Extract
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Introduction
Several studies have reported that plant extracts have protective effects against ischemic damage in several organs such
as the brain, heart and kidneys[1_3]. Licorice or
Glycyrrhiza inflata, which is a commonly used herb, has a history of
consumption for a few thousand years in both Eastern and Western cultures. It has been reported that major bioactive
components of licorice are saponins, such as glycyrrhizin and glycyrrhetinic acids. In previous studies, it has been reported
that glycyrrhizin has various desirable pharmacological properties such as anti-inflammatory
effects[4], anti-viral
effects[5] and free radical scavenging
activity[6]. In recent studies it has been shown that
18-b glycyrrhetinic acid, a major component of licorice,
reduces infarct size in isolated rabbit
hearts[7], and that glycyrrhrizin shows a protective effect on ischemia/reperfusion or
nephrotoxic injuries[8,9]. However, in some studies it was found that licorice interferred with steroid metabolism and might
cause edema and hypertension[10].
The roasting process of licorice modifies the chemical composition and reduces the toxicity
level[11]. Roasting of licorice under controlled conditions, including the roasting temperature and duration, results in the conversion of glycyrrhrizin to
18-b glycyrrhetinic acid[12]. Despite the considerable number of physiological studies that have been done on licorice, a
comparative study between licorice and roasted licorice during
in vivo ischemia has not been carried out. In the present
study, therefore, we investigated the neuroprotective effects of licorice and roasted licorice on neuronal injury in the gerbil
hippocampus induced by transient ischemia.
Materials and methods
Reagents Dried licorice roots were obtained from Dea Guang Medical (Chunchon, South Korea). Acetic acid was
obtained from Merk (Ger, Germany). Glycyrrhetic acid (GA), glycyrrhetic acid monoglucuronide (GM) and glycyrrhizin (GL)
were obtained from Sigma-Aldrich (St Louis, MO, USA). Acetonitrile was of HPLC grade, and all other chemicals were of
analytical grade.
Roasting of licorice and GL Roasting of licorice was performed in a controlled manner using an oil bath. For dry-roasting,
20 g of sliced licorice was weighed into a rotary glass bottle (30 r/min) and placed in the heated oil bath at 150
ºC for 100 min. To determine whether roasting of licorice caused the thermal decomposition of GL, the decomposition of GL in powder form was
also studied by heating. For roasting, 10 mg of powder was weighed into a glass vial and placed in the heated oil bath at 150
ºC for 30 min.
Preparation of samples of licorice and GL before and after roasting
The GL samples (raw and roasted) were dissolved in
methanol and the solutions were membrane-filtered (0.45 µm). After dilution with methanol, the final concentrations of the
prepared raw and roasted GL solutions for HPLC analysis were 1 and 5
mg/mL, respectively. Roasted licorice roots were first
ground into fine powder using a laboratory blender (Waring Model 51BL30). The licorice powder (10 g) was refluxed in 50 mL
of 95% ethanol for 2 h. Boiling stones were used during reflux to minimize bumping and sample loss. This extraction
procedure was repeated three times. For comparison, 10 g of unprocessed licorice (raw licorice) was refluxed similarly. The
ethanol extract was dried under vacuum at a low temperature (below 40 ºC). The final concentrations of the prepared licorice
solutions for HPLC analysis were 5 mg/mL.
HPLC analysis HPLC system consisted of two models of P-580 pumps, a model of ASI-100 Automated sample injector,
a model of STH-585 column oven, and a model of UVD 170S UV-detector (Dionex, USA). The sample was separated on a
reverse-phase Phenomenex Luna C18 column (4.6 mm×250 mm ID; 5
µm). The mobile phases consisted of 2% acetic acid and
acetonitril as mobile phase A and B, respectively, which were degassed ultrasonically prior to use. The gradient was as
follows: an isocratic elution with 20% acetonitril for 5 min, followed by a linear gradient elution with 20%_90% acetonitril for
50 min. The overall analysis time including re-equilibration was 65 min. The column was thermostated at 28 ºC and a flow-rate
of 0.8 mL/min was used. UV detection was operated at 254 nm.
Culture of PC12 cells PC12 cells were grown in Dulbecco¡¯s modified Eagle¡¯s
medium.
