The Impact of Adding a Cationic Metal Salt and Curcumin to Monoammonium Glycyrrhizic Acid on Its Solubilizing Capacity and Gelation

Abstract

Monoammonium glycyrrhizic acid (MAG), a glycyrrhizic acid monoammonium salt, is a naturally derived low-molecular-weight gelling agent with surface-active properties. It has the capacity to individually facilitate the preparation of gel-solubilized drugs. As MAG is an anionic surfactant with carboxyl groups, the addition of counterions may affect micelle formation and gelation. In this study, the solubilization and gelling properties of MAG were investigated following the addition of metal salts (NaCl and KCl). The addition of metal salts resulted in a decrease in the critical micelle concentration and an increase in gel hardness. Supersaturation of curcumin (CUR) was maintained by the addition of metal salts because of increased micelle number and viscosity. When the gel hardness was compared between formulations with and without CUR, a significant reduction in hardness was observed with the solubilization of CUR. The addition of KCl prevented the decrease in the hardness of gels containing CUR compared to the addition of NaCl. Put together, the addition of metal salts had a noteworthy impact on micelle and gel formation of MAG. In particular, the addition of KCl was more effective in the preparation of gel-solubilized CUR.

Introduction

Glycyrrhizic acid is a naturally occurring component of licorice, extracted from the dried root of Glycyrrhiza glabra.^1,2) It is an oleanane-type triterpene glycoside, commonly used as a flavoring and sweetening agent, as well as a pharmaceutical excipient.³⁾ Glycyrrhizic acid has various pharmacological effects such as anti-inflammatory, antimicrobial, and antiviral activities.^4–6) It is a polyprotic organic weak acid with three carboxyl groups, and is composed of one glycyrrhetic acid molecule and two glucuronic acid molecules. The three dissociation constants of the free acid determined through titration have been reported to be 3.98 (pK_a1) and 4.62 (pK_a2) for the glucuronic acid molecules, and 5.17 (pK_a3) for glycyrrhetic acid.⁵⁾ Because the dissociation of the three carboxylic acids is restricted in water, glycyrrhizic acid exhibits poor water solubility.⁷⁾ Therefore, it is used as various salt forms with water-soluble properties, including ammonium, sodium, and potassium salts. Glycyrrhizic acid salts are widely used as sweeteners, as well as anti-inflammatory and anti-allergy components in topical delivery.^8,9) Among the glycyrrhizic acid salts, we focused on the ammonium salt in terms of solubilization capacity and gelling properties.

Structurally, monoammonium glycyrrhizic acid (MAG) has a glucuronic acid directly linked to glycyrrhetic acid in form of an ammonium salt¹⁰⁾ (Fig. 1). It is used therapeutically in pharmaceutical dosage forms for liver disease and anti-allergy preparations.¹¹⁾ In addition, it has anti-oxidative and anti-inflammatory effects.^12,13) A common method for preparing an aqueous solution of MAG is to heat it at 70 °C or higher under stirring.¹⁴⁾ During the cooling process of the heated solution, it forms micelles at concentrations above the critical micelle concentration (CMC).¹⁵⁾ The solubility of solids generally increases with increasing temperature, and if excessively dissolved, precipitation occurs during cooling. The apparent solubility of MAG increases from micelle formation, allowing the preparation of highly concentrated solutions without precipitation. It has also been reported that MAG forms fibrillar hydrogels through micelle formation.¹⁶⁾ In other words, MAG is a low-molecular-weight gelator with the potential to solubilize drugs via micelle formation and the preparation of gels for drug encapsulation. Although there have been several reports on micelle formation and gelation with MAG, to the best of our knowledge, there have been no reports on the effects of the addition of cationic metal salts to the aforementioned processes. The addition of metal salts to ionic surfactants decreases their CMC due to their binding to the surfactant head group as a counter ion.^17,18) MAG, an anionic surfactant with three carboxyl groups, can interact with cationic metal salts. Therefore, the micellization and gelation of MAG may be influenced by the addition of cationic metal salts such as Na⁺, K⁺, Ca²⁺, and Mg²⁺. The purpose of this study was to elucidate the effect of adding cationic metal salts to MAG on its solubilizing capacity and gelation. The changes in the CMC of the MAG solution and gel hardness were evaluated in the presence and absence of metal salts. The solubilizing capacity of MAG was studied using curcumin (CUR) as a model drug. Although CUR has pharmacological properties as anti-inflammatory¹⁹⁾ and antibacterial,²⁰⁾ its application is limited by extremely low water-solubility.²¹⁾ If CUR is solubilized with MAG, it can be applied as oral jelly-formulation and skin gel-formulation. The solubilization of CUR in MAG micelles and the effect of CUR solubilization on MAG gelation were investigated in the presence or absence of metal salts. This study provides useful information on the application of MAG in gel formulations and the solubilization of poorly soluble drugs.

