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

Article abstract of DOI:10.1038/aps.2013.32

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

Full text of DOI:10.1038/aps.2013.32

Acta Pharmacologica Sinica (2013) 34: 893–900
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Original Article
Enhancing the potency of lithospermate B for
inhibiting Na+/K+-ATPase activity by forming
transition metal ion complexes
1, 5, 6,
Nan-Hei LIN1, #, Tse-Yu CHUNG1, #, Feng-Yin LI2, Hsin-An CHEN3, 4, , Jason TC TzEN
*
*
1Graduate Institute of Biotechnology and 2Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan,
China; 3Division of General Surgery, Department of Surgery, Shuang Ho Hospital, Taipei Medical University, Taipei 11031, Taiwan,
China; 4Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan, China; 5School of
Chinese Medicine, China Medical University, Taichung 40402, Taiwan, China; 6Agricultural Biotechnology Research Center, Academia
Sinica, Taipei 11529, Taiwan, China
Aim: To determine whether replacing Mg2+ 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+, Mg2+, Cr3+, Mn2+, Co2+, Ni2+, and zn2+) 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 Ca2+ 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 Cr3+, Mn2+, Co2+, or Ni2+ 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 Ca2+ level was substantially elevated, and the cells were viable for one day. The transition metals, as
exemplified by Co2+, appeared to be coordinated by two carboxylate groups and one carbonyl group of LSB. Titration of LSB against
Co2+ demonstrated that the Co-LSB complex was formed with a Co2+:LSB molar ratio of 1:2 or 1:1, when [Co2+] was less than half of the
[LSB] or higher than the [LSB], respectively.
Conclusion: LSB complexed with Cr3+, Mn2+, Co2+, or Ni2+ 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.
Keywords: cardiac glycoside; lithospermate B; Salvia miltiorrhiza; transition metal; metal complex; Na+/K+-ATPase; congestive heart
failure; traditional Chinese medicine
Acta Pharmacologica Sinica (2013) 34: 893–900; doi: 10.1038/aps.2013.32; published online 20 May 2013
(ATP) energy generated in animal cells at rest to actively
Introduction
Na+/K+-ATPase is responsible for the active transport of transport three Na+ out of the cell and two K+ into the cell[2].
sodium and potassium ions, and it is essential for maintaining The Na+/K+-ATPase structure comprises three subunits,
membrane potentials, cell volume, and the active transport termed the α, β, and γ subunits, that execute distinct biological
of other solutes in animal cells[1]. In addition, this enzyme functions[3–5]. The sites for ATP binding, phosphorylation and
is a P-type ATPase, also known as a sodium pump, which ion occlusion are located in the α subunit. This subunit also
commonly consumes 20%–30% of the adenosine triphosphate contains an inhibitor-binding cavity that serves as the primary
target for many pharmacological agents, such as cardiac
glycosides[6–8]
.
# These two authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail dtsurga6@gmail.com (Hsin-An CHEN);
TCTzEN@dragon.nchu.edu.tw (Jason TC TzEN)
Received 2012-12-13 Accepted 2013-03-11
The therapeutic effect of cardiac glycosides in the treatment
of congestive heart failure derives from their reversible
inhibition of the Na+/K+-ATPase located in the cell membrane
of the human myocardium[9, 10]. This inhibition leads to the
*

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accumulation of sodium in cardiac cells, the promotion of from Amresco (Solon, OH, USA). Penicillin, streptomycin,
the sodium-calcium exchange system in the cell membrane, Dulbeco’s modified Eagle’s medium (DMEM), Roswell Park
and ultimately, a higher level of intracellular and myocardial Memorial Institute (RPMI) medium 1640, and Dulbecco's
calcium[11]. The elevated intracellular calcium concentration phosphate buffered saline (DPBS) were purchased from
results in an increased inotropism, accentuating the force of GIBCO (Grand, Island, NY, USA). Fluo-4-AM was purchased
myocardial contractions by increasing the velocity and extent from Invitrogen (Burlington, Ontario, Canada). Fetal bovine
of sarcomere shortening. In other words, there is increased serum (FBS) was purchased from Hyclone (Logan, UT, USA).
stroke work for a given filling volume of pressure[12]. Although Water soluble tetrazolium (WST-1) was purchased from
inhibition of the Na+/K+-ATPase produces beneficial effects in Biovision (Mountain View, CA, USA).
patients with congestive heart failure, cardiac glycosides have
severe side effects and a narrow therapeutic index, limiting Preparation of metal-LSB complexes
their clinical applications[13]
.
