Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp. 369ñ377, 2017
ISSN 0001-6837
Polish Pharmaceutical Society
POTENTIOMETRIC STUDY OF Pd(II) COMPLEXES OF SOME FLAVONOIDS
IN WATER-METHANOL-1,4-DIOXANE-ACETONITRILE (MDM) MIXTURE
ANNA KUèNIAR*, JANUSZ PUSZ and URSZULA MACIO£EK
Department of Inorganic and Analytical Chemistry, Faculty of Chemistry, RzeszÛw University of
Technology, Al. PowstaÒcÛw Warszawy 6, 35-959 RzeszÛw, Poland
Abstract: The potentiometric method was used to determine the protonation (dissociation) constants for morin,
rutin and chrysin and the composition and formation constants of the Pd(II)-flavonoid complexes in the
water/methanol/acetonitrile/1,4-dioxane mixture (water/MDM). All investigations were carried out at a constant ionic strength of I = 0.2 (KCl) at 298 K. Morin, rutin and chrysin are polyprotic acids (polihydroxyflavones) practically insoluble in water, but they are soluble in organic solvents. The mixture of water/MDM is
interesting because several solvents were mixed together to produce a solvent having a physical and chemical
properties different of the pure components. The two (chrysin) and three (morin, rutin) dissociation constants
were obtained by the Bjerrum method (graphic approximations) and numeric data analysis by Hyperquad2008
computer program. Obtained results provide evidence to show that investigated flavonoids in water-MDM solutions form with Pd(II) complexes of composition ML and ML2. The values of the stability constants suggest
that the complexes of chrysin, rutin and morin with Pd(II) ions are of medium stability. Moreover, obtained
result implies that morin forms the strongest complexes with Pd(II), subsequently rutin and chrysin.
Keywords: morin, rutin, chrysin, palladium complexes, stability constants, potentiometric method
tumors (4). The ability to chelate metal ions is of
great importance for analytical and pharmaceutical
applications of flavonoids.
In review (5) it was stated, that Pd-morin complexes exhibit greater antioxidative effects (scavenging superoxide radicals) than the morin itself.
Moreover, the Pd-morin compound shoved an
inhibitory effect on lipid peroxides which was
greater than that of free ligand.
The spectrophotometric investigations performed by Maleöev, co-author of (5), helped designate of stability constants of Pd-rutin and Pd-morin
complexes; the obtained values of log β are 10.15
(pH = 8.0) and 4.55 (pH = 4.0), respectively. The
Morin, rutin and chrysin (Fig. 1) belong to the
group of flavonoids, a very important class of phenolic compounds occurring in all parts of plants.
Flavonoids have received increasing attention during the last years because of their wide range of biological activities. Beneficial effects of flavonoids
have been described for diabetes mellitus, cancer,
allergy, viral infections and inflammations.
Polihydroxyflavones can bind biomolecules, such as
hormone carriers, DNA and enzymes.
Hydroxyflavones catalyze electron transport
and scavenge free radicals (1-3).
Also transition metal complexes have a tracked
much attention as therapy for various types of
Figure 1. Structural formulae of chrysin, morin and rutin
* Corresponding author: e-mail: [email protected]; phone: +48 017 865 15 64
369
370
ANNA KUèNIAR et al.
trile, as organic solvents) that dissolves a wide range
of poorly water soluble compounds. This universal
co-solvent system has a combination of polar and
nonpolar properties so the solubility is improved for
hydrophobic compounds but is still good for polar
molecules what is very important for early phase of
drug research. This system enables pK measurements by potentiometric and UV titration (11).
The previous papers (12-16) presented the
investigations of chrysin, rutin and morin dissociation constants in a different solvents (Table 1). The
constants were determined by the potentiometric
and some computational methods.
In this study, potentiometric titration was used
to determine the stoichiometric protonation constants
of chrysin, morin and rutin and the stability constants
of Pd(II) complexes of those flavonoids. A co-solvent mixture consisting of water (50%) and equal
volumes of methanol, dioxane and acetonitrile
referred to as MDM (50%), was tried and found to be
efficient for pKa measurements of these flavonoids.
The protonation constants of polihydroxyflavones and stability constants of their
Pd(II)-complexes were analyzed by Bjerrum method
and Hyperquad2008 computer program.