(DMEM) supplemented with 7% fetal calf serum, 7% horse serum,
100 µg/mL streptomycin, and 100
U/mL penicillin. The cell cultures were
incubated at 37 °C in an atmosphere of 6%
CO2. Medium was changed twice
weekly, and the cultures were split at a 1:6 ratio once a week. Cells were washed twice with warm DMEM (without phenol red), then treated with serum-free
medium. In all experiments, cells were treated with licorice before hypoxia.
Hypoxia On the day of experiment, culture media were replaced with glucose-free DMEM, then gassed with 85%
N2, 10 % H2, and 5 %
CO2 for various periods in the absence or presence of various doses of licorice extract (raw and roasted).
Lactate dehydrogenase (LDH) release
assay After hypoxia for 1 or 2 h, the supernatant of cultured PC12 cells was
collected for assay of LDH release. The reaction was initiated by mixing 0.1 mL of cell-free supernatant with potassium
phosphate buffer containing nicotinamide adnine dinucleotide (NADH) and sodium pyruvate in a final volume of 0.2 mL in a
96-well plate. The rate of absorbance was read at 490/630 nm on an automated SpectraMAX 340 microtiter plate reader. Data
were expressed as the mean percent of viable cells versus hypoxia control.
Experimental animals The progeny of male Mongolian gerbils
(Meriones unguiculatus) were obtained from the
Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were housed in a temperature
(23 ºC) and humidity (60%) controlled room with a 12-h light/12-h
dark cycle and provided with food and water ad
libitum. Experimental procedures and animal care conformed to the Institutional Guidelines that are in compliance with current
international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication
No 85_23, 1985, revised 1996) and are approved by the Hallym¡¯s Medical Center Institutional Animal Care and Use Committee. All of the
experiments were conducted to minimize the number of animals used and the amount of suffering.
Induction of ischemia The gerbils were divided into 4 groups: normal group, vehicle (saline)-treated group, raw
licorice-treated group and roasted licorice-treated group. At least 21 d before surgery, 50 and 100 mg/kg of the extract of raw or
roasted licorice were injected orally using the jonde every day until gerbils were killed. Gerbils weighing 65_75 g were
anesthetized with a mixture of 2.5% isoflurane (Baxtor, USA) in 33% oxygen and 67% nitrous oxide. A midline ventral incision
was made in the neck. Both common carotid arteries were isolated, freed of nerve fibers, and occluded with non-traumatic
aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in the eyeball using an
ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. Restoration
of blood flow (reperfusion) was observed under the ophthalmoscope. We maintained the body (rectal) temperature under
free-regulating or normothermic (37±0.5 ºC) conditions with a rectal temperature probe (TR-100; YSI, USA) and thermometric
blanket before, during, and after the surgery until the animals fully recovered from anesthesia. Normal animals served as
controls.
Histological analysis by cresyl violet staining
Seven animals in each group were anesthetized with pentobarbital sodium
and perfused transcardially with 0.1 mol/L phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1
mol/L PBS (pH 7.4) at the designated times after the surgery. Brains were removed and post-fixed in the same fixative for 6 h.
The brain tissues were cryoprotected by infiltration with 30% sucrose solution overnight. Thereafter the tissues were frozen
and serially cut into 30-µm thick coronal sections on a cryostat and the sections were
collected in 6-well plates containing PBS. The sections were stained with cresyl violet acetate according to the previously published
procedures[13].
The number of cresyl violet-positive neurons was counted by two blinded observers at the same time using an image
analyzing system equipped with a computer-based CCD camera (Software: Optimas 6.5, CyberMetrics, USA). The number of
cresyl violet-positive neurons in a 1-mm diameter of the hippocampus was counted in 10 sections for each animal. The
number of cresyl violet-positive neurons was compared to that of the sham-operated group.
Assay of SOD1 activity Seven animals in each group were used for SOD1 activity measurement. The activity was
measured by monitoring the capacity to inhibit the reduction of ferricytochrome c by xanthine/xanthine oxidase as described
by McCord and Fridovich[14]. The samples were separated by electrophoresis in 10% native polyacrylamide
gels and visualized as described by Beauchamp and
Fridovich[15]. Briefly, the gel was soaked in 2.45 mmol/L nitroblue tetrazolium solution for 15 min,
and then in 28 mmol/L
N,N,N¡¯,N¡¯-tetramethylethylene diamine, 28 µmol/L riboflavin, and 0.36 mmol/L potassium phosphate
buffer (pH 7.8) for 30 min. The gel was then exposed to a fluorescence light source until the bands showed maximum
resolution.