Fig. 1. Chemical Structures of (a) MAG and (b) CUR

Results and Discussion

MAG Micelle and Gel Formation in the Presence or Absence of Cationic Metal Salts

When the divalent metal salts CaCl₂ (Ca²⁺) and MgCl₂ (Mg²⁺) were added to the MAG solution, MAG precipitation was observed. This phenomenon is interpreted as salting out. Therefore, the monovalent metal salts NaCl (Na⁺) and KCl (K⁺) were used as cationic counterions. The changes in the surface tension of the MAG solutions were evaluated with and without the addition of a cationic metal salt in distilled water (Fig. 2). The surface tension of the MAG solution decreased with increasing MAG concentration and reached a constant value. The CMC values of the MAG solutions with and without metal salts were calculated using the analytical method described by Lin et al.²²⁾ (Table 1). Herein, the CMC of the MAG solution without metal salts was 0.055%. Contrarily, in a previous study, the CMC values of MAG solutions were reported as approx. 0.13 and 0.31% in pH 5 and 6 buffer solutions, respectively.¹⁵⁾ The dissociation constants of the free acid are 3.98 (pK_a1) and 4.62 (pK_a2) in glucuronic acid and 5.17 (pK_a3) in glycyrrhetic acid.¹⁶⁾ The ionized moiety has a high aqueous solubility compared to unionized moiety. In this study, the CMC of MAG appeared to be low in distilled water because of the limited dissociation of carboxylic acids. The addition of metal salts decreased the CMC in a concentration dependent manner. It has been reported that the addition of metal salts as counterions decreases the CMC of ionic surfactants.^23–25) A repulsion between the heads of ionic surfactants is disadvantageous for micelle formation. Ionic surfactant micelles bind to counterions through electrostatic interactions between the surface charge of the micelle heads and the counterion via dissociation of the surfactants in the solution. The binding of counterions can contribute to the suppression of electrostatic repulsion at the micelle surface, causing a decrease in the CMC of the ionic surfactants. The micellar properties of ionic surfactants are affected not only by the counterion concentration but also by the type of the added counterion.²⁶⁾ It has been reported that the CMC of dodecyl sulfate is lower when KCl is added than when NaCl is added; the CMC increases with an increase in the size of the hydrated counterion.²⁷⁾ In the case of MAG, although the addition of K⁺ showed a tendency to reduce the CMC compared to the addition of NaCl, there was no significant difference between the two.

Fig. 2. Changes in Surface Tension of MAG Solution at 37 °C

(a) After addition of KCl. (b) After addition of NaCl. Surface tension measurements were performed under the same temperature as the stability test.

Table 1. Critical Micelle Concentration (CMC) of MAG Solution Following (a) Addition of KCl and (b) Addition of NaCl

(a)		(b)
	CMC (%)		CMC (%)
MAG solution	0.055	MAG solution	0.055
MAG solution with 0.1% KCl	0.033	MAG solution with 0.1% NaCl	0.037
MAG solution with 0.5% KCl	0.028	MAG solution with 0.5% NaCl	0.027
MAG solution with 1.0% KCl	0.021	MAG solution with 1.0% NaCl	0.024

The micellar structure of MAG was observed at different concentrations using transmission electron microscope (TEM) (Fig. 3). Rod- or string-like micelles were barely observed at a CMC of approximately 0.05%. Although the micellar structure may not be fully reflected by drying process, rod- or string-like micelles were observed above 0.1%. Matsuoka et al. reported that MAG forms rod-like micelles in a pH 5 buffer, which have a similar structure to those formed in distilled water.¹⁵⁾ Although a change in pH affects the CMC of MAG solution, it does not appear to significantly affect the micelle structure. Supplementary Fig. S1 shows a TEM image of MAG solution containing KCl. During preparation of the test samples, precipitation of the added metal salt was observed. The effect of salt addition may not be fully reflected in the TEM image because of salt precipitation; however, the rod- or string-like structures appeared to be maintained. Further investigation is required to determine the structural changes caused by salt addition.

