Five racemic phthalides from the aerial parts of Lycopodiastrum casuarinoides and their neuroprotective activities

Five racemic phthalides (1–5), including four undescribed phthalides monomers [(+)-1, (+)-2, (−)-2 and (−)-3], four undescribed phthalide dimers [(+)-4, (−)-4, (+)-5 and (−)-5], together with two known compounds [(−)-1 and (+)-3], were isolated from the aerial parts of Lycopodistrum casuarinoides. Their chemical structures were delineated by extensive spectroscopic data (UV, 1D/2D NMR, HRESIMS), in combination with the comparison of the experimental and calculated electronic circular dichroism spectra, calculated spin-spin coupling constants, and calculated NMR. All compounds were reported from Lycopodiaceae family for the first time. In addition, all isolates were tested for their neuroprotective effects on HT-22 cell injury induced by glutamate. Interestingly, among the five racemic phthalides, only the homologous dimers [(±)-5] displayed significant differences in neuroprotective effects, and (−)-5 exhibited the best neuroprotective activity against glutamate-induced HT-22 cells damage, with 29.3% increase rate in cell survival at 5 μM concentration. The neuroprotective effect of (−)-5 at different concentrations is equivalent to that of the positive control drug D/L-3-n-butylphthalide (racemic NBP). Furthermore, the biological evaluation revealed that (−)-5 could ameliorate glutamate-induced neuronal cell death via the Bax/Bcl-2 anti-apoptotic pathway.