[Objective] The study aimed to explore the antibacterial activity and stability of antagonistic bacteria P72 against wheat scab.[Method] The Bacillus subtilis P72 was inoculated into fermentation media with the inocul...[Objective] The study aimed to explore the antibacterial activity and stability of antagonistic bacteria P72 against wheat scab.[Method] The Bacillus subtilis P72 was inoculated into fermentation media with the inoculation amount 5%,then cultured on shaking table at 28 ℃ for 48 h and centrifuged at 8 000 r/min for 10 min.The supernatant of fermentation broth was purified and then the genetic stability,thermal stability and pH stability were detected.[Result] By DEAE-52 ion exchange chromatography,the protein eluted by 0.5 mol/L NaCl solution possessed the strongest antagonistic ability against wheat scab pathogen.The SDS-PAGE electrophoresis showed that the molecular weight of the purified protein with antibacterial effect was 40 kD.By stability test,the antibacterial substance produced by P72 strain showed heritable antagonistic activity,high stability below 60 ℃,stability to acid and unstability to alkali.[Conclusion] The antagonistic bacteria P72 had strong antagonistic ability to wheat scab pathogen,stable antibacterial activity and thermal stability,so it would possess a wide development prospect.展开更多
A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathi...A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.展开更多
Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl ...Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.展开更多
The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous...The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the ceils were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli ceils were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while fxozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol. L^-1 trehalose had the best effect in sertoli cells crvooreservation.展开更多
In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid ...In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid and 6% terpene trilactones) was used,After a pretreatment step,optimized by the uniform design method ,the concentrated intermediate extract with high content of GA and gb(+90%) was separated into the individual terpenes by preparative liquid chromatography eluted with petroleum ether-ethylacetate,Analysis of products was carried out by means of HPLC-ELSD(evaporative light -scattering detector),The results show that ginkgolides A and B are obtained in higher yield and better purity.展开更多
An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matog...An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.展开更多
Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chrom...Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography(RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC_(50) values of the purified peptides for ACE inhibition activity were 34.72 μmol L^(-1), 20.55 μmol L^(-1) and 22.77 μmol L^(-1), respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.展开更多
To further utilize bioactive substance such as bovine colostrum sIgA and IgG, sIgA and IgG were isolated and purified simultaneously by salting out, ultrafiltration and gel chromatography, etc. The analysis of results...To further utilize bioactive substance such as bovine colostrum sIgA and IgG, sIgA and IgG were isolated and purified simultaneously by salting out, ultrafiltration and gel chromatography, etc. The analysis of results were showed quantitatively by nonhydrogenized SDS-PAGE, and quanlities of sIgA and IgG were respectively detected by Western Blot. The results showed that the purity and yield of bovine colostrum sIgA were 85.3% and 42.8%, respectively, while the purity and yield of bovine colostrum IgG were respectively 97.2% and 64.4%. This preparative method provides theoretical and experimental foundation for sIgA industrial production.展开更多
BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics o...BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil (E. coil) JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION: From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.展开更多
A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.
