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Acid-base behaviour of sanguinarine and dihydrosanguinarine


Abstract and Figures

Acid-base and optical properties of sanguinarine and dihydrosanguinarine were studied in the presence of HCl, HNO3, H2SO4, H3PO4, CAPSO and acetic acid (HAc) of different concentrations and their mixtures. The equilibrium constants pK(R+) of the transition reaction between an iminium cation Q(+) of sanguinarine and its uncharged QOH (pseudo-base, 6-hydroxy-dihydroderivative) form were calculated. A numerical interpretation of the A-pH curves by a SQUAD-G computer program was used. Remarkable shifts of formation parts of absorbance-pH (A-pH) curves to alkaline medium were observed. The shifts depend on the type and concentration of inert electrolyte (the most remarkable for HNO3 and HCl). The corresponding pK(R+) values ranged from 7.21 to 8.16 in the same manner (Delta pK(R+) = 0.81 and 0.73 for HNO3 and HCl, respectively). The priority effect of ionic species and ionic strength was confirmed in the presence of NaCl and KCl. The strength of interaction of SA with bioactive compounds (i.e. receptors, transport proteins, nucleic acids etc.) may be affected because of the observed influence of both cations and anions of the inert electrolytes.
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Received 13 February 2008; Accepted 27 May 2009
Central European Journal of Chemistry
1Department of Chemistry and Biochemistry,
Mendel University of Agriculture and Forestry,
CZ-613 00 Brno, Czech Republic
2Department of Chemistry, Masaryk University,
CZ-603 00 Brno, Czech Republic
3Department of Medical Chemistry and Biochemistry,
Palacky University Olomouc,
CZ-77515 Olomouc, Czech Republic
4Department of Food Biochemistry and Analysis,
Tomas Bata University in Zlín,
CZ-672 72 Zlín, Czech Republic
Acid-base behaviour of sanguinarine
and dihydrosanguinarine
Helena Absolínová1, Luděk Jančář2, Irena Jančářová1, Jaroslav Vičar3,
Vlastimil Kubáň1,2,4*
Abstract: Acid-base and optical properties of sanguinarine and dihydrosanguinarine were studied in the presence of HCl, HNO3, H2SO4, H3PO4,
CAPSO and acetic acid (HAc) of different concentrations and their mixtures. The equilibrium constants pKR+ of the transition reaction
between an iminium cation Q+ of sanguinarine and its uncharged QOH (pseudo-base, 6-hydroxy-dihydroderivative) form were calcula-
ted. A numerical interpretation of the A-pH curves by a SQUAD-G computer program was used. Remarkable shifts of formation par ts of
absorbance-pH (A-pH) curves to alkaline medium were observed. The shifts depend on the type and concentration of inert electrolyte
(the most remarkable for HNO3 and HCl ). The corresponding pKR+ values ranged from 7.21 to 8.16 in the same manner (ΔpKR+ =
0.81 and 0.73 for HNO3 and HCl, respectively). The priority effect of ionic species and ionic strength was confirmed in the presence of
NaCl and KCl. The strength of interaction of SA with bioactive compounds (i.e. receptors, transport proteins, nucleic acids etc.) may
be affected because of the observed influence of both cations and anions of the inert electrolytes.
© Versita Warsaw and Springer-Verlag Berlin Heidelberg.
Keywords: Sanguinarine • Dihydrosanguinarine • UV-VIS Spectrophotometry • Equilibrium constants
* E-mail:
Research Article
1. Introduction
Sanguinarine (SA) is one of the most important members
[1] of the family of secondary plant metabolites,
quaternary benzo[c]phenanthridine alkaloids (QBAs),
displaying a wide spectrum of biological activities, i.e.
antimicrobial and anti-inflammatory effects [2,3]. It is
used as an antiplaque component [4], as remedies
in myopathy [2] and as a constituent of veterinary
preparations [5]. Dihydrosanguinarine (DHSA), its
dihydro-derivative, has been recently identified as the
first product in sanguinarine reductive metabolism [6] in
The beneficial biological effects and/or adverse side
effects are evidently connected with the occurrence
of SA in the two structurally different forms at the
physiological pH 7.4 [7]. The pH-dependent (acid-base)
equilibrium (Fig. 1) between the charged iminium cation
Q+ and the uncharged QOH (pseudo-base, 6-hydroxy-
dihydroderivative) forms of the alkaloid is totally
reversible at very low concentrations. The reaction may
be characterized by an equilibrium constant KR+ = [H+]
[QOH]/[Q+] in analogy to the acid-base dissociation
constant Ka of Brønsted acids. The pKR+ values range
from 6.3 to 8.2 as determined by spectrophotometry,
fluorimetry, potentiometry and capillary electrophoresis
Cent. Eur. J. Chem. • 7(4) • 2009 • 876–883
DOI: 10.2478/s11532-009-0079-y
H. Absolínová et al.
[7] indicates dependence on experimental conditions
(ionic strength, liquid medium composition etc.).
The quaternary cationic form of SA is well soluble
in water. Upon alkalization the intensive brown color of
the solution disappears and a white opalescence and/
or precipitate of a hydrophobic and sparingly soluble [7]
uncharged pseudobase of SA appears at concentrations
above 25 μmol L-1. The limited solubility of the uncharged
form of SA (not mentioned in most papers) influences
the behaviour of the alkaloids in aqueous solution.
The pseudobase easily dissolves in organic solvents
of medium polarity [8,9]. Spontaneous dimerization of
the uncharged form, which was found in polar organic
solvents [8,9], fortunately, does not take place in
aqueous solutions [10]. In addition, the pseudobase
is photochemically oxidized on the C6 carbon atom to
oxysanguinarine in strongly alkaline solutions [11].
These reactions may not only distort the determination
of its pKR+ values but also seriously influence interaction
studies of SA with bio-macromolecules (proteins,
peptides etc.). The exact knowledge of acid-base
(protolytic) behaviour and the pKR+ values of SA and
DHSA are necessary for the interpretation of any
investigation of the interactions of these alkaloids with
biological macromolecules, e.g. receptors, transport
proteins, nucleic acids, etc.
The main goals of our effort were (i) recognizing
of the acid-base behaviour and time stability of SA
and DHSA as the function of experimental conditions
(pH, ionic strength, concentration of electrolyte and
its composition, etc.), (ii) determination of true pKR+
constants, (iii) identification of experimental conditions
and requirements that qualify the possibility and
correctness of interaction studies with these compounds
in nearly neutral and weakly basic solutions.
