Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine

Central European Journal of Chemistry 1. Introduction Quaternary benzo[c]phenanthridine alkaloids (QBAs) are present mainly in plants of Fumariaceae, Papaveraceae, Ramunculaceae and Runaceae families. Sanguinarine (SA) and chelerythrine (CHE) are the only commercially available members of QBAs, in addition to minor members (i.e., sanguirubine, chelirubine, sanguilutine, chelilutine and macarpine). QBAs, a group of low molecular compounds with N-heteroatom, belong to the highly bioactive substances [1]. Some side effects were reported (toxic effect on some cellular level [2-5]) in addition to the beneficial pharmacological actions (antimicrobial, antiapoptotic, antiinflamatory etc. effects [6,7]). Standardized plant extracts have been exploited as anti-plaque components of dental hygiene preparations (mouth-wash, tooth paste etc.), as active components for myopathy preparations and in veterinary preparations. Many very extraordinary properties were observed in the frame of detail studies of acidobasic and electrophoretic behavior of sanguinarine (SA) and chelerythrine (CHE) [8-11] and mainly their dihydroderivatives (dihydrochelerythrine, dihydrosanguinarine, etc., the former being recognized as the first metabolite of detoxification of sanguinarine in rats [2]) in the presence of inert electrolytes [3,4,10,11]. They exhibit very characteristic spectral features that can be used for their direct determination, for studies of their interactions with biomacromolecules using UV-VIS spectrophotometry, fluorimetry, mass spectrometry etc. [12-15] The exact knowledge of physico-chemical characteristics is very important for correct interpretation of experiments concerning studies of interactions and behavior of the alkaloids at nearly physiological conditions. It is evident that the study of interactions [3-6] with biologically important macromolecules (i.e., receptors, transport proteins, nucleic acids etc.), as E-mail: [email protected]* Research Article Received 14 August 2009; Accepted 6 March 2010 1 Department of Chemistry and Biochemistry, Mendel University, 613 00 Brno, Czech Republic 2 Department of Chemistry, Masaryk University, 611 37 Brno, Czech Republic 3Department of Medical Chemistry and Biochemistry, Palacky University, 775 15 Olomouc, Czech Republic 4 Department of Biochemistry and Food Analysis, Tomas Bata University, 762 72 Zlín, Czech Republic Helena Absolínová1, Luděk Jančář2, Irena Jančářová1, Jaroslav Vičar3, Vlastimil Kubáň2,4* Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine Keywords: Chelerythrine•Dihydrochelerythrine•Time stability•UV-VIS spectral characteristics• Equilibrium constants Abstract: 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 ∆pKR+ (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. © Versita Sp. z o.o. Cent. Eur. J. Chem. • 8(3) • 2010 • 626–632 DOI: 10.2478/s11532-010-0038-7 626 Amresco®, Solon, Ohio, USA) and sodium hydroxide (reagent grade purity, Penta, Chrudim, Czech Republic) solutions were used for the pH adjustment in a pH interval pH = 2 – 11 with steps ∆pH = 0.2 – 0.5. All other experimental conditions were in accordance to previous paper [17]. All spectrophotometric measurements were performed using a UV-VIS Lambda 25 double beam spectrophotometer controlled by UV WinLab software (range 190 -1100 nm, slit 1 nm, fixed, quartz cuvette 10 mm, Perkin Elmer, Shelton, USA) or a Helios Beta UV-VIS single beam spectrophotometer (range 190 – 1100 nm, slit 1 nm, fixed, quartz cuvette 10 mm, Unicam, Cambridge, UK) controlled by a Vision software. 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 six selected wavelengths between 268 and 405 nm (268, 272, 280, 316, 339 and 405 nm) using the SQUAD-G computer program [18]. The absorbance values of both alkaloids were measured three times at each pH and the mean values from these measurements were used to calculate of pKR+. For more details see our previous paper [17]. 