• <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 ?

    Structural characterization and immunostimulatory activity of a water-soluble polysaccharide from abalone (Haliotis discus hannai Ino) muscle

    2023-01-21 05:02:24LinfnShiGengxinHoJunChenJunlingWngWuyinWeng

    Linfn Shi,Gengxin Ho,Jun Chen,Junling Wng,Wuyin Weng,b,*

    a College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China

    b Engineering Research Center of the Modern Technology for Eel Industry,Ministry of Education,Xiamen 361021,China

    Keywords:Polysaccharide Abalone muscle Structural characterization Immunostimulatory activity Nuclear magnetic resonance spectra

    ABSTRACT A water-soluble polysaccharide from abalone muscle (AMPP) was isolated.The contents of carbohydrate,protein,uronic acid,and sulfate in AMPP were 83.5%,0.5%,2.7%,and 2.6%,respectively.High-performance liquid chromatography analysis indicated that AMPP was homogeneous and had an average molecular weight of approximately 3.2 kDa.The main monosaccharides of AMPP were glucose (Glc) and mannose with a molar ratio of 99.7:0.3.The structural characteristics of AMPP were elucidated through methylation analysis,Fourier transform infrared spectroscopy,and nuclear magnetic resonance spectroscopy.The linkages of AMPP consisted of terminal,1,4-linked,1,6-linked,and 1,4,6-linked Glcp with a molar ratio of 3.1:7.2:1.0:2.5.In one repeat unit of the proposed AMPP structure,the backbone chain was composed of eight 1→4 glycosidic bonds and one 1→6 glycosidic bond,with three branch chains linked by 1→6 glycosidic bond.In addition,AMPP was found to possess potent immunostimulatory activity via rising phagocytosis of RAW264.7 cells and promoting secretion of TNF-α.

    1.Introduction

    Abalone is an “archeogastropod” mollusk with a characteristic single shell,and it is considered a seafood delicacy with a high commercial value in Asian countries [1,2].Abalone is also regarded as “ginseng in the ocean” and has health-promoting effects because of its nutrients and bioactive components,including polysaccharides [3].Various polysaccharides isolated from the abalone muscle and viscera have also been studied.Zhu et al.[4]purified a neutral polysaccharide consisting of 1-,1,4-,1,6-,1,4,6-linked Glc and 1-,1,3-,1,6-,1,4,6-linked galactose (Gal) from the abalone muscle.Furthermore,several types of polysaccharides extracted from the gonad or viscera of abalone have been reported [5-8].Differences have also been found in the monosaccharide compositions of abalone polysaccharides from different regions [9].

    In a previous study [10],a crude polysaccharide was extracted from abalone (Haliotis discus hannaiIno) muscle (AMP) through protease hydrolysis and ethanol fractional precipitation.Three subfractions (AMP-1,AMP-2,and AMP-3) eluted with 0,0.1,and 0.3 mol/mL sodium chloride solutions were separated from AMP via DEAE-52 column chromatography.Among the subfractions,AMP-1 shows the highest inhibitory activities against the growth of human breast cancer MDA-MB-231 and hepatocellular carcinoma HepG2 cells.Furthermore,the carbohydrate and Glc contents (percentage of the carbohydrate) of the obtained AMP-1 reach 83.8% and 99.3%,respectively [10],which differ from those of the reported polysaccharide extracted from an abalone pleopod [6].The role of the immune system is to eliminate potentially harmful substances and prevent the growth of cancer cells [11].Thein vitrocell culture studies are often used to evaluate the immunomodulatory effects of seafood derived polysaccharides.A glucan fromCyclina sinensisexhibited significant ability to stimulate RAW264.7 cells to release tumor necrosis factor-alpha (TNF-α) [12].The release of TNF-α could be significantly promoted by a polysaccharide purified from jellyfish [13].However,information on the molecular structure and immunomodulatory activity of these polysaccharides has not been published.

    In the present study,an abalone polysaccharide (AMPP) was further purified from AMP-1 by using a Sephadex G-25 column.Then,the molecular weight and monosaccharide composition of AMPP were investigated.The structural characteristics of AMPP were revealed through methylation analysis,Fourier transform infrared spectroscopy (FTIR),and 1D (1H and13C),and 2D nuclear magnetic resonance (NMR) spectroscopy,including COSY,TOCSY,HSQC,HMBC,and ROESY.Last,the immunomodulatory activity of the obtained polysaccharide was also investigated to elucidate the antitumor mechanism.Our results indicated that the abalone muscle was a valuable source for polysaccharide,which exhibits immune function.

    2.Materials and methods

    2.1 Materials and chemicals

    Glc,Gal,glucuronic acid,galacturonic acid,glucosamine,galactosamine,sodium tetraborate,sodium borohydride,acetic anhydride,pyridine,and 1-phenyl-3-methyl-5-pyrazolone (PMP)were purchased from Sigma Chemical Co.(St.Louis,MO,USA).DEAE-52 cellulose and Sephadex G-25 were procured from Shanghai Yuanye Biological Technology Co.,Ltd.(Shanghai,China).All other chemicals used in the study were analytical grade.

    2.2 Purification of polysaccharide

    AMP-1,which was extracted in accordance with our previous study [10],was purified on a Sephadex G-25 column (400 mm ×16 mm) and eluted with distilled water at a flow rate of 0.5 mL/min.The collected eluent was monitored using the phenol-sulfuric acid method [14].The obtained polysaccharide fraction was dialyzed and lyophilized for further analyses.

    2.3 Component analysis

    Carbohydrate content was determined by phenol-sulfuric acid method by usingD-Glc as a reference [14].Protein content was determined with the Lowry method by using bovine serum albumin as a reference [15].Uronic acid content was measured with them-hydroxydiphenyl method withD-glucuronic acid as a reference [16].Sulfate content was determined with the barium chloride-gelatin method by using K2SO4as a reference [17].