Na-LSB, K-LSB, Mg-LSB, and Zn-LSB complexes were
Recently, a number of steroid-like compounds from diverse prepared in 1 mL of H2O by mixing LSB (to a final
Chinese medicinal products used for promoting blood concentration of 10 mmol/L) with NaOH (20 mmol/L),
circulation were demonstrated to be effective inhibitors KOH (20 mmol/L), Mg(OH)2 (10 mmol/L), or Zn(OH)2
of Na+/K+-ATPase; thus, these compounds could exert (10 mmol/L), respectively. To prepare Cr-LSB, Mn-LSB,
therapeutic cardiac effects via the same molecular mechanisms Co-LSB, and Ni-LSB complexes, LSB (10 mmol/L) was first
as cardiac glycosides[14–18]. Exceptionally, no appreciable precipitated with NaOH (20 mmol/L) in 1 mL of ethanol, and
steroid-like compound content was found in danshen (Salvia the precipitate was then dissolved by adding CrCl3, MnCl2,
miltiorrhiza), a well-known Chinese herb traditionally used CoCl2, or NiCl2 (10 mmol/L) to form a metal-LSB complex.
for promoting blood circulation[19]
. Instead, magnesium
lithospermate B (Mg-LSB), the major soluble ingredient in Inhibition of Na+/K+-ATPase by metal-LSB complexes
danshen, was demonstrated to be a strong inhibitor of Na+/ The activity of Na+/K+-ATPase was determined by measuring
K+-ATPase and is regarded as the active ingredient responsible the amount of inorganic phosphate (Pi) liberated from ATP[20]
.
for the therapeutic cardiac effect of this herb[20]. Mg-LSB may A commercial Na+/K+-ATPase from porcine cerebral cortex
have the potential to be a substitute for cardiac glycosides in (Sigma, 0.3 units/mg) was added to 500 μL of the reaction
the treatment of congestive heart failure because it is a non- solution containing 1 mmol/L ATP, 3 mmol/L MgCl2, 48
toxic antioxidant and appears to have no adverse effects; mmol/L NaCl, 12 mmol/L KCl, and 24 mmol/L Tris-HCl
however, clinical trials assessing the efficacy of this compound (pH 7.8). The enzymatic reaction was terminated by adding
are lacking[21]
.
250 µL of 30% (w/v) trichloroacetic acid after the incubation
Mg-LSB possesses a relatively rigid structure as a result period. After centrifugation at 10 000×g for 10 min at 4 °C,
of the formation of salt bridges between Mg2+ and the four the supernatant was diluted 12.5-fold with deionized water.
oxygen atoms of the carboxyl groups that originated from Next, 50 μL of color development reagent provided by the
the four caffeic acid fragments[22]. The rigid structure around phosphate assay kit was added to the solution. After 30 min
the salt bridges formed between Mg2+ and the carboxyl of incubation at room temperature, the color intensity was
groups partially mimics the core steroid structure of cardiac measured at 620 nm on a SpectraMax M2 reader (Molecular
glycosides. To develop more effective drugs for the treatment Devices, USA). Sodium pump activity was expressed as μmol
of congestive heart failure, we sought to determine whether Pi liberated from ATP by 1 mg of Na+/K+-ATPase in 1 h. The
the magnesium ion of Mg-LSB could be replaced with other relative activity was calculated as the level of sodium pump
metal ions to form complexes with a higher potency for activity in the presence of LSB, metal-LSB complexes, and
inhibiting Na+/K+-ATPase activity. In this study, abundant metal ions when normalized to a control of deionized water
metal ions and trace transition metal ions found in the human only (100%).
body were used to form soluble complexes with LSB. The
potency of these complexes for inhibiting Na+/K+-ATPase Cell cultures
activity and their cytotoxicity was evaluated. As exemplified The human adrenergic neuroblastoma cell line SH-SY5Y[23] was
by the formation of a Co-LSB complex, the transition metal kindly provided by Dr Tin-yun HO of the Graduate Institute
binding site in LSB and the binding modes with different of Chinese Medical Science at China Medical University in
ratios of Co2+ to LSB were also examined.
