Food Chemistry 136 (2013) 513517

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

Chemical analysis and anti-inammatory comparison of the cell culture

of Glycyrrhiza with its eld cultivated variety
Shuli Man a,b, Juan Wang a,b,c, Wenyuan Gao a,b,c,, Songbo Guo a,b, Yuanyuan Li a,b, Liming Zhang a,b,
Peigen Xiao d
a
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
b
Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
c
Tianjin Key Laboratory for Modern Drug Delivery and High Efciency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
d
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

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

Article history: Suspended cells of Glycyrrhiza (CG) possessed a similar content of avonoids and a lower content of tri-
Received 29 April 2012 terpenes, when compared with its eld cultivated equivalent (NG). Xylene-induced ear oedema and oval-
Received in revised form 31 July 2012 bumin-induced mouse paw oedema were applied, to compare the effects of CG and NG on the acute
Accepted 8 August 2012
inammatory response. Extracts of the cell culture of Glycyrrhiza possessed a similar anti-inammatory
Available online 17 August 2012
effect to those of NG, through the enhancement of the SOD activity of plasma and liver tissues. The use of
a cell culture of liquorice instead of eld cultivation would be potentially protable.
Keywords:
2012 Elsevier Ltd. All rights reserved.
Glycyrrhiza
Suspension cell
Field cultivation
Anti-inammatory
Comparison

1. Introduction farms. However, demand for liquorice is high and supply is limited.
Cell culture has been exploited as an alternative for more efcient
The roots and rhizomes of liquorice (Glycyrrhiza) species have and controllable production of liquorice and its active metabolites
long been used worldwide as an important agricultural commod- (Wang, Qi, & Wang, 2010). The avonoid productivity of the cells
ity. This species contains a large number of active components, which are cultured for 3 years is higher than that of the 3-year-
including avonoids, isoavonoids, chalcones and triterpene sapo- old plant, which suggests that avonoid production by cell cultures
nins (Asl & Hosseinzadeh, 2008). Flavonoids, secondary metabo- of liquorice is potentially protable (Yang, He, Yu, Ji, & Wang,
lites of the plant, are especially used for anti-oxidative (Li et al., 2009).
2011), anti-inammation (Kim et al., 2008), and skin-whitening Until now, there are no reports regarding the comparison of
properties. Triterpenes, also main active compounds in liquorice, chemical and pharmacological properties between cell-cultured
exhibit anticancer, antiviral, anti-inammatory (Schrofelbauer and eld-cultivated products. In this research, we compared the
et al., 2009) and immunoregulative (Cao et al., 2006) properties. compositions of triterpenes and avonoids in cultured cells and
In recent years, liquorice has been increasingly used as a health eld cultivated liquorice, in relation to the observed anti-inamma-
additive formulated into a variety of commercial products, such as tory effects.
drugs, foods, drinks and cosmetics, which are marketed in Asia as
well as many other countries around the world (Iida et al., 2007). 2. Materials and methods
Generally, almost all commercial liquorice root is collected from
2.1. Plant material
Abbreviations: 6-BA, 6-benzylaminopurine; CG, crude extract of cell cultural
Glycyrrhiza; MS, Murashige and Skoog; NAA, naphthaleneacetic acid; NG, Crude Native roots and seeds of Glycyrrhiza uralensis were supplied by
extract of eld cultivated Glycyrrhiza; SOD, superoxide dismutase; tR, retention Beijing Institute of Shizhen Chinese Herbal Medicine.
time.
Corresponding author at: Key Laboratory of Industrial Microbiology, Ministry of
Education, College of Biotechnology, Tianjin University of Science & Technology, 2.1.1. Callus induction and proliferation
Tianjin 300457, China. Tel./fax: +86 22 87401895. Seed surfaces were sterilised with 70% EtOH for 30 s, immersed
E-mail address: pharmgao@tju.edu.cn (W. Gao). in 5% NaOCl solution for 20 min and rinsed three times with sterile

0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.08.010
514 S. Man et al. / Food Chemistry 136 (2013) 513517

