2019 年 67 巻 12 号 p. 1284-1292
The purpose of the study was to prepare a poly-γ-glutamic acid hydrogel (PGA gel), to evaluate physicochemical properties, its ease of swallowing using texture profile analysis (TPA) and its taste-masking effects on amlodipine besylate (AML) using the artificial taste sensor and human gustatory sensation testing. Using TPA, 0.5 and 1.0% (w/v) PGA gels in the absence of drug were within the range of acceptability for use in people with difficulty swallowing according to permission criteria published by the Japanese Consumers Affairs Agency. The elution of AML from prepared PGA gels was complete within an hour and the gel did not appear to influence the bioavailability of AML. The sensor output of the basic bitterness sensor AN0 in response to AML mixed with 0.5 and 1.0% PGA gels was suppressed to a significantly greater degree than AML mixed with 0.5 and 1.0% agar. In human gustatory sensation testing, 0.5 and 1.0% PGA gels containing AML showed a potent bitterness-suppressing effect. Finally, 1H-NMR spectroscopic analysis was carried out to examine the mechanism of bitterness suppression when AML was mixed with PGA gel. The signals of the proton nearest to the nitrogen atom of AML shifted clearly upfield, suggesting an interaction between the amino group of AML and the carboxyl group of PGA gel. In conclusion, PGA gel is expected to be a useful excipient in formulations of AML, not only increasing ease of swallowing but also masking the bitterness of the basic drug.
Taste and ease of swallowing are important factors used to determine the acceptability of oral pharmaceutical formulations. The bitter taste of an active pharmaceutical ingredient may cause poor palatability1) and consequently reduce drug efficacy through non-compliance. As many active pharmaceutical ingredients exhibit bitterness, taste masking is an important step in formulation development. There are various strategies for this. Traditionally, bitterness has been masked by the addition of sweeteners or flavours.2,3) At the cognitive level, the perceived inhibition of bitterness occurs in the central nervous system in the brain via taste–taste interactions. An alternative approach is to prevent bitterness perception peripherally, using techniques such as encapsulation, molecular inclusion of cyclodextrins,4,5) complexation with ion-exchange resins,6) tannate,7,8) fatty acids9) or food proteins10) or the addition of bitterness-masking substances such as chlorogenic acid. Electrostatic interactions between basic bitter active ingredients and chlorogenic acid attenuate the binding of the bitter active ingredients to bitterness receptors.11,12) A third strategy is the application of bitter taste receptor blockers.13,14)
Swallowing is another critical factor influencing adherence for oral medicines, especially in geriatric populations. The elderly have been reported to exhibit less accurate speech movements,15) more variable orofacial movements,16,17) and slower and more variable tongue movements during swallowing.18) Aspiration pneumonia is a common form of pneumonia with especially high mortality in the elderly who are more susceptible due to a deterioration of the swallowing function. It is therefore necessary to develop oral formulations which can be easily swallowed by elderly patients.
Oral jellies are generally easy to swallow, but their manufacturing method is complicated due to the need for transition from a solid by heating to a gel by cooling, while being molded into an appropriate shape.
Murakami et al. reported the preparation of a biodegradable poly-γ-glutamic acid (PGA) gel by cross-linking of PGA with L-lysine (L-Lys) by amide linkage in an environmentally benign aqueous solvent. PGA is a biodegradable polymer, produced by Bacillus subtilis var. natto. The key feature of PGA gel is that gelation occurs immediately on the addition of water to a powdered form of the PGA gel. It has been reported that the water absorption of PGA gel ranged from 300 to 2100 g/g.19)
Therefore, active pharmaceutical ingredients which are vulnerable to heat, can be made into solid oral formulations using the powdered form of PGA gel and be gelated at the time of use by the simple addition of water.
PGA is already known to be a safe excipient and is used as a food additive to assist the absorption of minerals.20,21) To our knowledge, however, there have been no reports in which PGA gel has been used in a pharmaceutical preparation to enable taste-masking and improve ease of swallowing.