D-allulose has very little content in nature,and it needs to be synthesized artificially and meet the purity requirements of industrial grade.The basic physical and chemical properties of D-allulose,its preparation me...D-allulose has very little content in nature,and it needs to be synthesized artificially and meet the purity requirements of industrial grade.The basic physical and chemical properties of D-allulose,its preparation methods and many different ways of isolation and purification were described.In order to achieve the goal of industrial production of D-allulose as soon as possible,the research progress of D-allulose isolation and purification technologies at home and abroad in recent years was classified and discussed,so as to provide useful reference for the practical improvement of D-allulose isolation and purification process technologies.展开更多
A strain (T3) of Bacillus has been screened from paddy field. It secretes large amount of antibacterialproteins which show a strong inhibiting activity against several pathogens of rice. This paper presentsa systemati...A strain (T3) of Bacillus has been screened from paddy field. It secretes large amount of antibacterialproteins which show a strong inhibiting activity against several pathogens of rice. This paper presentsa systematic study of the inhibition spectrum and characteristics of T3 proteins. Total proteins wereprecipitated with ammonium sulfate at 70% saturation from cell-free culture. One of the proteins(Tzl) was purified from the crude extracts with Sephadex G-100, DEAE52 and FPLC Superose 12columns. A single band was demonstrated in both 15% SDS-PAGE and IEF, with an apparent MWof 6,9 kd and a pI of 7.8. Its amino acid composition was analyzed and part of its sequence,determined.展开更多
Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solu...Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solution containing collagenase,then digested and isolated.展开更多
Ampelopsis grossedentata(Hand-Mazz) W. T. wang is a typical edible plant with important physiological health functions. Dihydromyricetin is the main active ingredient of A. grossedentata, which has good pharmacologica...Ampelopsis grossedentata(Hand-Mazz) W. T. wang is a typical edible plant with important physiological health functions. Dihydromyricetin is the main active ingredient of A. grossedentata, which has good pharmacological effects such as antibacterial, anti-inflammatory, anti-oxidation, anti-tumor and liver protecting effects. This paper summarized the research progress on the extraction materials, extraction methods and separation and purification processes of dihydromyricetin in A. grossedentata to understand the best experimental method of dihydromyricetin, aiming to provide an important theoretical basis for the comprehensive development and utilization of dihydromyricetin.展开更多
Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and disc...Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...展开更多
Phytochemical investigation of the vinegar-prepared Corydalis yanhusuo led to the isolation of one aristolactam derivative,1,2,8,9-tetramethoxy-5-methyldibenzo[cd,f]indol-4-(5H)-one(1),and seven aporphine alkaloids,in...Phytochemical investigation of the vinegar-prepared Corydalis yanhusuo led to the isolation of one aristolactam derivative,1,2,8,9-tetramethoxy-5-methyldibenzo[cd,f]indol-4-(5H)-one(1),and seven aporphine alkaloids,including 2,9,10-trimethoxydibenz[de,g]quinolin-7-one(2),1-hydroxy-2,9,10-trimethoxy-7H-dibenzo(de,g)quinoline-7-one(3),oxoglaucine(4),N-methyloxoglaucine trifluoroacetate trifluoroacetate(5),corunine acetate(6),pontevedrine(7),and oxoglaucidaline trifluoroacetate(8).The structures of the isolated compounds were elucidated by extensive spectroscopic data analysis and comparison with the previous reports.Among them,compounds 1 and 2 were obtained as natural products for the first time,and their NMR data were unambiguously assigned.In addition,compound 1 exhibited moderate cytotoxic activity against HepG2 cells with an IC50 value of 16.0±6.4μM.展开更多
Water-soluble polysaccharides were prepared from Panax japonicus by hot water extraction and ethanol precipitation.The polysaccharides were further purified by ion exchange chromatography to obtain neutral and acidic ...Water-soluble polysaccharides were prepared from Panax japonicus by hot water extraction and ethanol precipitation.The polysaccharides were further purified by ion exchange chromatography to obtain neutral and acidic polysaccharides.The neutral polysaccharide fraction mainly contained Glc(90.2%),which was a glucan fraction.The acidic polysaccharide fraction mainly contained GalA(43.6%),Gal(21.7%),and Ara(15.4%),with a degree of methyl-esterification of 20.3%,which was a pectic polysaccharide.The acidic polysaccharide of Panax japonicus could effectively inhibit the replication of human seasonal influenza virus H1N1 and canine influenza virus H3N2 in MDCK cells and A549 cells and significantly reduce the virus titer in infected cells.It also effectively inhibited the number of infected cells of the SARS-CoV-2 South Africa strain and the Omicron strain.The acid polysaccharide of Panax japonicus showed good efficacy against influenza virus and COVID-19 infection,which could be used as a potential antiviral candidate drug molecule in the future.展开更多
