A reliable ultrasound-assisted extraction （UAE） method combined with HPLC-UV for quantification of eight active alkaloids in fruits of Macleaya cordata （Willd） R. Br. was developed. The optimization conditions of ...A reliable ultrasound-assisted extraction （UAE） method combined with HPLC-UV for quantification of eight active alkaloids in fruits of Macleaya cordata （Willd） R. Br. was developed. The optimization conditions of UAE were obtained by using Box-Behnken design of response surface methodology. Chromatography was carried out using a Kromasil C18 column by gradient elution with 0.1% phosphoric acid aqueous solution for HPLC-UV. All calibration curves showed good linear correlation coefficients （R^2〉0.999 6） and recoveries （from 97.3% to 104.9%） were acceptable. 1,1-diphenyl-2-picrylhydrazyl （DPPH） method was employed to test the antioxidant activity of the extract from the samples. The proposed method was successfully applied to quantifying eight components in nine samples of M.cordata, and significant variations of alkaloid contents and antioxidant aetivity of the samples from different habitats were demonstrated. It presents a powerful proof for the selection of the best sources to extract eight kinds of alkaloids.展开更多
OBJECTIVE Activation of the PINK1/Parkin-mediated autophagy pathway has been proposed to play a protective role in the development of neurological disorders through the elimination of damaged macromolecules or organel...OBJECTIVE Activation of the PINK1/Parkin-mediated autophagy pathway has been proposed to play a protective role in the development of neurological disorders through the elimination of damaged macromolecules or organelles.Exposure to excessive manganese(Mn) causes neurotoxicity and can produce a Parkinson disease(PD)-like neurological disorder.Mitochondrial dysfunction and oxidative stress are implicated in the mechanism of Mn induced neurotoxicity.The present study was designed to determine whether Dendrobium nobile Lindl.alkaloids(DNLA),a Chinese medicinal herb extract,confers protective function over Mn-induced cell toxicity,and to investigate whether the modulation of PINK1/Parkin-mediated autophagy is involved in the mechanism of DNLA-mediated cell protection over Mn toxicity.METHODS AND RESULTS Rat adrenal pheochromocytoma PC12 cells were utilized as an in vitro model of Mn cell toxicity.It was found that the treatment of the PC12 cells with Mn resulted in concentration-dependent cell death,accompanied by a decrease in mitochondrial respiration capacity and an increase in ROS generation,whereas pretreatment of cells with DNLA significantly alleviated cell toxicity induced by Mn and improved mitochondrial function and oxidative status.Mn treatment enhanced apoptotic cells along with a marked increase in the protein expression of Bax and a decrease in the expression of Bcl-2 protein.On the contrary,DNLA increased Bcl-2 expression,and concomitantly dramatically decreased the Bax/Bcl-2 ratio.Analysis of the expression of PINK1 and Parkin revealed that pretreatment of cells with DNLA significantly alleviated the decrease in protein levels of both PINK1 and Parkin caused by Mn.Furthermore,cells treated with Mn exhibited increased expression of LC3-Ⅱ and a decrease in accumulation of P62,which was noticeably reversed by the pretreatment of cells with DNLA.CONCLUSION DNLA inhibits Mn induced cytotoxicity,which may be mediated through modulating PINK1/Parkin-mediated autophagic flux and improving mitochondrial function.展开更多
