micelle formation of monoammonium glycyrrhizinate

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Journal of Oleo ScienceCopyright ©2021 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess21046J. Oleo Sci. 70, (7) 911-918 (2021)

Micelle Formation of Monoammonium Glycyrrhizinate Keisuke Matsuoka1＊ , Mamoru Arima1, Yusuke Goto1, Shiho Yada2, and Tomokazu Yoshimura2

1 Faculty of Education, Laboratory of Chemistry, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, JAPAN 2 Department of Chemistry, Faculty of Science and Graduate School of Science, Nara Women’s University, Kitauoyanishi-machi, Nara, 630-8506,

JAPAN

1 IntroductionGlycyrrhizic acid is found in the roots of licorice, a legu-

minous plant1）. Licorice is native to the Asian continent and has been used as crude drug and anti-inflammatory agent2, 3）. In addition, glycyrrhizic acid is also used as a sweetener because its sweetness is several tens of times that of sugar2, 4）. However, glycyrrhizic acid has very low aqueous solubility because the hydrophilic groups are car-boxylic acids. Therefore, glycyrrhizic acid is typically dis-solved in a buffered aqueous solution or neutralized as an ammonium salt or alkali salt in crystalline form for practical use. Crystals of ammonium glycyrrhizinate are easily ob-tained by the extraction of glycyrrhizic acid from licorice root in aqueous ammonia solution1, 5）. This ammonium glycyrrhizinate can be obtained relatively inexpensively at high purity and has applications in toothpastes and other oral care and cosmetic products2, 6, 7）.

＊Correspondence to: Keisuke Matsuoka, Faculty of Education, Laboratory of Chemistry, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, JAPANE-mail: [email protected] March 5, 2021 (received for review February 1, 2021)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

As shown in Fig. 1, the molecular structure of monoam-monium glycyrrhizinate has an aglycon skeleton as the hy-drophobic part and two glucuronic acids（the carboxylic acids in this part are labeled A and B in Fig. 1）and a car-boxylic acid（C in Fig. 1）as hydrophilic groups at both ends of the hydrophobic group. In general, the formation of mo-lecular aggregates is more likely when the hydrophobic group and hydrophilic group are clearly separated, such as in synthetic surfactants. The glycyrrhizic acid is an am-phipathic substance having a molecular structure in which the distribution of the hydrophobic group and the hydro-philic group is somewhat poor, as shown in Fig. 1. On the other hand, there are reports that bio-surfactants form ag-gregates even if they have an unbalanced molecular struc-ture. Plant-derived surfactants such as saponin and human-derived bile salts can form unique aggregates based on their amphipathic molecular structure. For example, soy

Abstract: Monoammonium glycyrrhizinate is produced by the neutralization of glycyrrhizic acid from plant licorice with ammonia. In this study, the physicochemical properties of aqueous monoammonium glycyrrhizinate were investigated from the viewpoint of surface chemistry. The structure of the amphiphilic molecule is bola type, comprising two glucuronic acid moieties having two carboxylic acids groups and an aglycone part having a carboxylic acid at the opposite end of the molecule from the glucuronic acids. We found that the physicochemical properties of aqueous monoammonium glycyrrhizinate are dependent on the ionization of the carboxylic acid groups. The solubility of monoammonium glycyrrhizinate gradually increased above pH 4 in the buffer solution. The critical micelle concentration (CMC) and surface tension at the CMC (γCMC) of monoammonium glycyrrhizinate were determined by the surface tension method to be 1.5 mmol L–1 and 50 mN m–1 in pH 5 buffer and 3.7 mmol L–1 and 51 mN m–1 in pH 6 buffer, respectively. The surface tension gradually decreased with increasing concentration of monoammonium glycyrrhizinate in the pH 7 buffer, but the CMC was not defined by the curve. Light scattering measurements also did not reveal a clear CMC in the pH 7 buffer. The ionization of the carboxylic acid groups in the bola-type amphiphilic molecule with increasing pH is disadvantageous for micelle formation. Cryo-transmission electron microscopy showed that monoammonium glycyrrhizinate forms rod-like micelles in pH 5 buffer, and small angle X-ray scattering experiments confirmed that the average micellar structure was rod-like in pH 5 buffer. Thus, it was found that monoammonium glycyrrhizinate can form micelles only in weakly acidic aqueous solutions.