The knowledge of acid dissociation constant
values of compounds is a very necessary parameter
in ADMET studies (Absorption, Distribution,
Metabolism, Excretion, Toxicity) for it allows to
determine chemical aspects of absorption, distribu-
Bjerrum method was modified and simplified and
used in this research.
Palladium catalyzed cross-coupling reactions
have revolutionized the way in which molecules are
constructed. These reactions are used in the synthesis,
medicinal chemistry, materials science and others. In
medicine palladium is already often used in dental
applications, where its biocompatibility has proven
to be satisfactory (6). PdCl2 is widely used as a color
forming reagent in spectrophotometric determinations of many drugs (7).
Flavonoids are sparingly soluble in water, but
they are soluble in organic solvents. Water solubility of morin at 303 K is 1.25 ∑ 10-4 mol/L (8); rutin
0.66 ∑ 10-5 mol/L (298 K) (9), chrysin: 1.86 ∑ 10-5
mol/L (298 K) and 2.38 ∑ 10-5 mol/L (303 K) (10).
Not all compounds dissolve in any single organic
solvent + water mixtures. In connection with this
problem, multicomponent co-solvent mixtures, consisting of equal volumes of methanol, dioxane and
acetonitrile, were prepared. This mixture, termed
MDM, improves the solubility of the hydrophobic
compounds, is a good solvent for polar molecules,
and fulfils all the requirements that are needed for
the application of the pH-metric method. The validation of pKa values determined in MDM-water
mixtures was presented in (11).
The MDMñwater mixture is a relatively new
multicomponent co-solvent mixture (consisting of
equal volumes of methanol, dioxane and acetoni-
Table 1. Dissociation constants of chrysin, morin and rutin in different solvents.
Solvent
pKa1
pKa2
pKa3
pKa4
Method
Ref.
Chrysin
50 v%
DMSO/water
6.97
8.22
-
potentiometric
(10)
50 v%
ethanol/water
7.90
11.40
-
potentiometric
(11)
50 v%
dioxane/water
8.57 ± 0.04
12.37 ± 0.03
-
potentiometric
(12)
Rutin
4 v%
methanol/water
7.35 ± 0.02
8.8 ± 0.1
11.04 ± 0.1
11.9 ± 0.1
6.84 ± 0.60
(single pKa)
7.19 ± 0.60
(single pKa)
9.76 ± 0.35
(single pKa)
9.42 ± 0.10
(single pKa)
6.84 ± 0.60
(approx. pKa)
8.10 ± 0.60
(approx. pKa)
9.46 ± 0.10
(approx. pKa)
5.18 ± 0.010
8.34 ± 0.008
10.24 ± 0.010
AM1 quantum
chemistry
method
(13)
12.55 ± 0.35
(approx. pKa)
Calculated using
the ACD/pKa
DBv7.0 program
-
potentiometric
(14)
Morin
49.8 mass%
methanol/water
Potentiometric study of Pd(II) complexes of some flavonoids in...
tion and excretion of substances with biological
meaning (14).
EXPERIMENTAL
Reagents and apparatus
Chrysin and morin were purchased from
Sigma-Aldrich (USA); rutin was bought from
KOCH-Light Laboratories Ltd. Stock solutions of
chrysin (2.75∑10-3 mol/L), morin (4.94∑10-3 mol/L),
rutin (5.12∑10-3 mol/L) were prepared in MDM. The
MDM mixtures were prepared by mixing equal volumes of the three solvents: MeOH, dioxane and
MeCN.
All flavonoids were weighed accurately and
dissolved in MDM.
PdCl2 from POCH (pure p.a., Poland) was used
in this investigation. Solutions (0.00984 mol/L) of
palladium(II) chloride were obtained by dissolving
the appropriate weighed amounts of compound in
redistilled water and acidifying them with a concentrated hydrochloric acid. The solution was standardized by complexometric titration with Na2H2EDTA
solution and xylenol orange as indicator (17).
Stock solution of hydrochloric acid (0.1 mol/L)
was obtained by direct dilution of the commercial
hydrochloric acid with water. The exact concentration of HCl was determined by acid-base titration.
Stock solution of carbonate-free sodium
hydroxide (0.0550 mol/L) was prepared from concentrated solutions and standardized with potassium
hydrogen phthalate from POCH.
Potassium chloride (2 mol/L) was added to
standardize the ionic strength of solvent-water mixtures.
Acetonitrile (LC-MS Chromasolv, Fluka),
methanol (spectroscopic grade) and 1,4-dioxane
(pure p.a.) were supplied by POCH.
