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Nov 17, 2017 - Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, ... interesting biological effects.1 Among them, xantho...
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Acid−Base Properties of Xanthohumol: A Computational and Experimental Investigation Marta Arczewska,*,† Daniel M. Kamiński,‡ Barbara Gieroba,§ and Mariusz Gagoś§ †

Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland Department of Chemistry, Maria Curie-Skłodowska University, pl. Marii Curie-Skłodowskiej 2, 20-031 Lublin, Poland § Department of Cell Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland ‡

S Supporting Information *

ABSTRACT: UV−vis spectrophotometry has been applied to determine acid dissociation constants of the prenylated chalcone xanthohumol. The pKa values were compared with those derived from pH-metric titrations. The order of the deprotonation site in the xanthohumol molecule was estimated by quantum mechanical calculations as 2′-OH, 4′-OH, and 4OH. Furthermore, the electronic and spectroscopic properties of xanthohumol have been investigated on the basis of the time-dependent density functional theory (TDDFT). The TDDFT method, combined with a hybrid exchange− correlation functional using the B3LYP and CAM-B3LYP levels of theory in conjunction with the SMD solvation model, was used to optimize all geometries and predict the excitation energies of the neutral form and ionized species of the chalcone depending on pH value. The computed results were in good agreement with the experimental data. Consideration of the acid− base profile in conjunction with other molecular properties has a great importance and has the potential to be used to further improve the bioavailability of xanthohumol.

N

less than 0.69 mg of XN/L has been found, which is not sufficient to cause the disease-preventive effects.13 Recently, Miranda et al.14 showed the antiobesity effect of XN on a mouse model at doses of 30 and 60 mg/kg/day, corresponding to a human equivalent dose of 350 mg/day for a 70 kg person. Such an amount of XN can only be obtained in the form of dietary supplements.3b Xanthohumol was isolated in 1913, and its structure was later confirmed using partial synthesis and X-ray crystallography.15 Chemically, XN belongs to the chalcone class of flavonoids and is composed of acetophenone (A-ring) and benzene (B-ring) moieties linked by a trans-(E)-configured double bond and carrying a 3′-isoprenyl unit (3,3-dimethylallyl) (Figure 1).15c In

umerous studies indicate that hop-derived prenylflavonoids are becoming more popular due to their potentially interesting biological effects.1 Among them, xanthohumol (2′,4,4′,6′-tetrahydroxy-3′-prenylchalcone; XN), the most abundant prenylflavonoid present in the female hop plant (Humulus lupulus),2 has gained great attention because of its multiple health-promoting properties.3 A number of recent studies have reported its beneficial influences on health, including anticarcinogenic,3b,4 antiangiogenic,5 anti-inflammatory,1,6 and antioxidative bioactivities,7 and offers therapeutic benefits for a number of metabolic syndromes.8 In fact, there is recent evidence suggesting the ability of XN to act as a mitochondrial uncoupler8,9 and to induce intracellular reactive oxygen species (ROS) generation, which are responsible for the triggering of apoptosis in cancer cells.10 Xanthohumol is produced in the secretion glands of female flowers,11 but the XN concentration in hop cones has been estimated at about 0.95% of their dry weight.3b The availability of XN in natural products is insufficient to have a significant effect through oral administration.12 Beer consumption is still the most significant dietary source of XN and other prenylflavonoids. However, due to the fact that the brewing process causes the thermal isomerization of XN into the flavanone isoxanthohumol (IXH), the amount of XN represents only a small percentage of the hop-enriched products.2,3b For example, xanthohumol-rich beers contain about 3.4 mg of XN/L, while in commercially available ones © 2017 American Chemical Society and American Society of Pharmacognosy

Figure 1. Structure of xanthohumol (XN) with atom numbering. Received: June 20, 2017 Published: November 17, 2017 3194