Fig. 3. TEM Images of MAG at Concentrations of (a) 0.05%, (b) 0.1%, (c) 0.2%, and (d) 0.3%

Figure 4 shows the presence or absence of gelation at different MAG concentrations with or without 0.5% metal salts. The images were taken after 15 s of inversion immediately after removal of samples stored under 4 °C conditions. Without metal salt addition, MAG solution at a concentration greater than 0.5% did not flow after the container was inverted, indicating the start of gelation. The formation of rod- or string-like micelles has been reported to cause gelation. The rod- or string-like micelles entangle with each other and form the gel by holding water molecules in entanglements.²⁸⁾ On the other hand, it is difficult to form the gel in spherical micelles because they does not entangle with each other. Gel formation of MAG solution may be induced by the formation of entangled rod- or string-like micelles as shown in TEM images. The addition of metal salts reduced the gelation concentration of MAG. The MAG solution with 0.5% metal salts formed a gel even at a concentration of 0.3%. MAG gel hardness was evaluated at various concentrations of MAG and metal salts, as shown in Fig. 5. An increase in the hardness was observed with increasing MAG concentration. The addition of 0.1% NaCl showed a slight increase in hardness compared to MAG alone. Conversely, the addition of 0.1% KCl resulted in a slight decrease in hardness. Significant increases in hardness were observed with addition of 0.5 or 1% metal salts. The significant increase in hardness may be attributed to two factors. The first is the decrease in CMC due to the addition of metal salts. A decrease in CMC increased the number of micelles. Consequently, the hardness of the MAG gel increased with an increase in the number of entangled micelles. The second is the reduced repulsion between micelles owing to the binding of the metal salt to the surfactant headgroup. It is reported that anionic surfactants that form rod- and string-like micelles increase the entanglement between micelles following the addition of counterions.²⁹⁾ In anionic polymer gels such as carrageenan, the addition of metal salts also increases gel hardness.³⁰⁾ This is because the addition of metal salts suppresses electrostatic repulsion between polymers and increases entanglement between them. MAG is an anionic surfactant that is negatively charged, even when it is difficult to dissociate in distilled water. The addition of cationic metal salts reduces the charge repulsion between micelles, increases micelle entanglement, and increases gel strength.

Fig. 4. Photo Images of MAG Sol or Gel after 15 s of Inversion at 25 ± 3 °C

Fig. 5. Effect of (a) KCl and (b) NaCl Addition on Hardness of MAG Gels at 25 ± 3 °C

* p < 0.05 compared with MAG without metal salts and ^# p < 0.05 compared with MAG with 0.1% KCl and ⁺ p < 0.05 compared with MAG with 0.1% NaCl.

Preparation of MAG Gel Containing CUR

The effect of adding metal salts to MAG solution on the solubilization of CUR and subsequent gel formation was investigated. Figure 6 shows the change in CUR solubility at different MAG concentrations with or without 0.5% metal salts immediately after preparation and 7 d after preparation. CUR is a poorly-water soluble compound, and its solubility was approx. 0.013 µg/mL in distilled water. As shown in Fig. 6(a), when CUR was added to MAG, solutions in which CUR was completely dissolved immediately after preparation were obtained. The amount of CUR dissolved in the prepared solutions was approximately 75000 times higher its aqueous solubility. This result indicates that CUR was solubilized in MAG micelles. CUR precipitation was observed in 0.8% MAG solution after storage for 7 d. CUR in the 0.8% MAG solution with metal salts remained in complete solution after 7 d. As shown in Table 1, the CMC of the MAG solution decreased with the addition of metal salts and the number of micelles increased. As the micelle number increased, the amount of solubilized CUR in the micelles also increased, thus maintaining the supersaturated state of CUR. Increased viscosity due to salt addition may also have contributed to the maintenance of CUR supersaturation. It has been reported that the viscosity of the dispersant contributes to the generation and maintenance of a high degree of supersaturation.^31,32) As depicted in Fig. 4, the addition of metal salts promoted MAG gel formation. The enhanced gelling properties of MAG may have contributed to the maintenance of CUR supersaturation.