Key words: ammonium glycyrrhizinate, glycyrrhizic acid, aggregation, micelle, surface tension

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lecithin forms sub-micrometer of vesicles8）, whereas human sodium taurocholate forms small aggregates of dimer and trimer molecules in aqueous solution9, 10）.

We have investigated the aggregation behavior of glycyr-rhizic acid and glycyrrhetic acid 3-O-glucuronide11, 12）. As a result, we have reported that glycyrrhizic acid forms rod-shaped micelles only in weakly acidic aqueous buffer solu-tions11）, and glycyrrhetic acid 3-O-glucuronide forms spherical micelles in neutral aqueous solution12）. In this study, we investigated the micelle formation of ammonium glycyrrhizinate in several buffer solutions and clarified the differences by comparison with the acid form: glycyrrhizic acid.

2 Experimental and Procedure2.1 Materials

Monoammonium glycyrrhizinate（＞98％）was obtained from EXTRASYNTHESE. Pure water was obtained by ion-exchange treatment（PRA-0015, ORGANO）. Each buffer solution（pH 4–7）was prepared by the addition of the ap-propriate quantity of acetic acid（0.05 mol/L）or sodium hy-droxide（0.05 mol/L）to a neutral phosphate salt（0.05 mol/L）solution. The pH was measured with a pH meter（HM-25R, TOA DKK）. Crystals of monoammonium glycyrrhiz-inate were finally dissolved in each buffer solution with sonication at ca. 298.2 K.

2.2 Solubility of monoammonium glycyrrhizinate in the buffer solution

Excess crystalline monoammonium glycyrrhizinate was placed in 2–3 mL of each buffer solution（pH＝3.5, 4.0, 4.5, and 5.0）. The suspension was placed in a thermostat and stirred by a magnetic string bar at 298.2±0.3 K. Saturation was reached within 24 h. Then, filtration was performed using a membrane filter having a pore size of 0.65 μm（Du-rapore, DVPP01300）and a 10-mL glass injector. The mono-ammonium glycyrrhizinate solution has maximum ultravio-let absorbance at ca. 258–260 nm. The molar absorption coefficient was defined as 9.98×103 L mol－1 cm－1 at the maximum ultraviolet absorbance determined using a ultra-violet-visible spectrophotometer（JASCO V-630 iRM）. The solubility of monoammonium glycyrrhizinate was deter-mined from calculating the absorbance of the filtrate and the molar absorption coefficient. These operations were performed 3–5 times to determine the mean value.

2.3 Surface tension measurement The surface tension of the monoammonium glycyrrhiz-

inate solution was measured using the Wilhelmy plate technique and a surface tensiometer（DyneMaster DY-300YM, KYOWA）at 298.2±0.3 K. A relatively high concen-tration of monoammonium glycyrrhizinate was added step-

wise into the respective buffer solution at half-hour intervals. The critical micelle concentration（CMC）is defined as the point of intersection of the tangents to the two slopes of the surface tension curve.

2.4 Light scattering measurement for CMC determinationThe CMC of monoammonium glycyrrhizinate in each

buffer solution was determined by using a laser light scat-tering photometer（ALV-5000, Germany）at 298.2±0.3 K. The light source was a 200 mW Nd:YAG laser with a wave-length of 532 nm. The CMC determination is based on the phenomenon that the scattering intensity of the laser in the 90° direction increases with increase in micelle forma-tion. The monoammonium glycyrrhizinate solution was fil-tered through a membrane filter with a pore size of 0.8 μm（MILLEX）. The light scattering intensity was measured by adding the concentrated monoammonium glycyrrhizinate solution in each buffer solution.