2. Experimental
2.1. Chemicals
Stock solutions of sanguinarine chloride (SA, Sigma
Aldrich) were prepared using freshly boiled distilled
water acidified with hydrochloric acid to pH below 5.
Working solutions (c = 13 μmol L-1) were prepared by
dilution of the stock solution by freshly boiled distilled
water. Dihydrosanguinarine (DHSA, 99% purity, MP
189-191°C) was prepared from SA by reduction with
NaBH4 in methanol [12]. Stock solutions of DHSA were
prepared by dissolution of DHSA in ethanol or methanol.
Working solutions (c = 12 μmol L-1) were prepared freshly
by dilution of the stock solution by ethanol or methanol
and/or freshly boiled distilled water. All the solutions
were stored in the refrigerator and were protected from
Stock solutions of electrolytes were prepared from
HCl, HNO3, H2SO4, NaCl, KCl, (all Penta, Chrudim,
Figure 1. The structural formulas of alkaloids sanguinarine (SA) and dihydrosanguinarine (DHSA) and the equilibrium between their charged and
uncharged forms.
Acid-base behaviour of sanguinarine
and dihydrosanguinarine
Czech Republic), CH3COOH, H3PO4 (Lach-Ner,
Neratovice, Czech Republic) all of p.a. purity and
acid (CAPSO, Sigma-Aldrich). Tris-(hydroxymethyl)
aminomethane (TRIS – ultra pure grade, Amresco®,
Solon, Ohio, USA), potassium or sodium
hydroxide (both Penta, Chrudim, Czech
Republic) solutions (1 – 0.001 mol L-1)
were used for the pH adjustment in a pH interval
pH = 2 – 11 with steps ∆pH = 0.3 – 0.5. All solutions
were degassed with helium and kept under nitrogen.
2.2. Apparatus and conditions
All spectrophotometric measurements were performed
using a UV/VIS Lambda 25 (Perkin Elmer, Shelton, USA)
or Helios Beta UV-VIS spectrophotometers (Unicam,
Cambridge, UK). The final pH of the solutions was
controlled using a pH-meter model WTW pH 527 with a
WTW SenTix 21 combined electrode. The electrode was
regularly calibrated (several times per day, at least at the
beginning and at the end of A-pH curve measurement)
using a set of standard buffer solutions of pH = 4.01,
7.00, and 9.01 (all WTW GmbH, Wilheim, Germany).
The pKR+ constants were calculated from the
absorbance values at selected wavelengths between
270 and 350 nm using the SQUAD-G computer program
[13]. The absorbance values of both alkaloids were
measured three times at each pH and the mean values
from these measurements were used for the calculation
of pKR+.
2.3. The SQUAD-G program
For a system comprising up to five interacting basic
components, the SQUAD-G [13] program assembly
makes it possible, based on a matrix analysis of
absorbance data, to determine the number of absorbing
species, the dissociation constants of the compound
or the equilibrium constants, stability constants of
complexes, the molar absorptivities of individual species
and their standard deviations. The concentration
proportions of the complexes present at a given pH,
spectra of individual complexes or reagent species
(even those that do not enable direct measurements),
distribution diagrams of all species in the solution (with
respect to the basic components of the system) and
contributions of colored species to the total measured
absorbance are also computed and printed out.
The input data include spectra in the form of an
absorbance matrix for up to 170 solutions and 2 – 50
wavelengths, pH values for pH dependent reactions,
total concentrations of components, composition of
expected species and their constants estimates. The
criteria used for adopting a model or for including the
species are: i) convergence of the calculation, ii) minimal
value of the sum of squares of absorbance residuals
U = Σi (Aexp,iAcalc,i)2, where i = 1-n is the total number of
absorbance data for all solutions and wavelengths used,
iii) minimal value of the average standard deviation of
absorbance s(A) over the whole data set, iv) for the
standard deviation of calculated constant k (KR+, β),
validity of the condition s(log k) < 0.1 log k.
3. Results and discussion
3.1. Time stability of SA and DHSA solutions
Time stability A = f(time) of the working solutions of
DHSA was evaluated measuring absorption spectra
(200-600 nm interval) in the aqueous solution with 4, 10,
30, 60, 100% (v/v) ethanol or methanol. The data were
(in addition) collected at 274, 284, 322, 327 and 350 nm
as the means of absorbance values (in relative %) for
five replicates in 4% (v/v) ethanol at pH = 2 (0.01 mol L-1
HCl) and pH = 7 (HCl + NaOH) and in the presence of
formiate (pH = 2.8), acetate (pH = 4.2) and phosphate
(pH = 6.7) buffers in addition.
The time stability of the DHSA working solutions was
very short (a decrease to 93 and 85% of initial absorbance
values in 30 min, respectively) in the less concentrated
ethanol solutions (4 and 10%). The solutions were
very stable at higher ethanol contents (≥ 30%) with a
non-significant increase of absorbance values at
350 nm (up to 115% of the initial value) probably due
to the increase of solubility in the mixed solvent. The
highest stability of DHSA solution was observed in
the strongly acidic medium (pH = 2, 10 mmol L-1 HCl)
while in all other cases (formiate, pH = 2.8, acetate, pH
= 4.2, phosphate, pH = 6.7 and HCl/NaCl, pH 7.0) the
stability was lower. A very similar behaviour was found
for ethanol (≤ 30%) and methanol (≤60%).
On the contrary, working solutions of SA were stable
over the whole pH intervals of 2 through 8. In the alkaline
solutions (pH > 8) the solutions were less stable (ca.
10% decrease of the initial absorbance values in 60 min
at pH 10.8) and a slow reduction of the intensity of the
absorption bands was observed for at least two hours
(a sharp isosbestic point (IP) at 346 nm indicates a
reversible reaction). The instability of the DHSA solutions
can be most probably explained by photochemical
oxidation of DHSA similar to that described [14] for SA
in a strongly alkaline medium. Absorption spectra of the
final products of the photooxidation of SA and DHSA are
very similar. The reactions take place in the pH regions
in which the uncharged pseudobases prevail.