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Figure 2. (A) UV−vis spectroscopic evaluations occurring upon a pH jump from a stock solution of xanthohumol (2.63 × 10−5 M) at pH 12 to acidic pH values (only selected spectra). All spectra were recorded immediately after the pH changes ( 7, along with the increasing pH, the intensity of the main band gradually increased and was shifted at a pH of ca. 9 toward longer wavelengths, which was accompanied by a change in the solution color from light to intensive yellow. This process turned out to be reversible, which occurred immediately with the pH change. As shown in Figure 1, the ionizable groups of XN are part of the chromophore system in aromatic rings, and the ionization gives intensive pH-dependent spectral shifts. When deprotonation was completed, a strong absorption band located at 438 nm was observed. The intensity of this band increased with the growth of the solution pH and reached a maximum at pH 12 (ε = 35 490 M−1 cm−1). A similar bathochromic shift as a result of deprotonation was reported in the case of the related chalcones phloretin and phlorizin.30 Acid−Base Properties. In general, the bioavailability of active molecules is correlated with the degree of dissociation of their ionizable groups. More importantly, only a nonionized form of compounds can diffuse passively across biological membranes. The dissociation constants for XN have not yet been determined in the aqueous solutions. However, to consider the potential of XN to be employed as a pharmaceutical, it is crucial to estimate its pKa values. There are three ionizable hydroxy groups positioned at C-2′, C-4′, and C-4, which are able to undergo deprotonation reactions leading to formation of a monoanion XN−1, a dianion XN−2, and a trianion XN−3. The classical analytical and spectrophotometric methods were used for the pKa determination of XN. First, the pKa values were estimated by means of a pH-titration procedure in a 30% EtOH/H2O mixture (v/v). This method is limited to a relatively high concentration of approximately 10−4 M. Additionally, the poor solubility of XN in water may lead to precipitation. Therefore, a cosolvent is used, which

additionally complicates the interpretation of results. We found that a 30% ethanol solution is an optimal mixture for this kind of experiment, and the original pH values in EtOH/H2O mixture were subjected to minor variations in the case of a high percentage of a cosolvent.31 It was possible to determine the pKa values for XN, which are very close to each other, with the use of a model fitting method in HyperQuad 2003.32 Without that, it was impossible to find the pKa values from the inflection of the titration curve (Figure S1, Supporting Information). The addition of EtOH was compensated for in the calculations by an increase in the solution volume, so the curve overlapped the points at low pH. The fitting was done for eight forms of XN. In the model, the following variables were fitted: three stability constants, pβ1 = pKa3, pβ2 = pKa3 + pKa2, and pβ3 = pKa3 + pKa2 + pKa1, and the pKw of the aqueous solution. In every fitting cycle, the rest of the five pβ constants were recalculated from the best pβ1, pβ2, and pβ3 values and set as a constant. It is important to mention that the contribution of the rest of the pβ values had a minor effect on the fitting χ2. The fitting procedure with the corrected pβ4−8 was repeated several times until no changes in the parameters were observed. The final χ2 for 51 points was 1.5. The final pKa values of XN for aqueous solutions were linearly scaled form the values obtained for a pKw of 14.6. The final results with standard deviation are shown in Table 1. The level of the standard deviations obtained is expected for XN due to its low water solubility and three ionizing groups that lie within a pH range of 1 pKa unit. Based on the calculated pKa values, the concentration of different XN forms for a given pH value was determined (Figure 3).

Figure 3. Concentration changes in XN ionized forms as a function of pH. Numbers in brackets denote deprotonated phenolic groups. 3196

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calculations. The graphic representation of eq 1 determined at 352 nm for the first linear region is illustrated in Figure 5. In an

Analysis of the spectra shown in Figure 2A revealed that distinct spectroscopic changes were observed in the pH range of 7−9, thus suggesting that the three-step process of ionization take places in this region. For the compounds with more than two ionization values that lie within 1 pH unit of the pKa values, the estimation of dissociation constants might be possible by fitting the experimental data and using the graphical methods.31,33 The first step of our experiments was dedicated to plotting the absorbance vs pH solution at three particular wavelengths where the spectroscopic changes were the most significant. Figure 2B presents the results of fitting using a dose−response curve. The general procedure for improving the accuracy of numerical approximations is to interpolate additional points located between the experimental ones.34 Therefore, the determination of an approximate pKa value was performed based on interpolated data by locating the inflection point in the fitted curves. The results of these calculations are collated in Table 1. Another approach to this issue was to take the wavelengths of maximum absorbance at extreme pH values located at 438 nm (predominance of XN−3) and 352 nm (neutral form prevalence) and create an absorbance diagram. As can be seen in Figure 4, there are three linear regions in the

