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Journal of Ethnopharmacology
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a r t i c l e i n f o a b s t r a c t
Article history: Ethnopharmacological relevance: Panax ginseng (family Araliaceae) is traditionally used as a remedy for
Received 2 November 2012 cancer, inammation, stress and aging.
Received in revised form Aim of study: To explore whether ginsenosides Rg5 and Rh3, the main constituents of heat-processed
27 December 2012
ginseng (the root of Panax ginseng), could protect memory decit.
Accepted 29 December 2012
Materials and methods: We isolated ginsenosides Rh3 and Rg5 from heated-processed ginseng treated
Available online 9 January 2013
with and without human feces, respectively. Then we investigated their protective effects on memory
Keywords: impairment using the passive avoidance, Y-maze and Morris water maze tasks in mice. Memory decit
Panax ginseng was induced in mice by the intraperitoneal injection of scopolamine.
Araliaceae
Results: Ginsenosides Rg5 or Rh3 increased the latency time reduced by scopolamine in passive
Ginsenoside Rg5
avoidance test. Treatment with ginsenoside Rg5 or Rh3 signicantly reversed the lowered spontaneous
Ginsenoside Rh3
Memory decit-protecting effect alteration induced by scopolamine in Y-maze task. Ginsenoisde Rg5 or Rh3 (10 mg/kg) signicantly
shortened the escape latencies prolonged by treatment with scopolamine on the last day of training
trial sessions in Morris water maze task. Furthermore, ginsenosides Rg5 and Rh3 inhibited acetylcho-
linesterase activity in a dose-dependent manner, with IC50 values of 18.4 and 10.2 mM, respectively. The
inhibitory potency of ginsenoside Rh3 is comparable with that of donepezil (IC50 9.9 mM). These
ginsenosides also reversed hippocampal brain-derived neurotrophic factor (BDNF) expression and
cAMP response element-binding protein (CREB) phosphorylation reduced by scopolamine. Of them,
ginsenoside Rh3 more potently protected memory decit.
Conclusions: Ginsenoside Rg5 and its metabolite ginsenoside Rh3 may protect memory decit by
inhibiting AChE activity and increasing BDNF expression and CREB activation.
& 2013 Published by Elsevier Ireland Ltd.
0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.jep.2012.12.047
E.-J. Kim et al. / Journal of Ethnopharmacology 146 (2013) 294299 295
2.2. Animals
ginsenoside Rg3 and ginsenoside Rg5/Rk1 mixture (1:1, w/w) Passive avoidance task was performed according to previously
isolated from heat-processed ginseng show memory enhancing described (Yang et al., 2009). The apparatus consists of a two-
effect in scopolamine-treated mice (Bao et al., 2005). Ginsenoside compartment acrylic box in which a lighted compartment
Rg3 isolated from red ginseng, and its metabolite ginsenoside (20 20 20 cm) is connected to a dark compartment
Rh2, as well as ginsenoside Rg1 also protected scopolamine- (20 20 20 cm) by an entrance hole (5 5 cm). Briey, in the
induced memory decit in mice (Yang et al., 2009; Fang et al., acquisition trial a mouse was placed in the lighted chamber, and
2012). However, when ginsenoside Rg5 was incubated with when the mouse entered the dark chamber, a 0.3 mA electrical
human feces, it was metabolized to ginsenoside Rh3 (Bao et al., shock of 2 s durations was delivered through oor grids. Ginseno-
2005; Shin et al., 2006). Nevertheless, the neuroprotective effect side Rg5, Rh3 (5, 10 and 20 mg/kg, p.o.) or donepezil (5 mg/kg,
of ginsenoside Rh3 has not been studied. p.o.) as a positive control were orally given 1 h before treatment
During a screening program for Chinese traditional medicines to with scopolamine. Memory impairment was induced by treat-
protect memory impairment, heat-processed ginseng and its main ment with scopolamine (1 mg/kg, i.p.) and the maze task was
constituent ginsenoside Rg5 potently protected scopolamine- performed 30 min after treatment with scopolamine. A retention
induced memory decit in mice. Therefore, we investigated the trial was performed 24 h after the acquisition trial, and latency
protective effect of ginsenoside Rg5 or its metabolite ginsenoside times to reenter the dark chamber were measured. The success
Rh3 against memory decit in scopolamine-treated mice. rate was then calculated as the number of mice that did not enter
the dark compartment divided by the total number of mice and
expressed as a percentage (%). The maximum entry latency time
2. Materials and methods allowed in the acquisition session and retention session was 180
and 300 s, respectively.
2.1. Materials
2.5. Y-maze task
() Scopolamine hydrobromide, acetylthiocholine (ATCh), 5,50 -
dithiobis-(2-nitrobenzoic acid) (DTNB), acetylcholine esterase Y-maze is a three-arm horizontal maze (40 cm-long and 3 cm-
(AChE, electric eel type VI-S) and dimethylsulfoxide (DMSO) wide with 12 cm-high walls) in which the arms are symmetrically
were purchased from Sigma (St. Louis, MO, USA.). Antibodies disposed at 1201 angles from each other. The maze oor and walls
against brain-derived neurotrophic factor (BDNF), cAMP response were constructed from dark opaque polyvinyl plastic as has been
element-binding protein (CREB), p-CREB and b-actin were pur- described previously (Joh et al., 2012). Mice were initially placed
chased from Santa Cruz Biotechnology (Santa Cruz, L.A. U.S.A.). within one arm, and the sequence (i.e., ACABC, etc) and number of
The protease inhibitor cocktail was purchased from Roche Applied arm entries were recorded manually for each mouse over 8 min
Science (Mannheim, Germany). Immobilon-P nylon membrane was period. An actual alternation was dened as entries into all three
from Millipore Co. (Billerica, MA, U.S.A.). Polyvinylidene diuoride arms on consecutive choices (i.e., ABC, CAB or BAC but not ABA).
