Food Chemistry 141 (2013) 1681–1689

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Comparison of three officinal Chinese pharmacopoeia species

of Glycyrrhiza based on separation and quantification of triterpene
saponins and chemometrics analysis
Weiwei Tao a, Jinao Duan a,⇑, Runhuai Zhao b, Xueyu Li c, Hui Yan a, Jianping Li a, Sheng Guo a,
Nianyun Yang a, Yuping Tang a
a
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210046, China
b
Technology Development Center, China National Group Corp. of Traditional & Herbal Medicine, Beijing 102600, China
c
Institute of Licorice, Shihezi University, Shihezi 832003, China

a r t i c l e i n f o a b s t r a c t

Article history: The dried roots and rhizomes of Glycyrrhiza species, named licorice, have been utilized as food as well as
Received 2 February 2013 crude drugs in China for thousands of years. Glycyrrhiza species can be differentiated based on the mor-
Received in revised form 27 March 2013 phologic features of their aerial part, i.e. leaf and fruit morphologies, but not on their root morphology,
Accepted 16 May 2013
even though that is the medicinal part. In this paper, a fast and effective UPLC–TQ-MS/MS method was
Available online 24 May 2013
explored for better identification and quantitative investigation of triterpene saponins in licorice, which
laid basis for chemical comparison of three officinal Chinese pharmacopoeia Glycyrrhiza species. The
Keywords:
results showed that all of these licorice samples were rich in triterpene saponins but with significant dif-
Glycyrrhiza inflata
Glycyrrhiza glabra
ference corresponding to different Glycyrrhiza species. The proposed method could be useful in quality
Glycyrrhiza uralensis control and standardization of licorice raw materials and its products.
Licorice Ó 2013 Elsevier Ltd. All rights reserved.
Triterpene saponins
UPLC–TQ-MS/MS

1. Introduction effects, such as antioxidant (Lee et al., 2008; Oganesyan, 2002),

Licorice, the dried roots and rhizomes of Glycyrrhiza species
(Leguminosae family), represents an important agricultural com-
modity (Chin et al., 2007). The name Glycyrrhiza originates from
the Greek words ‘‘glykos rhiza’’, which mean ‘‘sweet root’’ (Hayashi
& Sudo, 2009). Licorice has been used as a sweetener and a flavour
enhancer for foods in China and other countries (for example, it is
approved by Food and Drug Administration as a food additive, re-
garded with the ‘‘GRAS’’ label; it is registered as CFR 184.1408 in
the USA) (Liu, Sugimoto, Akiyama, & Maitani, 2000; Liut, Akiyama,
Sugimoto, & Maitani, 2001). It is also considered one of the oldest
and most widely used herbal drugs in both Eastern and Western
countries. The Glycyrrhiza genus consists of about 30 species, and
is widely distributed all over the world. In China, three species
including G. uralensis, G. glabra and G. inflata are officially used as
licorice and recorded in Chinese Pharmacopoeia. Among them, G.
uralensis is the mainstream species, which constitutes more than
90% of total licorice production (Zhang & Ye, 2009). Biological stud-
ies have demonstrated that licorice has a variety of biological
⇑ Corresponding author. Tel./fax: +86 25 8581 1116.
E-mail address: dja@njutcm.edu.cn (J. Duan).
antiviral (Fiore et al., 2008), antidepressant (Dhingra & Sharma,
2006), anti-inflammatory (Kim et al., 2006), anti-carcinogenesis
(Wang, 1994), and hepatoprotective bioactivities (Lee et al., 2009).
Phytochemical studies have revealed that licorice contains two
major types of secondary metabolite, including triterpene saponins
and flavonoids. Among them, triterpene saponins are responsible
for the sweet taste, which are used as natural sweetners due to
their unique properties such as higher solubility and a long-lasting
sweetness (Liu et al., 2000). In recent years, licorice and major sap-
onins have been used as a botanical dietary supplement ingredient
in products marketed for detoxification (Chin et al., 2007). Glycyr-
rhizin is the principal saponin in licorice and was reported to pre-
vent the development of hepatic carcinoma from C hepatitis. In
addition, Glycyrrhizin finds application in inhibiting unwanted ef-
fects of contraceptive formulations, such as alterations in blood
coagulation and thrombosis (Staehli et al., 2007). Pharmacological
studies demonstrated that licorice saponins exhibited multiple
biological effects such as hepatoprotective (Gao, Zheng, Sun, Wang,
& Yang, 2011), anti-cancer (Zheng et al., 2010), antiviral (Ashfaq,
Masoud, Nawaz, & Riazuddin, 2011), anti-inflammatory (Li et al.,
2010) and neuroprotective activities (Cherng et al., 2006).
However, overconsumption of licorice saponins may result in side

