Simultaneous determination of glycyrrhizin metabolites formed by the incubation of glycyrrhizin with rat feces by semi-micro high-performance liquid chromatography

Article abstract of DOI:10.1248/bpb.26.658

A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of 3α- hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid together with glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide formed by incubation of glycyrrhizin with rat feces. The analysis was accomplished within 25 min with a TSKgel ODS-80TsQA (150 x 2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml·min^-1, a thermostatic oven at 25°C, and detection at 254 nm. The detection limits of these compounds were 0.2 pmol per injection (5 μl). The metabolites of glycyrrhizin, by anaerobic or aerobic incubation with rat fecal suspension over 48 h, were determined. Glycyrrhizin was almost completely converted to metabolite glycyrrhetic acid, and metabolites 3α-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid in negligible amounts in anaerobic conditions. However, the metabolic time courses of 3-dehydroglycyrrhetic acid when incubated in aerobic conditions revealed that it apparently continued increasing during the whole incubation period.

Full text of DOI:10.1248/bpb.26.658

6
58
Biol. Pharm. Bull. 26(5) 658—661 (2003)
Vol. 26, No. 5
Simultaneous Determination of Glycyrrhizin Metabolites Formed by the
Incubation of Glycyrrhizin with Rat Feces by Semi-micro
High-Performance Liquid Chromatography
Nobuyuki OKAMURA,* Hidetoshi MIYAUCHI, Tominari CHOSHI, Takashi ISHIZU, and Akira YAGI
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; 1 Sanzo, Gakuen-cho, Fukuyama, Hiroshima
7
29–0292, Japan. Received December 5, 2002; accepted February 10, 2003
A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the si-
multaneous determination of 3a-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid together with gly-
cyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide formed by incubation of glycyrrhizin with
rat feces. The analysis was accomplished within 25 min with a TSKgel ODS-80TsQA (150؋2.0 mm i.d.) column
by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow
؊1
rate of 0.2 ml·min , a thermostatic oven at 25 °C, and detection at 254 nm. The detection limits of these com-
pounds were 0.2 pmol per injection (5 ml). The metabolites of glycyrrhizin, by anaerobic or aerobic incubation
with rat fecal suspension over 48 h, were determined. Glycyrrhizin was almost completely converted to metabo-
lite glycyrrhetic acid, and metabolites 3a-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid in negligible
amounts in anaerobic conditions. However, the metabolic time courses of 3-dehydroglycyrrhetic acid when incu-
bated in aerobic conditions revealed that it apparently continued increasing during the whole incubation period.
Key words 3a-hydroxyglycyrrhetic acid; 3-dehydroglycyrrhetic acid; glycyrrhizin; glycyrrhetic acid; semi-micro HPLC; rat
feces
1
4)
The root of Glycyrrhiza sp. (licorice root) is the most com- licorice root and peony root). However, no HPLC method
mon herb used in Kampo medicine. Glycyrrhizin is a involved in the determination of glycyrrhizin and its metabo-
saponin, a major effective ingredient of licorice root, which lites containing 3a-hydroxyglycyrrhetic acid and 3-dehydro-
1
)
2)
3)
has steroid-like, antiviral and interferon-inducing effects. glycyrrhetic acid was reported.
It has been used as a medicine in the treatment of chronic
The present study describes a simple, sensitive and repro-
hepatitis, gastric ulcers, inflammation and allergic disor- ducible semi-micro HPLC method for the simultaneous de-
4
)
5)
6)
7
)
der, and as a sweetener in many foods. However, gly- termination of glycyrrhizin, glycyrrhetic acid, glycyrrhetic
cyrrhizin has been reported to produce adverse effects, such acid mono-glucuronide, 3a-hydroxyglycyrrhetic acid and 3-
8
)
as pseudo-aldosteronism, when over consumed. Gly- dehydroglycyrrhetic acid (Fig. 1). This method was applica-
cyrrhizin is known to be hydrolyzed to glycyrrhetic acid, an ble to determine metabolites of glycyrrhizin by anaerobic
active metabolite and also a minor ingredient of licorice root, and aerobic incubations with rat fecal suspensions.
9
)
by human intestinal bacteria. Glycyrrhetic acid is responsi-
ble for the adverse reaction of pseudo-aldosteronism after
1
0)
glycyrrhizin intake.
