16298
J. Phys. Chem. 1996, 100, 16298-16301
Reduction of the Bitter Taste Intensity of Propantheline Bromide by Cyclodextrins As Predicted by Surface Tension Measurements Noriaki Funasaki,* Yasuko Uemura, Sakae Hada, and Saburo Neya Kyoto Pharmaceutical UniVersity, Misasagi, Yamashina-ku, Kyoto 607, Japan ReceiVed: May 7, 1996; In Final Form: July 28, 1996X
The surface tension and the bitter taste intensity of aqueous solutions of propantheline bromide alone and of complexes with R-, β-, and γ-cyclodextrin have been determined, and a unique correlation between these quantities has been found to hold regardless of the kind and concentration of the cyclodextrin. The surface tension of an aqueous solution of propantheline bromide increases on addition of one of the cyclodextrins, and the effectiveness in surface tension enhancement decreases in the order β-, γ-, R-cyclodextrin. Analysis of these surface tension data yields the equilibrium constants of 1:1, 1:2, and 2:1 complexations of propantheline bromide and R-, β-, γ-cyclodextrin. These binding constants are in excellent agreement with those estimated by spectrophotometry. As well as the equimolar complex, β-cyclodextrin forms the 1:2 complex, and γ-cyclodextrin forms the 2:1 complex. The dimer of propantheline bromide can be incorporated into γ-cyclodextrin. Although the dimer is not included into R- and β-cyclodextrins, the extent of complexation is decreased by the dimerization. The bitter taste of propantheline bromide is suppressed by adding one of the cyclodextrins. The order of this suppression agrees with that of surface tension enhancement. From the relationship between the surface tension and the bitter taste intensity of aqueous solutions of propantheline bromide and the observed value of surface tension for an aqueous solution of propantheline bromide and one of the cyclodextrins, we can predict the bitter taste intensity of the mixed solution. Molecular models of some complexes of propantheline bromide and cyclodextrins have been proposed on the basis of size and shape of these molecules and the affinity of each group of propantheline bromide to the cyclodextrin cavity.
Introduction Cyclodextrins (CDs) are capable of forming inclusion complexes with compounds having a size compatible with dimensions of their cavity. Geometrical rather than chemical factors are decisive in determining the kind of guest molecules which can penetrate into the CD cavity.1-3 The extent of complex formation also depends on the polarity of the guest molecule. Strongly hydrophilic molecules and strongly hydrated and ionized groups are not, or are very weakly, complexable. The included molecules are normally oriented in the host in such a position as to achieve the maximum contact between the hydrophobic part of the guest and the apolar CD cavity. The hydrophilic part of the guest molecule remains, as far as possible, at the outer face of the complex.3 The equilibrium binding constant of a guest and one of the CDs has been determined by a number of methods, e.g., NMR, EPR, spectrophotometry, spectrofluorometry, calorimetry, polarography, electric conductometry, solubility analysis, circular dichroism, polarimetry, catalytic kinetics, the sound velocity method, chromatography,1-3 and the surface tension method.4-6 The surface tension method is based on the fact that CDs are surface-inactive. This method has been applied to surfactants.4-6 In general, a surfactant has a long alkyl chain and can form ternary and quaternary complexes with CDs. The binding constants reported for a surfactant and one of the CDs range over three orders, depending on the methods used, e.g., for sodium dodecyl sulfate and β-CD.5 The surface tension method provides reliable binding constants even for ternary and quaternary complexes.5,6 A large number of drugs self-associate in aqueous media by hydrophobic interactions.7-9 Drugs are generally less surfaceactive than surfactants. The dimerization constants of drugs X
Abstract published in AdVance ACS Abstracts, September 1, 1996.