Figure 5. Plot of eq 1 to determine the pKa1 of xanthohumol in water at 21 °C.

analogous manner, three linear segments were analyzed at wavelengths 352, 398, and 438 nm (data not shown). When the linear regression (R2 > 0.997) was performed, the following average values of Ka were obtained from the slopes of straight lines: Ka1 = 5.438 × 10−8 ± 2.438 × 10−9, Ka2 = 4.560 × 10−9 ± 1.366 × 10−10, and Ka3 = 1.056 × 10−9 ± 7.505 × 10−11. The pKa values calculated on the basis of these graphical investigations are summarized in Table 2. As can be seen, the Table 2. Approximated pKa Values Determined with the ACD/pKa Percepta DB v 14.00 Program (pKa Classic Module)

pH range 7 to 9.2 that give information about the presence of three XN species in equilibrium. Moreover, from the intersection points, it was possible to evaluate pH ranges in which the successive deprotonations occurred. The equilibrium constants Ka1, Ka2, and Ka3 that characterize the following ionization for XN species were calculated on the basis of the following equations:34a (1)

(Aλ − Aλ XN1−) × 10−pH = −K a2Aλ + K a2Aλ XN2−

(2)

(Aλ − Aλ XN2−) × 10−pH = −K a3Aλ + K a3Aλ XN3−

4′-OH

4-OH

pKa1 7.59 ± 0.40

pKa2 8.81 ± 0.30

pKa3 9.67 ± 0.30

pKa values are almost the same in the error bars as the data obtained from the titration method. Thus, there is excellent agreement between all the methods employed. Moreover, the pKa values obtained here are also close to those predicted with the ACD/pKa Percepta DB v 14.00 software. These values were almost comparable to those obtained by the traditional methods, except for pKa3 (Table 2). However, it should be borne in mind that the above pKa estimation does not take into account the intramolecular bonds and cannot properly define the order of protons undergoing dissociation. Nevertheless, the computational method gives information about the expected values and allows establishing the stages in which hydrogen atoms dissociate from phenolic groups as 2′-OH > 4′-OH > 4OH. The differences in the dissociation constants may be related to the fact that computations are performed on the basis of databases of compounds with a similar structure at zero ionic strength and 25 °C.17b The order of XN ionization was also estimated from the excited state energy, which was calculated for all possible ionized forms of XN (vide infra). The calculated pKa1 value (∼7.4) is similar to that reported for the structurally related dihydroxychalcones and assigned to the 2′-hydroxy group.30,35 The third dissociation constant, which is related to dissociation of the proton at the 4-OH group, has a value similar to quercetin (pKa2 = 8.7) and rutin (pKa2 = 8.8).17b Generally, the pKa values for deprotonation of hydroxy groups,

Figure 4. Absorbance diagram of xanthohumol in the pH range 7 to 9.2.

(Aλ − Aλ XN) × 10−pH = −K a1Aλ + K a1Aλ XN1−

2′-OH

(3) 1−

where Aλ is the absorbance of species XN and XN (or XN2− and XN3−) in the buffer solution, at the selected wavelength; AλXN is the absorbance of XN (measured at pH 2); and AλXN1−, AλXN2−, and AλXN3− are the absorbance of the conjugate forms XN1−, XN2−, and XN3−, respectively. The values of AλXN1−, A λXN 2− , and A λXN 3− were evaluated after plotting the experimental data according to eqs 1−3) in each stage of the 3197

