Acid-base behaviour of sanguinarine and dihydrosanguinarine

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 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.

Full text

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: kuban@ft.utb.cz
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
rats.
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
876

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
light.
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.
877

Acid-base behaviour of sanguinarine
and dihydrosanguinarine
Czech Republic), CH3COOH, H3PO4 (Lach-Ner,
Neratovice, Czech Republic) all of p.a. purity and
3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic
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,i – Acalc,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.
878

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
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
250 270 290 310 330 350 370
nm
A
1
2
3
4
5
7
6
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
0
0,05
0,1
0,15
0,2
0,25
0,3
220 260 300 340 380
nm
A
1
2
3
4
5
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
Conditions
ε1
(274 nm)
[L mol–1 cm–1]
ε2
(274 nm)
[L mol–1 cm–1]
ε1
(327 nm)
[L mol–1 cm–1]
ε2
(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
a
b
879

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 ~
PO4
3– ~ SO4
2–.
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.
[21]).
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
0,2
0,22
0,24
0,26
0,28
0,3
0,32
0,34
0,36
34567891011
pH
A
1
2
3
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
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]
880

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
H3PO4.
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,
respectively))
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
881

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
purposes.
7,50
7,70
7,90
8,10
8,30
8,50
-2 -1,5 -1 -0,5 0
log I
pK
KCl
a
2
3
1
4
7,50
7,70
7,90
8,10
8,30
8,50
-2 -1,5 -1 -0,5 0
log I
pK
NaCl
a
4
3
2
1
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
Conditions
pKR+
0.01 mol L-1
HCl
0.01 mol L-1
HNO3
0.01 mol L-1
H2SO4
0.01 mol L-1
H3PO4
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
882

H. Absolínová et al.
Acknowledgments
Financial support from the Grant Agency of the Czech
Republic (GA ČR), grant No. 525/07/0871 is gratefully
acknowledged.
The paper is dedicated to prof. RNDr. Lumír Sommer´s
80th birthday.
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