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This research established a high-performance liquid chromatography(HPLC) method for simultaneous determination of isoorientin, orientin, vitexin, and isovitexin in Commelina communis to conduct content difference analysis and quality evaluation of 62 batches of C. communis from different origins. The HPLC content determination was performed on a Dikma Platisil ODS chromatographic column(4.6 mm×250 mm, 5 μm), with acetonitrile-0.1% formic acid(14∶86) as the mobile phase. The detection wavelength was set at 348 nm, the flow rate was 1.0 mL·min~(-1), and the column temperature was 35 ℃. The differences in origins and quality of 62 batches of C. communis were studied by chemometrics. The results showed that the determination of four components mani-fested a good linear relationship in the range of mass concentration(r>0.999 9), and the average recovery rate was 96.17%-103.0%. The relative standard deviations(RSDs) of precision, stability, and repeatability were all less than 2.0%. The content of four components from high to low was isoorientin>isovitexin>orientin>vitexin. Forty-seven batches of C. communis with clear origins were classified into six categories by chemometrics. C. communis from different origins had different qualities. Generally, C. communis from Western China, Central China, and South of China had superior qualities. The HPLC method established in this study is specific, simple, and efficient, which provides references for the comprehensive evaluation of the quality of C. communis. The chemometrics shows that the qualities of C. communis from different origins are largely different. Isoorientin can be used as an index to determine the content of C. communis, and its content limit should be set no less than 0.023%.
Subject(s)
Commelina , Chemometrics , Drugs, Chinese Herbal/chemistry , China , Chromatography, High Pressure Liquid/methodsABSTRACT
AIM:To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose depriva-tion/reoxygenation(OGD/R). METHODS:The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was iden-tified by PCR,and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass,reactive oxygen species (ROS) and ATP production,cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining,flow cytometry,ATP metabolic assay kit and TUNEL. RESULTS:Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons(P<0.05),en-hanced the ability of ATP synthesis (P<0.01),inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION:PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis,inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.
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Aim To observe the effects of realgar nano-particles on B cell non-Hodgkin's lymphoma Raji cells in vitro. Methods Realgar nanoparticles and crude realgar particles were characterized with a laser particle size analyzer, a transmission electron microscopy (TEM)and an atomic force microscopy(AFM). The morphological changes of proliferation of Raji cells brought about by the use of realgar naoparticles and crude realgar particles were observed with a light mi-croscope. The membrane changes of Raji cells treated with realgar naoparticles and crude realgar particles were observed with AFM. The ultrastructures of Raji cells were observed with TEM. The inhibitory effects of Raji cells treated with realgar naoparticles and crude realgar particles were measured with MTT. The nuclear apoptosis morphologies of Raji cells were observed with fluorescence microscopy. The apoptosis rates and the cell cycle distributions of Raji cells treated with real-gars were measured with flow cytometry. Results The size of realgar nanoparticles and crude realgar particles was (79 ± 8)nm and (1. 89 ± 0. 2)μm,respectively. Light microscopy showed that realgar nanoparticles could inhibit the aggregation growth of Raji cells. AFM showed that Raji cells treated with realgar nanoparticle became shrank, had smaller volume and lost the growth state of stretching out. Raji cells treated with crude realgars did not change significantly. TEM showed Raji cells treated with realgar nanoparticle had damaged subcellular organelles and mitochondria with increased vacuoles. The Raji cells treated with crude realgar did not change significantly. MTT assay showed that when treated with the final concentration of 50 mg ·L - 1 of realgar nanoparticle for 24 h,the cell survival rate of Raji cells was (40 ± 2)% . When treated with the same concentration of crude realgar,its survival rate was (65 ± 3)% . When treated with 50 mg·L - 1 of realgar nanoparticle for 48 h,its survival rate was only 10 % ,and when treated with crude realgar ,its survival rate was (42 ± 2 )% . Fluorescence micro-scope indicated that the Raji cells treated with realgar nanoparticle had obvious nuclear apoptosis,which was not obvious in crude realgar group. Flow cytometry showed that the total apoptosis rate of Raji cells in-duced by realgar nanoparticles and by crude realgar was 11. 14%,15. 9%,respectively. Compared with those treated with crude realgar,the Raji cells treated with realgar nanoparticles presented a significantly higher ratio cell distribution in G1 phase and an obvious decreased ratio in S phase. Conclusion Compared with crude realgar particles,the same dose of realgar nanoparticles can significantly inhibit the proliferation of Raji cells,destroy their sub-cellular structure,and induce the cell apoptosis of Raji cells.
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In order to increase awareness of infection of Penicillium marneffei combined with Pneumocystis carinii pneu monia,we analysed and discussed the diagnosis and treatment of a P.marneffei combined with Pneumocystis carinii pneumonia and reviewed relevant literaure.Pharynx and larynx P.marneffei infection uozhes logy and molecullar was confirmed by physical examination,sputum culture and biopsy.Pneumocystis carinii pneumonia was diagnosed by CT findings and tested positive by PCR for P.carinii (PC).After antifungal treatment,the patient's symptoms and signs showed significant im provement.In conclusion,to achieve early diagnosis and appropriate treatment,sputum culture,biopsy and chest radiograph are suggested for pharynx and larynx recurrent ulcer which is difficult to heal.
