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The protective effect of blackberry anthocyanins against free radical-induced oxidation and cytotoxicity… Elisia, Ingrid 2006

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The Protective Effect of Blackberry Anthocyanins against Free Radical-Induced Oxidation and Cytotoxicity in Multiple Cell Lines B y I N G R I D E L I S I A B.Sc. The University of British Columbia, 2003 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R O F S C I E N C E in The F A C U L T Y OF G R A D U A T E S T U D I E S (Food Science) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A November 2005 © Ingrid Elisia, 2005 A B S T R A C T Anthocyanins are the blue and red pigments found in berries, with known antioxidant properties that may be associated with several health benefits, such as a reduction in the risk of heart disease and cancer. Blackberry in particular, is a rich source of anthocyanins and has notable antioxidant activity. Although the antioxidant capacity of anthocyanins has been well established in cell free in vitro system, there is very little evidence that links this antioxidant activity with protection against free radical associated oxidative damage in biological systems. The ultimate goal of this thesis, therefore, was to evaluate the protective effect of blackberry anthocyanins against free radical-induced oxidative stress and the resulting cytotoxicity using multiple cultured cell lines. Anthocyanins of both crude blackberry extracts as well as an anthocyanin enriched fraction were identified and quantified using H P L C . Different cytotoxicity assays ( M T T , CellTiterGlo, BrdU) were validated against cell counting method to determine the most appropriate cytotoxicity assay(s) for the evaluation of blackberry anthocyanins in cultured cells. The effect of blackberry anthocyanins were individually evaluated in five distinct cell lines: two breast cancer lines ( M D A - M B - 4 5 3 and M C F - 7 ) , two intestinal cell lines (Caco-2 and INT-407), and one prostate cancer cell line (LNCaP) , using M T T and CellTiter-Glo assay. In other tests, A A P H (2, 2 ' -azobis (2-amidinopropane) dihydrochloride), a free radical generator, was used to initiate intracellular oxidation and induce cytotoxicity. The effect of the blackberry extracts against A A P H initiated intracellular oxidation was monitored with a dichlorofluorescin diacetate ( D C F H - D A ) probe. The protective effect of blackberry anthocyanins against AAPH-induced cytotoxicity was measured with M T T and CellTiterGlo assays. Ce l l cycle analysis was also performed to determine possible protective mechanisms of blackberry anthocyanins against free radical associated damages. Cyanidin-3-glucoside was found to be the major anthocyanin (85 %) in blackberry. Blackberry anthocyanins were demonstrated to have no cytotoxic properties at physiological concentration. In addition, blackberry anthocyanins were found to suppress AAPH-ini t ia ted intracellular oxidation. This effect corresponded a protection effect against free radical-induced cytotoxicity. Ce l l cycle analysis with propidium iodide staining demonstrated that blackberry anthocyanins prevented cytotoxicity by scavenging the generated peroxyl radicals, thus alleviating AAPH-induced apoptosis. T A B L E OF C O N T E N T S A B S T R A C T , i i T A B L E O F C O N T E N T S iv LIST O F T A B L E S v i L I S T O F F I G U R E S v i i L I S T O F A B B R E V I A T I O N S v i i A C K N O W L E D G E M E N T ix I N T R O D U C T I O N 1 L I T E R A T U R E R E V I E W 4 Anthocyanins 4 Reactive Oxygen Species and Oxidative Stress 7 Oxidative Stress on Cellular Component 8 Antioxidants 10 Measurement of Antioxidant Capacity 11 Antioxidant Capacity of Anthocyanins 12 Blackberry 15 Bioavailability and Metabolism of Anthocyanin 18 Diverse Effect Associated to Anthocyanins 20 Anticancer Property of Anthocyanins 21 Protective Effect of Anthocyanins 24 Ce l l Proliferation and Death 27 Cel l Lines 28 iv Cytotoxicity Assays 28 A . M T T assay 28 B . B r d U assay 29 C. CellTiter-Glo assay 29 R E S E A R C H H Y P O T H E S I S A N D O B J E C T I V E S 30 E X P E R I M E N T 1: Characterization, identification, quantification of and the antioxidant capacity of blackberry anthocyanins 33 E X P E R I M E N T 2: Comparison of four cytotoxic assays to assess the cytotoxicity of blackberry anthocyanins in multiple cell lines 46 E X P E R I M E N T 3: The protective effect of blackberry anthocyanins against free radical-initiated intracellular oxidation and free radical-induced cytotoxicity 72 E X P E R I M E N T 4: A proposed protective mechanism of blackberry anthocyanins against free radical-induced cytotoxicity I l l O V E R A L L C O N C L U S I O N A N D F U T U R E S T U D I E S 120 R E F E R E N C E S 122 A P P E N D I X A 134 v L I S T O F T A B L E S Table 1: Substitution pattern of six most commonly found anthocyanidins 5 Table 2: Total antioxidant activity of various fruit extracts 13 Table 3: Total anthocyanin content of some common fruits and vegetables 16 Table 4: Total anthocyanin content of blackberry extracts 40 Table 5: Anthocyanin profile and associated antioxidant activity o f blackberry extracts 41 Table 6: LC50 o f blackberry crude extracts on five distinct cell lines evaluated using four different assays 62 Table 7: Correlation coefficient between cell counting and alternative assays 63 Table 8: Concentration dependent inhibition of AAPH-induced intracellular oxidation in various cell lines by blackberry extracts 84 Table 9. IC50 o f blackberry extracts in the inhibition of AAPH-induced intracellular oxidation