A water-soluble polysaccharide, HM(41), was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM(41)...A water-soluble polysaccharide, HM(41), was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM(41) was composed of rhamnose(Rha), arabinose(Ara), xylose(Xyl), mannose(Man),galactose(Gal), glucose(Glc) with a molar ratio of 1.0:5.5:1.8:3.0:9.4:21. The average molecular weight of HM(41) was approximately 1.17 * 10~4. Periodate oxidation, Smith degradation, methylation and GC, IR,NMR, XRD, GC–MS analysis were used for the structural analysis of HM(41). Its main chain was composed mainly of β-(1→4)Gal, β-(1→4)Glc and β-(1→6)Glc. β-(1 →4)Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues;(1→ 4)Glc had no branch;(1→6)Glc were substituted at 3-O and on average there were 9 branches among 14 main chain residues. The side chain was composed of(1→3,6)-Rha,(1→4)/(1→5)-Ara,(1 →4)/(1→5)-Xyl,(1→4,6)-Man and(1→2)-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM(41) had strong scavenging activities in vitro hydroxyl. Overall, HM and HM(41) may have potential applications in the antioxidants for medical and food industry.展开更多
OBJECTIVE: To evaluate the antioxidant and immunomodulatory activities of a unique polysaccharide from the medicinal fungus Flammulina velutipes in vitro.METHODS: Using water extraction and alcohol precipitation, crud...OBJECTIVE: To evaluate the antioxidant and immunomodulatory activities of a unique polysaccharide from the medicinal fungus Flammulina velutipes in vitro.METHODS: Using water extraction and alcohol precipitation, crude polysaccharides were obtained. After purification by DEAE-cellulose 52 ion exchange chromatography and Sephacryl S-300 HR gel filtration chromatography, High performance liquid chromatography equipped with evaporative light-scattering detector, Infrared radiation and Nuclear magnetic resonance were used to evaluate the structure of the polysaccharide. Its immunomodulatory activity was measured by examining the production of nitric oxide(NO) and cytokine secretion, and via lymphocyte proliferation experiments. Its effects on the scavenging activities of hydroxyl radical, superoxide anion and reducing power were also measured.RESULTS: A water-soluble polysaccharide, Flammulina velutipes polysaccharide I-A(FVP I-A), was obtained with a molecular mass of 8.14×104Da determined by high performance gel permeation chromatography. An in vitro antioxidant assay indicated that FVP I-A could scavenge hydroxyl radical, superoxide anion and possessed reducing power and could largely promote NO production and augment the interleukin-1β, interleukin-6, and tumor necrosis factor-α secretion by RAW264.7 macrophages(P<0.05). Moreover, FVP I-A could promote lymphocyte proliferation(P<0.05), and synergistically enhance the augmentation of the proliferation of mouse lymphocytes by concanavalin A and lipopolysaccharides(P<0.01, P<0.05).CONCLUSION: The FVP I-A obtained from Flammulina velutipes possessed antioxidant activity and could enhance non-specific and specific immune responses in vitro.展开更多
文摘[Objective] The study aimed to explore the antibacterial activity and stability of antagonistic bacteria P72 against wheat scab.[Method] The Bacillus subtilis P72 was inoculated into fermentation media with the inoculation amount 5%,then cultured on shaking table at 28 ℃ for 48 h and centrifuged at 8 000 r/min for 10 min.The supernatant of fermentation broth was purified and then the genetic stability,thermal stability and pH stability were detected.[Result] By DEAE-52 ion exchange chromatography,the protein eluted by 0.5 mol/L NaCl solution possessed the strongest antagonistic ability against wheat scab pathogen.The SDS-PAGE electrophoresis showed that the molecular weight of the purified protein with antibacterial effect was 40 kD.By stability test,the antibacterial substance produced by P72 strain showed heritable antagonistic activity,high stability below 60 ℃,stability to acid and unstability to alkali.[Conclusion] The antagonistic bacteria P72 had strong antagonistic ability to wheat scab pathogen,stable antibacterial activity and thermal stability,so it would possess a wide development prospect.
文摘A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.