3. Results and Discussion 3.1. UV-VIS spectral and acid-base behavior of dihydrochelerythrine (DHCHE) Very complex absorption spectra of DHCHE were registered in the wavelength interval 220 – 380 nm in presence of 60% (volume per volume v/v percentage is well as the often mentioned formation of intercalation complexes with cyclodextrins and biopolymers, belongs to this class of experiments. The main goals of our effort were (i) study of the acidbase behavior and time stability of CHE and DHCHE as the function of experimental conditions (pH, ionic strength, concentration of electrolyte and its composition, etc.), (ii) determination of UV-VIS spectral characteristics and their “true” pKR+ constants, (iii) identification of experimental conditions and requirements that qualify the possibility and correctness of interaction studies with these compounds in almost neutral and weakly basic solutions (close to physiological conditions). 2. Experimental Procedure 2.1. Materials and methods Stock solution of chelerythrine (c = 130 µmol L-1) was prepared by dissolution of chelerythrine chloride (Sigma Aldrich) in distilled water. Working solutions of CHE (c = 6.5 or 9.4 µmol L-1) were prepared by dilution of the stock solution with cold freshly boiled distilled water. Dihydrochelerythrine (DHCHE, MP 189-191°C, 99% purity – checked by HPLC and controlled by calculation using molar absorptivity) was prepared from CHE by reduction with NaBH4 in methanol [16]. Stock solutions of dihydrochelerythrine (cm = 60 μg mL-1) were prepared by dissolution of DHCHE in methanol or ethanol. Working solutions (cm = 2.4 µg mL-1) were prepared freshly by dilution of the stock solution with methanol, ethanol 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, Czech Republic), H3PO4, ethanol and methanol (LachNer, Neratovice, Czech Republic), all of p.a. purity. Tris- (hydroxymethyl)aminomethane (TRIS – ultra pure grade, Figure 1. Absorption spectra of dihydrochelerythrine (cDHCHE = 2.4 µg mL-1, cHCl = 0.01 mol L-1, the pH was adjusted by NaOH) in 60% methanol (A) and in 60% ethanol (B): A: 1 – pH = 2,10; 2 – pH = 3,11; 3 – pH = 4,05; 4 – pH = 8,97; B: 1 – pH = 2,08; 2 – pH = 2,95; 3 – pH = 3,64; 4 – pH = 9,58. 2 H. Absolínová, L. Jančář, I. Jančářová, J. Vičar, V. Kubáň 627 12% after 30, 60 and 120 min, respectively, in all cases. The same trend (but much slower) was observed for higher concentrations of methanol and ethanol (10 – 30%). The solutions in 60% methanol and 60% ethanol were mostly stable in the time interval (≤ 120 min) and insignificant increase of absorbance at 317 and 353 nm was observed (approx. up to 104 % of their initial values) in methanolic solutions. The graphs for time stability of DHCHE in 4, 10, 30 and 60% methanol (A) and methanol (B) at λ = 274 nm are depicted in Fig. 2. Higher time stability of DHCHE in ethanol is evident from the Fig. 2B. 3.1.2. Influence of acidity on time stability. The solutions of DHCHE are relatively stable only in strongly acidic milieu at all contents of methanol or ethanol. At pH 2 in the presence of 0.01 mol L-1 HCl absorbance decreased between 95-92% of their initial values after 30 min in 4% methanol and ethanol, respectively. In the presence of formiate, acetate and phosphate buffers (pH 2.8, 4.2 and 6.7, respectively) the decreases were more evident (see Fig. 3 for 4% water/methanol – A - and water/ethanol mixtures - B) due to lower stability at higher pH values and probably due to presence of components of buffers. used through the text if not stated differently) methanol or ethanol (Fig. 1) and 0.01 mol L-1 HCl in the pH range 2 – 10 with ∆pH = 0.3 – 0.5 steps (pH was