for different cell lines 86 Table 10: The protective effect of the crude extract on various cell lines as evaluated by CellTiter-Glo 94 Table 11: The protective effect of the crude extract on various cell lines as evaluated by M T T assay 95 Table 12: The protective effect of the anthocyanins extract on various cell lines as evaluated by CellTiter-Glo 96 Table 13: The protective effect of the anthocyanin enriched extract on various cell lines as evaluated by M T T assay 97 v i L I S T O F F I G U R E S Figure 1: The flavylium cation 4 Figure 2: Structural transformation of anthocyanins in aqueous acidic solution at room temperature 6 Figure 3: H P L C profile blackberry anthocyanins and cyanidin-3-glucoside standards 42 Figure 4: Interference of blackberry crude extracts and a cell free CellTiter-Glo assay 58 Figure 5: Concentration response curve of blackberry crude extract incubated for 24 hours evaluated with four different cytotoxicity assays 59 Figure 6: LC50 o f blackberry crude extracts on five distinct cell lines evaluated using M T T and CellTiter-Glo assay 64 Figure 7: The effect of anthocyanins rich extract in multiple cell lines as evaluated with M T T and CellTiter-Glo assays 65 Figure 8-12: Suppression of intracellular oxidation for five distinct cell lines by blackberry anthocyanins 79 Figure 13: Percent inhibition of intracellular oxidation by blackberry anthocyanins 85 Figure 14-18: Protective effect of anthocyanin enriched extract and blackberry crude extract against AAPH-induced cytotoxicity for various cell lines : .. 98 Figure 19: Histogram of cell cycle distribution of Caco-2 cells upon treatment with A A P H and/or an anthocyanins rich extract 118 Figure 20: Percentage of cells in each cell cycle phase in Caco-2 cells upon treatment with A A P H and/or anthocyanins rich extract 119 L I S T O F A B B R E V I A T I O N S A A P H 2, 2 ' -azobis (2-amidinopropane) dihydrochloride A B T S * 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) p - P E P-phycoerythrin Caco-2 Human intestinal adenocarcinoma cancer cell line D C F H - D A dichlorofluorescin diacetate D P P H 1, l-diphenyl-2-picrylhydrazyl i N O S inducible nitric oxide INT-407 Human embryonic intestinal cell line L D L L o w density lipoprotein L N C a P Hormone sensitive prostate cancer cell line M C F - 7 Hormone sensitive breast cancer cell line M D A - M B - 4 5 3 Hormone insensitive breast cancer cell line M T T 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide 'o2 singlet oxygen O H ' hydroxyl radicals (h, superoxide radicals O R A C Oxygen radical absorbance capacity PI Propidium iodide R- alkyl radical R 0 2 ' peroxyl radicals R O S Reactive oxygen species T B A R S thiobarbituric acid-reactive substances A C K N O W L E D G E M E N T I would like to acknowledge first and foremost, my parents, Samuel El is ia and Utami Soebiantoro, for their full support, sacrifices and their ceaseless prayers. M y utmost gratitude goes to Dr. David Kitts, for being the most supportive supervisor I could possibly have. Ultimately, I am indebted to his wisdom, provision, comrnitment to my learning process and his determination to refine my characters and research skills. No words of appreciation are sufficient to describe my thankfulness to Dr . David Popovich, for being a great mentor. His counsel, technical assistance and confidence in me have contributed greatly to the completion of this thesis. I would also like to appreciate my committee members, Dr. Susan Cheung and Dr. Murray Isman for their valuable assistance in my attempt to produce a worthy thesis. M y special thanks for Dr. Susan Cheung for her listening ears, prayers, technical counsels and encouraging words. I am grateful to Dr. Charles H u and Amirreza Faridmoayer, whose technical assistance and scientific questionings have shaped my thesis greatly into what it is today. Lastly, I would like to thank Admond Siow for his sacrifices, love and patience which allowed me to persevere throughout the roller coaster ride of graduate school. i x INTRODUCTION Reactive oxygen species (ROS) have been implicated to be central in the pathogenesis of more than 50 diseases, including atherosclerosis (Gey, 1993), cancer (Halliwell , 1991), diabetes mellitus (Hayakawa and Kuzuya, 1990; Nerup et al., 1988), Alzheimer's disease (Simonian and Coyle, 1996) and aging (Meydani et al., 1998). R O S , such as superoxide radicals (O2'"), hydroxyl radicals (OH"), hydrogen peroxide (H2O2) and peroxyl ( R 0 2 ' ) radicals are constantly formed as byproducts of normal metabolism and are removed by cellular antioxidant defenses system (Hall iwell and Gutteridge, 1989; Wang and Jiao, 2000). Increased levels of these highly reactive and unstable molecules, however, may overwhelm the antioxidant defense mechanisms and cause oxidative damage to cellular lipids, proteins and nucleic acids, hence impairing cell metabolism that ultimately results in cell death (Packer, 1996; Pol i et al., 2004; Stadtman, 1994). Cumulative oxidative damage has been thought to be the underlying reason for the biochemical and physiological change which leads to the development of the various diseases and disorders (Wang and Jiao, 2000). Therefore antioxidants, which can neutralize free radicals, may be of central importance in combating the prevalence of these disease states (Wang et al., 1996). Dietary antioxidants from natural sources such as fruits and vegetables in particular are of interest as increased dietary intake of these agents may lead to protection against free radical-induced diseases (Dol l , 1990; Gey, 1990). Anthocyanins are pigments that give rise to the red to blue colors observed