distilled water. Disinfected seeds were inoculated into Murashige crude triterpenes extract was dissolved in methanol to a designed
and Skoog (MS) solid medium containing 3% sucrose (Murashige concentration (1 mg/mL) and 2 mL of the solution were added to a
& Skoog, 1962). Each conical ask (100 mL) was cultured at 10-mL tube. After the solution was evaporated to dryness in a
23 2 C with a light intensity of 25003000 lx in a biochemical water bath, 0.2 mL of freshly-prepared 5% (w/v) vanillinacetic
incubator. After 1 week of culture, seedlings were observed. The acid solution and 0.8 mL perchloric acid were added, mixed and
middle tissue of hypocotyls were cut into sections of 5 mm and incubated at 55 C for 15 min. The tubes were taken out and cooled
inoculated into MS solid medium containing 1.0 mg L 1 2,4-dichlo- in running water. Then 5.0 mL acetic acid were added and
rophenoxyacetic acid (2,4-D), 0.2 mg L 1 6-benzylaminopurine absorbance of the solution was measured at 595 nm. Glycyrrhizin
(6-BA) and 3% sucrose. Each Petri dish (9 cm in diameter and was chosen as a standard. Using a ve-point standard curve
1.5 cm in height) was sealed and cultured at 23 2 C in the dark. (50250 lg), the total triterpene contents in NG and CG were
After 4 weeks of culture, calli were induced, which proliferated un- determined in triplicate, respectively. The data were expressed as
der the same conditions for a further 4 weeks. milligram glycyrrhizin equivalents/100 mg lyophilised powder
(NG and CG). The regression equation was y = 0.0007x 0.0073
2.1.2. Shake ask culture of suspension cells (y, the absorption of the mixture; x, triterpenes concentration of
Suspension cells were initiated from callus and cultivated in 1-L samples (%)).
conical asks with 400 mL 3/4MS medium supplemented with
1.0 mg L 1 2,4-D, 1.0 mg L 1 1-napthaleneacetic acid (NAA), 2.4. HPLC analysis of the main compounds
0.2 mg L 1 6-BA and 3% w/v sucrose. Suspension cells were cul-
tured on a rotary shaker (120 rpm) at 23 2 C under dark/light 2.4.1. Calibrations
conditions (illumination (7:0019:00)). Subculture was routinely Each standard was accurately weighed: 2 mg were dissolved in
conducted at 3-week intervals. 10 mL of methanol and diluted with methanol to an appropriate
concentration. The mixed solution of eight standards was prepared
2.2. Sample preparation in methanol and stored in at 4 C until required for analysis.
Validation of this analytical method was performed in accor-
After 18 days culture, cell suspensions were ltered and washed dance with International Conference on Harmonization (ICH)
several times with distilled water. The fresh cells were dried at guidelines. The method was validated in terms of linearity, limit
50 2 C for 2 days and then ground into powder. The powders of detection and quantication, precision and accuracy. The regres-
(30 g) were extracted twice with 750 mL 50% methanol containing sion equation is shown in Table 1.
0.3% ammonia for 1 h at 65 C (Huang, Zhou, & Song, 2011a). Fol-
lowing ltration, the extract was evaporated to dryness. 2.4.2. Sample preparation for HPLC analysis
Dried, native roots (40 g) of G. uralensis were also ground into Extracts of NG and CG (each 20 mg/mL) were analysed using an
powder. The powder (30 g) was then extracted using the same pro- Agilent 1100 high-performance liquid chromatograph (Agilent,
cedure as for the cell suspensions. Santa Clara, CA). Chromatographic separations were performed
on a Promosil RP-C18 column (4.6  250 mm, 5 lm, Agela, China).
2.3. Analyses of total avonoids and triterpenes The analytical column temperature was kept at 30 C; acetonitrile
(A) and 0.04% formic acid (B) under gradient conditions (04 min,
2.3.1. Determination of total avonoids 20% B; 420 min, 2038% B; 2025 min, 3855% B) was the mobile
NG and CG (0.5 g) were dissolved in methanol in separate 250- phase at a ow rate of 1 mL min 1. The wavelength of UV detector
mL volumetric asks. After mixing 1 mL of each solution with 1 mL was 260 nm. The injection volume was 20 lL.
of 10% KOH for 5 min with shaking, absorbance was read at 400 nm
against distilled water blank on a spectrophotometer (Tianjin, Chi- 2.5. Animals
na). Rutin was chosen as a standard. Using a ve-point standard
curve (20100 lg), the levels of total avonoids contents in NG Male Kun-Ming mice (China), weighing 2328 g were used in all
and CG were determined in triplicate, respectively. The data were experiments. Animals were obtained from the Institute of Labora-
expressed as milligram rutin equivalents/100 mg lyophilised pow- tory Animal of Chinese Academy of Medical Sciences, Beijing, Chi-
der (NG and CG). The regression equation was y = 0.0013x 0.0024 na. The animals were fed a standard rodent diet with free access to
(y, the absorption of the mixture; x, avonoids concentration of water, and were housed in rooms maintained at 25 1 C with a
samples (%)). 12 h light/dark cycle following international recommendations.
The Animal Ethics Committees of the Faculty of Medicine approved
2.3.2. Determination of total triterpenes all experimental protocols, in accordance with Principles of Labo-
A colorimetric method with vanillinacetic acid system was ratory Animal Care and Use in Research (Ministry of Health, Bei-
performed for the quantication of total triterpenes. Briey, the jing, China).

