• <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 ?

    Dietary Supplementation with Black Garlic Powder Exerts Immunostimulatory Activity in Cyclophosphamide Induced BALB/c Mice

    2019-12-03 01:08:20YANGMingQINYeHAOJunyuWUTaoLIURuiSUIWenjieZHANGMin
    食品科學(xué) 2019年21期

    YANG Ming, QIN Ye, HAO Junyu, WU Tao, LIU Rui, SUI Wenjie, ZHANG Min*

    (Engineering Research Center of Food Biotechnology, Ministry of Education, Institute for New Rural Development,College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China)

    Abstract: The present study aimed to investigate the immunostimulatory activity of black garlic powder on immunosuppressed BALB/c mice. Cyclophosphamide was injected to establish an immunosuppressed model. Then, the effect of black garlic powder at doses of 100, 300 and 900 mg/kg on splenic and thymus index, the transformation of splenic lymphocytes, delayed type hypersensitivity, quantity of antibody-producing cells, half hemolysis value (HC50), carbon clearance rate, interleukin (IL)-8 and IL-12 gene expression levels in splenic cells of mice were investigated. The results indicated that all three doses of black garlic powder significantly improved thymus index, antibody production, splenic lymphocyte proliferation and the expression of IL-8 and IL-12 in splenic cells when compared with the model control(MC) group (P < 0.05). In addition, compared with the MC group, black garlic powder at the middle and high doses could significantly enhance splenic index and delayed-type hypersensitivity response (P < 0.05). Moreover, compared with the MC group, black garlic powder at the high dose effectively improved serum hemolysin level and mononuclear macrophage phagocytosis activity (P < 0.05). Thus, black garlic powder appears to be an excellent candidate to improve immune function in BALB/c mice.

    Keywords: black garlic powder; BALB/c mouse model; immunostimulatory

    Black garlic is obtained from fresh garlic(Allium sativumL.)that has been fermented for a period of time at a high temperature (60-90 ℃) and relative humidity (80%-90%).Compared with fresh garlic, black garlic exhibits more nutritional and physiological effects because of the changes of physicochemical properties[1-2]. In the development of black garlic, oligosaccharides converted into fructose[3], which not only increased the sweetness of black garlic but also reduced the content of volatile sulfide.

    Several studies reported that black garlic extract activates helper T cell (Th)1 and Th2 cells by stimulating T lymphocyte of mice splenic and promotes immune regulation by activating cellular and humoral immunity. Feng Yonghui et al[4]found black garlic extracts exhibited better activity for natural killer cells and better secretion ability of the non-specific immune molecule-nitric oxide. In addition, the extract of black garlic inhibited the promotion of Th1 type cytokines, interleukin (IL)-2, interferon-γ and tumor necrosis factor-α. Besides, several studies have reported that black garlic contains kinds of immunological substances such as serine, lysine, VC and trace element zinc etc.[5].

    Black garlic powder (BGP) is a powder product of black garlic. As a new garlic product, BGP not only can prolong the storage period, but also reduce transportation, packaging and other costs, and expand its application range. In addition,BGP is nutritional and easy to eat. It can be widely used as ingredients for processed foods. Thus, the objective of this work is to determine whether BGP can improve the immune function of mice and to further provide basic information in the immune regulation ability of BGP.

    1 Materials and methods

    1.1 Materials, animals and reagents

    Black garlic were purchased from Dawn Food Co. Ltd..As shown in Fig. 1, BGP (B) processed from black garlic (A)is dark brown powder. In this study, mice were given BGP to verify its immune efficacy.

    BALB/c mice were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People’s Liberation Army (Production license number: 2012-0004) and kept in Animal Laboratory of Central Barrier system (Use license number: 2006-0005).

    BGP was prepared by differential pressure expansion in laboratory (Tianjin University of Science & Technology);3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), concanavalin A (Con A) and Red Blood Cell Lysis Buffer America Sigma Co. Ltd.; TRIzol reagent America Invitrogen Co. Ltd.; RNA Extraction Kit Beijing Kangwei century Co. Ltd.; SuperRT cDNA first-chain synthesis kit and SYBR Green PCR mixture Beijing Transgen Co. Ltd.. All the other chemicals used in the experiment were of analytical-purity grade.

    Fig. 1 Photographs of black garlic (A) and BGP (B)

    1.2 Instruments and equipment

    DH-101 Oven Tianjin Zhonghuan Experimental Electric Furnace Co. Ltd.; SX2-25-10 Fluorination furnace Tianjin Sanshui Scientific Instrument Co. Ltd.;WK21027 Electromagnetic furnace Guangdong Midea Life Appliance Manufacturing Co. Ltd.; SOX-406 Soxhlet extractor, K9840 Kjeldahl nitrogen determinator Jinan Haineng Instrument Manufacturing Co. Ltd.; TY-150 Flour mill Wuyi Haina Electric Appliance Co. Ltd.;TU-1810PC Ultraviolet spectrophotometer Beijing Puxi General Instrument Co. Ltd.; JX-1000 Differential pressure extruder Shandong Delico Industrial Equipment Co. Ltd..