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adopt positions above and below the A-ring. To distinguish these conformations, we introduced two conformations, namely, 1a and 1b (Figure 6). First, the calculations in the gas phase for 1b were performed at the B3LYP level of theory and 6.31++(2d,2p) as a basis set. The results indicated that the order of ionization was identical to the above-mentioned results. To improve the numerical results, we introduced the SMD solvation model, which is known to give good results for predicting the energy of solvated molecules when no specific interactions exist between solvent and solvate.44 The results obtained by B3LYP and CAM-B3LYP functionals with the 6.31++(2d,2p) basis set for two conformations are shown in Table 3. Obviously, the energy strongly changes with the next ionized OH group, but the order is always the same, i.e., XN1−(1) > XN1−(2) > XN1−(3) and XN2−(1,2) > XN2−(1,3) > XN1−(2,3), and it is independent of the functional (B3LYP or CAM-B3LYP). This suggests that the first XN dissociation will occur for 2′-OH, and 4-OH will be the last. These results are in good agreement with those obtained for the chalcone form of naringin based on a kinetic measurement in an aqueous solution.45 Although the difference in energy between XN1−(1) and XN1−(2) is 0.0005 hartree, it does not mean that the difference is not significant because the error (16 kJ/mol) of the method used is only systematic in this case.44 For this reason, the order of ionization should be maintained. The energies calculated for 1a are similar within the first ionization (0.0001 hartree difference). For 1b the biggest difference is 0.002 hartree between XN−1(2) and XN1−(3). The CAMB3LYP functional indicates almost equal dissociation constants for the 2′-OH and 4′-OH groups, which is not observed in the experimental data. It could be that the CAM-B3LYP, commonly applied for the calculation of higher excited states, fails in the case of low excitations of XN in combination with the SMD model. In both levels of theory the similar energies for XN1−(1) and XN1−(2) could be explained by the quasisymmetrical position of these OH groups. Other important information on the acid−base properties of the ionized form of XN may arise from the frontier orbital energies, EHOMO and ELUMO. The eigenvalue of the HOMO is used to evaluate the electron donor ability of molecular systems, whereas the LUMO eigenvalue is directly connected with the electron acceptor ability. The HOMO and LUMO eigenvalues and their energy gaps (ΔEH‑L) provide a reasonable qualitative indication of the chemical stability and the chemical reactivity of the molecules46 (Table S2, Supporting Information). The sequence of XN deprotonation was investigated based on the changes of these orbital energies computed after the geometrical optimization of different species of XN in 1b. Among the XN monoanions, XN−1(3) gives the greatest difference between HOMO and LUMO (ΔEH‑L = 3.27 and 5.59 obtained with B3LYP and CAM-B3LYP, respectively), and the value of the HOMO was minimal. Generally, the large energy gap implies high stability, and given the results presented in Table S2, the most likely form of the XN monoanion will be XN1−(3). On the other hand, the XN1−(2,3) dianion has the lowest HOMO energy, the largest ΔEH‑L gap, and the smallest dipole moment, making it the most stable form among the dianions. This implies that the dissociation of the 4′OH proton will probably be the second to take place. The predicted hydrogen dissociation sequence, according to ΔEH‑L, was found to be the same as that provided with the solvation energies (Table S2, Supporting Information).

particularly hydroxyflavones, may be within the physiological pH range.36 On the other hand, the overlapping of pKa values observed here may be related to the quasi-symmetrical position (C-4′ and C-2′) of OH groups in the A-ring of XN.34a Since XN is a weak acid with a pKa close to physiological pH, the 2′-monoanion is in equilibrium with the neutral form of XN (at 1:1 ratio) at pH 7.4. Its maximal membrane activity is at pH values higher than pKa due to more difficult diffusion across the cell plasma membranes of a negatively charged form than that of a neutral form. Recently, it has been suggested that mitochondria are the principle cellular target of XN action.10b XN might act as mitochondrial uncouplers able to collapse the mitochondrial transmembrane potential.8 Being also a weak acid with a pKa of ca. 7.4, XN could be expected to possess protonophoric activity under physiological conditions, able to dissipate the proton gradient by binding a proton at the acidic side of the membrane (in the outer membrane space), diffusing through the membrane, and releasing the proton in the mitochondrial matrix where the pH is basic.8,37 Moreover, several studies reported that more acidic extracellular pH (6.2−6.9) is a characteristic feature of cancer phenotype.38 Being a weakly acidic compound, XN can exploit the tumor pH gradient, because the acidic extracellular conditions will lower the total charge, and its cytotoxic activity would be improved by acidic microenvironments. In this context, XN might interact differently with biological membranes according to their charge state.39 Chalcones, in particular hydroxychalcones, are known to be effective inducers of apoptosis.40 It has been suggested that a 2′-OH group is essential for the reactivity of the chalcone.41 The presence of a reactive 2′-OH group in XN has been suggested to be crucial for the interaction with the mitochondrial electron transfer chain complex I (NADH dehydrogenase). XN directly inhibits the activity of complex I and causes the overproduction of ROS responsible for apoptosis in HeLa and A549 cancer cells in vitro.10b Energy of XN Ionized Forms. In order to support our experimental findings and estimate the energy of the different XN ionized forms, several calculations were performed for two possible conformations of XN (Figure 6). Generally, it is