2.5 Small angle X-ray scattering measurementsThe shapes of the aggregates of monoammonium glycyr-

rhizinate in aqueous solution were determined by small angle X-ray scattering（SAXS）at the BL40B2 beamline at SPring-8（Hyogo, Japan）. The wavelength（λ）of the X-rays was 0.7 Å. The monoammonium glycyrrhizinate solution was placed in a mark tube type of quartz cell（φ＝2 mm）. The sample-to-detector distance was fixed at 2 m. The ex-posure time was 3 min. The scattered X-rays were detected using a Rigaku image plate. The scattering data were ob-tained by subtracting the scattering from the buffer solvent to that from the sample scattering. The scattering profiles（I（Q））obtained within the q range of 0.01 to 5 nm－1, where q＝（4π/λ）sin（θ /2）and θ represents scattering angle.

2.6 Transmission electron microscopy（TEM）2.6.1 Cryo-TEM.

A small amount of monoammonium glycyrrhizinate solu-tion was placed on a copper grid covered by a porous carbon film. The excess liquid was removed by touching one end of the grid with a filter paper. The copper grid with sample was immersed into liquid ethane and placed in a freezing device（Leica, EM CPC）. The ice embedded sample on the grid was transferred into the specimen chamber of the TEM using a cryo-transfer system. The sample was cooled by liquid nitrogen during observation using a cryotransfer specimen holder（Gatan model 626）. The cryo-TEM observation was carried out on a Technai G2 20 operated at an acceleration voltage of 200 kV. The images of the aggregates were taken with a charge-coupled device（CCD）camera attached to the TEM.2.6.2 Negative staining method

A droplet of monoammonium glycyrrhizinate solution was placed on a copper grid whose surface covers the porous carbon film. The excess liquid was removed by

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touching one end of the grid with a filter paper. After the grid had partially dried, a drop of a staining solution（2％ uranyl acetate）was placed on the grid. The excess liquid was removed by filter paper and the grid was dried at room temperature. All micrographs were obtained using a Hitachi H-7500 operated at 80 kV.

3 Results and Discussion3.1 Aqueous solubility of monoammonium glycyrrhizinate

in the buffer solutionGlycyrrhizic acid is poorly soluble in aqueous solution at

low pH（＜pH 4）because the hydrophilic group in the mol-ecule has three carboxylic acid groups11）. In contrast, monoammonium glycyrrhizinate is expected to have rela-tively high solubility in water because some of the carbox-ylic acids form carboxylate ammonium salts. As shown in Fig. 1, glycyrrhizic acid has two carboxylic acid groups in the glucuronic acids and a carboxylic group on the top of molecule as a hydrophilic group.

For convenience, the three carboxylic acids are indicated by abbreviations A, B, and C, as shown in Fig. 1. According to 13C-NMR and infrared absorption results reported previ-ously, the ionization of the three carboxylic acids proceeds in the order of A, B→C13, 14）. In addition, X-ray crystal structure analysis of monoammonium glycyrrhizinate re-vealed that the ammonium ion is present close the A group15）. Therefore, the carboxylic acids in the couple of A and B bind to ammonium in a certain ratio in monoammo-nium glycyrrhizinate. The solubility of monoammonium glycyrrhizinate was measured at different pH values using different buffer solutions, as shown in Fig. 2（a）. The solu-bility in each pH buffer solution was determined using a

spectrophotometer, as described in Section 2.2. Figure 2（a）also shows the aqueous solubility of glycyrrhizic acid in each buffer solution at 298.2 K for comparison11）. The solu-bility of monoammonium glycyrrhizinate increased gradu-ally at pH 4.0 and then increased rapidly over pH 4.5. As expected, monoammonium glycyrrhizinate was dissolved more easily in the lower pH buffer solution than glycyrrhi-zic acid. Therefore, based on these results, the solvents used for further experiments had pH values greater than pH 5. In fact, Kvasnicka et al. reported that glycyrrhizic

Fig. 1　 Structure of monoammonium glycyrrhizinate. A, B, and C indicate the labeling scheme used for the three carboxylic groups.