The metabolic procession of gly-
cyrrhizin to glycyrrhetic acid by human intestinal bacteria
has been considered to follow two pathways: the main path-
way directly metabolizing glycyrrhizin to glycyrrhetic acid,
and the minor pathway metabolizing glycyrrhizin to gly-
1
1)
cyrrhetic acid via glycyrrhetic acid mono-glucuronide.
Moreover, glycyrrhetic acid is converted to 3a-hydroxygly-
cyrrhetic acid through 3-dehydroglycyrrhetic acid by human
1
2)
and rat intestinal bacteria. Glycyrrhizin is detected in the
plasma when glycyrrhizin is orally administered to rats, but
1
3)
its bioavailability is lower than that of glycyrrhetic acid.
Therefore, quantification of glycyrrhizin and its metabolites
is necessary to investigate the biotransformation of gly-
cyrrhizin. Although there have been many reports on the
high-performance liquid chromatography (HPLC) determi-
nation of glycyrrhizin or glycyrrhetic acid independently,
none of these methods were sufficiently sensitive for the si-
multaneous determination of glycyrrhizin and its metabo-
lites. We have been studying a biotransformation of gly-
cyrrhizin in Kampo medicine and developed a method for
semi-micro HPLC for the simultaneous determination of gly-
cyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glu-
curonide in incubation mixtures of rat fecal flora, by the ad-
dition of Shakuyaku-kanzo-to decoction (combination of Fig. 1. Structures of the Investigated Compounds
*
To whom correspondence should be addressed. e-mail: okamura@fupharm.fukuyama-u.ac.jp
© 2003 Pharmaceutical Society of Japan

May 2003
659
MATERIALS AND METHODS
of excess EtOH stopped the reaction. The reaction mixture
was filtered, the filtrate was evaporated in vacuo, and the
Materials Glycyrrhizin, glycyrrhetic acid and gly- residue was recrystallized from MeOH to afford 3-dehydro-
cyrrhetic acid mono-glucuronide were purchased from Wako glycyrrhetic acid as colorless needles (869 mg), which were
1
13
Pure Chemical Industries (Wako, Osaka, Japan). Gly- identified with H- and C-NMR spectroscopy.
cyrrhizin dipotassium salt was kindly provided by Maruzen
Synthesis of 3a-Hydroxyglycyrrhetic Acid A solution
Pharmaceuticals Co., Ltd. (Hiroshima, Japan). HPLC grade of 3-dehydroglycyrrhetic acid (300 mg) in dry tetrahydrofu-
acetonitrile and analytical reagent grade phosphoric acid ran (THF) (20 ml), in a nitrogen atmosphere, was cooled in a
(
85%) were purchased from Wako. Ultrapure distilled water dry ice-acetone bath (Ϫ74 °C), and a 1 M solution of K-selec-
with a resistivity greater than 18 MW was prepared with tride (15 ml, potassium tri-sec-butylborohydride, Aldrich
deionized-distilled water. The organic solvents and other Chemical Company, Milwaukee, WI, U.S.A.) was added with
chemical reagents were of analytical reagent grade, and were a syringe. After cooling at the same temperature for 2 h, the
supplied by Wako.
reaction mixture was left to stand overnight at room tempera-
Instrumentals The HPLC apparatus was an Agilent ture. The reaction was quenched with 1 M HCl to pH 2.0 in
Technologies 1100 Series system (Waldbronn, Germany) ice cooling conditions, and extracted with CHCl (50 ml).
3
consisting of a binary pump, an autosampler, a thermostated The extract was washed with brine, dried over anhydrous
1
7)
column compartment and a photodiode array detector. All MgSO , and evaporated in vacuo. The residue was recrys-
4
modules and data collection were controlled by Agilent tallized from MeOH to afford 3a-hydroxyglycyrrhetic acid
1
Chemstation software. The column was a TSKgel ODS- as colorless needles (176 mg), which were identified with H-
1
3
8
5
0TsQA reversed-phase column, particle size of the packing and C-NMR spectroscopy.
mm, 150ϫ2.0 mm i.d. (Tosoh, Tokyo, Japan).