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Mitochondrion is an organelle containing its own genome in eukaryotic cells , which encodes 37 genes involved in mitochondrial functions .Mitoepigenetic regulation is a major form of mitochondrial genome-encoded genes that regulates the expression levels without altering the sequences of the genes .The mitoepigenetic regulation is involved in the occurrence and development of various diseases .This paper reviews the progress of mitoepigenetic regulation and Alzhe-imer disease.
ABSTRACT
Mitochondrion is an organelle containing its own genome in eukaryotic cells , which encodes 37 genes involved in mitochondrial functions .Mitoepigenetic regulation is a major form of mitochondrial genome-encoded genes that regulates the expression levels without altering the sequences of the genes .The mitoepigenetic regulation is involved in the occurrence and development of various diseases .This paper reviews the progress of mitoepigenetic regulation and Alzhe-imer disease.
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OBJECTIVE: To develop the high-performance thin-layer chromatographic (HPTLC) fingerprints of triterpenoid from Ilex pubescens and compare the fingerprints similarity with its related species, such as Ilex rotunda and Ilex asprella. METHODS: The separation was performed on pre-coated HPTLC GF254 silica-gel plate stationary phase with trichloromethane-ethyl acetate-methanol-water-formic acid(3:22:5:2:0.2) as solvent system and 10% sulfuric acid alcoholic solution as color development reagent. The common pattern of HPTLC fingerprints were obtained through Chromafinger2005 solution software, and authentication and quality assessment were analyzed by similarity and Principle Commonent Analysis. RESULTS: The common pattern of the pileus of Ilex pubescens consists of 13 characteristic peaks. There are notable differences among the fingerprints of different parts of Ilex pubescen, and those between Ilex pubescens and its related species. CONCLUSION: The different parts of Ilex pubescens can be distinguished by comparison of the HPTLC chromatographs. The other relatives can be easily differentiated from Ilex pubescens too. Copyright 2012 by the Chinese Pharmaceutical Association.
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It is one of the key points for modernization and internationalization of traditional Chinese medicines to construct the Good Agricultural Practice (GAP) base of Chinese materia medica (CMM).GAP helps to minimize contamination and improve the quality of CMM during the plantation and the production of Chinese crude drugs.In this article,the status and development of CMM production bases of GAP in Guangdong Province,China,are presented.The suggestions upon the problems during the development of GAP for Chinese crude drugs are also provided.
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<p><b>BACKGROUND</b>To evaluate expression of human lactoferrin gene by high-density fermentation in recombinant Pichia pastoris on the premise of maintaining its biological activities.</p><p><b>METHODS</b>The neutrophil was isolated from human peripheral blood and its total RNA was prepared. Full-length cDNA of human lactoferrin gene was then obtained by RT-PCR, cloned into expression vector pPIC 3.5 K and transformed into Pichia pastoris strain KM71. With two-layer filter method, the transformants with high-productivity of human lactoferrin were screened out into fed-batch high-density fermentation. And later, the physical, chemical and biological activities of fermentation product were detected preliminarily.</p><p><b>RESULTS</b>The strain p3.5-k-7 with better productivity of human lactoferrin was screened out into fed-batch high-density fermentation. The fermentation lasted nearly for nine days, with A-600 of culture once above 260 and the highest productivity of human lactoferrin being 115 mg/L, 7.67 times the amount of that in shake flask cultivation.</p><p><b>CONCLUSION</b>The authors successfully realized high-density fermentation expression of human lactoferrin gene in recombinant Pichia pastoris.</p>
Subject(s)
Humans , Cloning, Molecular , Fermentation , Lactoferrin , Genetics , Pichia , Genetics , Metabolism , Recombinant ProteinsABSTRACT
Southern blot analysis with probe from mini-Tn5 gfp-km transposon indicated that 5 non-pathogenic mutants which were generated by insertion of mini-Tn5 gfp-km mutagenesis contained a single copy of the transposon. Using genomic DNA of each mutant as a template, TAIL-PCR was performed with seven arbitrary degenerate (AD) primers pairing with 3 nested specific primers designed based on the sequence of GFP toward outside in mini-Tn5 gfp-km. After 3-step PCR reactions, the flanking sequence of each mutant was obtained. The PCR product was ligated with pGEM-T EASY vector and then was transformed into E. coli DH5 alpha by electroporation. Positive clones were selected by white/blue colony and plasmid was isolated, then digested with EcoRI. Plasmid was sequenced if its insert was longer than 300 bp. Our results indicated that TAIL-PCR was proved to be a simple and efficient approach in identification of gene using insertion mutagenesis.