Fig. 2　（a）Aqueous solubi l i ty of monoammonium glycyrrhizinate and glycyrrhizic acid in buffer solution at 298.2 K12）. （b）Neutralization titration curve and the change of maximum absorption of wavelength for monoammonium glycyrrhizinate solution（0.10 mmol L–1）on the addition of NaOH solution at 298.2 K.


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acid is easily soluble in alcohol, but, in the case of water, the solubility only increased over pH 4.516）. The result is consistent with the neutralization titration curve of aqueous monoammonium glycyrrhizinate（fixed to 0.1 mmol L－1）on the addition of NaOH solution（Fig. 2（b））. The titration curve gradually changed from pH 5 to 8 with increasing addition of NaOH. The moderate increase in the titration curve over pH 8 means that the hydrogen ions of the carboxylic acid group are completely dissociated. The effect of pH on the monoammonium glycyrrhizinate solu-tion was also confirmed from the change in the absorption spectrum of the molecule. In general, a change in the ab-sorption spectrum corresponds to a structural change or ionization of molecules. In fact, the maximum absorption wavelength of the aqueous solution also changed, as shown by the second axis of Fig. 2（b）. This result indicates that the ionization of the monomer molecules gradually pro-ceeds up to pH 8. Therefore, this phenomena indicates that the hydrophobic–hydrophilic balance for monoammonium glycyrrhizinate changes as ionization proceeds. Thus, when using monoammonium glycyrrhizinate, careful attention to the pH of the aqueous solution is required.

3.2 Surface properties and CMC of monoammonium glycyrrhizinate in buffer solutions

Based on the chemical structure in Fig. 1, monoammoni-um glycyrrhizinate is an amphiphilic molecule. Like surfac-tants, amphiphiles can adsorb at gas–liquid interfaces de-pending on the balance between the hydrophobic group and the hydrophilic group and may form micelles at a certain concentration. On the basis of X-ray structure anal-ysis, the molecular volume is several times as large as that of a normal linear surfactant molecule15）. In addition, monoammonium glycyrrhizinate has a non-flexible bulky aglycone moiety and the hydrophilic group is quite sepa-rated from the hydrophobic part（Fig. 1）. This relatively large molecular volume may be not advantageous for ad-sorption at gas–liquid interfaces. The surface tension of monoammonium glycyrrhizinate solution was measured by changing the pH of the buffer solution（pH＝5, 6, and 7）. Figure 3 shows the surface tension（γ）as a function of the logarithm of the concentration of monoammonium glycyr-rhizinate（pH＝5, 6, and 7）. The CMC was determined as the concentration where the two lines in the surface tension curve intersect. As shown in Fig. 3, the surface tension gradually decreased until the CMC, after which the surface tension value remained almost constant in the weakly acidic aqueous buffers（pH 5 and 6）. On the other hand, in the neutral aqueous solution（pH 7）, the surface tension decreased with increase in concentration, but there was no inflection point indicating a clear CMC. In ad-dition, no minimum was observed in the surface tension–concentration curves, which indicates an absence of sur-face-active impurities. On the basis of these results,

monoammonium glycyrrhizinate has a certain surface ac-tivity.

Next, the results were analyzed using Gibbs adsorption equation. The amount of adsorbed surfactant at the surface（Γ）can be calculated using Equation（1）.

Γ＝－（1/iRT）［dγ /（dln C）］（1）

Here, γ is the surface tension, R is the gas constant（8.314 J・mol－1・K－1）, T is the absolute temperature, C is the sur-factant concentration, and i is the number of molecular species17）. In the present system, i＝1 was used for mono-ammonium glycyrrhizinate solution because 50 mmol L－1 of the salt is included in the buffer solution18）. Subsequent-ly, the area（ACMC）of a surfactant molecule at the air–water interface was obtained from the saturation adsorption value at the CMC using Equation（2）.