In this study, we focused on the use of PGA gel in formulations for elderly patients as the gel was expected to enable easy swallowing. In addition, Tsuji et al. reported that gel formulations could suppress drug bitterness by protecting the active ingredient from direct exposure to the tongue by creating a physical barrier.22)
Amlodipine besylate (AML)23–25) is a basic drug and well-established to be a bitter medicine. It is widely used in the elderly to treat hypertension, at doses of 2.5–10 mg as amlodipine (3.47–13.87 mg as AML).
The use of an ‘electronic tongue’ or taste sensor for pharmaceutical purposes is an innovation which reduces dependence on human gustatory sensation testing. We have previously evaluated the bitterness of several registered basic medicines using the taste sensor.
The purpose of this study was to prepare a PGA hydrogel, to evaluate physicochemical properties, its ease of swallowing using texture profile analysis (TPA) and its taste-masking effect when loaded with AML, both using the artificial taste sensor and in human gustatory sensation testing.
As physicochemical properties of powdered PGA gel, the melting-point and Fourier transform (FT)-IR spectra of PGA and powdered PGA gel were evaluated. And the swelling behavior was observed.
In TPA, the hardness, adhesiveness and cohesiveness of 0.5 and 1.0% (w/v) PGA gels, 0.5 and 1.0% (w/v) agar and 1.0% (w/v) ι-carrageenan were examined in the absence of drug, and determined whether or not these formulations were within the range of permission criteria for people with difficulty swallowing, as published by the Japanese Consumers Affairs Agency. The stress–strain curve for five different gels were also evaluated in the absence of drug.
The elution of AML from the PGA gel was determined in order to demonstrate the lack of influence of the gel on the bioavailability of AML.
In order to examine the bitterness intensity of five different gels containing AML, the sensor outputs of AN0, the basic bitterness-sensitive sensor in the artificial taste sensor, were measured and human gustatory sensation testing was performed. The results of these tests confirmed the potent bitterness-suppressing effect of PGA gel containing AML.
Finally, 1H-NMR spectroscopic analysis was carried out to examine the mechanism of the bitterness suppression of AML when mixed with PGA gel.
PGA, average molecular weight 200000–500000, L-Lys monohydrochloride, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM) were obtained from Wako Pure Chemical Corporation, Ltd. (Japan), amlodipine besylate (AML) from LKT Labs, Inc. (U.S.A.), ι-carrageenan from Tokyo Chemical Industry Co., Ltd. (Japan), and agar purified powder from Nacalai Tesque, Inc. (Japan). Quinine hydrochloride (Qui), used as a standard of bitterness, was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). The structure of AML is shown in Fig. 1.
PGA gel was prepared according to the method described in the report of Murakami et al.19) The method can be summarized as follows: PGA was placed in a glass vessel and dissolved in 1.0 M aqueous sodium hydroxide (NaOH) solution. L-Lys was dissolved in water and adjusted to pH 8.9 by the addition of 1.0 M NaOH. The solution of L-Lys was added to the solution of PGA and the mixture stirred for 30 min. DMT-MM dissolved in water was then added to the solution of PGA/L-Lys and the mixture stirred for four hours at 25°C. The reaction product was then immersed in 0.1 M phosphate buffer (pH 7.0) for 24 h, before adjusting the pH to 8.0 by adding 1.0 M NaOH. The swelled product was transferred into a nylon mesh bag and filtered. The product, which became PGA gel, was washed by immersing the bag in purified water, which was changed once a day for a week. Powdered PGA gel was obtained by lyophilization of the PGA gel. As physicochemical properties of powdered PGA gel, the melting-point and FT-IR spectra of PGA and powdered PGA gel were evaluated.
