Modulation of Neuronal Activity on Intercalated Neurons of Amygdala Might Underlie Anxiolytic Activity of a Standardized Extract of Centella asiatica ECa233.

GABAergic intercalated neurons of amygdala (ITCs) have recently been shown to be important in the suppression of fear-like behavior. Effects of ECa233 (a standardized extract of Centella asiatica), previously demonstrated anxiolytic activity, were then investigated on ITCs. Cluster of GABAergic neurons expressing fluorescence of GFP was identified in GAD67-GFP knock-in mice. We found that neurons of medial paracapsular ITC were GABAergic neurons exhibiting certain intrinsic electrophysiological properties similar to those demonstrated by ITC neurons at the same location in C57BL/6J mice. Therefore, we conducted experiments in both C57BL/6J mice and GAD67-GFP knock-in mice. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of the external capsule during the whole cell patch-clamp recordings from ITC neurons in brain slices. ECa233 was found to increase the EPSC peak amplitude in the ITC neurons by about 120%. The EPSCs in ITC neurons were completely abolished by the application of an AMPA receptor antagonist. Morphological assessment of the ITC neurons with biocytin demonstrated that most axons of the recorded neurons innervated the central nucleus of the amygdala (CeA). Therefore, it is highly likely that anxiolytic activity of ECa233 was mediated by increasing activation, via AMPA receptors, of excitatory synaptic input to the GABAergic ITC leading to depression of CeA neurons.

Comparative pharmacokinetics between madecassoside and asiaticoside presented in a standardised extract of Centella asiatica, ECa 233 and their respective pure compound given separately in rats.

1. ECa 233, the standardised extract of Centella asiatica, contains not less than 80% triterpenoid glycosides, in a madecassoside:asiaticoside ratio of 1.5 (±0.5):1. 2. The pharmacokinetic comparison of madecassoside and asiaticoside was performed in rats following intravenous and oral administration of ECa 233, or an equivalent dose of the individual compounds. Blood, tissues, urine and faeces were collected after dosing to determine drug and metabolite levels using liquid chromatography-tandem mass spectrometry. 3. Our study demonstrated that plasma levels of madecassoside, and to a lesser extent asiaticoside, were higher after administration of ECa 233 than the corresponding values for the pure compounds. There was a bidirectional interconversion between asiaticoside and madecassoside consistent with the increased exposure of madecassoside and asiaticoside in ECa 233. 4. Both madecassoside and asiaticoside appeared to be widely distributed in several organs and metabolized extensively; following intravenous administration of either compound, approximately 80-90% of the dose was recovered as madecassic acid and asiatic acid in the faeces.

Pharmacokinetics of a Standardized Extract of Centella asiatica ECa 233 in Rats.

ECa 233, a standardized extract of Centella asiatica, has been found to exhibit various positive neurological effects and to have a good safety profile. The present study aimed to explore the disposition kinetics of ECa 233, containing madecassoside (53.1 %) and asiaticoside (32.3 %), in rats. The extract was intravenously or orally administered at doses from 50 to 200 mg/kg. Plasma, tissues, urine, and feces were collected at time points from 0 to 48 h after dosing. The levels of madecassoside and asiaticoside, as well as their postulated triterpenic metabolites, madecassic acid and asiatic acid, in biological samples, were simultaneously measured by liquid chromatography-tandem mass spectrometry. The results showed that all animals had a good tolerability for ECa 233, whereas madecassic and asiatic acids were found in negligible amounts after pharmacokinetic assessment. Madecassoside and asiaticoside demonstrated rather similar absorption and tissue distribution profiles. They were rapidly absorbed, reaching maximum levels within 5-15 min after oral administration, but they had poor oral bioavailability, less than 1 %. Both triterpenoids were extensively distributed in the brain, stomach, and skin within 1 h and remained there for at least 4 h after dosing. Madecassoside and asiaticoside in ECa 233 were mainly excreted as an unchanged form after being injected, and exclusively as triterpenic acid metabolites in feces after oral administration. The pharmacokinetic results obtained could provide some guidance for an appropriate dosing regimen of ECa 233 in future studies. This study also provided the first evidence demonstrating the presence of madecassoside and asiaticoside in their target tissues.

Study of ameliorating effects of ethanolic extract of Centella asiatica on learning and memory deficit in animal models.