(PVDF) membrane and enhanced chemiluminescence (ECL) detection Maze arms were thoroughly cleaned between tasks to remove
296 E.-J. Kim et al. / Journal of Ethnopharmacology 146 (2013) 294299
residual odors. Ginsenoside Rg5, Rh3 (5, 10 and 20 mg/kg, p.o.) or (ANOVA) followed by the StudentNewmanKeuls test for multi-
donepezil (5 mg/kg, p.o.) as a positive control were orally given ple comparisons. Latency in the Morris water maze task was
1 h before treatment with scopolamine. Memory impairment was analyzed using two-way ANOVA followed by Tukeys analysis
induced by treatment with scopolamine (1 mg/kg, i.p.) and the with the day as one variable and treatment as a second. Statistical
maze task was performed 30 min after treatment with scopola- signicance was set at p o0.05, po0.001 and po0.001.
mine. The percentage of alternations was dened according to the
following equation: % alternation[(number of alternations)/
(total arm entries 2)] 100. The number of arm entries served 3. Results
as an indicator of locomotor activity.
In the preliminary experiment, we found that red ginseng, of
2.6. Morris water maze task which main constituent is ginsenoside Rg3, and heat-processed
ginseng, of which main constituent is ginsenoside Rg5, potently
Morris water maze is a circular pool (90 cm in diameter and protect scopolamine-induced memory decit in mice. Of them,
45 cm in height) with a featureless inner surface (Morris, 1989). heat-processed ginseng more potently improved the memory
The circular pool was lled to a depth height of 30 cm with water decit. Therefore, we isolated ginsenoside Rg5 from heat-
(20 71 1C), in which 500 mL of milk was mixed. A white platform processed ginseng and its metabolite ginsenoside Rh3 and mea-
(6 cm in diameter and 29 cm in height) was centered in one of sured their AChE-inhibitory effects. Ginsenosides Rg5 and Rh3
four quadrants of the pool (southwest area) and submerged 1 cm inhibited AChE activity in a dose-dependent manner, with IC50
below the water surface so that it was invisible at water level. The values of 18.4 and 10.2 mM, respectively (Fig. 1B). The inhibitory
rst day of the experiment was dedicated to swimming training potency of ginsenoside Rh3 is comparable with that of donepezil
for 60 s in the absence of the platform. In the following days the (IC50 9.9 mM).
mice were given the training trial sessions of four trials each day Next, we investigated their protective effects against
for four consecutive days. During each trial, the mouses escape scopolamine-induced memory decit in the passive avoidance
latency, measured with a stop watch, were recorded. This para- task. The step-through latency of mice treated with scopolamine
meter was averaged for each session of trials and for each mouse. alone (control group) was signicantly shorter than that of
Once the mouse located the platform, it was permitted to remain vehicle-treated mice (normal group) (po0.001) (Fig. 2A). Treat-
on it for 10 s. If the mouse did not locate the platform within 60 s, ment with ginsenoside Rg5 or Rh3 (5, 10 and 20 mg/kg) increased
it was placed on the platform for 10 s. During this period, the
platform was located in a xed position. The animal was taken to
its home cage and was allowed to dry up under an infrared lamp
after each trial. The time interval between each trial was 30 s. On
the day following the last day of training, mice were given a probe
trial session which consisted of removing the platform from the
pool and allowing the mice to swim for 60 s in search of it. A
record was kept of the swimming time in the pool quadrant
where the platform had previously been placed. Ginsenoside Rg5,
Rh3 (5 and 10 mg/kg, p.o.) or donepezil (5 mg/kg, p.o.) as a
positive control was given 1 h before treatment with scopola-
mine. Memory impairment was induced by treatment with
scopolamine (1 mg/kg, i.p.) and the rst trial session was per-
formed 30 min after treatment with scopolamine.
2.7. Immunoblotting
Fig. 4. Effects of ginsenosides Rg5 and Rh3 on the expressions of p-CREB, CREB and BDNF in the hippocampi of scopolamine-treated mice. Effects on CREB phosphorylation
and BDNF expression were detected by immunoblotting. Ginsenoside Rg5, Rh3 (5, 10 and 20 mg/kg, p.o.) or donepezil (DNZ, 5 mg/kg, p.o.) was orally given 1 h before
treatment with scopolamine. Memory impairment was induced by treatment with scopolamine (1 mg/kg, i.p.) 1 h after the nal administration of test agents and the
passive avoidance task was performed 30 min after treatment with scopolamine. Then mice were sacriced 30 min after passive avoidance task. Then the mice were
sacriced within 30 min and then the hippocampus tissues were isolated. The levels of BDNF, p-CREB and b-actin were measured by immunoblotting. Values are expressed
as mean 7 SEM (n 4). #Signicantly different from normal group in the level of BDNF, CREB or p-CREB (]P o0.05). nSignicantly different from scopolamine-treated
control group in the level of BDNF, CREB or p-CREB (nPo 0.05).
298 E.-J. Kim et al. / Journal of Ethnopharmacology 146 (2013) 294299
4. Discussion
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