in many soft fruits and flowers (Pericles, 1982). There is an increased interest for the use of anthocyanins in the functional food and nutraceutical industry, as it has been associated 1 with various potential health benefits (Zhang et al., 2004). Anthocyanins consumption has been reported to promote a reduced risk to coronary heart disease (Renaud and Lorgeril, 1992), visual improvement (Matsumoto et al., 2003), in addition to having potential anti-carcinogenic (Bomser et al., 1996; Kamei et al., 1995), anti-mutagenic (Tate et al., 2003) and anti-inflammatoric (Hu et al., 2003; Wang and Mazza, 2002) effects. Some of these health benefits have been attributed to the antioxidant property of the pigments (Wu et al., 2002). Anthocyanins have been known to possess potent antioxidant capacity (Kahkonen et al., 2001; Kahkonen and Heinonen, 2003). Anthocyanin-containing fruits were demonstrated to have the highest antioxidant capacity in comparison to 30 other commonly encountered fruits, as evaluated by three different chemical assays (Pellegrini et al., 2003). Blackberry (Rubus fruticosus), in particular, was found to have the highest antioxidant capacity of all . Blackberry is unique from other anthocyanin-containing fruit, in that the only predominating anthocyanin is cyanidin-3-glucoside, the most commonly occurring type of anthocyanin in nature (Pericles, 1982). Cyanidin-3-glucoside was shown to have the highest antioxidant activity among the 14 different anthocyanins tested and with a comparable activity that is three to four times stronger than Trolox, the water soluble analogue of vitamin E (Rice-Evans et al., 1995; Wang et al., 1997). Despite the fact that the antioxidant capacity of blackberry anthocyanins has been well demonstrated in various in vitro cell free systems, there is still very little evidence regarding its efficacy in biological systems, which may then lead to the prevention of free radicals associated diseases. 2 The purpose of this thesis therefore was to first characterize, identify, quantify and evaluate the antioxidant capacity of the blackberry anthocyanins. Moreover, the effect of the blackberry anthocyanins on multiple cell lines was assessed with two different viability assays. The primary objective of this thesis, however, is to evaluate the protective effect of blackberry anthocyanins against free radical-initiated intracellular oxidation as well as free radical-induced cytotoxicity using five distinct cultured cell lines. In addition, the mechanism by which blackberry anthocyanins may confer a protective effect in selected cell lines was investigated. 3 LITERATURE REVIEW Anthocyanin Anthocyanins belong to a class of phenolics known as the flavonoids. The term anthocyanin is derived from Greek word anthos for flower and kyanos for blue (Mazza and Miniat i , 1993). Anthocyanins function mainly to attract animals for the purpose of pollination and seed dispersal of plants (Kong et al., 2003). Moreover, anthocyanins may contribute to the developed resistance in plants from insect attacks as well protecting of leaves from ultraviolet radiation. Structurally, anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavylium salts (Figure 1). Figure 1. The flavylium cation. RI and R2 are -H, -OH, or -OCH3; R3 is a glycosyl or -H; and R4 is -OH or a glycosyl (Mazza and Miniati, 1993) The anthocyanin molecule consists of two or three parts; the basic flavylium salts, sugar and acyl group. The number or position of the hydroxyl or methoxyl groups (e.g. the nature, number and position of sugar attached to the flavylium salt, and the nature and number of aliphatic or aromatic groups attached to the sugar in the molecule), w i l l govern which anthocyanin is produced. A l l these variables increase the number of individual compounds that belong to the anthocyanin family (Galvano et al., 2004). It has been R4 4 estimated that more than 400 anthocyanins are found in nature (Kong et al., 2003). Nevertheless, only six anthocyanidins (i.e. aglycone or the anthocyanins without the sugars), namely cyanidin, pelargonidin, peonidin, delphinidin, petunidin and malvidin are commonly found in plants. Table 1 identifies the 6 different aglycones and the unique compositional differences for each compound. Table 1. Substitution Pattern of Six Most Commonly Found Anthocyanidins (Francis, 1989] Name 3 5 6 7 3' 4' 5' Color Cyanidin OH OH H OH OH OH H orange red Delphinidin OH OH H OH OH OH OH bluish red Malvidin OH OH H OH OMe OMe OMe bluish red Peonidin OH OH H OH OMe OH H red Petunidin OH OH H OH OMe OH OH bluish red Pelargonidin OH OH H OH H OH H orange Despite the wide range of colors that are displayed by individual anthocyanins, most are generally reddish when in acidic solution, colorless at intermediate p H and bluish in a basic condition. Anthocyanins exist in equilibrium between four anthocyanin species, the quinonoidal base (A), flavylium cation (AH+), pseudobase or carbinol (B) and chalcone (C). In acidic medium, the anthocyanin exists predominantly as the reddish flavylium cation (AH+). With increasing p H , the flavylium cation loses a proton and forms the bluish quinonoidal base (A). Upon hydration, the reddish flavylium cation is converted to the colorless carbinol base. This may subsequently equilibrate to form the open chalcone forms (C), which are also colorless. The interconversion between the four structures can be described in Figure 2. The conversion of the reddish flavylium cation at low p H to the colorless hemiketals at intermediate p H (e.g. 4.5) has been used as a tool for measuring total anthocyanin content. The p H differential method determines total anthocyanin content based on colour loss with increasing p H . 