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.05.073
1682 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689
effects like salt retention and hypokalaemic hypertension though
licorice is generally considered safe (Heikens, Fliers, Endert, Acker-
mans, & van Montfrans, 1995; Zhang & Ye, 2009). Therefore, triter-
pene saponins are of great importance for the food and medicinal
use of licorice, being closely related to the organoleptic and phar-
maceutical properties, which makes their analysis of considerable
interest. Furthermore, experiments focusing on those triterpene
saponins might be useful in discrimination and classification of
the three officinal Chinese pharmacopoeia Glycyrrhiza species.
Wide use of licorice extract has also prompted studies to im-
prove its quality control. Several methods have been developed
for analysis of licorice by thin-layer chromatography (TLC) (Cui,
Fu, Lee, & Wang, 2005), high-performance liquid chromatography
(HPLC)–ultraviolet detection (UV) or mass spectrometry (MS)
(Andrisano, Bonazzi, & Cavrini, 1995; Ye et al., 2007), gas chroma-
tography–flame ionization detection (FID) or mass spectrometry
(MS) (Guillaume, van der Molen, Kerstens, Dullaart, & Wolthers,
1999; Zhang & Ye, 2009). Most of these studies, however, were fo-
cused on quantitative analysis of one or a few triterpene saponins
in licorice. Recent success with the use of liquid chromatography
coupled with triple quadrupole mass spectrometry (LC–TQ-MS/
MS) for characterizing and quantifying a wide variety of com-
pounds in complex samples suggests that LC–TQ-MS/MS might
be a powerful technique in the comprehensive determination of
multiple triterpene saponins in complex plant extracts. Ultra-
high-performance liquid chromatography coupled with a triple
quadrupole electrospray tandem mass spectrometry (UPLC–TQ-
MS/MS) is a powerful tool to solve the problems of above methods
because of its high sensitivity and rapid resolution. Due to the high
selectivity of multiple reaction monitoring (MRM) mode, optimiza-
tion of chromatographic separation is greatly simplified. Further-
more, precursor and production ion monitoring can be used to
increase specificity of detection and identification of the known
molecules. In this study, a fast and effective UPLC–TQ-MS/MS
method was explored for better identification and quantitative
investigation of triterpene saponins in licorice, which laid basis
for chemical comparison of three officinal Chinese pharmacopoeia
Glycyrrhiza species.
Despite licorice widespread medicinal and culinary uses, the
authentication of ground licorice samples and standardization of
its extracts poses a problem due to heterogeneity of the plant
material and contamination or purposeful adulteration with other
Glycyrrhiza species. It is difficult to identify Glycyrrhiza species
accurately based on their root or rhizome morphology. In addition,
it is well known that herbs collected from different species or var-
ious regions are discrepant in the types and quantities of chemical
constituents, which influence their therapeutic effects. Thus sev-
eral methods have been already applied for the discrimination of
Glycyrrhiza species (Farag, Porzel, & Wessjohann, 2012; Liao, Lin,
Chang, & Huang, 2012). However, batch number from some species
was relatively small, which may cause false-positive or false-nega-
tive results. Moreover, batch effects that arise from laboratory,
temporal, or other experimental variation can be addressed in sev-
eral ways. In our present study, a large batch of licorices was col-
lected from three officinal Chinese pharmacopoeia Glycyrrhiza
species. Finally, principal component analysis (PCA) was per-
formed to evaluate and classify the three species according to the
contents of the detected triterpene saponins.
2. Materials and methods
2.1. Chemicals, reagents, and materials
Acetonitrile and methanol were HPLC-grade from Merck
(Darmstadt, Germany) and deionized water was purified by an
EPED super purification system (Eped, Nanjing, China). Other re-
agent solutions were analytical grade (Nanjing Chemical Plant,
Nanjing, PR China). Reference standards of uralsaponin C (1), ural-
saponin F (2), uralsaponin D (3), 22b-acetoxyl-glycyrrhizin (4), 24-
hydroxy-licorice-saponin E2 (5), licorice-saponin G2 (6), licorice-
saponin E2 (7), 22b-acetoxyl-glycyrrhaldehyde (8), glycyrrhizin
(9), and licorice-saponin J2 (10) were isolated and purified in our
laboratory. On the basis of UV, NMR and MS analysis, the structures
of isolated reference standards were confirmed, and their purities
determined using UPLC–PDA–MS were over 98.0% (Supplementary
material). Their structures are presented in Fig. 1.
The raw materials of G. inflata (samples 1–11), G. glabra (sam-
ples 12–22) and G. uralensis (samples 23–82) were collected from
their main production regions in China. All information on col-
lected samples and their origins is recorded in Table 1S and
Fig. 1S. The crude drugs were authenticated by one of the authors,
Prof. Runhuai Zhao and the voucher specimens are deposited at
Herbarium in Nanjing University of Chinese Medicine, China.
2.2. Chromatographic conditions and instrumentation
Chromatographic analysis was performed on a Waters Acquity
UPLC system (Waters, Corp., Milford, MA, USA), consisting of a bin-
ary pump solvent management system, an online degasser, and an
autosampler. An ACQUITY UPLC BEH C18 column
(100 mm  2.1 mm, 1.7 lm) was employed and the column tem-
perature was maintained at 35 °C. The mobile phase was composed
of A (0.1% formic acid/water) and B (acetonitrile/methanol 3:1, v/v)
using a gradient elution of 30–48% B at 0–2 min, 48–65% B at 2–
6 min with a flow rate set at 0.40 mL min 1. The autosampler
was conditioned at 4 °C and the injection volume was 2 lL.
Mass spectrometry detection was performed using a Xevo Tri-
ple Quadrupole MS (Waters Corp., Milford, MA, USA) equipped
with an electrospray ionization source (ESI). The ESI source was
set in negative ionization mode. The parameters in the source were
set as follows: capillary voltage 3.0 kV; source temperature 150 °C;
desolvation temperature 550 °C; cone gas flow 50 L h 1; desolva-
tion gas flow 1000 L h 1. The analyte detection was performed by
using multiple reaction monitoring (MRM). The cone voltage and
collision energy were optimized for each analyte and selected val-
ues are given in Table 1. Dwell time was automatically set by the
software.
2.3. Preparation of sample solutions
The dried roots and rhizomes were pulverized to homogeneous
powders (40 mesh). The dried powder (0.5 g) was weighed accu-
rately into a 50 mL conical flask with a stopper, and 30 mL of
50% aqueous methanol was added. After accurate weighing, reflux-
ing was performed at 110 °C for 1 h, and then the same solvent was
added to compensate for the weight lost during the extraction.
After centrifugation (10,000 rpm, 10 min), the supernatant was fil-
tered through a 0.22 lm membrane prior to use. The filtrates were
diluted fifty times with 50% aqueous methanol, and then 2 lL ali-
quot was injected into the UPLC system for analysis.
2.4. Preparation of standard solutions
A mixed standard stock solution containing compounds 1–10
was prepared in 50% aqueous methanol. Working standard
solutions for calibration curves were prepared by diluting the
mixed standard stock solution with 50% aqueous methanol at dif-
ferent concentrations within the following ranges: 0.0125–
2.5000 lg mL 1; 0.0068–1.3500 lg mL 1; 0.0404–8.0750 lg mL 1;
0.0513–10.2500 lg mL 1; 0.0057–1.1375 lg mL 1; 0.0569–11.37
50 lg mL 1; 0.0200–4.0000 lg mL 1; 0.0064–1.2750 lg mL 1;
 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689 1683

