PhD Dissertation Defense by Md Sagir Mia
Committee Members Name
Dr. Catherine Neto
Professor, Department of Chemistry and Biochemistry
Dissertation Advisor
Dr. Shuowei Cai
Professor, Department of Chemistry and Biochemistry
Dissertation Committee
Dr. Tracie Ferreira
Professor and Chair, Department of Bioengineering, University of Massachusetts Dartmouth
Dissertation Committee
Dr. Hang Ma
Assistant Professor, Department of Biomedical and Pharmaceutical Sciences
University of Rhode Island, Kingston, RI
Dissertation Committee
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 (IC50 = 0.726 µg/mL) and contained 34% ursolic acid, 18% oleanolic acid, and 26% their hydroxycinnamoyl esters by weight. 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) which contained 55.6% ursolic acid, 15% oleanolic acid, and 29% their hydroxycinnamoyl esters by weight. Several fractions significantly suppressed LPS-induced IL-1β expression in THP-1 monocytes, especially fraction D (MIC = 1.25 µg/mL) which was rich in α- and β-amyrins and sitosterol, and fractions J, K, and M which contain varying concentrations of ursolic acid, oleanolic acid, and their HCA esters.
Preliminary screening of POM-ACE for anti-proliferative activity over 24 h using the MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) showed mild to moderate anti-cancer potential. The IC50 for HT-29 (colorectal adenocarcinoma), A375 (melanoma), and HEK 293T (human embryonic kidney) ranged from 22.9 to 329 µg/ml. Factors such as dosage form, delivery, formulation, solubility, and dissolution may influence the overall cell membrane permeability of POM-ACE. We hypothesized that specific components (e.g., triterpenoids, sterols), or combinations of these, could be absorbed more effectively or be more efficient at inhibiting cell proliferation. POM-ACE fractions were then evaluated in HT-29 colon cancer cells by MTT assay, and their relative anti-proliferative effects were found more potent than those of the original POM-ACE extract. Several purified fractions demonstrated dose-dependent anti-proliferative activity, especially fractions 2F, 2G, and 2K, which were also rich in ursolic acid, oleanolic acid, and their esters. These fractions showed moderate, dose-dependent reduction in cell proliferation over 24 h (IC₅₀ = 12–17 µg/mL) based on the MTT assay. For the most effective fractions (2F, 2G, and 2K), cell death in 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 from colon cancer cell culture indicate that the combination of triterpenoids found in cranberry pomace can suppress cell proliferation in part by triggering apoptosis.
Meeting ID: 994 1554 5046
Passcode: 904975
SENG-311
Rachel White
508/999-8232
rwhite@umassd.edu
https://umassd.zoom.us/j/99415545046?pwd=v6YYk0cF53AbcVygbu1cTrezDJbH9O.1