    2.4 Monosaccharide composition

    Monosaccharide composition was analyzed in accordance with the reported method through reversed-phase high-performance liquid chromatography (HPLC) [18].Briefly,2 mg of each sample was hydrolyzed with 1.0 mL of 2.0 mol/L trifluoroacetic acid at 110 °C for 6 h.The resulting hydrolysate was derivatized with PMP.Then,the PMP-labeled carbohydrates were analyzed using an HPLC system(Agilent Technologies) equipped with an Atlantis?dC18column(4.6 mm × 250 mm,5 μm) and detected at UV 245 nm and a flow rate of 0.5 mL/min.The mobile phase was 0.1 mol/L phosphate buffer(pH 6.7) containing 17% acetonitrile.Mannose (Man),rhamnose,glucosamine,galactosamine,glucuronic acid,galacturonic acid,Glc,Gal,arabinose,and frucose were used as monosaccharide standards.

    2.5 FTIR spectroscopy

    FTIR spectroscopy was carried out at room temperature by using a Thermo Nicolet iS50 FTIR spectrometer (Thermo Scientific,Waltham,MA,USA).The spectra in the range of 4 000–400 cm?1were rationed,and the automatic signals gained were collected in 32 scans at a resolution of 4 cm?1.

    2.6 Homogeneity and molecular weight

    The molecular weight of the purified polysaccharide sample was determined through high-performance gel filtration chromatography(HPGFC) on a Waters 1525 instrument equipped with a 2410 refractive index detector.The samples dissolved in the mobile phase were loaded onto an HPGFC system with tandem analytical UtrahydrogelTMLinear (300 mm × 7.8 mm,2 μm).Elution was carried out with 0.1 mol/L NaNO3at a flow rate of 0.8 mL/min.Dextran T-150,Dextran T-40,Dextran T-10,and Dextran T-5 were used as standards.

    2.7 Methylation analysis and gas chromatography-mass spectrometry (GC-MS) assay

    Methylation analysis was carried out in accordance with the method of Needs and Sevendran [19]with minor modifications.The purified polysaccharide sample (5 mg) was decomposed in 2 mL of dimethyl sulfoxide before NaOH was added,and the mixture was stirred at room temperature for 90 min.The mixture was added with 1.5 mL of methyl iodine and stirred in darkness for 1 h.Then,methylation was terminated by adding 4 mL of distilled water.The methylated polysaccharides were extracted with chloroform and dried through evaporation under reduced pressure.The completely methylated polysaccharides were hydrolyzed with 2 mol/L trifluoroacetic acid at 110 °C for 6 h.The obtained hydrolysates were dissolved in 2 mL of 0.2 g/100 mL NaOH,and 10 mg of NaBH4was added to reduce uronic acid.After the obtained mixture was incubated at 25 °C for 4 h,glacial acetic acid (100 μL) was added to terminate the reduction.The reaction products were dried under reduced pressure and acetylated by adding 2 mL of acetic anhydride and 2 mL of pyridine at 100 °C for 1 h.After the remaining acetic anhydride in the reaction mixture was decomposed with distilled water,the acetylated derivatives were extracted with methylene chloride.The glycosidic linkage of the acetylated derivatives was analyzed through gas chromatography-mass spectrometry (GCMS-QP 2010,Shimadzu,Kyoto,Japan) equipped with an RTX-5 capillary column (60 m ×0.32 mm,0.25 μm,Restek Co.,Bellefonte,PA,USA).The temperature program was set as follows: the initial column temperature was 150 °C and held for 1 min,increased to 180 °C at 5 °C/min,further increased to 210 °C at 1.5 °C/min and held for 5 min,and increased to 280 °C at 40 °C/min.The injection temperature was 230 °C.The ion source of the mass spectrometer was set at 240 °C.

    2.8 NMR spectroscopy analysis

    The purified polysaccharide sample was dried in a vacuum oven with dried silica gel for several days.Approximately 30 mg of polysaccharide samples were then dissolved in 0.5 mL of D2O.1H and13C NMR experiments were recorded on a Bruker Avance III (400 MHz) spectrometer (Bruker,Rheinstetten,Germany) by using sodium 3-trimethysilyl propionate as a chemical shift reference.The spectra of HSQC,COSY,TOCSY,HMBC,and ROESY were also obtained.Experimental data were analyzed using MestReNOVA 5.3 (Mestrelab Research SL,CA).

    2.9 Immunostimulatory activity test in vitro

    2.9.1 Preparation of murine peritoneal macrophages

    Male BALB/c mice (18-22 g) were injected with 2 mL sterile thioglycollate medium.After 3 days,the peritoneal macrophages were harvested by peritoneal lavage and centrifugation at 4 °C,1 500 ×gfor 5 min.The precipitated cells were resuspended in RPMI-1640 and centrifuged to remove non-adherent cells.The cell viability of obtained macrophages was measured by trypan blue exclusive assay and reached up to 97% .

    2.9.2 Cell viability

    The peritoneal macrophages (1 × 106cells/mL) were plated in 96-well plates and incubated at 37 °C for 3 h in a 5% CO2incubator.After non-adherent cells were removed,the RPMI-1640 medium(without 10% FBS) containing series of AMPP concentrations (125,250,500,1 000 μg/mL) were added and incubated at 37 °C for 24 h in a 5% CO2incubator.The negative control and positive control groups were treated with RPMI-1640 medium containing 10% FBS and 1 μg/mL LPS,respectively.The cell viability was detected with a MTT kit at a wavelength of 570 nm and calculated using the following equation:

    Where the cell viability of peritoneal macrophages from control group was calculated as 100% .