H. Absolínová et al.
3.2. Absorption spectra
Marked precipitation of sanguinarine was observed
when its more concentrated (≥50 μmol L-1) stock solution
was prepared by dissolution of sanguinarine chloride in
neutral or alkaline solution or in sodium phosphate buffer
of pH 7.4. The precipitate steadily dissolved at the lower
concentrations (10 μmol L-1 and lower) within several
days while at the concentration 25 μmol L-1 remained
opalescent for several weeks. More concentrated
solutions (≥50 μmol L-1) did not change. This finding
indicates that the total solubility of sanguinarine is below
the 25 μmol L-1. Thus 13 μmol L-1 working solutions
in water acidified to pHs of 1 through 5 were used as
starting conditions in all experiments.
Absorption spectra of SA were registered
in wavelength intervals from 250 to 600 nm at
pH = 2.5 – 11.0 in the presence of HCl (see
Fig. 2a), HNO3, H2SO4, H3PO4, HAc and CAPSO (initial
concentration c = 10 mmol L-1). The spectra exhibited
two distinct UV absorption maxima at 274 and 327 nm,
three less promoted absorption maxima at 260, 398 and
470 nm and a broad shoulder at 350 nm in acidic media.
The short wavelength maximum at 260 nm shifted to
shorter wavelengths (≈ 245 nm) while the maximum
at 274 nm shifted to longer wavelengths (284 nm,
288 nm for CAPSO) in alkaline medium of pH = 8 – 10.
The absorption band with a maximum at 327 nm exhibited
a broadening with a new maximum at 322 nm and with a
broad shoulder in the 345 – 355 nm range. The intensity
of other UV-VIS bands was reduced. The presence
of isosbestic points at 286 and 307 nm confirmed a
reversible equilibrium between cationic iminium Q+
and the uncharged QOH forms in the pH interval of
5 through 10. The equilibrium is partly influenced by
limited solubility of the uncharged QOH form. The molar
absorptivities ε1 a ε2 of both forms of sanguinarine in HCl
medium calculated by the SQUAD-G program are given
in Table 1.
The absorption spectra of DHSA registered in the
range 200 – 600 nm in the presence of 60% (Fig. 2b)
and 4% (v/v) methanol or 4% (v/v) ethanol exhibited
250 270 290 310 330 350 370
pH: 1 – 2.99; 2 – 3.97; 3 – 5.69; 4 – 7.46; 5 – 8.48; 6 – 8.96; 7 – 9.93
cSA = 1.3×10–5 mol L-1
220 260 300 340 380
pH: 1 – 1,16; 2 – 2,16; 3 – 3,11; 4 – 4,12; 5 – 6,16 cDHSA = 6.10–6 mol L-1
Figure 2. Absorption spectra of sanguinarine (a) in 100 mmol L-1 HCl
and dihydrosanguinarine (b) in 60% (v/v) methanol and
100 mmol L-1 HCl
Table 1. Molar absorptivities ε1 and ε2 (and their standard deviations) of cationic form (ε1) and pseudobase (ε2) of sanguinarine measured at
different experimental conditions
(274 nm)
[L mol–1 cm–1]
(274 nm)
[L mol–1 cm–1]
(327 nm)
[L mol–1 cm–1]
(327 nm)
[L mol–1 cm–1]
0.001 mol L-1 HCl 25 380 ± 290 18 300 ± 240 17 840 ± 230 10 520 ± 180
0.01 mol L-1 HCl 24 610 ± 80 18 460 ± 110 18 460 ± 70 10 150 ± 110
0.1 mol L-1 HCl 23 900 ± 50 19 540 ± 80 18 509 ± 60 10 810 ± 110
30 700 L mol–1 cm–1 was given by [21] at 327 nm
Acid-base behaviour of sanguinarine
and dihydrosanguinarine
eight distinct absorption maxima at 238, 253, 268, 274,
308, 322, 338 and 355 nm and a broad shoulder at
214 nm with isosbestic points at 270, 303, 330 and
364 nm in the interval pH = 1 – 4. Due to the gradual
destruction of the DHSA molecule the intensity of
absorption bands continuously decreased and the
isosbestic points disappeared at higher pH values.
Absorption maxima at 237, 284, 327 nm, a less distinct
maximum at 350 nm and a broad shoulder at 210 –
220 nm were present in the pH interval 4 – 10. With
the increasing content of organic solvent (4, 10, 30, 60,
100% (v/v) ethanol or methanol) a distinct maximum at
284 nm and a less distinct maximum at 237 nm appear.
The absorption maximum at 327 nm (1 – 10% (v/v)
ethanol or methanol) was shifted to shorter wavelengths
(322 nm) with increasing content of organic solvent (60
– 100% (v/v) ethanol or methanol).
3.3. Absorbance-pH curves
Influence of experimental conditions on acid-base
behaviour of SA (type and concentration of anions of
inorganic/organic acids) was studied by interpretation
of absorbance-pH curves (A-pH curves) measured at a
constant concentration of sanguinarine c = 13 μmol L-1.
The data were collected for HCl (see Fig. 3), HNO3,
H2SO4, H3PO4, acetic acid and CAPSO (not graphically
presented) starting at the initial concentrations of acids
1, 10 and 100 mmol L-1.
The formation parts of the A-pH curves (and of
course the corresponding pKR+ values) were shifted to
the more alkaline medium with increasing concentration
of acids and in dependence on type of anion. The
corresponding pKR+ values calculated using a numerical
interpretation of the A-pH curves by the SQUAD-G
program (see Table 2) changed from 7.21 to 8.16 in
the same manner (in agreement with published data
[15-22]). The most remarkable shift of pKR+was observed
in the presence of the strongest mineral acids HNO3
(∆pKR+ = 0.81) and HCl (∆pKR+ = 0.73) and acetic acid
(∆pKR+ = 0.68) while a less remarkable one was recorded
in the presence of CAPSO (∆pKR+ = 0.34), H3PO4
(∆pKR+ = 0.29) and H2SO4 (∆pKR+= 0.23). Thus the pKR+
values were influenced in increasing/decreasing order
by the following anions: NO3
~ Cl ~ Ac > CAPSO ~
3– ~ SO4
Due to the low stability of solutions, the A-pH curves
for DHSA were measured at a constant concentration
of dihydrosanguinarine c = 12 μmol L-1 in the presence
of HCl in 60% (v/v) methanol and pKR+ value of 2.32
was estimated in agreement with the value 2.3-2.6 acc.