1 2 3 4

5 6 7 8

9 10

Fig. 1. Chemical structures of uralsaponin C (1), uralsaponin F (2), uralsaponin D (3), 22b-acetoxyl-glycyrrhizin (4), 24-hydroxy-licorice-saponin E2 (5), licorice-saponin G2
(6), licorice-saponin E2 (7), 22b-acetoxyl-glycyrrhaldehyde (8), glycyrrhizin (9), and licorice-saponin J2 (10).

Table 1
Precursor/product ion pairs and parameters for MRM of compounds used in this study.

Analyte Retention time (min) [M H] (m/z) MRM transitions (Precursor ? product) Cone voltage (V) Collision energy (eV)
Uralsaponin C 1 2.36 823 823 ? 351 74.0 40.0
Uralsaponin F 2 2.69 895 895 ? 351 62.0 40.0
Uralsaponin D 3 2.93 849 849 ? 351 70.0 42.0
22b-Acetoxyl-glycyrrhizin 4 3.12 879 879 ? 351 68.0 40.0
24-Hydroxy-licorice-saponin E2 5 3.32 835 835 ? 351 64.0 38.0
Licorice-saponin G2 6 3.76 837 837 ? 351 56.0 40.0
Licorice-saponin E2 7 3.78 819 819 ? 351 68.0 38.0
22b-acetoxyl-glycyrrhaldehyde 8 3.95 863 863 ? 351 66.0 38.0
Glycyrrhizin 9 4.17 821 821 ? 351 64.0 40.0
Licorice-saponin J2 10 5.03 823 823 ? 351 66.0 36.0

0.2425–48.5000 lg mL 1; and 0.0146–2.9250 lg mL 1. The stan-
dard solutions were filtered through a 0.22 lm membrane prior
to injection. All solutions were stored in a refrigerator at 4 °C
before analysis.
2.5. UPLC method validation
Stock solution containing 10 reference compounds was diluted
to a series of appropriate concentrations with 50% aqueous meth-
anol. The limits of detection (LOD) and quantification (LOQ) were
determined at signal-to-noise (S/N) ratios of 3 and 10, respectively.
The intra-day and inter-day precisions were investigated by deter-
mining the 10 analytes in six replicates during a single day and by
duplicating the experiments on three consecutive days. To confirm
the reproducibility, six different working solutions prepared from
licorice were analysed, and one of them was injected into the appa-
ratus at 0, 2, 4, 8, 12, and 24 h, respectively, to evaluate the stability
of the solution. Variations were evaluated using relative standard
deviations (RSD). A recovery test was used to evaluate the accuracy
of this method. Accurate amounts of reference compounds were
added to the licorice separately and then extracted and analysed
in accordance with the methods mentioned above. The average
recovery percentage was calculated using the ratio of contents de-
tected (actual) to those added (theoretical).
2.6. Identification and quantification
Identification of the triterpene saponins was carried out by
comparing the UPLC retention times and mass/charge ratios of tar-
get peaks with those of the standards. To further confirm the struc-
tures of the constituents, standards and samples were analysed by
UPLC–ESI–MS/MS in negative ion mode. Quantification was per-
formed on the basis of linear calibration plots of the peak areas ver-
sus the concentration.
1684 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689
2.7. Method for PCA of samples
The PCA was done by SPSS 13.0 software (SPSS, Chicago, IL,
USA). In this study, the contents of the 10 markers analysed from
the 82 samples composed a data matrix with 82 rows and 10 col-
umns, which was used for PCA analysis after normalization. The
first three principal components (PCs) were extracted, and the
scatter plot were obtained by plotting the scores of PC1 versus
PC2 and PC3.
3. Results and discussion
3.1. Optimization of extraction procedure
In order to obtain the best extraction efficiency, variable factors
involved were carefully optimized. After comparison of different
extraction methods (reflux and sonication), different solvents
(30%, 50%, and 70% aqueous methanol, and 100% methanol), and
Fig. 2. Electrospray ion MS/MS spectra of compounds 1–10.
different time (0.5, 1, and 2 h), it was finally determined that the
samples would be extracted with 50% aqueous methanol for 1 h
by reflux method.
3.2. Optimization of mass spectrometry
A standard solution of the analytes was directly infused along
with the mobile phase into the mass spectrometer with ESI as
the ionization source. The response observed in the negative ioni-
zation mode was higher than that in positive ionization mode. In
the precursor ion full-scan spectra, the most abundant ions were
deprotonated molecules [M H] for all the analytes. Parameters
such as desolvation temperature, ESI source temperature, capillary
and cone voltage, flow rate of desolvation gas and cone gas were
optimized to obtain the highest intensity of deprotonated molecule
of analytes. The ion pairs of precursor ? production for MRM
detection were generated by the Intellistart procedure, which
was embedded in the MassLynx software. All the MRM transitions
 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689 1685

