A standardized extract of Centella asiatica (ECa 233) strengthened the hippocampal synapses, resulting in the memory-enhancing effect. The ratio of the major active compounds in the extract – madecassoside [MDS] and asiaticoside [ASS] – is at 1.5 ± 0.5:1. However, the interactions between MDS and ASS are still unknown. To delineate the role of MDS and ASS in ECa 233 on synaptic enhancement by mimicking the applied MDS and ASS concentrations in ECa 233. The hippocampal slices obtained from healthy 8-week-old male Wistar rats and differentiated SH-SY5Y cells were treated with DMSO, ECa 233, MDS, ASS, or MDS+ASS. The hippocampal long-term potentiation (LTP was monitored for 3 h. The cell extracted proteins were examined by proteomic analysis. ECa 233 demonstrated the highest hippocampal synaptic response, followed by the MDS+ASS and the MDS, which expressed a stronger synaptic enhancement effect than the ASS. A number of mitochondrial proteins, including NDUFB6, were also expressed in the differentiated SH-SY5Y cells after treatment with ECa 233, ASS, MDS, and MDS+ASS. Our data revealed that the synergistic effect of MDS and ASS in the combined treatment was necessary to achieve the same level of responses obtained from ECa 233 treatment. MDS majorly contributed to the synaptic enhancement action of ECa 233. In addition, all substances were involved in mitochondrial metabolism, a process necessary for providing energy and sustaining synaptic Ca2+ levels in synaptic transmission. This discovery is crucial for dementia drug development because it demonstrated the role of major parent compounds in ECa 233 and their interaction.