基金supported by the Marine S&T Fund of Shandong Province for the Pilot National Laboratory for Marine Science and Technology (Qingdao) (Nos. 2018 SDKJ0304-4-2, 2018SDKJ0303-1)the Central Public-Interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences (Nos. 2017HY-XKQ01-01, 2016ZD0902, 2018GH10)+3 种基金the Central Public-Interest Scientific Institution Basal Research Fund, YSFRI, CAFS (No. 20603022018025)the Aoshan S&T Innovation Project from Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02-02-04)the Antarctic Marine Biological Resources Development and Utilization Project from the Ministry of Agriculture and Rural Affairs, People’s Republic of China (2017)the Financial Fund of the Ministry of Agriculture and Rural Affairs, People’s Republic of China (Nos. NFZX2018, FSTICE2019)
文摘Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.
基金Supported by Scientific Research Foundation for Doctors of Northeast Agricultural University (2012RCB27)Postdoctoral Fund of Heilongjiang Provincial Academy of Agricultural Sciences (LRB04-185)
文摘The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the ceils were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli ceils were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while fxozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol. L^-1 trehalose had the best effect in sertoli cells crvooreservation.
基金Supported by the Natural Science Foundation of Tianjin(No.993606911).
文摘In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid and 6% terpene trilactones) was used,After a pretreatment step,optimized by the uniform design method ,the concentrated intermediate extract with high content of GA and gb(+90%) was separated into the individual terpenes by preparative liquid chromatography eluted with petroleum ether-ethylacetate,Analysis of products was carried out by means of HPLC-ELSD(evaporative light -scattering detector),The results show that ginkgolides A and B are obtained in higher yield and better purity.
基金Supported by the Science and Technology Plan of Liaoning Province, China(No.2006226002)the Project of the Doctor Fund of Hebei University of Science and Technology, China(No.005121)
文摘An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.
基金supported by research grant of Guangxi Key Laboratory Traditional Chinese Medicine Quality Standards (No. GXGZZK201501)the Open Research Fund Program of Guangxi Key Laboratory of Marine Biotechnology (No. GLMBT-201407)+1 种基金partly supported by Shanghai Fengxian District Science and Technology Project (Nos. 20141001 and 20151205)Shanghai No. 6 People’s Medical Group Project and research project of Shanghai municipal health and Family Planning Commission (No. 201540027)
文摘Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography(RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC_(50) values of the purified peptides for ACE inhibition activity were 34.72 μmol L^(-1), 20.55 μmol L^(-1) and 22.77 μmol L^(-1), respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.
文摘To further utilize bioactive substance such as bovine colostrum sIgA and IgG, sIgA and IgG were isolated and purified simultaneously by salting out, ultrafiltration and gel chromatography, etc. The analysis of results were showed quantitatively by nonhydrogenized SDS-PAGE, and quanlities of sIgA and IgG were respectively detected by Western Blot. The results showed that the purity and yield of bovine colostrum sIgA were 85.3% and 42.8%, respectively, while the purity and yield of bovine colostrum IgG were respectively 97.2% and 64.4%. This preparative method provides theoretical and experimental foundation for sIgA industrial production.
基金Supported by:the National Natural Science Foundation of China,No.30600224Supported by:the National Natural Science Foundation of China,No.30700438+2 种基金China's Post-doctoral Science Fund, No.20060390886Hunan Province Natural Science Foundation,No.06JJ30014 Hunan Province Scientific Program,No.2008FJ3138
文摘BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil (E. coil) JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION: From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.
文摘A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.
基金Shandong Province Key R&D Program(Major Innovation Project)(No.2020CXGC010603,No.2019JZZY011003)National Natural Science Foundation of China(No.31801527)Taishan Industry Leading Talent Project(No.tscy20180103).
文摘D-allulose has very little content in nature,and it needs to be synthesized artificially and meet the purity requirements of industrial grade.The basic physical and chemical properties of D-allulose,its preparation methods and many different ways of isolation and purification were described.In order to achieve the goal of industrial production of D-allulose as soon as possible,the research progress of D-allulose isolation and purification technologies at home and abroad in recent years was classified and discussed,so as to provide useful reference for the practical improvement of D-allulose isolation and purification process technologies.