OBJECTIVE: The present study investigated the effect of Centella asiatica ethanolic extract (CE) on learning and memoly imnpairment induced by either transient bilateral common carotid arteries occlusion (T2 VO) or an intraperitoneal injection of scopolamine in mice. MATERIAL AND METHOD: CE (100, 300, 1000 or 1500 mg/kg, p.o.) were administered to learning and memory impaired mice once daily for 8 consecutive days. Learning and memory performance were evaluated by Morris water maze (MWM) and step-down passive avoidance (PA) test. Changes in malondialdehyde (MDA) levels in the brain were determined by lipid peroxidation assay. RESULTS: T2 VO mice exhibited learning and memory impairment in the MWM and PA tests. Treatment with CE ameliorated the learning and memory impairment of T2VO mice. Furthermore, CE significantly reduced MDA level in the brain of T2VO mice. On the other hand, administration of CE did not attenuate learning and memory impairment induced by scopolamine in mice. CONCLUSION: The present study demonstrated ameliorating effect of CE on learning and memory impairment in T2VO mice. Furthermore, it is likely that the positive effect of CE observed could be, at least partly, accounted by its antioxidative property. Thus, CE might be beneficial for memory impairment in which oxidative stress is an underlying cause.

Reorganization of sensory pathways after neonatal hemidecortication in rats.

We investigated ascending somatosensory pathways in neonatally hemidecorticated rats. Injection of an anterograde tracer, biotinylated dextran amine (BDA), into the contralesional dorsal root ganglions revealed ipsilateral projections to the dorsal column nuclei (DCN) in hemidecorticated rats as well as in normal rats. Injection of BDA into the DCN on the same side revealed that while most axons projected to the contralateral thalamus, some axons were detected in the ipsilateral thalamus in hemidecorticated rats while such projections were rarely detected in normal rats. The results suggest that aberrant ipsilateral projections of DCN neurons contralateral to the lesion developed after the hemidecortication.

Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells.

BACKGROUND: In order to gain insight into neuroprotective effects of ECa 233, a standardized extract of Centella asiatica, previously demonstrated in animal models of memory impairment induced by transient global ischemia or intracerebroventricular injection of ß-amyloid, the effect of ECa 233 on neurite outgrowth of human IMR-32 neuroblastoma cell line was investigated. METHODS: Cells were seeded and incubated with various concentrations of ECa 233. Morphometric analysis was carried out by a measurement of the longest neurite growth of cells at 24 and 48 h. Contributing signaling pathways possibly involved were subsequently elucidated by western blot analysis. RESULTS: While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1-100 µg/ml. Western blot analysis revealed that ECa 233 significantly upregulated the level of activated ERK1/2 and Akt of the treated cells suggesting their involvement in the neuritogenic effect observed, which was subsequently verified by the finding that an addition of their respective inhibitors could reverse the effect of ECa 233 on these cells. CONCLUSIONS: The present study clearly demonstrated neurite outgrowth promoting activity of ECa 233. ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed. In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases.

Anxiolytic effects of standardized extract of Centella asiatica (ECa 233) after chronic immobilization stress in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica has long been used for various neurological disturbances in Southeast Asian countries. The present study aims to demonstrate the anxiolytic effect of ECa 233, a standardized extract of C. asiatica containing triterpenoids not less than 80%, in comparison to diazepam. MATERIALS AND METHODS: The test compound was given orally to non-stressed mice and mice subjected to chronic immobilization stress. Anxiolytic effect was assessed by an elevated plus maze (EPM), a dark-light box and an open-field tests. RESULTS: Anxiolytic effect of ECa 233 was clearly demonstrated in non-stressed mice subjected to acute stress in all behavioral tests employed. In the EPM test, chronically stressed mice showed significant decrease in the number of open arm entries, shortening the time spent in open arms and an increase of the latency to leave the central area, suggesting their release from the stress. In addition, ameliorating effect of ECa 233 was observed on the body weight and serum corticosterone which were adversely affected by immobilization stress. Madecassoside and asiaticoside, equal to their respective contents of the effective doses of ECa 233, exclusively presented anxiolytic effects in EPM, while no distinct effect was observed on the body weight and serum corticosterone. CONCLUSIONS: The present study demonstrated anxiolytic effect of ECa 233 in both acutely and chronically stressed animals. These effects could be mainly accounted by madecassoside and asiaticoside, suggesting a possible use of ECa 233 for the treatment of both acute and chronic anxiety in the pathological state.

Wound healing activities of different extracts of Centella asiatica in incision and burn wound models: an experimental animal study.