5 The stability of an anthocyanin containing solution is affected by the concentration of the four forms of the anthocyanins at equilibrium. Anthocyanin is most stable when the flavylium cation (AH+) predominates in an acidic condition. The formation of the colorless chalcone (C) however, has been hypothesized to be the precursor of anthocyanin degradation. OR" Flavylium Cation (AH+) Reddish at pH = 1 OR" Carbinol Base (B) Colorless at pH = 4.5 + H + OR" Quinonoidal Base (A) Bluish at pH = 7 OH O „ OH R2 OR' OR" Chalcone (C) Colorless at pH = 4.5 Figure 2. Structural Transformation of Anthocyanins in Aqueous Acidic Solution at Room Temperature. R1 and R2 are usually H, OH or OCH3. R' is glycosyl and R" is H or glycosyl. Factors that drive the equilibrium to the formation of chalcones therefore potentially promote anthocyanin degradation. Intermediate p H , elevated temperature and hydration 6 by water (or nucleophilic attack to the flavylium cation in general) are some of the factors that cause the equilibrium to shift from flavylium cation to carbinol base, which then quickly forms an unstable tautomer, the chalcone. Reactive Oxygen Species and Oxidative Stress Reactive oxygen species (ROS) are the underlying cause for the development of numerous diseases, such as cancer, cardiovascular, and neurodegenerative diseases (Halliwell , 1994a). R O S is a collective term that includes both oxygen centered free radicals and non-radical oxidants (Halliwell and Whiteman, 2004). Free radicals are molecules with one or more unpaired electrons and are thus highly reactive and unstable (Packer, 1996). Examples of oxygen free radicals are superoxide (O2'"), hydroxyl (OH*), alkoxyl radicals (RO*) and peroxyl (RO2"). Non-radical oxidants, such as hydrogen peroxide (H2O2) and singlet oxygen ('02) are not technically a radical but are strong oxidizing agents and may or may not be easily converted into free radical. R O S , such as those listed above are continuously generated as byproducts of cell metabolism. The high reactivity of R O S with nearby cellular components results in oxidative damage to D N A , lipid and protein, which leads to a number of biochemical and physiological changes that can result in cell death (Wang and Jiao, 2000). This deleterious effect can be counterbalanced by the cell defense and repair mechanisms (Plumb et al., 1997). Endogenous antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase, in addition to the low molecular weight antioxidants, such as tocopherols and ascorbic acid act to neutralize the excess R O S produced (Yuan and Kitts, 1996). 7 A moderate concentration of R O S is however also necessary for life, despite its potential damaging effect, since R O S have important roles as regulatory mediators in cell signaling processes (Yoshida et al., 2004). Organisms are however exposed to exogenous sources of free radicals (e.g. U V light, smoking, chronic alcohol consumption and exercise) in addition to those produced by ordinary metabolic processes (Stadtman, 1994). Elevated concentrations of free radicals may overwhelm the antioxidant and repair mechanism of the cell which wi l l alter the balance between the oxidant and antioxidant in favor of the oxidant, thereby creating a condition referred to as oxidative stress. This condition has been implicated in the progression of more than fifty diseases (Halliwell , 1991). Oxidative Stress on Cellular Components Cel l injuries caused by oxidative stress arise from interrelated damage to several key cellular components: the D N A , protein and lipids. Free radicals attack D N A and cause D N A strand breakage, base lesions, DNA-protein cross-links, and D N A adducts with reactive aldehydes derived from lipid oxidation (Cadet et al., 1994). These oxidative D N A lesions have been linked to D N A mutations in carcinogenesis (Cooke et al., 2003). Oxidative stress also elicits various modifications to protein molecules, which includes oxidation of amino acid residues as well as the cleavage of peptide bonds in the protein structure (Stadtman, 1994). Further reactions of the oxidized amino acids with carbohydrate or l ipid peroxidation products form protein carbonyl products. Oxidized proteins that contain the carbonyl group are generally dysfunctional, and accumulation of protein oxidation products may lead to disruptions in cellular function (Levine and 8 Stadtman, 2001). L ip id peroxidation results in the production of hydroperoxides, which readily transform to various secondary oxidation products, such as reactive aldehydes (e.g. malondialdehyde), alkanes, l ipid epoxides and alcohols (Paulet et al., 1994). The lipid oxidation products may cause alteration in biomembrane, interfere with cell signaling pathways, and ultimately, cellular function and survival (Poli et al., 2004). The introduction of a free radical into a system is generally followed by a free radical chain reaction. L ip id peroxidation is one of the oldest studied free radical chain reactions in food chemistry. The mechanism can be divided into several stages; an initiation, propagation and termination phase. The chain reaction is initiated when a reactive species (R») abstract a hydrogen atom from a lipid molecule (LH) which produces an alkyl radical (L»). In the propagation step, the elimination of one radical results in the generation of another or more radicals. The alkyl radical formed earlier w i l l be oxidized to form peroxyl radical ( L O O ) , which readily interacts with nearby l ipid molecules causing the formation of more radicals. The chain reaction wi l l not cease until the