    2.9.3 Phagocytosis assay

    The effect of AMPP on the phagocytic activity of peritoneal macrophages was measured using neutral red assay according to the method established by Sun et al.[20].The macrophage cells cultured according to the method described in Section 2.9.2 were added with neutral red solution (0.1 g/100 mL) and incubated at 37 °C for 60 min.After discarding supernatant,the neutral red was removed by washing with PBS three times.Then cell lysis buffer (acetic acid/ethanol,1:1;V/V) was added and kept at room temperature for 2 h.The absorbance of resulting solution was measured at 540 nm by a microplate reader.On the other hand,the TNF-α existed in the peritoneal macrophages were measured by ELISA kit according to the manufacturers’ manner.

    2.10 Statistical analysis

    Results of immunostimulatory activity testin vitrowere recorded as mean ± standard deviation (n=5),and significant differences between groups were tested using one-way ANOVA and Duncan’s test atP<0.05.Statistical analysis was conducted by using SPSS version 18.0 software (SPSS,Inc.,Chicago,IL,USA).

    3.Results and discussion

    3.1 Purification of polysaccharide

    The polysaccharide of AMP-1 was purified with the Sephadex G-25 column,and only one symmetrical fraction of AMPP was obtained from distilled water elution (Fig.1a).The obtained AMPP was white,and its carbohydrate,protein,uronic acid,and sulfate contents were (83.5 ± 3.0)%,(0.5 ± 0.1)%,(2.7 ± 0.3)%,and(2.6 ± 0.1)%,respectively.The recovery rate of AMPP based on the amount of AMP-1 was 77.7% .After HPGFC analysis,AMPP showed a single and symmetrical sharp peak (Fig.1b),indicating that AMPP was a highly homogeneous polysaccharide.The calculated average molecular weight of AMPP was approximately 3.2 kDa based on the calibration curve of dextran.

    Fig.1 Elution curve of polysacchride on a Sephadex G-25 column chromatography (a) and HPGFC profile of AMPP (b).

    3.2 Monosaccharide composition

    Fig.2 shows the HPLC profiles of PMP-labeled monosaccharide standards and AMPP.After comparing with the standard monosaccharide,it was found that the obtained AMPP was composed of Glc and Man with a molar ratio of 99.7:0.3.A neutral polysaccharide extracted from abalone pleopods through a proteolytic method with papain and followed by precipitation with ethanol and found that this polysaccharide is composed of Glc and Gal with a molar ratio of 1:1 [6].The obvious difference in the monosaccharide compositions of the polysaccharides between this study (Fig.2) and the previous report [6]may be associated with different extraction methods and cultivated regions of abalone [9].

    Fig.2 HPLC profiles of PMP derivatives of standard monosachhrides (a) and AMPP from abalone muscle (b).

    3.3 FTIR analysis

    The FTIR spectrum of AMPP is presented in Fig.3.The absorption peaks in the spectrum of AMPP were similar to those of polysaccharides from abalone pleopods [6].The absorption peaks around 3 335,2 928,1 644,and 1 024 cm?1in this study corresponded to the stretching vibration of O–H,asymmetrical stretching vibration of C–H,asymmetric stretching vibration of C=O,and stretching vibration of C–O,respectively [21].A previous study reported that a monosaccharide in a polysaccharide of Hawk mature leaf tea has a pyranose ring when strong absorption peaks appear in the range of 1 200–1 000 cm?1[22].The weak band at 1 078 cm?1and the intense band at 1 024 cm?1in AMPP were assigned to C–O and C–C stretching vibrations of a pyranose ring (Fig.3a),respectively.This characteristic of AMPP was similar to that of a polysaccharide from edible brown and red seaweeds [23].Furthermore,the characteristic absorption at 849 cm?1indicated thatα-configuration glycosidic bonds existed in the obtained AMPP (Fig.3a).When the polysaccharide is successfully methylated,the OH absorption peak around 3 400 cm?1decreases,whereas the CH3– absorption peak around 2 900 cm?1increases [24].In our study,the absorption band in the range of 3 000–2 800 cm?1strengthened after AMPP was methylated,whereas the absorption band around 3 335 cm?1decreased significantly (Fig.3b).However,when the AMPP was further methylated,a weak peak around 3 335 cm?1could be found due to the slight moisture content.Therefore,the result of Fig.3 suggested that AMPP was methylated completely.

    Fig.3 FTIR spectra of AMPP (a) and the methylated polysaccharide (b).

    3.4 Methylation and GC-MS spectra

    Generally,the position of glycosidic linkage in a polysaccharide can be observed through methylation analysis [25].The methylated AMPP was hydrolyzed with acid,converted into alditol acetate,and analyzed through GC-MS.The partially methylated monosaccharides were analyzed on the basis of retention times and mass spectra(Table 1).The linkages of AMPP consisted of terminal,1,4-linked,1,6-linked,and 1,4,6-linked Glcpwith a molar ratio of 3.1:7.2:1.0:2.5.The number of 1,4-linked and 1,4,6-linked Glcpresidues accounted for 70.18% of the total methylated monosaccharide residues,suggesting that the two residues could be the backbone of AMPP,and the branches were likely attached to the O-6 position of 1,4,6-linked Glcpresidues.This was similar to the report previously described by Hu et al.[26],who elucidated a water-soluble polysaccharide isolated from the stem barks ofAcanthopanax brachypus.Three polysaccharides were isolated fromDictyophora echinovolvata,which were conjectured to (1→4)-linked-glucans branched atO-6 based on the results of methylation and GC-MS analysis [27].The terminal Glcpwas also possibly the branch chain attached to the backbone of AMPP via a 1,6-glycosidic bond in present study.

    Table 1 Methylation analysis of AMPP from abalone muscle.