Preparation of Calibration Curves An original stan-
Chromatographic Conditions The mobile phase was a dard solution was prepared by dissolving 3a-hydroxygly-
Ϫ1
gradient system with 200 ml·l (ca. 0.017%) phosphoric cyrrhetic acid (2.65 mg) and 3-dehydroglycyrrhetic acid
acid in water (A) and acetonitrile (B), which was deaerated (2.67 mg) in MeOH (2 ml), and diluted to make methanolic
Ϫ1
Ϫ1
by sonication before use. The flow rate was 0.2 ml·min . standard solutions (0.081—167 mg·ml ). Calibration curves
The separation was performed for 25 min from 38 to 50% were generated using ten different concentrations of standard
solvent B in 3 min, 50 to 52% solvent B in 7 min, 85 to 90% solutions from least-squares regression of peak area in tripli-
solvent B in 10 min, and 90% solvent B in 5 min. A re-equili- cate assays.
bration period of 12 min was used between individual runs.
The injection volume was 5 ml. The column temperature was hydroxyglycyrrhetic acid (1.35 mg) and 3-dehydroglycyr-
5 °C. The detection wavelength was set at 254 nm for deter- rhetic acid (1.85 mg) was dissolved in MeOH (100 ml) con-
mination and in the range of 200 to 500 nm for validation of taining 0.85% phosphoric acid, and diluted to 5- and 25-fold
peak purity. dilutions with the same solvent. The standard mixture was
Validation of HPLC Analysis A mixture of 3a-
2
Incubation of Glycyrrhizin with Rat Fecal Suspension prepared from a combination of the diluted solution (5.0 ml),
Fresh feces (1.0 g) obtained from male Wistar rats (200 g) fecal suspension (4.0 ml), as above prepared, and water
were homogenized in 0.1 M potassium phosphate buffer (1.0 ml). The within-day and between-day precision of the
(
20 ml, pH 7.4) by bubbling with CO gas to eliminate air, method for detection of 3a-hydroxyglycyrrhetic acid and 3-
2
and the sediments were removed by filtration through dehydroglycyrrhetic acid was evaluated by using the standard
1
5)
gauze. The fecal suspension prepared as above was divided mixture and the incubation mixture, as above prepared. All
into eleven aliquots (1 ml each). These aliquots were mixed mixtures were filtered through a Minisart-RC 15 filter and
with glycyrrhizin dipotassium salt (0.25 ml) in aqueous solu- subjected to HPLC. The within-day precision was examined
tion, and sorted into three groups: one control aliquot and with ten-replicate assays on the same day. The between-day
two incubation mixtures composed of five aliquots. The con- precision was assessed by analyzing a mixture stored at 4 °C
centration resulted in 84 mM of glycyrrhizin in each sample. on five different days over 2 weeks.
To the control mixture, 0.85% phosphoric acid in methanol
1.25 ml) was added immediately. Each incubation mixture droxyglycyrrhetic acid (10.8 mM) and 3-dehydroglycyrrhetic
was investigated at 37 °C for different incubation periods (2, acid (13.2 mM) with a mixture (diluent) of water, 0.1 M potas-
, 8, 24, 48 h) in anaerobic and aerobic conditions. Anaero- sium phosphate buffer (pH 7.4) and MeOH containing 0.85%
The standard solution was prepared by diluting 3a-hy-
(
4
bic procedures were accomplished by using anaerobic jar phosphoric acid (1 : 4 : 5), to 2-, 4-, 8-, 16- and 32-fold dilu-
with an AnaeroPack (Mitsubishi Gas Chemical). The reac- tions. The test solutions were prepared by the addition of the
tion was stopped after 2, 4, 8, 24 or 48 h incubation and incubation mixture or diluent (0.5 ml) to the standard solu-
mixed immediately with 0.85% phosphoric acid in methanol tion (0.5 ml). All test solutions were filtered through a Minis-
(
1
1.25 ml). Each mixture was then centrifuged (3000 rpm, art-RC 15 filter and subjected to HPLC. The recoveries were
0 min) and the supernatant was passed through a Sartorius determined from the slope ratios of regression equations,
membrane (Minisart RC 15, pore size: 0.45 mm, Japan Sarto- with and without the incubation mixture.
rius, Tokyo, Japan) and subjected to HPLC.