Objective To develop a liquid chromatography-tandem mass spectrometry(LC-MS/MS) screening method for 45 poisonous alkaloids in blood. Methods Identification was based on the compound's retention time and two precu...Objective To develop a liquid chromatography-tandem mass spectrometry(LC-MS/MS) screening method for 45 poisonous alkaloids in blood. Methods Identification was based on the compound's retention time and two precursor-to-production transitions. The method involved a liquid-liquid extraction(LLE) followed by LC-MS/MS with multiple-reaction monitoring(MRM). When 1 m L of blood was extracted with diethyl ether at p H=9.2 with SKF525 Aas the internal standard, the target compounds were analyzed with LC-MS/MS in the positive ionization mode. Results The target alkaloids had good linearity(r>0.995 1), both the intra-day precision and inter-day precision being less than 14.77%. The limits of detection ranged from 0.05 to 25 ng/m L in blood. Conclusion The method is selective and sensitive in detecting poisonous alkaloids with a total running time of 12 minutes; therefore it was successfully applied to some actual cases of suspected alkaloids poisoning.展开更多
OBJECTIVE o facilitate the basic acquaintance with the bioactive lycodine-type alkaloids biosynthetic pathways,we conducted the transcriptome analysis of L.casuarinoides by illumina sequencing.METHODS The plant of L.c...OBJECTIVE o facilitate the basic acquaintance with the bioactive lycodine-type alkaloids biosynthetic pathways,we conducted the transcriptome analysis of L.casuarinoides by illumina sequencing.METHODS The plant of L.casuarinoides was collected and was subjected to RNA isolation,cDNA library construction,and illumina sequencing before bioinformatics analysis.After sequencing,the clean reads were obtained for de novo assembly by using Trinity software,and then further processed with TGICL sequencing clustering software to generate unigenes,The unigenes are aligned by Blast X alignment to six public protein database.In addition,all unigenes are functionally annotated by GO,KEGG and characterized putative genes involved in lycopodium alkaloids biosynthesis.RESULTS In total,124,524 high-quality unigenes were obtained with an average sequence length of 601 bp.Among the L.casuarinoides transcripts,61,304 showed significant similarity(E-value<1 e-5) to the known proteins in the public database.Among the total 124 524 unigenes,47,538 open reading frame(ORFs) were predicted.Based on the bioinformatics analysis,all possible enzymes involved in the Lycodine-type alkaloids biosynthetic pathway of L.casuarinoides were identified,including primary amine oxidase(PAO),and Malonly-CoA decarboxylase.In addition,a total of 64 putative cytochrome P450(CYP450) and 827 transcription factors were selected as the candidates of Lycodine-type alka.loids modifiers.Furthermore,a total of 13 352 simple sequence repeats(SSRs) were identified from the 124,524 unigenes,of which dinucleotide motifs AG/CT(50.1%),were the most abundant.CONCLU.SION This transcriptome analysis of L.casuarinoides,provides many valuable candidate genes involv.ing in the biosynthesis of novel secondary metabolites but also lays the foundation for genetic diversity analysis via SSRs markers in L.casuarinoides.展开更多
基金Project(20576142) supposed by the National Natural Science Foundation of China Project(2009DFA31270) supported by the International Cooperation Project of Ministry of Science and Technology of China
文摘A reliable ultrasound-assisted extraction （UAE） method combined with HPLC-UV for quantification of eight active alkaloids in fruits of Macleaya cordata （Willd） R. Br. was developed. The optimization conditions of UAE were obtained by using Box-Behnken design of response surface methodology. Chromatography was carried out using a Kromasil C18 column by gradient elution with 0.1% phosphoric acid aqueous solution for HPLC-UV. All calibration curves showed good linear correlation coefficients （R^2〉0.999 6） and recoveries （from 97.3% to 104.9%） were acceptable. 1,1-diphenyl-2-picrylhydrazyl （DPPH） method was employed to test the antioxidant activity of the extract from the samples. The proposed method was successfully applied to quantifying eight components in nine samples of M.cordata, and significant variations of alkaloid contents and antioxidant aetivity of the samples from different habitats were demonstrated. It presents a powerful proof for the selection of the best sources to extract eight kinds of alkaloids.