    3.5 NMR analysis

    The structural features of AMPP from the abalone muscle were further established through 1D and 2D NMR spectral analysis.The chemical shift of the anomeric proton inα-configuration residues was atδ4.8–5.3,and the vicinal coupling constant value was in the range of 2–4 Hz [28-30].Meanwhile,the chemical shift ofβ-configuration residues was atδ4.0–4.8,and the vicinal coupling constant value was in the range of 7–10 Hz [28-30].Considering that the sugar residues of AMPP were linked via anα-configuration glycosidic bond based on the results of FTIR analysis (Fig.3a),four anomeric proton signals atδ5.35 (d,3JH-1,H-2=4.13 Hz),5.33 (unresolved),5.24 (d,3JH-1,H-2=4.00 Hz),and 4.98 (d,3JH-1,H-2=4.13 Hz) in the1H-NMR spectrum(Fig.4a) were selected for analysis.

    Fig.4 1H NMR (a),13C NMR (b),HSQC (c),COSY (d) and TOCSY (e) spectra of AMPP from abalone muscle.

    The anomeric carbon signals atδ102.83,101.63,100.76,and 94.68 were observed in the13C NMR spectrum (Fig.4b),corresponding to theδ5.35,4.98,5.33,5.24 in the anomeric regions of HSQC (Fig.4c).The anomeric carbon signal atδ98.58 corresponds to theδ4.68,indicating that the residue isβ-configuration moiety.This is not consistent with the results of FTIR and1H-NMR analysis.Therefore,four glycosyl residues were assigned atδ5.35/102.83 (residue A),5.33/100.76 (residue B),5.24/94.68 (residue C),and 4.98/101.63 (residue D).The anomeric carbon signals in the13C NMR spectra (Fig.4b) also indicated that the obtained AMPP contained pyranose rings,and this observation was consistent with the FTIR result (Fig.3a).The results of the1H-NMR spectrum (Fig.4a)and methylation analysis (Table 1) suggested that the residues of A,B,C,and D were tentatively assigned to →4)-α-D-Glc-(1→,→4,6)-α-DGlcp-(1→,→6)-α-D-Glcp-(1→,and T-α-D-Glcp-(1→,respectively.

    The signals in1H NMR and13C NMR spectra were assigned on the basis of the results of HSQC,COSY,and TOCSY experiments and previous literatures [31-33],and they are and summarized in Table 2.The resonance in the region ofδ80.80 and 80.87 in the13C NMR spectrum indicated that substitutions at position C-4 of residues A and B were present.Similarly,substitutions at position C-6 of residues B and C were also observed.These results further indicated that the assignment of residues A,B,C,and D was reasonable.

    Table 2 Chemical shift assignment of 1H NMR and 13C NMR spectra of AMPP based on the data of COSY,TOCSY and HSQC.

    The sequence and linkage of the glycosyl residues of AMPP were completely elucidated by the HMBC and ROESY spectra.The cross peaks of both anomeric protons and carbons from the HMBC experiment of the sugar residues were examined,and the residual connectivities were observed (Fig.5a).Cross peaks were found between H-1 (δ5.35) and C-4 of residue A (A H-1/A C-1),H-4 of residue B (δ3.66) and C-1 of residue A (B H-4/A C-1),and H-1 of residue B (δ5.33) and C-4 of residue A (B H-1/A C-4).These results suggested that residue A was linked to the residues of A and B via a 1,4-glycosidic bond.

    Fig.5 HMBC (a) and ROESY (b) spectra of AMPP from abalone muscle.

    ROESY experiment was also carried out (Fig.5b).Cross peaks were identified between H-1 of residue A (δ5.35) and H-6 of residue C(A H-1/C H-6) and between H-1 of residue D (δ4.98) and H-6 of residue B (D H-1/B H-6).This result indicated that 1,6-glycosidic linkages were present in residues A and C and residues D and B of AMPP from abalone muscle.The cross peaks of A H-1/A H-4 and A H-1/B H-4 were also observed in the ROESY spectrum.

    A possible molecular structure of AMPP from abalone muscle(Fig.6) was proposed on the basis of the chemical and spectroscopic evidence presented above.In one repeat unit of the proposed AMPP structure,the backbone chain consisted of eight 1→4 glycosidic bonds and one 1→6 glycosidic bond,with three branch chains linked by a 1→6 glycosidic bond.Therefore,it can be concluded that AMPP belongs to theα-type glucan.Special glucans derived from mollusks have beneficial effects on immunomodulatory and antitumor activities and have been focused by many researchers,but it is still unknown how glucans generated in mollusks and what their biological activities are [34].We determined the monosaccharide composition of abalone viscera,connective tissues,pui-muscle and pleopod muscle (Fig.7),suggesting that the seaweeds fed by abalone are digested by various enzymes in the abalone viscera and gradually converted into Glc,which is absorbed and involved in the metabolism,and converted to glucans.Therefore,the sampling parts are very important for the analysis and comparison of abalone polysaccharides from different sources or cultivated regions.The next studies will be focused on the possible generation mechanisms of glucans in the abalone muscle.

    Fig.6 Proposed model for AMPP from abalone muscle.

    Fig.7 Monosaccharide compositions of abalone viscera,connective tissues,pui-muscle and pleopod muscle.

    3.6 Immunomodulatory activity in vitro

    The macrophages are important immunomodulatory effector cells,and have been widely used as cell model to investigate the immunomodulatory activity of food derived extracts [35].The effect of AMPP on the proliferation of Raw264.7 cells was investigated and shown in Fig.8a.No significant effects (P>0.05) of cell viability in the AMPP concentration range from 125 to 1 000 μg/mL,which was selected to assess the potential immunomodulatory activity of AMPP.

    Fig.8 Effect of AMPP on the proliferation (a),phagocytic activity (b),and TNF-α (c) of RAW264.7 cells.LPS (1 μg/mL) was used as positive control.Results were presented as mean ± SD from five replicates (n=5).Means with different superscript letters are significantly different (P <0.05) by Duncan’s test.

