The effect of Ginseng Jawa (Talinum paniculatum Gaertn.) root on the thickness of CA1 pyramidal lamina of rat hippocampus

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, Indonesia

³Department 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 CA₁ 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 system^{1, 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 acetylcholin^{11, 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 bregma^19. Brain tissue grown in tragachant then immersed into nitrogen liquid²⁰ for 25 to 35 seconds, then placing inside the refrigerator at a temperature of 25^oC. 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 mm(P3)  and 64.58 ± 11.25mm(P2), but only 42.98± 9.54mm (KN1) and  46.55 ± 9,99mm (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±12.81mm.

FIGURE 3. Comparison of average (x ± SD) thickness (mm) 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.

Simak

Baca secara fonetik

Kamus

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.  

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