Figure 6. Possible conformations of XN: the conformation 1a (on the left) with the prenyl group and the B-ring opposite the A-ring; the conformation 1b (on the right) with the prenyl group and the B-ring on the same side of the A-ring.

known that chalcones possess three approximately planar parts and adopt the trans- or cis-type configuration.42 According to Xue et al., the most stable form is the trans-isomer due to the strong steric effects between the B-ring and the carbonyl group.43 Additionally, according to our calculations the prenyl unit and the B-ring are not coplanar with the A-ring but can 3198

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Table 3. Dissociation Energy of XN Species (Hartrees) for the Gas Phase and the SMD Solvation Model Performed at the B3LYP and CAM-B3LYP Levels of Theorya gas phase

SMD water B3LYP

CAM-B3LYP

species

1b

1b

1a

1b

1a

XN XN−1(1) XN1−(2) XN1−(3) XN2−(1,2) XN2−(1,3) XN2−(2,3) XN3−

−1189.6787 −1189.1319 −1189.1347 −1189.1530 −1188.4767 −1188.5123 −1188.5283 −1187.7664

−1189.7123 −1189.2386 −1189.2391 −1189.2443 −1188.7568 −1188.7686 −1188.7690 −1188.2842

−1189.7114 −1189.2405 −1189.2409 −1189.2428 −1188.7614 −1188.7685 −1188.7694 −1188.2868

−1189.1173 −1188.6455 −1188.6454 −1188.6484 −1188.1648 −1188.1735 −1188.1747 −1187.6921

−1189.1162 −1188.6466 −1188.6467 −1188.6467 −1188.1686 −1188.1743 −1188.1749 −1187.6940

a

Calculations for 1a and 1b conformations of XN; see Figure 6. The systematic error of this method in the prediction of solvation energy for single ionized molecules was 0.0064 hartree and increased with higher ionizations.

Figure 7. Superposition of experimental (Exp.) spectra and calculated results for absorption bands expressed as the oscillator strength f obtained with B3LYP (A) and CAM-B3LYP (B) levels of theory. The calculations with the CAM-B3LYP functional evidently overestimate the band energies. This is an effect of insufficient geometry optimization with the use of this functional together with the SMD solvation model. The experimental spectra for the XN species were normalized to 1. The oscillator strength f is indicated by the vertical lines.

Figure 8. Experimental (Exp.) and calculated (Cal.) UV−vis spectra for the XN and XN3− in water. The two forms of XN were chosen because the neutral (XN) and trianion (XN3−) forms predominate at pH 2 and pH 12, respectively. The band structures are indicated by the vertical lines and are summarized in Table S1. The theoretical fwhm for the bands was set to 0.25 eV. For the neutral form, it was possible to determine approximately the contribution of 1b and 1a to be 1:4, respectively. In the case of the XN trianion, the calculated spectrum is red-shifted because strongly charged molecules are poorly described by the SMD solvation model.

Calculations of UV−Vis Spectra. In recently published papers, the DFT method has been successfully used to study different substituted chalcones.34a,43,47 It is known that solvents have a significant influence on spectral shifts;48 therefore, we directly performed the calculations of spectral properties using the SMD model. First, we used previously optimized geometries to calculate electron absorption spectra with the TDDFT method with B3LYP and CAM-B3LYP calculations including the SMD solvation model. Because the accuracy of

B3LYP for higher excited states is not entirely satisfactory (usually underestimated), we also tested the CAM-B3LYP level of theory. The B3LYP functional was chosen to best fit the experimental data, especially for the neutral form (Figure 7A). Figure 7 illustrates the comparison between the calculated (the B3LYP functional) and experimental UV−vis spectra. The contribution in the spectra of 1a is higher than that of 1b for the neutral form despite the lower energy of the latter. This can be explained by the effect of intermolecular hydrogen bonds 3199

