美九色日韩亚洲蝌蚪91| av天堂在线播放| 亚洲国产av新网站| 欧美日韩亚洲高清精品| 日本av免费视频播放| 精品国产超薄肉色丝袜足j| 只有这里有精品99| 欧美成人午夜精品| 在现免费观看毛片| 99精国产麻豆久久婷婷| 大片免费播放器 马上看| 美女高潮到喷水免费观看| 脱女人内裤的视频| 亚洲男人天堂网一区| 国产精品 欧美亚洲| 在线观看免费视频网站a站| 久久久久国产一级毛片高清牌| 亚洲少妇的诱惑av| 99精品久久久久人妻精品| 国产日韩欧美视频二区| 亚洲激情五月婷婷啪啪| 亚洲欧美一区二区三区国产| 伊人亚洲综合成人网| 中文乱码字字幕精品一区二区三区| 国产有黄有色有爽视频| 精品久久久精品久久久| 色网站视频免费| 精品国产国语对白av| 久久精品人人爽人人爽视色| 在线观看免费高清a一片| 看免费成人av毛片| 午夜免费鲁丝| 人妻一区二区av| 男女国产视频网站| 两个人免费观看高清视频| 日本猛色少妇xxxxx猛交久久| 日本欧美视频一区| 日韩中文字幕视频在线看片| 国产精品麻豆人妻色哟哟久久| 十分钟在线观看高清视频www| 男女午夜视频在线观看| 18禁黄网站禁片午夜丰满| 伊人久久大香线蕉亚洲五| 国产极品粉嫩免费观看在线| 久久人人97超碰香蕉20202| 操出白浆在线播放| 成人国产一区最新在线观看 | 亚洲精品中文字幕在线视频| 如日韩欧美国产精品一区二区三区| 国产精品一二三区在线看| 成年人黄色毛片网站| 超碰97精品在线观看| 精品少妇黑人巨大在线播放| 免费不卡黄色视频| 国产成人欧美| 91国产中文字幕| 性色av一级| 两性夫妻黄色片| 亚洲欧美激情在线| 国产精品成人在线| 欧美精品亚洲一区二区| 日韩av在线免费看完整版不卡| 久热爱精品视频在线9| 视频在线观看一区二区三区| 国产精品免费视频内射| 中国美女看黄片| 99热全是精品| 国产一区二区三区av在线| 飞空精品影院首页| bbb黄色大片| 国产在线一区二区三区精| 精品久久久久久电影网| 久久久国产欧美日韩av| 爱豆传媒免费全集在线观看| 精品国产一区二区三区四区第35| 少妇的丰满在线观看| 巨乳人妻的诱惑在线观看| 大片电影免费在线观看免费| 男女无遮挡免费网站观看| 国产精品三级大全| 少妇被粗大的猛进出69影院| 制服人妻中文乱码| 捣出白浆h1v1| 国产成人精品久久二区二区免费| 99精国产麻豆久久婷婷| 超碰成人久久| cao死你这个sao货| 亚洲五月色婷婷综合| 亚洲色图 男人天堂 中文字幕| 午夜精品国产一区二区电影| 国产极品粉嫩免费观看在线| av线在线观看网站| 爱豆传媒免费全集在线观看| 男女无遮挡免费网站观看| 精品一区在线观看国产| 亚洲,欧美精品.| 日日夜夜操网爽| 亚洲成色77777| 高清欧美精品videossex| av网站免费在线观看视频| 侵犯人妻中文字幕一二三四区| 在线观看免费日韩欧美大片| 亚洲精品国产区一区二| 国产一区二区在线观看av| 亚洲欧美色中文字幕在线| 国产亚洲午夜精品一区二区久久| 国产午夜精品一二区理论片| 久久国产精品人妻蜜桃| 女人久久www免费人成看片| 中文字幕精品免费在线观看视频| 日本a在线网址| 亚洲欧美日韩另类电影网站| 欧美亚洲 丝袜 人妻 在线| 黄色 视频免费看| bbb黄色大片| 在线观看人妻少妇| 亚洲欧洲日产国产| 宅男免费午夜| 久久99热这里只频精品6学生| 90打野战视频偷拍视频| 亚洲精品国产av成人精品| 天天操日日干夜夜撸| 又紧又爽又黄一区二区| 波多野结衣一区麻豆| 亚洲av在线观看美女高潮| 777米奇影视久久| 亚洲第一青青草原| 91精品国产国语对白视频| 狠狠婷婷综合久久久久久88av| 国产老妇伦熟女老妇高清| 国产成人影院久久av| 亚洲精品中文字幕在线视频| 精品一区二区三区四区五区乱码 | 夫妻性生交免费视频一级片| 免费日韩欧美在线观看| 麻豆av在线久日| 欧美 日韩 精品 国产| 国产97色在线日韩免费| 日韩欧美一区视频在线观看| 人人妻人人添人人爽欧美一区卜|