and parameters applied in the study are listed in Table 1. Under the
optimized UPLC and MS/MS conditions, all 10 compounds in lico-
rice were identified and quantified. As shown in Fig. 2, all saponins
could fragment into m/z 351 [diglucuronic acids – H2O] and 193
[monoglucuronic acid] . These ions were highly characteristic for
the identification of licorice saponins from complicated extracts.
3.3. Optimization of the UPLC chromatographic conditions
To achieve good peak shape, good separation, and short analyt-
ical time for the 10 compounds, the chromatographic conditions
were optimized. Considering all of the reference compounds were
the derivatives of oleanane-type triterpene diglucuronide, formic
acid was added into the mobile phase to improve the peak shape
of the target peaks. Different combinations of methanol, acetoni-
trile, and 0.1% formic acid in water as mobile phase were tried
on two different HPLC columns. Finally, an Acquity UPLC BEH C18
(100 mm  2.1 mm, 1.7 lm) column was selected for the whole
analysis, and a mixture of solution A (0.1% formic acid in water)
and solution B (acetonitrile–methanol, 3:1, v/v) was used for the
mobile phase. The flow rate and column temperature were set at
0.4 mL min 1, and 35 °C, respectively. The analysis was carried
out under a gradient program as follows: (30–48% B at 0–2 min,
48–65% B at 2–6 min). Representative chromatograms for the
mixture of standards and samples are shown in Fig. 3.
3.4. Analytical method validation
The proposed UPLC–TQ-MS/MS method for quantitative
analysis was validated to determine the linearity, LOD, LOQ, in-
tra-day and inter-day precisions, stability, and accuracy. As shown
in Table 2S, all calibration curves showed good linearity

1
A 2

7
8

100 10
%

0 T im e
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

1
B 2

100 10
%

0 Tim e
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

Fig. 3. Representative MRM chromatograms of solution of standards (A) and samples (B).
1686 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689