Swelling Behavior of PGA GelThe water absorption of PGA gel was measured using the method described in a previous report.19) The powdered PGA gel was weighed and placed in a nylon mesh bag, similar to that used for washing the gel. The bag was immersed in purified water at 25°C. After 0.5, 1, 3, 6 and 24 h, it was hung for 10 min to remove excess water and weighed. In order to exclude water absorption by the nylon mesh bag, the operation was carried out using an empty nylon bag as reference (n = 3). The water absorption (g/g) of PGA gel was calculated using the following equation:
 |
TPA was carried out to evaluate the physical properties of the gel using TA.XT plusC texture analyzer (Stable Micro Systems, Surrey, U.K.). Samples tested were PGA gel (0.5 and 1.0%), agar (0.5 and 1.0%) and ι-carrageenan (1.0%). TPA was performed according to the method used to determine permission criteria for persons with difficulty swallowing, as published by the Japanese Consumers Affairs Agency. This involves measurement of the texture parameters hardness, adhesiveness and cohesiveness. Textural properties were analyzed by performing two sequential ‘two-bite’ compression tests with a cylindrical-shaped probe, diameter 20 mm, height 100 mm. All samples were compressed to 70% of their original length. The test speed was set to 10 mm/s. The values of hardness, adhesiveness and cohesiveness were calculated from the obtained profiles using software provided by Stable Micro Systems. The stress–strain curve of five different gels was shown using measured the data by TA.XT plusC texture analyzer.
In Vitro Conventional Elution Tests of AML from PGA GelThe elution test was measured with a conventional elution tester (PJ-62N, Riken’s elution tester, Miyamoto Riken Ind., Co., Ltd., Osaka, Japan) according to the Japanese Pharmacopoeia, 17th edition. In vitro elution tests of AML from PGA gel were carried out using the paddle method at 37°C and 50 rpm using 900 mL of both the first fluid (pH 1.2) and the second fluid (pH 6.8) for the elution test. At various time intervals, 2 mL aliquots were withdrawn and replaced by the same volume of fresh medium. The concentration of AML was determined using HPLC (LC-2010C, Shimadzu Corp., Japan). The HPLC method used for AML was based on the Japanese Pharmacopoeia and the previous report.24) For HPLC, an integrator (LC solution; Shimadzu Corp., Japan) and reverse-phase column (CAPCELL PAK C18 UG120 S5: 150 × 4.6 mm i.d.; Shiseido Co., Ltd., Japan). The column temperature was kept at 25°C. The mobile phase composition was methanol: potassium dihydrogen phosphate (41→10000) (13 : 7, v/v) and the flow rate was 1.0 mL/min. Run time was 10 min. The ultraviolet detection wavelength was set at 237 nm.
Taste Sensor Measurement of PGA Gel in the Absence of Drug and AML Alone, and of Five Different Gels Mixed with AMLTaste sensor SA501 (Intelligent Sensor Technology Inc., Atsugi, Japan) was used to measure the electric potential of the sample solutions in order to evaluate their taste. Sensors have been developed specifically to detect various tastes: AAE to detect umami, CA0 to detect sourness, CT0 to detect saltiness, AN0 to detect the bitterness of basic substances,12,24,26,27) C00 to detect the bitterness of acid substances and AE1 to detect astringency. The taste sensor measurements were performed as follows, according to our previous articles.28–31)
The electrode set was attached to a mechanically controlled robot arm. The detecting sensor part of the equipment consists of a reference electrode and a working electrode composed of lipid/polymer membranes. The electrodes have an internal cavity filled with 3.3 M KCl solution. The difference between the electric potential of the working electrode and the reference electrode was measured using a high-input impedance amplifier connected to a computer. Fresh 30 mM KCl solution containing 0.3 mM tartaric acid (corresponding to saliva) was used as the reference solution and also to rinse the electrode after every measurement.
