Clove Oil Prevents Glycyrrhizin Gel Formation in Aqueous Solution
Kenjiro KOGA,*,a Kumiko TAKEKOSHI,

a Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokuriku University; Ho-3, Kanagawa-machi,
Kanazawa 920–1181, Japan: and bKyoto Research Laboratory, Amato Pharmaceutical Products; 134 Minami-machi,
Chudoji, Shimogyo-ku, Kyoto 600–8813, Japan.
Received July 30, 2004; accepted September 4, 2004; published online September 14, 2004

One persistent problem with using therapeutic concentrations of glycyrrhizin (GZ) is that, at these high
concentrations, it forms a gel in an aqueous solution. We previously solved this problem by dissolving GZ in a
highly concentrated phosphate buffer. Unfortunately, the resulting GZ solution has a hyperosmotic pressure that
renders it unsuitable for use in patients. The aim of this study was to prepare a highly concentrated GZ solution
having an osmotic pressure ratio of 1 and a pH of 7.4. By adding small amounts of oil and using a 100 mM phosphate buffer, we achieved an emulsified GZ solution that is stable at room temperature and has a physiological
osmotic pressure and pH. When clove oil was used as an emulsifier, the gel formation temperature of GZ solution
decreased appreciably compared to that of GZ solution without clove oil. Using scanning electron microscopy
(SEM), we examined the detailed characteristics of GZ gels prepared from solutions with or without clove oil.
SEM of cross sections of GZ gels revealed an irregular structure in gels prepared with clove oil, indicating that
clove oil prevented the formation of the intermolecular GZ networks typically characterized by gels derived from
pure GZ solutions.
Key words glycyrrhizin; gel; clove oil; fennel oil; mentha oil; scanning electron microscopy (SEM)

　

Glycyrrhizin (GZ), a major component of licorice (Glycyrrhiza glabra L.), is used as a remedy for chronic hepatitis,
allergies, and other maladies. Because GZ is poorly absorbed by the intestinal tract and readily forms gels, the only way it can be therapeutically delivered is by parenteral, low-dose injection. Indeed, prescription grade GZ is currently available in only a 2 mg/ml solution. To obtain a sufficient therapeutic dose to treat chronic hepatitis, however, a patient must receive 100 mg GZ per day or 50 ml of a 2 mg/ml solution per day.1,2) We prelycyrrhizin (GZ), a major component of licorice (Glycyrrhiza glabra L.), is used as a remedy for chronic hepatitis, allergies, and other maladies. Because GZ is poorly absorbed by the intestinal tract and readily forms gels, the only way it can be therapeutically delivered is by parenteral, low-dose injection. Indeed, prescription grade GZ is currently available in only a 2 mg/ml solution. To obtain a sufficient therapeutic dose to treat chronic hepatitis, however, a patient must receive 100 mg GZ per day or 50 ml of a 2 mg/ml solution per day.1,2) We previously showed that GZ aqueous solutions prepared in 300 mM phosphate buffer (pH 7—8) did not form gels at room temperature.3) In fact, with this procedure, we achieved a 200 mg/ml solution of GZ. However, this came at a high cost. The phosphate salt concentration required to prepare such a highly concentrated GZ solution causes it to have a high osmotic pressure, making it difficult to deliver via injection. The purpose of this study was to prepare a 100 mg/ml GZ solution that can be used under physiological conditions. Thus, we aimed to prepare a GZ solution having an osmotic pressure ratio of 1 and a pH of 7.4. Experimental Chemicals GZ (purity 98%) and Glycyrrhetic acid were gifts from Koki Co. (Tokyo, Japan) and Maruzen Pharmaceutical Co. (Hiroshima, Japan), respectively. Soybean oil, olive oil, fennel oil, and clove oil were purchased from Wako Pure Chemical Industries (Osaka, Japan). Mentha oil was purchased from Hoei Pharmaceutical Co. (Osaka, Japan). All other materials were reagent grade. Osmolarity Measurement The osmolarity of phosphate buffered solutions (50—300 mM) and GZ solutions (1—100 mg/ml) was measured using a Freezing point osmometer (model OM-802, VOGEL GmbH, Germany). Preparation of GZ Solution After 1.0 g GZ was added to a screw vial containing 5 ml of phosphate buffer and 0—150 ml of oil, the samples were stirred for 16 h at 100 rpm at 60 °C on a Hotplate stirrer (model DP-1L, Asone Co., Osaka, Japan). Any oil drops that separated from the GZ aqueous solution were carefully removed using a micropipette. Measurement of Gel Formation Temperature After preparing the GZ solution, we placed all samples into a 60 °C water bath equipped with a Cool-pipe (model 80LF, Taitec, Tokyo, Japan), and gradually decreased theviously showed that GZ aqueous solutions prepared in 300 mM phosphate buffer (pH 7—8) did not form gels at room temperature.3) In fact, with this procedure,we achieved a 200 mg/ml solution of GZ. However, this came at a high cost. The phosphate salt concentration required to prepare such a highly concentrated GZ solution causes it to have a high osmotic pressure, making it difficult to deliver via injection. The purpose of this study was to prepare a 100 mg/ml GZ solution that can be used under physiological conditions. Thus, we aimed to prepare a GZ solution having an osmotic pressure ratio of 1 and a pH of 7.4.