Table 2
Contents of 10 investigated compounds in different samples.
1
Sample no contents of analytes (mg g , n = 3a) Total
1 2 3 4 5 6 7 8 9 10
1 0.123 ndb 0.343 0.473 nd 8.889 0.168 nd 47.259 3.315 60.569
2 0.156 0.802 0.337 2.608 nd 10.299 0.188 0.439 41.304 5.684 61.817
3 0.321 nd 0.514 0.386 nd 10.401 0.188 nd 50.128 2.792 64.731
4 0.152 nd 0.535 0.334 nd 5.286 0.127 nd 38.016 1.146 45.595
5 0.187 0.133 0.241 0.945 nd 5.899 0.151 0.169 42.148 0.926 50.799
6 0.191 0.172 1.266 0.950 nd 6.744 0.392 0.100 39.448 1.733 50.996
7 0.382 0.433 0.451 2.581 nd 8.691 0.279 0.572 59.569 2.597 75.557
8 0.117 nd 0.124 0.430 nd 6.577 nd nd 70.424 6.278 84.095
9 0.170 0.329 0.574 2.123 nd 5.476 0.405 0.252 35.037 1.689 46.055
10 0.344 0.684 0.000 1.553 nd 5.941 0.124 0.357 19.311 1.944 30.259
11 0.473 0.838 0.000 2.045 nd 7.503 0.151 0.472 21.652 2.357 35.490
12 0.758 nd nd nd nd 6.770 nd nd 38.861 2.012 48.401
13 0.644 nd nd nd nd 5.477 nd nd 28.342 1.247 35.710
14 0.520 nd nd nd nd 4.147 nd nd 23.858 0.858 29.383
15 0.396 nd nd nd nd 5.014 nd nd 22.255 1.293 28.958
16 0.544 nd nd nd nd 4.536 nd nd 24.523 1.107 30.710
17 0.509 nd nd nd nd 4.028 nd nd 21.435 1.373 27.346
18 0.549 nd nd nd nd 1.093 nd nd 37.143 0.978 39.763
19 0.493 nd nd nd nd 4.982 nd nd 37.081 1.240 43.796
20 1.170 nd nd nd nd 2.983 nd nd 41.831 1.820 47.804
21 0.384 nd nd nd nd 0.741 nd nd 59.025 2.014 62.164
22 0.452 nd nd nd nd 2.932 nd nd 40.032 0.862 44.278
23 2.784 3.045 0.423 17.615 0.290 9.080 1.566 3.431 47.091 1.800 87.126
24 1.166 0.080 6.815 0.443 0.757 6.709 3.078 trc 29.282 1.877 50.268
25 5.186 0.510 9.350 2.523 2.616 16.535 10.433 0.546 62.074 1.355 111.128
26 0.889 0.400 1.646 1.846 0.235 9.373 0.887 0.335 36.441 1.349 53.400
27 0.760 0.132 5.905 0.734 0.485 4.930 2.137 trc 20.665 0.714 36.541
28 1.646 0.262 12.749 1.184 0.855 6.236 3.546 0.157 27.554 0.554 54.743
29 2.644 0.648 20.740 3.353 1.173 9.549 4.573 0.446 40.281 1.686 85.093
30 1.841 0.480 15.536 2.353 0.830 7.243 3.288 0.333 30.007 1.282 63.193
31 1.255 0.577 5.500 1.846 0.653 6.318 2.915 0.238 21.359 0.919 41.580
32 3.565 1.727 5.395 13.613 0.278 7.339 1.831 2.839 50.367 2.447 89.402
33 1.156 0.974 4.086 5.556 0.556 6.395 2.370 0.865 27.754 0.888 50.601
34 0.683 1.229 1.511 6.674 0.174 9.476 0.714 1.314 33.818 2.064 57.658
35 1.624 1.465 15.146 6.655 0.511 7.881 2.252 1.084 35.413 1.489 73.520
36 1.138 0.668 2.648 3.392 1.113 10.472 6.067 0.565 44.489 3.587 74.141
37 3.441 0.407 1.170 1.663 1.427 7.494 5.436 0.350 22.703 1.577 45.667
38 1.861 2.582 4.820 13.589 0.500 10.523 2.313 2.225 34.201 3.084 75.698