The measurement procedure is as follows: The electrodes are dipped first into the reference solution and the electric potential obtained (mV) is defined as Vr0. Then a sample solution is measured and the electric potential obtained defined as Vs. The relative sensor output (R), represented by the difference between the potentials of the sample and the reference solution (Vs − Vr0), corresponds to the ‘taste immediately after putting in the mouth.’ The electrodes are subsequently rinsed with a fresh reference solution for 6 s. When the electrodes are dipped into the reference solution again, the new potential of the reference solution is defined as Vr1. The difference between the potentials of the reference solution before and after sample measurement (Vr1 − Vr0) is defined as the ‘Change in the membrane Potential caused by Adsorption’ (CPA), and corresponds to the so-called ‘aftertaste.’ CPA is a specific expression of bitterness. In this study, the CPA of AN0 and C00 were used to evaluate bitterness and the R was used to evaluate each tastes except bitterness. 0.03% (w/v) monosodium glutamate (MSG) for umami, 0.0012% (w/v) tartaric acid (Tar) for sourness, 0.25% (w/v) sodium chloride (NaCl) for saltiness, 0.001% (w/v) Qui for bitterness of basic substances, 0.016% (w/v) diclofenac sodium (Dic) for bitterness of acid substance and 0.075% (w/v) tannic acid (Tan) for astringency. These concentrations represent the threshold for control substances for each taste.
Firstly, we measured the predicted taste (umami, sourness, saltiness, bitterness of basic and acidic substances and astringency) of 0.5 and 1.0% PGA gel using 1.0% agar as a reference and AML alone. Secondly, we evaluated bitterness-masking of AML (3.47 mg) by the five different gels individually.
For prediction of taste of PGA gel, PGA gels at concentrations of 0.5 and 1.0% were prepared by adding purified water. Agar (1.0%) was prepared by dissolving 50 mg of agar purified powder in 5 mL purified water and heating.
For evaluation of bitterness-masking, AML (3.47 mg) was added to PGA gel, agar and ι-carrageenan at concentrations of 0.5, 1.0 and 1.0%, respectively, and mixed for 10 s. In order to examine simulated bitterness in the oral cavity in a time-dependent manner, mixtures of AML and gel were placed in a mesh filter and immersed in 20 mL purified water (37°C). Solutions were tested in the taste sensor after immersion for 5, 15 and 30 s.
Determination of Concentrations of AML Eluted from Five Different GelsIn order to examine AML elution from the five gels in a time-dependent manner, the concentration of AML was determined using HPLC. The HPLC method is the same as that used in the in vitro conventional elution tests of AML from PGA gel, described above. Samples were prepared in the same way as for taste sensor measurement.
Human Gustatory Sensation TestThe human gustatory sensation test to measure bitterness intensity was performed with six well-trained subjects, 31 ± 9 years old, according to a modified, previously described, method.12,24) No subject reported having a cold or other respiratory tract infection in the week prior to testing. The subjects were asked to refrain from eating, drinking, or chewing gum for at least 1 h prior to testing. All subjects were non-smokers and signed an informed consent form before the experiments. The experimental protocol of this study (No. 19-19) was approved in advance, on May 18, 2019, by the ethical committee of Mukogawa Women’s University.
Quinine hydrochloride solutions at concentrations of 0.01, 0.03, 0.1, 0.3 and 1.0 mM were prepared as standards of bitterness. Bitterness intensities (τ) of 0, 1, 2, 3 and 4 were allocated to these increasing concentrations of standard solution. Before testing, the subjects were asked to keep 2 mL of these quinine hydrochloride solutions in their mouths for 15 s and were told the concentration and bitterness intensity (τ) of each solution. In the test, the subjects were asked to keep 5 mL of sample solution in their mouth for 15 s and to evaluate the bitterness intensity of each sample. After testing each sample, subjects gargled well and waited for at least 20 min before testing the next sample. Samples of each gel were prepared as described for TPA and the quantity of AML per sample was the same as that used in evaluations by the taste sensor.
