*3.7. Determination of the Absolute Configuration of the Sugar Unit*

Compounds **6** (1 mg) was hydrolyzed with HCl (1 N) at 80 ◦C (1 mL) over 2 h, followed by a liquid-liquid partition with EtOAc (2 × 1 mL). The aqueous layer was neutralized with Ag2CO3, and the supernatant was dried. L-cysteine methyl ester (2 mg) in pyridine (1 mL) was added and heated for 1 h at 60–70 ◦C. Phenylisothiocyanate (150 μL) was added and heated for an additional hour at 60–70 ◦C to form the thiocarbamoyl thiazoline derivative. The reaction mixture was analyzed by the HPLC method previously reported [44]. The absolute configuration of the sugar was determined by comparing the HPLC retention times of the prepared thiocarbamoyl thiazolidine derivatives to appropriate standards.

### *3.8. Physicochemical Parameters of Charantoside XV (6)*

Amorphous white solid, [α] 25D—71 (*c* 0.1, MeOH). HRESIMS of compound **1** showed a molecular ion at *m*/*z* 687.3967 [M + Na]+ (calculated 687.4047 *m*/*z* [M + Na]<sup>+</sup> for C37H60O10). 1H- and 13C-NMR chemical shifts are presented in Table 1.

### *3.9. Bioassays*

3.9.1. Cell Culture and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

The anti-inflammatory activity of compounds purified from bitter melon was carried out using RAW 264.7 murine macrophage cells (ATCC, Rockville, MD, USA). The cells were cultured in RPMI 1640 medium supplemented with 10% (*v*/*v*) fetal bovine serum. Additionally, 100 U/mL of penicillin and 100 μg/mL streptomycin were added to the growth medium. Cultured cells were maintained at 37 ◦C in an incubator with 5% CO2. After 80% cell confluency, spent media was replaced with fresh media. For the analysis of anti-inflammatory gene expression, RAW 264.7 cells were seeded into 6 well plates (5.0 × <sup>10</sup><sup>5</sup> cells/well). After a 24 h incubation period, the cells were treated with a <sup>50</sup> <sup>μ</sup><sup>M</sup> solution of purified compounds for 1 h followed by the addition of LPS (1 μg/mL) to all cells except the control cells. The concentration of compounds used in this assay was determined according to previously published research articles [45]. The cells were incubated for an additional 18 h after which the total RNA was extracted from the cells. Total RNA extraction was carried out using the Aurum Total RNA Mini Kit. The quantity of RNA extracted from the cells was determined using a Nanodrop Spectrophotometer.

(Thermo-Fisher, Waltham, MA, USA) [19]. The purified RNA obtained from cell lysates were used as templates to synthesize cDNA. The synthesis procedure was carried out using the specified manufacture's protocols (iScript cDNA Synthesis Kit, Bio-Rad Inc, Hercules, CA, USA).

Further, real-time PCR was carried out using the manufacturer's specification for the Bio-Rad SYBR Green PCR Master Mix. The relative expression of *IL-6*, *TNF-α*, *COX-2*, and *iNOS* was compared and normalized to the expression of *GAPDH* from the respective treatment groups. Primer sequences for this study are available upon request. The negative control constituted of untreated cells, while the positive control consisted of LPS-stimulated cells.
