Comparative secondary metabolites profiling and biological activities of aerial, stem and root parts of Salvadora oleoides Decne (Salvadoraceae)

In this study, different parts (aerial, stem and root) of Salvadora oleoides Decne were investigated in order to explore their phytochemical composition and biological potential. The bioactive contents were evaluated by conventional spectrophotometric methods. Additionally, the secondary metabolite compounds were identified by UHPLC-MS analysis. Biological potential was evaluated by determining antioxidant (DPPH, FRAP and Phosphomolybdenum) and enzyme inhibitory (butrylcholinesterase and lipoxygenase) effects. Higher total bioactive contents were found in methanolic extracts which tend to correlate with higher radicalscavenging and reducing potential of these extracts. LC/MS spectrum revealed the presence of 16 different secondary metabolites belonging to terpene, glucoside and sesquiterpenoid dervivatives. Glucocleomin and emotin A were the main compounds present in all three parts. The strongest butrylcholinesterase and lipoxygenase inhibitory activity was observed for root and stem DCM extracts. Demonstrated biological potential of S. oleoides plant can trace a new road map for developing newly designed bioactive pharmaceuticals.


Plant collection and extraction
Aerial, stem and root parts of S. oleoides were collected in July from Bahawalpur, Pakistan and identified by Dr. H. Waris, Taxonomist at Cholistan Institute of Desert Studies, The Islamia University of Bahawalpur, Pakistan. In addition a voucher specimen number  were also deposited in the herabarium of Department of Pharmacy and Alternative Medicines, The Islamia University of Bahawalpur. The shade-dried parts were subjected for extraction by maceration (72 hrs) successively with dichloromethane and methanol at room temperature with occasionally shaking for 24 hrs. The resultant extracts were concentrated by Rotavapor-R20 at 35 °C.

Total phenolic and flavonoid contents
Total phenolic content assay was done by utilizing well-established Folin-Ciocalteu reagent method (Kahkonen et al. 1999). The total phenolic content was expressed as mg gallic acid equivalent per gram of fresh sample (mg GAE/g). Total flavonoid contents was determined with the aluminium chloride colorimetric method as described by Chew et al. (Chew et al. 2009).
The flavonoid concentration was expressed as mg quercetin equivalent per gram sample (mg QE/100).

LC-MS analysis
UHPLC Accurate-Mass Q-TOF (Agilent 1290 Infinity LC system coupled to Agilent 6520) mass spectrometer with dual ESI source was used. Column specifications were as: XDB-C18 Agilent Zorbax Eclipse, narrow-bore 2.1 x 150 mm, 3.5 micron (P/N: 930990-902). The temperature of column was maintained at 25 °C, while auto-sampler temperature was 4 °C. The following two mobile phases used were: A (0.1% formic acid in water), B (0.1% formic acid in acetonitrile) at flow rate of 0.5 mL/min. Injection volume was 1.0 µL. Run time was 25 min and post-run time was 5 min. MS analysis full scan was carried out over a range of m/z 100-1000 employing electrospray ion source in the negative ionization mode. Flow rate for nitrogen as nebulizing and drying gas was 25 and 600 L/hour, respectively with drying gas temperature of 350 °C. The fragmentation voltage was optimized to 125. Capillary voltage for analysis was 3 4. Antioxidant assays

FRAP assay
Plant sample (1000 µg/mL) was added to 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1% w/v), incubated for 20 min at 50°C. After 20 min, trichloroacetic acid (2.5 mL, 10% w/v) was added. The contents were divided into two halves; equal volume of water was added in one half of 2.5ml and then 0.5 mL of FeCl 3 solution (0.1% w/v) was added. The contents were incubated for 30 min at 25 o C and the absorbance was measured at 700 nm (Chan et al. 2010). The results were expressed as mg GAE/g.

Phosphomolybdenum assay
Total antioxidant capacity (TAC) of was determined by phosphomolybdenum method (Prieto et al. 1999). Briefly, Plant extract solution (0.3 mL, 1 mg/mL) was mixed with 3 ml of molybdate reagent solution, incubated at 95 ºC for 90 min and the absorbance of the solution was measured at 695 nm against blank. TAC was expressed as equivalent of gallic acid (mg GAE/g) (Prieto et al. 1999).

Butrylcholinesterase assay
The BChE inhibition activity was performed according to the method (Ellman et al. 1961) with slight modifications. Total volume of the reaction mixture was 100 µl. It contained 60 µl Na 2 HPO 4 buffer with concentration of 50 mM and pH 7.7. 10 µl test compound (0.5 mM well -1 ) was added, followed by the addition of 10 µl (0.005 unit/ well) enzyme. The contents were mixed and prread at 405 nm. Then, contents were pre-incubated for 10 min at 37 ºC. The reaction was initiated by the addition of 10 µl of 0.5 mM/well substrate (butyrylthiocholine chloride), followed by the addition of 10 µl DTNB (0.5 mM/well). After 30 min of incubation at 37 ºC absorbance was measured at 405 nm using 96-well plate reader (Synergy HT, Biotek, USA). All experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM/well) was used as a positive control. The percent inhibition was calculated with the help of following equation 1.
Where, Control = Total enzyme activity without inhibitor.
Test = Activity in the presence of test compound.
IC 50 values were calculated using EZ-Fit Enzyme kinetics software (Perrella Scientific Inc. Am herst, USA).

Statistical analysis
All the experiments were carried out in triplicates to calculate the mean values which are expressed as the mean ± standard deviation (SD). The results were analysed employing one way analysis of variance (ANOVA). Tukey's test was used for the post hoc treatment using SPSS (Statistical Package for Social Science) 24.0 for windows.