    1.3 Methods

    1.3.1 Determination of basic nutritional components of BGP

    The water content of BGP was determined according to the method of GB 5009.3–2016. The protein content of BGP was determined according to the method of GB 5009.5–2016.The fat content of BGP was determined according to the method of GB 5009.6–2016. The ash content of BGP was determined according to the method of GB 5009.4–2016. The crude fiber content of BGP was determined according to the method of GB/T 5515–2008.

    1.3.2 Grouping of mice and immune model

    The establishment of immune model was determined according to the method of Liu Jiancheng et al[6]. After one-week adaptation, the mice were randomly divided into 5 groups consisting of 12 mice each. One group of healthy mice was used as a normal control (NC) group and was treated once per day with physiological saline for 30 days. For Days 1–3,the other 4 groups of mice were given cyclophosphamide at 80 mg/(kg·d) via intraperitoneal injection. For Days 4–30,the mice were administered as follows: model control (MC)group, physiological saline was administered via gavage;three BGP (BGP-L, BGP-M and BGP-H) groups, at 100, 300,and 900 mg/kg BGP were administered via gavage.

    1.3.3 Calculation of splenic and thymus indices

    After 30 days of gavage, the mice were weighed and killed immediately. The immune organs, splenic and thymus of each animal of all experimental groups were collected after sacrificing. Splenic and thymus of each animal were weighed upon collection. Splenic and thymus indices were calculated based on splenic or thymus mass and animal body mass[7].The calculating formula as formula (1) and (2).

    1.3.4 Con A induced splenic lymphocyte transformation

    The splenic was removed aseptically and placed in a plate with a proper amount (3-5 mL) of aseptic Hanks solution. Grinding the splenic and making a single cell suspension. Erythrocytes in the splenic lymphocytes were lysed with double distilled water (DDW), splenic lymphocytes were washed twice in sterile Hanks solution and centrifuged at 1 000 r/min for 10 min, and finally suspended in 2 mL complete culture solution.

    In 24-well plates, 3 × 106splenocytes from each mice were seeded into two wells, one with 75 μL Con A and the other without Con A. After incubation for 72 h, 0.7 mL supernatant was gently removed from each well, then 0.7 mL of RPMI-1640 (no bovine foetal serum) containing 50 μL of MTT (5 mg/mL) was added into each well. The cells were continuously cultured for further 4 h. Finally, 1 mL of acid isopropyl alcohol was added into each well to fully solubilise the formazan precipitate. Then it was packed into 96-well culture plate. The optical density (OD) value was measured at 570 nm[8].

    1.3.5 Analysis of delayed type hypersensitivity

    Abdominal skin of each mice was depilated with barium sulfide in a range of 3 cm × 3 cm and sensitized with 50 μL dinitrofluorobenzene (DNFB) solutions for 5 days. 10 μL DNFB solution was evenly applied to the right ear of the mouse (two sides) to attack for 24 h. After the attack, the mice were killed by cervical dislocation and the left and right ear shells were cut off, punched holes to remove ear slices 8 mm in diameter and weighed them. The results were expressed as the mass difference between left and right ears[9].

    All the animals were immunized with 2% washed sheep red blood cells (SRBCs) 0.2 mL for 5 days. On Day 6 mice were sacrificed by cervical dislocation. Single cell suspension of splenocytes was made. Splenic cells were counted under the microscope and its count was adjusted up to 1 × 107cells/mL.50 μL of 10% SRBCs and 200 μL of splenocytes were taken in a test tube containing 0.5 mL top layer. Rapidly mixed,pour into the 6-well plate and smooth the top mixture. These culture plates were incubated at 37 ℃ for 1 h. Then 500 μL of complement (1:10) was added to each pore and incubated for 2 h. The plaques were observed under an automatic image analyzer. The average number of plaques in parallel wells was taken as the hemolytic plaque value of the sample, which was expressed as plaque number/106splenic cells[10].

    1.3.7 Serum hemolysin analyses

    Five days before the end of the administration, mice were injected intraperitoneally with 0.2 mL/d 10% SRBCs and blood from mice orbit was collected. Briefly, both the serum and the guinea-pig serum were diluted with 0.85%saline before used. One millilitre of diluted serum was mixed with 0.5 mL 5% SRBCs in a tube. Then 1 mL diluted guinea pig serum was added into the reaction.

    After incubation at 37 ℃ for 30 min, the reaction was terminated in ice. Samples were centrifuged at 3 000 r/min at 4 ℃ for 5 min to remove intact erythrocytes. One millilitre supernatant was collected and mixed uniformly with 3 mL of Kathmandu’s reagent for 10 min. The OD value of the reaction was measured at 540 nm[11]. Then another 0.25 mL 10% SRBC suspension was mixed with 3.75 mL of Kathmandu’s reagent. The OD value was determined as half of the hemolysis value (HC50). The calculation of HC50value is shown in the equation (3).

    1.3.8 Determination of mononuclear-macrophage phagocytosis activity

    After 30 days of gavage, India ink (0.01 mL/g) was injected into the caudalis vein of the mice. Twenty microlitres of blood sample were collected after 2 min (t1) and 10 min(t2), and was mixed with 2 mL 0.1% Na2CO3solution. The OD of the blood was measured at a wavelength of 600 nm[12].The mononuclear-macrophages phagocytic activity (α) was calculated as formula (4) and (5).