ACMC＝1/（N ΓCMC）（2）

Here, N is Avogadro’s number. Table 1 lists the CMC, surface tension at the CMC（γCMC）, amount of adsorbed sur-factant（ΓCMC）, and area occupied by a surfactant molecule（ACMC）, as well as the other values relevant to typical sur-factants11, 12, 19－21）.

As shown in Table 1, the γCMC for monoammonium glyc-yrrhizinate solution in the pH 5 and 6 solutions were 50 and 51 mN/m, respectively, whereas, those for glycyrrhizic acid were 55 and 57 mN/m, respectively. The slight differ-ence in the surface tension is a result of the different coun-terion（ammonium cation or proton）. The results show that

Fig. 3　 Change in surface tension with increasing monoammonium glycyrrhizinate concentration in pH 5, 6, and 7 buffer solutions at 298.2 K.


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monoammonium glycyrrhizinate has higher surface activity than glycyrrhizic acid. In addition, glycyrrhetic acid 3-O-glucuronide, a similar compound, and the bio-surfactant sodium cholate have bulky aglycone and steroid structures, respectively. These compounds have also relatively high γCMC, values: 54 and 50 mN/m, respectively, as shown in Table 112, 19）. In contrast, the linear surfactants sodium do-decylsulfate（SDS）and sodium myristate can lower the surface tension to 40 and 25 mN/m, respectively, as shown in Table 120, 21）. These results indicate that, compared to linear surfactants, bulky surfactants are relatively disad-vantageous for reducing the surface tension. Similarly, the Γ values for monoammonium glycyrrhizinate are relatively lower than those of linear surfactants. In general, the degree of ionization of the carboxylic acid in hydrophilic group affects the amount of adsorption（ΓCMC）in the system of ionic surfactants. Badban et al. reported that nonanoic acid effectively adsorbed at the gas-liquid interface rather than nonanate with NaOH in pure water22）. The result means that ionization of carboxylic acid is not advanta-geous for adsorption（Γ CMC）at the surface. On the other hand, Γ CMC in the system of monoammonium glycyrrhiz-inate was increased with increasing pH（molecular ioniza-tion proceed）. The difference in the tendency seems to be derived from pure water and relative high-concentration buffered aqueous solution. The amount of adsorption is ex-pected to increase in a near neutrality buffered solution because the electrostatic repulsion decreases between molecules at the surface. ACMC was obtained using Equation（2）, where A indicates the area occupied by a single mole-cule at the gas–liquid interface. For monoammonium glyc-yrrhizinate, ACMC was 1.8 nm2 molecule－1 at pH 5, and this is larger than that at pH 6（ACMC: 1.0 nm2 molecule－1）. A larger value of ACMC represents ineffective adsorption. That is, the monomer molecules are finally used for micelle for-mation in the bulk rather than for adsorption at the inter-face in pH 5 solution. However, on increasing the solvent

