Phytochemical profiling, antioxidant, enzyme inhibition and cytotoxic potential of Bougainvillea glabra flowers

In this study, phytochemical composition, antioxidant, enzyme inhibition and cytotoxic activities of methanol and dichloromethane (DCM) extracts of Bougainvillea glabra (B. glabra) flowers were investigated. Methanol extract was found to have higher total bioactive contents and UHPLC-MS analysis of methanol extract revealed the presence of well-known phenolic and flavonoid compounds. Antioxidant activities were performed by radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC), phosphomolybdenum (TAC) and metal chelating assays. From our result, we observed that methanol extract had many antioxidant compounds. The DCM extract exhibited higher cholinesterases and α-glucosidase enzyme inhibition, while methanol extract showed significant urease inhibition. Both extracts exhibited strong to moderate cytotoxicity against MCF-7, MDA-MB-231, CaSki, DU-145 and SW-480 cancer cells with IC 50 values ranging from 88.49 to 304.7 µg/mL. The findings showed the B. glabra to possess considerable antioxidant, enzyme inhibition and cytotoxic potentials and therefore has potential to discover novel bioactive molecules.


Total bioactive components
Total phenolics were determined by employing standard Folin-Ciocalteu method (Kahkonen et al. 1999) using gallic acid as standard and the results were expressed as mg GAE/g (gallic acid equivalents). Total flavonoid content assay was done by Aluminium chloride colorimetric method (Chew et al. 2009). Quercetin was used as standard and the results were expressed as mg QE/g (quercetin equivalent).
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 In this method, 1 mL of plant extract of different concentrations (1000-15.625 µg/mL) was added to 2 ml of DPPH solution (0.059 mg/mL methanol). Absorbance was measured at 517 nm after 30 min incubation, (Miliauskas et al. 2004).
Firstly, ABTS solution was diluted in methanol until the absorbance reached the value of 0.700 0.02 at 734 nm. The test solution (1 mL), mixed with ABTS solution (2 mL), was mixed and the absorbance was recorded at 734 nm after 30 min of incubation at room temperature. The results were expressed as milligrams of trolox equivalents per gram of dry extract (TEs/g extract) (Grochowski et al. 2017).

Ferric reducing power method
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 o 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.

Cupric ion reducing (CUPRAC) method
Extract solutions (0.5 mL) were added to reaction mixture [CuCl 2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM), NH 4 Ac buffer (1 mL, 1 M, pH 7.0)] and the absorbance was recorded at 450 nm after 30 min of incubation at room temperature. Similarly, a blank sample (prepared in the same manner but without the extract) was prepared and analysed according to this procedure. Milligrams of trolox equivalents per gram of dry extract (TEs/g extract) were the measurement unit (Grochowski et al. 2017).

Phosphomolybdenum assay (TAC)
Total antioxidant capacity (TAC) was determined by phosphomolybdenum method (Prieto et al. 1999). Briefly, the extract solution (0.3mL, 1 mg/mL) was mixed with 3 mL of molybdate reagent solution, incubated for 90 min 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).

Metal chelating activity on ferrous ions
Extract solution (2.0 mL) was added to FeCl 2 (0.05 mL, 2 mM), and the reaction was started using 0.2 mL of 5 mM ferrozine. Similarly, a blank sample for each sample (prepared in the same manner but without ferrozine) was prepared, and all the absorbances were recorded after 10 min of incubation (room temperature) at 562 nm. Milligrams of EDTA equivalents per gram of dry extract (EDTAEs/g extract) (Grochowski et al. 2017).

Cholinesterase inhibition activity
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity were determined spectrophotometrically according to the method Ellman et al (Ellman et al. 1961). Total reaction mixture in 96-well plate was 100 µL containing 60 µL of phosphate buffer (50 mM, pH 7.7), 10 µL plant sample of 10 mg/mL stock solution. Then 10 µL enzyme (0.005 units AChE or 0.5 units BChE) was added. The reaction mixture was mixed, incubated at 37 ºC for 10 min and its absorbance was taken at 405 nm using Synergy HT, Biotek, USA 96-well plate reader followed by addition of 10 µL of 0.5 mM substrate (acetylthiocholine iodide for AChE and butyrylthiocholine chloride for BChE) and 10 µL of 0.5 mM DTNB was added to the above reaction mixture to initiate the reaction and incubated again at 37 ºC for 30 min.
Absorbance was again measured at 405 nm. Eserine was used as a control.
The inhibition (%) was calculated as EZ-Fit Enzyme kinetics software was used to calculate IC 50 values (Perrella Scientific Inc. Amherst, USA).

Urease inhibition activity
The total assay mixture of 85 µL in 96 well plates contained phosphate buffer (50 mM, pH 7.0) 10 µL sample and jackbean urease enzyme (25 µL of 0.14 units). Contents were incubated at 37 °C for 5 min. After incubation, 40 µL of urea substrate (20 mM) was added and incubation continued for further 10 min. Then, 115 µL of freshly prepared phenol hypochloride reagent was added in each well and further incubated for 10 min at 37 °C for colour development. Absorbance was measured at 625 nm (Weatherburn 1967). Kojic acid was used as a control. The inhibition (%) and IC 50 results were determined as given above for cholinesterases.

α-Glucosidase inhibition activity
100 µL reaction mixture in 96-well plate contained 70 µL of phosphate buffer (50 mM, pH 6.8), 10 µL plant sample (0.5 mM) and 10 µL of baker's yeast enzyme (0.057 units). The reaction mixture was mixed, incubated for 37 °C for 10 min and its absorbance was taken at 400 nm. Reaction was initiated by adding 10 µL of substrate, p-nitrophenyl-Dglucopyranoside (0.5 mM) (Chapdelaine et al. 1978). Incubation was continued for further 30 min and after-read. Acarbose was used as a control. The inhibition (%) and IC 50 values were calculated as given above for cholinesterases.

Cytotoxicity assays
Cell lines and culture medium Cell viability (%) = Abs s -Abs c × 100.
Abs s = absorbance of sample, Abs c = absorbance of control.

Statistical data analysis
All the experiments were repeated three times and analysis was done in triplicates. The obtained results were expressed as mean value and standard deviation (mean±SD). One-way analysis of variance (ANOVA) was used to calculate the differences, followed by Tukey's significant difference post hoc test (p < 0.05). Graph Pad Prism software (San Diego, CA, USA, Version 6.03) was used to calculate IC 50. SPSS v22.0 software was used to carry out all experimental analysis.     Values are expressed as means of three replicates, IC 50 value represents concentration that reduces cell viability to 50%. **The IC 50 value was higher than 500 µg/mL