Screening and Identification of Compounds in Pomegranate Peel to Ameliorate 5‐Fluorouracil Induced Intestinal Mucositis in Rats and Induce HT‐29 Colorectal Cancer Cell Death through Reactive Oxygen Species Mediated Apoptosis and Cell Cycle Arrest

Abstract

Upon searching the Chinese Pharmacopoeia, it is found that 97 herbs belong to the large intestine meridians among which 23 of them are traditionally used for intestine‐related cancer treatment. Pomegranate peel, in particular, is used to treat diarrhea, dysentery, and bloody stool which are similar to symptoms of colorectal cancer and intestinal mucositis. As such, pomegranate peel is selected for network pharmacology analysis. Pathways related to colorectal cancer, VEGF, toll‐like receptor and mTOR, together with gene‐associated diseases like cancer, immune disorders and infection were found to be significantly related to compounds in pomegranate peel. DPPH and ABTS free‐radical scavenging activity assays coupled with HPLC‐DAD‐MS/MS analysis were employed to identify the pomegranate peel extracts and corresponding compounds with the strongest antioxidant activities. Various concentrations of acetone, methanol, and ethanol were used to extract compounds from pomegranate peel with 60 % solvent extracts exhibited the strongest free‐radical scavenging activities among the three solvents used. Nine most consistently appeared chromatographic peaks were identified, namely gallic acid, gallagyl‐hexoside (punicalin), digalloly‐hexoside, galloyl‐HHDP‐hexoside, bis‐HHDP‐hexoside (pedunculagin I), punicalagin‐I, punicalagin‐II, granatin B, ellagic acid. Punicalagin was identified as the major component contributing to the potent free‐radical scavenging activities. All compounds except gallic acid and ellagic acid could directly react with the free‐radicals and thus exhibiting free‐radical scavenging activities. Pomegranate peel ellagitannins (PETs, mainly containing punicalagin), pomegranate peel ethyl acetate extracts (PPEEs, mainly containing granatin B and punicalagin), and 20% aqueous methanol extracts (M20, mainly containing punicalin, digalloly‐hexoside, galloyl‐HHDP‐hexoside, and pedunculagin I) were prepared and their anti‐inflammatory and anti‐colorectal cancer activities were examined in RAW 264.7 and HT‐29 colorectal cancers cells. PPEEs exhibited both superior anti‐inflammatory and anticolorectal cancer activities. Anti‐HT‐29 cancer cell and anti‐intestinal mucositis activities of PETs, PPEEs, and M20 were further examined in xenograft nude mice and chemotherapy 5‐FU treated rats. Consistent with the in vitro results, in vivo results showed that PPEEs exhibited the strongest anti‐HT‐29 cancer cell and anti‐intestinal mucositis activities. Cell death mechanisms of PPEEs on HT‐29 cells were further examined. PPEEs induced dose‐ and time‐dependent apoptosis in HT‐29 cells as shown by Annexin V‐FICT and PI double staining, together with western blotting of cleaved caspase 3, 8, and 9. Interestingly, autophagy was inhibited. Cell cycle analysis revealed that the cells were arrested in S phase. Moreover, ROS production was dose‐dependently stimulated by PPEEs. Anti‐oxidant N‐acetyl‐L‐cysteine pre‐incubation ameliorated PPEEs‐induced ROS stress, apoptosis, and cell cycle arrest. Collectively, the cell death might be initiated through ROS mediated autophagy inhibition, cell cycle arrest and apoptosis induction. Moreover, PPEEs enhanced cytotoxicity and apoptosis‐induction capability of 5‐FU against HT‐29 cells. Taken together, the results have provided evidence for developing PPEEs as a potential therapeutic agent for colorectal cancer and 5‐FU‐induced intestinal mucositis.Support or Funding InformationThis study was supported financially by Seed Fund for Translational and Applied Research (Project code: 201511160027) and Seed Fund for Basic Research (Project code: 201611159232) of The University of Hong Kong.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.