Taiwan, China. SH-SY5Y cells grown in RPMI-1640 culture
medium supplemented with 10% FBS and 1% L-glutamine
were maintained at 37°C in a humidified atmosphere of 95%
air/5% CO2. Cell passages were performed every other day
Materials and methods
Chemicals and reagents
Purified LSB was obtained from KO DA Pharmaceutical by trypsinization. For fluorescence imaging, cells were plated
Company (Taiwan, China). Mg(OH)2 was purchased from in 3-cm cell culture dishes and grown to 80% confluency
Showa Chemical Co (Japan). NaOH, KOH, CrCl3, MnCl2, (approximately 48 h). H9c2 cells (rat cardiomyoblast cell line)
CoCl2, and NiCl2 were purchased from Sigma-Aldrich (St were obtained from the Bioresource Collection and Research
Louis, MO, USA). The phosphate assay kit was purchased Center (Hsinchu, Taiwan, China). H9c2 cells grown in DMEM
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supplemented with 10% FBS, 1% penicillin/streptomycin, quartz cuvette. The spectroscopic changes in the solution
and 1.5 g of sodium bicarbonate were maintained at 37°C in were monitored. Visible spectra were obtained using a Jasco
a humidified atmosphere of 95% air/5% CO2. Cell passages V500 spectrometer (Jasco Corporation, Tokyo, Japan).
were performed every other day by trypsinization. For WST-1
staining, cells were plated in a 96-well culture plate at a Statistical analysis
density of 1×104 cells.
Data are expressed as the mean±standard error of mean (SEM)
of 4 replicates. An analysis of variance (One-way ANOVA)
was performed using SPSS 12.0 for Windows. Differences
Intracellular Ca2+ imaging
Fluctuations in the intracellular Ca2+ levels of SH-SY5Y cells were considered statistically significant at P<0.05.
were tracked and visualized with the preloaded fluorescent
Ca2+-sensitive dye Fluo4-AM[21, 24]. Cell-permeable Fluo4-AM
Results
was dissolved in DMSO at a concentration of 4 mmol/L. Potency of metal-LSB complexes for inhibiting Na+/K+-ATPase
The dye was then further diluted to 4 µmol/L in RPMI- Three relatively abundant metal ions (Na+, K+, and Mg2+)
1640 culture medium. The cells were washed once with found in the human body and five transition metal ions (Cr3+,
DPBS, and then RPMI-1640 culture medium supplemented Mn2+, Co2+, Ni2+, and Zn2+) regarded as trace essential elements
with 4 µmol/L Fluo4-AM was added to the cells for 30 min in human biological functions were used to form soluble
in a humidified 5% CO2 incubator at 37 °C. After washing complexes with LSB. The potency of these eight metal-LSB
in DPBS and RPMI-1640 culture medium, 25 µmol/L of complexes (50 µmol/L) for inhibiting Na+/K+-ATPase activity
Mg-LSB, Cr-LSB, Mn-LSB, Co-LSB, Ni-LSB, or ouabain was was determined and compared with that of LSB (Figure 1A).
added to the cells. Ca2+ fluorescence imaging was monitored The results showed that the inhibitory potency of LSB was
at multiple intervals over 20 min. Time-lapse images of significantly enhanced by forming a complex with any of the
live cells loaded with Fluo4-AM were collected using IX71 five transition metal ions but not with Na+, K+, or Mg2+. To
inverted microscopy (Olympus, Tokyo, Japan). Fluctuations determine whether the enhancement of inhibitory potency
in fluorescence intensity of SH-SY5Y cells treated with metal- for Na+/K+-ATPase was due to the presence of metal ions,
LSBs and ouabain were analyzed frame by frame with a Time eight metal ions of equivalent concentrations were subjected
Series Analyzer[25, 26]. This plugin was used to analyze time- to the same assay. The results showed only Zn2+ affected
lapse image stacks. Cells were selected in a region of interest Na+/K+-ATPase activity (Figure 1B). Therefore, Zn-LSB was
(ROI), and the fluorescence intensity of each time point was excluded in the subsequent studies of metal-LSB complexes.
measured.
