Enhancing the potency of lithospermate B for inhibiting Na + /K + -ATPase activity by forming transition metal ion complexes

Aim:To determine whether replacing Mg 2+ in magnesium lithospermate B (Mg-LSB) isolated from danshen (Salvia miltiorrhiza) with other metal ions could affect its potency in inhibition of Na + /K + -ATPase activity.Methods:Eight metal ions (Na +, K +, Mg 2+, Cr 3+, Mn 2+, Co 2+, Ni 2+, and Zn 2+) were used to form complexes with LSB. The activity of Na + /K + -ATPase was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Human adrenergic neuroblastoma cell line SH-SY5Y was used to assess the intracellular Ca 2+ level fluctuation and cell viability. The metal binding site on LSB and the binding mode of the metal-LSB complexes were detected by NMR and visible spectroscopy, respectively.Results:The potencies of LSB complexed with Cr 3+, Mn 2+, Co 2+, or Ni 2+ increased by approximately 5 times compared to the naturally occurring LSB and Mg-LSB. The IC 50 values of Cr-LSB, Mn-LSB, Co-LSB, Ni-LSB, LSB, and Mg-LSB in inhibition of Na + /K + -ATPase activity were 23, 17, 26, 25, 101, and 128 μmol/L, respectively. After treatment of SH-SY5Y cells with the transition metal-LSB complexes (25 μmol/L), the intracellular Ca 2+ level was substantially elevated, and the cells were viable for one day. The transition metals, as exemplified by Co 2+, appeared to be coordinated by two carboxylate groups and one carbonyl group of LSB. Titration of LSB against Co 2+ demonstrated that the Co-LSB complex was formed with a Co 2+:LSB molar ratio of 1:2 or 1:1, when [Co 2+ ] was less than half of the [LSB] or higher than the [LSB], respectively.Conclusion:LSB complexed with Cr 3+, Mn 2+, Co 2+, or Ni 2+ are stable, non-toxic and more potent in inhibition of Na + /K + -ATPase. The transition metal-LSB complexes have the potential to be superior substitutes for cardiac glycosides in the treatment of congestive heart failure.