Experimental
Chemicals GZ (purity 98%) and Glycyrrhetic acid were gifts from Koki Co. (Tokyo, Japan) and Maruzen Pharmaceutical Co. (Hiroshima,Japan), respectively. Soybean oil, olive oil, fennel oil, and clove oil were purchased from Wako Pure Chemical Industries (Osaka, Japan). Mentha oil was purchased from Hoei Pharmaceutical Co. (Osaka, Japan). All other materials were reagent grade.

Osmolarity Measurement

The osmolarity of phosphate buffered solutions (50—300 mM) and GZ solutions (1—100 mg/ml) was measured using a
Freezing point osmometer (model OM-802, VOGEL GmbH, Germany).

Preparation of GZ Solution

After 1.0 g GZ was added to a screw vial containing 5 ml of phosphate buffer and 0—150 ml of oil, the samples were
stirred for 16 h at 100 rpm at 60 °C on a Hotplate stirrer (model DP-1L, Asone Co., Osaka, Japan). Any oil drops that separated from the GZ aqueous solution were carefully removed using a micropipette.Measurement of Gel Formation Temperature After preparing the GZ solution, we placed all samples into a 60 °C water bath equipped with a Cool-pipe (model 80LF, Taitec, Tokyo, Japan), and gradually decreased the water bath temperature at a rate of 1 °C every 10 min (range: 60 °C—3 °C). We determined the starting point of gel formation in each sample by tilting the vial and noting the temperature at which a thin layer of gel formed on the boundary surface of the tube.

HPLC Assay of GZ

We determined GZ concentration according to an HPLC method previously described.4) Briefly, a Nucleosil 5C18 column
(inner diameter: 4.6 mm; length: 250 mm) was used and maintained at a temperature of 40 °C during separation. Detection was performed at a UV wavelength of 254 nm. The solvent used for the mobile phase was comprised of
methanol, distilled water, 25% ammonia solution, and 60% perchloric acid(53 : 47 : 0.5 : 0.5, v/v); flow rate was 0.8 ml/min. The correlation coefficients of the calibration curves were 0.999 or better; the detection limit was
0.2 mg/ml.

Particle Size Assay

We measured the average particle size of GZ
micelles in GZ solutions prepared in the absence or presence of clove oil using a Laser particle analyzer (model DLS-900, Otsuka Electronics Co.,Osaka, Japan). Prior to analysis, the samples were filtered with a Millipore
membrane filter (0.45 mm pore size). Accumulation times were 100, the incidence angle of the laser beam was set to 30°, and the average particle diameter was evaluated as a Z-average using a monomodal method (a cumulant
analysis)5,6) at 25 °C.Scanning Electron Microscopy (SEM) in GZ Gel Sections GZ solutions (100 mg/ml) were prepared in the absence and presence of 2% clove oil, then incubated for 16 h at 60 °C. After incubation, excess clove oil drops were removed. The solutions were transferred into chloroethylene tubes (2 mm inside diameter), which were briefly immersed in liquid nitrogen to quickly freeze the samples. The samples were subsequently freeze-dried at
20 °C and 26 Pa by touching them to cooled metal to avoid disrupting gel formation. The gel specimens were sectioned and prepared for SEM analysis.