文摘A strain (T3) of Bacillus has been screened from paddy field. It secretes large amount of antibacterialproteins which show a strong inhibiting activity against several pathogens of rice. This paper presentsa systematic study of the inhibition spectrum and characteristics of T3 proteins. Total proteins wereprecipitated with ammonium sulfate at 70% saturation from cell-free culture. One of the proteins(Tzl) was purified from the crude extracts with Sephadex G-100, DEAE52 and FPLC Superose 12columns. A single band was demonstrated in both 15% SDS-PAGE and IEF, with an apparent MWof 6,9 kd and a pI of 7.8. Its amino acid composition was analyzed and part of its sequence,determined.
文摘Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solution containing collagenase,then digested and isolated.
基金Supported by Project of Guilin Science and Technology Bureau(20140105-12)
文摘Ampelopsis grossedentata(Hand-Mazz) W. T. wang is a typical edible plant with important physiological health functions. Dihydromyricetin is the main active ingredient of A. grossedentata, which has good pharmacological effects such as antibacterial, anti-inflammatory, anti-oxidation, anti-tumor and liver protecting effects. This paper summarized the research progress on the extraction materials, extraction methods and separation and purification processes of dihydromyricetin in A. grossedentata to understand the best experimental method of dihydromyricetin, aiming to provide an important theoretical basis for the comprehensive development and utilization of dihydromyricetin.
基金supported by grants from National Natural Sciences Foundation of China(No.30672243,30671093)
文摘Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...
基金This work was financially supported by Beijing Natural Science Foundation(Grant No.JQ18026)the National Key R&D Program of China(Grant No.2017YFC1700400)+1 种基金the National Natural Science Foundation of China(Grant No.82073978)the Fundamental Research Funds for the Central Universities(Grant No.2021-BUCMXJKY007).
文摘Phytochemical investigation of the vinegar-prepared Corydalis yanhusuo led to the isolation of one aristolactam derivative,1,2,8,9-tetramethoxy-5-methyldibenzo[cd,f]indol-4-(5H)-one(1),and seven aporphine alkaloids,including 2,9,10-trimethoxydibenz[de,g]quinolin-7-one(2),1-hydroxy-2,9,10-trimethoxy-7H-dibenzo(de,g)quinoline-7-one(3),oxoglaucine(4),N-methyloxoglaucine trifluoroacetate trifluoroacetate(5),corunine acetate(6),pontevedrine(7),and oxoglaucidaline trifluoroacetate(8).The structures of the isolated compounds were elucidated by extensive spectroscopic data analysis and comparison with the previous reports.Among them,compounds 1 and 2 were obtained as natural products for the first time,and their NMR data were unambiguously assigned.In addition,compound 1 exhibited moderate cytotoxic activity against HepG2 cells with an IC50 value of 16.0±6.4μM.
基金supported by National Key Research and Development Program of China(2023YFC0871100)
文摘Water-soluble polysaccharides were prepared from Panax japonicus by hot water extraction and ethanol precipitation.The polysaccharides were further purified by ion exchange chromatography to obtain neutral and acidic polysaccharides.The neutral polysaccharide fraction mainly contained Glc(90.2%),which was a glucan fraction.The acidic polysaccharide fraction mainly contained GalA(43.6%),Gal(21.7%),and Ara(15.4%),with a degree of methyl-esterification of 20.3%,which was a pectic polysaccharide.The acidic polysaccharide of Panax japonicus could effectively inhibit the replication of human seasonal influenza virus H1N1 and canine influenza virus H3N2 in MDCK cells and A549 cells and significantly reduce the virus titer in infected cells.It also effectively inhibited the number of infected cells of the SARS-CoV-2 South Africa strain and the Omicron strain.The acid polysaccharide of Panax japonicus showed good efficacy against influenza virus and COVID-19 infection,which could be used as a potential antiviral candidate drug molecule in the future.
