title: Enhancing the potency of lithospermate B for inhibiting Na+/K+-ATPase activity by forming transition metal ion complexes.
abstract: OBJECTIVE
  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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, China.
authors.forename: Nan-Hei
authors.initials: NH
authors.lastname: Lin
authors.forename: Tse-Yu
authors.initials: TY
authors.lastname: Chung
authors.forename: Feng-Yin
authors.initials: FY
authors.lastname: Li
authors.forename: Hsin-An
authors.initials: HA
authors.lastname: Chen
authors.forename: Jason T C
authors.initials: JT
authors.lastname: Tzen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D056831:Coordination Complexes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013552:Swine; D014131:Trace Elements / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D028561:Transition Elements / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D056831:Coordination Complexes; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D014131:Trace Elements; D028561:Transition Elements; C061091:lithospermate B; D000254:Sodium-Potassium-Exchanging ATPase