Synthesis of 3-Dehydroglycyrrhetic Acid The follow- RESULTS AND DISCUSSION
9
)
ing method was modified from a previous report. Gly-
cyrrhetic acid (1.0 g) was dissolved in acetone (100 ml), to
which 1.0 ml of Johnes reagent was added portionwise Dehydroglycyrrhetic Acid by HPLC (Fig. 2) In an at-
Determination of 3a-Hydroxyglycyrrhetic Acid and 3-
1
6)
while stirring at room temperature. After 20 min, the addition tempt to conduct biopharmaceutical studies on glycyrrhizin,

6
60
Vol. 26, No. 5
Table 1. Within-Day and Day-to-Day Relative Standard Deviations (RSD)
for 3a-Hydroxyglycyrrhetic Acid (1) and 3-Dehydroglycyrrhetic Acid (2) in
Standard and Incubation Mixtures
a)
b)
Within-day
Between-day
Marker substance
c)
c)
MeanϮS.D. RSD (%) MeanϮS.D. RSD (%)
Standard mixture
1
2.63Ϯ0.003
.54Ϯ0.003
3.55Ϯ0.002
.80Ϯ0.006
0.11
0.47
0.06
0.76
2.64Ϯ0.007
0.55Ϯ0.011
3.55Ϯ0.014
0.91Ϯ0.017
0.25
2.03
0.38
1.90
0
2
0
Incubation mixture
1
2
0.98Ϯ0.007
1.05Ϯ0.007
0.70
0.69
0.94Ϯ0.016
1.00Ϯ0.017
1.71
1.66
a) nϭ10, b) nϭ5, c) mM.
Table 2. Slopes of Regression Equations of 3a-Hydroxyglycyrrhetic Acid
1) and 3-Dehydroglycyrrhetic Acid (2) with and without Incubation Mix-
ture
(
a)
Slope of standard solution
Initial amount
of incubation
mixture (mM)
b)
Marker
substance
Recovery
(%)
With
incubation
mixture
Without
incubation
mixture
Fig. 2. HPLC Profiles of Standard (A), Glycyrrhizin Metabolites (B) and
Blank Incubation Mixtures (C)
1
2
0.609
0.793
9.045
10.70
8.754
10.21
103.3
104.7
For sample preparation and analytical conditions, see the Materials and Methods sec-
tion. Peaks: 1ϭglycyrrhizin, 2ϭglycyrrhetic acid mono-glucuronide, 3ϭglycyrrhetic
acid, 4ϭ3a-hydroxyglycyrrhetic acid, 5ϭ3-dehydroglycyrrhetic acid.
a) Concentration of standard (mM): 1ϭ0.170, 0.341, 0.681, 1.36, 2.73, 5.45;
ϭ0.207, 0.413, 0.826, 1.65, 3.31, 6.61. b) Values are means of three experiments.
2
we have already reported the method for determination of
glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono- acid were 0.2 pmol per injection (5 ml). 3a-Hydroxygly-
glucuronide by reversed-phase HPLC using an ODS semi- cyrrhetic acid and 3-dehydroglycyrrhetic acid were clearly
1
4)
micro column. This method is applicable to the determina- separated without any pre-purification and determined within
tion of these compounds in an incubation mixture of rat fecal 25 min, without any pre-cleaning of the column (Fig. 2A).
flora by the addition of Shakuyaku-kanzo-to decoction. Gly- Glycyrrhizin was anaerobically incubated with fecal suspen-
cyrrhetic acid, the main metabolite of glycyrrhizin, was fur- sions obtained from fresh rat feces for 4 h. As shown in Fig.
ther converted to 3a-hydroxyglycyrrhetic acid through 3-de- 2B, adequate separation of 3a-hydroxyglycyrrhetic acid and
hydroglycyrrhetic acid by human and rat intestinal bacte- 3-dehydroglycyrrhetic acid was achieved. Distinct elution
1
2)
ria. Conventionally, these metabolites have been examined profiles of these compounds were obtained without any inter-
by thin-layer chromatography, and no HPLC method in- ference from the endogenous peak of the blank incubation
volved in the determination of glycyrrhizin and its metabo- mixture (without glycyrrhizin) (Fig. 2C). The identification
lites containing 3a-hydroxyglycyrrhetic acid and 3-dehydro- and purity of the chromatographic peaks were estimated by a
glycyrrhetic acid was reported. However, the simultaneous photodiode array detector. The UV spectra of the peaks at
determination of 3a-hydroxyglycyrrhetic acid and 3-dehy- five different points (up-, top- and down-slope) were com-
droglycyrrhetic acid, together with glycyrrhizin, glycyrrhetic pared with those of synthetic specimens.