    The phagocytic activity of peritoneal macrophages RAW264.7 cells treated with AMPP was measured using neutral red assay and shown in Fig.8b.The phagocytosis of RAW264.7 cells treated with AMPP was increased (P<0.05) with increasing AMPP concentration,and all phagocytosis indices for AMPP at the test concentrations were higher than 1.0,indicating that the obtained AMPP had ability to activate phagocytic activity of RAW264.7 cells.A similar trend was also reported by Xiong et al.[36],who investigated inflammatory effect ofCyclocarya paliuruspolysaccharide in RAW264.7 macrophage.

    It is reported that TNF-α is a mediator of immune stimulation and inflammatory response,which plays an important role in the resistance of host cells to malignant tumor growth [37].Thus,the effect of AMPP on TNF-α was assayed and presented in the Fig.8c.AMPP could promote the production of TNF-α in a dose-dependent manner(P<0.05),which is similar to a glucan fromCyclina sinensis[8].This result further suggests that AMPP has immunostimulatory activity on macrophages,thus promoting the secretion of TNF-α to enhance the antitumor activity of polysaccharide.

    4.Conclusion

    In this study,AMPP was purified and the obtained AMPP was a highly homogeneous polysaccharide with an average molecular weight of approximately 3.2 kDa.Based on the results of methylation and NMR spectral analyses,our conclusion was that the main backbone of the proposed AMPP structure was composed of 1→4 and 1→6 linked Glc,with branches consisting ofα-D-Glcp-(1→6).The obtained structural information would be important for further studies on the structure–activity relationship of polysaccharide from abalone muscle.Immunological assaysin vitroshowed that AMPP has the ability to activate phagocytic activity of RAW264.7 cells and stimulate cells to release TNF-α.

    Conflicts of interest

    The authors declare that they have no conflicts of interest.

    Acknowledgments

    The authors are grateful for financial support received from the National Key R&D Program of China (2021YFD2100200/2021Y FD2100202),National Natural Science Fund (31571835),Fujian Key Project of Natural Science Foundation (2019J02013),and the Opening Project of Fujian Provincial Engineering Technology Research Center of Marine Functional Food (Z820239).

