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

1Department of Anatomy, Embryology and Anthropology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia 2Department of Pharmacy, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia3Department 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 Kruskal-Wallis and Mann Whitney test.Results: The thickness of CA1 pyramidal laminas of hippocampus were 42,98±9,54 µm (KN1); 46,55±9,99 µm (KN2); 46,19±11,39 µm (P1), 64,58±11,25 µm (P2) and 70,00±14,56 µm (P3);and 62,62±12,81 µm (KP).There was significant effect for the thickness of CA1 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-inflamation3, increasing motility of spermatozoa4,

anti-diarrhea5, and as stimulant nervous system6. 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 CA3 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 CA1 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 μm, 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 3-

4 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 of62.62±12.81m.

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 1KN2: negative control group 2P1: the treatment group dose of 6 mg / ratP2: the treatment group dose of 12 mg / ratP3: treatment group 24 mg dose / ratKP: 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 memory22. 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-term–potentiation (LTP) in the Schaffer’s collateral pathway

involves NMDA receptors (N-methyl D-aspartate) and non-NMDA receptors14. 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 rat11. Ginsenosida

Rb1 enhances mRNA expression nerve growth factor in hippocampus26. 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 hippocampus27.

Ginsenosida also been shown to increase influx calcium ion28, 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 CA128 hippocampus at a

dose of 12 mg and 24 mg / rat. The increasing of the thickness of CA1 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 CA1 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.

REFERENCES

1. Khaled Radad, Gabriele Gille, Wolf-Dieter Rausch. Use of Ginseng In Medicine: Perspectives on CNS Disorders. Iranian Journal of Pharmacology & Therapeutics.2004

2. Owen RT. Ginseng in the Nineties : More Than A Roborant (cited 10 Februari 2002). Available from URL: http://www.nimh.btinternet.co.uk/ejhm/1_2_mm3.htm

3. Sumastuti R. Efek Anti Radang Infus Daun dan Akar Som Jawa (Thalinum paniculatum Gaertn.) pada Tikus Putih in Vivo. Warta Tumbuhan Obat Indonesia Vol.IV No.5. Jakarta: Kelompok Kerja Nasional Tumbuhan Obat Indonesia, 1999

4. Sa’roni, Yun Astuti N, Adjirni. Pengaruh Infus akar Thalinum paniculatum Gaertn. terhadap Jumlah dan Motilitas Spermatozoa pada Mencit. Buku Panduan Seminar Nasional Tumbuhan Obat Indonesia XI: 4-5 September 1996: Surabaya, 1996

5. Rahayu L, Purwanggana A. Uji Efek Antidiare Akar Som Jawa. Buku Panduan Seminar Nasional Tumbuhan Obat Indonesia XI:4-5 September 1996: Surabaya, 1996

6. Widowati L, Pudjiastuti, Nuratmi B. Efek Stimulan Susunan Saraf Pusat Infus Talinum paniculatum Gaertn. pada Mencit Putih. Buku Panduan Seminar Nasional Tumbuhan Obat Indonesia XI:4-5 September 1996: Surabaya, 1996

7. Syamsuhidayat SS, Hutapea JR. Inventaris Tanaman Obat Indonesia I. Badan Penelitian dan Pengembangan Kesehatan Departemen Kesehatan Republik Indonesia.1991.

8. Wahjoedi B, Martono WP, Shelvia D. Efek Androgenik Sari Akar Ginseng Jawa (Talinum paniculatum Gaertn.) pada Anak Ayam Jantan. Warta Tumbuhan Obat Indonesia Vol IV No.5 1999.

9. Wijayakusuma H, Dalimartha S, Wirian AS. Tanaman Berkhasiat Obat di Indonesia. Jilid 3. Jakarta: Pustaka Kartini.1994: 124.

10. Sukardiman. Perbandingan Profil Kandungan Kimia dari Akar Talinum paniculatum Gaertn. dan Panax ginseng dengan metode KLT-densitometri. Buku Panduan Seminar Nasional Tumbuhan Obat Indonesia XI Surabaya.1996.

11. Hsou Lin J, Leang SW Ginseng: A New Look At An Old Story [cited May 2001]: [4 screens] avaliable from URL: http:// users.med.arth.gr/~karanik/english/articles/ginseng.html

12. Sari DCR. Pengaruh Estrogen terhadap Memori: Kajian Keterlibatan Sistem Serotonergik dalam Memori pada Tikus (Rattus norvegicus). Tesis. Yogyakarta: Program Pascasarjana UGM. 2000.

