3.1. Isolation of chemical compounds
The constituents from S. graciliflorus were isolated following bioactivity guided isolation and purification methodology. Both the shoot and root extracts were first screened for cell viability against corticosterone induced toxicity in differentiated human neuroblastoma cell line (SH-SY5Y)
The extracts were screened for cell viability both in presence and absence of corticosterone. Contrary to shoot, the root extract showed either similar or better cell viability than control at two different concentrations (10µM and 50µM) (Fig. 1). So only root extract was undertaken for isolation and identification of active constituents. Thus 60.0g of the ethyl-acetate extract of S. graciliflorus DC root was subjected to fractionation and purification of constituents using normal phase column chromatography.
Four fractions Fr-1, Fr-2, Fr-3, and Fr-4 of root extract were obtained using Hexane-EtOAc as eluent with increasing polarity of 10, 30, 50 and 80% ethyl acetate respectively. Purification of Fr-1 yielded two constituents C-01 and C-02. Similarly C-03 and C-04; C-05 and C-06 were obtained from Fr-2 and Fr-3 respectively. Repeated column chromatography of Fr-4 yielded C-07, C-08 and C-09 as pure isolates. The structures of isolated constituents were characterised based on spectral data analysis in the light of literature. Thus the isolated natural products were identified as 3,4-di-tert-butyl toluene (C-01), stigmasterol (C-02), 2β-{[(Z)-2-Hydroxymethylbut-2-enoyl]oxy}furanoeremophilane (C-03), gallic acid (C-04), β-sitosterol (C-05),2β- (Angeloyloxy)furanoeremophilane (C-06), 1-Hydroxypentan-2-yl-4-methylbenzoate (C-07), sarcinnic acid (C-08), and sitosterol 3-O-β-D-glucopyranoside (C-09) (Fig. 2) [20–27]. All the compounds were isolated for the first time from S. graciliflorus DC.
3.2. Natural compounds as potential pro survival entities against neural cells(SH-SY5Y)
To search for natural molecules with potential cell survival activity, cell line based assay was employed. Among the library of compounds screened only three compounds showed significant cell survival activities from the root part of plant (Fig. 2).
3.3. Neuroprotective activity of selected hits against corticosterone induced cell toxicity
Human neuroblastoma cell lines were pre incubated with positive hits (24 hrs.), later subjected to high dose Corticosterone (400 µM) treatment again for 24hrs (Fig. 3). Corticosterone induces impairment in differentiated cell lines showing obvious injury, reversed by the selected hits thereby increasing cell survival. Initially differentiated human neuroblastoma cell lines were incubated with root and shoots extracts in presence and absence of corticosterone for 48hrs. Though most of the constituents showed positive effects on cell viability but three constituents (C-04, C-05 and C-08) were capable of restoring cell survival (Fig. 1).
3.4. Protective Effects of Natural products (C-04, C-05 and C-08) against corticosterone induced cytotoxicity in human neuroblastoma cell lines (SHSY5S)
The effect of natural isolates (C-04, C-05 and C-08) on viability of Human neuroblastoma cell lines SHSY5S cells was studied in presence and absence of corticosterone. The viability of SHSY5S cells was determined using MTT assay. Human neuroblastoma cell lines were preincubated with three natural products C-04, C-05 and C-08, which showed the best cell viability in preliminary screening, for 24 hours and later subjected to corticosterone treatment for another 24 hours. As expected corticosterone treatment at 400µM concentration induced cell impairment which resulted into decreased cell viability. Similar to DHF (5nM) treatment the cell viability remained unaffected, compared to control, when the cells were treated with C-04, C-05 and C-08 only at 20µM concentration. On the other hand the cell viability increased after the treatment of the cells with 20µM solution of C-04, C-05 or C-08 for 24 hours prior to corticosterone treatment (Fig. 3). These results clearly indicate that the isolated constituents (C-04, C-05 and C-08) from S. graciliflorus show neuroprotective effects on SHSY5S cell lines.
In another experiment SHSY5S cells were treated only with different concentrations of C-04, C-05 and C-08 without the treatment of corticosterone. The treatment of SH-SY5S cells with C-04, C-05 and C-08 showed a dose dependent response. The cell viability increased up to 31.25 µM concentration of all the three natural products C-04, C-05 and C-08 but decreased beyond this concentration and proved to be toxic at higher concentrations. Though lower concentrations of the positive compounds enhanced cell viability but their effects after a certain dose level provoke neuronal damage in differentiated neuroblastoma cell lines (Fig. 4).
3.5. Screening of C-04, C-05 and C-08 for activation of various survival proteins
All the three natural isolatesC-04, C-05 and C-08 which showed neuroprotection were screened for activation of survival proteins using differentiated neuroblastoma cells under standard optimised conditions of temperature and humidity. Earlier SHSY5Y cells were subjected to differentiation 5 days prior to incubation with compounds in concentration dependent manner for 48hrs.Multiple line experiments were performed to study the effect of positive hits (C-04, C-05 and C-08) on neuronal cell survival and activation of various survival pathways.
Since AKT/ PI3K signalling pathway plays an important role in promoting neuronal survival, the mechanism by which C-04, C-05 and C-08 protect SHSY5Y cells from corticosterone induced neurotoxicity was particularly explored by studying the effect of these constituents on AKT, ERK activation. We examined the phosphorylation of AKT and ERK. Though all the three isolates showed phosphorylation of AKT/ ERK but C-08 lead to maximum increase in the phosphorylation of AKT/ ERK in SHSY5Y neuroblastoma cells (Fig. 5). Thus these results clearly indicate that C-04, C-05 andC-08 cause neuroprotection against corticosterone induced neurotoxicity by expressing AKT/ ERK pathway.
The effect of different concentrations of positive hits (C-04, C-05 and C-08) on AKT/ ERK expression was also studied and the resulted showed that all the three constituents showed maximum AKT/ ERK pathway activation at 20µM concentration compared to 10µM or 40µM (Fig. 6/Fig. 7 ).
3.6. C-08 induces AKT, ERK activation in differentiated neuroblastoma cells in time dependent manner
In order to study whether the neurotropic activity is time dependent, we determined the phosphorylation reaction at different time intervals after differentiating SHSY5Y cells for a period of 5 days and later incubated with C-08 at optimum conditions of humidity and temperature. C-08 activated the survival pathway (p-ERK and p-AKT at 20µM) in time dependent manner (Fig. 7).