1H-NMR Spectroscopic Analysis1H-NMR spectra were measured on a JEOL 500 MHz spectrometer using dimethyl sulfoxide (DMSO-d6) as solvent and tetramethylsilane (TMS) as internal standard. Sample solutions of AML were prepared with/without powdered PGA gel. The mixing ratios of powdered PGA gel to AML in the sample solution were 1 : 7 w/w, corresponding to AML in 0.5% PGA gel, and 1 : 14 w/w, corresponding to AML in 1.0% PGA gel.
Statistical AnalysisBellCurve for Excel® (Social Survey Research Information Co., Ltd., Japan) was used for statistical analysis. Tukey’s test and Dunnett’s test were used for multiple comparisons. Correlation was examined using the Pearson product-moment correlation coefficient. The 5% level of probability was considered significant.
The obtained PGA gel was colorless solid and be confirmed to decompose around 300°C by the micro melting-point apparatus (MP 500D, Yanaco Co., Ltd., Japan). FT-IR spectra of PGA and powdered PGA gel were measured using an IRAffinity-1 spectrometer (Shimadzu Corp., Japan) and shown in Fig. 2. FT-IR spectrum of PGA showed a characteristic peak around 1740 cm−1 representing the C=O stretch vibration of carboxy group. On the other hand, FT-IR spectrum of PGA gel showed a characteristic peak at 1635 cm−1 representing the C=O stretch vibration of amide group. These peaks were suggested that carboxy groups of PGA were cross-linked by the amino group of L-Lys and amide linkage formed.
The time-dependent swelling behavior of powdered PGA gel was shown in Fig. 3. The water absorption of powdered PGA gel at 24 h after starting water absorption was 614 ± 69 g/g (n = 3, mean ± standard deviation (S.D.)) which was within the range described in a previous report.19) The water absorption at 0.5 h after starting water absorption was 452 ± 14 g/g which was about 74% of the water absorption at 24 h after starting water absorption. These results suggest that powdered PGA gel obtained in this study showed excellent water absorbency and prepared successfully according to the method described in a previous report.19)
n = 3, mean ± S.D.
Table 1 (a) shows the permission criteria according to which foodstuffs may be considered suitable for persons with difficulties swallowing, as published by the Japanese Consumers Affairs Agency.32) The permission criteria are divided into three grades, defined as I, II and III, with grade I describing the most suitable conditions. The overall grade of each sample is decided on the permission criteria of its highest grade for any parameter (hardness, adhesiveness or cohesiveness).
| (a) | ||||
|---|---|---|---|---|
| The permission criteria | Hardness (N/m2) | Adhesiveness (J/m2) | Cohesiveness | |
| I | 2500–10000 | 400 or less | 0.2–0.6 | |
| II | 1000–15000 | 1000 or less | 0.2–0.9 | |
| III | 300–20000 | 1500 or less | — | |
| (b) | ||||
| Hardness (N/m2) | Adhesiveness (J/m2) | Cohesiveness | Grade in the permission criteria | |
| 0.5% PGA gel | 2327.12 ± 111.19 | 973.10 ± 97.06 | 0.81 ± 0.01 | II |
| 1.0% PGA gel | 3087.38 ± 142.31 | 1266.45 ± 57.17 | 0.83 ± 0.01 | III |
| 0.5% agar | 853.08 ± 91.44 | 281.37 ± 71.72 | 0.62 ± 0.01 | III |
| 1.0% agar | 3568.88 ± 398.65 | 621.97 ± 130.06 | 0.61 ± 0.07 | II |
| 1.0% ι-carrageenan | 572.01 ± 46.10 | 44.11 ± 6.45 | 0.78 ± 0.01 | III |
(n = 3, mean ± S.D.)
The hardness, adhesiveness and cohesiveness of the five gels and their grades are shown in Table 1 (b). All five gels were within the range of permission criteria for persons with difficulty swallowing, with grades varying between I and III for the various parameters. Overall, 1.0% PGA gel, 0.5% agar and 1.0% ι-carrageenan were considered grade III, while 0.5% PGA gel and 1.0% agar were grade II.