Fig. 6. Dissolved CUR in MAG/CUR/Metal Salt Solution (a) after Preparation (b) after 7 d at 37 °C

* p < 0.05 compared with MAG (0.8%) without metal salts.

The effect of CUR solubilization on the gelation of MAG was studied in the presence or absence of 0.5% metal salts (Fig. 7). No precipitation of CUR was observed in the prepared samples, indicating the successful preparation of MAG gel or sol with complete dissolution of CUR. Solubilization of CUR inhibited MAG gelation. When the gel hardness was compared between formulations with and without CUR, a significant reduction in hardness was observed with CUR solubilization. The structure of certain micelles has been reported to be altered by solubilizing compounds.^33,34) For example, the shape of sodium lauryl ether sulfate and cocamidopropyl betaine micelles changes from worm-like to disk-like by solubilizing dodecanoic acid, resulting in reduced viscosity of the micellar solution.³⁴⁾ MAG gelation is caused by entanglement of rod- or string-like micelles. The solubilization of CUR may change the structure of MAG micelles, resulting in the inhibition of gelation. In the absence of CUR, no significant differences in hardness were observed between the formulations. In contrast, the hardness of formulations containing CUR differed between the metal salts. The decrease in gel hardness between the formulations with and without CUR was smaller when KCl was added. The differences between KCl and NaCl addition may be estimated as follows. The CUR, with a planar chemical structure, would be solubilized by penetrating to the hydrophobic part of the MAG micelles. The solubilization of CUR increased the distance between headgroups in MAG micelles. The increased distance between headgroups affected the interaction between metal ions and headgroups. The K⁺ with a larger ionic radius than Na⁺ was advantageous for binding between spread headgroups. Therefore, the addition of KCl could contribute to the maintenance of gel hardness compared to the addition of NaCl.

Fig. 7. Hardness of MAG/CUR/Metal Salts Gel at 25 ± 3 °C

* p < 0.05 compared with MAG without metal salts and CUR. ^# p < 0.05 compared with MAG without metal salts. ⁺ p < 0.05 compared with MAG with 0.5% KCl without CUR. ^& p < 0.05 compared with MAG with 0.5% KCl. ^$ p < 0.05 compared with MAG with 0.5% NaCl without CUR.

We finally investigated the effect of solubilized CUR content on gel hardness (Fig. 8). The hardness of the MAG gel decreased as the content of solubilized CUR in micelles increased. An increase in solubilized CUR correlated with the number of MAG micelles in the system, thus a reduction in the number of free MAG micelles. We hypothesized that MAG micelles containing CUR were less likely to form gels because of structural changes in the micelles themselves. Although the effect of the addition of metal salts requires further investigation, the results show that the addition of KCl is more effective than NaCl for the formation of MAG gel when CUR is solubilized.

Fig. 8. Hardness of MAG Gels with Various Concentrations of CUR at 25 ± 3 °C

Conclusion

In the study, we demonstrated the effect of metal salts on the solubilization and gelation of MAG. The CMC of MAG solution was reduced by increasing the concentration of the investigated metal salts. The gel hardness of MAG increased with increasing concentrations of added metal salts. The effect of the type of metal salt on gel hardness was irrelevant between KCl and NaCl. The supersaturated state of CUR was stabilized by the addition of metal salts when the MAG concentration was 0.8%. Solubilization of CUR inhibited MAG gel formation. The MAG solution containing CUR had lower gel hardness than that without CUR. When CUR was solubilized in MAG, differences in hardness were observed from the addition of KCl and NaCl. The addition of KCl was more effective for preparing MAG gels with solubilized CUR. This study highlights the usefulness of metal salts for use with MAG as a gelling and solubilizing agent. The addition of metal salts allows MAG to achieve drug solubilization and gelation at lower concentrations.