Results
Osmolarity The osmolarity of the phosphate buffered solution (pH 7.4) increased linearly with increasing concentrations of phosphate buffer (Fig. 1A). To determine the osmolarity of GZ (1—100 mg/ml), we used GZ solutions
prepared in 200 mM phosphate buffer, because a 100 mg/ml GZ solution immediately forms a gel when dissolved in
100 mM phosphate buffer. The osmolarity increased exponentially with increasing concentrations of GZ (Fig. 1B).
Temperature of GZ Gel Formation We measured the gel formation temperature of GZ solutions containing different kinds of oil (Table 1). Unadulterated GZ solution (control) formed a gel at 32 °C. GZ solutions containing either
olive oil or soybean oil formed gels at the same temperature as that of the control solution. On the other hand, the gel formation temperature of GZ solutions containing mentha oil,fennel oil, or clove oil decreased with increasing oil content.The decrease in gel formation temperature related to the addition of clove oil was characteristic of several different essential oils. In fact, the gel formation temperatures of GZ solutions containing 2% and 3% clove oil were significantly lower (4 °C and 3 °C, respectively) than the control. Interestingly, GZ solutions containing clove oil turned a light dark-brown, even though very little separated oil remained.
　

To better understand the structural features of GZ gel formation, we examined whether glycyrrhetic acid, a hydrolytic metabolite of GZ, forms a gel in aqueous solution. Glycyrrhetic acid (100 mg/ml) failed to form a gel.

GZ Stability

We were interested in determining whether the GZ-clove oil solution was a true emulsion. To rule out the
possibility that GZ and clove oil chemically react with each other in aqueous solution, we measured the concentration of GZ in a 100 mg/ml GZ solution containing 3% clove oil.

Measurements were determined using an HPLC.

The GZ concentration was 99.9 2.4 mg/ml, suggesting that the GZclove oil solution was an emulsion.
GZ Micelle Particle Size To verify that GZ and clove oil form an emulsion, we used a Laser particle analyzer to
measure the micelle particle size of control and GZ solutions containing clove oil. In control solutions (without clove oil), we were unable to measure the average diameter of GZ micelles, suggesting that their diameters were less than 3 nm,the detection threshold of the DLS-900. On the other hand, the average particle diameter of GZ micelles in solutions containing 3% clove oil was 44.1 nm.

SEM of GZ Gel

To clarify why clove oil prevented GZgel formation in aqueous solution, we examined with SEM cross sections of freeze-dried GZ solutions prepared with or without clove oil. The structure of specimens of GZ prepared without clove oil had lamellar cavities (Figs. 2A, B), while those of GZ solutions with 2% clove oil did not have these
cavities (Figs. 2C, D). This suggests that clove oil alters the structure of GZ solutions, lowering the temperature at which GZ gels form.

Discussion
Highly concentrated GZ rapidly forms gels in aqueous solution. To our knowledge, no reports exist that describe the
formation of GZ gel in aqueous solution. GZ is a triterpenoid saponin with two glucuronic acids. Interestingly, glycyrrhetic acid, a GZ-derived compound lacking two glucuronic acids, fails to form gels in aqueous solution. Thus, the presence of the two glucuronic acids may be an important structural characteristic of GZ that underlies its ability to form gels in aqueous solution. Indeed, recent findings indicate that sugars may promote gelation of certain compounds in aqueous solutions.7,8) Generally, most gelatinizing agents are polymers, and very few low molecular weight gelatinizers exist. However, recently developed low molecular weight organic compounds containing sugar chains have proven to be effective gelatinizers,7,8) suggesting that one or more sugar chains are important for gel formation in aqueous solutions. These findings motivated us to find a way to inhibit GZ gel formation
by altering the osmolarity of GZ solutions, with the aim of developing a concentrated GZ solution for therapeutic use. First, to prepare a highly concentrated GZ solution, we systematically varied the diluent phosphate buffer concentration until we could obtain a 100 mg/ml GZ solution. To obtain an isoosmotic 100 mg/ml GZ solution, we calculated that a phosphate concentration of 100 mM was necessary.After preparing a 100 mg/ml GZ solution in 100 mM phosphate buffer, we determined the osmolarity of this concentrated GZ solution to be 275 mOsm, with an osmotic pressure ratio of approximately 1 (physiological osmotic pressure 285 mOsm). This confirmed that a 100 mM phosphate
buffered solution was needed to prepare an isoosmotic 100 mg/ml GZ solution.