Western blotting of SOD1 protein Seven animals in each group (the same animals as in the analysis of SOD1 activity)
were used for an immunoblotting study. The hippocampus were removed and sectioned into
400-mm thick coronal slices on a Vibratome (Leica, Germany), and the hippocampal CA1 region was dissected with a surgical blade. The tissues were
homogenized using an electrical homogenization machine in 50 mmol/L PBS (pH 7.4) containing 0.1 mmol/L egtazic acid (pH
8.0), 0.2% NP-40, 10 mmol/L edetic acid (pH 8.0), 15 mmol/L sodium
pyrophosphate, 100 mmol/L b-glycerophosphate, 50
mmol/L NaF, 150 mmol/L NaCl,
2 mmol/L sodium orthvanadate, 1 mmol/L PMSF, and 1
mmol/L DTT. After centrifugation at 10
000×g, the protein concentration was determined in the supernatants by using the Micro BCA protein assay kit with bovine serum albumin as the standard
(Pierce Chemical, USA). Aliquots containing 20 µg total protein were boiled in
loading buffer containing 150 mmol/L Tris (pH 6.8), 3
mmol/L DTT, 6% SDS, 0.3% bromophenol blue, and 30% glycerol. Each aliquot was then loaded onto a 10% polyacryamide
gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher and Schuell, USA). To
reduce background staining, the filters were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min,
followed by incubation with rat anti-mouse SOD1 antiserum (1:400) with peroxidase conjugated horse anti-mouse IgG (Sigma,
USA), and then with ECL kit (Amersham, USA).
For quantitative analysis of the Western band of SOD1 in the hippocampus, video images were digitized into an array of
512 pixels¡Á512 pixels. Each pixel resolution was of 256 gray levels. The intensity of western band of SOD1 was
expressed as a relative optical density (ROD) value which was transformed from mean gray values using the formula: ROD=lg
(256/mean gray). A background parameter was obtained from each section out of the immunolabeled structures and
subtracted from obtained ROD values of each group. ROD values are informed as ROD units. The bands of Western blot study
were scanned and a ROD value was
obtained using Scion Image software (Scion Corp, USA).
Statistical analysis Inter-animal differences in each group, as well as inter-experiment differences, were not statistically
significant. Values shown represent the mean of experiments performed for each hippocampal area. All data obtained from
the quantitative data are expressed as mean±SD and analyzed using one-way ANOVA to determine statistical significance.
Bonferroni¡¯s test was used for post-hoc comparisons.
P<0.05 or 0.01 was considered statistically significant.
Results
Effects of roasting on licorice and GL
The major peaks in licorice extracts were completely separated by HPLC analysis.
The comparative non-polar components which were eluted after 30 min were significantly increased. The untreated GL
showed only the GL peak. After being roasted at 150
°C for 30 min, two new peaks appeared (Figure 1). Upon spiking with
standard solution (GA and GM), the new peak was identified to have resulted from GA and GM (data not shown). The
appearance of GM and GA was also confirmed by the UV contour plot obtained by the PDA detector (data not shown). The
fact that the formation of GM and GA was attributed to thermal decomposition of GL was the same as previous
reports[12,15]. It was reported that sugar chains in the saponin and glycosidic flavonoid constituents in licorice were hydrolyzed
step-by-step during roasting through
hydrothermolysis[16].
LDH release inhibition by raw and roasted licorice
Raw and roasted licorice effectively protected PC12 cells from
hypoxic damage. In the raw licorice-treated group, LDH release was decreased by 9%_33% at concentrations of 10_1000
µmol/L, while in the roasted licorice-treated group, LDH release was decreased by 17%_49% at concentrations of 10_1000
µmol/L (Figure 2).
Protective effects of raw and roasted licorice on ischemic pyramidal cells
In the vehicle-treated group, the percentage of cresyl violet-positive pyramidal cells in the CA1 region was 11.5% compared with the control group at d 4 after
ischemia/reperfusion (Figures 3B, 4B, 5). In 50 mg/kg raw licorice-treated group, the number of cresyl violet-positive neurons showed
no difference to that of the vehicle-treated group
(P>0.05; Figures 3E, 4E, 5). In 100 mg/kg raw licorice-treated group, the
number of cresyl violet-positive neurons was slightly increased
(P<0.05; Figures 3F, 4F, 5).
