COMPARISON OF BIOLOGICAL ACTIVITIES OF GLYCYRRHIZA GLABRA AND

The biological activities of Glycyrrhiza (GLs) extracts (GL-1, Glycyrrhiza glabra from Eumseong, Korea; GL-2, G. uralensis from Eumseong, Korea; GL-3, G. uralensis from Yeongcheon, Korea; GL-4, G. uralensis from Neimenggu, China: GL-5, G. uralensis purchased from Korea Medicine Herbal Association, Korea) were investigated. G. uralensis (GLs-2, -3, -4, and -5) extracts exhibited higher free radical scavenging activity against DPPH and OH radicals than G. glabra (GL-1). In addition, all GLs had antibacterial activity against E. coli, S. aureus, and H. pylori. GL-3 inhibited the growth of E. coli and S. aureus, while GL-1 had antibacterial activity against H. pylori. All GL extracts tested inhibited the lipopolysaccharideand interferon-γ-induced inflammatory activity of RAW 264.7 cells. G. glabra and G. uralensis reduced NO generation. GL-3 also inhibited the growth of AGS human gastric adenocarcinoma cells. GLs-3 and -4 showed the inhibition of rat lens aldose reductase. GL-4 had a higher total content of glycyrrhizin (1), glycyrrhetinic acid (2), glabridin (3), and isoliquiritigenin (4). G. uralensis (GLs-2, -3, -4, and -5) is thus more effective than G. glabra (GL-1).


Introduction
Licorice (liquorice) is the name given to the roots and stolons of some Glycyrrhiza species that have been used worldwide as herbal medicine for over 4 000 years.The pharmaceutical effects of licorice are known to include anti-inflammatory, antivirus, antiulcer and anticarcinogenic activities (Wang and Nixon 2001).In addition, licorice extract can be used as a food addictive in tobaccos, chewing gums and candies (Fu et al., 2005).
There are different species of licorice, including G. glabra (European licorice) and G. uralensis (Chinese licorice), that contain species-specific flavonoids (Nomura et al., 2002).G. uralensis, G. glabra and G. inflate are recognized as medicinal plants in China, while G. uralensis and G. glabra are considered as beneficial herbs in Japan (Kondo et al., 2007) and G. uralensis is recognized as a medicinal plant in Korea.In previous studies, G. glabra has been shown to have antiulcer, expectorant, diuretic, antipyretic (Lata et al., 1999), antimicrobial, and anxiolytic ac-tivities (Ambawade et al., 2001).Moreover, an ethanol extract from G. uralensis has been shown to be beneficial in diabetes, abdominal obesity and hypertension in animal models (Mae et al., 2003).
In this study, we compare the licorice species G. glabra and G. uralensis by measurement of their biological activities and analysis of active components.

Plant materials
GLs-1 and -2 were G. glabra and G. uralensis, respectively, cultivated at the National Institute of Horticultural and Herbal Science, RDA, Eumseong, Korea.GLs-3 and -4 were G. uralensis collected at Yeongcheon, Korea and Neimenggu, China, respectively.GL-5 was G. uralensis purchased from Korea Medicine Herbal Association, Korea.

Preparation of methanol (MeOH) extracts of GLs
Ten grams of dried GLs were extracted with MeOH (200 mL × 3) under reflux conditions and the solvent was evaporated in vacuo.Each individual MeOH extract (1.0 mg) was dissolved in DMSO (1 mL).

DPPH and hydroxyl (OH) radical-scavenging activity
In a 96-well microplate, 100 μL of each sample were added to an ethanol solution of DPPH (60 μM) according to the method described by Hatano et al., (1989).After vortexing, the mixture was incubated for 30 min at room temperature and absorbance was measured at 540 nm.The DPPH radical-scavenging activity was recorded as a percentage (%) compared to the control.Scavenging of OH radicals was measured according to the method of Chung et al., (1997).The reaction mixture contained 10 mM FeSO 4 •7H 2 O 2 -EDTA, 10 mM 2-deoxyribose and the sample solutions.After incubation at 37°C for 4 h, the reaction was stopped by adding 2.8% trichloroacetic acid and 1.0% thiobarbituric acid solution.The solution was boiled for 20 min and then cooled in a water bath.OH scavenging activity was measured at 490 nm.

Antibacterial activity
Escherichia coli and Staphylococcus aureus were provided by the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea).Trypticase Soy Agar (TSA) was purchased from BD Difco (NJ, USA), and disc paper was obtained from Adabantec (Tokyo, Japan).The TSA culture medium contained 15 g pancreatic digest of casein, 5 g papaic soybean digest, 5 g NaCl, 15 g sodium chloride and 15 g agar in 1 L of distilled water.Microaerophilic conditions were maintained at 37°C.Helicobacter pylori, provided by the Korean Type Culture Collection (KTCC, Daejeon, Korea), were cultured in Brucella broth (Difco, NJ, USA) containing 10% horse serum (Welgene, Daegu, Korea) and, for testing, were grown on a medium prepared with (per liter) BD Bactodextrose (1 g), BD Bactoyeast extract (2 g) (Becton, Dickinson and Company [BD], Franklin Lakes, NJ, USA), sodium chloride (5 g), and sodium bisulfate (0.1 g).Antibacterial activity against S. aureus, E. coli and H. pylori was measured by the disc agar method (Davidson and Parish, 1989).Plates of medium were spread with 0.1 mL of culture broth, and 15 and 30 μg/30 μL of the fractions and compounds were pipetted onto sterile filter paper discs (8 mm).Inhibition zones were determined after 24 h at 37°C.