39 1.616 1.301 3.446 6.227 0.564 5.192 2.256 0.968 19.073 2.107 42.749
40 5.023 0.677 0.901 2.515 2.137 11.098 7.009 0.512 33.523 1.703 65.097
41 1.214 0.265 4.206 1.223 0.654 9.673 2.099 0.263 28.618 2.637 50.853
42 1.714 0.694 3.844 2.076 1.214 23.225 2.332 0.347 56.670 2.968 95.083
43 1.330 0.230 2.090 1.137 0.706 8.631 2.216 0.221 29.976 1.046 47.582
44 3.741 0.174 2.638 0.922 1.602 10.435 6.536 0.156 46.587 2.251 75.042
45 1.262 2.093 2.352 9.966 0.458 7.785 1.957 1.585 29.364 2.831 59.653
46 0.520 1.581 12.337 7.696 0.439 9.932 1.504 0.790 38.378 1.546 74.722
47 0.745 0.399 22.927 1.129 0.918 13.239 2.017 0.375 31.615 1.742 75.107
48 0.909 1.607 15.561 6.592 0.608 10.379 1.612 1.019 23.901 0.975 63.163
49 0.971 1.644 8.344 12.170 0.323 9.067 1.621 1.835 43.569 1.566 81.110
50 0.950 2.278 3.821 13.892 0.249 7.050 1.410 2.389 27.276 0.926 60.241
51 1.961 1.949 12.464 15.905 0.733 11.971 6.315 2.603 61.588 3.265 118.754
52 1.354 1.662 13.404 15.175 0.567 9.422 3.924 1.894 42.376 2.007 91.787
53 1.377 1.279 1.629 6.155 1.714 22.067 7.534 0.965 67.949 3.128 113.797
54 0.532 1.986 4.982 12.676 0.205 7.618 1.389 1.689 39.646 1.006 71.729
55 1.069 1.169 13.452 6.973 0.486 8.246 2.333 1.134 30.054 2.233 67.148
56 1.377 1.355 5.897 7.922 0.534 8.166 2.567 1.310 26.519 2.084 57.731
57 1.402 2.645 1.897 13.761 0.544 11.950 2.733 2.090 50.544 1.933 89.501
58 0.861 0.276 4.573 1.419 0.970 11.180 3.965 0.285 43.908 3.064 70.503
59 0.760 1.387 4.280 8.731 0.416 7.885 2.323 1.363 33.128 0.846 61.118
60 0.950 1.481 9.867 9.797 0.364 7.237 2.136 1.905 33.271 1.069 68.078
61 2.176 1.346 7.373 9.494 1.246 11.386 6.769 1.915 51.764 2.283 95.751
62 0.949 1.461 10.068 9.239 0.549 7.776 2.911 1.832 37.144 1.199 73.129
63 5.883 1.306 0.781 10.061 2.643 16.695 14.624 2.815 62.196 2.746 119.750
64 1.835 1.954 1.238 10.352 0.527 8.115 2.218 3.131 29.443 1.053 59.866
65 2.537 0.877 0.246 3.309 1.617 10.261 7.153 1.332 32.978 1.041 61.350
66 2.821 2.630 0.883 14.665 0.833 12.309 4.079 4.144 41.881 1.246 85.490
67 1.300 2.352 3.057 10.313 0.463 12.003 2.006 2.528 36.977 0.786 71.785
68 2.740 2.273 1.111 11.362 0.786 16.992 3.769 3.855 51.521 2.588 96.998
69 3.168 1.610 2.706 9.213 1.629 17.936 6.965 2.008 54.654 2.310 102.199
70 1.468 1.415 1.392 4.914 1.271 13.659 3.962 1.358 39.245 1.181 69.864
71 0.944 2.452 1.878 8.422 0.375 12.920 1.274 2.492 32.647 1.007 64.409
72 2.836 0.622 0.841 4.768 1.209 10.472 8.633 1.197 59.301 0.965 90.844
73 1.155 1.501 4.743 7.870 0.306 8.848 1.316 1.471 33.582 1.597 62.387
 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689 1687