3.4. Effect of electrolyte composition (M+, X-)
To confirm the influence of the type of anion, the A-pH
curves were measured in the mixtures of acids at
their constant total concentration 100 mmol L-1. The
pKR+ values increased if the HCl, H2SO4 and H3PO4
(7.42±0.09, 7.79±0.09, 8.09±0.11) as the anions were
Figure 3. Absorbance-pH curves of sanguinarine at different HCl
concentrations. Experimental conditions: 1 – 0.001 mol L-1
HCl; 2 – 0.01 mol L-1 HCl; 3 – 0.1 mol L-1 HCl, λ = 274 nm,
cSA = 1.3.10–5 mol L-1
Table 2. pKR+ constants of sanguinarine (and their standard
deviations) measured at different experimental
Conditions pKR+ s(A)1) U2)
0.001 mol L-1 HCl 7.33 ± 0.042 0.0063 0.0017
0.001 mol L-1 HNO37.21 ± 0.065 0.0102 0.0046
0.001 mol L-1 H2SO47.52 ± 0.026 0.0039 0.0011
0.001 mol L-1 H3PO47.50 ± 0.026 0.0041 0.0008
0.001 mol L-1 CH3COOH 7.48 ± 0.037 0.0063 0.0001
0.001 mol L-1 CAPSO 7.25 ± 0.027 0.0030 0.0003
0.01 mol L-1 HCl 7.69 ± 0.0323) 0.0004 0.0013
0.01 mol L-1 HNO37.56 ± 0.065 0.0125 0.0069
0.01 mol L-1 H2SO47.68 ± 0.039 0.0048 0.0015
0.01 mol L-1 H3PO47.68 ± 0.031 0.0050 0.0014
0.01 mol L-1 CH3COOH 7.88 ± 0.020 0.0032 0.0006
0.01 mol L-1 CAPSO 7.30 ± 0.032 0.0058 0.0011
0.1 mol L-1 HCl 8.06 ± 0.026 0.0023 0.0004
0.1 mol L-1 HNO38.02 ± 0.027 0.0030 0.0005
0.1 mol L-1 H2SO47.75 ± 0.055 0.0088 0.0038
0.1 mol L-1 H3PO47.79 ± 0.041 0.0035 0.0006
0.1 mol L-1 CH3COOH 8.16 ± 0.051 0.0100 0.0028
0.1 mol L-1 CAPSO 7.59 ± 0.066 0.0084 0.0027
1)minimal value of the average standard deviation of absorbance
s(A) over the whole data set; 2) the sum of squares of absorbance
residuals U = Σi (Aexp,i – Acalc,i)2; 3) 7.65±0.04 is given for
0.01 mol L-1 HCl,pKR+ constants of SA 7.32–8.16 [8,9,11,15,16]
H. Absolínová et al.
changed. The same trend was observed for 10 mmol L-1
HCl with addition of 10 and 100 mmol L-1 H
2SO4 or
Due to the highest differences in pKR+values and
in the most remarkable shifts of formation parts of A-pH
curves, the mixtures of HCl + H2SO4, HCl + H3PO4,
HNO3 + H2SO4, HNO3 + H3PO4, HCl + HNO3 and H2SO4
+ H3PO4 at the constant concentrations 0.1 mol L-1 and
at the concentration ratios 0.07 mol L-1 + 0.03 mol L-1
and vice versa were tested in further experiments. The
increasing pKR+ values and also their differences pKR+
(see Table 3) documented the priority effect of anions
of inorganic acids (HCl and HNO3) in their mixtures with
H2SO4 or H3PO4 with increasing concentration of HCl
and HNO3. The priority effects were less remarkable
for the acids with very close pKR+ values, i.e. HCl
(pKR+ = 8.06) and HNO3 (pKR+= 8.02), or H2SO4
(pKR+= 7.75) and H3PO4 (pKR+ = 7.79)
To verify the influence of cationic species, the pH of the
sanguinarine solutions in HCl, HNO3, H2SO4 and H3PO4
at the concentrations c = 10 mmol L-1 and 100 mmol L-1
was adjusted with TRIS and KOH. The corresponding
pKR+ values (see Table 4) were again shifted to the more
alkaline medium with increasing concentration of acids
and they were significantly higher for TRIS compared to
the values obtained for the A-pH curves with solutions
neutralized with NaOH. The lowest differences pKR+
were in the presence of HCl and, on the other hand, the
differences were comparable for the other acids.
3.5. Influence of ionic strength
The changes in acid-base behaviour of sanguinarine
in dependence on ionic strength were evaluated in the
presence of NaCl and KCl at I = 0.01, 0.10 and 1.0 and
at the constant concentration of HCl, HNO3, H2SO4,
H3PO4 (c = 0.01 mol L-1). The corresponding pKR+ values
are presented in Table 5 and in a graphical form in
Figs. 4a and 4b. The formation parts of A-pH curves and
also the corresponding pKR+ values were shifted to the
alkaline region with increasing ionic strength in the range
∆pKR+ = 0.35 – 0.78. The highest influence was observed
in the presence of HNO3 while the lowest in the presence
of H3PO4. A slightly more remarkable effect was observed
in the presence of KCl (∆pKR+= 0.38 – 0.89).