文摘OBJECTIVE Activation of the PINK1/Parkin-mediated autophagy pathway has been proposed to play a protective role in the development of neurological disorders through the elimination of damaged macromolecules or organelles.Exposure to excessive manganese(Mn) causes neurotoxicity and can produce a Parkinson disease(PD)-like neurological disorder.Mitochondrial dysfunction and oxidative stress are implicated in the mechanism of Mn induced neurotoxicity.The present study was designed to determine whether Dendrobium nobile Lindl.alkaloids(DNLA),a Chinese medicinal herb extract,confers protective function over Mn-induced cell toxicity,and to investigate whether the modulation of PINK1/Parkin-mediated autophagy is involved in the mechanism of DNLA-mediated cell protection over Mn toxicity.METHODS AND RESULTS Rat adrenal pheochromocytoma PC12 cells were utilized as an in vitro model of Mn cell toxicity.It was found that the treatment of the PC12 cells with Mn resulted in concentration-dependent cell death,accompanied by a decrease in mitochondrial respiration capacity and an increase in ROS generation,whereas pretreatment of cells with DNLA significantly alleviated cell toxicity induced by Mn and improved mitochondrial function and oxidative status.Mn treatment enhanced apoptotic cells along with a marked increase in the protein expression of Bax and a decrease in the expression of Bcl-2 protein.On the contrary,DNLA increased Bcl-2 expression,and concomitantly dramatically decreased the Bax/Bcl-2 ratio.Analysis of the expression of PINK1 and Parkin revealed that pretreatment of cells with DNLA significantly alleviated the decrease in protein levels of both PINK1 and Parkin caused by Mn.Furthermore,cells treated with Mn exhibited increased expression of LC3-Ⅱ and a decrease in accumulation of P62,which was noticeably reversed by the pretreatment of cells with DNLA.CONCLUSION DNLA inhibits Mn induced cytotoxicity,which may be mediated through modulating PINK1/Parkin-mediated autophagic flux and improving mitochondrial function.
文摘Objective To develop a liquid chromatography-tandem mass spectrometry(LC-MS/MS) screening method for 45 poisonous alkaloids in blood. Methods Identification was based on the compound's retention time and two precursor-to-production transitions. The method involved a liquid-liquid extraction(LLE) followed by LC-MS/MS with multiple-reaction monitoring(MRM). When 1 m L of blood was extracted with diethyl ether at p H=9.2 with SKF525 Aas the internal standard, the target compounds were analyzed with LC-MS/MS in the positive ionization mode. Results The target alkaloids had good linearity(r>0.995 1), both the intra-day precision and inter-day precision being less than 14.77%. The limits of detection ranged from 0.05 to 25 ng/m L in blood. Conclusion The method is selective and sensitive in detecting poisonous alkaloids with a total running time of 12 minutes; therefore it was successfully applied to some actual cases of suspected alkaloids poisoning.
基金supported by Jiangxi′s Major International Science and Technology Cooperation Projects(20151BDH80020)
文摘OBJECTIVE o facilitate the basic acquaintance with the bioactive lycodine-type alkaloids biosynthetic pathways,we conducted the transcriptome analysis of L.casuarinoides by illumina sequencing.METHODS The plant of L.casuarinoides was collected and was subjected to RNA isolation,cDNA library construction,and illumina sequencing before bioinformatics analysis.After sequencing,the clean reads were obtained for de novo assembly by using Trinity software,and then further processed with TGICL sequencing clustering software to generate unigenes,The unigenes are aligned by Blast X alignment to six public protein database.In addition,all unigenes are functionally annotated by GO,KEGG and characterized putative genes involved in lycopodium alkaloids biosynthesis.RESULTS In total,124,524 high-quality unigenes were obtained with an average sequence length of 601 bp.Among the L.casuarinoides transcripts,61,304 showed significant similarity(E-value<1 e-5) to the known proteins in the public database.Among the total 124 524 unigenes,47,538 open reading frame(ORFs) were predicted.Based on the bioinformatics analysis,all possible enzymes involved in the Lycodine-type alkaloids biosynthetic pathway of L.casuarinoides were identified,including primary amine oxidase(PAO),and Malonly-CoA decarboxylase.In addition,a total of 64 putative cytochrome P450(CYP450) and 827 transcription factors were selected as the candidates of Lycodine-type alka.loids modifiers.Furthermore,a total of 13 352 simple sequence repeats(SSRs) were identified from the 124,524 unigenes,of which dinucleotide motifs AG/CT(50.1%),were the most abundant.CONCLU.SION This transcriptome analysis of L.casuarinoides,provides many valuable candidate genes involv.ing in the biosynthesis of novel secondary metabolites but also lays the foundation for genetic diversity analysis via SSRs markers in L.casuarinoides.