adjusted by a stepwise addition of a NaOH solution). Distinct absorption maxima at 230, 274, 317 and 353 nm are present in acidic media pH < 3. Only two distinct absorption maxima appear at 227 and 280 nm in pH region 4 – 10. The absorption maximum at 230 nm is shifted to shorter wavelengths (λmax = 227 nm) and the absorption maximum at 274 nm is shifted to longer wavelengths (λmax = 280 nm) when pH was increased (pH > 3). Presence of isosbestic points at 232, 268, and 333 nm confirmed reversible acidbasic equilibrium between the charged iminium Q+ and uncharged QOH forms of DHCHE at pH 4 - 10. 3.1.1. Influence of alcohol content on time stability Time stability of DHCHE solutions was registered in 4, 10, 30 and 60% methanol or ethanol at pH = 2 (0.01 mol L-1 HCl), in the presence of formiate (pH = 2.8), acetate (pH = 4.2) and phosphate buffers (pH = 6.7) at four selected wavelengths 274, 278, 317 and 353 nm. The mean values (three replicates) of absorbance rapidly decreased in 4% aqueous methanol to about 38, 14 and Figure 3. Time stability of DHCHE solutions containing 2.4 µg mL-1 DHCHE in 4% aqueous methanol (A) and in 4% aqueous ethanol (B) measured at 274 nm. Curve 1 – 0.01 mol L-1 HCl (pH = 2); 2 – formiate buffer (pH = 2.8), 3 – acetate buffer (pH = 4.2); 4 – phosphate buffer (pH = 6.7) Figure 2. Time stability of solutions containing 2.4 µg mL-1 DHCHE in aqueous methanol (A) and ethanol (B) media and measured at 274 nm. Alcohol content (in % v/v): 1 – 4%; 2 – 10%; 3 – 30%; 4 – 60% 3 Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine 628 reaction between a charged iminium Q+ and an uncharged QOH (pseudo-base, 6-hydroxy-dihydroderivative) forms of chelerythrine is confirmed by the presence of three sharp isosbestic points at 239, 275 nm and 302 nm. The molar absorption coefficients ε1 a ε2 for both acidobasic forms of CHE in 10 and 100 mmol L-1 HCl (calculated using SQUAD-G) are given in Table 1. 3.2.2. Absorbance-pH curves Absorbance-pH curves (A-pH curves) were measured at constant concentration of chelerythrine c = 6.5 µmol L-1 and 10-100 mmol L-1 HCl (see Fig. 5), HNO3, H2SO4 and H3PO4 (not graphically presented). The data were collected in pH interval pH = 3 – 11 with steps ∆pH = 0.3 – 0.5 for six wavelengths (268, 272, 280, 316, 339 and From the results it is clearly evident that the solutions of DHCHE are stable only in more then 60% methanol/ ethanol and with a decreasing content of alcohols the stability decreases. Time stability is also seriously increased by acidity. The highest time stability was observed in strongly acidic media (0.01 mol L-1 HCl for methanol and ethanol and in formiate buffer of pH 2.8 for ethanol). In slightly acidic, neutral and alkaline media the solutions are unstable and stability decreases with decreasing content of alcohols and increasing pH. 3.2. UV-VIS-spectral and acid-base properties of chelerythrine 3.2.1. Absorption spectra Absorption spectra of CHE (c = 6.5 µmol L-1) were registered between 220 – 430 nm and pH 2.5 - 11 in the presence of 10 mmol L-1 concentrations of HCl, HNO3, H2SO4 and H3PO4. Two distinct absorption maxima at 268 and 316 nm and less promoted maxima at 339 and 405 nm are present in the pH interval pH = 2.5 – 8. Absorption maxima shifted to longer wavelengths in alkaline media. The two distinguished absorption bands at 280 nm and a less distinguished one at 316 nm characterize the spectrum of the alkaline solutions (pH 8 – 11). Absorption maxima at 339 and 405 nm disappear with increasing pH (see Fig. 4). The reversible transition Figure 4. Absorption spectra of chelerythrine (cCHE = 6.5 µmol L-1, cHCl = 0.01 mol L-1, pH adjusted by NaOH). Curve - pH: 1 – 3.16; 2 – 5.16; 3 – 7.07; 4 – 8.03; 5 – 9.05; 6 – 10.03 Table 1. Values of molar absorptivity coefficients ε1 and ε2 and their average