Table 3. pKR+ values of sanguinarine in mixtures of inorganic acids (c = 0.1 mol L-1, concentrations ratios 0.03 + 0.07 mol L-1 and 0.07 + 0.03 mol L-1,
Conditions pKR+ s(A)1) U2)
0.03 mol L-1 HCl + 0.07 mol L-1 H2SO47.96 ± 0.039 0.0046 0.0016
0.07 mol L-1 HCl + 0.03 mol L-1 H2SO48.17 ± 0.022 0.0028 0.0006
0.03 mol L-1 HCl + 0.07 mol L-1 H3PO48.06 ± 0.035 0.0049 0.0017
0.07 mol L-1 HCl + 0.03 mol L-1 H3PO48.11 ± 0.023 0.0027 0.0007
0.03 mol L-1 HNO3 + 0.07 mol L-1 H2SO47.89 ± 0.074 0.0067 0.0031
0.07 mol L-1 HNO3 + 0.03 mol L-1 H2SO48.12 ± 0.033 0.0043 0.0009
0.03 mol L-1 HNO3 + 0.07 mol L-1 H3PO48.15 ± 0.030 0.0052 0.0016
0.07 mol L-1 HNO3 + 0.03 mol L-1 H3PO48.30 ± 0.067 0.0056 0.0017
0.03 mol L-1 HCl + 0.07 mol L-1 HNO38.11 ± 0.020 0.0021 0.0003
0.07 mol L-1 HCl + 0.03 mol L-1 HNO38.05 ± 0.031 0.0034 0.0007
0.03 mol L-1 H2SO4+ 0.07 mol L-1 H3PO47.81 ± 0.048 0.0060 0.0027
0.07 mol L-1 H2SO4+ 0.03 mol L-1 H3PO47.89 ± 0.022 0.0029 0.0006
1) minimal value of the average standard deviation of absorbance s(A) over the whole data set; 2) the sum of squares of absorbance residuals
U = Σi (Aexp,i – Acalc,i)2
Table 4. pKR+ values of sanguinarine in the presence of TRIS
Conditions pKR+ s(A)1) U2)
0.01 mol L-1 HCl 8.17 ± 0.037 0.0090 0.0051
0.10 mol L-1 HCl 8.24 ± 0.047 0.0059 0.0021
0.01 mol L-1 HNO38.27 ± 0.065 0.0098 0.0056
0.10 mol L-1 HNO38.66 ± 0.041 0.0095 0.0039
0.01 mol L-1 H2SO48.05 ± 0.080 0.0090 0.0028
0.10 mol L-1 H2SO48.44 ± 0.032 0.0055 0.0021
0.01 mol L-1 H3PO47.96 ± 0.037 0.0055 0.0018
0.10 mol L-1 H3PO48.26 ± 0.047 0.0083 0.0047
1) minimal value of the average standard deviation of absorbance s(A) over the whole data set; 2) the sum of squares of absorbance residuals
U = Σi (Aexp,i – Acalc,i)2
Acid-base behaviour of sanguinarine
and dihydrosanguinarine
4. Conclusions
This study showed that interaction measurements are
possible with SA in almost neutral and weakly alkaline
solutions despite their limited solubility in such solutions.
The necessary prerequisite for obtaining correct values is
the use of the lowest possible concentrations of SA. The
applied SA concentration should be below the solubility
limit in the used buffer or, if this is impossible, close to
it. Samples dissolved in water acidified with hydrochloric
acid pHs of 1 through 5 are recommended to obtain
further improvement in the studies.
The reported pKR+ values of SA (7.32 – 8.16)
[8,9,11,15-22] and our previous experience with
electrophoretic behaviour of SA [20,23] imply that at least
some of the published pKR+ values are distorted by the
abovementioned experimental conditions. Thus, in some
cases an organic solvent was added to the solutions
to improve the alkaloid solubility. Knowledge on the
behaviour of QBAs in the solutions, particularly the ones
containing an organic solvent, is therefore irrelevant to
measurements performed in almost neutral and weakly
basic solutions where the concentrations of uncharged
forms of QBAs become comparable or prevail [24,25].
Different behaviour and solubility of SA in different
media, which were adjusted to identical pH and ionic
strength, must result from interactions of ionic species
of acids and/or bases with investigated alkaloids.
Recently we have found that MOPS and CAPSO anions
form pseudo-micelles that can enhance dissolution of
uncharged SA [23,26]. It has to be therefore expected
that analogical interaction occurs between uncharged
hydrophobic sanguinarine not only with complex
molecular structures but similarly with the components
of buffer solutions, electrolytes and non-electrolytes
and other compounds present in solutions etc. Also
adsorption/desorption processes on colloidal species,
solid particles surfaces etc. can play an important role.
The low solubility of the uncharged form of SA is the most
probable reason for their extraordinary unfavourable
behaviour we have observed in neutral and weakly
alkaline region. To prevent precipitation of SA, the use of
sufficiently sensitive techniques, e.g. fluorimetry, MS etc.
(allowing application of the lowest concentration of SA)
is recommended for the measurements for biological
-2 -1,5 -1 -0,5 0
log I
-2 -1,5 -1 -0,5 0
log I
Figure 4. pKR+ values of sanguinarine in dependence on a type and concentration of inert electrolyte (ionic strength) in the presence of NaCl (right)
or KCl (left). Experimental conditions: 1 – 10 mmol L-1 HCl, 2 – 10 mmol L-1 HNO3, 3 – 10 mmol L-1 H2SO4, 4 – 10 mmol L-1 H3PO4
Table 5. Influence of ionic strength (NaCl and KCl as inert electrolytes) on pKR+ of sanguinarine
0.01 mol L-1
0.01 mol L-1
0.01 mol L-1
0.01 mol L-1
0 7.69 ± 0.032 7.56 ± 0.065 7.68 ± 0.039 7.68 ± 0.031
0.01 mol L-1 NaCl 7.84 ± 0.014 7.60 ± 0.027 7.78 ± 0.018 7.65 ± 0.047
0.10 mol L-1 NaCl 7.97 ± 0.020 8.09 ± 0.023 8.07 ± 0.024 7.78 ± 0.049
1.00 mol L-1 NaCl 8.29 ± 0.024 8.34 ± 0.017 8.25 ± 0.026 8.04 ± 0.057
0.01 mol L-1 KCl 7.87 ± 0.056 7.62 ± 0.027 7.82 ± 0.020 7.62 ± 0.034
0.10 mol L-1 KCl 7.92 ± 0.065 8.05 ± 0.032 8.01 ± 0.030 7.80 ± 0.037
1.00 mol L-1 KCl 8.18 ± 0.019 8.46 ± 0.019 8.42 ± 0.030 8.07 ± 0.060
H. Absolínová et al.
Financial support from the Grant Agency of the Czech
Republic (GA ČR), grant No. 525/07/0871 is gratefully
The paper is dedicated to prof. RNDr. Lumír Sommer´s
80th birthday.
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... Because of its aptitude to target various components embedded in a cell [16][17][18][19][20][21], SGR can impede proliferation and stimulate apoptosis in malignant cells [22][23][24][25]. Of the two forms of SGR, the iminium (SA) and alkanolamine (SGROH) which reversibly convert depending on medium pH, the SA form is biologically more active having electronically less dense C atom at position 6 (and thus being susceptible to nucleophilic addition) [26][27][28][29]. SGR is able to modulate several molecular targets in mammalian cells [30,31] and its salts are extremely effective for dental cares [7,12]. ...