Second, we sought to inhibit gelation of this 100 mg/ml GZ solution by systematically adding small amounts of various oils to emulsify the GZ solution. The ability of these oils to prevent gelation of concentrated GZ was assessed by examining the temperature at which the GZ-oil solutions form gels. We found that clove oil remarkably decreased the gel formation temperature of GZ. Because about one-third of the initial quantity of clove oil separated from the GZ solution after 16 h of incubation at 60 °C, it is possible that the remaining clove oil may have formed an emulsion with GZ or may have chemically reacted with GZ to form a water soluble reaction product. The former scenario seems reasonable because GZ has been demonstrated to be surface active and water soluble.9) Because of these characteristics, GZ has been used to increase the solubility of drugs that have poor water solubility.10,11) If clove oil inhibits GZ gelation by forming a water soluble GZ-clove oil reaction product, then one would expect the GZ content in the aqueous solution to decrease in the presence of clove oil. HPLC analysis of GZ solutions with and without 3% clove oil revealed no differences in the GZ concentration of these two solutions, suggesting that clove oil does not react with GZ in the aqueous solution to form a water soluble reaction product. To verify that clove oil and GZ formed an emulsion by means of the surface activity of GZ, we measured the mean particle size of GZ-oil micelles.

Although laser particle size analysis failed to measure particles in GZ solutions lacking clove oil, this analysis positively identified the presence of particles in the GZ-clove oil emulsion, confirming that clove oil was indeed emulsified in the GZ solution. As part of our analyses, we also sought to determine whether the absence or presence of clove oil affected the crystalline structure of GZ gels. This was accomplished by examining freeze-dried GZ sections by SEM. It is difficult to accurately assess the molecular structure of gels derived from aqueous solutions because of the ice crystals that form during the freeze-drying process can obscure the true structure of the gel. To avoid this pitfall, ice crystal inhibitors such as glycerol are usually added to the solution. However, we avoided the addition of ice crystal inhibitors to the GZ sample, because their addition may affect GZ gel structure.

For our SEM analyses, therefore, we postulated that the formation of ice crystals may be closely related to the intermolecular network that underlies GZ gel formation. SEM examination of GZ gels revealed that GZ solutions
form gels comprised of a network of regularly spaced GZ molecules arranged as lamellar cavities. In contrast, GZ gels derived from GZ-clove oil solutions formed gels comprised of a network of irregularly spaced GZ molecules; lamellar cavities were absent. These observations strongly indicate that the addition of clove oil to the GZ aqueous solution blocked the formation of the intermolecular networks of lamellar cavities that generally occurs during the normal gelforming process. Although we could not definitively decipher the mechanism by which clove oil alters these networks, we proposed the following sequence of events. First, clove oil is either dissolved or dispersed in GZ solution by means of the surface active action of GZ. Second, clove oil suppresses GZ
gelation by forming hydrogen bonds with the glucuronic acids of GZ.

Because individual clove oil droplets were too small to resolve with SEM, it was not possible to show that clove oil
exists as an emulsion with GZ and that it inhibits GZ gelation. However, when taken together, our data lead us to conclude that clove oil inhibits GZ gel formation by collapsing the intermolecular network of GZ.

Conclusions
We have developed a new procedure for preparing concentrated GZ solutions that can be used under physiological conditions. These solutions have an osmotic pressure ratio of 1 and a pH of 7.4. Addition of clove oil to concentrated GZ aqueous solutions effectively inhibits the formation of GZ gels by preventing the expansion of intermolecular networks of GZ molecules. The prevention of GZ gelation by clove oil

Chem. Pharm. Bull. 52(12) 1507-1510 (2004)
https://www.jstage.jst.go.jp/article/cpb/52/12/52_12_1507/_pdf

　

　

Investigations into the structure-function relationship of plant-based surfactant glycyrrhizin: Interfacial behavior & emulsion formation

a
Department of Food Physics and Meat Science, University of Hohenheim, Garbenstrasse 25, 70599, Stuttgart, Germany
b
Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich, Lise-Meitner-Strasse 34, 85354, Freising, Germany
Received 5 August 2019, Revised 28 November 2019, Accepted 30 November 2019, Available online 2 December 2019.