Table 1
Linearity of calibration curve for eight standards and their mean contents in two samples (mg/g, n = 3).

Peaks Standards tRa [min] Regression equationb R2 Field Cell

1 Liquiritin 8.20 Y = 1.376x + 93.64 0.9987 4.051 2.384
2 Isoliquiritin 14.4 Y = 1.062x 64.78 0.9977 1.439 1.903
3 Liquiritigenin 18.3 Y = 2.690x 182.6 0.9972 0.229 1.653
4 Licochalcone A 22.5 Y = 2.508x 173.0 0.9966 0.251 0.778
5 Glycyrrhizin 24.2 Y = 0.9129x 55.69 0.9961 3.702 1.936
6 Isoliquiritigenin 25.7 Y = 2.266x 149.4 0.9964 0.278 0.765
7 Glabridin 32.6 Y = 1.575x 74.69 0.9965 1.321 0.715
8 Glycyrrhetinic acid 38.1 Y = 1.350x 99.47 0.9942 0.988 0.235
a
tR, Retention time.
b
Y, Peak area; x, amount injected (ng).
 S. Man et al. / Food Chemistry 136 (2013) 513517 515

2.5.1. Experimental groups sample preparation, we obtained cell Glycyrrhiza extract (CG) and
The mice were randomly divided into ve groups, including native Glycyrrhiza extract (NG). The yield of CG (15.0%) is higher than
Control (0.9% sodium chloride/0.1 mL/10 g); NGH (3 mg of NG/ that of NG (12.7%). According to the 400 nm UV detection and rutin
0.1 mL/10 g); NGL (1 mg of NG/0.1 mL/10 g); CGH (3 mg of CG/ standard indication, the content of avonoids in NG was 31.2 1.5%,
0.1 mL/10 g; CGL (1 mg of CG/0.1 mL/10 g). They were adminis- while that in CG was up to 30.6 1.9%. Meanwhile, using glycyrrhi-
tered orally once daily for 6 consecutive days. zin as the standard, the content of triterpenes in NG reached
45.4 1.1%, while that in CG was only 7.0 0.2%. Therefore, sus-
2.5.2. Xylene-induced auricular oedema pended cells of Glycyrrhiza were good for avonoids accumulation.
One hour after the sixth administration, 20 lL xylene were ap-
plied to the anterior and posterior surface of the right ear of each 3.1.2. HPLC analysis of main active compounds
mouse. The left ear was considered as a control. One and a half To compare the detailed composition of NG with CG, eight com-
hours after xylene application, mice were killed (cervical disloca- pounds were used as avonoids and triterpenes standards (Table 1).
tion) and both ears were removed. Circular sections were taken As Fig. 1 shows, the effective anti-inammatory components liquir-
using a cork borer with a diameter of 9 mm, and weighed. The in- itin (Seo et al., 2011), liquiritigenin (Kim et al., 2008), licochalcone A
crease in weight caused by the irritant was measured by subtract- (Franceschelli et al., 2011; Kolbe et al., 2006; Kwon et al., 2008b), gly-
ing the weight of the untreated left ear section from that of the cyrrhizin (Wang, Kao, Lo, & Yen, 2011), isoliquiritigenin (Lee, Kim,
treated right ear. Seo, Kim, & Sohn, 2009), glabridin (Kwon, Oh, & Kim, 2008a) and
glycyrrhetinic acid (Xiao et al., 2010) all existed in NG and CG. The
2.5.3. Ovalbumin-induced mouse paw edema contents of the glycosides liquiritin, glycyrrhizin and glycyrrhetinic
The mouse paw oedema was induced on the conclusion of the acid were much higher in NG than in CG; suspended cells of Glycyr-
6 days treatment, and autopsies were performed after the mea- rhiza produced a lower concentration of triterpenes than eld culti-
surement. Oedema was induced on the right hind paw by intrapl- vated material. In contrast, the content of aglycones in liquiritigenin,
antar injection of ovalbumin (10 w/v% in physiological saline, licochalcone A and isoliquiritigenin were much lower in NG than in
0.1 mL). Paw volume was measured before and after ovalbumin CG. The reason may be that the formation of glycoside from aglycone
injection after 30 min, 1, 2 and 4 h, using a sliding caliper. Swelling in plant cell cultures was much more difcult than in eld cultivated
rate was assessed in terms of the mean thickness increase of each material. Glucuronylation is an exogenous reaction in plant cell
paw in comparison with the control group. cultures (Hayashi, Fukui, & Tabata, 1990; Shams-Ardakani,
Mohagheghzadeh, Ghannadi, & Barati, 2007).
2.5.4. Blood and liver tissues collection
3.2. Effects of CG and NG on the inammatory models
After measurement of the paw oedema, blood was processed for
plasma by centrifugation at 3500 rpm for 15 min. Plasma samples
Glycyrrhiza avonoids have an anti-inammatory effect (Thiy-
were frozen and maintained at 20 C until the various assays de-