standard deviation over the whole data set for the charged iminium Q+ (ε1) and the uncharged QOH (pseudo-base, 6-hydroxy-dihydroderivative, ε2) forms of chelerythrine calculated using a numerical interpretation of the A-pH curves by a SQUAD-G program. (cCHE = 6.5 µmol L-1, cHCl = 0.01 or 0.1 mol L-1, pH adjusted by NaOH). Medium ε1(268 nm) [L mol–1 cm–1] ε2(268 nm) [L mol–1 cm–1] ε1(316 nm) [L mol–1 cm–1] ε2(316 nm) [L mol–1 cm–1] 0.01 mol L-1 HCl 59 660 ± 220 33 520 ± 370 37 060 ± 120 16 790 ± 260 0.1 mol L-1 HCl 59 970 ± 200 34 350 ± 270 38 230 ± 150 17 990 ± 200 Figure 5. Absorbance-pH curves of chelerythrine (cCHE = 6.5 µmol L-1) in the presence of mineral acids at c = 0.01 mol L-1 measured at λ = 268 nm, neutralization with NaOH (a) or TRIS (b). Curve – acid: 1 – HCl; 2 – HNO3; 3 – H2SO4; 4 – H3PO4 H. Absolínová, L. Jančář, I. Jančářová, J. Vičar, V. Kubáň 629 4 405 nm) corresponding to the absorption maxima of both acidobasic forms. The formation parts of the A-pH curves (and of course the corresponding pKR+ values) were shifted to the more alkaline medium with increasing initial concentration of acids (ionic strength) and depended on the character of the corresponding anion of the acid. The corresponding pKR+ values calculated using a numerical interpretation of the A-pH curves by the SQUAD-G program (see Table 2) varied from 8.51 – 9.31. The most serious shift of the pKR+ was found in the presence of H3PO4 (∆pKR+ = 0.75) and H2SO4 (∆pKR+ = 0.53), while the less distinct shift was observed for HCl (∆pKR+ = 0.50), HNO3 (∆pKR+ = 0.46) and the smallest one for CH3COOH (∆pKR+ = 0.37). From the results it can be concluded that the corresponding pKR+ values are influenced by the character (type) of anions and also by total mass concentration of anions in the order PO4 3– > SO4 2– > Cl– > NO3 – > CH3COO–. To verify the influence of cationic species on pKR+ values, the pH of the CHE solutions in HCl, HNO3, H2SO4 and H3PO4 at the concentrations c = 10 mmol L-1 was adjusted by stepwise addition of NaOH or TRIS solutions in the interval 2 - 10. The corresponding pKR+ values (see Table 3 and Fig. 5) were again shifted to the more alkaline medium with increasing concentration of acids and they were significantly higher for TRIS (mainly in the presence of H2SO4 and H3PO4) compared to the values obtained for the A-pH curves in presence of NaOH. The lowest differences ∆pKR+ between values for A-pH curves neutralized with TRIS or NaOH were observed in the presence of HCl and, on the other hand, the differences were comparable in the presence of other acids. To confirm the influence of the type of anions on corresponding pKR+ values, the A-pH curves were measured in the mixtures of acids at constant total concentration 100 mmol L-1 and the corresponding pKR+ values were again calculated using a numerical interpretation of the A-pH curves by the SQUAD-G program (see Table 4). Mixtures of HCl + HNO3, HCl + H2SO4, HCl + H3PO4, HNO3 + H2SO4, HNO3 + H3PO4 and H2SO4 + H 3PO4 for mass concentration ratios 0.07 mol L-1 + 0.03 mol L-1, 0.05 mol L-1 + 0.05 mol L-1 and 0.03 mol L-1 + 0.07 mol L-1, respectively, were tested. The corresponding pKR+ values confirm that the priority effect of dominant anion of mineral acid is highly questionable since the pKR+ chelerythrine are practically the same. 3.3. Influence of ionic strength and character of the electrolyte The changes in acid-base behavior of CHE depending on ionic strength and character of inert electrolyte 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 the graphical form in Fig. 6. 