Experimental

Materials

MAG was supplied by Maruzen Pharmaceuticals Co., Ltd. (Hiroshima, Japan). KCl and acetonitrile were purchased from Wako Pure Chemical Corporation (Osaka, Japan). NaCl was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). CUR was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Phosphoric acid and dimethyl sulfoxide were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Ethanol was purchased from Japan Alcohol Trading Co., Ltd. (Tokyo, Japan).

Surface Tension Measurements

MAG stock solution was diluted to a concentration range of 0–0.15% in a total volume of 10 mL. The surface tensions of the solutions were measured using the bubble pressure method with a SITA Dyno Tester+ (Eko Instruments, Tokyo, Japan), and the average of three measurements was recorded. The bubble lifetime was set to 20 s. The sample temperature was maintained at 37 °C. The same method was used to measure surface tension following the addition of KCl and NaCl.

Transmission Electron Microscopy (TEM)

TEM images of MAG (0.05–0.3%) were obtained using an HT7800 (Hitachi, Tokyo, Japan) transmission electron microscope with an 80 kV accelerating voltage. After glow-discharging the collodion-coated copper grid, the solutions were deposited and it was then negatively stained with 1% uranyl acetate.

Preparation of MAG Gel Containing CUR

After dissolving MAG and CUR in mixture of distilled water and ethanol [50 : 50 (v/v)], the MAG solution was added to the ethanol solution and stirred for 30 min at 25 ± 3 °C. Ethanol was removed from the resultant solution using a rotary evaporator R-3 (Buchi, Tokyo, Japan) at a pressure of 102 mbar in a water bath at 50 °C to achieve MAG concentrations of 0.3, 0.5, 0.8, 1.0, 1.25, and 1.5% and a CUR concentration of 0.1%. Stability tests were performed by storing MAG gels containing CUR at 37 °C for 7 d. Gel hardness tests were performed after the MAG gels containing CUR were stored at 4 °C for 24 h. The same method was used following the addition of NaCl and KCl.

CUR Solubility Test

MAG gels containing CUR immediately after preparation and after storage at 37 °C for 7 d were heated at 50 °C for 1 min. Its heated samples were filtered through 0.2-µm polytetrafluoroethylene membrane filters (Hawach Scientific Co., Ltd., Xi’an, China). The solubility of CUR was determined by HPLC (Prominence; Shimadzu, Kyoto, Japan). A COSMOSIL 5C18-MS-II column (5 µm, 4.6 × 50 mm, Nacalai Tesque Inc.) was used for analysis at a column temperature of 40 °C. The mobile phase constituting acetonitrile: 0.1% phosphoric acid aqueous solution [45 : 55 (v/v)] was maintained at a flow rate of 1.0 mL/min, and the injection volume was 10 µL. CUR was detected at a wavelength of 480 nm, and the retention time was approximately 3.7 min.

Measurement of Gel Hardness

The hardness of MAG gels was determined using a TEX-100N (Japan Instrumentation System Co., Ltd., Nara, Japan) texture analyzer.³⁵⁾ MAG gels (0.5, 0.8, 1.0, 1.25, and 1.5%) were filled into polystyrene screw tube bottles (diameter; 35.8 mm) to a height of 15 mm and stored at 4 °C for 24 h. Measurements were taken at room temperature (25 ± 3 °C) immediately after removal of samples stored under 4 °C conditions. The hardness test was conducted by lowering and raising a duracon plunger (cross-sectional area 314 mm²) twice to a clearance of 5 mm at a compression speed of 10 mm/s, three times in succession, and the average value was recorded. The same method was used following the addition of KCl, NaCl, and CUR.

Statistical Analysis

Texture measurement results are expressed as the mean of three measurements ± standard deviation (S.D.). Statistical analyses were performed using the Tukey’s multiple comparison method. Statistical significance was set at p < 0.05 and is indicated as follows: *, ^#, ⁺, ^& and ^$.

Acknowledgments

The authors thank Maruzen Pharmaceuticals Co., Ltd. (Hiroshima, Japan) for supplying monoammonium glycyrrhizic acid (MAG).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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