terminating reactions take place. This usually involves the combination of free radicals to form non reactive species. The l ipid peroxidation mechanism can be briefly described by the following equations (Equation 1-7) (Nawar, 1996; Pryor, 1994). Initiation + L H R H + L« (Equation 1) Propagation L ' L O O ' L O O -+ 0 2 + L H L O O * L O O H + L ' LO» + HO« (Equation 2) (Equation 3) (Equation 4) Termination 2 LOO« L ' 2 L -+ L O O ' Non-radical products (Equation 5) Non-radical products (Equation 6) Non-radical products (Equation 7) 9 -1 s The lipid radicals have one of the longest half lives (10 to 10 times) amongst all radicals produced in biological systems (Pryor, 1994). The lipid peroxidation products therefore have a greater chance to attack cellular components thus increasing the potential to exert more oxidative damage than other free radicals (X in et al., 1996). Antioxidants Dietary consumption o f antioxidants has been promoted to prevent free radical associated disease. Antioxidants have been defined as 'any substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate' (Halliwell and Gutteridge, 1989). There are several antioxidants which are classified based on active mechanisms to neutralize free radicals. Preventive enzymatic antioxidants (e.g. glutathione peroxidase, superoxide dismutase) suppress free radical formation. N o n enzymatic radical scavenging antioxidants also suppress chain initiation (e.g. vitamin C, uric acid and albumin) or break chain propagation (e.g. vitamin E , ubiquinol). Lastly, repair factors (lipase, protease, D N A repair enzyme) restore cellular damage by recycling salvageable components and reconstructing cell membranes (Etsuo et al., 1996). Antioxidants may prevent the formation of free radicals by acting as a metal sequestering agent, reducing agent or oxygen scavenger. Chain breaking antioxidants scavenge free radicals by donating a hydrogen or electron to nearby free radicals, which consequently slows the uncontrolled propagation of free radical and the damage caused by them. 10 Measurement of Antioxidant Capacity In light of the current escalating interest in peroxidation reactions and human health, knowledge regarding the antioxidant capacity of various food samples, or its constituents, is of great importance. Researchers have developed numerous different assays that measure the antioxidant capacity of food or biological samples, but there remains no single validated and reliable method. The O R A C (Oxygen Radical Absorbance Capacity) assay, has recently gained inter-laboratory (three laboratory) validation and industrial recognition, thus emerging to be one of the few commonly accepted assays to measure antioxidant capacity (Huang et al., 2005). Essentially, O R A C measures the peroxyl radical scavenging activity of an antioxidant(s) molecule. The original O R A C assay employed P-phycoerythrin (P-PE), a fluorescing protein which decays upon exposure to A A P H (2,2' -azobis (2-amidinopropane) dihydrochloride ), thus generating a free radical (Cao et al., 1993). A A P H is a water soluble free radical initiator that undergoes spontaneous thermal decomposition at 37°C (Peyrat-Maillard et al., 2003; Yoshida et al., 2004). A A P H dissociates to form two carbon centered radicals which quickly combine with oxygen to form peroxyl radical and initiate l ipid oxidation (equation 8-9) (Nik i , 1990). R-N=N-R -> (l-e) R : R + 2eR» + N 2 (Equation 8) R« + 0 2 -> ROO« (Equation 9) :where R-N=N-R is the radical initiator, ROO* is the peroxyl radical and e is the efficiency of free radical production. The radicals formed may then alter P - P E conformation which results in a decrease in fluorescence intensity. Antioxidants that scavenge the peroxyl radicals thus 11 reduce the loss of fluoresence (Niki , 1990). The antioxidant capacity of test compound(s) is assessed by quantitating the area under the fluorescence decay curve ( A U C ) of unknown sample, in comparison to the blank which has no antioxidant is present. Trolox (the water soluble counterpart of vitamin E) is used as a positive control. Recent studies have replaced the use of p -PE with fluorescein (3', 6'-dihydroxyspirol [isobenzofuran-1[3H], 9'[9H]-xanthen]-3-one) since P -PE varies lot to lot, interacts with polyphenols with non specific binding and is photobleached under plate reader conditions (Naguib, 2000; Ou et al.,2001). Antioxidant Capacity of Anthocyanins The antioxidant capacity of anthocyanins has been well demonstrated in the literature. The unique structure of anthocyanins lack electrons thereby reacting readily with R O S to form a stable radical, and thus reducing the risk of developing free radical associated diseases. Upon reaction with free radicals, anthocyanins can form phenoxyl radicals; its conjugated structure wi l l allow electron derealization which forms stable radical. Out of 30 fruits tested, three fruits containing anthocyanins (blackberries, raspberries and red currant) were consistently found to have the highest antioxidant activity, regardless of the three different assays used (Table 2) (Pellegrini et al., 2003). Phenolic extracts from various berries (blackberries, red raspberries, sweet cherries, blueberries and strawberries) exhibited effective inhibition of human L D L oxidation and liposome oxidation (Heinonen et al., 1998). Berries also demonstrated high scavenging activity against chemically generated free radical (Hu et al., 2005; Meyers et al., 2003). 