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

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.

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.

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.

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 IC50 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.