*2.1. Materials*

Allopurinol, xanthine, xanthine oxidase, potassium di-hydrogen phosphate (KH2PO4), di-potassium hydrogen phosphate (K2HPO4), DPPH, and ascorbic acid were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Dimethylsulphoxide (DMSO), hydrochloric acid (HCl), absolute ethanol, methanol, chloroform, n-butanol, hexane, and other reagents of analytical grade were obtained from Merck (Darmstadt, Germany).

#### *2.2. Plant Collection and Processing*

Fresh leaves (1 kg) of *A. bilimbi* were obtained in July 2016 from the garden at Indera Makhota in the neighbourhood of International Islamic University Malaysia (IIUM), Kuantan Campus. The plant was identified and authenticated by the taxonomist at Taman Pertanian, Indera Makhota, 25200 Kuantan, Pahang Darul Makmur, Malaysia. Subsequently, the sample was deposited in the herbarium of Kulliyyah of Pharmacy, IIUM, Kuantan, Pahang DM, Malaysia (voucher no: NMPC-QAAB-12). The fresh plant material was initially dried in a PROTECH laboratory air dryer (FDD-720-Malaysia) at 45 ◦C. Thereafter, the dried plant material was pulverised into a powdered form using Fritsch Universal Cutting Mill-Pulverisette 19-Germany. The powdered material was kept in airtight polythene bags at 4 ◦C prior to further use.

#### *2.3. Extraction and Fractionation*

Total leaf extract was prepared by soaking powdered leaves (350 g) in methanol (2 L) at room temperature for 48 h, followed by filtration and concentration of the filtrate under reduced pressure using a rotary evaporator (Buchi, Switzerland). The entire procedure was repeated 3 times to yield 40.5 g (11.6%) of crude methanolic leaves extract. Afterwards, the dried crude methanolic leaves extract was subjected to liquid-liquid fractionation. The extract was first suspended in 10% aqueous ethanol, then transferred into the separating funnel and sequentially extracted with hexane, chloroform, n-butanol, and double distilled water to afford hexane fraction (11.67 g), chloroform fraction (8.26 g), n-butanol (8.30 g), and aqueous fraction (12.14 g), respectively.

#### *2.4. Preliminary Phytochemical Screening*

The crude methanolic leaves extract of *A. bilimbi* was initially subjected to various phytochemical tests to detect the occurrence of a different class of secondary metabolites which are associated with the existence of flavonoids (Shinoda test) [16], terpenoids (Salkowski test), saponins (frothing test) [17], alkaloids (Dragendorff's and Mayer's tests) and anthraquinones (free and combined) [18].

#### *2.5. LC-QTOF-MS and LC-QTOF-MS/MS Analyses of Bioactive Constituents of n-Butanol Fraction*

The LC-QTOF-MS and LC-QTOF-MS/MS analyses of the active n-butanol fraction, which showed significant antiradical (DPPH radical scavenging activity) and xanthine oxidase inhibitory effects, were carried out using an Agilent 1290 Infinity Ultra high performance liquid chromatographic system (LC) (Agilent, Santa Clara, CA, USA) coupled to an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with dual electrospray ionization (ESI) source. The n-butanol fraction was prepared at a concentration of 0.01 mg/mL in methanol, and was first filtered using a 0.45 μm nylon syringe filter before an aliquot was collected (3.0 μL) and injected into the LC system. The sample was separated on a reverse phase column [Agilent Zorbax Eclipse XDB-C18, Narrow-Bore 2.1 × 150 mm, 3.5 micron (P/N: 930990-902)] (Agilent, Santa Clara, CA, USA). The mobile phase included A–0.1% formic acid in water, and B–0.1% formic acid in acetonitrile. The column was eluted using a gradient mode under the following conditions. Auto sampler temperature: 6 ◦C; column temperature: 25 ◦C; flow rate: 0.5 mL/min; linear gradient from 5% B to 100% B from 0 min to 25 min. Full scan mass spectrometry was carried out using ESI positive ionization mode resulting in full mass spectrum with mass range ( *m*/*z*) of 100–3200. The system workstation was equipped with a spectra library of several compounds which was used in the tentative identification of major, possibly bioactive constituents based on their accurate mass and elemental composition.

#### *2.6. DPPH Free Radical Scavenging Activity*

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity of *A. bilimbi* crude methanolic leaves extract, and its fractions viz. hexane, chloroform, and n-butanol fractions, was evaluated according to the protocol described by Nickavar et al. and Barontini et al. [19,20] with some modifications. Briefly, 1 mL of different concentrations of test samples in methanol (4.8 μg/mL–1000 μg/mL) were prepared and respectively treated with 2 mL of 0.1 mM of freshly prepared DPPH solution in methanol, and finally, diluted using 1 mL of deionized water. The mixture was allowed to stay in an incubator at 30 ◦C for 30 min and absorbance at 517 nm was recorded using a microplate reader (Infinite M200 Nanoquant Tecan, Männedorf, Switzerland). Methanol was used as the blank, whereas DPPH, methanol, and water (2:1:1) served as controls. Ascorbic acid was used as positive standard and IC50 values in μg/mL were determined. The IC50 values were calculated by the linear regression of plots. The percentage DPPH free stable radical scavenging activity was computed using the following equation:

$$\% \text{ radical scaveringing activity} = (Abs\_{\text{control}} - Abs\_{\text{sample}}/Abs\_{\text{control}}) \times 100.$$

where, *Abs*control = absorbance of control, *Abs*sample = absorbance of extract, fraction or ascorbic acid.

#### *2.7. Xanthine Oxidase Inhibitory Activity Assay*

The xanthine oxidase inhibitory activity of *A. bilimbi* crude methanolic leaves extract and its fractions viz. hexane, chloroform and n-butanol fractions was determined by following the protocol described by Thiombiano et al. [21]. The assay was carried out in 96 well plates. The reaction mixture was prepared in such a way that it contains 50 μL of test solution together with 30 μL of freshly xanthine oxidase solution (0.2 units/mL). The assay mixture was pre-incubated at 37 ◦C for 15 min. Thereafter, the reaction was initiated by the addition of 60 μL of substrate solution (0.15 mM of xanthine). The mixture was placed in the incubator set at 37 ◦C and allowed to stay for 30 min. The reaction was terminated by the addition of 50 μL of 0.5 M HCl. A known potent xanthine oxidase inhibitor, viz. allopurinol, was used as positive control. The absorbance at 295 nm was measured using microplate reader (Infinite M200 Nanoquant Tecan, Tecan Group Ltd., Männedorf, Switzerland). The percentage of xanthine oxidase inhibition was calculated using the following equation:

> % xanthine oxidase inhibition = (*Abs*control − *Abs*sample/*Abs*control) × 100.

where, *Abs*control = activity of enzyme without extract/fraction, and *Abs*sample = enzyme activity in presence of extract/fraction or allopurinol.