All water solutions were prepared in deionized
water with conductivity lower than the unit of 0.05
µS/cm obtained with a purification system (SolPure
7 Elkar).
The potentiometric titrations were performed
using an automatic titrator T-70 (Mettler-Toledo,
Switzerland) and TitroLine Alpha TL 20X (Schott,
Germany). The electrodes were calibrated with
standard buffer solutions (pH 4.0 and 9.0; Merck)
before and after each series of pH-potentiometric
titrations. The titrators were connected to a personal computer and the factory titration software was
used to control the titration and data acquisition.
Combined pH electrode, was calibrated according
to the Gran method and adapted for the given solvent (18, 19).
371
The pH titrations were carried out in a titration
cell consisted of a double-walled glass beaker. The
cell was thermostated externally at 298 ± 0.1 K with
a water bath cooler system (thermostat PGW E-1,
Medingen).
The titration data were numerically analyzed
by Origin and the Hyperquad2008 computer program; the distribution species diagrams were produce using HySS program.
Titrations in water-MDM media
The basic procedures in pH-potentiometric
determination are titrating the basic ligand alone
first, and then titrating in the presence of both Pd(II)
metal ion and ligand. Each solution was left to stand
15 min before titration. During the process, the solution temperature was maintained at 298 ± 0.1 K and
kept stirring.
The solutions of flavonoids were prepared in
the range of contents from 50 to 80 mM. The cell
was filled with 50 mL of flavonoid solution and
other components, where solution composition was
50% vol. MDM and 50% vol. of water.
The titrations were performed using the
described cell apparatus and titrators in pH (mV)
measurements. The ionic strength was maintained at
0.2 with KCl. The autoprotolysis constant for the
given solvent system has been determined previously and is 10-14.93 (20).
Determination of the dissociation constants and
stability constants of the complexes of Pd(II) with
chrysin, rutin and morin
The classical Bjerrum method belongs to the
most willingly applied ones due to its capability
to determine the number of forming complexes,
as well as to determine the stability constants and
indicate whether the chelate complexes are
mono- or multi-nuclear. The potentiometric
measurement of changes in hydrogen ions activity during protonation or complexing reactions
was applied as the experimental method for all
complexing systems. The stability constants of
all investigated complexes were determined by
the Bjerrum method (graphic approximations)
(21, 22), and numeric data analysis by Hyperquad2008.
Morin, rutin and chrysin are polyprotic acids,
and their protonation constants can be expressed by
pH measurement during their titration with a base of
known concentration.
The mean number of j protons connected with
the appropriate base was calculated for each experimentally determined pH value from the formula:
372
ANNA KUèNIAR et al.
Ht ñ [H+] + Kw,s [H+]-1
j = ñññññññññññññññññññññ
(1)
At
+
where (H ) is equilibrium concentration of
hydronium ions, Kw,s is autoprotolysis constant for
the given solvent system, Ht is concentration of protons capable of dissociation, At is total anion concentration of hydronium ions, respectively (19).
It follows from Eq. 1 that j is only a function
of (H+) and is independent of the investigated compound concentration. Figures 2 and 3 present the
curves of j = f(pH) for morin and rutin solutions,
respectively.
The relation between the mean number of protons, equilibrium concentration of hydronium ions
and protonation constants Ki is expressed by equation (2):
[H+] ∑ K1 + 2 [H+]2 ∑ K1 ∑ K2 + ...
j = ññññññññññññññññññññññññññññññññ
(2)
1 + [H+] ∑ K1 + [H+]2 ∑ K1 ∑ K2 + ...
Based on j and pH values, the protonation constants Ki were read from the graph for half values of
j , for which the dependence between pH = logKi was
realized. Determined dissociations constants of
chrysin, rutin and morin are given in Table 2.
Investigations of the equilibria of complexation reactions were carried out within pH range 2-12
for all flavonoids. The equilibria were determined
on the base of data of the titration curve of chrysin,
morin and rutin in the presence of Pd(II) ions. On
the Figures 4 and 5 are shown examples of potentiometric titration curves ligands and ligands in a presence of metal ions.
The titration was carried on at 298 K and at an
ionic strength equal to 0.2 (KCl). Chrysin/morin/rutin concentration in the titrated solution was
changed in relation to the metal ion concentration: cL
: cM = 1 : 1; 2 : 1 and 3 : 1.