基金supported by the First-Class Discipline Construction Project for First-Class University of Minzu University of China (No. YLDX01013),111 Project (No. B08044)The ecological conservation technology integration and demonstration in north of Dzungaria (No. 2014BAC15B04)
文摘A water-soluble polysaccharide, HM(41), was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM(41) was composed of rhamnose(Rha), arabinose(Ara), xylose(Xyl), mannose(Man),galactose(Gal), glucose(Glc) with a molar ratio of 1.0:5.5:1.8:3.0:9.4:21. The average molecular weight of HM(41) was approximately 1.17 * 10~4. Periodate oxidation, Smith degradation, methylation and GC, IR,NMR, XRD, GC–MS analysis were used for the structural analysis of HM(41). Its main chain was composed mainly of β-(1→4)Gal, β-(1→4)Glc and β-(1→6)Glc. β-(1 →4)Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues;(1→ 4)Glc had no branch;(1→6)Glc were substituted at 3-O and on average there were 9 branches among 14 main chain residues. The side chain was composed of(1→3,6)-Rha,(1→4)/(1→5)-Ara,(1 →4)/(1→5)-Xyl,(1→4,6)-Man and(1→2)-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM(41) had strong scavenging activities in vitro hydroxyl. Overall, HM and HM(41) may have potential applications in the antioxidants for medical and food industry.
基金the Fungus Medicine Research and Development-Innovation Team of Science and Technology in Sichuan Province,the Key Project of Deep Processing Research Positions Construction in Sichuan Province[No.Sichuan Agriculture(2009)75]Fungi Herbs Research and Development of Scientific and Technological Innovation in Sichuan Province Team,Second Five Breeding Research Project in Sichuan Province"Collection of Mushrooms Medicinal Quality Germplasm Resources and Breeding of New Materials"(No.2011nz0098-12-04)+2 种基金The Sichuan Province Microbial Resource Sharing Platform-Agricultural Microbiology Platform ProjectA Major Science And Technology Projects of Sichuan Province"Industrial Chain Integration of Key Technology Research and Industrialization Demonstration of Edibleand Medicinal Fungi Modern"The Project of Eight Technology Industries in Chengdu:the Key Technology Research and Application of Edible And Medicinal Mushroom With Deep Processing
文摘OBJECTIVE: To evaluate the antioxidant and immunomodulatory activities of a unique polysaccharide from the medicinal fungus Flammulina velutipes in vitro.METHODS: Using water extraction and alcohol precipitation, crude polysaccharides were obtained. After purification by DEAE-cellulose 52 ion exchange chromatography and Sephacryl S-300 HR gel filtration chromatography, High performance liquid chromatography equipped with evaporative light-scattering detector, Infrared radiation and Nuclear magnetic resonance were used to evaluate the structure of the polysaccharide. Its immunomodulatory activity was measured by examining the production of nitric oxide(NO) and cytokine secretion, and via lymphocyte proliferation experiments. Its effects on the scavenging activities of hydroxyl radical, superoxide anion and reducing power were also measured.RESULTS: A water-soluble polysaccharide, Flammulina velutipes polysaccharide I-A(FVP I-A), was obtained with a molecular mass of 8.14×104Da determined by high performance gel permeation chromatography. An in vitro antioxidant assay indicated that FVP I-A could scavenge hydroxyl radical, superoxide anion and possessed reducing power and could largely promote NO production and augment the interleukin-1β, interleukin-6, and tumor necrosis factor-α secretion by RAW264.7 macrophages(P<0.05). Moreover, FVP I-A could promote lymphocyte proliferation(P<0.05), and synergistically enhance the augmentation of the proliferation of mouse lymphocytes by concanavalin A and lipopolysaccharides(P<0.01, P<0.05).CONCLUSION: The FVP I-A obtained from Flammulina velutipes possessed antioxidant activity and could enhance non-specific and specific immune responses in vitro.