    He was very careful not to leave enough space for the dragon to jump out, but unluckily there was just room for his great mouth, and with one snap the king vanished down his wide red jaws16

    1.3.9 Quantitative real-time polymerase chain reactions analysis

    RNA extraction from splenocytes: The splenic of 0.1 g mice was weighed and ground into powder in liquid nitrogen without enzyme. Moved the powder to the centrifugal tube,adding 1 mL trazole to each tube. After repeated absorption,it was kept for 5 min and dissolved sufficiently. According to general RNA Extraction Kit manual to extract and ensured the ratio of OD260nm/OD280nmof all RNA samples was 1.6–1.8[13].

    Establishment of reverse transcription (RT) system: A Kit for First Chain Synthesis of Trans Gen Super RT cDNA.The sample adding system: 5 μL template + 1 μL enzyme +1 μL buffer + 3 μL DDW. The mixture was incubated in polymerase chain reactions (PCR) instrument at 37 ℃ for 30 min. Then the mixture was denatured subsequently at 85 ℃ for 5 min. The synthesized cDNA is stored in the refrigerator of -80 ℃.

    Quantitative real-time PCR (qPCR): 12.5 μL mixed SYBR,1 μL cDNA, upstream and downstream primers (Table 1)1 μL each, 9.5 μL DDW. Internal reference isGAPDH. The template is cDNA.

    Table 1 Primer sequences used for qPCR

    The operating procedure is based on the instructions of qPCR mixture kit. The reaction system of 25 μL qPCR was used in this experiment.

    1.4 Statistical analysis

    Results are presented as the mean ± standard error of the mean. Data were analyzed using one-way analysis of variance followed by the least significant difference test for multiple comparisons among groups. Statistical analysis and image processing were conducted using Origin 8.0 software.P< 0.05 was considered to indicate a statistically significant difference.

    2 Results and Analysis

    2.1 Basic nutritional components of BGP

    The water content in black garlic was greatly reduced by pressure differential expansion technology, and the basic nutritional components of BGP were obtained as shown in the Table 2.

    Table 2 Compositions of the BGP

    2.2 Effects of BGP on the splenic and thymus indices in BALB/c mice

    Fig. 2 Effect of BGP on splenic index (A) and thymus index (B) of BALB/c mice

    As shown in Fig. 2, BGP increased the splenic and thymus indices as compared to MC group at the end of the experiment. Compared with the MC group, splenic index in the middle and high dosage of BGP were remarkably increased (P< 0.05). Meanwhile, insignificant variation was found between the splenic index in the low dosage of BGP and the MC group (P> 0.05). On the other hand, all the dosage of BGP displayed a significant enhancement in the thymus index (P< 0.05). Besides, the thymus index also showed a significantly increase with the increasing dosage of the BGP (P< 0.05).

    2.3 Effects of BGP on Con A-induced splenic lymphocyte transformation and DNFB-induced delayed type hypersensitivity in BALB/c mice

    To investigate the effects of different dosage of BGP to cellular immune function in mice, Con A-induced splenic lymphocyte transformation and DNFB-induced delayed type hypersensitivity were determined. As shown in Fig. 3, all the dosage of BGP displayed a significant enhancement in splenic lymphocyte transformation when compared with MC group(P< 0.05) and a significant difference among each dosage of BGP was observed (P< 0.05). On the other hand, the middle and high dosage of BGP significantly reduced the mass difference between left and right ears in a DNFB-induced delayed type hypersensitivity model (P< 0.05). However, no significant difference was observed among the low and MC groups (P> 0.05).

    Fig. 3 Effects of BGP on splenic lymphocyte transformation activity (A)and delayed-type hypersensitivity response (B) in BALB/c mice

    2.4 Effects of BGP on hemolytic plaque value and serum haemolysin level in BALB/c mice

    Fig. 4 Effect of BGP on hemolytic plaque value (A) and HC50 (B) of BALB/c mice

    The ability of antibody-producing in mice splenic are presented in Fig. 4A, all the dosage of BGP displayed a significant enhancement in the number of the antibody-producing cells compared with the MC group (P< 0.05).Among the BGP groups, the number of the antibody-producing cells dramatically increased with the dosage increasing(P< 0.05). As shown in Fig. 4B, the maximum value of HC50was achieved in the high dosage of BGP, which significantly higher than other groups (P< 0.05). However, the low and middle dosage of BGP presented an insignificant difference in the production of serum haemolysin when compared with the MC group (P> 0.05).

    2.5 Effects of BGP on mononuclear-macrophages phagocytic index in BALB/c mice

    Fig. 5 Effect of BGP on mononuclear macrophage phagocytic index of BALB/c mice

    As shown in Fig. 5, the phagocytic index of the high dosage of BGP was significantly higher than that of other groups(P< 0.05). However, the low and middle dosage of BGP did not present an increased tendency of mononuclear-macrophages phagocytic index when compared with the MC group and no significant difference was observed among them.

    2.6 Effects of BGP on the mRNA levels of cytokines of splenocyte in BALB/c mice

    Fig. 6 Effect of BGP on the expression of IL-8 (A) and IL-12 (B)mRNA in BALB/c mice

    As shown in Fig. 6, the expression levels of cytokines(IL-8,IL-12) mRNA in the MC group decreased significantly when compared to the NC group (P< 0.05), and the administration of BGP group remarkably up-regulated the mRNA expression levels ofIL-8andIL-12compared to the MC group (P< 0.05).