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Thus, these results will contribute to a continuous interest in understanding the role of natural chalcones and open up new perspectives of the rational use of chalcones in the fields of pharmacy and food chemistry.

between water and XN that causes a decrease in the energy of 1a compared to 1b in water. Additionally, this method correctly predicts the spectral shifts for all forms of XN from the short wavelengths of XN to the long wavelengths for XN3−. This shows that the calculated longest-wavelength band positons are in good accord with the experimental measurements (Figure 8A). Additionally, it has also been reported that the deprotonation of the 2′-OH and 4-OH groups of chalcones produces the strong absorption band.49 The CAM-B3LYP seems to overestimate the spectral shifts in all cases (Figure 7B). This is caused by the on average 0.015 hartree higher energy after geometry optimization for all forms in comparison to the B3LYP functional. Moreover, the single ionized XN molecule at 4-OH, which has the lowest energy (should be observed in the spectra) for the first ionization, is strongly redshifted (∼132 nm), which is not observed in the experimental measurements (Figure 8B). This suggests that the functional together with the SMD model does not adequately describe or optimize the geometry of ionized XN molecules. The prediction of absorption bands with this functional is available in Table S1 of the Supporting Information. It can be expected that both functionals insufficiently describe the XN3− spectra (Figure 8B). This is understandable due to the error in the SMD solvation model, which increases with the charge of solvated molecules. Additionally, water can interact specifically in this case through H-bonding with the carbonyl oxygen of XN. All observed bands in the range from 300 to 500 nm are related to n → π* transitions where nonbonding orbitals are assigned to the free electron pairs localized close to the oxygen atoms. In conclusion given the potential of XN to be employed as an effective antiproliferative and cytotoxic agent in many types of cancer cell lines, it was important to investigate its dissociation constant. The cytotoxicity of XN to the cancer cells is likely due to its ability to generate pro-oxidant phenoxy radicals and uncouple mitochondria. Knowledge of the acid−base properties of XN in aqueous solution is a basic step toward understanding its structure and bioavailability. As evident from a large number of studies, XN has been extensively studied for its health-promoting properties, but little is known about its interactions with membrane components at the molecular level. In this work, the pKa values using both spectrophotometric and potentiometric methods were determined. Additionally, the stages at which hydrogen atoms dissociate from phenolic groups, i.e., 2′-OH > 4′-OH > 4-OH, were established by computational methods. The sequence in which hydrogen atoms dissociate shows an excellent agreement with the experimental measurements. Moreover, the ionization constants obtained from the analysis of electronic absorption spectra and the pH-titration procedure also show good accuracy with the computationally estimated acidity constant. The B3LYP functional together with the SMD solvation model describes the energies and thus spectra for ionized XN molecules in water much better than the CAM-B3LYP functional. The B3LYP/6-31++(2d,2p) method correctly predicts the order of the OH group dissociations and the spectral shifts. As can be expected, the error in band absorption prediction increases with the charge of the XN molecules. Therefore, the comparable results between the calculated and experimental absorption wavelengths were in reasonable agreement for the neutral form of XN when the presence of 1a and 1b in water was considered.