Cell culture
AGS cells were maintained in RPMI-1940 medium and RAW 264.7 cells were cultured in DMEM containing 100 U•mL -1 of penicillin/streptomycin and 10% FBS at 37°C in a 5% CO 2 incubator.Cells were sub-cultured weekly with 0.05% trypsin-EDTA in phosphate buffered saline.

Cell viability assay
After confluence had been reached, the cells were plated at a density of 5 × 10 4 cells/well into 96-well plates, incubated for 2 h and then treated with LPS (1 μg/mL) and IFN-γ (10 ng/mL).Samples were treated for 24 h.After incubation, cell viability was determined using the MTT assay.MTT solution was added to each 96-well plate, the plates were incubated for 4 h at 37°C, and the medium containing MTT was removed.The incorporated formazan crystals in the viable cells were solubilized with 100 mL of DMSO and the absorbance of each well was read at 540 nm (Mosmann 1983).

Measurement of nitrite
Nitric oxide (NO) production was assayed by measuring the accumulation of nitrite using a microplate assay method based on the Griess reaction (Sreejayan and Rao 1997).RAW 264.7 cells were seeded in 96well plates (5 × 10 4 cells/well) and LPS (1 μg/mL) and IFN-γ (10 ng/mL) were added.After incubating the samples for 24 h, 100 μL of culture supernatant was allowed to react with 100 μL of Griess reagent and the mixture was incubated at room temperature for 15 min.The optical density of the samples was measured at 540 nm using a microplate reader (Chiou et al., 1997).

Inhibition of aldose reductase (AR)
Rat lenses (one lens per 0.5 mL of sodium buffer) were removed from Sprague-Dawley rats (weighing 250-280 g) and preserved until use by freezing.The rat lenses were homogenized and centrifuged at 10 000 rpm (4°C, 20 min) and the supernatant was used as an enzyme source.AR activity was spectrophotometrically determined by measuring the decrease in the absorption of β-NADPH at 340 nm for a 4 min period at room temperature in a quartz cell with dlglyceraldehydes as the substrate (Sato and Kador 1990).The assay mixture contained 0.1 M potassium phosphate buffer (pH 7.0), 0.1 M sodium phosphate buffer (pH 6.2), 1.6 mM β-NADPH, and the test samples (in DMSO), with 0.025 M dl-glyceraldehyde as the substrate.

HPLC analysis
The residue was dissolved in 1 mL of MeOH and then filtered with a Whatman 0.2-μm nylon syringe filter.The resulting solution was used for HPLC analysis.A µBondapak C18 (3.9 × 300 mm, 10 μm) column was used for simultaneous analysis.The mobile phase was 0.05% trifluoroacetic acid + acetonitrile (solvent A) and 0.05% trifluoroacetic acid (solvent B).The elution program used a flow rate of 1 mL/ min and decreased solvent B from 55% to 35% for 35 min, eluted with 35% B for 10 min, and then ran at 0% B for 25 min.The injection volume was 10 μL.The UV chromatograms were recorded at 254-350 nm for analysis.All the injections were performed in triplicate.

Antioxidant activities
The DPPH radical and hydroxyl radical scavenging assay has been used extensively in recent years for evaluating antioxidant activity.DPPH is a stable free radical at room temperature, and produces a violet solution in ethanol.The presence of the antioxidants leads to a less strongly colored solution (Mensor 2001).The DPPH radical contains an odd electron that is responsible for absorbance at 540 nm and for the visible deep purple color.A higher percent in our assay indicates better scavenging activity or an antioxidant potential.As shown in Table 1, G. uralensis (GLs-2, -3, -4, and -5) had a DPPH radical-scavenging activity of more than 70% at concentrations of 100 μg/mL.G. glabra (GL-1) had less scavenging activity (33.33%) at these concentrations.OH is the most reactive species of activated oxygen produced from superoxide (O 2 -) and H 2 O 2 under a variety of stress conditions and is involved in numerous cellular processes including inflammation, cell death and killing of micro-organisms in pathogen-defense reactions (Winterbourn 1981;Halliwell and Gutteridge 1984;Babbs et al., 1989;McCormick et al., 1994).The •OH scavenging activity of GL extracts at a concentration of 100 μg/mL was greater than 75%.G. uralensis (GLs-2, -3, -4, and -5) in particular had a scavenging activity greater than 80% under the same conditions.These results suggest that G. uralensis may be effective scavengers of DPPH and OH radicals.