When values for hardness and adhesiveness are small, the property of the sample is similar to that of a liquid. The values for hardness and adhesiveness of 0.5% agar and 1.0% ι-carrageenan were both small. A grade I product should ideally have a high value for hardness and a low value for adhesiveness. For the five gels studied, 0.5% PGA gel and 1.0% agar were closest to grade I.
Agar gel can be transformed into agar that have melting temperatures close to physiological temperatures, while gelation of ι-carrageenan needs not only water but also cation. Conversely, PGA gels are easy to prepare by the addition of water to the powdered form. PGA gel preparation is also a safe procedure, glutamic acid and L-Lys, its main components, being naturally occurring products.
The stress–strain curve obtained using texture analyzer was shown in Fig. 4. It was indicated that the initial slope of the stress–strain curve increased in the order of 1.0% ι-carrageenan, 0.5% agar, 0.5% PGA gel, 1.0% PGA gel and 1.0% agar. This result was identical with the result of hardness by TPA, because the steep slope of the stress–strain curve shows high hardness. Furthermore, it was confirmed that the strain of each sample did not completely restored after removing the stress. It suggests that all of five gels showed elasto-plasticity as physical property.
Figure 5 shows elution profiles of AML from mixtures of AML and PGA gel as determined by a conventional elution test. The full elution of AML from all samples was reached after about 10 min for the first fluid (pH 1.2).
(a) First fluid (pH 1.2); (b) Second fluid (pH 6.8). n = 3, mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. AML (Tukey’s test).
The elution of AML from the PGA gel was the same as AML alone for the first fluid (pH 1.2). Significant differences in elution of AML from 0.5% PGA gel and 1.0% PGA gel after 60 min compared with the elution of AML alone were not found for the second fluid (pH 6.8). These results suggest that the elution of AML from mixtures with PGA gel are complete within an hour and that mixing AML in a PGA gel would not influence the bioavailability of AML in the stomach.
Taste Evaluation of PGA Gel in the Absence of Drug and AML Alone by Taste Sensor MeasurementFigure 6 shows sensor responses to PGA gel (0.5 and 1.0%) using agar (1.0%) as reference and AML alone for all six sensors. The largest response was from AAE, which was developed specifically to detect umami in PGA gel. The sensor output of AAE was equivalent to the threshold for umami (0.03% MSG). It was considered that the umami was probably derived from the glutamic acid component of PGA. The sensor outputs of the other five sensors were below the thresholds for each taste. Consequently, PGA gel was suggested to have a slight flavor of umami, and confirmed the acceptability of PGA gel for use in oral pharmaceutical preparations. The AAE, CT0, C00 and AE1 sensor outputs of AML alone were below the threshold for umami (0.03% MSG), saltiness (0.25% NaCl), bitterness of acidic substance (0.016% Dic) and astringency (0.075% Tan), respectively. Whereas the AN0 and CA0 sensor outputs of AML alone were significantly increased than the threshold for bitterness of basic substance (0.001% Qui) and sourness (0.0012% Tar). These results showed AN0 sensor was capable to use in sensitive evaluation for the bitterness of AML. It was considered that the sourness was probably derived from besylate of AML.
(a) AAE, (b) CA0, (c) CT0, (d) AN0, (e) C00, (f) AE1. n = 3, mean ± S.D. * p < 0.05, *** p < 0.001 vs. control substances for each taste (Dunnett’s test).
Figure 7(a) shows that AN0 sensor outputs in response to AML were in direct proportion to the logarithm of the concentration of AML, according to the Weber–Fechner law.33) Figure 7(b) shows the influence of mixtures of AML with the five different gels on AN0 sensor outputs. The sensor outputs in response to AML (3.47 mg per sample) were significantly reduced in mixtures with each gel. The CPA value of AN0 for a quinine solution with τ = 1, the bitterness threshold, was 1.99 ± 0.05 mV. The sensor outputs in response to AML mixed with 0.5 or 1.0% PGA gel were equivalent to or beneath the bitterness threshold at all times.
