A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Tradi...A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concen-tration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detec-tion (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.展开更多
DNA topoisomerases （topo） I and II are molecular targets of several potent anticancer agents. Thus, inhibitors of these enzymes are potential candidates for anticancer development. Traditionally, Nerita albicilla ha...DNA topoisomerases （topo） I and II are molecular targets of several potent anticancer agents. Thus, inhibitors of these enzymes are potential candidates for anticancer development. Traditionally, Nerita albicilla had been used in Kei Island, Southern Maluku, Indonesia to treat liver disease including cancer. The paper reports on the chemical composition ofNerita albicilla and its topo I inhibitor ofhexane, ethyl acetate and methanol extracts. Topoisomerase-I inhibitor activity was determined using the method reported by TopoGEN. The proximate analysis described that Nerita albicilla dried powder contained 12.45% ± 0.05% moisture; 9.17% ± 0.03% ash; 62.05% ± 0.10% protein; 5.58% ± 0.08% fat; 6.60% ± 0.02% crude fiber and 4.15% ± 0.24% carbohydrate （by difference）. Furthermore, the protein consisted of 11 essential amino acids and six non-essential amino acids. It contained significant amount of branched-chain amino acids （BCAA） valine, leucine, isoleucine （a total of 187.8 mg g-1 protein） and lower content of aromatic amino acids phenylalanine, tyrosine and histidine （a total of 111.26 mg .g-1 protein）. The protein score was 92.2. The yield of hexane, ethyl acetate and methanol extracts ofNerita albicilla were 2.05% ± 0.05%, 1.56% ± 0.06% and 6.99% ± 0.14%, respectively. All extracts showed topoisomerase-I inhibitor activities. Minimum inhibitory concentration （MIC） of methanol extract was 2.50 ug mL-1. Chemical screening of the extracts showed that they contained steroidal and alkaloid compounds. The investigation revealed that Nerita albicilla contains active compounds that could be potential for nutraceutical or pharmaceutical development.展开更多
基金Project supported by the National Natural Science Foundation oChina (No. 20375036) and the Natural Science Foundation of Zhejiang Province (No. RC 0042) China
文摘A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concen-tration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detec-tion (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.
文摘DNA topoisomerases （topo） I and II are molecular targets of several potent anticancer agents. Thus, inhibitors of these enzymes are potential candidates for anticancer development. Traditionally, Nerita albicilla had been used in Kei Island, Southern Maluku, Indonesia to treat liver disease including cancer. The paper reports on the chemical composition ofNerita albicilla and its topo I inhibitor ofhexane, ethyl acetate and methanol extracts. Topoisomerase-I inhibitor activity was determined using the method reported by TopoGEN. The proximate analysis described that Nerita albicilla dried powder contained 12.45% ± 0.05% moisture; 9.17% ± 0.03% ash; 62.05% ± 0.10% protein; 5.58% ± 0.08% fat; 6.60% ± 0.02% crude fiber and 4.15% ± 0.24% carbohydrate （by difference）. Furthermore, the protein consisted of 11 essential amino acids and six non-essential amino acids. It contained significant amount of branched-chain amino acids （BCAA） valine, leucine, isoleucine （a total of 187.8 mg g-1 protein） and lower content of aromatic amino acids phenylalanine, tyrosine and histidine （a total of 111.26 mg .g-1 protein）. The protein score was 92.2. The yield of hexane, ethyl acetate and methanol extracts ofNerita albicilla were 2.05% ± 0.05%, 1.56% ± 0.06% and 6.99% ± 0.14%, respectively. All extracts showed topoisomerase-I inhibitor activities. Minimum inhibitory concentration （MIC） of methanol extract was 2.50 ug mL-1. Chemical screening of the extracts showed that they contained steroidal and alkaloid compounds. The investigation revealed that Nerita albicilla contains active compounds that could be potential for nutraceutical or pharmaceutical development.
