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The effect of Ginseng Jawa (Talinum paniculatum

Gaertn.) root on the thickness of CA1 pyramidal


lamina of rat hippocampus
Dwi Cahyani Ratna Sari1, Soedjono Aswin1, Sri Suharmi2, Untung Tranggono3, Mansyur Romi1
1
Department of Anatomy, Embryology and Anthropology, Faculty of Medicine, Universitas Gadjah
Mada, Yogyakarta, Indonesia
2
Department of Pharmacy, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia
3
Department of Surgery, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

ABSTRACT
Introduction: Ginseng Jawa (Talinum paniculatum Gaertn.) is the traditional plant which has similar
component to Panax ginseng. It is assumed that it has the same effect to the memory processing in
the brain especially in the hippocampus neuron.
Objective: To reveal the effect of ethanol extract of Ginseng Jawa root on the thickness of CA 1
pyramidal lamina of the hippocampus.
Methods: Three-month old male rats (n=42) 150-200 g body weight, were divided randomly into six
groups, i.e. the control group (KN1 and KN2) administered with aquadest and propylen glikol
respectively. Treated group (P1, P2, and P3) administered with etanol extract of Ginseng Jawa root
orally, at doses 6, 12, and 24 mg/rat, the positive control group (KP) received ethanol extract of
Panax ginseng root at 12 mg/rat. After three days of rehearsal followed by twelve days of test of
eight-arm radial maze, the rats were decapitated, and the brains were collected for histological
preparation. The hippocampal sections were stained with Tholuidin Blue 1 % and the thickness of
CA1 pyramidal lamina of hippocampus were measured. Data was analyzed statistically with KruskalWallis and Mann Whitney test.
Results: The thickness of CA1 pyramidal laminas of hippocampus were 42,989,54 m (KN1);
46,559,99 m (KN2); 46,1911,39 m (P1), 64,5811,25 m (P2) and 70,0014,56 m (P3);and
62,6212,81 m (KP).There was significant effect for the thickness of CA 1 pyramidal lamina of
hippocampus (p<0,05) at 12 mg/rat and 24 mg/rat doses of Ginseng Jawa root compared with the
negative control group. Using thin layer-chromatography, terpenoid saponins were found as the main
active constituents in Ginseng Jawa root
Conclusions: Ginseng Jawa root administered orally increases the thickness of pyramidal lamina
CA1 of the hippocampus at 12 mg/rat and 24 mg/rat.
Key words: Ginseng Jawa, memory, CA1 pyramidal lamina, Hippocampus, Rat

INTRODUCTION
Panax ginseng has been known since thousands years ago as traditional herb
plant used as tonic and aphrodisiac. In addition, a variety of research has proven the
influence of Panax ginseng on the cardiovascular system, central nervous system, and
immune system1, 2.
In Indonesia, traditional herb that similar to Panax ginseng is Ginseng Jawa
(Talinum paniculatum Gaertn.). Research on Ginseng Jawa roots have been done, and
proved to have the benefits of anti-inflamation 3, increasing motility of spermatozoa4,
anti-diarrhea5, and as stimulant nervous system 6. Syamsuhidayat and Hutapea7 (1997)

classify Talinum paniculatum Gaertn. as the following: Division: spermatophyta;


Subdivision: angiospermae; class: dicotyledone; subclass: monochlamydaeae; nation:
caryophyllales; tribe: portulaceae; genus: talinum; type: Talinum paniculatum Gaertn.
In taxonomy, the tribe of Ginseng Jawa and Panax ginseng are portulaceae and
araliaceae, respectively. However, there are some similarities between them, i.e: 1)
plant morphology, particularly the root of Ginseng Jawa shows similarities with Panax
ginseng8, 2) both commonly used as medications (aphrodisiac and tonic) 9, 3) Analysis
method with thin-layer-chromatography (TLC) showed at least there are two
compounds that are almost the same between them, terpenoid and steroid group, both
included in the saponin compound 10.
Panax ginseng contains three kinds of specific classes of terpenoid compounds
called ginsenosida, panaxosida and chiketsusaponin. Panax ginseng at least contained
77 of ginsenosida. The ginsenosida Rb-1 and ginsenosida Rg-1 have been known to
improve the learning and memory function, increase the synthesis and release of
acetylcholin11, 12, whereas the number of synapses in the hippocampus retained by
acetylcholin and serotonin13. Ginsenosida Rb-1 and Rg-1 also spurred the metabolism of
nucleotide and protein of the brain, as well as serve as a antioxidant11.
Hippocampus was involved in the change of short-term memory (more than
sixty minutes) into long-term memory (several days or more) 12, 13. There was evidence
that in patients which have been taken their hippocampus bilaterally shows amnesia
anterograd, where event before surgery was maintained but the new long-term memory
could not be construct13. Hippocampus also suggested storing long-term memory for
several weeks and transferring it to a specific memory region in the cerebral cortex 14,
involving the long-term potentiation (LTP). LTP is stimulation on line afferen to the
hippocampus,