... This present work is focused on a detailed investigation of the interaction of the bio-active fluorescent alkaloid Sanguinarine (SGR) [24][25][26][27][28][29][30][31] with niosome prepared by the combination of cholesterol (Scheme 1b) and nonionic surfactant Triton X-100 (TX100) (Scheme 1c) [30]. The steady-state absorption and emission spectroscopic results reveal the inter-conversion between the two prototropic forms of SGR following interaction with the niosome membrane. ...
... And studies showed that SA could induce apoptosis in prostate cancer cells by regulating ERK1/2 Par-4, and inhibit epithelial ovarian cancer development through NT-Κb signaling or PI3K/AKT/mTOR pathway (Rahman et al., 2019;Zhang et al., 2018). DHSA, the first product from SA reductive metabolism in vivo, has been studied along with SA in drug residue and pharmacokinetics, but there are few explorations of DHSA in antineoplastic values (Absolínová et al., 2009;Xie et al., 2015). ...
... As a representative quaternary benzophenanthridine alkaloid, DHSA can also be exacted from plenty of plants belong to papaveraceae, such as Hypecoumleptocapum and Macleaya cordata (Navarro and Delgado, 1999;Yao et al., 2011). As the first metabolite of SA, DHSA has a shorter stability time in working solution than Sanguinarine (Absolínová et al., 2009), as well as better toleration and faster elimination in the rat (Vrublova et al., 2008). It has also been shown that DHSA could induce apoptosis in HL-60 cells by regulating caspase activities, including caspase-3, caspase-8 and caspase-9 (Vrba et al., 2009). ...
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Background: There have been some reports implicating the pharmacologic action of Dihydrosanguinarine (DHSA), but little research including the effects of it on cancer cells. PANC-1 cells have mutations in K-Ras and TP53, which respectively express mutant K-Ras and p53 protein, and the mutations in Ras/p53 have been believed with closely relationship to the occurrence of various tumors. Purpose: To reveal the inhibition of Dihydrosanguinarine on pancreatic cancer cells (PANC-1 and SW1990) proliferation by inducing G0/G1 and G2/M phase arrest via the downregulation of mut-p53 protein, inducing apoptosis and inhibiting invasiveness through the Ras/Mek/Erk signaling pathway. Methods: Human pancreatic cancer cell lines were cultured with cisplatin and DHSA. Then, cell proliferation, the cell cycle and apoptosis were measured by CCK-8 and flow cytometry. The migratory and invasive abilities of pancreatic cancer cells were evaluated by transwell assay. The expression levels of mRNA and protein were measured by RT-PCR and western blotting. Results: The results showed that DHSA treatment inhibited cell proliferation, migration and invasion in a time- and dose-dependent manner and led to induction of cell cycle arrest and apoptosis. G0/G1 and G2/M phase arrest inhibited the viability of PANC-1 cells by downregulating the expression of mut-p53 protein. Decreased levels of C-Raf and Erk phosphorylation in DHSA-treated PANC-1 and SW1990 cells were observed in a time- and dose-dependent manner. However, the total expression of p53 and Ras proteins had a different change in PANC-1 and SW1990 cells. Conclusions: Our findings offer the novel perspective that DHSA inhibits pancreatic cancer cells through a bidirectional regulation between mut-p53/-Ras and WT-p53/-Ras to restore the dynamic balance by Ras and p53 proteins.
... While the fluorescence of sanguinarine and chelerythrine is well described [20][21][22][23], knowledge of the fluorescence properties of other QBAs is limited. Fluorescence spectra in aqueous solution and their change upon binding to DNA has previously been reported [24] and their potential use as probes in fluorescence microscopy and flow cytometry was proposed [19]. ...
... Previous studies of sanguinarine and chelerythrine showed that the fluorescence of QBAs comes from two distinct species-neutral alkanolamine and positively charged iminium [20,21]. In aqueous solution typical values of pK ROH for QBAs are between 7.7 (chelirubine) and 9 (chelerythrine) [30]. ...
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Quaternary benzo[c]phenanthridine alkaloids are secondary metabolites of the plant families Papaveraceae, Rutaceae, and Ranunculaceae with anti-inflammatory, antifungal, antimicrobial and anticancer activities. Their spectral changes induced by the environment could be used to understand their interaction with biomolecules as well as for analytical purposes. Spectral shifts, quantum yield and changes in lifetime are presented for the free form of alkaloids in solvents of different polarity and for alkaloids bound to DNA. Quantum yields range from 0.098 to 0.345 for the alkanolamine form and are below 0.033 for the iminium form. Rise of fluorescence lifetimes (from 2–5 ns to 3–10 ns) and fluorescence intensity are observed after binding of the iminium form to the DNA for most studied alkaloids. The alkanolamine form does not bind to DNA. Acid-base equilibrium constant of macarpine is determined to be 8.2–8.3. Macarpine is found to have the highest increase of fluorescence upon DNA binding, even under unfavourable pH conditions. This is probably a result of its unique methoxy substitution at C12 a characteristic not shared with other studied alkaloids. Association constant for macarpine-DNA interaction is 700000 M-1.
... Argemone mexicana Linn (Mexican poppy, Papaveracea) is an annual weed which grows at the side of agricultural fields and is found mostly in Mexico and Florida of South America as well as in Asian, African and Caribbean countries (namkeleja et al. 2014 The plant shows the presence of fatty acids, alkaloids, phenolics and other phytoconstituents (Apu et al. 2012). There are two types of alkaloids present namely Sanguinarine and Dihydrosanguinarine that are proven toxic to humans, by simple process of reduction and oxidation both the alkaloids are interconvertible to each other (Sarkar 1948;Vrba et al. 2009;Absolínová et al. 2009). The seeds of Argemone mexicana has similar physical appearance as mustard oil seeds, thus after the maturation of the plant, the light weight black colour seeds of Argemone mexicana gets contaminated with mustard seeds and during the oil processing, the oil forms argemone seeds have been mixed with oil of mustard seeds. ...
