Acetylcholinesterase, oxidative stress, and inflammation inhibitory potentials of raw-extract Centella asiatica (RECA) on cell lines and brain tissue homogenates of Sprague dawley rats / Zetty Zulikha Hafiz

The present study was performed to investigate the effect of ethanolic extract of C.asiatica, designated as raw-extract of C.asiatica (RECA) on reducing the acetylcholinesterase (AChE), oxidative stress, and inflammation activities via both in vitro (SH-SY5Y and RAW 264.7 cells) and in vivo (Sprague Dawley rats). Selected major triterpenes of RECA were identified and quantified using high-performance liquid chromatography (HPLC), which further reveals that RECA contains significantly high proportion of glycosides than the aglycones with madecassoside is the highest component, followed by asiaticoside. RECA showed no toxicity effect at the concentrations tested since its IC50 could not be determined in concentrations ranging from 3.91 μg/mL to 1000 μg/mL, at 24 h and 48 h incubation times. Treatment of RECA on SH-SY5Y cells significantly reduced the AChE activity in a concentration-dependent manner with an IC50 value of 31.09 ± 10.07 μg/ml. The antioxidant and anti-inflammatory activities of RECA were also evaluated in vitro, by using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The production of reactive oxygen species (ROS) and reduced glutathione (GSH) levels were evaluated to measure oxidative stress in vitro. Meanwhile, the production of nitrite, prostaglandin E2 (PGE2), and tumor necrosis factor alpha (TNF-α) levels were quantified as inflammatory parameters. Results showed that treatment with RECA significantly suppressed the level of oxidative stress and pro-inflammatory cytokine/mediators released in concentration-dependent manner. Among all, RECA displayed remarkable inhibition against nitrite with IC50 value of 5.205 ± 3.13 μg/mL. Interestingly, these patterns of inhibition were consistent as observed in the LPS-induced neuroinflammation Sprague Dawley rats’ model, in both pre- and post-treatment of RECA. AChE activity, marker of oxidative stress (GSH) and pro-inflammatory cytokine/mediator (PGE2 and TNF-α) were studied by using rats’ brain. It was found that inhibitory effects of RECA in both different time of oral administration (pre- and post-treatment) on LPS-induced rats’ model successfully suppressed the AChE and inflammation activities, while the GSH level was up regulated, with RECA at highest dosage (350 mg/kg) producing the most profound effects. The AChE activity was also investigated in salt-soluble (SS) and detergent-soluble (DS) fractions of cerebral cortex, hippocampus, and cerebellum, which consists of predominantly G1 an G4 molecular isoforms of AChE respectively. In general, RECA preferentially inhibited the G4 form AChE in cerebral cortex and cerebellum, whereas G1 form AChE in hippocampus. The fractionation of RECA was accomplished by using vacuum liquid chromatography (VLC). Ten fractions were obtained and pooled based on the same solvent used for elution. Its selected major triterpenes contents were quantified using HPLC. Combined fractions (F1C-F5C) at concentration 200 μg/ml were subjected to AChE assay and F5C was found as the most active fraction in inhibiting AChE activity. Subsequently, the antioxidant and anti-inflammatory activities of F5C in vitro were further evaluated. Results showed that F5C significantly suppressed the level of oxidative stress and pro-inflammatory cytokine/mediators released in concentration-dependent manner. Herein, findings from present study strongly suggest that RECA may offer therapeutic potential for the treatment of Alzheimer’s disease through inhibiting the AChE, oxidative stress, and inflammatory activities.