12 TABLE 2. Ferric reducing-antioxidant power (FRAP), total radical-trapping antioxidant parameter (TRAP) and Trolox equivalent antioxidant capacity (TEAC) of fruit extracts* FRAP TRAP TEAC Rank Fruit Value Fruit Value Fruit Value (mmol Fe2+/kg Fresh Weight} (mmol Trolox/kg Fresh Weight) 1 Blackberry 51.53 Blackberry 21.01 Blackberry 20.24 2 Redcurrant 44.86 Olive (black) 18.08 Raspberry 16.79 3 Raspberry 43.03 Olive (green) 14.64 Olive (black) 14.73 4 Olive (black) 39.99 Redcurrant 12.14 Redcurrant 14.05 5 Strawberry (wild) 28.00 Raspberry 10.48 Strawberry (wild) 11.34 6 Olive (green) 24.59 Strawberry (wild) 10.34 Cultivated strawberry 10.94 7 Cultivated strawberry 22.74 Blueberry 9.30 Olive (green) 10.43 8 Orange 20.50 Cultivated strawberry 8.56 Pineapple 9.91 9 Blueberry 18.61 Plum (red) 8.09 Orange 8.74 10 Pineapple 15.73 Pineapple 5.92 Blueberry 7.43 11 Plum (red) 12.79 Orange 5.65 Plum (red) 5.11 12 Grape (black) 11.09 Cherry 4.17 Tangerine 4.16 13 Grapefruit (yellow) 10.20 Grapefruit (yellow) 4.04 Grape (black) 3.85 14 Tangerine 9.60 Pear 3.87 Clementine 3.10 15 Clementine 8.88 Tangerine 2.76 Grapefruit (yellow) 3.05 16 Cherry 8.10 Clementine 2.74 Cherry 2.69 17 Kiwi fruit 7.41 Grape (black) 2.50 Grape (white) 2.48 18 Prickly pear 6.97 Kiwi fruit 2.30 Fig 2.47 19 Peach (yellow) 6.57 Apricot 2.29 Kiwi fruit 2.28 20 Fig 5.82 Apple (red Delicious) 2.23 Pear 2.19 21 Melon (cantaloupe) 5.73 Prickly pear 2.06 Peach (yellow) 1.67 22 Pear 5.00 Fig 2.06 Apple (red Delicious) 1.59 23 Apricot 4.02 Loquat 1.73 Prickly pear 1.46 24 Apple (red Delicious) 3.84 Grape (white) 1.59 Apricot 1.44 25 Grape (white) 3.25 Apple (yellow Golden) 1.54 Apple (yellow Golden) 1.31 26 Apple (yellow Golden) 3.23 Peach (yellow) 1.49 Melon (cantaloupe) 1.20 27 Loquat 2.70 Melon (honeydew) 1.12 Loquat 0.75 28 Banana 2.28 Banana 1.05 Watermelon 0.69 29 Melon (honeydew) 2.27 Melon (cantaloupe) 0.95 Melon (honeydew) 0.65 30 Watermelon 1.13 Watermelon 0.46 Banana 0.64 * adapted from Pellegrini et al. (2003) Strawberry juice extract and lingonberries, for example, are potent scavengers of hydroxyl and superoxide radicals generated from Fenton reaction and xanthine-xanthine oxidase system, respectively (Wang et al., 2005; Wang and Jiao, 2000). Saskatoon berries also showed direct scavenging activity towards D P P H * and A B T S * radicals (Hu et al., 2005). 13 The antioxidant capacity of these berries has been attributed to the anthocyanin content. Prior et al., (1998) discovered a linear relationship between antioxidant capacity, as measured by O R A C , and anthocyanin content in several varieties of Vaccinium species. H u et al., (2003) demonstrated that the anthocyanin rich fraction of black rice extract significantly prevented supercoiled D N A strand scission induced by peroxyl or hydroxyl radicals. They were also successful at showing the suppression of human L D L oxidation upon exposure to the same anthocyanin rich fraction. The major anthocyanidins that make up pomegranate anthocyanins were found to contribute to the overall antioxidant activity of the fruit extract against hydroxyl and superoxide radicals, as evaluated by electron spin resonance (Noda et al., 2002). It was hypothesized that anthocyanidins bind the free metal transition ion involved in the Fenton reaction (Fe 2 + ) thus inhibiting the generation of hydroxyl radicals. It appears that a relationship exists between the chemical structure o f anthocyanin and antioxidant activity (Stintzing et al., 2002a). It has been well established that glycosylation significantly influences the antioxidant property of a compound (Rice-Evans and Mil ler , 1998). Depending on the anthocyanidin, glycosylation may increase or decrease the antioxidant activity of the respective anthocyanin (Fukumoto and Mazza, 2000). Glycosylation results in lower antioxidant activity for peonidin, pelargonidin and cyanidin. The reverse effect was observed for malvidin. The degree of hydroxylation, methoxylation in the B-ring has also been thought to contribute to the different antioxidant activity of each anthocyanin (Pereira et al., 1997; Zheng and Wang, 2003). Cyanidin-3-glucoside, in particular, has been determined by Wang et.al., (1997) to have the highest antioxidant activity among the 14 anthocyanins tested, with an 14 activity that is three to four times stronger than Trolox (Rice-Evans et al., 1995; Wang et al., 1997). Cyanidin has also been shown to form a co-pigmentation complex with D N A , which protects both the D N A and the anthocyanin from damage brought forth upon exposure to hydroxyl radicals (Sarma and Sharma, 1999). Seeram et al., (2001) showed that cyanidin-3-rutinoside exhibited antioxidant activity that is comparable to several synthetic antioxidants (tert-butylhydroquinone, butylated hyroxyanisole, and butylated hydroxytoluene). Blackberry Blackberry (Rubus fruticosus sp.) belongs to the large and diverse Rose family (Rosaceae). It may grow native in many parts o f the world, but cultivation and commercialization is only common in North America (Markham and Mabry, 1975). Blackberry is of particular importance in the Pacific Northwest (western Oregon, western Washington and southwestern British Columbia), parts of coastal California, Texas, Missouri , Arkansas and New Zealand (Hollman et al., 1996; Markham, 1975). Blackberry was used for medicinal purposes up to the 16 t h century. The juice was recommended for mouth and eye infections. Blackberry contains approximately 83-326 mg of anthocyanins per 100 g (Table 3) (Mazza and Miniat i , 1993). 