    3 Discussion

    At present, research in nutritional immunology has focused on the role of natural food and its ability in improving the immunological response[14]. Nutritional research is targeted towards the development of health food formula to reduce the risk of infection. Among plant derived food, BGP is found to be a rich source of unique bioactive molecules such as flavonoids, organosulfur compounds, dietary fiber,phenolic compounds and others[15]. Recent animal studies have shown that supplementation of BGP extract to mice activates Th1 and Th2 cells by stimulating T lymphocytes in mouse splenic cells and leads to immunomodulation by activating cellular and humoral immune responses of the immune system[16].

    Splenic and thymus are the main immune organs of the body so that changes of organ indices are the key indicators of immunity in body[17-18]. Thymus plays an important role in the establishment of immune function and the recovery of immune regulatory function after the loss of immunity. A large number of thymocytes produced by thymus can be used as backup for T lymphocyte when T cells are insufficient.Mature thymocytes can also reach the peripheral organs of the body with blood to assist B lymphocyte forits cellular immune function[19]. The splenic is a large immune organ of the body. The specific immunity of the body mostly occurs in the splenic. It has a certain filtering effect on all kinds of tumor cells and harmful microorganisms[20]. In the present study, the levels of both thymus and splenic indices revealed markedly increased in mice treated with BGP which was able to increase lymph contents in both splenic and thymus so that the mass of thymus and splenic significantly increased in mice.

    Splenic lymphocyte transformation activity and delayed type hypersensitivity are two important parameters of cellular immunity. Con A is a non-specific mitotic source. It can promote lymphocyte proliferation and transformation when it acts on lymphocyte alone. When Con A acts on some specific antigens, it has synergistic effect on lymphocyte proliferation and transformation[21]. Mice models of delayed type hypersensitivity (DTH) reactions were generated by DNFB-induced subcutaneous sensitization to the ears of mice. By feeding with BGP, the swelling degree of mice’s ears were observed to determine whether BGP could alleviate local inflammation induced by DTH[22]. The present study revealed that there is a significantly increase of splenic lymphocyte transformation activity by feeding with BGP and BGP has a positive effect on attenuating DTH reactions when compared with MC groups.

    The ability of secreting antibodies is an important aspect for evaluation of the host humoral immune response. The hemolytic plaque formation assay and serum haemolysin analysis are well-documented models to measure antibody production in response to an antigenic stimulation[23-24]. On one hand, the number of hemolytic plaques reflects the total number of antibody-producing cells in the splenic. The number of haemolytic plaques in antibody level increased significantly, suggesting that the host ability to defend against foreign body invasion was enhanced[25]. On the other hand,the content of hemolysin in the serum of mice was estimated by the content of Hb released by erythrocyte. HC50can reflect the level of antibody produced by immunizing mice, and the content of hemolysin in animal serum can be reflected by measuring the content of hemoglobin[26]. In the present study the number of splenic antibody-producing cells increased significantly in all doses of BGP groups, but only in high doses of BGP group, HC50increased significantly.

    As important immune cells in the body, macrophage phagocytosis targeting on xenobiotic acts as a crucial role for early host defence against foreign invasion. Removal of carbon particles from the blood circulation is correlated with an enhanced phagocytic activity[27-28]. Results from this project suggest that the high dose of BGP can effectively improve the phagocytosis ability of mononuclear-phagocyte in mice.

    Cytokine is a kind of polypeptide molecule, which can act on the immune system and play an important role in regulating cell interaction, cell proliferation and differentiation. IL-8 is a chemokine derived from a variety of cells[29]. It has chemotaxis to T lymphocyte and basophil[30].After activation, the expression ofIL-8gene was significantly enhanced, and a large number ofIL-8genes were clustered together. The gene clustering can induce resistance of host defense system to bacterial invasion and play an important role in regulating inflammation and immune function[31-33].IL-12is a multifunctional immunoregulatory factor that can stimulate the proliferation of activated T cells[34-36]. In order to evaluate whether BGP activated splenocyte produced cytokines, the mRNA levels ofIL-8,IL-12in splenic were analyzed using qPCR. In this study. The results presented here suggested that the expression ofIL-8andIL-12mRNA suppressed by cyclophosphamide in mice gradually returned to normal level upon treatment with BGP.

    4 Conclusion

    In summary, BGP, as a new fruit-vegetable powder, showed potential immunostimulatory properties and could reverse immunosuppression by enhancing immune organ’s development,lymphocyte proliferation and monocyte-macrophages phagocytosis. BGP could also induce the expression ofIL-8andIL-12mRNA. Therefore, we suggested that BGP could be used as a novel immunological fruit-vegetable powder for numerous fields of food processing.

    精品国产一区二区久久| 午夜激情av网站| 午夜精品国产一区二区电影| 国产不卡一卡二| 淫秽高清视频在线观看| 国产av又大| 丰满人妻熟妇乱又伦精品不卡| 国产精品亚洲美女久久久| 亚洲精品在线美女| 国产精品久久久久久精品电影 | 亚洲av电影在线进入| 国产色视频综合| 午夜福利成人在线免费观看| 午夜福利影视在线免费观看| 91成人精品电影| 日韩欧美三级三区| 这个男人来自地球电影免费观看| 级片在线观看| 激情视频va一区二区三区| 国产xxxxx性猛交| 黄色视频,在线免费观看| 真人一进一出gif抽搐免费| 亚洲国产精品久久男人天堂| 在线观看66精品国产| 久久欧美精品欧美久久欧美| videosex国产| 免费在线观看亚洲国产| 香蕉久久夜色| 亚洲国产精品999在线| 久久久久久大精品| 非洲黑人性xxxx精品又粗又长| 欧美大码av| 国产一区二区三区在线臀色熟女| 无人区码免费观看不卡| 成人欧美大片| 成人av一区二区三区在线看| 日韩三级视频一区二区三区| 午夜老司机福利片| 91精品国产国语对白视频| 不卡av一区二区三区| 国产男靠女视频免费网站| 欧美日韩中文字幕国产精品一区二区三区 | 午夜精品久久久久久毛片777| 91大片在线观看| 亚洲一区中文字幕在线| 久久亚洲精品不卡| 国产精品久久视频播放| xxx96com| 国产精品综合久久久久久久免费 | 国产精品久久电影中文字幕| 亚洲五月色婷婷综合| 巨乳人妻的诱惑在线观看| 欧美激情高清一区二区三区| 精品日产1卡2卡| 国产亚洲精品综合一区在线观看 | 亚洲人成网站在线播放欧美日韩| 岛国视频午夜一区免费看| 丝袜人妻中文字幕| 国产人伦9x9x在线观看| 欧美日韩黄片免| 亚洲成人国产一区在线观看| 欧美人与性动交α欧美精品济南到| 国产成人啪精品午夜网站| 搡老熟女国产l中国老女人| 欧美不卡视频在线免费观看 | 给我免费播放毛片高清在线观看| 欧美日韩福利视频一区二区| 日本在线视频免费播放| 欧美最黄视频在线播放免费| 成人永久免费在线观看视频| 激情视频va一区二区三区| 久久狼人影院| 国产av精品麻豆| 高清毛片免费观看视频网站| 国产熟女午夜一区二区三区| 一二三四社区在线视频社区8| 1024视频免费在线观看| 校园春色视频在线观看| 国产精品电影一区二区三区| 亚洲五月色婷婷综合| 色在线成人网| 一a级毛片在线观看| 亚洲性夜色夜夜综合| 欧美成人一区二区免费高清观看 | 9191精品国产免费久久| 亚洲av五月六月丁香网| 99国产极品粉嫩在线观看| 国产真人三级小视频在线观看| 波多野结衣高清无吗| 男女做爰动态图高潮gif福利片 | 欧美成人免费av一区二区三区| 精品国产乱子伦一区二区三区| 老司机在亚洲福利影院| 亚洲欧美日韩无卡精品| 亚洲av日韩精品久久久久久密| 给我免费播放毛片高清在线观看| 最新在线观看一区二区三区| 中文字幕av电影在线播放| 国产一区二区三区视频了| 婷婷六月久久综合丁香| 日本精品一区二区三区蜜桃| 欧美日韩乱码在线| 国产色视频综合| 国产亚洲欧美精品永久| 精品无人区乱码1区二区| 久久亚洲精品不卡| 国产黄a三级三级三级人| 免费高清在线观看日韩| 人成视频在线观看免费观看| 国产精品 国内视频| 国产精品国产高清国产av| 亚洲男人天堂网一区| 成人国产一区最新在线观看| 少妇熟女aⅴ在线视频| 精品国产超薄肉色丝袜足j| 亚洲欧美日韩无卡精品| 99在线人妻在线中文字幕| 日韩欧美免费精品| 日本 欧美在线| 最新在线观看一区二区三区| 怎么达到女性高潮| 亚洲欧美精品综合一区二区三区| 91老司机精品| 国产精品免费视频内射| 日韩中文字幕欧美一区二区| 免费在线观看完整版高清| 电影成人av| 精品国产一区二区久久| 校园春色视频在线观看| 精品国内亚洲2022精品成人| 搡老熟女国产l中国老女人| 免费在线观看影片大全网站| 97人妻精品一区二区三区麻豆 | 中出人妻视频一区二区| 国产99久久九九免费精品| 国产精品秋霞免费鲁丝片| 69av精品久久久久久| 一级作爱视频免费观看| 久久热在线av| 日日夜夜操网爽| 老熟妇乱子伦视频在线观看| 国产亚洲精品一区二区www| 日本黄色视频三级网站网址| 国产视频一区二区在线看| 99久久久亚洲精品蜜臀av| 美女免费视频网站| 亚洲久久久国产精品| 久久精品国产清高在天天线| 国产三级黄色录像| 免费人成视频x8x8入口观看| 一边摸一边抽搐一进一小说| 操美女的视频在线观看| 亚洲黑人精品在线| 美女国产高潮福利片在线看| 又紧又爽又黄一区二区| 男女之事视频高清在线观看| 国产真人三级小视频在线观看| 国产精品,欧美在线| 91成年电影在线观看| 国产精品 国内视频| 亚洲五月婷婷丁香| 精品国产一区二区久久| svipshipincom国产片| 9191精品国产免费久久| ponron亚洲| 欧美av亚洲av综合av国产av| 亚洲男人天堂网一区| 俄罗斯特黄特色一大片| 久久精品91无色码中文字幕| 日韩高清综合在线| 精品一区二区三区四区五区乱码| 亚洲欧美激情综合另类| 中文字幕色久视频| 我的亚洲天堂| 99国产精品99久久久久| av免费在线观看网站| 91成人精品电影| 宅男免费午夜| 琪琪午夜伦伦电影理论片6080| 亚洲精华国产精华精| 在线观看免费午夜福利视频| 很黄的视频免费| www.精华液| 亚洲国产日韩欧美精品在线观看 | 成在线人永久免费视频| 亚洲aⅴ乱码一区二区在线播放 | www.自偷自拍.com| 国产精品久久久久久精品电影 | 亚洲一码二码三码区别大吗| 丝袜人妻中文字幕| 亚洲男人的天堂狠狠| 岛国在线观看网站| 亚洲成人精品中文字幕电影| 琪琪午夜伦伦电影理论片6080| 最近最新中文字幕大全免费视频| 人人妻人人澡人人看| 国产精品久久电影中文字幕| 欧美乱色亚洲激情| 亚洲av五月六月丁香网| 村上凉子中文字幕在线| 视频区欧美日本亚洲| 欧美成人一区二区免费高清观看 | 亚洲男人的天堂狠狠| 麻豆一二三区av精品| 成人永久免费在线观看视频| 色在线成人网| 亚洲成av片中文字幕在线观看| 91成年电影在线观看| 欧美成狂野欧美在线观看| 免费在线观看黄色视频的| 久久影院123| 国产亚洲精品av在线| 麻豆久久精品国产亚洲av| 免费在线观看完整版高清| 黄片大片在线免费观看| 欧美黄色淫秽网站| 亚洲色图综合在线观看| 女同久久另类99精品国产91| 国产三级在线视频| 亚洲国产精品成人综合色| 日本在线视频免费播放| 亚洲精品在线美女| 深夜精品福利| 日韩大码丰满熟妇| 久久久久国产一级毛片高清牌| 精品久久久精品久久久| 乱人伦中国视频| 久久精品人人爽人人爽视色| 一区二区三区精品91| 最新在线观看一区二区三区| 国产aⅴ精品一区二区三区波| 午夜日韩欧美国产| 午夜视频精品福利| 欧美黑人精品巨大| 免费观看人在逋| 欧美日韩福利视频一区二区| 高清在线国产一区| 色综合婷婷激情| 久久久久久人人人人人| 婷婷六月久久综合丁香| 69av精品久久久久久| 国产麻豆69| 精品国产一区二区久久| 国产成人av激情在线播放| 在线观看免费视频日本深夜| 久久久久亚洲av毛片大全| 欧美日韩中文字幕国产精品一区二区三区 | 久久精品国产清高在天天线| 18禁黄网站禁片午夜丰满| 亚洲专区字幕在线| 男女做爰动态图高潮gif福利片 | 国产人伦9x9x在线观看| 精品不卡国产一区二区三区| 国产精品二区激情视频| 黄色片一级片一级黄色片| 久久青草综合色| 久久久久久人人人人人| 国产精品爽爽va在线观看网站 | 久久久久久久精品吃奶| 一级作爱视频免费观看| 一级毛片精品| 国产成人欧美| 亚洲成人国产一区在线观看| 一级片免费观看大全| 在线av久久热| 99久久99久久久精品蜜桃| 悠悠久久av| 欧美黑人欧美精品刺激| 欧美成狂野欧美在线观看| 久久人人爽av亚洲精品天堂| 国产一区二区三区视频了| 午夜免费观看网址| 一级片免费观看大全| 久久精品国产99精品国产亚洲性色 | 级片在线观看| 国产99白浆流出| 宅男免费午夜| 大香蕉久久成人网| 我的亚洲天堂| 丝袜在线中文字幕| 在线天堂中文资源库| 欧美成人一区二区免费高清观看 | 免费在线观看视频国产中文字幕亚洲| 91字幕亚洲| or卡值多少钱| 99在线视频只有这里精品首页| 免费观看精品视频网站| 999久久久国产精品视频| 久久精品人人爽人人爽视色| 日本一区二区免费在线视频| 久久 成人 亚洲| 每晚都被弄得嗷嗷叫到高潮| 深夜精品福利| 欧美成狂野欧美在线观看| 国产成人精品久久二区二区免费| 99久久久亚洲精品蜜臀av| 亚洲无线在线观看| 女人精品久久久久毛片| 亚洲一区二区三区不卡视频| 亚洲精品在线观看二区| 黄色视频,在线免费观看| 日韩有码中文字幕| 一本大道久久a久久精品| 在线观看免费视频网站a站| 男女之事视频高清在线观看| 国产精品 欧美亚洲| 麻豆成人av在线观看| www.999成人在线观看| 精品无人区乱码1区二区| 亚洲欧美精品综合一区二区三区| 国产蜜桃级精品一区二区三区| 丝袜人妻中文字幕| 国产激情欧美一区二区| 好男人电影高清在线观看| 90打野战视频偷拍视频| 久99久视频精品免费| 久久精品国产综合久久久| 国产av在哪里看| 成在线人永久免费视频| 老司机午夜十八禁免费视频| 成人国产综合亚洲| 亚洲av美国av| 一区在线观看完整版| 午夜日韩欧美国产| av欧美777| 久久午夜亚洲精品久久| 丝袜美足系列| 午夜福利欧美成人| 一级片免费观看大全| 两个人看的免费小视频| 国产一区二区三区视频了| 精品国产美女av久久久久小说| 嫁个100分男人电影在线观看| 亚洲一区二区三区不卡视频| 天堂动漫精品| 美女 人体艺术 gogo| 精品福利观看| 变态另类丝袜制服| 中出人妻视频一区二区| 动漫黄色视频在线观看| 女生性感内裤真人,穿戴方法视频| 成人国语在线视频| 欧美精品亚洲一区二区| 国产欧美日韩一区二区三| 日本一区二区免费在线视频| 色综合婷婷激情| 丁香欧美五月| 亚洲五月天丁香| 脱女人内裤的视频| 精品人妻1区二区| 国产精品 国内视频| 久久影院123| 精品久久久久久,| 嫁个100分男人电影在线观看| 亚洲欧美日韩高清在线视频| 美女午夜性视频免费| 一区二区三区精品91| 真人做人爱边吃奶动态| 日韩 欧美 亚洲 中文字幕| 最近最新中文字幕大全免费视频| 九色国产91popny在线| 午夜福利高清视频| 亚洲国产精品成人综合色| 久久影院123| 国产高清视频在线播放一区| 免费高清在线观看日韩| 法律面前人人平等表现在哪些方面| 丝袜在线中文字幕| av视频在线观看入口| 看免费av毛片| 久久久国产欧美日韩av| 麻豆av在线久日| 91老司机精品| 欧美激情久久久久久爽电影 | 国产午夜福利久久久久久| 男女床上黄色一级片免费看| 国产成人一区二区三区免费视频网站| 亚洲欧美日韩高清在线视频| 午夜福利高清视频| 黄色视频不卡| 欧美黄色淫秽网站| 一边摸一边做爽爽视频免费| 国产一区二区激情短视频| 大香蕉久久成人网| a级毛片在线看网站| 亚洲第一电影网av| 身体一侧抽搐| 国产亚洲欧美在线一区二区| 亚洲免费av在线视频| 9热在线视频观看99| 宅男免费午夜| 操出白浆在线播放| 国产视频一区二区在线看| 日韩av在线大香蕉| 精品国内亚洲2022精品成人| 亚洲自拍偷在线| 亚洲五月婷婷丁香| 亚洲精品av麻豆狂野| 国产成人系列免费观看| 大香蕉久久成人网| 国产1区2区3区精品| 黑人巨大精品欧美一区二区蜜桃| 满18在线观看网站| 日日摸夜夜添夜夜添小说| 一进一出好大好爽视频| xxx96com| 国产一区二区在线av高清观看| 可以在线观看毛片的网站| 国产一区二区激情短视频| 成人精品一区二区免费| 欧美激情高清一区二区三区| 老熟妇乱子伦视频在线观看| 嫁个100分男人电影在线观看| 国产99白浆流出| 午夜精品久久久久久毛片777| 999久久久国产精品视频| 精品一区二区三区四区五区乱码| 美女大奶头视频| 国产亚洲欧美精品永久| 成人av一区二区三区在线看| 天天添夜夜摸| 精品一区二区三区av网在线观看| 国产不卡一卡二| 日本在线视频免费播放| 88av欧美| xxx96com| 国产精品香港三级国产av潘金莲| 夜夜夜夜夜久久久久| 亚洲美女黄片视频| 欧美中文综合在线视频| 免费在线观看完整版高清| 精品国产乱子伦一区二区三区| 国产精品九九99| 欧美日本视频| 亚洲七黄色美女视频| 国产精品98久久久久久宅男小说| 欧美成狂野欧美在线观看| 纯流量卡能插随身wifi吗| 久久这里只有精品19| 在线视频色国产色| 午夜成年电影在线免费观看| 美女高潮喷水抽搐中文字幕| 波多野结衣巨乳人妻| 亚洲性夜色夜夜综合| 精品国产国语对白av| 99精品欧美一区二区三区四区| 最近最新中文字幕大全电影3 | 99国产极品粉嫩在线观看| av超薄肉色丝袜交足视频| 国产成+人综合+亚洲专区| 男女下面插进去视频免费观看| 免费无遮挡裸体视频| 久久精品国产亚洲av高清一级| 亚洲中文日韩欧美视频| 亚洲中文av在线| 欧美日本视频| 俄罗斯特黄特色一大片| 精品卡一卡二卡四卡免费| 久久久国产精品麻豆| 亚洲自偷自拍图片 自拍| 老鸭窝网址在线观看| 9热在线视频观看99| 夜夜爽天天搞| 757午夜福利合集在线观看| 国产蜜桃级精品一区二区三区| 亚洲精品美女久久av网站| 欧美黄色淫秽网站| 亚洲精品国产区一区二| 亚洲av熟女| 9191精品国产免费久久| 亚洲第一青青草原| 精品国产一区二区久久| 国产99白浆流出| 一级,二级,三级黄色视频| 老司机深夜福利视频在线观看| 人人妻人人爽人人添夜夜欢视频| 高清在线国产一区| 国产主播在线观看一区二区| 亚洲第一欧美日韩一区二区三区| 日本五十路高清| 久99久视频精品免费| 亚洲一区二区三区色噜噜| 视频在线观看一区二区三区| 女性被躁到高潮视频| 国产亚洲精品久久久久5区| 午夜日韩欧美国产| 久久香蕉国产精品| 啦啦啦观看免费观看视频高清 | 美女 人体艺术 gogo| 久久久久久人人人人人| 国产欧美日韩一区二区精品| 一区二区三区激情视频| 国产成人影院久久av| 亚洲专区国产一区二区| cao死你这个sao货| 亚洲国产精品合色在线| 日本a在线网址| 两性午夜刺激爽爽歪歪视频在线观看 | 久9热在线精品视频| 老汉色∧v一级毛片| 一级作爱视频免费观看| 亚洲精品在线美女| 男人的好看免费观看在线视频 | 久久性视频一级片| 侵犯人妻中文字幕一二三四区| 国产伦人伦偷精品视频| 一个人观看的视频www高清免费观看 | 精品国产一区二区三区四区第35| 成年女人毛片免费观看观看9| 亚洲自拍偷在线| 久久久久国产精品人妻aⅴ院| 亚洲中文av在线| 亚洲色图综合在线观看| 日本精品一区二区三区蜜桃| 久久午夜综合久久蜜桃| 欧美老熟妇乱子伦牲交| 色尼玛亚洲综合影院| av欧美777| 亚洲国产精品久久男人天堂| 精品一品国产午夜福利视频| 法律面前人人平等表现在哪些方面| 亚洲精品久久成人aⅴ小说| 国产成人啪精品午夜网站| 一区福利在线观看| www.精华液| 日韩欧美一区视频在线观看| 桃色一区二区三区在线观看| 免费看a级黄色片| 欧美中文日本在线观看视频| 99re在线观看精品视频| 精品欧美一区二区三区在线| 美女免费视频网站| 国产亚洲欧美精品永久| 日本 av在线| 黄色丝袜av网址大全| 黄片小视频在线播放| 两个人看的免费小视频| 亚洲久久久国产精品| 国产成人av激情在线播放| 在线天堂中文资源库| 色综合婷婷激情| 久久人妻福利社区极品人妻图片| 色播在线永久视频| 大陆偷拍与自拍| 熟妇人妻久久中文字幕3abv| 久久人妻熟女aⅴ| 久久久久国产精品人妻aⅴ院| 电影成人av| 午夜两性在线视频| 制服诱惑二区| 亚洲精品国产区一区二| 两个人视频免费观看高清| 久久九九热精品免费| 国产亚洲av高清不卡| 天天添夜夜摸| 丝袜美足系列| 黄色视频,在线免费观看| 一本大道久久a久久精品| av视频在线观看入口| 亚洲色图av天堂| 欧美成人一区二区免费高清观看 | 欧美日韩黄片免| 无人区码免费观看不卡| 涩涩av久久男人的天堂| 性欧美人与动物交配| 一区二区三区高清视频在线| 午夜福利欧美成人| 国产精品久久电影中文字幕| 日韩欧美一区二区三区在线观看| www.熟女人妻精品国产| 日韩欧美一区二区三区在线观看| 国产日韩一区二区三区精品不卡| 日韩 欧美 亚洲 中文字幕| 亚洲欧美精品综合久久99| 久久婷婷人人爽人人干人人爱 | 欧美色欧美亚洲另类二区 | 两性夫妻黄色片| 激情视频va一区二区三区| 欧美成狂野欧美在线观看| 男人舔女人的私密视频| 久久久久久亚洲精品国产蜜桃av| 国产xxxxx性猛交| 国产片内射在线| 免费高清视频大片| 99精品在免费线老司机午夜| 一个人免费在线观看的高清视频| 久久精品亚洲精品国产色婷小说| 免费人成视频x8x8入口观看| 亚洲人成网站在线播放欧美日韩| 一级作爱视频免费观看| 国产男靠女视频免费网站| 欧美日韩亚洲国产一区二区在线观看| 久久久国产欧美日韩av| 黄色视频不卡| 波多野结衣巨乳人妻| 黄频高清免费视频| 免费在线观看视频国产中文字幕亚洲| 亚洲国产精品成人综合色| 在线观看66精品国产| 后天国语完整版免费观看| 欧美成人午夜精品| 好男人在线观看高清免费视频 | 亚洲午夜理论影院| 亚洲av熟女| 国产亚洲av高清不卡| 一区在线观看完整版| 亚洲国产欧美日韩在线播放| 天堂动漫精品| a级毛片在线看网站| 老熟妇乱子伦视频在线观看| 91国产中文字幕| 亚洲av成人不卡在线观看播放网| 狂野欧美激情性xxxx| 精品人妻在线不人妻| 脱女人内裤的视频|