which

increase

excitatory

potential

synaptic

on

postsynaptic

hippocampus neuron, which can end up until a few hours, days, or weeks. Those aferen
pathway which started in the subiculum to CA1 regions, namely: (1) the perforant
pathway (from the subiculum to cell granuler gyrus dentatus), (2) the mossy fiber
pathway (of the granuler gyrus dentatus to CA 3 pyramidal cells hippocampus), (3) the
Schaffer's collaterals (pyramidal cells of CA3 to CA1 hippocampus) 14. Changes to the
LTP may lead to changes in memory. Previous research has proven the correlation
between the memory and hippocampus, which the increase of memory accompanied

with an increase in thickness in pyramidal lamina CA 1 hippocampus on the awarding of


estrogen12.
The similarity of components between Ginseng Jawa and Panax Ginseng on
contents of the saponin10 raises an argument about the possibility of their similar effect
on the process of memory in the brain. Research on Ginseng Jawa has been done so far
mostly to identify the compounds contained in it and

has not been yet identify the

active substances which could enhance memory similar to that of in Panax ginseng.
Therefore this study aimed to reveal the effect of ethanol extract of Ginseng Jawa roots
on the thickness of CA1 pyramidal lamina of the rat hippocampus.
METHODS
Three-month old male rats (Rattus norvegicus) obtained from the Development
Unit of Animal Experimental (UPHP), strain Wistar, n=42, 150-200g body weight, were
kept in wire cages sized 40x40x20 cm3, each containing three and four rats. Food, in the
form of pellets and drinking water provided daily for ad libitum.
The root of Ginseng Jawa is taken from the Medicinal Plant Research Center
(BPTO) Tawangmangu, Surakarta. Ethanolic extraction of the root of Ginseng Jawa was
performed in the way specified in the Pharmacopoeia Indonesia Ed. III with some
modification15. The root of Ginseng Jawa was cleaned then dried with a dryer at
temperatures of 50 degrees Celsius, than it was made into powder with a certain degree
of fineness.
Panax ginseng root extractdose16 given to rats was 8 mg per day for 12-33 days.
Based on that experiment, this study administered variation doses of ethanol extract of
Ginseng Jawa root at 6 mg, 12 mg, and 24 mg per day orally for 18 days, using a simple
experimental post-test only control group design17. A total of 42 rats were divided
randomly into six groups as follows: (1) negative control group 1 (KN1) with 1 ml of
distilled water orally, (2) negative control group 2 (KN2) with 1 ml of propylene glycol
orally, (3 ) treatment group (P1) ethanol extract of ginseng Jawa root 6 mg / rat orally,
(4) treatment group (P2) ethanol extract of ginseng Jawa root 12 mg / rat orally, (5)
treatment group (P3) ethanol extract of ginseng Jawa root 24 mg / rat orally, and (6)
positive control group (KP) with ethanol extract of the roots of Panax ginseng 12 mg /
rat orally. After treatment for 18 days, experimental animals underwent 8-radial arm-

