Year of Publication

2009

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Dr. Russell Mumper

Second Advisor

Dr. Jurgen Rohr

Abstract

Blackberries are rich in polyphenols including anthocyanins. Polyphenols are hypothesized to have biological activities that impact positively on human health. The purpose of these studies was to develop phenolic extracts from selected cultivars of blackberries currently grown in Kentucky as potential Botanical Drug Products for the treatment and prevention of cancer and inflammatory diseases.

An ultrasound-assisted ethanol extraction method was employed to obtain anthocyanin-containing extracts (ACEs) from puree or powder (lyophilized puree) of blackberries. ACEs were analyzed for total anthocyanin and phenolics content, polymeric color, and total antioxidant capacity (TAC). The influence of water content in the extraction system was evaluated. A 90 day stability study of the extract and a 48 h stability study of the extract in biologically relevant buffers were completed. HPLC-MS results showed the anthocyanins in ACE were mainly cyanidin-based. As compared to powder-derived ACEs, puree-derived ACEs contained similar amounts of anthocyanins, but greater levels of phenolics and increased TAC.

The in vitro antiproliferative effects of ACEs were evaluated in human leukemia (HL- 60), colon (HT-29), and breast (MCF-7) cancer cells. The anticancer mechanism involving reactive oxygen species (ROS) generation was investigated. It was found puree-derived ACEs significantly enhanced production of H2O2 and cytotoxicity in all cell lines as compared to powder-derived ACEs. Cyanidin 3-glucoside exerted anticancer effect by acting synergistically or additively with other active components in the extracts. Furthermore, the phenolic-enriched fractions were separated from non-phenolic fractions in ACEs and found to have potent antioxidant and antiproliferative activities. Pureederived ACE and corresponding phenolic-enriched methanol fraction (MF) induced cell death through ROS-independent caspase 3 pathway whereas the cytotoxicity induced by powder-derived ACE and corresponding MF is related to ROS mechanisms.

The in vitro anti-inflammatory studies showed ACEs inhibited Lipid A-induced Interleukin-12 (IL-12) release from mouse dendritic cells, and modulated lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) from murine macrophages.

These studies have important implications for the potential use of blackberry extracts for the treatment and prevention of cancer and inflammation diseases and provide essential information for the development of Botanical Drug Products from extracts derived from blackberries and other fruits.

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