pH to 6, the monoammonium glycyrrhizinate monomers move from bulk solution to the surface in the direction dis-advantageous for aggregation, resulting in an increase in Γ . Finally, no inflection point indicating micelle formation was found for the pH 7 monoammonium glycyrrhizinate solu-tion, as shown in Fig. 3. The increase in pH results in a stepwise ionization of monoammonium glycyrrhizinate, as shown in Fig. 2（b）. The influence of the ionization also affects the surface properties. Thus, the effect of the pH on micelle formation was evaluated from light scattering mea-surements. The intensity of the scattered light from the aqueous solution greatly increases with increase in micelle formation. As shown in Fig. 4, the scattered light intensity was measured in each aqueous buffer solution（pH 5, 6, and 7）as the concentration of monoammonium glycyrrhizinate was changed. The intensity of the scattered light from the aqueous solution greatly increases with increase in aggre-gate formation. In pH 5 and 6 aqueous buffer, the CMCs were 1.0 and 3.3, respectively（Table 1）, which are consis-tent with those determined by the surface tension mea-surement method. There was no inflection point in the plot of data obtained in the pH 7 solution over a wide concen-tration range, which indicates that monoammonium glycyr-rhizinate exists as a monomer at pH＞7. These results cor-respond to the surface tension results for monoammonium glycyrrhizinate solutions（Fig. 3）. As shown in Fig. 2（a）, monoammonium glycyrrhizinate is poorly soluble below pH 4.5. Therefore, the monoammonium glycyrrhizinate forms micelles within the weakly acid pH range of pH 5 to 6. On the other hand, with increasing pH, the hydrophilicity in-creases because of the dissociation of the carboxylic acid groups. As a result, complete ionization results in an in-crease in hydrophilicity, and the molecule changes to a bo-la-type surfactant having hydrophilic groups at both ends. This bola-type surfactant structure has a disadvantage in terms of micelle formation because the hydrophobic part exists between two hydrophilic parts23）. Thus, monoammo-

Table 1　 Surface tension properties and CMC for monoammonium glycyrrhizinate and typical surfactants at 298.2 K.

surfactant γCMC

(mN m–1)ΓCMC

(10–6 mol m–2)ACMC

(nm2 molecule–1)CMCa

(mM)CMCb

(mM)Monoammonium glycyrrhizinate (pH5) 50 0.95 1.8 1.5 1.0 Monoammonium glycyrrhizinate (pH6) 51 1.5 1.0 3.7 3.3 Glycyrrhizic acid (pH5)c 55 1.3 1.3 2.9 2.5Glycyrrhizic acid (pH6)c 57 2.9 0.57 5.3 5.0 Glycyrrhetic acid 3-O-glucuronide (pH7)d 54 2.2 0.77 1.6 2.0 Sodium cholatee 50 1.2 1.4 13.8 －Sodium dodecylsulfatef 40 3.2 0.52 8.2 －Sodium myristateg 25 4.0 0.41 2 －

aSurface tension method, bLight scattering method, cRef.11）, dRef.12）, eRef.19）, fRef.20）, and gRef.21）. mM: mmol L–1


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nium glycyrrhizinate cannot form micelles at pH＞7.

3.3 Aggregation of monoammonium glycyrrhizinate in buffer solutions

The molecular structure of an amphiphile affects the shape and size of the aggregates in aqueous solution. It has been reported that glycyrrhizic acid forms rod-like micelles in weakly acidic buffer solutions11）, and glycyrrhetic acid 3-O-glucuronide, which has one less glucuronic acid mole-cule than glycyrrhizic acid, forms large spherical mi-celles12）. In the present study, monoammonium glycyrrhiz-inate only contains a single ammonium ion associated with one of the carboxylic acid groups. Therefore, the micellar properties of monoammonium glycyrrhizinate may be very similar to those of glycyrrhizic acid in aqueous buffer.

Monoammonium glycyrrhizinate has a large molecular volume of hydrophobic groups, and the hydrophilic parts are separated at both ends of the molecule（Fig. 1）. This unique structure results in clearly different properties to those of linear alkyl surfactants. Images of the monoammo-nium glycyrrhizinate aggregates were obtained by TEM. The shape of the micelles of monoammonium glycyrrhiz-inate in pH 5 buffer（5 mmol L－1）was confirmed by Cryo-TEM. As shown in Fig. 5（a）, high-contrast images could not be obtained, but, from the outlines, it seems that the rod-like micelles or string micelles exist. Therefore, TEM observation was additionally performed on the aqueous so-lution of monoammonium glycyrrhizinate by the negative staining method. Figure 5（b）shows TEM images of 10

mmol L－1 of monoammonium glycyrrhizinate formed in pH 6 buffer solution, showing many rod-like micelles. Similar aggregates were seen with pH 5 buffer. In the TEM obser-vation, the aggregates appeared to overlap, so only a rough image of the aggregates could be captured. Therefore, SAXS was applied to the monoammonium glycyrrhizinate solutions to clarify the detailed structure of the aggregate.

