ABSTRACT
Objective:To comprehensively evaluate the quality of Herba Clematidis Intricatae through HPLC multi-index components, chemometrics combined with EW-TOPSIS. Methods:A total of 18 batches of Herba Clematidis Intricatae samples from seven provinces were collected. Contents of luteolin-7-O-glucoside, rutoside, hyperoside, quercitrin, quercetin, luteolin, apigenin and kaempferol in Herba Clematidis Intricatae were simultaneously determined by HPLC. Chemometrics method was used to comprehensively analyze the content determination results, and the main potential markers affecting the quality of Herba Clematidis Intricatae were analyzed. The quality of Herba Clematidis Intricatae from different origins was evaluated. Results:The eight components showed good linear relationships within their respective ranges ( r≥0.999 1), and accuracy was good ( RSD<2.0%). The chemometrics method showed that 18 batches of Herba Clematidis Intricatae could be clustered into 3 categories, showing certain regional differences. Rutoside, hyperoside and luteolin-7-O-glucoside were the indicative components affecting the difference of chemical constituents in Herba Clematidis Intricatae; results of EW-TOPSIS method showed that the optimum quality of Herba Clematidis Intricatae from Inner Mongolia and Liaoning, followed by those of Hebei, Shanxi and Shanxi, and lowest in Qinghai and Gansu. Conclusion:The established HPLC method is convenient and accurate, and combined with chemometrics and EW-TOPSIS method can be used for the comprehensive evaluation of the quality of Herba Clematidis Intricatae.
ABSTRACT
Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC(50) <100 µM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K(i) value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.
Subject(s)
Botulinum Toxins, Type A/antagonists & inhibitors , Botulinum Toxins/chemistry , Protease Inhibitors/chemical synthesis , Succinates/chemical synthesis , Triterpenes/chemical synthesis , Animals , Botulinum Toxins/chemical synthesis , Botulinum Toxins/pharmacology , Botulinum Toxins, Type A/metabolism , Neurons/drug effects , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Rats , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Succinates/chemistry , Succinates/pharmacology , Triterpenes/chemistry , Triterpenes/pharmacologyABSTRACT
Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960's, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a K(i) of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.