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+ = 0.17 – 0.61. The highest effect was observed in the presence of H2SO4 while the lowest in the presence of HCl. A notable influence was observed in the presence of KCl (∆pKR+ = 0.69 – 1.01) with the extreme effects in the presence of H3PO4 (highest) and HNO3 (lowest), respectively. Medium pKR+ as(A)bUcMedium pKR+ as(A)bUc 0.01 mol L-1 0.1 mol L-1 HCl 8.80 ± 0.072 0.0100 0.0030 HCl 9.30 ± 0.037 0.0039 0.0005 HNO38.81 ± 0.046 0.0180 0.0183 HNO39.26 ± 0.017 0.0023 0.0002 H2SO48.77 ± 0.038 0.0158 0.0177 H2SO49.31 ± 0.021 0.0033 0.0004 H3PO48.51 ± 0.053 0.0240 0.0450 H3PO49.29 ± 0.031 0.0046 0.0011 a (pKR+ = 8.9 – 9.0 [6], pKR+ = 9.0 [9]; pKR+ = 8.77 ± 0.028 and 9.14 ± 0.040 were obtained for 0.01 and 0.1 mol L-1 acetic acid (this work); b the average standard deviation of absorbance (s(A)) over the whole data set; c the sum of squares of absorbance residuals U= Σ ( Aexp,i – Acalc,i ) 2 for i = 1 – n, where n is the total number of absorbance data for all solutions and wavelengths used Table 2. Values of pKR+ (and their average standard deviation over the whole data set) of chelerythrine in dependence on the type and concentration of inorganic acids calculated using a numerical interpretation of the A-pH curves by a SQUAD-G program. (cCHE = 6.5 µmol L-1, cHA = 0.01 or 0.1 mol L-1 mineral acid, pH adjusted by NaOH). Table 3. Values of pKR+ of chelerythrine in dependence on the type and concentration of inorganic acids (cCHE = 6.5 µmol L-1, cHA = 0.01 mol L-1 mineral acid, pH adjusted by NaOH or TRIS). medium pKR+pKR+ TRIS NaOH HCl 8.85 ± 0.064 8.80 ± 0.072 HNO38.89 ± 0.063 8.81 ± 0.046 H2SO49.33 ± 0.067 8.77 ± 0.038 H3PO49.27 ± 0.068 8.51 ± 0.053 Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine 630 5 Table 4. Values of pKR+ of chelerythrine in dependence on the type and concentration of inorganic acids mixtures calculated using a numerical interpretation of the A-pH curves by a SQUAD-G program (cCHE = 6.5 µmol L-1, cHA = 0.1 mol L-1 mineral acid, pH adjusted by NaOH). Medium pKR+ s(A)aUb 0.03 mol L-1 HCl + 0.07 mol L-1 HNO39.02 ± 0.028 0.0100 0.0076 0.05 mol L-1 HCl + 0.05 mol L-1 HNO39.03 ± 0.019 0.0081 0.0051 0.07 mol L-1 HCl + 0.03 mol L-1 HNO38.99 ± 0.021 0.0081 0.0051 0.03 mol L-1 HCl + 0.07 mol L-1 H2SO48.99 ± 0.031 0.0122 0.0115 0.05 mol L-1 HCl + 0.05 mol L-1 H2SO48.99 ± 0.030 0.0130 0.0134 0.07 mol L-1 HCl + 0.03 mol L-1 H2SO48.90 ± 0.033 0.0148 0.0180 0.03 mol L-1 HCl + 0.07 mol L-1 H3PO48.85 ± 0.041 0.0190 0.0290 0.05 mol L-1 HCl + 0.05 mol L-1 H3PO48.91 ± 0.034 0.0120 0.0190 0.07 mol L-1 HCl + 0.03 mol L-1 H3PO48.97 ± 0.032 0.0149 0.0172 0.03 mol L-1 HNO3 + 0.07 mol L-1 H2SO48.84 ± 0.027 0.0063 0.0031 0.05 mol L-1 HNO3 + 0.05 mol L-1 H2SO48.96 ± 0.032 0.0070 0.0034 0.07 mol L-1 HNO3 + 0.03 mol L-1 H2SO49.11 ± 0.037 0.0078 0.0047 0.03 mol L-1 HNO3 + 0.07 mol L-1 H3PO48.91 ± 0.031 0.0158 0.0193 0.05 mol L-1 HNO3 + 0.05 mol L-1 H3PO48.99 ± 0.021 0.0102 0.0080 0.07 mol L-1 HNO3 + 0.03 mol L-1 H3PO49.06 ± 0.014 0.0072 0.0040 0.03 mol L-1 H2SO4 + 0.07 mol L-1 H3PO48.87 ± 0.028 0.0060 0.0025 0.05 mol L-1 H2SO4 + 0.05 mol L-1 H3PO48.89 ± 0.023 0.0053 0.0022 0.07 mol L-1 H2SO4 + 0.03 mol L-1 H3PO48.91 ± 0.024 0.0058 0.0026 a the average standard deviation of absorbance (s(A)) over the whole data set; b the sum of squares of absorbance residuals U = Σ ( Aexp,i – Acalc,i ) 2 for i = 1 – n, where n is the total number of absorbance data for all solutions and wavelengths used Medium pKR+ HCl HNO3H2SO4H3PO4 no addition of Cl-8.80 ± 0.072 8.81 ± 0.046 8.77 ± 0.038 8.51 ± 0.053 0.01 mol L-1 NaCl 8.84 ± 0.021 8.82 ± 0.039 8.83 ± 0.035 8.80 ± 0.035 0.10 mol L-1 NaCl 8.97 ± 0.026 9.11 ± 0.028 9.04 ± 0.013 8.97 ± 0.021 1.00 mol L-1 NaCl 9.13 ± 0.031 9.33 ± 0.018 9.38 ± 0.014 9.00 ± 0.055 0.01 mol L-1 KCl 8.81 ± 0.088 8.88 ± 0.034 8.89 ± 0.038 8.65 ± 0.043 0.10 mol L-1 KCl 9.08 ± 0.017 9.15 ± 0.024 9.21 ± 0.019 9.09 ± 0.020 1.00 mol L-1 KCl 9.53 ± 0.055 9.48 ± 0.024 9.49 ± 0.022 9.52 ± 0.012 Table 5. Values of pKR+ of chelerythrine in dependence on the type