Abstract
Vaccinium macrocarpon (American cranberry) is commonly used to produce juice, supplements, and other edible products. A byproduct called pomace is left behind, consisting of skins, seeds, peels, and stems. The goal of this study was to develop ultrasound-assisted extraction, fractionation, and chromatographic methods to isolate and characterize pentacyclic triterpenoids, phytosterols, and tocopherols from cranberry pomace for bioactivity assessment, including anti-inflammatory pathways and anti-proliferative effects on colon cancer cells. Extracts and fractions were prepared and analyzed using HPLC-DAD, UPLC-MS, and GC-MS, enabling the identification and quantification of triterpenoid acids and esters, neutral triterpenoids, phytosterols, and tocopherols in pomace-derived preparations. Cranberry pomace acetone extract (POM-ACE), containing at least 51% triterpenoids and 4% or more phytosterols, was fractionated using silica gel column chromatography. Anti-inflammatory effects were evaluated in a THP-1 monocyte model using ELISA to measure IL-1β production. An initial screening for inhibition of pro-inflammatory enzymes sEH (soluble epoxide hydrolase) and LOX (lipoxygenase) was conducted. Fractions rich in triterpenoids exhibited dose dependent activity against both enzymes; among these, fraction F showed the highest sEH inhibitory activity . Fractions abundant in triterpenoids, such as ursolic and oleanolic acid and their esters, showed mild dose-dependent inhibition of lipoxygenase activity; notably, fraction 2G (IC₅₀ = 104 µg/mL) . Several fractions significantly suppressed LPS-induced IL-1β expression in THP-1 monocytes, especially fraction D (MIC = 1.25 µg/mL) fractions J, K, and M . In a docking study, we found that oleanolic acid-sEH exhibited favorable interactions. PLS regression identified oleanolic acid and cis-HCA derivatives as the primary chemical predictors of soluble epoxide hydrolase inhibition. Pearson correlation analysis shows that POM-ACE constituents are positively associated with LOX inhibition. Preliminary screening of POM-ACE for anti-proliferative activity over 24 h using the MTT assay (showed mild to moderate anti-cancer potential. Several purified fractions demonstrated dose-dependent anti-proliferative activity, especially fractions 2F, 2G, and 2K, which are also rich in ursolic acid, oleanolic acid, and their esters. These fractions showed moderate, dose-dependent activity (IC₅₀ = 12–17 µg/mL) in the MTT assay. Multivariate analysis found a strong correlation between UA and OA and their potent inhibition of HT-29. Docking study data support multivariate analysis and indicates involvement of apoptosis. For the most effective fractions (2F, 2G, and 2K), cell death in HT-29 cancer cells via apoptosis or necrosis was evaluated by flow cytometry using the Annexin V-FITC/PI assay. We observed that these fractions induced both early and late apoptosis at the IC₅₀ concentration after 24 hours of incubation. These studies indicate that cranberry pomace is a promising source of bioactive triterpenoids and phytosterols with anti-inflammatory and anti-proliferative properties; particularly ursolic and oleanolic acids, their hydroxycinnamoyl esters, amyrins and beta-sitosterol. The anti-inflammatory assay data improve our understanding of how pomace-derived terpenoids can inhibit inflammation by several routes including targeting the enzymes soluble epoxide hydrolase and lipoxygenase, and inhibition of inflammasome-linked cytokine production. Our data in HT-29 colon cancer cells indicate that the combination of triterpenoids found in cranberry pomace can suppress cell proliferation, in part by triggering apoptosis.