For every titration point, the fraction of titration a, the concentration of ligand (L) and the average number of ligands were calculated using the
Eqs. 3-5 (19):
(m ñ ac)cH
L
ñ [H+] + [OH-]
m
[L] = ññññññññññññññññññññññññññññññ
+
+
+
[H ]K1H + 2[H ]2K1HK2H + ... + m[H ]mK1H ...KmH
(3)
where m is number of protons in ligand, a is fraction
of titration, cHmL is concentration of ligand in solution titrated to half, K1 ,Ö, Km ñ protonation constant of ligand, (H+) and (OH-) are concentration of
hydrogen and hydroxide ions, respectively.
cHmL ñ [L] ∑ αL{H)
n- = ññññññññññññññ
(4)
cM
where cHmL is concentration of ligand in solution
titrated to half, cM is concentration of metal in solution titrated to half, αL{H) is ratio of side reactions of
ligand protonation described by Eq. 5:
αL{H) = 1+ [H+]K1H + 2[H+]2K1HK2H +
m[H+]mK1HK2H...KmH
(5)
On the basis of the experimentally defined set
and (L) formation curves of the investigated complexes were plotted in the n- = f(pL) system. The values of the following complexation stability constants
K1, K2, K3... were read directly from the graph for values n- = 0.5, 1.5, 2.5, ..., which fulfil dependence:
Figure 2. The formation curve of morin at 298 K (I = 0.2 KCl)
H
H
Potentiometric study of Pd(II) complexes of some flavonoids in...
373
Figure 3. The formation curve of rutin at 298 K (I = 0.2 KCl)
Figure 4. Potentiometric titration curves for chrysin and Pd(II) + chrysin (cM : cL = 1 : 2, T = 298 K, I = 0.2 KCl)
1
Kn = ñññññññññ
[L]n- = n ñ 0.5
(6)
Numeric data analysis
The potentiometric data were analyzed by
import the values into the Hyperquad2008 calcula-
tion software which is frequently used for obtaining more precise stability constants values. The
program computes protonation constants of ligands and stability constants from potentiometric
data by a non-linear least-square curve-fitting
analysis.
374
ANNA KUèNIAR et al.
The calculations consist in determining the
concentration of hydrogen ions (pH) for the next
potentiometric titration point. The program can calculate the value using such output data as: substratesí concentration, the initial volume of the system, the volume of the added titrant, corresponding
pH values and protonation and stability constants
determined by another method. Then, the calculated
pH values are compared with the determined ones
and the criterion for the best compatibility is the
minimal sum value of weighed squares of pH deviation:
U = Σ wi ∑ (pHieksp ñ pHiobl)
(7)
The weight indicator is calculated according to
the formula:
wi = (pHi+1 ñ pHi)
(8)
VNaOH, mL
Figure 5. Potentiometric titration curves for morin and Pd(II) + morin (cM : cL = 1 : 2, T = 298 K, I = 0.2 KCl)
Figure 6. The complex formation curve of chrysin at concentration 3.03∑10-4 mol/dm3 with Pd(II) ions (Hyperquad2008 screen, cM : cL = 1
: 3, pKw,s = 14.93, I = 0.2, T = 298 K)
375
Potentiometric study of Pd(II) complexes of some flavonoids in...
Figure 7. Speciation plots of chrysin and chrysin complexes with Pd(II) ions (Hyss screen)
Table 2. Dissociation constants of chrysin, rutin and morin at 298 K (I = 0.2; KCl).
Ligand
pKa1
pKa2
pKa3
Methods
Chrysin
8.01
12.29
-
Bjerrum
7.92 ± 0.03
11.89 ± 0.07
-
Hyperquad2008
4.83
6.20
8.45
Bjerrum
5.10 ± 0.12
5.92 ± 0.17
8.26 ± 0.17
Hyperquad2008
5.00
6.35
8.66
Bjerrum
5.16 ± 0.05
6.18 ± 0.06
8.63 ± 0.02
Hyperquad2008
Rutin
Morin
Table 3. Stability constants of chrysin, rutin and morin complexes with Pd(II) ion at 298 K, at the ionic strength I = 0.2 (KCl).
Ligand
log fl1
Bjerrum method
log fl2
Hyperquad2008
Bjerrum method
Hyperquad2008
Chrysin
7.48
7.45 ± 0.02
13.97
13.96 ± 0.03
Rutin
9.69
9.48 ± 0,04
14.86
14.63 ± 0.04
Morin
10.26
10.37 ± 0.02
16.22
16.56 ± 0.02
376
ANNA KUèNIAR et al.