and concentration of inert electrolyte (ionic strength - as NaCl or KCl additions) calculated using a numerical interpretation of the A-pH curves by a SQUAD-G program (cCHE = 6.5 µmol L-1, cHA = 0.01 mol L-1 mineral acid, pH adjusted by NaOH). Figure 6. Dependence of pKR+ values of chelerythrine on the character of inert electrolyte and ionic strength (NaCl – A, KCl – B), in the presence of 10 mM HCl (curve 1), HNO3 (curve 2), H2SO4 (curve 3) and H3PO4 (curve 4) H. Absolínová, L. Jančář, I. Jančářová, J. Vičar, V. Kubáň 631 6 4. Conclusions Changes in acid-base characteristics of chelerythrine were studied by measuring the absorbance-pH curves at constant concentration of chelerythrine with varying character (type) and concentration of strong inorganic acids (HCl, HNO3, H2SO4, H3PO4). The formation parts of the absorbance-pH curves shifted to a more alkaline region depending on the character and initial concentration of acids and this resulted in a corresponding increase in equilibrium constants pKR+.The most remarkable shifts were observed in the presence of phosphoric acid (H3PO4). The abovementioned results indicate that it is practically impossible to identify the priority effect of anions since the pKR+ values of chelerythrine (for concentration of individual acids 0.1 mol L-1) differed very little from each other. The character and total concentration (ionic strength) of inert electrolytes (NaCl and KCl) also significantly influence the pKR+ values of chelerythrine. The pKR+ values increased with increasing ionic strength of solutions (NaCl and KCl additions I = 0.01 – 1.0) in HCl, HNO3, H2SO4 and H3PO4. DHCHE solutions in 4% methanol and ethanol are time stable only at pH = 2 (0.01 mol L-1 HCl). However in the presence of formiate (pH = 2.8), acetate (pH = 4.2) and phosphate buffers (pH = 6.7) their absorbance rapidly decreases from 92% – 17% in 4% methanol and 95-25% in 4% ethanol after 120 minutes (see Fig. 3). DHCHE solutions are stable at relatively high contents of methanol or ethanol (60%, not presented graphically). The results confirmed that the experimental conditions (pH, ionic strength, inert electrolyte concentration, type and concentration of buffers and organic solvents, etc.) seriously influence the acid-base and UV-VIS spectral properties of chelerythrine and mainly its dihydroderivative. None of the investigated components in solutions (anions, cations etc.) is indifferent to the alkaloids, thus, exact evaluation of the side effects as well as the determination of pH-dependencies of spectral and/or acidobasic properties and time stability of the alkaloids in different media is highly important before starting any interaction studies with biomacromolecules. Application of different types and concentrations of buffers, electrolytes and components of solution, acidbase properties and interactions could be very carefully studied. This study points out that the behavior of chelerythrine and especially its dihydro-derivative in aqueous solutions is more complex than be expected [7,8,10,11]. Acknowledgments Financial support from the Grant Agency of the Czech Republic (GA ČR), grant No. 525/07/0871 is gratefully References [1] V. Šimánek, in: A. Brossi (Ed.), The Alkaloids (Academic Press, Orlando, 1985) 26, 185 [2] J. Psotová et al., J. Chromatogr. B., Analyt. Technol. Biomed. Life Sci. 830, 427 (2006) [3] M. Maiti, G.S. Kumar, Med. Res. Rev. 27, 649 (2007) [4] R. Sinha, M. Hossain, G.S. Kumar, DNA Cell Biol. 28, 209 (2009) [5] J. Urbanová, P. Lubal, I. Slaninová, E. Táborská, P. Táborský, Anal. Bioanal. Chem. 394, 997 (2009) [6] I. Slaninová, J. Slanina, E. Táborská, Chem. Listy 102, 427 (2008) (in Czech) [7] Z. 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Chem. 7, 876 (2009) Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine 632 7