The dose-dependent increase in AN0 (CPA) of (a) AML, (b) AML mixed with five different gels. n = 3, mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0.001% quinine (τ1) (Tukey’s test).
Figures 8(a) and (b) show concentrations of AML (3.47 mg per sample) eluted from five gels in a time-dependent manner, (b) being an expansion of (a). The amount of AML eluted was confirmed to increase in a time-dependent manner from 1.0% agar and ι-carrageenan. 10.79 ± 0.35 µg/mL of AML in 0.5% agar was eluted within five seconds; the amount eluted did not change between five and 15 s, with 20.50 ± 0.21 µg/mL of AML in 0.5% agar being eluted between 15 and 30 s. A small amount of AML (about 0.03 µg/mL) was eluted from 0.5 and 1.0% PGA gel in the first five seconds; thereafter it was not eluted at all.
n = 3, mean ± S.D.
Figure 9(a) shows the effect of mixing AML (3.47 mg per sample) with each of the five gels on bitterness intensity by human gustatory sensation test. The bitterness intensities of AML mixed with 0.5%, 1.0% PGA gel and 1.0% ι-carrageenan were lower than that of AML alone and equal to or less than τ1, the bitterness threshold. Figure 9(b) shows the correlation between bitterness intensities obtained from human gustatory sensation testing and the taste sensor output (CPA) of AN0 for mixtures of AML with each of the five gels. The AN0 sensor outputs in response to AML (3.47 mg per sample) were about 50 mV (The data were shown in Figs. 6(d) and 7(b)). That was confirmed to corresponded to τ4 as bitterness intensity by human gustatory sensation test. There was a good correlation between bitterness intensities obtained from human gustatory sensation test results and taste sensor outputs of AN0 for all mixtures (r = 0.823, Pearson product-moment correlation coefficient, p < 0.05). These results show that suppression of the bitterness intensity of AML by all five gels was accurately predicted by the taste sensor. The results suggest that 0.5 or 1.0% PGA gel and ι-carrageenan may be the best gels for bitterness-masking.
(a) n = 6, mean ± S.E. ** p < 0.01, *** p < 0.001 vs. AML alone (Tukey’s test); (b) r = 0.823 (p < 0.05, Pearson product-moment correlation coefficient).
It is estimated that bitter substance establishes electrostatic interactions and hydrophobic interaction with bitter taste receptor by docking simulation of bitter taste receptor with bitter substance.34) AML which is a basic bitter drug, has an amino group which is positively charged and hydrophobic group in the structure. Electrostatic interaction and hydrophobic interaction which are formed in AML with bitter taste receptor on the mucous membrane in the oral cavity are expected to be main mechanism of bitterness caused by AML. AML is adsorbed on the negatively charged and hydrophobic part of the taste sensor membrane and causes a change in membrane potential by altering the charge density of the taste sensor output. In previous studies, we have demonstrated a taste-masking effect when a basic bitter drug is combined with an acidic compound, for example, diphenhydramine hydrochloride as basic bitter drug with chlorogenic acid as acidic compound11) or zopiclone as basic bitter drug with citric acid as acidic compound.35) In this study, 1H-NMR analysis suggested an electrostatic interaction between the amino group of the basic bitter drug and the carboxyl group of the acidic compound. It was suggested that the electrostatic interaction reduced the membrane adsorption of the basic bitter drug in the taste sensor and binding with the bitterness receptor in human gustatory sensation tests.
