Mitochondria regulate numerous crucial cell processes, including energy production, apoptotic cell death, oxidative stress, calcium homeostasis and lipid metabolism. Here, we applied an efficient mitochondria-based ce...Mitochondria regulate numerous crucial cell processes, including energy production, apoptotic cell death, oxidative stress, calcium homeostasis and lipid metabolism. Here, we applied an efficient mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry（LC/MS） method,also known as screening method for mitochondria-targeted bioactive constituents（SM-MBC）. This method allowed searching natural mitochondria-targeting compounds from traditional Chinese medicines（TCMs）, including Puerariae Radix（PR） and Chuanxiong Radix（CR）. A total of 23 active compounds were successfully discovered from the two TCMs extracts. Among these 23 hit compounds, 17 were identified by LC/MS, 12 of which were novel mitochondria-targeting compounds. Among these, 6 active compounds were analyzed in vitro for pharmacological tests and found able to affect mitochondrial functions. We also investigated the effects of the hit compounds on Hep G2 cell proliferation and on loss of cardiomyocyte viability induced by hypoxia/reoxygenation injury. The results obtained are useful for in-depth understanding of mechanisms underlying TCMs therapeutic effects at mitochondria level and for developing novel potential drugs using TCMs as lead compounds. Finally, we showed that SM-MBC was an efficient protocol for the rapid screening of mitochondria-targeting constituents from complex samples such as PR and CR extracts.展开更多
Single particle-inductively coupled plasma mass spectrometry (SP-ICP-MS) is a powerful tool for size-characterization of metal-containing nanoparticles (MCNs) at environmentally relevant concentrations,however,coexist...Single particle-inductively coupled plasma mass spectrometry (SP-ICP-MS) is a powerful tool for size-characterization of metal-containing nanoparticles (MCNs) at environmentally relevant concentrations,however,coexisting dissolved metal ions greatly interfere with the accuracy of particle size analysis.The purpose of this study is to develop an online technique that couples hollow fiber ultrafiltration (HFUF) with SP-ICP-MS to improve the accuracy and size detection limit of MCNs by removing metal ions from suspensions of MCNs.Through systematic optimization of conditions including the type and concentration of surfactant and complexing agent,carrier pH,and ion cleaning time,HFUF completely removes metal ions but retains the MCNs in suspension.The optimal conditions include using a mixture of 0.05 vol.%FL-70 and 0.5 mmol/L Na2S2O_(3)(pH=8.0) as the carrier and 4 min as the ion cleaning time.At these conditions,HFUF-SP-ICP-MS accurately determines the sizes of MCNs,and the results agree with the size distribution determined by transmission electron microscopy,even when metal ions also are present in the sample.In addition,reducing the ionic background through HFUF also lowers the particle size detection limit with SP-ICP-MS (e.g.,from 28.3 to 14.2 nm for gold nanoparticles).This size-based ion-removal principle provided by HFUF is suitable for both cations (e.g.,Ag+) and anions (e.g.,AuCl_(4)^(-)) and thus has good versatility compared to ion exchange purification and promising prospects for the removal of salts and macromolecules before single particle analysis.展开更多
基金Financial support from the National Natural Science Foundation of China (Grant 81660596, 81673395 and 81373921)the Research Fund for the Doctoral Program of Higher Education of China (Grant 20130001110057)the Application and Basis Research Project of Yunnan China (Grant 2017FF117-(013) and 2016FD050)
文摘Mitochondria regulate numerous crucial cell processes, including energy production, apoptotic cell death, oxidative stress, calcium homeostasis and lipid metabolism. Here, we applied an efficient mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry（LC/MS） method,also known as screening method for mitochondria-targeted bioactive constituents（SM-MBC）. This method allowed searching natural mitochondria-targeting compounds from traditional Chinese medicines（TCMs）, including Puerariae Radix（PR） and Chuanxiong Radix（CR）. A total of 23 active compounds were successfully discovered from the two TCMs extracts. Among these 23 hit compounds, 17 were identified by LC/MS, 12 of which were novel mitochondria-targeting compounds. Among these, 6 active compounds were analyzed in vitro for pharmacological tests and found able to affect mitochondrial functions. We also investigated the effects of the hit compounds on Hep G2 cell proliferation and on loss of cardiomyocyte viability induced by hypoxia/reoxygenation injury. The results obtained are useful for in-depth understanding of mechanisms underlying TCMs therapeutic effects at mitochondria level and for developing novel potential drugs using TCMs as lead compounds. Finally, we showed that SM-MBC was an efficient protocol for the rapid screening of mitochondria-targeting constituents from complex samples such as PR and CR extracts.
基金supported by the National Key Research and Development Project (No.2020YFA0907400)Strategic Priority Research Program of the Chinese Academy of Sciences (No.XDPB2005)+2 种基金National Natural Science Foundation of China(No.21777178)the National Young Top-Notch Talents (No.W03070030)Youth Innovation Promotion Association of the Chinese Academy of Sciences (No.Y202011)。
文摘Single particle-inductively coupled plasma mass spectrometry (SP-ICP-MS) is a powerful tool for size-characterization of metal-containing nanoparticles (MCNs) at environmentally relevant concentrations,however,coexisting dissolved metal ions greatly interfere with the accuracy of particle size analysis.The purpose of this study is to develop an online technique that couples hollow fiber ultrafiltration (HFUF) with SP-ICP-MS to improve the accuracy and size detection limit of MCNs by removing metal ions from suspensions of MCNs.Through systematic optimization of conditions including the type and concentration of surfactant and complexing agent,carrier pH,and ion cleaning time,HFUF completely removes metal ions but retains the MCNs in suspension.The optimal conditions include using a mixture of 0.05 vol.%FL-70 and 0.5 mmol/L Na2S2O_(3)(pH=8.0) as the carrier and 4 min as the ion cleaning time.At these conditions,HFUF-SP-ICP-MS accurately determines the sizes of MCNs,and the results agree with the size distribution determined by transmission electron microscopy,even when metal ions also are present in the sample.In addition,reducing the ionic background through HFUF also lowers the particle size detection limit with SP-ICP-MS (e.g.,from 28.3 to 14.2 nm for gold nanoparticles).This size-based ion-removal principle provided by HFUF is suitable for both cations (e.g.,Ag+) and anions (e.g.,AuCl_(4)^(-)) and thus has good versatility compared to ion exchange purification and promising prospects for the removal of salts and macromolecules before single particle analysis.