The calculations are conducted until the minimal U value is received, that is after the best compatibility between the calculated pH values and the
experimental data. The protonation constant values
or stability constants are the final result (23).
The stability constants are presented as βMLH,
where M, L, and H designate metal, ligand, and
hydrogen, respectively. Optimized Hyperquad fitted
the model titration curve (continuous curve) to the
experimental data (¯) showed in Figure 6.
The determined chrysin/morin/rutin dissociation constants values and the stability constants of
flavonoidsí complexes with Pd(II) ions calculated
by Hyperquad2008 were used to plot the dependencies of the mole fractions of the particular
flavonoids ionic forms and Pd(II) complexes on pH.
For the calculations, HySS2009 by Protonic
Software was used. The dependence for chrysin is
shown in Figure 7.
RESULTS AND DISCUSSION
In this investigation, the determination of dissociation constant of chrysin, rutin and morin and
their stability constants of complexes with Pd(II)
ions by potentiometric method was performed.
In the first stage, pKa (I = 0.2) values of three
flavonols sparingly soluble in water and with several ionizable hydroxyl groups were estimated by
potentiometric method. They were titrated in
water/MDM mixtures.
Figures 2ñ3 prove, that the function j = f(pH)
does not depend on flavonoid concentrations.
Therefore, it can be claimed that within pH 4ñ12.5
in water-MDM solutions of flavonoids, only the ligand dissociation takes place in the solution and no
other side reactions occur.
Chrysin (H2L) rutin (H4L) and morin (H5L) in a
solution undergo a gradual dissociation with the
increase in pH Appropriate equations for morin are
given below:
H5L + H2O‰
(9)
ŠH4L- + H3O+
H4L + H2O‰
(10)
ŠH3L2- + H3O+
H3L2- + H2O‰
(11)
ŠH2L3- + H3O+
3H2L + H2O‰
(12)
ŠHL4- + H3O+
HL4 + H2O‰
(13)
ŠL5- + H3O+
The Ka1 and Ka2 (chrysin) and Ka1, Ka2 and Ka3
(morin, rutin) dissociation constants were experimentaly determined. The last constants for morin
and rutin were not determined because, in these conditions (alkaline medium), flavonoids are probably
unstable. Quite good compatibility of the dissociation constant values was obtained by the two methods: graphical and computational (Table 2). In the
literature, there are no data exist for the dissociation
constants of the discussed flavonoids in the waterMDM system. The determination of acidic dissociation constants was necessary to investigate the equilibria of complexation with metal ions.
It follows from speciation plots of investigated
ligands (chrysin ñ Fig. 7) that in aqueous-MDM
solutions of morin and rutin for pH < 5.5, only H5L
and H4L- forms occur; while below pH < 8 H2L and
HL- forms of chrysin are existing.
In the next stage, the stability constants of
chrysin, rutin and morin with Pd(II) ions were determined. The titration curves of suitable flavonoid and
a solution of flavonoid containing Pd(II) ions in the
range of pH 6ñ12 (chrysin, Fig. 4); pH 3.5ñ12.5
(morin, Fig. 5) and pH 5.0ñ12.5 (rutin) were different on account of complex formation. Complex formation at pH values lower than 3.0 is difficult
because the flavonoids are predominantly present in
their undissociated form. Simultaneously, based on
the course of the curves (Figs. 4, 5), it can be
observed that the investigated flavonoids form
mononuclear complexes with Pd(II) ions.
In the presence of water, the aqua-complexes
of palladium may be formed. Otherwise, the limit
pH value of hydroxy-complex formation (pH[MOH] )
amounts to pKw ñ 2 ñ log β1, where β1 is a first stability constant of appropriate hydroxy-complex
(22). For the investigated metal ion the value in
water is equal 3.9 (log β1 = 8.1 (21)), but it appears
that in the water-MDM environment hydroxo-complexes are notformed.
Our present results provide evidence that investigated flavonoids in water-MDM solutions form with
Pd(II) complexes have composition ML and ML2.
The values of the stability constants of the investigated complexes (Table 3) suggest that the complexes of
chrysin, rutin and morin with Pd(II) ions are of medium stability. Moreover, obtained result implies that
morin forms the strongest complexes with Pd(II),
then subsequently are rutin and chrysin.
+
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Received: 5. 02. 2016