An interaction between the amino group of AML and the carboxyl group of glutamic acid in PGA gel was expected to reduce the membrane adsorption of AML in the taste sensor and binding with the bitterness receptor in human gustatory sensation tests. 1H-NMR was used to evaluate the interaction between AML and PGA gel. Chemical shifts (δ) of each proton of AML alone or with PGA gel (at ratios of 1 : 7 and 1 : 14) obtained from 1H-NMR analysis are shown in Table 2. The difference between the chemical shifts (Δδ) of AML alone and AML with PGA gel was calculated by the following equation:
 |
| Proton | AML | AML : PGA gel = 1 : 7 | AML : PGA gel = 1 : 14 |
|---|---|---|---|
| 1 | 1.103 | 1.100 | 1.100 |
| 2 | 3.977 | 3.976 | 3.976 |
| 6 | 5.310 | 5.311 | 5.310 |
| 8 | 7.297–7.339 | 7.281–7.339 | 7.282–7.339 |
| 9 | 7.220 | 7.209 | 7.209 |
| 10 | 7.128 | 7.117 | 7.117 |
| 11 | 7.272 | 7.260 | 7.258 |
| 16 | 3.505 | 3.504 | 3.504 |
| 18 | 2.296 | 2.300 | 2.304 |
| 19 | — | — | — |
| 21 | 4.565 | 4.544 | 4.556 |
| 4.697 | 4.683 | 4.681 | |
| 23 | 3.655 | 3.612 | 3.607 |
| 24 | 3.074 | 2.994 | 2.981 |
| 25 | — | — | — |
| 28 | 7.619 | 7.610 | 7.608 |
| 29 | 7.297–7.339 | 7.281–7.339 | 7.282–7.339 |
| 30 | 7.297–7.339 | 7.281–7.339 | 7.282–7.339 |
In the 1H-NMR spectra, the signal of proton 23 (proton No. shows in Fig. 1) of AML was at 3.655 ppm for AML alone, 3.612 ppm (Δ − 0.043) at a mixing ratio of AML to PGA gel of 1 : 7, and 3.607 ppm (Δ − 0.048) at a mixing ratio of 1 : 14, while the signal of proton 24 of AML was 3.074 ppm for AML alone, 2.994 ppm (Δ − 0.080) at a mixing ratio of 1 : 7, and 2.981 ppm (Δ − 0.093) at a mixing ratio of 1 : 14. The signals of proton 23 and 24, near to the nitrogen atom of AML, shifted clearly upfield when AML was mixed with PGA gel. The signal of other protons in AML were not clearly shifted. On the basis of these results, it is suggested that the upfield shift was due to a shielding effect. Lan et al. reported that the proton of cation moieties experiences a chemical shift and up-field drift caused by the addition of an anion, with the possible formation of an electrovalent bond between the cation and anion.36) Their results support the results described in the present paper.
It is postulated that PGA gel suppresses the bitterness of bitter substances such as AML by electrostatic interaction between a positive ion-charged amino group of AML and a negative ion-charged carboxyl group of PGA gel.
Taste and ease of swallowing are important factors determining the acceptability of oral pharmaceutical formulations. Therefore, taste-masking and minimum critical viscosity must be considered in dosage form design of oral pharmaceutical formulations. The PGA gel used in this study had a slight taste of umami and was without bitterness or astringency. The 0.5% PGA gel was assigned grade II in the permission criteria according to which foodstuffs may be considered suitable for persons with difficulties in swallowing by the Japanese Consumers Affairs Agency, and had adequate hardness and adhesiveness to enable easy swallowing. The 0.5% PGA gel sustainably suppressed the bitterness sensor outputs (CPA) of AN0 to AML to a greater degree than agar or ι-carrageenan and also suppressed the bitterness intensity of AML to a greater degree than agar in human gustatory sensation tests. It can therefore be concluded that the bitterness intensity of AML is suppressed by mixing with PGA gel. The mechanism underlying bitterness suppression of AML by PGA gel is suggested to be electrostatic interaction, confirmed by 1H-NMR spectroscopic analysis, in addition to creating a physical barrier by the PGA gel.
PGA gel is therefore a novel gelling agent which does not require heating or cations. Furthermore, it may be useful in oral pharmaceutical formulations to enable easy swallowing and the masking of bitterness of basic drugs such as AML.
The authors declare no conflict of interest.
