In Vitro Neuroprotective Activities of Natural Products Isolated from Senecio Graciliorus DC against Corticosterone Induced Impairment in SHSY5Y Cells

Senecio graciliorus DC root extract was studied for secondary metabolite composition following bioactivity guided isolation technique. The ethyl acetate extract of Senecio graciliorus root yielded nine chemical constituents: 3,4-di-tert-butyl toluene, stigmasterol, β-sitosterol, 2β-(angeloyloxy)furanoeremophilane, gallic acid, 2β-{[(Z)-2-hydroxymethylbut-2-enoyl]oxy}furanoeremophilane,1-Hydroxypentan-2-yl-4-methylbenzoate, sarcinnic acid, and sitosterol 3-O-β-D-glucopyranoside. The structures of the chemical constituents were elucidated on the basis of spectral data analysis in the light of literature. All the compounds are being reported for the rst time from this plant. The isolated constituents were screened for in-vitro neuroprotective effects against corticostereone induced impairment. Among various isolated compounds, three natural products (sarcinnic acid, gallic acid and β-sitosterol) displayed robust neurotropic activity. These three compounds increased neuronal cell survival in differentiated neuroblastoma cells from high dose corticosterone (400µM) induced cell death. The studies are aimed to explore small molecules for treating neurodegeneration underlying various neurological disorders to restore neuronal cell plasticity.


Introduction
Senecio (tribe Senecioneae), is the largest genus of family Asteraceae (Compositae) consisting of more than 1500 species with worldwide distribution. The genus has been extensively studied for secondary metabolite composition [1]. Pyrrolizidine alkaloids (PAs), eremophilanolides, and cacalolides are the characteristic classes of compounds contained in the genus [2]. Senecio species not only enjoy positive impact but also cherish a negative impact in many agricultural countries, because such plants cause deaths to livestock than any other poisonous plant. Their toxicity concerns are mainly attributed to their content of pyrrolizidine alkaloids and some furanoeremophilanes [3]. Senecio species were used in the folk medicine for the treatment of wounds, antiemetic, anti-in ammatory, and vasodilatory preparations [1]. Phytochemistry of a number of Senecio species have resulted into the identi cation of diverse class of constituents which include pyrrolizidines, eremophilanes, avonoids, diterpenes, triterpenes, sesquiterpenes [4][5][6][7][8][9]. Among the naturally occurring N-containing compounds, the PAs of macrocyclic senecionine type constitute an important group. These compounds are pharmacologically potent and characteristic secondary metabolites for most species of the genus Senecio which cause hepatotoxicity to animals [10]. Senecio species possess antibacterial and antifungal activities [1]. A thorough biological study of the genus Senecio has revealed that the extracts as well as compounds of the genus possess hepatotoxic, teratogenic or carcinogenic effects, antifeedant activity, anti-mitotic effect, and inhibition of angiotensinogen converting enzyme (ACE), insecticidal and neurotoxic activity [11][12][13][14][15][16].
Senecio gracili orus DC grows in the alpine forests and meadows of Kashmir valley. It is locally known as "Bhagghu". The water extract of the leaves is a folk medicine for the treatment of skin rashes and eruptions [17][18]. Earlier we have reported the essential oil analysis of differ parts of S. gracili orus DC using GC-FID and GC-MS. The essential oil contains α-pinene, cis-ocimene, 1,2,3-trimethylcyclohexane and β-pinenes as the major constituents [19].
However, to the best of our knowledge, there are no reports about the secondary metabolites and neuroprotective effects of S. gracili orus DC and its constituents. So, we have carried out bioactivity guided isolation of natural products from S. gracili orus and investigated the isolated components for neuroprotection against corticosterone induced neurotoxicity using SH-SY5Y cells.

Preparation of extract
The root part of the plant was processed separately, chopped, oven dried at 40°C and ground to a ne powder. The powdered roots (2.0 kg) were extracted sequentially by maceration with ethyl-acetate. The solvent was removed under vacuo on a rotary evaporator to afford crude ethyl acetate extract of root part (80.0 g).