BACKGROUND: The efficacy of Centella asiatica for incision and burn wounds are not fully understood. Here, we report the wound healing activities of sequential hexane, ethyl acetate, methanol, and water extracts of Centella asiatica in incision and partial-thickness burn wound models in rats. METHODS: Male Sprague-Dawley rats weighing 250-300 g were randomly divided into incision and burn wound groups. Each group was stratified into seven subgroups: (1) untreated; (2) NSS-; (3) Tween 20®- (vehicle control); (4) hexane extract-; (5) ethyl acetate extract-; (6) methanol extract-; and (7) aqueous extract-treated groups. The test substances were applied topically once daily. The tensile strength of the incision wound was measured on the seventh day after wound infliction. The general appearance and degree of wound healing of the burn wound were assessed on Days 3, 7, 10 and 14 after burn injury and prior to histopathological evaluation. RESULTS: On the seventh day after wound infliction, the tensile strength of incision wound in all extract-treated groups was significantly higher than that of the vehicle control (Tween 20®), but comparable to the NSS-treated group. The degrees of healing in the burn wound with the four extracts were significantly higher than that of the control on Days 3, 10 and 14. Histopathological findings on Day 14 after burn injury revealed prominent fibrinoid necrosis and incomplete epithelialization in the control and untreated groups, whereas fully developed epithelialization and keratinization were observed in all extract-treated groups. Analysis by thin layer chromatography demonstrated that the phyto-constituents ß-sitosterol, asiatic acid, and asiaticoside and madecassocide were present in the hexane, ethyl acetate and methanol extracts, respectively. CONCLUSIONS: All extracts of Centella asiatica facilitate the wound healing process in both incision and burn wounds. Asiatic acid in the ethyl acetate extract seemed to be the most active component for healing the wound.

γ-Mangostin increases serotonin 2A/2C, muscarinic, histamine and bradykinin receptor mRNA expression.

AIM OF THE STUDY: γ-Mangostin is a xanthone found in the fruit hulls of Garcinia mangostana L., which have long been used in Southeast Asia as a traditional medicine for the treatment of abdominal pain, dysentery, wound infections, fever and convulsions. Recent studies have revealed that γ-mangostin exhibits a variety of pharmacological activities, including serotonin 2 (5-HT(2)) receptor antagonism, anti-inflammatory effects and analgesic effects. To explore the mechanism of γ-mangostin responsible for these pharmacological activities, especially its effects on some related receptors, we investigated the effects of γ-mangostin on 5-HT(2), histamine (H(1)) and bradykinin (BK(2)) receptor gene expression in neuroblastoma (NG 108-15) cells in vitro. Additionally, to extend the study of the pharmacological properties, we examined the effect of γ-mangostin on the muscarinic (M(4)) receptor. MATERIALS AND METHODS: NG 108-15 cells were cultured in vitro and treated with γ-mangostin or a 5-HT(2) receptor antagonist (either imipramine or ketanserin). Then, the levels of mRNA for 5-HT(2A/2C) receptors were evaluated by semi-quantitative RT-PCR. The preventive effect of serotonin on the enhancement effects was also revealed. Additionally, the effects of γ-mangostin on the muscarinic, histamine and bradykinin receptors were determined. RESULTS: Chronic application of γ-mangostin at a concentration of 0.1 µM induced a significant increase in the level of 5-HT(2A/2C) receptor mRNA. These effects were prevented by serotonin. Moreover, γ-mangostin up-regulated the M(4), H(1) and BK(2) receptors. CONCLUSION: The ability of γ-mangostin to enhance the expression of 5-HT(2A/2C), muscarinic, histamine and bradykinin receptor mRNA suggests that this compound has antagonistic effects. These pharmacological properties may partly account for the benefits of using mangosteen in the treatment of inflammation, pain and neuropsychiatric symptoms.

Isobolographically additive anticonvulsant activity between Centella asiatica's ethyl acetate fraction and some antiepileptic drugs.

OBJECTIVE: To investigate interaction between orally given Centella asiatica's ethyl acetate fraction (EACA) and intraperitoneally administered antiepileptic drugs (AEDs), namely, phenytoin, valproate and gabapentin. MATERIAL AND METHOD: Isobolographic analysis was used to evaluate the interaction between EACA and AEDs in terms of protection of mice in the pentylenetetrazole test. Rotarod test was used to evaluate neurotoxicity. RESULTS: When given alone, the median effective dose of phenytoin, valproate and gabapentin were found to be 13, 104, and 310 mg/kg BW, respectively, whereas the corresponding values in the presence of EACA were 5, 29 and 79 mg/kg BW. Together with isobolographic analysis, the results obtained indicated an additive effect among all combinations tested. In relation to neurotoxicity, combination of gabapentin and EACA demonstrated a broader margin between the effective dose and the neurotoxic dose while the other two combinations did not. CONCLUSION: The present finding suggested a potential of Centella asiatica to be developed as an adjunctive medication for epileptic patients.