15 Table 3. Anthocyanin Content of Some Common Fruits and Vegetables (Wrolstad and Giusti, 2000) Source Pigment content (mg/100 g fresh weij Apples 10 Bilberries 300-320 Blackberries 83-326 Black chokeberries 560 Black currants 130-400 Black raspberries 300-400 Blueberries 25-495 Cherries 4-450 Cranberries 60-200 Elderberry 450 Grapes 6-600 K i w i 100 Plum 2-25 Red cabbage 25 Red onions 7-21 Red radishes 11-60 Red raspberries 20-60 Strawberries 15-35 Blackberry is unique from other anthocyanin containing fruits in that it only has one major anthocyanin, cyanidin-3-glucoside, which is also the most commonly occurring anthocyanin in nature (Pericles, 1982). A minor amount of other anthocyanins may also exist, depending on the fruit species (Mazza and Miniat i , 1993; Pericles, 1982). Cyanidin-3-rutinoside is the most reported minor anthocyanin present in blackberry (Pericles, 1982; Stintzing et al., 2002a). Cyanidin-3-xyloside, cyanidin-3-glucoside acylated with malonic acid, and cyanidin-3-dioxalylglucoside has also been identified in various blackberry cultivars in trace amount (Fan-Chiang and Wrolstad, 2005; Stintzing et al., 2002b). Other polyphenolics common to blackberry are quercetin glycosides, catechin and epicathechin, as well as ellagic acid derivatives (Siriwoharn et al., 2004; Siriwoharn et al., 2005). 16 Blackberry is a rich source of antioxidants. It has been shown to contain the highest amount of antioxidants of most fruits (Halvorsen et al., 2002). Ripe blackberry was also found to have the highest antioxidant capacity as measured by O R A C assay in comparison to black or red raspberry and strawberry (Wang and L i n , 2000). The marked antioxidant capacity exhibited by blackberry can be associated to its high anthocyanin content, in particular cyanidin-3-glucoside. Cyanidin-3-glucoside was found to have the highest O R A C activity among the 14 anthocyanins tested and was four times stronger than Trolox (Rice-Evans et al., 1995; Wang et al., 1997). Cyanidin-3-glucoside was also more potent than ascorbic acid or resveratrol at suppressing copper-induced L D L oxidation (Amorini et al., 2001). There are several proposed mechanisms by which cyanidin-3-glucoside exerts an antioxidant property. Tsuda et al., (1996) reported that cyanidin-3-glucoside was broken down to another radical scavenger upon reaction with biological radicals in vivo . In a later study, Tsuda et al., (1999b) pointed out that protocatechuic acid, also a radical scavenger, was produced upon oxidation of cyanidin-3-glucoside and was present at a concentration 8 times greater than cyanidin-3-glucoside. It was proposed that both cyanidin-3-glucoside and protocatechuic acid may contribute to total antioxidant activity. Sarma and Sharma (1999) hypothesized another possible antioxidant mechanism of cyanidin-3-glucoside. A co-pigmentation complex between ascorbic acid, metal and anthocyanin was formed, which explains the observation that ascorbic acid oxidation by copper ion can be prevented by the addition of anthocyanin from black rice (Sarma et al., 1997). A co-pigmentation between D N A and cyanidin was also observed by Sarma and Sharma (1999), who reasoned that co-pigmentation protected both D N A and the 17 anthocyanin from hydroxyl radical attacks. Exposure of the D N A , or the anthocyanidin alone, to hydroxyl radicals; however, can result in severe oxidative damage. Despite the many possible mechanisms, Amorini et al., (2001), using a copper-induced L D L oxidation model system, showed that the antioxidant activity of cyanidin-3-glucoside was due to its radical scavenging property rather than to a metal-chelating property. Bioavailability and Metabolism of Anthocyanin Anthocyanins were thought to be absorbed only in its aglycone form. Since no specific enzymes were capable of selectively cleaving the glycosidic bonds, the anthocyanins were believed to be poorly absorbed (Galvano et al., 2004). However, many recent studies have concluded that anthocyanins are absorbed in an intact glycosylated form (Miyazawa et al., 1999; Talavera et al., 2003). Cyanidin-3-glucoside, for example, has been detected in the plasma and urine of rats and humans, following oral consumption of various anthocyanin rich extracts (e.g. blueberry skin extract, elderberry, blackberry) (Matsumoto et al., 2001; Talavera et al., 2005; Tsuda et al., 1999b; W u et al., 2002). Matsumoto et al., (2001) discovered similar findings upon oral administration of delphinidin-3-rutinoside and cyanidin-3-rutinoside to rats and human. Anthocyanins are believed to form several metabolites upon absorption. In addition to the intact glycosylated form, methylated and glucuronidated derivative of the parent anthocyanin are found. Administration of delphinidin-3-glucoside, the most potent antioxidant in blueberries, w i l l result in the formation of 4'-0-methyl delphinidin-3-glucoside (methylation of the 4 ' - O H on the delphinidin B ring) in rats (Ichiyanagi et al., 2004). 18 Peonidin-3-glucoside and peonidin-3-glucuronide were often the metabolite products identified in rats when fed with cyanidin-3-glucoside rich extract (Talavera et al., 2003; Talavera et al., 2004). Peonidin-3-glucoside may arise from hepatic methylation at the 3' hydroxyl moiety of cyanidin-3-glucoside (Felgines et al., 2002). Ichiyanagi et al., (2005) recently identified four metabolites of cyanidin-3-glucoside in rats using tandem mass spectrometry; two of which were monomethylated cyanidin-3-glucoside (one was identified as peonidin-3-glucoside), while the other two were glucuronides of cyanidin and methylated cyanidin (Ichiyanagi et al., 2005). Miyazawa et al., (1999) detected the presence o f the methylated form, but not glucuronidated form o f cyanidin-3-glucoside in rats, and found neither the methylated nor glucoronidated metabolites in human. W u et al., (2002) was the first to show in vivo methylation of cyanidin to peonidin and glucuronide conjugate in humans after elderberry