Cell viability assay
H9c2 cells were treated with ouabain, LSB and metal-LSB
complexes at a concentration ranging from 10 nmol/L to
100 µmol/L for 24 h. Next, these cells were subjected to cell
viability assay using WST-1. The absorbance was measured at
440 nm on a SpectraMax M2 reader. Control cells underwent
the same conditions without the addition of any drugs. Cell
viability is expressed as a percentage of the absorbance of
treated cells over the absorbance of control cells.
NMR spectroscopic studies
LSB was dissolved in 1 mL of ethanol to a final concentration
of 15 mmol/L and then precipitated by titrating with NaOH
(30 mmol/L). The precipitated LSB was re-dissolved by
adding CoCl2 (15 mmol/L) to the ethanol solution. Ethanol
was removed in vacuum, and the dried sample was dissolved
in D2O. The 13C NMR spectra of LSB and Co-LSB dissolved
in D2O were obtained using a Varian Unity Inova-600 NMR
spectrometer (Blue Lion Biotech, WA, USA).
Figure 1. (A) The inhibition of porcine Na+/K+-ATPase by 50 µmol/L of
Visible spectroscopic studies
metal-LSB complexes. (B) The inhibition of porcine Na+/K+-ATPase by 50
µmol/L of the metal ions used in the metal-LSB complexes. The inhibitory
LSB was dissolved in 1 mL of methanol to a final concentration
of 15 mmol/L. Next, NaOH (30 mmol/L) was added. CoCl2
potency of the complexes and metal ions was determined by the reduction
of Pi liberation from ATP by a constant amount of commercial porcine Na+/
K+-ATPase. The data represent the mean±SEM of five replicates. cP<0.01
vs LSB only or control group (Con; deionized water only).
was dissolved in methanol to a final concentration of 400
mmol/L. A titration experiment was performed by sequential
addition of the CoCl2 solution to the LSB preparation in a
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Quantitatively, the IC50 value of LSB (101 µmol/L) and Mg- growth or decreased the necrosis of H9c2 cells[27]
.
LSB (128 µmol/L) was approximately 5 times higher than
that of Cr-LSB (23 µmol/L), Mn-LSB (17 µmol/L), Co-LSB (26 Identification of the Co2+-binding site in LSB by NMR spectroscopy
µmol/L), or Ni-LSB (25 µmol/L) (Figure 2).
Regarding the paramagnetic properties of the four transition
metal-LSB complexes examined, Co-LSB possessed a clear
signal pattern in 13C NMR spectroscopy; therefore, we used
this complex to study the interaction between a transition
metal and LSB. To identify the Co2+-binding site in LSB, the
13C NMR spectra of LSB and Co-LSB were compared (Figure
5A). In comparison with the 13C NMR spectrum of LSB, the
signals for C8″, C8′″, C9″, and C9′″ were completely eliminated.
The signal for C9’, but not C9, was significantly quenched
in the 13C NMR spectrum of Co-LSB. These data suggest
that two carboxylate groups and one carbonyl group of LSB
are responsible for coordinating to Co2+. The negatively
charged carboxylate groups of LSB seem to play a key role in
the chelation of the positively charged transition metal ions.
Based on our NMR spectral analysis, a 2D structure of Co-LSB
is depicted in Figure 5B.
Detection of binding modes of LSB and Co2+ by visible spectro-
scopy
Figure 2. The potency of LSB and metal-LSB complexes for inhibiting
porcine Na+/K+-ATPase. The inhibitory potency of various concentrations
of LSB, Mg-LSB and four transition metal-LSB complexes was determined
by the reduction of Pi liberation from ATP by a constant amount of
commercial porcine Na+/K+-ATPase.
LSB was incorporated with different concentrations of Co2+.