Table 2 (continued)
1
Sample no contents of analytes (mg g , n = 3a) Total
1 2 3 4 5 6 7 8 9 10
74 1.659 1.623 0.550 9.557 1.068 12.860 6.788 1.204 47.468 2.097 84.875
75 0.714 1.227 0.706 6.698 0.745 12.570 4.233 0.778 37.677 2.271 67.618
76 1.556 1.575 1.030 8.440 0.818 19.256 3.983 1.515 54.296 5.264 97.734
77 0.568 0.177 8.742 1.058 0.623 7.283 4.434 0.194 36.087 2.360 61.525
78 1.568 2.352 2.680 14.006 0.945 15.257 5.458 2.074 55.872 2.074 102.286
79 1.457 0.474 0.151 2.168 1.920 18.575 9.655 0.407 58.869 4.659 98.336
80 0.893 0.239 trc 0.717 1.544 11.768 6.888 0.178 35.493 1.550 59.337
81 0.628 1.122 trc 6.694 0.554 11.655 3.302 0.789 47.603 2.175 74.597
82 0.545 0.883 7.098 6.360 0.452 10.541 2.617 0.732 46.280 1.884 77.391
a
The data are presented as the average of three replicates (RSDs < 7%).
b
nd, not detected.
c
tr, under the limits of quantitation.