S0022-3654(96)01294-4 CCC: $12.00
Figure 1. Chemical structure of PB.
are much larger than those of surfactants.7 The dimer of a drug would complex with a few CDs. Even if the dimer does not complex with cyclodextrins, the extent of complexation will decrease because of the reduction in monomer concentration due to dimerization. Therefore, it is interesting to test the applicability of the surface tension method to such dimerizing drugs and to compare to other methods. The modification of taste is important mainly in food products and medicines. Many orally administrated drugs have an astringent bitter taste. This can cause a serious problem in pediatric preparations. The bitter taste can be greatly suppressed by complexaation with CDs.3 Since the assay of the intensity of bitter taste is not only time-consuming but also dependent on individual persons, it would be favorable if the extent of reduction in bitter taste intensity by addition of one of the CDs could be predicted by the measurement of a physical property. Most of the bitter compounds are hydrophobic and surfaceactive. Therefore we can expect that the bitter taste intensity of an aqueous solution containing a drug and one of the CDs is determined by the surface tension value for the solution, regardless of the kind and concentration of the CD. In this work we measure the surface tension and the bitter taste intensity of the aqueous solution of one of the CDs and propantheline bromide (PB), a bitter medicine, having the chemical structure shown in Figure 1. It will be demonstrated that the surface tension is an excellent method for the determination of the binding constant of this system and that the © 1996 American Chemical Society
Reduction of Bitter Taste Intensity
J. Phys. Chem., Vol. 100, No. 40, 1996 16299
Figure 3. Relationship between the surface tension and the bitter taste intensity of PB in the absence (b) and the presence of R-CD (O), β-CD (4), and γ-CD (0). Figure 2. (a) Surface tensions and (b) scores of bitter taste of aqueous PB solutions plotted against the PB concentration.
bitter taste intensity of this system is predictable by the surface tension data only. Experimental Section Materials. Propantheline bromide was purchased from Sigma Chemical Co. Since this sample was analyzed to be pure by reversed-phase liquid chromatography, it was used without purification. Sodium bromide and CDs of analytical grade from Nacalai Tesque Co. were used as received. Ion-exchanged water was used after double distillation. Measurements of Surface Tension and Absorbance. The surface tension of aqueous solution of PB and one of the CDs containing 154 mmol dm-3 sodium bromide was measured at 309.5 ( 0.2 K by the Wilhelmy method. Since the surface tension of a fresh surface of the aqueous solution reduced with elapsed time, we took a time-independent value as the equilibrium surface tension. The absorbance of a PB solution containing one of the CDs and 154 mmol dm-3 sodium bromide was determined with a Shimadzu MPS 2000 spectrophotometer at 309.5 ( 0.2 K. The reference cell contained the CD and sodium bromide at the same concentrations as those of the sample. Intensity of Bitter Taste. Five volunteers were involved in the sensory test. These panelists tasted the solutions. The bitter taste intensities of the drug and its complexes were evaluated by the following scores: 0 ) no bitter taste; 1 ) very slightly bitter taste; 2 ) slightly bitter taste; 3 ) appreciably bitter taste; 4 ) very bitter taste; 5 ) extremely bitter taste. Molecular Modeling of Three-Dimensional Structures. The literature crystal structures of R-,10 β-,11 and γ-CDs12 were used to model their complexes with PB. The HyperChem software suite (Hypercube, Inc., Canada) was used to model the molecular structures of PB and its CD complexes. The energy-minimized structure of PB was used as a rigid body, and its complexes with CDs were not energy-minimized. Results Surface Tensions and Bitter Taste Intensities of Aqueous Solutions of Propantheline Bromide. As Figure 2a shows, the equilibrium surface tension of a PB solution containing 154 mmol dm-3 sodium bromide decreased with increasing PB concentration. At higher concentrations the reduction in surface tension became gradual. One of the reasons for this result is due to the self-association of PB, as has already been established by frontal chromatography.13 The intensity of bitter taste of a PB solution is shown as a function of PB concentration, Cp, in Figure 2b. From the results shown in Figure 2, we can determine the relationship between
Figure 4. Effects of CDs on the surface tension of PB. (a) Dependence on PB concentration in the absence (b) and the presence of a 4.74 mmol dm-3 R-CD solution (O), a 4.38 mmol dm-3 β-CD solution (4), and a 4.50 mmol dm-3 γ-CD solution (0). (b) Dependence of the surface tension of a 1.50 mmol dm-3 PB solution on the concentrations of R-CD (O), β-CD (4), and γ-CD (0). The solid lines are calculated using the equilibrium constants of dimerization and CD binding shown in Table 1.