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In this present study, a simple, rapid, cheap, sensitive and reproducible HPTLC-MS Method has been developed for the identification of two important bioactive compounds, Sanguinarine and Dihydrosanguinarine in Argemone Mexicana Linn seeds. The work further discussed and developed a sensitive HPTLC – MS method to analyse the adulteration and/or contamination of argemone oil in the edible mustard oil by spiking Sanguinarine and Dihydrosanguinarine as biomarkers. The n-hexane: diethyl ether (1:1 v/v) solvent system has been used as an extraction medium to extract the Sanguinarine and Dihydrosanguinarine from the seeds followed by HPLC-MS detection. The CAMAG HPTLC system modules consisted with ATS-4, ADC-2, visualizer-2, TLC scanner-4; Derivatizer and TLC-MS interface-2 have been used for sample application, HPTLC plate development, plate photo documentation, scanning of plate, spraying of derivatization reagent and elution of biomarkers directly from HPTLC plate respectively. The n-hexane: acetone (23:7, v/v) and dragendorff’s reagent has been used as mobile phase and derivatization reagent respectively. The UV- spectra and MS data confirmed the detection of selected biomarkers in the samples/spiked samples. Thus, the work highlights the use of HPTLC-MS to develop simple and routine method for the sensitive detection of Sanguinarine and Dihydrosanguinarine in Argemone Mexicana seeds and able to become bases to these biomarkers’ evaluations in other samples such as various edible oils, agro-products, drugs and biomedical products.
The use of natural products derived from plants as medicines precedes even the recorded human history. In the past few years there were renewed interests in developing natural compounds and understanding their target specificity for drug development for many devastating human diseases. This has been possible due to remarkable advancements in the development of sensitive chemistry and biology tools. Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of the plant species Sanguinaria canadensis. The alkaloid can exist in the cationic iminium and neutral alkanolamine forms. Sanguinarine is an excellent DNA and RNA intercalator where only the iminium ion binds. Both forms of the alkaloid, however, shows binding to functional proteins like serum albumins, lysozyme and hemoglobin. The molecule is endowed with remarkable biological activities and large number of studies on its various activities has been published potentiating its development as a therapeutic agent particularly for chronic human diseases like cancer, asthma, etc. In this article, we review the properties of this natural alkaloid, and its diverse medicinal applications in relation to how it modulates cell death signaling pathways and induce apoptosis through different ways, its utility as a therapeutic agent for chronic diseases and its biological effects in animal and human models. These data may be useful to understand the therapeutic potential of this important and highly abundant alkaloid that may aid in the development of sanguinarine-based therapeutic agents with high efficacy and specificity.
We previously identified cysteine 475 as a key residue for the inhibitory action of sanguinarine on the human glycine transporter GlyT1c. To define potential benzophenanthridine binding pocket more closely, we created a structural homology model of GlyT1 and also mutated several amino acids in the vicinity of cysteine 475. Even though this area contains the molecular determinants of the glycine and sodium permeation pathways, and several mutations resulted in an inactive transporter, we found that the mutation of a polar aromatic tyrosine 370 to purely aromatic phenylalanine, but not to an aliphatic leucine, significantly increased the sensitivity of GlyT1 to both sanguinarine and chelerythrine. The reduction of sanguinarine to dihydrosanguinarine completely eliminated the alkaloid's inhibitory potency. Both these results suggest that aromaticity is important in the interaction of benzophenanthridines with GlyT1. Even though tyrosine 370 is part of the conformationaly highly flexible glycine binding site, and is accesible during the transport process from both intra and extracellular sites, the cytoplasmic location of the second alkaloid sensitive residue, cysteine 475, suggests that the benzophenanthridines might attack the area of the GlyT1 intracellular gates. Copyright © 2015. Published by Elsevier B.V.
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Time stability, acid-base and UV-VIS spectral properties of dihydrochelerythrine (DHCHE) were studied spectrophotometrically in water:methanol and water:ethanol media. DHCHE is stable in strongly acid milieu (pH < 3) and at the higher amounts (60% v/v) alcohol. Acid-base characteristics and UV-VIS spectral properties of chelerythrine (CHE) were studied in aqueous solutions in the presence of different concentrations of HCl, HNO3, H2SO4, H3PO4 and their mixtures. Remarkable shifts of formation parts of absorbance-pH (A-pH) curves to the alkaline medium were observed depending on the type and concentration of inert electrolyte (most remarkable for HNO3 and HCl). The corresponding equilibrium constants pKR+ of the transition reaction between charged iminium Q+ and uncharged QOH (pseudo-base, 6-hydroxy-dihydro derivative) forms of chelerythine were calculated using a numerical interpretation of A-pH curves by a SQUAD-G computer program which ranged from 8.51–9.31. The highest changes of ΔpK R+ (0.75 and 0.53) were observed for H3PO4 and H2SO4, respectively. The priority effect of ionic species and ionic strength was confirmed in the presence of additions of NaCl and KCl. The strength of interaction of CHE with biomacromolecular compounds (i.e., peptides, proteins, nucleic acids etc.) may be affected because of the observed influence of both cation and anion of the inert electrolyte on acid-base behavior.
We previously demonstrated that glycine transporters GlyT1 and GlyT2 are differentially affected by toxic benzophenanthridine alkaloids. Using a combination of homology modeling, knowledge of the sensitivity of sanguinarine to sulfhydryl reagents and site directed mutagenesis we show here that the increased sensitivity of human GlyT1c to sanguinarine is abolished by the mutation of only cysteine 475. Inhibition requires the membrane permeable alkaloid alkanolamine, which is consistent with the intracellular location of the targeted cysteine.
The inclusion of sanguinarine, a biologically active natural benzophenanthridine alkaloid, in cucurbit[7]uril (CB7) was studied by NMR and ground-state absorption spectroscopy, as well as steady-state and time-resolved fluorescence measurements in aqueous solution. The iminium form of sanguinarine (SA(+)) produces very stable 1 : 1 inclusion complex with CB7 (K = 1.0 × 10(6) M(-1)), whereas the equilibrium constant for the binding of the second CB7 is about 3 orders of magnitude smaller. Marked fluorescence quantum yield and fluorescence lifetime enhancements are found upon encapsulation of SA(+) due to the deceleration of the radiationless deactivation from the single-excited state, but the fluorescent properties of 1 : 1 and 1 : 2 complexes barely differ. The equilibrium between the iminium and alkanolamine forms is shifted 3.69 pK unit upon addition of CB7 as a consequence of the preferential encapsulation of the iminium form and the protection of the 6 position of sanguinarine against the nucleophilic attack by hydroxide anion. On the basis of thermodynamic cycle, about 225 M(-1) is estimated for the equilibrium constant of the complexation between the alkanolamine form of sanguinarine (SAOH) and CB7. The confinement in the CB7 macrocycle can be used to impede the nucleophilic addition of OH(-) to SA(+) and to hinder the photooxidation of SAOH.