Next, the d-d orbital electron transition was monitored by
visible spectroscopic changes in different binding modes of
LSB and Co2+. The absorbance of LSB (15 mmol/L) at 400–800
nm was rapidly elevated when the [Co2+] increased from 0 to
Effects of metal-LSB complexes on intracellular Ca2+ levels in SH- 7.5 mmol/L; however, further elevation of the absorbance was
SY5Y cells
slowed as the [Co2+] increased from 7.5 to 15 mmol/L (Figure
To examine the effects of the four transition metal-LSB 6A). No apparent elevation of absorbance was observed when
complexes (Cr3+, Mn2+, Co2+, and Ni2+) on intracellular Ca2+ the [Co2+] was increased from 15 to 30 mmol/L, except for the
levels, SH-SY5Y cells were preloaded with Fluo4-AM and absorbance between 500 and 550 nm. This result was observed
then incubated with 25 μmol/L of a metal-LSB complex or presumably because of the absorbance of non-coordinated
ouabain. Cells were monitored for intracellular fluorescence Co2+, which has a maximal absorbance at 529 nm in methanol.
fluctuations at multiple intervals over 20 min. Compared with The titration curve of the absorbance at 420 nm suggested that
cells treated with buffer alone (control), SH-SY5Y cells treated complexes were formed with a Co2+:LSB molar ratio of 1:2 and
with any of the metal-LSB complexes displayed a significantly 1:1 when [Co2+] was lower than 7.5 mmol/L and higher than
elevated fluorescence intensity (Figure 3). These results 15 mmol/L, respectively (Figure 6B). The absorbance at 420
indicate that the metal-LSB complexes and ouabain increased nm was minimally elevated and plateaued when the [Co2+]
the intracellular Ca2+ levels of SH-SY5Y cells.
was higher than [LSB] (15 mmol/L). Obviously, excess Co2+
could not bind to LSB after forming the Co-LSB complex at a
1:1 molar ratio. The presence of excess Co2+ existing in its free
Cytotoxicity of metal-LSB complexes on H9c2 cells
To evaluate the cytotoxicity of the four transition metal- form is in agreement with the unique elevation of absorbance
LSB complexes on cardiomocytes, H9c2 cells were treated between 500 and 550 nm when the [Co2+] was increased from
with various concentrations (0.01–100 µmol/L) of these four 15 to 30 mmol/L, as shown in Figure 6A.
complexes for 24 h. Next, the cell viability was examined
and compared with that of cells treated with the same
Discussion
concentrations of LSB or Mg-LSB (Figure 4). No apparent In light of the narrow safety margin and severe side effects of
cytotoxicity was observed in H9c2 cells treated with any cardiac glycosides in the treatment of congestive heart failure,
of the four transition metal-LSB complexes, except for cells researchers have extensively searched for potential substitutes
treated with 100 µmol/L of Mn-LSB. Based on these data, isolated from natural sources or novel drugs rationally
H9c2 cells were viable for at least one day when treated developed through chemical synthesis and modification[15, 28–32]
.
with metal-LSB complexes of concentrations lower than 10 Side effects are unlikely to be eliminated for candidate
µmol/L. Interestingly, the viability of H9c2 cells treated with drugs that possess the same steroid backbone, or a similar
100 µmol/L of Co-LSB was substantially higher compared one, as that of cardiac glycosides. In our recent screening of
with other treatments. It is possible that Co-LSB increased the many Chinese medicines used for the promotion of blood
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Figure 3. Fluctuations in intracellular Ca2+ levels of SH-SY5Y cells
treated with metal-LSB complexes. SH-SY5Y cells were loaded with
Fluo4-AM prior to incubation with 25 µmol/L of a metal-LSB complex
or ouabain. The intensity of fluorescence was recorded at multiple
time intervals over 20 min (A). Each point is representative of time-
lapse images in three independent experiments. Serial images of
cells treated with metal-LSB complexes and ouabain for 1, 5, 9, and
20 min were captured to display the fluctuation of intracellular Ca2+
levels (B). The scale bar represents 20 µm.
circulation, numerous analogues of steroid-like compounds that some of these transition metal-LSB complexes may be
were identified as the active ingredient responsible for the superior substitutes for cardiac glycosides in the treatment of
therapeutic effect; furthermore, these compounds utilized congestive heart failure, provided that they undergo necessary
the same molecular mechanism triggered by cardiac clinical trials.