agarajan, Chandrasekaran, Deepak, & Agarwal, 2011). In order to
scribed below. Meanwhile, liver tissues were obtained for further
compare the anti-inammatory effects of CG and NG, we chose
analysis. They were rinsed with saline solution and stored at
two models: xylene-induced ear oedema and ovalbumin-induced
20 C for further assays. Hepatic tissues were homogenised in
paw oedema. As shown in Fig. 2, CG and NG exhibited varying de-
1:10 volumes of saline solution. The homogenate was centrifuged
grees of anti-inammatory activities. They could mildly dose-
at 3000 rpm for 10 min. and its supernatant was used as a total li-
dependently reduce the level of the xylene-induced ear oedema.
ver homogenised sample.
Especially for the CGH, its inhibitory effect reached 33.53%, com-
pared with NGHs inhibition rate 16.85%. The reduction of ovalbu-
2.5.5. Determination of superoxide dismutase (SOD) levels in plasma
min-induced paw oedema was measured for 4 h (Fig. 3). CGH
SOD was measured in a spectrophotometer (723C, Shanghai)
signicantly inhibited ovalbumin-induced paw oedema in a
using spectrophotometric diagnostic kits obtained from Nanjing
dose-dependent manner (p < 0.05). The inhibition rates of CG and
Jiancheng Bioengineering Research Institute (Shen et al., 2009).
NG at dose of 300 mg/kg bw after 4 h were up to 34.54% (CGH)
and 20.58% (NGH), respectively.
2.5.6. Statistical treatment of data Although the content of triterpenes in CG was lower than in NG,
All the data were expressed as means SD of three replications, triterpene saponin glycyrrhizin might exhibit no efcacy in the
and the Students test was used for the statistical analysis. The val- mechanism of anti-inammatory effect of glycyrrhizin (Rackova
ues were considered to be signicantly different when the P value et al., 2007). Meanwhile, liquiritigenin and isoliquiritigenin (agly-
was less than 0.05. cones) were more effective than liquiritin and glycyrrhizin (glyco-
sides) in suppressing eotaxin secretion, which could induce
3. Results and discussion inammatory reaction (Jayaprakasam et al., 2009). Through inhib-
iting the production of inammatory mediators, liquiritigenin (Kim
3.1. Chemical composition et al., 2008) and licochalcone A (Kolbe et al., 2006; Kwon et al.,
2008b) may be potential natural products for treatment of various
3.1.1. Determination of total avonoids and total triterpenes inammatory diseases. Therefore, although there was a similar
Plant tissue culture technology, as an alternative approach, has content of avonoids in NG and CG, the higher content of liquiri-
been used for the production of valuable secondary metabolites tigenin, isoliquiritigenin and licochalcone A (aglycones) in CG
(Wang, Man, Gao, Zhang, & Huang, 2013). There are some reports caused CG to possess a more powerful anti-inammatory action
on the production of avonoids by G. uralensis cell culture (Yang than NG has (Fig. 3).
et al., 2009). In our previous research, cell growth, avonoid and tri-
terpene production in cell suspension cultures of Glycyrrhiza were 3.3. Mechanism of NG and CG on inhibition of ovalbumin-induced paw
investigated under various initial inoculum densities, and sucrose oedema mice
and nitrogen concentrations, to develop an optimisation method
for improved secondary metabolites production (Bian, Gao, & Wang, In the paw oedema mice, the anti-inammatory mechanism
2008a,b). Through the cell culture optimisation and the same was based on the inhibition of malondialdehyde (MDA), via
516 S. Man et al. / Food Chemistry 136 (2013) 513517

Fig. 1. HPLC analysis of eld cultivated Glycyrrhiza extract (NG) and cell cultural Glycyrrhiza extract (CG). 1 Liquiritin, 2 Isoliquiritin, 3 Liquiritigenin, 4 Licochalcone A, 5
Glycyrrhizin, 6 Isoliquiritigenin, 7 Glabridin, 8 Glycyrrhetinic acid.

Fig. 2. Effect of CG and NG antagonism on xylene-induced ear oedema. NGH, 3 mg Fig. 3. Effect of CG and NG antagonism on ovalbumin-induced mouse paw oedema.
of NG/0.1 mL/10 g treated group; NGL, 1 mg of NG/0.1 mL/10 g treated group; CGH, Results were expressed as the mean SEM (n = 8). p < 0.05 was compared with the
3 mg of CG/0.1 mL/10 g treated group; CGL, 1 mg of CG/0.1 mL/10 g treated group. control group.
 S. Man et al. / Food Chemistry 136 (2013) 513517 517

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