    免费看十八禁软件| 黑人欧美特级aaaaaa片| 黑人操中国人逼视频| 动漫黄色视频在线观看| 一进一出好大好爽视频| 亚洲七黄色美女视频| 久久性视频一级片| 精品日产1卡2卡| 丁香六月欧美| 午夜视频精品福利| 黄片小视频在线播放| 国产精品av久久久久免费| 成年人黄色毛片网站| 日本vs欧美在线观看视频| 黑人欧美特级aaaaaa片| 久久精品91蜜桃| 国产不卡一卡二| 91成年电影在线观看| 欧美在线一区亚洲| aaaaa片日本免费| 淫妇啪啪啪对白视频| a级毛片黄视频| 人人妻,人人澡人人爽秒播| 美女高潮喷水抽搐中文字幕| 久久天躁狠狠躁夜夜2o2o| 国产97色在线日韩免费| 国产熟女xx| 热re99久久精品国产66热6| 午夜a级毛片| 国产精品九九99| 日韩大码丰满熟妇| 自线自在国产av| 午夜福利一区二区在线看| 亚洲性夜色夜夜综合| 欧美日韩视频精品一区| 丰满人妻熟妇乱又伦精品不卡| 香蕉久久夜色| 亚洲一区二区三区色噜噜 | 国产精品二区激情视频| 狠狠狠狠99中文字幕| 亚洲人成伊人成综合网2020| 亚洲国产欧美网| 亚洲精品中文字幕在线视频| 人人妻人人爽人人添夜夜欢视频| 在线观看免费日韩欧美大片| 两个人看的免费小视频| 日韩中文字幕欧美一区二区| 在线免费观看的www视频| 精品福利永久在线观看| 国产成人欧美在线观看| 亚洲专区字幕在线| 亚洲国产精品sss在线观看 | 精品一区二区三区av网在线观看| 少妇 在线观看| 精品国产一区二区三区四区第35| 亚洲aⅴ乱码一区二区在线播放 | 欧美激情 高清一区二区三区| 满18在线观看网站| 亚洲精品国产一区二区精华液| 成人永久免费在线观看视频| 91字幕亚洲| 亚洲久久久国产精品| 中国美女看黄片| 亚洲国产中文字幕在线视频| 热99国产精品久久久久久7| 午夜免费成人在线视频| 久久人妻福利社区极品人妻图片| 少妇的丰满在线观看| 精品卡一卡二卡四卡免费| 国产欧美日韩一区二区三区在线| 国产免费现黄频在线看| 很黄的视频免费| 欧洲精品卡2卡3卡4卡5卡区| 水蜜桃什么品种好| 欧美午夜高清在线| 成人手机av| 久久久久精品国产欧美久久久| 又黄又爽又免费观看的视频| 丁香欧美五月| 夜夜爽天天搞| 中文字幕最新亚洲高清| 999久久久精品免费观看国产| 他把我摸到了高潮在线观看| a在线观看视频网站| svipshipincom国产片| 一边摸一边抽搐一进一出视频| 国产成人系列免费观看| 欧美在线一区亚洲| 欧美大码av| 一二三四在线观看免费中文在| 在线观看一区二区三区| 午夜福利在线观看吧| 日韩欧美在线二视频| 757午夜福利合集在线观看| 亚洲欧美日韩无卡精品| 精品福利永久在线观看| 最新美女视频免费是黄的| 亚洲人成网站在线播放欧美日韩| 免费在线观看完整版高清| 午夜福利在线观看吧| 夫妻午夜视频| 国产亚洲精品一区二区www| 亚洲伊人色综图| 久久久久久大精品| 少妇的丰满在线观看| 免费在线观看视频国产中文字幕亚洲| 男女下面进入的视频免费午夜 | 一区二区日韩欧美中文字幕| 中国美女看黄片| 国产高清videossex| 欧美精品亚洲一区二区| 日本精品一区二区三区蜜桃| 18禁黄网站禁片午夜丰满| 国产1区2区3区精品| 午夜视频精品福利| 黄色视频,在线免费观看| 免费在线观看亚洲国产| 欧美 亚洲 国产 日韩一| 在线视频色国产色| 日韩人妻精品一区2区三区| √禁漫天堂资源中文www| 一二三四在线观看免费中文在| 黄色女人牲交| 亚洲一卡2卡3卡4卡5卡精品中文| 91九色精品人成在线观看| 最好的美女福利视频网| 人成视频在线观看免费观看| 国产在线精品亚洲第一网站| 大型黄色视频在线免费观看| 黄频高清免费视频| 精品久久久久久电影网| 色播在线永久视频| 美女午夜性视频免费| 久久精品国产亚洲av高清一级| 老司机午夜福利在线观看视频| 亚洲va日本ⅴa欧美va伊人久久| 亚洲成人精品中文字幕电影 | 男男h啪啪无遮挡| 成人国产一区最新在线观看| 亚洲精品中文字幕一二三四区| 久久精品国产99精品国产亚洲性色 | 婷婷丁香在线五月| 亚洲三区欧美一区| 国产成人精品在线电影| 成人手机av| 高清av免费在线| 欧美国产精品va在线观看不卡| 巨乳人妻的诱惑在线观看| 日韩av在线大香蕉| 国产在线精品亚洲第一网站| 亚洲精品在线观看二区| 老鸭窝网址在线观看| 黄色丝袜av网址大全| 丰满饥渴人妻一区二区三| 亚洲熟妇中文字幕五十中出 | 丁香欧美五月| 国产精华一区二区三区| 18禁裸乳无遮挡免费网站照片 | a在线观看视频网站| 我的亚洲天堂| 午夜影院日韩av| 国产成人精品无人区| 亚洲精品国产一区二区精华液| 欧美乱码精品一区二区三区| 一区二区日韩欧美中文字幕| 久99久视频精品免费| 99国产精品99久久久久| 久久精品aⅴ一区二区三区四区| www.www免费av| 91老司机精品| 村上凉子中文字幕在线| 国产黄色免费在线视频| 我的亚洲天堂| 在线观看一区二区三区激情| 国产精华一区二区三区| 国产伦一二天堂av在线观看| 国产一卡二卡三卡精品| 极品人妻少妇av视频| 无限看片的www在线观看| 色在线成人网| 人成视频在线观看免费观看| 丁香六月欧美| 国产精品香港三级国产av潘金莲| 欧美人与性动交α欧美精品济南到| 麻豆一二三区av精品| 91成人精品电影| 在线看a的网站| 高清在线国产一区| 自线自在国产av| 日本a在线网址| 欧美中文综合在线视频| 午夜视频精品福利| 一个人观看的视频www高清免费观看 | 97超级碰碰碰精品色视频在线观看| 咕卡用的链子| 欧美老熟妇乱子伦牲交| 欧美人与性动交α欧美精品济南到| 99久久99久久久精品蜜桃| 一级a爱片免费观看的视频| 夜夜爽天天搞| 色综合婷婷激情| 免费少妇av软件| 亚洲精华国产精华精| 黄色怎么调成土黄色| 亚洲伊人色综图| 欧美在线一区亚洲| 看片在线看免费视频| 