the bitter taste intensity and the surface tension of the PB solution. This relationship is shown by the closed circles in Figure 3. As the surface tension of PB solution decreases, the PB solution becomes more bitter. Effects of Cyclodextrins on the Surface Tension of an Aqueous Solution of Propantheline Bromide. In the presence of a fixed concentration of one of the CDs, the surface tension of PB solution was measured as a function of PB concentration. As Figure 4a shows, the surface tension, γ, of PB solution increased by addition of one of the CDs. The extent of increase is greatest for β-CD, followed by γ-CD and is smallest for R-CD. It is noted that the shape of the γ vs Cp curve for β-CD is more complicated than those for R- and γ-CDs. As Figure 4b shows, the surface tension of a 1.50 mmol dm-3 PB solution was enhanced by addition of one of the CDs. This extent of enhancement by the kind of CD is consistent with the result of Figure 4a. Binding Constants of Propantheline Bromide for Cyclodextrins. The surface tension data in Figures 2-4 was analyzed on the basis of the scheme illustrated in Figure 5. We assume that the three CDs used are surface-inactive and their complexes with PB would be also surface-inactive, as has been demonstrated for surfactants.5,6 Under these conditions the surface tension of an aqueous solution of PB and one of the CDs is determined by the concentration of the uncomplexed monomer of PB. Propantheline bromide self-associates to form the dimer and higher aggregates, and the formation constants of these aggregates have been determined by chromatography at 298 K.13 The dimerization of PB is the most important multimer over
16300 J. Phys. Chem., Vol. 100, No. 40, 1996
Funasaki et al.
Figure 5. Interrelationship among the equilibria of surface adsorption, self-association, CD complexation, and receptor binding of PB.
Figure 6. Ultraviolet spectra of a 0.1472 mmol dm-3 PB solution at γ-CD concentrations of 0, 1.85, 3.71, 18.54, 37.08, and 64.89 mmol dm-3.
TABLE 1: Dimerization Constant and the Binding Constants of Propantheline Bromide for Cyclodextrins method
k2 (dm3 mol-1)
STa UVc
31 ( 20b 31
ST UV
31 ( 20 31
β-CD 3400 ( 400 3200 ( 300
ST UV
31 ( 20 31
γ-CD 230 ( 30 200 ( 150
a
K1 (dm3 mol-1)
K2 (dm3 mol-1)
K3 (dm3 mol-1)
R-CD 80 ( 11 90 ( 15 1200 ( 400 1800 ( 100 70 ( 40 50 ( 190
Surface tension. b Standard deviation. c Ultraviolet absorption.
the PB concentrations investigated in the present work. The effects of the higher multimerizations on the equilibrium constants are negligible (data not shown). Considering the size and shape of PB (P) and CDs (Ds), we presumed 1:1, 1:2, and 2:1 complexes formed between PB and one of the CDs. The equilibrium constants of these complexations are defined as
K1 ) [PD]/[P][D]
(1)
K2 ) [PD2]/[D][PD]
(2)
K3 ) [P2D]/[P][PD]
(3)
All the surface tension data shown in Figures 2 and 4 were used to determine best fit equilibrium constants by nonlinear least-squares methods.14 The dimerization constant of PB determined by chromatography at 298 K was used as an initial value.13 The best fit values are shown in Table 1. Negative binding constants were neglected, since these values are of no physical meaning. The fitting procedure has already been reported in detail elsewhere.5 The absorption spectra of PB at different concentrations of γ-CD are shown in Figure 6. There are no clear isosbestic points, suggesting that more than two complexes of PB and γ-CD coexist in the solutions. We employed the absorbance data at 290 and 240 nm to determine the binding constants and the molar absorption coefficients of complexes at these wavelengths by nonlinear least-squares methods. Then the dimerization constant of PB was kept fixed at the value determined from the surface tension data, although the effect of dimerization on the absorption spectra was negligible. The binding constants for R- and β-CDs have also been determined by the UV method. As Table 1 shows, the binding constants determined by the UV method are close to those from the surface tension method. This agreement demonstrates the validity of the surface tension method as applied to a moderately surface-active compound.