Analytical techniques
NMR spectra were obtained on 400 MHz Bruker spectrometer in CDCl 3 and MeOD with TMS as internal standard. Mass analysis was carried out on LC-MS 8030 mass spectrometer (Shimadzu Corporation, Kyoto, Japan). All the compounds were analysed in full scan mode with nitrogen serving as an interface gas. Column chromatography was carried on normal silica gel (60-120 mesh) (Merck grade) and precoated TLC plates with silica gel 60 F 254 (Merck, 0.25 mm) were used for monitoring the column chromatograph.

SH-SY5Y cell culture
Human neuroblastoma cell lines were propagated in DMEM-F12 media supplemented with 10% FBS, penicillin, and streptomycin in CO 2 atmosphere in humidi ed incubator. Cells were subjected to differentiation using retinoic acid. Cells were collected and subjected to digestion in 0.05% trypsin -EDTA solution. Experiments were performed with cell within 12-18 passages.

Cell survival analysis
Cells were incubated in 96 well plates (200µL/well) with seeding density of 1 × 10 cells/ml. After treatment, 20µL MTT dye (5 mg/ml) was added to each well and kept for 2 hrs. Formazon crystals formed were dissolved in cell culture grade DMSO and the values were measured using plate reader at 570 nm. All experiments were performed in triplicates, signi cance value (P < 0.01 ***).

Corticosterone induced cell impairment model
Cell based toxicity assay was established to induce cell impairment in cells subjected to Corticosterone (10µL of 400 µmol/L) treatment; cell survival was mainly dependent upon addition of certain external neurotropic factors (DHF/molecules). MTT assay was used to determine percentage cell survival after 48 hr of compound treatment.

Statistical analysis
Graph Pad Prism software was implied for statistical analysis followed by one way ANOVA variance.
Experiments were replicated in set of triplicates and the results were assessed as mean of ± SD of three independent experiments and the signi cance was expressed at P < 0.01 **.

Isolation of chemical compounds
The constituents from S. gracili orus were isolated following bioactivity guided isolation and puri cation methodology. Both the shoot and root extracts were rst 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 identi cation of active constituents. Thus 60.0g of the ethyl-acetate extract of S. gracili orus DC root was subjected to fractionation and puri cation of constituents using normal phase column chromatography. 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 signi cant cell survival activities from the root part of plant (Fig. 2).

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).

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. gracili orus 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 ).

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).

Conclusion
In conclusion S. gracili orus DC was studied both for its secondary metabolite composition and neuroprotection. The secondary metabolite composition of the roots of S. gracili orus DC yielded nine chemical constituents which were identi ed as 3,4-di-tert-butyl toluene, stigmasterol, β-sitosterol), gallic acid, 2β-(Angeloyloxy)furanoeremophilane, 2β-{[(Z)-2-Hydroxymethylbut-2enoyl]oxy}furanoeremophilane, sarcinnic acid, sitosterol 3-O-β-D-glucopyranoside, and 1-Hydroxypentan-2-yl-4-methylbenzoate. All the compounds are being reported for the rst time from S. gracili orus DC. The in-vitro screening of the isolated constituents for neuroprotective effects against corticosterone induced impairment resulted in the identi cation of three natural products (gallic acid, sarcinnic acid and β-sitosterol) with promising neuroprotective activity. All the three positive constituents enhanced cell survival /cell viability in corticosterone induced toxicity in SHS5YS cells through AKT/ ERK pathway activation in concentration and time dependent manner.   Protective effects of natural isolates (C-04, C-05, and C-08) on SHSY5Y cells in corticosterone induced impairment using MTT assay. Cells were subjected to corticosterone induced toxicity for 24hrs after preincubating with compounds. (P < 0.01 **) Figure 4 Cell viability after treatment of cells with increased concentration of C-04, C-05 and C-08 Screening of natural molecules as potent activators of survival pathways