Highlights
•
Licorice root glycyrrhizin and its aglycone showed low surface-activity.

•
Viscoelasticity of formed interface was absent for both molecules.

•
Both molecules formed nano-sized oil-in-water emulsions efficiently.

•
Molecular computing showed differences in hydrophobicity between the molecules.


Abstract
This study describes the interfacial and emulsifying properties of a plant-based saponin glycyrrhizin and its aglycone 18β-glycyrrhetinic acid derived from licorice root (Glycyrrhiza glabra L.) to enable first insights into a potential structure-function relationship. First, the surface activity of glycyrrhizin and its aglycone at air-water and oil-water interfaces, and the viscoelastic behavior at an oil-water interface was examined. Second, the emulsifying properties by preparing oil-in-water emulsions (100 g oil/kg emulsion, pH 7) by high-pressure homogenization was evaluated. Both glycyrrhizin and its aglycone reduced surface and interfacial activity only <19%, and did not form any viscoelastic interfaces. Nevertheless, glycyrrhizin and its aglycone formed stable oil-in-water emulsions around 0.2 μm at a very low molecule-to-oil ratio. Therefore, the low interfacial activity or interfacial viscoelasticity does not necessarily correlate with the emulsifying capability. Furthermore, molecular modelling of the compounds revealed different hydrophilic-lipophilic balance -values ranging from 1 to 10, and that hydrophobic surfaces dominated over the polar surfaces, indicating that hydrophobic interactions contribute to the stabilizing forces at the interface. The results are relevant for novel ‘all-natural’ emulsion-based foods, beverages, as well as pharmaceuticals, and cosmetics to enable a potential structure-function relationship for the promising surfactant group of saponins.

植物基表面活性剂甘草酸的结构-功能关系研究:界面行为与乳液形成

Author links open overlay panelTheoRallaaHannaSalminenaKatharinaBraunaMatthiasEdelmannbCorinnaDawidbThomasHofmannbJochenWeissa

一个

霍恩海姆大学食品物理和肉类科学系，加本斯特拉塞25,70599，德国斯图加特

b

慕尼黑工业大学食品化学和分子感觉科学讲座教授，德国弗雷辛市利斯-迈特纳大街34,85354

2019年8月5日收稿，2019年11月28日修订，2019年11月30日接受，2019年12月2日在线提供。



突出了

• 甘草甘草酸及其苷元的表面活性较低。

• 两种分子的形成界面都没有粘弹性。

• 两种分子都能有效地形成纳米水包油乳液。

• 分子计算显示了分子间的疏水性差异。


摘要

本研究描述了从甘草根(glycyrhiza glabra L.)中提取的一种植物皂苷甘草酸及其苷元18β-甘草次酸的界面和乳化特性，使人们能够首次了解其潜在的结构-功能关系。首先，考察了甘草酸及其苷元在空气-水和油水界面上的表面活性以及在油水界面上的粘弹性行为。其次，通过高压均质法制备油包水乳液(100 g油/kg乳液，pH 7)，考察其乳化性能。甘草酸及其苷元的表面和界面活性均低于19%，且未形成粘弹性界面。然而，甘草酸及其苷元在非常低的分子油比下，在0.2μm左右形成了稳定的水包油乳液。因此，低的界面活性或界面粘弹性并不一定与乳化能力有关。此外，化合物的分子模型显示了不同的亲水亲脂平衡值从1到10，疏水表面主导极性表面，表明疏水相互作用有助于在界面的稳定力。这一结果对于新型的“纯天然”乳化剂食品、饮料、药品和化妆品具有重要意义，它使皂素表面活性剂基团具有潜在的结构-功能关系。

Investigations into the structure-function relationship of plant-based surfactant glycyrrhizin: Interfacial behavior & emulsion formation - ScienceDirect
https://www.sciencedirect.com/science/article/abs/pii/S0023643819312526