Figure 7. Reduction of the bitter taste intensity of PB by addition of CDs. (a) Dependence on PB concentration in the absence (b) of the CDs and the presence of a 4.74 mmol dm-3 R-CD solution (O), a 4.38 mmol dm-3 β-CD solution (4), and a 4.50 mmol dm-3 γ-CD solution (0). (b) Dependence of the bitter taste intensity of a 1.50 mmol dm-3 PB solution on the concentrations of R-CD (O), β-CD (4), and γ-CD (0).
The UV method is applicable only for dilute solutions of PB. As a result, the formation constant of the 2:1 complex of PB and one of the CDs is less reliable than other constants. Effects of Cyclodextrins on the Bitter Taste Intensity of Propantheline Bromide. Figure 7a shows the intensity of bitterness of PB in the presence of one of the CDs as a function of the PB concentration. The intensity of bitter taste of PB is reduced by CDs. The increasing order of reduction of the bitter taste intensity by CDs is β-, γ-, and R-CDs. This order is in agreement with that of the magnitude of the equimolar complexation constant. The PB concentration administrated for adult patients is ca. 1.5 mmol dm-3, and this solution is very bitter, as shown in Figure 7b. The bitter taste intensity of this solution is decreased by addition of one of the CDs. The order of the magnitude of reduction among the CDs is consistent with the order of the equimolar binding constant. All the data of bitter taste intensities shown in Figures 2 and 7 and surface tensions shown in Figures 2 and 4 are included in Figure 3. These data on solutions containing PB and one of the CDs are close to the data on solutions containing PB only. The surface tension (γ) and the bitter taste intensity of an aqueous solution of PB and CD will be determined by the concentration ([P]) of uncomplexed monomer of PB in the solution, irrespective of the kind and concentration of one of the CDs. That is,
γ ) f ([P])
(4)
bitter taste intensity ) g{[P]} ) g{f -1(γ)}
(5)
This important result enables us to predict the intensity of bitter
Reduction of Bitter Taste Intensity
J. Phys. Chem., Vol. 100, No. 40, 1996 16301 The dimerization constant of PB at 298.2 K had been determined to be 13.4 ( 0.5 dm3 mol-1 by chromatography,13 and the value 31 ( 20 at 309.5 K was estimated here by the surface tension method. On the basis of these data, we can determine the thermodynamic parameters for the dimerization of PB: the standard entropy and enthalpy of dimerization are 210 J K-1 mol-1 and 56 kJ mol-1, respectively, although these values are not accurate, because of the uncertainty in k2 at 309.5 K. The positive entropy suggests that PB dimerizes by hydrophobic interactions. The dimer of PB can be incorporated in the γ-CD cavity, and the formation constant of this change is defined as
P2 + D ) P2D K3′ ) [P2D]/[P2][D] Figure 8. Proposed structures of three complexes of PB with R-CD, β-CD, and γ-CD.
