*2.1. Chemicals*

All reagents were purchased from Merck (Darmstadt, Germany), Sigma–Aldrich (Steinheim, Germany), and Acros Organics (Thermo Fisher Scientific, Reel, Belgium), while all solvents used were of analytical grade.

## *2.2. Plant Materials*

Sun-dried *D. indum* fruits were obtained from Bukit Ibam, Muadzam Shah, Pahang, Malaysia and identified by Shamsul Khamis, a botanist from Universiti Kebangsaan Malaysia Herbarium (UKMB). A voucher specimen (PIIUM 0257) was deposited in the Herbarium of Kulliyyah of Pharmacy, International Islamic University of Malaysia, Kuantan. Healthy and uninfected fruits were carefully selected and the exocarp (skin), mesocarp (pulp), and seed of the fruits were separated manually. Each fruit part was further dried in the drying oven (Memmert GmbH + Co. KG, Büchenbach, Germany) at 40 ◦C for three days, then immediately ground into powder form and kept in plastic containers at room temperature until further use.

#### *2.3. Preparation of Extracts and Fractions*

One crude extract and three fractions were prepared from each part of the *D. indum* fruit. For the crude extract, 25 g of each fruit part was macerated in 100% methanol (500 mL) at room temperature for 48 h. The extraction procedure was repeated three times. Next, the solvent was removed under reduced pressure using a rotary evaporator (RV 10, IKA®, Staufen im Breisgau, Germany) at 40 ◦C. Using a similar extraction procedure, 75 g of each fruit part was extracted successively with 1.5 L *n*-hexane, dichloromethane (DCM), and methanol to obtain hexane, DCM, and methanol fractions [18]. All extracts and fractions were stored at 4 ◦C until further use.

#### *2.4. Reduction of Phosphomolybdic-Phosphotungstic Acid Reagents*

Capability of the extracts and fractions to reduce phosphomolybdic-phosphotungstic acid reagents, otherwise known as Folin–Ciocalteu reagent, was determined according to a previous report with slight modifications [3]. Briefly, 90 μL diluted Folin–Ciocalteu reagen<sup>t</sup> in deionized water (20% *v*/*v*) was placed in each well of a 96-flat-bottomed-well microplate. Then, an 18 μL sample solution in methanol (1000 μg/mL) was added and incubated at room temperature for 5 min, followed by the addition of 90 μL sodium carbonate in deionized water (75 g/L). The mixture was incubated for 2 h at room temperature. The absorbance was then read at 725 nm using a microplate reader (Tecan, Infinite M200 Nanoquant, Männedorf, Switzerland). Gallic acid equivalence (GAE) was determined using a gallic acid calibration curve. All samples were tested in triplicate. Results were expressed as μmol GAE per gram dry weight crude extract or fraction ± standard error of the mean (*SEM*).

#### *2.5. Reduction of Neocuproine*

The extent of the conversion of light blue-colored bis(neocuproine)copper(II) chelate to yellow-orange-colored bis(neocuproine)copper(I) chelate by antioxidants was measured using the method described by Apak, Güçlü, Özyürek, Bekta¸soglu, and Bener [ ˇ 19]. An aliquot of 48.8 μL copper(II) chloride in deionized water (10 mM), 48.8 μL neocuproine in 96% ethanol (7.5 mM), and 48.8 μL ammonium acetate buffer (1 M, pH 7.0) were added into wells of a 96-flat-bottomed-well microplate. Then, a 24.4 μL sample solution in 96% ethanol (1000 μg/mL) and 29.3 μL deionized water were added to make up the final volume of 200 μL in each well. The absorbance was measured at 450 nm after 30 min incubation at room temperature. The total antioxidant capacity (TAC) was determined using the standard calibration curve of trolox. All samples were measured in triplicate. Results were expressed as μmol trolox equivalence (TE) per gram dry weight crude extract or fraction ± standard error of the mean (*SEM*).

#### *2.6. Scavenging of DPPH Radical*

Serial dilution from stock solution of the samples and positive standard (quercetin) in 100% methanol (1000 μg/mL) was done in 30 mL glass vials using a micropipette, then 100 μL of each concentration was transferred to each well of the microplate. A total of 100 μL DPPH in methanol (80 μg/mL) was then added. The mixture was incubated at room temperature for 30 min and the absorbance of the test mixture was then read at 517 nm against the blanks: Ablank sample (100 μL of 7.8125–1000 μg/mL sample without DPPH and 100 μL methanol) and Ablank methanol (200 μL of 100% methanol). The percentage of inhibition of the DPPH radical was calculated using the equation:

#### DPPH radical scavenging activity (%) = [1 − (Acontrol − Asample)/Acontrol] × 100

where Acontrol is the absorbance of DPPH without the sample after subtraction with Ablank methanol while Asample is the absorbance of samples with DPPH after subtraction with Ablank methanol and Ablank sample. Ablank sample was included in the equation to minimize the effect of varying visible colors of the different extracts to the absorbance readings. For each sample, the assay was conducted in triplicate. The concentration of extracts required to inhibit the DPPH radicals by 50% (IC50) was calculated using either the logarithmic or exponential or linear regression model equation that best fitted the data for each sample (*r* > 0.9) [20].

#### *2.7. Inhibition of Linoleic Acid Peroxidation*

Briefly, 62.5 μL of varying concentrations of the samples (7.8125–1000 μg/mL) and standard (quercetin) were prepared in 1.5 mL Eppendorf tubes. The linoleic acid emulsion was freshly prepared by the emulsification of 100 μL of linoleic acid with 200 μL Tween 20 and 19.7 mL deionized water [21]. Next, 62.5 μL of varying concentrations of the *D. indum* extracts, fractions and standard were mixed with 62.5 μL of the linoleic acid emulsion, 62.5 μL of phosphate buffer (100 μM, pH 7.4), and 12.5 μL of ferrous sulfate solution (4 mM in deionized water). Linoleic acid peroxidation was started by the addition of 12.5 μL ascorbic acid (2 mM in deionized water, freshly prepared for each extract), incubated for 30 min at 37 ◦C, and terminated by the addition of 187.6 μL of trichloroacetic acid (10% in deionized water). The mixture was then added to 100 μL of thiobarbituric acid solution (1% in 50 mM NaOH), followed by heating in a 100 ◦C water bath for 10 min. The mixtures were centrifuged at 3500× *g* for 10 min. Then, 100 μL of the supernatant was transferred into the well of a 96-flat-bottomed-well microplate and the absorbance of thiobarbituric acid-reacting substances (TBARS) in the supernatant was measured at 532 nm. For each sample concentration, the assay was conducted in triplicate [22]. The percentage of linoleic acid peroxidation inhibition and IC50 were calculated using a similar equation and method described in Section 2.6.