久久国产乱子伦精品免费另类| 人成视频在线观看免费观看| 国产成人免费无遮挡视频| 色在线成人网| 欧美乱妇无乱码| 亚洲av成人一区二区三| 国产一卡二卡三卡精品| 国产精品亚洲av一区麻豆| 一级a爱视频在线免费观看| 亚洲av成人av| 成熟少妇高潮喷水视频| 国产欧美日韩一区二区三区在线| 老鸭窝网址在线观看| 免费观看精品视频网站| 亚洲国产看品久久| 99热国产这里只有精品6| 免费日韩欧美在线观看| 亚洲国产看品久久| 无人区码免费观看不卡| 欧美不卡视频在线免费观看 | 国产亚洲精品综合一区在线观看 | 看免费av毛片| 国产高清激情床上av| 如日韩欧美国产精品一区二区三区| 韩国av一区二区三区四区| 精品人妻在线不人妻| 一区在线观看完整版| 亚洲中文日韩欧美视频| 国产欧美日韩一区二区三| 成人黄色视频免费在线看| 午夜成年电影在线免费观看| 99久久久亚洲精品蜜臀av| 女性生殖器流出的白浆| 在线播放国产精品三级| www.自偷自拍.com| 又大又爽又粗| 免费在线观看视频国产中文字幕亚洲| 在线免费观看的www视频| 日本撒尿小便嘘嘘汇集6| 欧美精品啪啪一区二区三区| 久久香蕉国产精品| 伦理电影免费视频| 亚洲国产精品sss在线观看 | 国产精品亚洲一级av第二区| 国产成年人精品一区二区 | 一区福利在线观看| 精品一区二区三区四区五区乱码| 日韩中文字幕欧美一区二区| 国产成人精品无人区| 脱女人内裤的视频| 日韩欧美一区视频在线观看| 免费在线观看亚洲国产| 亚洲一区二区三区色噜噜 | 亚洲国产欧美网| 日韩欧美一区二区三区在线观看| 桃色一区二区三区在线观看| 色哟哟哟哟哟哟| 免费女性裸体啪啪无遮挡网站| 久久人人爽av亚洲精品天堂| 亚洲国产看品久久| 操美女的视频在线观看| 悠悠久久av| 国产成人精品久久二区二区91| xxxhd国产人妻xxx| 一进一出好大好爽视频| 精品午夜福利视频在线观看一区| 91字幕亚洲| av超薄肉色丝袜交足视频| 免费一级毛片在线播放高清视频 | av超薄肉色丝袜交足视频| 一区二区三区国产精品乱码| 99热只有精品国产| 成人亚洲精品av一区二区 | 成人亚洲精品av一区二区 | 欧美日韩亚洲国产一区二区在线观看| 自线自在国产av| 久久精品成人免费网站| 免费在线观看日本一区| 波多野结衣av一区二区av| 欧美乱妇无乱码| 亚洲国产精品合色在线| 叶爱在线成人免费视频播放| 国产成人免费无遮挡视频| 少妇被粗大的猛进出69影院| 欧美一级毛片孕妇| 亚洲伊人色综图| 国产精品成人在线| 亚洲精品一卡2卡三卡4卡5卡| 成人亚洲精品一区在线观看| 日韩大尺度精品在线看网址 | 免费搜索国产男女视频| 午夜福利影视在线免费观看| 欧美日本中文国产一区发布| 欧美日韩亚洲综合一区二区三区_| 午夜影院日韩av| 欧美日本中文国产一区发布| 成人手机av| 免费在线观看日本一区| 三上悠亚av全集在线观看| 久久久国产精品麻豆| 很黄的视频免费| 欧美人与性动交α欧美软件| 国产一区在线观看成人免费| 免费搜索国产男女视频| 欧美大码av| 国产高清视频在线播放一区| videosex国产| 99久久精品国产亚洲精品| 99国产精品一区二区三区| 久久狼人影院| 日韩大码丰满熟妇| 亚洲精品av麻豆狂野| av视频免费观看在线观看| 国产区一区二久久| 欧美+亚洲+日韩+国产| 亚洲五月婷婷丁香| 国产免费男女视频| av视频免费观看在线观看| 一进一出抽搐动态| 老司机靠b影院| 18美女黄网站色大片免费观看| 国产成+人综合+亚洲专区| 在线天堂中文资源库| 高清av免费在线| 国产精品98久久久久久宅男小说| 亚洲自拍偷在线| 精品高清国产在线一区| 日韩中文字幕欧美一区二区| 亚洲熟妇中文字幕五十中出 | 欧美性长视频在线观看| 99精品欧美一区二区三区四区| 免费不卡黄色视频| 香蕉久久夜色| 在线观看免费视频网站a站| 又大又爽又粗| 热re99久久国产66热| 亚洲国产精品sss在线观看 | 国产精品 欧美亚洲| 久久精品91蜜桃| 久久久国产精品麻豆| www国产在线视频色| 一级黄色大片毛片| 天堂动漫精品| 午夜免费激情av| 精品国产乱子伦一区二区三区| 亚洲av成人不卡在线观看播放网| 男女之事视频高清在线观看| 免费av中文字幕在线| 欧美日韩瑟瑟在线播放| 少妇被粗大的猛进出69影院| 亚洲色图 男人天堂 中文字幕| 亚洲国产欧美网| 啦啦啦免费观看视频1| 男人的好看免费观看在线视频 | 不卡一级毛片| 精品久久久久久久毛片微露脸| 亚洲精品国产一区二区精华液| 涩涩av久久男人的天堂| 国产成人一区二区三区免费视频网站| 男人舔女人的私密视频| 日本一区二区免费在线视频| 成人av一区二区三区在线看| 亚洲一卡2卡3卡4卡5卡精品中文| 亚洲国产欧美日韩在线播放| 香蕉国产在线看| 一级黄色大片毛片| 国产黄色免费在线视频| 在线观看免费视频日本深夜| 免费看十八禁软件| 侵犯人妻中文字幕一二三四区| 狠狠狠狠99中文字幕| 午夜福利影视在线免费观看| 久久中文字幕一级| 国产熟女xx| 精品久久久久久久毛片微露脸| 黄色毛片三级朝国网站| 国产精品日韩av在线免费观看 | 国产欧美日韩一区二区三区在线| 欧美色视频一区免费| 日韩中文字幕欧美一区二区| 在线观看免费日韩欧美大片| 在线观看www视频免费| 麻豆av在线久日| 欧美日韩av久久| 亚洲av第一区精品v没综合| 级片在线观看| 午夜免费观看网址| 丰满迷人的少妇在线观看| 国产欧美日韩一区二区三| 多毛熟女@视频| 天天影视国产精品| 久久久国产成人精品二区 | 国产精品永久免费网站| 国产真人三级小视频在线观看| 国产成人欧美在线观看| 久久国产精品男人的天堂亚洲| 性欧美人与动物交配| 99国产极品粉嫩在线观看| 久久这里只有精品19| 精品电影一区二区在线| 日本一区二区免费在线视频| 日韩欧美国产一区二区入口| 老司机午夜十八禁免费视频| 久久久久亚洲av毛片大全| 