maze test, to investigate on memory performance, for three days followed by a real test
for twelve days18.
After measurements of memory performance, the experimental animals were
decapitated, than taken for preparation of microscopic specimen. Location of cerebral
cutting was on the cerebrum area 3.3 mm posterior to bregma19. Brain tissue grown in
tragachant then immersed into nitrogen liquid20 for 25 to 35 seconds, then placing inside
the refrigerator at a temperature of 25oC. Tissue was cut using a microtome with a
thickness of approximately 6 to 8 m, then attached it on the object glass, to be stained
with toluidine blue 1%. Pyramidalis cells will appear bright blue. The thickness of the
lamina pyramidalis CA1 hippocampus was measured using a micrometer on a light
microscope. Measurements made on the medial, top, and the lateral part of CA1
hippocampus. From each rat were made 12 observations in the 4-5 slides that contain 34 preparations lamina pyramidalis CA1 hippocampus layer, which then be averaged.
Data were analyzed with the Kruskal-Wallis analysis, followed by the Mann-Whitney
test to see the significance of differences between groups.
RESULTS
This study used 42 rats as experimental animals but only 41 rats (n = 41) are
eligible for analysis, 1 rat was dead. Histological hippocampus appearance shown in
Figure 1 and Figure 2, while the average measured thickness of the lamina pyramidalis
CA1 hippocampus of each group shown in Figure 3.

FIGURE 1. Hippocampus histological appearance of control group. The CA1


hippocampus, CA2, CA3, hippocampal fissure (HIF), and polymorphic layer of gyrus
dentatus (PoDG).
FIGURE 2. Histological hippocampus (magnification 200X) with toluidine blue
staining. Signs showed a thick layer of the lamina pyramidalis CA1.
Description:
(a) negative control group 1(KN1)
(b) negative control group 2(KN2)
(c) treatment group dose of 6 mg / rat(P1)
(d) treatment group dose of 12 mg / rat(P2)

(e) treatment group dose 24 mg / rat(P3)


(f) positive treatment group(KP)
In Figure 3, it appears that the average thickness of CA1 hippocampus in the
treatment of group 12 mg (P2) and 24 mg / rat (P3) shows the differences with the
negative control group aquades (KN1) or propylene glycol (KN2). The average
thickness of CA1 hippocampus is 70 14.56 m(P3) and 64.58 11.25m(P2), but
only 42.98 9.54m (KN1) and 46.55 9,99m (KN2). While in P1 that is for doses of
6 mg / rat did not show any significant difference with negative control group (KN1 and
KN2). The thickness of the layer of CA1 hippocampus at P2 and P3 groups actually
show a higher yield compared to the positive control group (KP) which had an average
thickness of62.6212.81m.

FIGURE 3. Comparison of average (x SD) thickness (m) layer of the lamina


pyramidalis CA1 hippocampus in the group of KN1, KN2, P1, P2, P3, and KP.
Description:
KN1: negative control group 1
KN2: negative control group 2
P1: the treatment group dose of 6 mg / rat
P2: the treatment group dose of 12 mg / rat
P3: treatment group 24 mg dose / rat
KP: positive treatment group
Statistical analysis performed with the Kruskal-Wallis test produces F count> F
table with 95% confidence level. This suggests that administration of ethanol extract of
ginseng Jaw root in rat causes a significant difference (p <0.05) in the thickness of CA1
hippocampus. Significance of differences between groups using Mann Whitney test can
be seen in Table 1.

TABLE 1. Statistical analysis (Mann Whitney test) on the thickness of CA1


hippocampus in rats after 18 days treatment
The table shows the presence of significant differences between the hippocampal
CA1 layers between these groups: KN1-KN2, KN1-P2, KN1-P3, KN1-KP, KN2-P2,
KN2-P3, KN2-KP, P1-P2, P1-P3, P1-KP, P2-P3, and P3-KP. While between KN1-P1
and P2-KP does not showany significance differences.This is study revealed that the
ethanol extract of ginseng Jawa root increased the thickess of CA1 at a dose of 12 mg /
rat (P2) and 24 mg / rat (P3). Further research required to investigate the influence of
propylene glycol to the process of cells in the hippocampus, because based on the
results above show there are significant differences between KN1-KN2 (both a negative
control groups).
In this study also obtained the results of analysis of bioactive compounds of ethanol
extract of ginseng Jawa root and Panax ginseng, conducted by the Center for Medicinal
Plant Research (PPOT) Faculty of Pharmacy UGM. Results of bioactive compounds
analysis shows there are similarities on terpenoid compounds, while the steroid
compounds is not found in both plants.