13. Bear NF, Connors BW, Paradiso MA. Neuroscience: Exploring the Brain. Philadelphia: Williams and Wilkins. 1996.

14. Kandel ER, Schwartz JH, Jessel TM. Essentials of Neural Science and Behavior. International ed. Stamford.1995

15. Anonim. Farmakope Indonesia. Edisi III. Departemen Kesehatan Republik Indonesia, Jakarta.1979.

16. Nitta H, Matsumoto K, Shimizu M, Ni XH, Watanabe H. Panax Ginseng Extract Improves the Scopolamine Induced Disruption of 8-arm Radial Maze Performance in Rats. Biol Pharm Bull 18(10) Oct 1995:1439-1442.

17. Pratiknya AW. Dasar-dasar Metodologi Kedokteran dan Kesehatan. Cetakan I.Jakarta: CV Rajawali. 1986.

18. Puspitasari DA, Sari DCR, Suharmi S Pengaruh Fraksi Eter Ekstrak Etanol Akar Ginseng Jawa (Talinum paniculatum Gaertn.) Terhadap Kesalahan Memasuki Lengan Maze Radial Pada Tikus (Rattus norvegicus). Laporan Skripsi. Yogyakarta: Fakultas Kedokteran UGM. 2005.

19. Paxinos, Watson. The Rat Brain In Stereotaxic Coordinates. 4th Edition. San Diego: Academic Press. 1998.

20. Handari. S. Metode Pewarnaan Histologi dan Histokimia. Bagian Anatomi dan Mikroteknik Hewan Fakutas Biologi UGM. Penerbit Bharata Karya Aksara, Jakarta. 1983.

21. Huang YY, Kandel ER, Varshavsky L, Brandon EP, Qi M, Idzerda RL, McKnight GS, Bourtchouladze R A. Genetic Test of the Effect of Mutations in PKA on Mossy Fiber LTP and Its relation to spatial and contextual learning. Cell 83: 1211-1222.1995

22. Pyapali GK, Turner DA, Williams CL, Meck WH, Swartzwelder HS. Prenatal Dietary Choline Supplementation Decrease the Threshold for Induction of Long-Term Potentiation in Young Adults Rats. J. Neurophysiol. 79: 1790-1796, 1998.

23. Matsukawa M, Ogawa M, Nakadate K, Maeshima T, Ichitani Y, Kawai N, Okado N. Serotonin And Acethylcholine Are Crucial To Maintain Hippocampal Synapses And Memory Acquisition In Rats.Neurosci. Lett 230(1) 1997: 13-16.

24. Carlson NL. Learning and Memory: Basic Mechanism. Physiology of Behaviour. 5th Ed. Boston: Allyn and Bacon.1994.

25. Squire LR, Kandel ER. Memory: From Minds to Molecules. New York: Scientific American Library.2000.

26. Salim KN, McEwen BS, Chao HM Ginsenoside Rb1 Regulates ChAT, NGF and trkA mRNA expression in The Rat Brain. Brain Res July 47(1-2) 1997:177-182

27. Mook-Jung I, Hong HS, Boo JH, Lee KH, Yun SH, Cheong MY, Joo I, Huh K, Jung MW Ginsenoside Rb1 and Rg1 Improve Spatial Learning and Increase Hippocampal Synaptophysin Level in Mice. J Neurosci Res Mar 63(6) 2001: 509-515

28. Abdrasilov BS. The Effect of Total Saponins from Panax ginseng CA Meyer on the Intracellular Signalling System in Erlich Ascites Tumour Cells. Biochem Mol Biol Int 38(3) Mar 1996: 519-526.

29. Chen X, Lee TJ. Ginsenosides-induced Nitric Oxide-mediated Relaxation of the Rabbits Corpus Cavernosum. Br J. Pharmacol 115(1) May 1995: 15-18.

CA2

CA3PoDG

HiF

CA1

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.

(a) (b)

(c) (d)

10µm 10µm

10µm 10µm

(e) (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 1KN2: negative control group 2P1: the treatment group dose of 6 mg / ratP2: the treatment group dose of 12 mg / ratP3: the treatment group dose of 24 mg / ratKP: positive treatment group

10µm 10µm

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 0,021*

KN1-P1 0,077KN1-P2 0,000*

KN1-P3 0,000*

KN1-KP 0,000*

KN2-P1 0,718KN2-P2 0,000*

KN2-P3 0,000*

KN2-KP 0,000*

P1-P2 0,000*

P1-P3 0,000*

P1-KP 0,000*

P2-P3 0,022*

P2-KP 0,248P3-KP 0,001*

Notes: * significant at p <0.05