The scattering intensity（I）for a monoammonium glycyr-rhizinate solution is expressed by the multiplication of the form factor（P（q））and structure factor（S（q））, as shown by Equation（3）.

I（q）＝n（Δρ）2V2P（q）S（q）（3）

Here, q is scattering vector, n is the number of particles per unit volume in solution, Δρ is the scattering contrast, and V is the volume of a single particle24）. P（q）is the aver-aged scattering profile of a single particle and is derived from each geometrical model. S（q）corresponds to the in-teractions between particles in the solution. If the solution system has only weak interactions between particles at rel-atively low concentrations, the scattering intensity is given

Fig. 4　 Change in the light scattering intensity with increasing monoammonium glycyrrhizinate concentration in pH 5, 6, and 7 buffer solutions at 298.2 K.

Fig. 5　（ a）C r y o - T E M i m a g e s o f 5 m m o l L – 1 o f monoammonium glycyrrhizinate in a pH 5 buffer solution. （b）TEM images of 10 mmol L–1 of monoammonium glycyrrhizinate in a pH 6 buffer solution using negative staining. Many rod-like aggregates can be seen in the micrograph（seen by outline or differences in contrast）.


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by the form factor25, 26）. The SAXS profiles for monoammonium glycyrrhizinate

in the buffer solutions are shown in Fig. 6 for pH 5 at 6 mmol L－1 and pH 6 at 8 mmol L－1. As shown in Fig. 6, the scattering intensities I（q）at both pH 5 and 6 gradually de-crease with increase in the scattering vector for 0.01 Å－1＜q＜0.1 Å－1. Further, the profiles obtained for pH 5 and 6 solutions showed I（q） ≈ q－1 in the low q-range（q＜0.05 Å－1）. Generally, a linear slope of -1 in the low angle region means that the aggregate is cylindrical（rod）. This is consistent with the appearance of the aggregates observed by TEM.

4 ConclusionMonoammonium glycyrrhizinate is obtained by replacing

one of the carboxylic acid groups of glycyrrhizic acid with an ammonium salt. Therefore, it has slightly higher water solubility than glycyrrhizic acid. Moreover, monoammoni-um glycyrrhizinate is a relatively inexpensive reagent com-pared to glycyrrhizic acid because of its easy extraction from licorice. In this study, the aqueous solutions of prop-erties for monoammonium glycyrrhizinate and glycyrrhizic acid were compared with respect to surface chemistry. Our analysis of the solution properties of monoammonium glyc-yrrhizinate revealed the lower surface tension and CMC than glycyrrhizic acid. Thus, monoammonium glycyrrhiz-inate has excellent surface activity. In addition, the micelles form rod-like aggregates, as does glycyrrhizic acid. There-fore, monoammonium glycyrrhizinate could be used as a

bio-surfactant. However, the pH of the solvent must be controlled because the micelle formation is strongly de-pended on the solvent pH. The monoammonium glycyrrhi-zinate should be used over pH 5 in the solution.

Acknowledgements This study was supported by JSPS KAKENHI Grant

Number 25460043. The SAXS experiments were performed at the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute（JASRI）. The proposal number for SAXS measurement was 2018A1688. The TEM work was performed at the Comprehensive Analysis Center for Science in Saitama University.

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Fig. 6　 SAXS profiles of monoammonium glycyrrhizinate in buffer solutions at pH 5（6 mmol L–1）and pH 6（8 mmol L–1）, respectively. The slopes in each figure were obtained from Guinier plots and the slope values are suggestive of rod-like aggregates.


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