Anti-bacterial activities
S. aureus is one of the most common Gram-positive bacteria causing food poisoning.It does not originate in the food itself, but from the humans who contaminate foods after they have been processed.E. coli, a Gram-negative bacterium, can also cause serious cases of food poisoning (Rauha et al., 2000).Infection with H. pylori, another Gram-negative bacterium, can lead to a variety of gastrointestinal disorders, including chronic gastritis, peptic ulcer disease and gastric cancer (Kusters et al., 2006); therefore, preservatives to eliminate its growth are needed.The antibacterial activities of the GL extracts against E. coli, S. aureus and H. pylori are shown in Table 2. Results represent the antibacterial effects of GL extracts at concentrations of 15 and 30 μg/30 μL given for 24 h.The largest inhibition zone was observed after treatment with the GL-3 extract (20 mm), which was similar to penicillin (25 mm), used as a positive control against E. coli.GLs-3 and -5 produced S. aureus growth inhibition zones greater than 15 mm.Overall, the largest inhibition zones were observed with the application of 30 μg/30 μL GL-3 against E. coli and S. aureus, which produced 20 and 19 mm clearing, respectively.GLs-1, -3 and -5 produced zones of H. pylori inhibition greater than 13 mm.GL-1 in particular showed significant antibacterial activity, producing an inhibition zone of 15 mm against H. pylori at a concentration of 15 μg/30 μL.These results indicate that GL-3 at the concentration of 30 μg/30 μL is potent against E. coli and S. aureus.GL-1 at a concentration of 15 μg/30 μL had the strongest antibacterial activity against H. pylori.

Anti-inflammatory activity
LPS, a principle component of the outer membrane of Gram-negative bacteria, is an endotoxin (Hewett and Roth 1993).Macrophages activated by inflammatory agents such as LPS are known to produce NO (Moncada and Higgs 1993).LPS and IFN-γ can synergistically stimulate cells to produce a large amount of NO (Nathan 1992).NO is involved in various biological processes including inflammation and immunoregulation (Ialenti et al., 1992;Stichtenoth and Frolich 1998).In order to screen the GL extracts for anti-inflammatory activity, we tested their ability to inhibit the expression of proinflammatory cytokines stimulated by LPS/IFN-γ.As shown in Table 3, there was no significant difference between the normal and control groups in terms of cell viability, which was measured at about 100% and 99.46%, respectively.RAW 264.7 cells were incubated with the inflammatory mediators, LPS and IFN-γ, which induced the generation of NO.Secretion of NO from RAW264.7 macrophage cells in the normal group decreased 47.77% as compared to the control (100%).All extracts markedly inhibited LPS/IFN-γ-induced NO generation to less than 22%.Thus, our results suggest that G. glabra and G. uralensis may be useful in the treatment of inflammatory disease.

Anticancer activity
In previous experiments, we confirmed that the extracts significantly inhibited the growth of H. pylori.We used the MTT method to test whether these extracts moderated the growth of AGS cells.Cell viability was measured by detecting purple formazan that was metabolized from MTT by mitochondrial dehydrogenases, which are active only in live cells.Cells were incubated for one day and then treated with GL extracts (100 μg/mL), which all caused some inhibition of gastric cancer cell growth.In particular, GL-3 had the greatest cancer cell growth inhibiting effect (45.73%) of the various extracts.GL-1 had the least effect, inhibiting AGS cell growth rate by 7.47%.These experiments show that GL-3 (G.uralensis) has  a significant antiproliferative effect on AGS cells (Table 4).

AR inhibition
The MeOH extracts of the GLs were tested for their ability to inhibit rat lens AR activity, and the results are shown in Table 5. GLs-1, -2, -3, -4 and -5 inhibited AR activity by 1.92, 22.78, 61.44, 66.59 and 55.72%, respectively.G. uralensis was a better inhibitor of AR than G. glabra.GLs-3 and -4 were more effective inhibitors than the other GL species, but were still less effective than the positive control, TMG.In a previous study, Daehwanggamchoeumja (Rhei Radix et Rhizoma, Glycyrrhizae Radix, or Glycine max) reduced diabetic metabolic dysfunction (glucose, triglyceride, total cholesterol, HDL cholesterol, total protein, albumin, creatine and blood urea nitrate) (Go et al., 2002).GLs are thus expected to have a certain therapeutic effect on diabetic metabolic dysfunction.
The extracts from G. glabra and G. uralensis display various biological benefits including antioxidant, antibacterial, anti-inflammatory, anticancer and AR inhibitory activities.Taken together, our data suggest that the biological activities of G. uralensis may be stronger than those of G. glabra, except for its antibacterial activity against E. coli.Considering these results, G. uralensis could be useful in the food industry and helpful as a natural supplement in the treatment of a number of diseases.

Table 2 .
Antibacterial activities of GLs.

Table 3 .
Anti-inflammatory activities of GLs.

Table 4 .
Anticancer activities of GLs.