(r2 > 0.9960) within the test ranges, and the overall LODs and LOQs
were in the range of 0.91–4.35 ng mL 1 and 2.05–13.24 ng mL 1,
respectively. The RSD values of intra- and inter-day variations,
repeatability and stability of the 10 analytes were all less than
5% (Table 3S). The overall recoveries lay between 91.4% and
107.2% with RSD less than 6.10%. All the results mentioned above
indicated that the established method was accurate.
3.5. Quantitative analysis of samples
Significant differences in the chemical composition, in terms of
the contents and ratios of the triterpene saponins, were observed
in the different licorice samples. (G. inflata, G. glabra, and G. uralen-
sis) (Table 2, Fig. 2S1–S5). For all batches of licorice, content of
glycyrrhizin was higher than that of other triterpene saponins, var-
ied from 19.073 to 70.424 mg g 1. The average ratios of glycyrrhi-
zin in total content of our investigated triterpene saponins were
76.8% for G. inflata, 83.29% for G. glabra, and 51.10–56.15% for G.
uralensis (Fig. 2S1–S5), respectively. In recent studies, glycyrrhizin
evidences profound anti-inflammatory activities, such as the inhi-
bition of LPS/D-galactosamine-induced liver injury (Yoshida et al.,
2007), the prevention of free fatty acid-induced hepatic lipotoxic-
ity (Wu et al., 2008), and the attenuation of carrageenan-induced
lung injury (Menegazzi et al., 2008). Furthermore, glycyrrhizin is
one of the leading natural compounds for clinical trials on chronic
viral hepatitis and human immunodeficiency virus infections
(Cirillo et al., 2011). Based on these considerations, the determina-
tion of glycyrrhizin and analogous compounds is of great
importance for the dietary and medicinal use of licorice.
In the present study, the overall chemical profile of licorice in
terms of the types and contents of the triterpene saponins is
mainly determined by the species of origin (Table 2). The 60
batches of G. uralensis from different regions showed a high simi-
larity in the contents and ratios of chemical constituents. Com-
pounds 1–10 were found as common constituents in all the G.
uralensis samples, which is in full agreement to previous studies
(Kitagawa, Hori, Sakagami, Zhou, & Yoshikawa, 1993; Kitagawa,
Hori, Taniyama, Zhou, & Yoshikawa, 1993; Kitagawa et al., 1993).
In addition, compounds 3, 4, 6, and 9 were four abundant compo-
nents in G. uralensis, which accounted for more than 80% of the
content of total saponins. For G. uralensis, samples from Gansu
province were quantitatively lower to average value. The signifi-
cant differences were found between the samples of the three
investigated species, and some of the major compounds may serve
as chemotaxonomic markers. For instance, compounds 4 and 7
were detectable in G. uralensis and G. inflata only, while compound
5 only in G. uralensis. And compounds 2, 3, and 8 were present in
some G. inflata, but not in any G. glabra.
3.6. PCA of the samples
To evaluate the variation of licorice, PCA was performed on the
basis of the contents of 10 tested compounds from UPLC profiles.
The first three principal components (PC1, PC2, and PC3) with
>79% of the whole variance were extracted for analysis. Among
them, PC1 accounted for 42.46% of total variance, whereas PC2
and PC3 explained 22.17 and 15.04% of total variance, respectively.
The remaining principal components, which had a minor effect on
the model, were discarded.
The components loading matrix is shown in Table 4S. According
to their loadings, PC1 had good correlation with compounds 1, 4, 5,
6, 7 and 8, and PC2 exhibited relationship with compound 2,
whereas PC3 was related mainly to compound 10. The results men-
tioned above suggested that most of the compounds may contrib-
ute to the classification of the samples. The scatter plot is shown in
Fig. 4, where each sample is represented as a marker. Eighty-two
sample dots were successfully classified into group I, group II,
and group III corresponding to G. inflata, G. glabra, and G. uralensis.
Dots in group I were relatively nearer to each other, indicating a
closer relationship among 11 batches of G. inflata. Overlapping of
dots in group II indicated the genetic origins to be the major ele-
ment influencing triterpenes of G. glabra. Dots in group III were rel-
atively scattered, suggesting diversification of the 60 batches of G.
uralensis. From 3D plots obtained from the contents of triterpene
saponins in licorice, discrimination of three different Glycyrrhiza
species was feasible.
Beside the successful distinction of three species, PCA was also
tried in discrimination of geographical origins. The result was
meaningful despite not to the satisfaction. That could be explained
by several reasons. Firstly, geographical origin might not be the
only factor influencing contents of triterpenes. For example, G.
uralensis is a perennial plant, it was reported that growth age
increasing is the main factor for the increment of saponin content
in licorice (Peng, Zhang, & Hu, 2006). But it was hard to obtain such
information from the wild samples. Secondly, other active compo-
nents in licorice exist except for triterpenes (flavonoids for exam-
ple), thus information of other active components might be
useful. Thirdly, for PCA of content, not all triterpenes in licorice
were taken into consideration.
4. Conclusion
In present study, triterpene saponins in 82 batches of licorice
belonging to three Glycyrrhiza species were analysed by UPLC–
TQ-MS/MS method. The assay had good performance in selectivity,
recovery, precision and accuracy, and proved to be highly sensitive
compared with the reported methods. Significant differences
among species in both the type of triterpene saponins and their
1688 W. Tao et al. / Food Chemistry 141 (2013) 1681–1689
Fig. 4. The scatter plot obtained by PCA of the 82 samples of licorice.
contents have been revealed. To a certain extent, difference in
chemicals may mean possible difference in their biological func-
tions. Since they are all officially used as licorice in Chinese medi-
cine, further detailed scientific investigations to these Glycyrrhiza
species are therefore strongly suggested to verify whether they
are actually equivalent. Furthermore, the established method in
this study could be useful in providing chemically supportive infor-
mation for Glycyrrhiza species authentication, as well as in quality
control and standardization of licorice raw materials and its
products.
Acknowledgements
This research was financially supported by National Basic Re-
search Program of China (973 Program) (2011CB505300,
2011CB505303), 2009’ Program for Excellent Scientific and Tech-
nological Innovation Team of Jiangsu Higher Education, the Priority
Academic Program Development of Jiangsu Higher Education Insti-
tutions (ysxk-2010).
We are pleased to thank Waters China Ltd. for technical
support.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/
j.foodchem.2013.05.073.
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