In contrast, in the roasted licorice-treated group, abundant CA1 pyramidal cells were detected in the hippocampal CA1
region after ischemia/reperfusion. In the 50 mg/kg roasted licorice-treated group, approximately 71.4% of CA1 pyramidal cells
were stained with cresyl violet (Figures 3C, 4C, 5). In the 100 mg/kg roasted licorice-treated group, approximately 66.4% cresyl
violet-positive neurons were detected in the striatum pyramidal of the CA1 region (Figures 3D, 4D, 5).
Changes in SOD1 activity In the vehicle-treated group, SOD1 activity was significantly decreased compared with the
control group. The SOD1 activity was slightly increased in the raw licorice-treated groups and significantly increased in the
roasted licorice-treated groups compared with that in vehicle-treated group (Figure 6).
Changes in SOD1 protein contents In the vehicle-treated and raw licorice-treated groups, SOD1 protein contents were
significantly decreased compared with the control group
(P<0.05). But in the roasted licorice-treated groups, SOD1 protein contents were not significantly altered compared with the
control group (P>0.05; Figure 7).
Discussion
In the present study, we examined the effects of raw or roasted licorice on LDH release in normoxic and hypoxic PC12 cells
because the release of LDH implied neuronal damage. We found that roasted licorice at 50_500 µmol/L significantly reduced
LDH release in hypoxic PC12 cells. A weak, but statistically significant protection was also observed in hypoxic PC12 cells
treated with 100 µmol/L of raw licorice. In addition, 100_1000 µmol/L of raw or roasted licorice caused cytotoxic effects in
normoxic PC12 cells. Therefore, the optimal concentration of neuroprotection for raw and roasted licorice was 50_100
µmol/L. In addition, we found that 50 mg/kg or 100 mg/kg of raw or roasted licorice, which is the commonly used dose in herbal
medicine, exerted neuroprotective effects in an
in vivo ischemic model.
We found that roasted licorice had a neuroprotective effect against transient forebrain ischemia. However, raw licorice
had no significant effect against ischemic damage. In the process of roasting licorice, almost all polar components were not
altered, while the non-polar components were significantly increased. In previously published studies, it has been reported
that licorice root has an effect on the anti-apoptotic protein Bcl-2, which is a 26-kDa protein that blocks cell death by
inhibiting cytochrome c release from mito-chondria, a critical event in the apoptotic
pathway[17, 18]. In our study, SOD1 activity and protein content in the roasted licorice-treated group were significantly increased compared to those in the
vehicle-treated group. This result suggests that roasted licorice has neuroprotective effects through antioxidant activity.
The biopharmaceutical properties of roasted licorice have been previously examined to clarify the influence of roasting on
the bioavailability of glycyrrhizin after oral administration of the extract. Among polar components of licorice, glycyrrhizin is
a major component. It is taken orally and is transformed (hydrolysed) by intestinal bacteria into the active metabolite
glycyrrhetic acid, non-polar
component[19]. This glycyrrhetinic acid is widely used as a gap junction inhibitor that is effective
in the micromolar concentration range. Glycyrrhetinic acid seems to act through changes in phosphorylation and/or connexin
assembly and inhibition of sarcolemmal
Ca2+ currents and of mRNA synthesis. In our study, we also observed that the
increased non-polar compounds found in roasted licorice contained glycyrrhizin-degraded products such as glycyrrhetinic
acid and glycyr-rhetinic acid monoglucuronide.
We observed in the present study that 18-b glycyrrhetinic
acid strongly protected hippocampal CA1 pyramidal neurons
from ischemic damage. This result is supported by a previous report that carbenoxolone, the succinyl ester of
18-b glycyrrhetinic acid has protective effects against
ischemic damage in middle cerebral artery occlusion
models[20].
In conclusion, roasted licorice has a significant neuro-protective effect against ischemic damage through anti-
oxidant effects. In addition, the neuroprotective effect of roasted licorice is associated with non-polar compounds including
glycyrrhetinic acid monoglucuronide and its degradation product, glycyrrhetinic acid.
Acknowledgements
The authors would like to thank Mr Suek HAN, Mr
Seung-uk LEE and Ms Hyun-sook KIM for their technical help during this
study.
References
1 Peng H, Li YF, Sun SG. Effects of Ginkgo
biloba extract on acute cerebral ischemia in rats analyzed by magnetic resonance spectroscopy.
Acta Pharmacol Sin 2003; 24: 467_71.
2 Zhang YY, Li PF, Li D. Effect of Ginkgo
biloba leaf extract on electroencephalography of rat with cerebral ischemia and reperfusion.
