[Objectives] To provide experimental basis for the effective development and utilization of Huoshan large-leaf yellow tea resources and the screening of safe and effective active ingredients of large-leaf yellow tea. ...[Objectives] To provide experimental basis for the effective development and utilization of Huoshan large-leaf yellow tea resources and the screening of safe and effective active ingredients of large-leaf yellow tea. [Methods] The active substances of Huoshan large-leaf yellow tea were extracted by hot-water extraction, and the freeze-dried powder of Huoshan large-leaf yellow tea was obtained by freeze drying. The antibacterial activity of the extract was preliminarily confirmed using the Oxford cup method, and its antimicrobial spectrum was analyzed using 14 strains. A xylene-induced mouse auricle swelling test was carried out to detect the swelling inhibition rate of the extract and analyze its in-vitro detumescent activity. Then, the antioxidant activity of the extract was identified through a DPPH free radical scavenging capacity test and a ferric reducing antioxidant power assay. [Results] The extract had significant inhibitory effects on various bacteria. The extract could effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, Enterococcus hirae, Acinetobacter baumannii, Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus subtilis , and other strains. The diameter of the inhibition zone increased with the increase of sample concentration. The extract had a significant inhibitory effect on auricle swelling induced by xylene in mice. When the concentration of the drug reached 1.0 mg/mL, its inhibition rate on mouse auricle swelling reached 55.2% ( P <0.01), slightly lower than the swelling inhibition rate of the aspirin group (66.52%, P <0.01). The results of the antioxidant test showed that large-leaf yellow tea extract also had strong activity. Within the concentration range of 0.1-1.0 mg/mL, its DPPH radical scavenging rate increased with the increase of sample concentration. Within the concentration range of 0.1-1.0 mg/mL, its DPPH radical scavenging rate increased with the increase of sample concentration. When the concentration reached 1.0 mg/mL, the scavenging rate reached 69.75%. The Fe 3+ -reduction capacity of the extract also increased with the increase of sample concentration within the concentration range of 0.1-2.5 mg/mL. When the concentration was 2.5 mg/mL, the reducing power of the extract reached 1.43±0.04. However, its DPPH free radical scavenging rate and reducing power were slightly lower than the capacity of V C at the same concentration. [Conclusions] The extract of Huoshan large-leaf yellow tea obtained by hot-water extraction had strong activity in many aspects, including inhibiting the growth of various microbes, subsiding swelling in vitro and resisting oxidation. These experimental results provide certain guiding significance for the basic research of Huoshan large-leaf yellow tea extract, as well as experimental data support for the subsequent development of functional foods and drugs of Huoshan large-leaf yellow tea.展开更多
[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different ...[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.展开更多
基金Supported by Natural Science Foundation of Anhui Higher Education Institutions of China(KJ2021A0922)Anhui Provincial Natural Science Foundation of China(2008085MC65)+1 种基金China Postdoctoral Science Foundation(2020T130117ZX,2020M671914)Open Fund of Anhui Provincial Engineering Laboratory of Exploitation and Utilization of Medicinal and Food Homologous Natural Resources(YSTY2022005).
文摘[Objectives] To provide experimental basis for the effective development and utilization of Huoshan large-leaf yellow tea resources and the screening of safe and effective active ingredients of large-leaf yellow tea. [Methods] The active substances of Huoshan large-leaf yellow tea were extracted by hot-water extraction, and the freeze-dried powder of Huoshan large-leaf yellow tea was obtained by freeze drying. The antibacterial activity of the extract was preliminarily confirmed using the Oxford cup method, and its antimicrobial spectrum was analyzed using 14 strains. A xylene-induced mouse auricle swelling test was carried out to detect the swelling inhibition rate of the extract and analyze its in-vitro detumescent activity. Then, the antioxidant activity of the extract was identified through a DPPH free radical scavenging capacity test and a ferric reducing antioxidant power assay. [Results] The extract had significant inhibitory effects on various bacteria. The extract could effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, Enterococcus hirae, Acinetobacter baumannii, Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus subtilis , and other strains. The diameter of the inhibition zone increased with the increase of sample concentration. The extract had a significant inhibitory effect on auricle swelling induced by xylene in mice. When the concentration of the drug reached 1.0 mg/mL, its inhibition rate on mouse auricle swelling reached 55.2% ( P <0.01), slightly lower than the swelling inhibition rate of the aspirin group (66.52%, P <0.01). The results of the antioxidant test showed that large-leaf yellow tea extract also had strong activity. Within the concentration range of 0.1-1.0 mg/mL, its DPPH radical scavenging rate increased with the increase of sample concentration. Within the concentration range of 0.1-1.0 mg/mL, its DPPH radical scavenging rate increased with the increase of sample concentration. When the concentration reached 1.0 mg/mL, the scavenging rate reached 69.75%. The Fe 3+ -reduction capacity of the extract also increased with the increase of sample concentration within the concentration range of 0.1-2.5 mg/mL. When the concentration was 2.5 mg/mL, the reducing power of the extract reached 1.43±0.04. However, its DPPH free radical scavenging rate and reducing power were slightly lower than the capacity of V C at the same concentration. [Conclusions] The extract of Huoshan large-leaf yellow tea obtained by hot-water extraction had strong activity in many aspects, including inhibiting the growth of various microbes, subsiding swelling in vitro and resisting oxidation. These experimental results provide certain guiding significance for the basic research of Huoshan large-leaf yellow tea extract, as well as experimental data support for the subsequent development of functional foods and drugs of Huoshan large-leaf yellow tea.
基金Supported by Anhui Provincial Natural Science Foundation of China(2008085MC65)Natural Science Foundation of Anhui Higher Education Institutions of China(KJ2021A0922)+1 种基金China Postdoctoral Science Foundation(2020T130117ZX,2020M671914)Research Activities of Postdoctoral Researchers Foundation of Anhui Province,China(2020B470)。
文摘[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.