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Sanguinarine and chelerythrine are intensively studied biologically active alkaloids for their potentially useful medicinal properties, such as antimicrobial, antiinflammatory, and antitumoral activities. This article aims to review critically recent literature published on the chemical behavior, synthesis, analytical methods and biotransformation of both alkaloids.
Quaternary benzo[c]phenanthridine alkaloids (QBAs) are natural products isolated from plants of Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. They are intensively studied for their biological activities, but they have also attractive fluorescence properties. Chromophores responsible for fluorescence are fused aromatic ring systems with electron-donor groups containing oxygen (OH, OCH3, OCH2O). Recently we have described fluorescent characteristics of QBAs - macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) - on interaction with living cells. All these alkaloids immediately enter the living cells and MA-, CHR-and SA-bound DNA; they showed a nucleus architecture similar to common DNA dyes. Moreover, MA binds to DNA stoichiometrically and can rapidly report the cellular DNA content in living cells at a resolution adequate for cell cycle analysis. QBAs could be excited by common argon lasers (488 nm) emitting light in the 575-755 nm range. Spectral characteristics of MA allow simultaneous surface immunophenotyping. These characteristics allow multiple applications of the above-mentioned QBAs with significant diagnostic utility. They can be used as supravital fluorescent DNA probes both in fluorescence microscopy and flow cytometry including multiparameter analysis.
The fluorescence properties of 28 isoquinoline alkaloids have been investigated. In most of them the chromophore responsible for fluorescence was the benzene ring with electron-donor substituents containing oxygen. The long-wave excitation peak practically coincides with the long-wave absorption peak of these substances, covering the region from 284 to 293 nm, the maximum emission being in the range 320-332 nm. With alkaloids having a number of conjugated rings, both excitation and emission were observed at higher wavelengths. Only protoberberine alkaloids behaved as hydrophobic probes, i.e. transfer of these compounds into a less polar medium produced a marked hypsochromic shift and a higher intensity of emission. The effect of polarity on the behaviour of tetrahydroprotoberberines, protopines, pavinanes, aporphines and benzophenanthridines was not so pronounced. Changes of pH manifested themselves most markedly in compounds with dissociable hydroxyl groups; the majority of phenolates did not fluoresce.The phenol group p K values of these compounds in the excited state were lower than in the ground state (which ranged between 8.4 and 10.4). The relations between the apparent p K , determined from fluorescence data, and the p K 's of these compounds in the ground and the excited states are discussed.
The structure of sanguinarine free base was examined. The base is either bis[6-(5,6-dihydrosanguinarinyl)] ether (3) or bis[6-(5,6-dihydrosanguinarinyl)]amine (4) depending on whether Na2CO3 or NH3, respectively, is used as an alkalizing agent. Oxysanguinarine (5) was identified as a side product formed by disproportionation of the pseudobase intermediate 2a.
We have studied the absorption, fluorescence, and surface-enhanced Raman scattering (SERS) spectra of sanguinarine using a silver hydrosol and an electrochemical cell with a silver working electrode for different pH values in the medium. We carried out quantum chemical calculations in order to interpret the electronic and vibrational spectra and to establish their correlations with the structure of the molecules. We optimized the structure and determined the spectral characteristics of the cationic and neutral forms of the sanguinarine molecules in solution and adsorbed on the surface of an anodized Ag electrode for different potentials.
There is compelling evidence that cellular DNA is the target of many small molecule anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of the rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA binding agents, ethidium and sanguinarine with calf thymus DNA. The study revealed non-cooperative binding phenomena for both the drugs to DNA with an affinity similar for ethidium and sanguinarine as observed from different techniques. The binding phenomena analyzed from isothermal titration calorimetry showed exothermic binding for both compounds that was favoured by negative enthalpy and positive entropy changes typical of intercalative binding. The binding of both the drugs was further characterized by strong stabilization of DNA against thermal strand separation in optical melting as well as differential scanning calorimetry studies. The data of the salt dependence of binding of sanguinarine and ethidium from the plot of log K versus log [Na+] revealed a slope of −0.711 and −0.875, respectively, consistent with the values predicted by the theories for the binding of monovalent cations and the binding free energy has been analyzed for contributions from polyelectrolytic and non-polyelectrolytic forces. The salt dependence of the binding was also evident from the conformational changes in the circular dichroism where both extrinsic and induced changes were lowered on increasing the salt concentration. The heat capacity changes obtained from temperature dependence of enthalpy change gave values of (−590 and −670) J · mol−1 · K−1, respectively for the binding of sanguinarine and ethidium to DNA. Overall the DNA binding of ethidium was slightly more favoured over sanguinarine.
Capillary electrophoresis was employed to determine the principal quaternary benzo[c]phenanthridine alkaloids, sanguinarine and chelerythrine, in two plant extracts and one oral hygiene product. Phosphate–Tris buffer of pH 2.5 was used as a background electrolyte, limits of detection were 3 μmol l−1 (sanguinarine) and 2.4 μmol l−1 (chelerythrine) using UV detection at 270 nm. The method, which correlated well with HPLC, is suitable for serial determination of sanguinarine and chelerythrine in plant products and pharmaceuticals.
The effects of quaternary protoberberine and benzophenanthridine alkaloids on acetylcholinesterase (ACHE, EC activity in vitro and on isolated rat duodenum and guinea pig ileum tissue preparations have been studied. The ACHE inhibitory activity of quaternary alkaloids depends on the cationpseudobase equilibrium of the studied alkaloids in solution. The relationship between pKR+ values and the biological activity is described. The influence of surfactants on pseudobase formation of benzo-phenanthridine alkaloids has been discussed.
The qualitative and quantitative aspects of capillary electrophoretic methods used to study drug-protein interactions, viz. the affinity capillary electrophoresis (ACE). Hummel-Dreyer (HD), frontal analysis (FA), vacancy peak (VP) and vacancy affinity capillary electrophoresis (VACE) methods have been investigated. In the ACE and the VACE methods the binding parameters can be calculated from the change in the electrophoretic mobility of the drug on complexation with a protein. In the frontal analysis and the vacancy peak method the free drug concentration is measured with UV detection. In the Hummel-Dreyer method the amount of drug bound is measured with UV detection. For the comparison of these five methods the warfarin-bovine serum albumin (BSA) system was used. Several factors that might influence the determination of association parameters were examined. With the FA, VP, HD and VACE methods the absolute numbers of the different binding sites involved in the complex formation can be determined, a major advantage in drug-binding studies.