Acta Pharmacol Sin 2003; 24: 157_62.
3 Wong TM, Wu S, Yu XC, Li HY. Cardiovascular actions of Radix Stephaniae Tetrandrae: a comparison with its main component,
tetrandrine. Acta Pharmacol Sin 2000; 21: 1083_8.
4 Kang JS, Yoon YD, Cho IJ, Han MH, Lee CW, Park SK,
et al. Glabridin, an isoflavan from licorice root, inhibits inducible nitric-oxide
synthase expression and improves survival of mice in experimental model of septic shock. J Pharmacol Exp Ther 2005; 312: 1187_94.
5 Ohuchi K, Kamada Y, Levine L, Tsurufuji S. Glycyrrhizin inhibits prostaglandin
E2 production by activated peritoneal macrophages from
rats. Prostaglandins Med 1981; 7: 457_63.
6 Abdugafurova MA, Li VS, Sherstnev MP, Atanaev TB, Isamuk-harmedov AS, Bachmanova GI. Antioxidative properties of glycyrrhyzic
acid salts and their effect on the liver monooxygenase system. Vopr Med Khim 1990; 36: 29_31.
7 Miura T, Ohnuma Y, Kuno A, Tanno M, Ichikawa Y, Nakamura Y,
et al. Protective role of gap junctions in preconditioning against
myocardial infarction. Am J Physiol Heart Cir Physiol 2003; 286: H214_21.
8 Nagai T, Egashira T, Yamanaka Y, Kohno M. The protective effect of glycyrrhizin against injury of the liver caused by
ischemia-reperfusion. Arch Environ Contam Toxicol 1991; 20: 432_6.
9 Yokozawa T, Liu ZW, Chen CP. Protective effects of
Glycyrrhizae Radix extract and its compounds in a renal hypoxia
(ischemia)-reoxygenation (reperfusion) model. Phytomedicine 2000; 6: 439_45.
10 Stewart PM, Wallace AM, Valentino R, Burt D, Shackleton CH, Edwards CR. Mineralocorticoid activity of liquorice: 11-beta-hydroxysteroid
dehydrogenase deficiency comes of age. Lancet 1987; 2: 821_4.
11 Majima T, Yamada T, Tega E, Sakurai H, Saiki I, Tani T. Pharmaceutical evaluation of liquorice before and after roasting in mice. J Pharm
Pharmacol 2004; 56: 589_95.
12 Sung MW, Li PC. Chemical analysis of raw, dry-roasted, and honey-roasted licorice by capillary electrophoresis. Electrophoresis 2004;
25: 3434_40.
13 Hwang IK, Eum WS, Yoo KY, Cho JH, Kim DW, Choi SH,
et al. Copper chaperone for Cu,Zn-SOD supplement potentiates the
Cu,Zn-SOD function of neuroprotective effects against ischemic neuronal damage in the gerbil hippocampus. Free Radic Biol Med 2005; 39:
392_402.
14 McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244:
6049_55.
15 Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971; 44:
276_87.
16 Kuwajima H, Taneda Y, Chen WZ, Kawanishi T, Hori K, Taniyama T,
et al. Variation of chemical constituents in processed licorice roots:
quantitative determination of saponin and flavonoid constituents in bark removed and roasted licorice roots. Yakugaku Zasshi 1999; 119:
945_55.
17 Reed JC. Double identity for proteins of the Bcl-2 family. Nature 1997; 387: 773_6.
18 Rafi MM, Vastano BC, Zhu N, Ho CT, Ghai G, Rosen RT,
et al. Novel polyphenol molecule isolated from licorice root (Glycrrhiza glabra)
induces apoptosis, G2/M cell cycle arrest, and Bcl-2 phosphorylation in tumor cell lines. J Agric Food Chem 2002; 50: 677_84.
19 Akao T, Hayashi T, Kobashi K. Intestinal bacterial hydrolysis is indispensable to absorption of
18b-glycyrrhetic acid after oral administration of glycyrrhizin in rats. J Pharm Pharmacol 1994; 46: 135_7.
20 Hosseinzadeh H, Nassiri Asl M, Parvardeh S. The effects of carbenoxolone, a semisynthetic derivative of glycyrrhizinic acid, on peripheral
and central ischemia-reperfusion injuries in the skeletal muscle and hippocampus of rats. Phytomedicine. 2005;12:
632-7.
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