taste of a PB and CD solution from the observed surface tension of the mixed solution and the relation between the bitter taste intensity and the surface tension of an aqueous PB solution. Proposed Structures of Complexes of Propantheline Bromide and Cyclodextrins. Possible three-dimensional structures of three complexes of PB with R-, β-, and γ-CDs are shown in Figure 8. The structures of the complexes were obtained by the trial and error method, by taking into consideration the inclusion of the benzene ring and the exclusion of the ammonium group of PB from the CD cavity. Since R-CD has a small cavity, the benzene ring of PB would be incorporated within the cavity. Thus, its binding constant is small (Table 1), and it cannot form any ternary complex. Although β- and γ-CDs can incorporate the xanthine ring of PB, the latter cavity is too large to exhibit effective van der Waals attraction, as compared to the former cavity. This difference in adaptability reflects the magnitude of the equimolar complexation constant, K1. The second β-CD molecule may bind the ethoxycarbonyl group of the PB molecule. A γ-CD molecule can bind the xanthine ring of the second PB molecule, whose ammonium group would be outside the cavity and oriented opposite the ammonium group of the first PB molecule, because of the electrostatic repulsion. Discussion Spectrophotometry is the most frequently used method for the equilibrium constant determination of CD complexation. In this method the entrapped compound must absorb ultraviolet or visible light, and its concentration is limited at rather low concentrations, depending on the extinction coefficient. At such low concentrations, the amount of the 2:1 ternary complex is small, so that it is rather unsuitable to determine the equilibrium constant of the ternary complexation. In fact, the 2:1 binding constant, K3, of PB and γ-CD by the UV method is not very certain, as shown in Table 1. The surface tension method for determining the binding constant is also applicable to concentrated solutions of a guest, so that we could determine the more reliable equilibrium constant of ternary complexation. Comparison between the surface tension method and a spectroscopic method has been carried out for the first time in the present work. Since most of the compounds incorporated in the CD cavity are surface-active, the surface tension method is applicable to many guests. Circular dichroism spectroscopy will be a good method to determine the binding constants of PB with one of the CDs and may also provide information about the structures of the complexes shown in Figure 8.
(6)
This formation constant can be calculated from
K3′ ) K1K3/k2
(7)
The K3′ value for γ-CD is 519 dm3 mol-1 from the surface tension data shown in Table 1. The dimerization of PB decreases the concentration of PB in monomeric form. This reduces the binding capability of PB for all CDs. This effect, though often neglected, should be taken into consideration for drugs and similar compounds. Although higher multimers of PB are present, they will be negligible in the concentration range of PB investigated in this work.13 The structures of the complexes proposed in Figure 8 are based on the size and shape of PB and CDs and the affinity of each group of a guest molecule to the CD cavity, viz., the preference of the benzene ring and the avoidance of the ammonium group. A further investigation is underway to confirm the structures proposed. The most important result in the present work is shown in Figure 3: for the mixture of PB and one of the CDs, the intensity of bitter taste of PB is a function of the surface tension only, irrespective of the kind and the concentration of CDs. This remarkable result stems from the fact that both the surface tension and the bitter taste intensity are unequivocally determined by the concentration of the uncomplexed and nondimerized PB molecule. This principle would generally hold true for other biological properties of PB and CDs and also for other drugs. References and Notes (1) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; Springer-Verlag: Berlin, 1978. (2) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344. (3) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, 1988, Chapters 2 and 3. (4) Saenger, W.; Muller-Fahrnow, A. Angew. Chem., Int. Ed. Engl. 1988, 27, 393. (5) Funasaki, N.; Yodo, H.; Hada, S.; Neya, S. Bull. Chem. Soc. Jpn. 1992, 65, 1323. (6) Dharmawardana, U. R.; Christian, S. D.; Tucker, E. E.; Taylor, R. W.; Scamehorn, J. F. Langmuir 1993, 9, 2258. (7) Funasaki, N. AdV. Colloid Interface Sci. 1993, 43, 87. (8) Mukerjee, P. In Physical Chemistry: Enriching Topics from Colloid and Surface Science; van Olphen, H., Mysels, K. J., Eds. Theorex: La Jolla, CA, 1975; Chapter 9. (9) Attwood, D.; Florence, A. T. Surfactant Systems; Chapman and Hall: London, 1983; Chapter 4. (10) Manor, P. C.; Saenger, W. J. Am. Chem. Soc. 1974, 96, 3630. (11) Lindner, K.; Saenger, W. Carbohydr. Res. 1982, 99, 103. (12) Harata, K. Bull. Chem. Soc. Jpn. 1987, 60, 2763. (13) Funasaki, N.; Uemura, Y.; Hada, S.; Neya, S. Langmuir 1996, 12, 2214. (14) Yamaoka, K. Analysis of Pharmacokinetics with Microcomputers; Nankodo: Tokyo, 1984; Chapter 4.
JP9612947
