Mayuree H. Tantisira

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

BackgroundThe 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.MethodsMale 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.ResultsOn 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 β-sitosterol, asiatic acid, and asiaticoside and madecassocide were present in the hexane, ethyl acetate and methanol extracts, respectively.ConclusionsAll 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.

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.

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC50) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC50 = 412.68 ± 15.44 μM) and CYP3A4 (IC50=343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC50 = 539.04 ± 14.18 μM) and CYP3A4 (IC50 = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (Ki=385.24 ± 8.75 μM) and CYP3A4 (Ki = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (Ki = 109.62 ± 6.14 μM) and CYP3A4 (Ki = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.

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

Abstract 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.

Acute effects of N-(2-propylpentanoyl)urea on hippocampal amino acid neurotransmitters in pilocarpine-induced seizure in rats

The present study aimed to investigate the anticonvulsant activity as well as the effects on the level of hippocampal amino acid neurotransmitters (glutamate, aspartate, glycine and GABA) of N-(2-propylpentanoyl)urea (VPU) in comparison to its parent compound, valproic acid (VPA). VPU was more potent than VPA, exhibiting the median effective dose (ED(50)) of 49 mg/kg in protecting rats against pilocarpine-induced seizure whereas the corresponding value for VPA was 322 mg/kg. In vivo microdialysis demonstrated that an intraperitoneal administration of pilocarpine induced a pronounced increment of hippocampal glutamate and aspartate whereas no significant change was observed on the level of glycine and GABA. Pretreatment with either VPU (50 and 100 mg/kg) or VPA (300 and 600 mg/kg) completely abolished pilocarpine-evoked increases in extracellular glutamate and aspartate. In addition, a statistically significant reduction was also observed on the level of GABA and glycine but less than a drastic reduction of glutamate and aspartate level. Based on the finding that VPU and VPA could protect the animals against pilocarpine-induced seizure it is suggested that the reduction of inhibitory amino acid neurotransmitters was comparatively minor and offset by a pronounced reduction of glutamate and aspartate. Therefore, like VPA, the finding that VPU could drastically reduce pilocarpine-induced increases in glutamate and aspartate should account, at least partly, for its anticonvulsant activity observed in pilocarpine-induced seizure in experimental animals. Some other mechanism than those being reported herein should be further investigated.

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.

Neuroprotective effect of a standardized extract of Centella asiatica ECa233 in rotenone-induced parkinsonism rats

BACKGROUND Mitochondrial dysfunction and reactive oxygen species (ROS) generation cause dopaminergic neurodegeneration in Parkinsons disease. The neuroprotective approach is a promising strategy to slow disease progression in Parkinsons disease. A standardized extract of Centella asiatica ECa233 has been previously reported to have pharmacological effects in the central nervous system. PURPOSE This study aimed to determine the neuroprotective effect and mechanisms of ECa233 in rotenone-induced parkinsonism rats. METHODS Rats were orally given either vehicle or ECa233 (10, 30 and 100 mg/kg) for 20 consecutive days. Rotenone (2.5 mg/kg i.p.) was given to parkinsonism (PD) and ECa-treated rats from day 15 to 20. Locomotor activity was recorded on day 1, 14, 17 and 20. Tyrosine-hydroxylase (TH) immunohistological staining was used to determine dopaminergic neurons in the substantia nigra and striatum. Furthermore, mitochondrial complex I activity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase protein expression were measured in brain tissue. RESULTS Rats receiving ECa233 30 mg/kg showed a significant increase in distances (p < 0.01) together with a higher number and intensity of dopaminergic neurons in the substantia nigra and striatum (p < 0.001) compared to PD rats. ECa233 (30 mg/kg) protected against mitochondrial complex I inhibition, decreased MDA levels (p < 0.05) and increased SOD (p < 0.01) and catalase (p < 0.05) expression. CONCLUSION ECa233 can protect against rotenone-induced motor deficits and dopaminergic neuronal death. These effects are mediated through the protection of mitochondrial complex I activity, the effects of antioxidants and the enhancement of antioxidant enzyme expression.

Effects of N(2-propylpentanoyl)urea on hippocampal amino acid neurotransmitters in spontaneous recurrent seizure rats

Abstract Background: N(2-propylpentanoyl) urea (VPU) is a new valproic acid (VPA) analog with higher anticonvulsant activity than its parent compound in various animal models including seizure acutely induced by pilocarpine. Objective: Investigate its effects on hippocampal amino acid neurotransmitters in spontaneous recurrent seizure (SRS) rats. Methods: Pilocarpine hydrochloride was used to induce status epilepticus (SE). Animals were visually observed for two hours/day for an episode of SRS for six weeks. Microdialysis experiment was performed to detect hippocampal amino acid neurotransmitters on those rats that developed SRS. Results: In comparison to normal rats, hippocampal glutamate, gamma-aminobutyric acid (GABA), and glycine, significantly increased in SRS rats. Occurrence of SRS in the faces of increased level of inhibitory neurotransmitters suggests the key role played by glutamate in the genesis and control of SRS. Based on the observation in pilocarpine-induced SE, the level of glutamate in SRS rats significantly decreased by a clinically effective anticonvulsant, VPA (300 and 600 mg/kg, i.p). Similar profile on hippocampal glutamate was also exhibited by VPU (50 and 100 mg/kg, i.p.). Conclusion: The possible role of VPU in controlling seizure in SRS rats and subsequently human temporal lobe epilepsy as VPA was suggested.

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt, ERK, and p38 MAPK signaling

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration‐ and time‐dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1–100 μg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound.