*2.1. Extraction of BG-4*

The 4 kDa peptide BG-4 was extracted from bitter gourd seeds as described previously [13].

#### *2.2. In Vitro Antioxidant Activity of BG-4*

The antioxidant capacity of BG-4 was tested via the oxygen radical absorbance capacity assay (ORAC) as described by Vernaza and coworkers [15] with slight modifications. In brief, 150 μL of fluorescein solution prepared in phosphate buffer (75 mM, pH 7.4) were plated in a black 96-well plate. Twenty-five microliters of Trolox standard curve (100–3.125 μM in phosphate buffer), blank or sample diluted in phosphate buffer were added and incubated at 37 ◦C for 30 min. Then, 25 μL of 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) (Sigma-Aldrich, St. Louis, MO, USA) solution at 41.5 mg/mL in phosphate buffer was added and fluorescence was read at 485 nm/20 nm excitation and 528 nm/20 nm emission wavelengths for 2 h every min. Each sample and standard curve point was measured in triplicate and results were expressed as μM of Trolox equivalents/g of BG-4. Radical scavenging activity was measured by quantifying the inhibition of 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH•). Briefly, 100 μL of sample and blank were plated in a clear 96-well plate, then 100 μL of 100 μM DPPH• (Sigma-Aldrich, St. Louis, MO, USA) dissolved in methanol were added and incubated in dark for 30 min. Simultaneously, a combination of sample and methanol, instead DPPH•, were tested as reference for background signal. Absorbance was read at 517 nm. The absorbance of methanol reference was subtracted and the percentage of inhibition was calculated against blank. Results are presented as percentage of scavenging rate of DPPH•.

#### *2.3. Measurement of Pro-Inflammatory Markers In Vitro*

Murine RAW 264.7 macrophages were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Life Tech, Carlsbad, CA, USA) and 1% penicillin/streptomycin at 37 ◦C in a humidified 5% CO2 incubator. Cells were seeded at 2 × 10<sup>5</sup> cells/well in 6-well plates in 2 mL media, or 5 × 10<sup>3</sup> cells/well in 96-well plates in 200 μL media and allowed to attach overnight. Cells were treated with BG-4 (0–500 μg/mL) for 8 h and stimulated with LPS (1 μg/mL) for 16 h. After which, supernatant was collected for TNF-α and IL-6 measurement via ELISA following the manufacturer's protocol (BioLegend, San Diego, CA) and nitric oxide (NO) production by Griess reagen<sup>t</sup> assay. Whole cell lysates were collected for immunoblotting of inducible nitric oxidase synthase (iNOS) and cyclooxygenase-2 (COX-2) (ProteinTech, Chicago, IL, USA) by chemiluminescence following standard protocol. Cell viability was tested by MTS assay following the manufacturer's protocol (Promega, Madison, WI, USA).

#### *2.4. Dosage Information and In Vivo Experimental Procedure*

The BG-4 dose used in the animal study (15 mg/kg body weight (bw)) is equivalent to the optimum concentration (375 μg/mL) of BG-4 in vitro producing significantly decreased expression of pro-inflammatory markers without affecting cell viability. This translation assumes an average mouse

weight of 25 g and circulating blood of 1 mL. This will be equivalent to a daily intake of two 500 mg capsules as a dietary supplement for a 70-kg person. The protocol for the animal experiment was approved by the Institutional Animal Care and Use Committee of the University of Tennessee Knoxville (Approval Protocol #2591-0418) and followed guidelines of the National Institutes of Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Twenty-two male C57BL/6 7-week old mice (Jackson Laboratories, Harbor, ME, USA) were randomized and housed in pairs in standard mouse cages with water and food provided ad libitum, under standard controlled conditions (23 ± 2 ◦C, 30–70% relative humidity with 12 h light and 12 h dark cycle). Mice were randomly divided into three groups and treated as follows: control group (CG, *n* = 6), DSS-treated group (DSS, *n* = 8), and DSS + BG-4-treated group (BG-4, *n* = 8). Mice in CG were administered with normal drinking water, while DSS and BG-4 groups received drinking water with 3% DSS (MW = 36–50 kDa, MP Biomedicals, Santa Ana, CA) to induce colitis. Two stages of DSS administration were performed allowing a recovery period in between, in order to simulate the periods of relapse and remission that IBD patients experience [16]. Daily intraperitoneal injection (IP) of 100 μL sterile water was performed over CG and DSS groups, while BG-4 received IP of 15 mg BG-4/kg bw dissolved in 100 μL sterile water. Food intake and body weight were recorded daily. Presence of visible blood (in stool or anus), as well as stool consistency, were monitored and scored daily. Stool samples were collected from the cages every three days to evaluate the presence of occult blood via quantification of hemoglobin in feces. At day 15, mice were anesthetized with isoflurane, blood collected by cardiac puncture, followed by cervical dislocation. Colon was removed, washed with PBS, length measured and cut longitudinally into two pieces: one for hematoxylin and eosin (H&E) staining, and the other frozen in liquid nitrogen for biochemical analysis.

#### *2.5. Myeloperoxidase Assay in the Colon*

Neutrophils infiltration was assessed by measuring myeloperoxidase (MPO) activity in colonic extracts as reported previously [17] with slight modifications. Colonic extracts were obtained by beads homogenization of 50 mg of colon samples with 1 mL bu ffer (5 g of hexadecyltrimethylammonium in 1 L of 50 mM potassium phosphate bu ffer at pH 6.0), followed by centrifugation at 20,000× *g* for 15 min at 4 ◦C. Supernatant was collected and further centrifuged to ensure total precipitation of solid tissue. Ten μL of supernatant was plated in triplicate in a 96-well plate and combined with 200 μL freshly prepared *o*-dianisidine solution (16.7 mg of o-dianisidine, 90 mL of deionized water and 10 mL of potassium phosphate bu ffer, combined with 50 μL of 1.2% H2O2). Absorbance at 450 nm was recorded every 30 s for 5 min. MPO activity was calculated as the amount needed to degrade 1 μmol H2O2/min at 21–22 ◦C per mg protein in the colonic extract.

#### *2.6. Measurement of Hemoglobin Content in the Feces*

Presence of occult blood in the feces was assessed by measuring hemoglobin content [18,19]. Twenty mg of freeze-dried pulverized samples were vortexed with 100 μL dH2O, and boiled for 10 min. Six hundred microliters of 30% acetic acid was added, vortexed for 2 min, then 900 μL of ethyl acetate was added, and organic layer was collected in a separate microcentrifuge tube after centrifugation at 2000× *g* for 3 min. In a quartz cuvette, equal parts of the organic phase and TMB solution (14.4 mg of 3,3,5,5-tetramethylbenzidine in 100 mL mixture of glacial acetic acid/dH2O/ethanol 20/30/50) were mixed and the reaction was started by adding the same amount of 3% H2O2. The absorbance was recorded at 660 nm at 30 s, 60 s, and 90 s. Hemoglobin content was calculated as the average for the three time points per mg of feces using bovine hemoglobin (Alfa Aesar, Ward Hill, MA, USA) standard curve.

#### *2.7. Measurement of Cytokines by ELISA*

Supernatants of treated macrophages, as well as blood serum and colonic extracts, were used to measure the expression of pro-inflammatory cytokines using ELISA kits according to manufacturer instructions (BioLegend, San Diego, CA, USA).