glycosides[19]. Surprisingly, Mg-LSB, a non-steroid antioxidant
The drastic difference in inhibitory potency for Na+/K+-
without apparent adverse effects, was identified as the active ATPase between Mg-LSB and Co-LSB (Figure 3) indicates that
ingredient in danshen, and it was proposed to be a potential Mg2+ and Co2+ might interact with LSB differently. Similar
substitute for cardiac glycosides[20]. In our current study, we to our observations (data not shown), it has been previously
successfully replaced the Mg2+ of Mg-LSB with transition shown that the 13C NMR spectra of LSB and Mg-LSB are essen-
metal ions. The resulting transition metal-LSB complexes tially identical[33]; more specifically, the signals for C8″, C8′″,
displayed an inhibitory potency approximately 5 times C9″ and C9′″ of Mg-LSB did not disappear, and the signal for
higher than LSB or Mg-LSB. No apparent cytotoxicity from C9’ of Mg-LSB was not significantly quenched compared with
these transition metal-LSB complexes (except for 100 µmol/L the 13C NMR spectrum of Co-LSB. Presumably, Co2+, but not
Mn-LSB) was observed in cardiomocytes. Our data suggest Mg2+, interacts strongly with the two carboxylate groups at
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Figure 4. The effects of LSB and metal-LSB complexes on the viability of
H9c2 cells. H9c2 cells were treated with various concentrations of LSB,
Mg-LSB or four transition metal-LSB complexes for 24 h. Cell viability
was measured using a WST-1 assay. The data are represented as the
mean±SEM (n=4; 4 wells in the same experiment).
Figure 6. (A) The visible spectra of LSB in complex with various concen-
trations of Co2+. (B) The absorbance intensity of LSB titrated against Co2+
at 420 nm. The change in LSB intensity titrated against Co2+ could be
separated into three parts, including a fast increment from 0 to 7.5 mmol/L,
a slow increment from 7.5 to 15 mmol/L, and no obvious change from 15
to 30 mmol/L.
C9″ and C9′″ of LSB and weakly with the carbonyl group at C9’
of LSB. These interactions likely lead to a more rigid structure
of Co-LSB that might be a better fit than Mg-LSB or LSB within
the binding pocket of Na+/K+-ATPase. The detailed structural
differences between Mg-LSB and Co-LSB as well as the vari-
ance of their molecular interaction with the residues around
the binding pocket of Na+/K+-ATPase needs to be investi-
gated.
In terms of chemical characteristics, many active ingredients
in Chinese herbs, particularly phenolic compounds clustered
with oxygen-containing functional groups, tend to chelate
metal ions. Chelation of metal ions might affect the
bioactivities of phenolic compounds, such as baicalein and
baicalin. These two major bioactive compounds in the Chinese
herb Scutellaria baicalensis were found to form complexes
with Fe2+ and Fe3+ that possessed distinctive anti-Fenton
properties[34]. Similar iron-binding properties of quercetin, the
major phenolic compound in cranberries, were also observed
and were proposed to be effective at modulating cellular iron
homeostasis under physiological conditions[35]. Moreover,
the biological activities of catechins, the active components in
green tea, have been reported to be influenced by metal ions,
especially transition metal ions. For example, Mn2+ was found
Figure 5. (A) The 13C NMR spectra of LSB and Co-LSB. The NMR signals
of C-8″, C-8′″, C-9″, and C9′″ in the LSB spectrum indicated by arrows
were quenched in the Co-LSB spectrum. The NMR signal of C-9′ in the
LSB spectrum was substantially reduced in the Co-LSB spectrum when
compared with that of C-9. (B) The proposed 2D structure of Co-LSB. The
solid and dashed lines between Co2+ and oxygen atoms of LSB represent
strong and weak interactions, respectively.
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on enhancing the potency of transition metal-LSB complexes
for inhibiting Na+/K+-ATPase activity, we suggest that the
chelation of active ingredients from Chinese herbs, by using
metal ions, may be an applicable approach to improving the
therapeutic effects of these herbs.
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Author contribution
Jason TC TZEN and Hsin-An CHEN designed the study; Nan-
Hei LIN and Tse-yu CHUNG performed the experiments;
Feng-yin LI contributed new analytical tools and reagents; and
Jason TC TZEN wrote the manuscript.
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