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EXPERIMENTAL SECTION

Materials. Xanthohumol was purchased from Sigma−Aldrich (St. Louis, MO, USA) and used with further purification by recrystallization from acetone/water/2-propanol. All other chemicals were of analytical grade quality. All experiments were performed in doubledistilled water under a nitrogen atmosphere. The pH was measured with a glass electrode (InLab Expert, Mettler Toledo, Switzerland) connected to a pH/ion-meter (CPI-502, Elmetron, Poland). The pH electrode was calibrated in standard buffer solutions before each experiment. UV−Vis Spectroscopy. Electronic absorption spectra of XN were recorded with a double-beam Cary 300 Bio (Varian) UV−vis spectrophotometer equipped with a thermostated cuvette holder with a 6 × 6 multicell Peltier block. The spectra were collected over a wavelength range 200−600 nm using a 2 nm step spacing with a 1 cm length, closed quartz cuvette (Helma). The temperature (21 ± 1 °C) was controlled with a thermocouple probe (Cary Series II from Varian) placed directly into the sample. Using the standard procedure described by Albert and Serjeant, the UV−vis spectroscopic method was applied for the determination of ionization constants (pKa).50 The approach used in this procedure involved taking advantage of the fact that each XN species absorbs at different wavelengths depending on pH due to the presence of a chromophore in proximity to the ionization centers.51 The value of pKa was graphically determined by plotting the absorbance at three wavelengths (before and after the isosbestic point) attributed to a neutral form (∼350 nm) and ionized species (∼400 and 440 nm) as a function of pH for a series of nonabsorbing buffers. After correcting the experimental data by subtraction of minimum absorbance at each wavelength and using the fitting of the sigmoidal curve (a dose response) to the measured points, it was possible to determine pKa values from the inflection points of each plot.52 The selected pH values were achieved using (i) NaOH−KCl, pH 12−10, (ii) Na2HPO4 − KH2PO4 for a pH 9−4 interval, and (iv) HCl−KCl, pH 3−2. The ionic strength of all these solutions had the same value of 0.1 M). The XN stock solution (2 mg/ mL) was prepared in a deoxygenated aqueous solution with 0.1 M NaOH to obtain a final pH of 12.2. Data analysis was performed using the Grams/AI (ThermoGalactic Ind., USA) and Origin Pro 8 (OriginLab Corp., USA) software programs. For quick prediction of the pKa values, the ACD/pKa software was also applied. Titration Procedure. The solution was prepared from freshly double-distilled water and 96% EtOH in a v/v ratio of 7:3. The solution was purged with a stream of N2 for 5 min, and XN (0.01 mmol) was injected to the mixture after dissolution in 2 mL of EtOH. The mixture was left for 10 min with continuous stirring under N2 flow. The pH of the mixture was changed by addition of 0.63 mmol of NaOH (0.1 M). The mixture was titrated from pH 11.5 (to minimize the concentration of isoxanthohumol) with 0.1 M HCl in a closed system under a water-saturated N2 flow. The titration error was estimated to be ±0.01 mL. The pH data were developed for a drift less than 0.02 pH unit per minute. The fitting curve was corrected for the CO2 content (8 × 10−4 mmol) predicted from another experiment. The apparent pKw of the titration mixture was 14.6 at 22 °C. This value was used to calculate the corrected pKa values related to pKw 14. The ionic strength of the mixture was not increased by the addition of salt to keep XN solubility as high as possible at neutral pH. The fitting of titration data was calculated in HyperQuad 2013. The model contains all possible neutral and ionized forms. The calculated overall association constants β were recalculated to appropriate pKa values.32 The final fitting χ2 was 1.5 for 51 points. Computation. The calculations were performed in the Gaussian 09 Rev. E.01 computational package (Gaussian Inc., Pittsburgh, PA, USA).53 The DFT theory with the B3LYP (Becke three-parameter 3200

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Lee−Yang−Parr) exchange−correlation functional and the Coulombattenuating method (CAM-B3LYP) with a 6-31G(2d,2p) basis set was used to optimize all geometries and predict the excitation state energies of the neutral form and ionized species of XN depending on pH value.54 The diffuse functions were used because of their better description of systems with free electron pairs and anions, as well as other systems with a large negative charge. All structures were optimized with tight convergence criteria. Since the solvent effect has a significant influence on the spectral shift and predicted energies, the SMD solvation model was applied with the dielectric constant ε of 78.39 for water. In this model, the surrounding media are unstructured, and therefore no specific interactions are taken into account. It is important to mention that protonated solvents can form hydrogen bonds especially with charged ions, which can introduce errors in calculations. The UV−vis spectra were calculated with the TDDFT method. For the geometry optimization and calculation of excited states, the same functional was used. Apart from the gas phase, all calculations were performed with the SMD solvation model.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00530. Titration curve obtained for XN in the H2O/EtOH mixture; DFT calculations of electronic transition and oscillator strengths for XN species; EHOMO, ELUMO levels, energy gap (ΔEH‑L), and dipole moment of different XN species (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +(48 81)445 69 05. E-mail: marta.arczewska@up. lublin.pl. ORCID

Marta Arczewska: 0000-0003-2553-0908 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Calculations were performed with the use of the resources provided by the Wroclaw Centre for Networking and Supercomputing (http://wcss.pl) under grant no. WCSS#10118851.

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