DISCUSSION
This study revealed that there was a significance difference between the
thickness of CA1 hippocampus after administration of ethanol extract of ginseng Jawa
root 12 mg and 24 mg/rat. Research conducted by Huang et al. 21 (1995) suggested that
area CA1 is the most important areas in spatial learning. Long-term-potentiation (LTP) is
an activity that causes changes in the thickness or the number of synapses found in the
hippocampus. LTP is important in the process of learning and memory 22. Increased

thickness of the pyramidal lamina CA1

28

hippocampus is associated with the events of

sprouting dendrites of neurons in the ventromedial region of the hippocampus and the
CA123, and the increase in dendrite spine number is associated with an increased number
of synapses that mediated by LTP24.
Induction of long-termpotentiation (LTP) in the Schaffers collateral pathway
involves NMDA receptors (N-methyl D-aspartate) and non-NMDA receptors 14. In the
resting state, the receptor is blocked by magnesium ions. Sensitization to the receptor
and postsynaptic membrane depolarization causes releasing magnesium and calcium
channel opened

25, 26

. Influx of calcium ions causes some calcium-dependent enzyme is

activated, including protein kinase C (PKC), calmodulin-dependent protein kinase II


(CaMKII), and Fyn kinase25, enzymes that cause the induction of LTP.
Panax ginseng extract has been shown to enhance memory in rat 11. Ginsenosida
Rb1 enhances mRNA expression nerve growth factor in hippocampus 26. Ginsenosida
Rb1 and Rg1 improve spatial learning and raise levels of sinaptofisin, a protein marker
in the synapse, which showed increased density of synapses in hippocampus 27.
Ginsenosida also been shown to increase influx calcium ion 28, increase the synthesis and
secretion of acetylcholine

11, 29

, increased production of NO28, increased levels of cyclic

adenosine monophosphate (cAMP) and cGMP11. Those mechanisms are known to


enhance LTP. The result of TLC analysis conducted in this study, showed that ethanol
extract of Panax ginseng and ginseng Jawa have the same compound, the terpenoid
saponins, which affect the thickness of the lamina pyramidalis CA 128 hippocampus at a
dose of 12 mg and 24 mg / rat. The increasing of the thickness of CA 1 after
administration of ethanol extract of ginseng Jawa roots due to the presence of
compounds assumed that terpenoid saponins have a role in LTP.

THE CONCLUSION
Administering ethanol extract of Ginseng Jawa (Talinum paniculatum Gaertn.)
root could increase the thickness of lamina pyramidalis CA 1 hippocampus in rats
(Rattus norvegicus). Effective doses of ethanol extract of Ginseng Jawa root to increase
the thickness of lamina pyramidalis CA1 hippocampus is 12 mg and 24 mg/rat.

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CA1
CA2
HiF

CA3

PoDG

FIGURE 1.Gambaran hippocampus histological control group (magnification


40X). Visible portions of the CA1 region of hippocampus namely, CA2, CA3,
hippocampal fissure (HIF), and polymorphic layer of gyrus dentatus (PoDG).
Signs indicate where measurements thick lamina pyramidalis CA1 layer.

10m
(a)

10m
(b)

10m
(c)

10m
(d)

10m
(e)

10m
(f)

FIGURE 2. Histological hippocampus (magnification 200X) with toluidine blue


staining. Signs showed a thick layer of the lamina pyramidalis CA1.
Description:
(a) negative control group 1
(b) negative control group 2
(c) treatment group dose of 6 mg / rat
(d) treatment group dose of 12 mg / rat
(e) treatment group dose 24 mg / rat
(f) positive treatment group

FIGURE 3. Comparison of average (x SD) thickness ( m) layer of the lamina


pyramidalis CA1 hippocampus in the group of KN1, KN2, P1, P2, P3, and KP.
Description:
KN1: negative control group 1
KN2: negative control group 2
P1: the treatment group dose of 6 mg / rat
P2: the treatment group dose of 12 mg / rat
P3: the treatment group dose of 24 mg / rat
KP: positive treatment group

Ginseng Jawa (talinum paniculatum gaertn.)

TABLE 1. Results Statistical analysis Mann Whitney test on a thick layer of CA1
hippocampus in rats after 18 days treatment
Kelompok

Mann Whitney test results

KN1-KN2
KN1-P1
KN1-P2
KN1-P3
KN1-KP
KN2-P1
KN2-P2
KN2-P3
KN2-KP
P1-P2
P1-P3
P1-KP
P2-P3
P2-KP
P3-KP
Notes: * significant at p <0.05

0,021*
0,077
0,000*
0,000*
0,000*
0,718
0,000*
0,000*
0,000*
0,000*
0,000*
0,000*
0,022*
0,248
0,001*