日本wwww免费看| 好男人电影高清在线观看| 国产精品美女特级片免费视频播放器 | 国产欧美日韩一区二区三区在线| 18禁黄网站禁片午夜丰满| 大型黄色视频在线免费观看| x7x7x7水蜜桃| 国产三级在线视频| www国产在线视频色| 亚洲国产精品sss在线观看 | 免费看a级黄色片| 色婷婷av一区二区三区视频| 国产三级在线视频| 老司机午夜福利在线观看视频| 国产成人系列免费观看| 校园春色视频在线观看| 久久亚洲精品不卡| 伦理电影免费视频| 亚洲av美国av| 人人澡人人妻人| 一a级毛片在线观看| 自拍欧美九色日韩亚洲蝌蚪91| 日韩成人在线观看一区二区三区| 99久久综合精品五月天人人| 在线观看免费视频网站a站| 国产成人啪精品午夜网站| 亚洲片人在线观看| 成人影院久久| 亚洲熟妇中文字幕五十中出 | 亚洲国产精品一区二区三区在线| 亚洲色图av天堂| 亚洲精华国产精华精| 又紧又爽又黄一区二区| 嫁个100分男人电影在线观看| 久久精品91蜜桃| 后天国语完整版免费观看| 国产一区二区三区视频了| 国产熟女午夜一区二区三区| 男人舔女人的私密视频| 久久久久亚洲av毛片大全| 色综合婷婷激情| 他把我摸到了高潮在线观看| 夜夜夜夜夜久久久久| 国产成人欧美| 超色免费av| 国产精品免费视频内射| 99国产综合亚洲精品| 高潮久久久久久久久久久不卡| 欧美在线黄色| 精品久久久久久电影网| 香蕉久久夜色| 国产黄a三级三级三级人| 在线播放国产精品三级| 美女大奶头视频| 欧美色视频一区免费| 黄色成人免费大全| 免费观看人在逋| 日韩大尺度精品在线看网址 | 亚洲自拍偷在线| 麻豆国产av国片精品| 久热爱精品视频在线9| 日本免费a在线| 一区二区三区国产精品乱码| 国产黄a三级三级三级人| 在线国产一区二区在线| а√天堂www在线а√下载| 亚洲av五月六月丁香网| www.www免费av| 国产成人欧美| 99久久精品国产亚洲精品| 搡老岳熟女国产| svipshipincom国产片| 岛国视频午夜一区免费看| 日日爽夜夜爽网站| 精品国产乱码久久久久久男人| 欧美激情久久久久久爽电影 | 久久人妻av系列| 一二三四社区在线视频社区8| 在线观看免费高清a一片| 极品教师在线免费播放| 日韩精品中文字幕看吧| 18禁裸乳无遮挡免费网站照片 | 国产1区2区3区精品| 国产精品av久久久久免费| 久久久久国产精品人妻aⅴ院| 午夜福利在线观看吧| 久久久久久免费高清国产稀缺| 精品熟女少妇八av免费久了| 国产野战对白在线观看| 日韩欧美在线二视频| 波多野结衣av一区二区av| 国产精品久久电影中文字幕| 免费在线观看完整版高清| 国产1区2区3区精品| 欧美日韩乱码在线| 水蜜桃什么品种好| 午夜免费激情av| 日本免费一区二区三区高清不卡 | 99国产精品一区二区三区| 啪啪无遮挡十八禁网站| 热re99久久精品国产66热6| 无人区码免费观看不卡| 美女午夜性视频免费| 国产高清videossex| 日本黄色日本黄色录像| 午夜福利影视在线免费观看| 一级,二级,三级黄色视频| 国产精品免费视频内射| 久久亚洲真实| 国产精品1区2区在线观看.| 成年人黄色毛片网站| 亚洲精品中文字幕在线视频| 两性夫妻黄色片| 天堂俺去俺来也www色官网| 国产精品美女特级片免费视频播放器 | 美女午夜性视频免费| 国产高清国产精品国产三级| 久久香蕉国产精品| 757午夜福利合集在线观看| 一区二区三区精品91| 自线自在国产av| 老司机福利观看| 美女高潮喷水抽搐中文字幕| 色播在线永久视频| cao死你这个sao货| 999精品在线视频| 久久久久久亚洲精品国产蜜桃av| 婷婷六月久久综合丁香| 一级毛片女人18水好多| 国产亚洲精品久久久久5区| 九色亚洲精品在线播放| 久久香蕉激情| 搡老岳熟女国产| 9色porny在线观看| av在线天堂中文字幕 | 日韩欧美一区二区三区在线观看| 日日爽夜夜爽网站| 最近最新中文字幕大全免费视频| 在线观看www视频免费| 日本撒尿小便嘘嘘汇集6| 两个人免费观看高清视频| 国产99久久九九免费精品| 国产有黄有色有爽视频| 国产亚洲av高清不卡| 精品无人区乱码1区二区| 一区福利在线观看| 50天的宝宝边吃奶边哭怎么回事| 精品国产乱码久久久久久男人| 黑人巨大精品欧美一区二区mp4| 18禁黄网站禁片午夜丰满| av在线播放免费不卡| 免费观看精品视频网站| 亚洲av第一区精品v没综合| 丰满饥渴人妻一区二区三| 极品人妻少妇av视频| 午夜福利一区二区在线看| 麻豆一二三区av精品| 亚洲黑人精品在线| 亚洲成人久久性| 成年女人毛片免费观看观看9| 精品乱码久久久久久99久播| 国产亚洲精品综合一区在线观看 | 男人舔女人的私密视频| 高清av免费在线| 成年人免费黄色播放视频| 一本大道久久a久久精品| 嫁个100分男人电影在线观看| 日本五十路高清| 高清在线国产一区| 午夜福利在线免费观看网站| 久热爱精品视频在线9| 精品国产一区二区久久| 80岁老熟妇乱子伦牲交| 久久久久久久久中文| 亚洲国产精品一区二区三区在线| 麻豆久久精品国产亚洲av | 亚洲av美国av| 又黄又粗又硬又大视频| 欧美乱妇无乱码| 国产成人系列免费观看| 亚洲精品久久午夜乱码| 精品免费久久久久久久清纯| 亚洲精品中文字幕在线视频| 国产精品美女特级片免费视频播放器 | 黄色毛片三级朝国网站| 在线观看一区二区三区| 久久久久精品国产欧美久久久| 久久精品国产亚洲av香蕉五月| 高清av免费在线| 久久中文看片网| 欧美最黄视频在线播放免费 | 中文字幕色久视频| 久久精品国产亚洲av香蕉五月| 9色porny在线观看| 久久久久久大精品| 免费在线观看视频国产中文字幕亚洲| 欧美成人性av电影在线观看| 美女国产高潮福利片在线看| 超色免费av| 伊人久久大香线蕉亚洲五| 十分钟在线观看高清视频www| 国产不卡一卡二| 精品欧美一区二区三区在线| 97碰自拍视频|