acid and glycyrrhetic acid mono-glucuronide, in rat fecal
suspension, was adopted at the present HPLC method in and 3-Dehydroglycyrrhetic Acid in the Incubation Mix-
which the monitoring time was extended up to 25 min under ture (Tables 1, 2) The within-day and between-day preci-
Validation of HPLC of 3a-Hydroxyglycyrrhetic Acid
the final gradient conditions.
sion of the method for 3a-hydroxyglycyrrhetic acid and 3-
The retention times (and capacity factors, kЈ) were 19.93 dehydroglycyrrhetic acid was evaluated by using the standard
3a-hydroxyglycyrrhetic acid, kЈϭ13.90) and 20.35 min (3- mixture and the incubation mixture. The within-day preci-
(
dehydroglycyrrhetic acid, kЈϭ14.21). The relative standard sion was examined with ten-replicate assays per day, and the
deviations (RSD, nϭ10) of the retention times were 0.03 and between-day precision by assays on five different days over
0
.02%. Linearity of these compounds was evaluated over the the 2 weeks. As shown in Table 1, the within-day and be-
range of 0.172 to 44.0 mM for 3a-hydroxyglycyrrhetic acid, tween-day RSDs were within 0.06 to 0.76% and 0.25 to
and 0.174 to 356 mM for 3-dehydroglycyrrhetic acid. The re- 2.03%, respectively. These compounds were shown to be sta-
gression equations were yϭ8.82xϪ0.26 for 3a-hydroxygly- ble in both the standard and the incubation mixtures for over
cyrrhetic acid, and yϭ10.26xϪ0.46 for 3-dehydrogly- 2 weeks at 4 °C by the between-day assay. Recovery was in-
cyrrhetic acid, with a correlation coefficient of 1.0000, where vestigated using the incubation mixture spiked with the stan-
y is the peak-area and x is the concentration (mM) of these dard solution. The slopes of the regression equations of the
compounds. Both detection limits (signal-to-noise ratioϭ3) standards and the recoveries by this method are shown in
of 3a-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic Table 2. The recoveries were 103.3% for 3a-hydroxygly-

May 2003
661
acid may be explained in terms of the acceleration by aerobic
condition.
Oral administration of Kampo medicines in which glyco-
side ingredients are involved, usually provides the corre-
sponding aglycone after hydrolysis by intestinal bacteria. In
fact, glycyrrhizin administered orally is absorbed in the form
of glycyrrhetic acid and shows various pharmacological ef-
1
9)
fects as a natural prodrug. Glycyrrhetic acid is further
transformed to 3a-hydroxyglycyrrhetic acid via a metabolic
intermediate, 3-dehydroglycyrrhetic acid. Since the method
described in this report is sufficiently sensitive and reliable to
be used for the simultaneous determination of glycyrrhizin
and its metabolites, it can be applied for the clinical evalua-
tion of glycyrrhizin in regards to the efficacy and safety of
Kampo and herbal medicines containing licorice root.
Acknowledgements The authors are grateful to
Maruzen Pharmaceuticals Co., Ltd. for supplying us with
glycyrrhizin dipotassium salt. We also thank Miss Nami
Kataoka, Miss Mayuko Kishimoto and Miss Chiharu Shi-
mada for technical assistance.
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(
Fig. 3. Metabolism of Glycyrrhizin by Rat Fecal Suspension in Anaerobic
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(
For sample preparation, see the Materials and Methods section. ᭿: glycyrrhizin, ᭹:
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1
1
(
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1
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3
mainly to glycyrrhetic acid, and negligibly to glycyrrhetic
acid mono-glucuronide, 3a-hydroxyglycyrrhetic acid and 3-
dehydroglycyrrhetic acid, by anaerobic incubation for 48 h.
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and 3-dehydroglycyrrhetic acid in approximate ratios of 1 : 1
(
1988).
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increased during the early phase, but gradually declined
thereafter, whereas 3-dehydroglycyrrhetic acid apparently
continued increasing during the whole incubation period.
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1