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The Stephania cephararantha HAYATA extract, and its constituent bisbenzylisoquinoline alkaloids, such as cycleanine, cepharanthine, isotetrandrine, berbamine, homoaromoline, and cepharanoline were studied for effects on Na+,K+-ATPase activity. The S. cephararantha HAYATA extract inhibited Na+,K+-ATPase activity with an apparent IC50 value of 540 μg/mL. Cycleanine markedly inhibited Na+,K+-ATPase activity with an IC50 value of 6.2×10-4 M. It slightly inhibited Mg2+-ATPase, H+-ATPase, and Ca2+-ATPase. No effects on alkaline and acid phosphatase activities were observed. The inhibition by isotetrandrine, homoaromoline, cepharanthine, and berbamine was less marked, and cepharanoline showed no effect. Five synthetic analogues of cepharanthine slightly inhibited the activity. The mechanism of inhibition by cycleanine on Na+,K+-ATPase activity was examined in detail, and the following results were obtained in the overall reaction: (1) the mode of inhibition was noncompetitive with respect to ATP; (2) the degree of inhibition was decreased with a decrease of K+ concentration; (3) it was not affected by Na+ concentration; (4) the inhibition mechanism was different from that of ouabain. The activity of K+-dependent p-nitrophenyl phosphatase, a partial reaction of Na+,K+-ATPase, did not appear to have been inhibited by cycleanine in the reaction mixture containing 15 mM K+ (optimum condition). However, cycleanine increased the K0.5 value for K+ and reduced the Ki values for Na+ and ATP, in K+-dependent p-nitrophenyl phosphatase. Cycleanine might interact with the enzyme in Na·E1-P form and prevents the reaction step from Na·E1-P to E2-P.