or blueberry consumption. In addition to the above metabolites, protocatechuic acid, which may be produced by degradation of cyanidin, was present in the plasma at a concentration that was 8 times higher than the detected cyanidin-3-glucoside (Tsuda et al., 1999b). Fleschhut et al., (2005) demonstrated that anthocyanin can be severely degraded after incubation with cecal microflora. It was proposed that anthocyanin may be metabolized by the bacteria or go through spontaneous degradation to form the phenolic acid descending from the B-ring of the anthocyanin skeleton. This might be the reason for the poor bioavailability observed in many anthocyanin pharmacokinetic studies. Anthocyanins and metabolites have been identified in the plasma, urine and in the following tissues; kidney, liver, jejunum, stomach and to a smaller extent, the brain (Talavera et al., 2005). The concentration found in these tissues however was different 19 depending on the types and concentration of anthocyanins fed to rats or humans. The total anthocyanins found in rat jejunum ranged from 0.15 pmol to 0.6 pmol of cyanidin-3-glucoside equivalence/ g of tissue (Talavera et al., 2005; Tsuda et al., 1999b). In rat and human plasma, cyanidin-3-glucoside was found to range from 5 nmol/L to 3.4 pmol/L. Only in a human study was the level narrowed down to 5 nmol/L to 24 nmol/ L (Ichiyanagi et al., 2005; Miyazawa et al., 1999). The concentration of anthocyanins in plasma and tissues generally increases before reaching peak levels in less than 30 minutes upon ingestion, thereafter declining slowly before being completely undetectable. Anthocyanins can be metabolized and excreted in the urine. Diverse Effects Associated to Anthocyanins Anthocyanins have been associated with numerous bioactivities, which may potentially protect against various diseases. Blackberry anthocyanins, for example can suppress chemical induced mutation in Salmonella typhimurium TA100 (Tate et al., 2003). In addition, anthocyanins have an anti-cancer effect, which can be mediated via inhibition of cancer cell proliferation or induction of apoptosis or programmed cell death (Olsson et al., 2004; Wang et al., 2005). Furthermore, anthocyanins obtained from mulberry were shown to have a dose dependent inhibition of migration and invasion of highly metastatic A549 lung cancer cells (Chen et al., 2005). Anthocyanins therefore may possess anti-metastatic property. H u et al., (2003) demonstrated that anthocyanins from black rice were effective at suppressing D N A strand scission induced by R O S as well as inducible nitric oxide ( iNOS) production, generally associated with inflammatory response. Wang and Mazza, 20 (2002) reported similar anti-inflammatoric effects with Saskatoon berries, blueberry, blackberry and black currant. Anthocyanins were also found to inhibit lipoprotein oxidation and platelet aggregation, both events associated to the progression of heart disease (Ghiselli et al., 1998; Kong et al., 2003; X i a et al., 2003). Anthocyanins consumption may therefore reduce the risk of heart disease. In addition to the above-mentioned bioactivities of anthocyanins, it has recently been discovered that this group of flavonoids may also up-regulate genes that are involved in l ipid metabolism, such as the hormone sensitive lipase in adipocytes. Anthocyanin therefore has the potential to be involved in the prevention of obesity or diabetes (Tsuda et al., 2005). Additionally, anthocyanins were found to facilitate the re-generation of rhodopsin, the light sensitive cells which function mainly for night vision (Matsumoto et al., 2003). Anticancer Property of Anthocyanins Despite the various potential bioactivities, anthocyanins have recently been thoroughly investigated as a possible chemopreventive agent. Bilberry extracts were shown to suppress the growth of cultured H L 6 0 human leukemia cell lines, through induction of apoptosis (Katsube et al., 2003). This growth inhibition was attributed to the anthocyanins, in particular the delphinidin and malvidin-glycosides. While bilberry extracts contained approximately a 30:36:13 ratio of cyanidin-: delphinidin-: malvidin-glycosides, respectively, delphinidin and malvidin aglycone and its glycosides exhibited the greatest apoptosis induction in H L 6 0 cells. The growth inhibitory effects of delphinidin-glycosides on both cell lines however were lower than those of the 21 delphinidin alone. This finding is supported by Zhang et al., (2005), who found that anthocyanidins in general exhibited greater cell growth inhibition in multiple cell lines than the anthocyanins. Katsube et al., (2003) also demonstrated that anthocyanins suppress the growth of normal cells (human dermal fibroblast H N D F ) to an extent less than that observed in cancerous cells (HCF116 human colon carcinoma cells). Mal ik et al., (2003) and Zhao et al., (2004) discovered similar findings, where chokeberry anthocyanins exhibited more growth suppression in HT-29 colon cancer cells than to normal colon cells (NCM460) . Different anthocyanins appeared to perturb the cell cycle at different phases (Malik et al., 2003). Exposure of cyanidin to human fibroblasts cells was found to induce cytotoxicity as well as to decrease the amount of cells in the S phase. Delphinidin treated fibroblasts cells on the other hand, caused a cell cycle arrest at S phase, which results in accumulation of cells in the S phase (Lazze et al., 2004). Anthocyanins have been reported to suppress the growth of cancer cells by inducing apoptosis and/or by inhibiting cell proliferation. Cyanidin-3-glucoside for example, reduces cells in the S phase thus exerted an anti-proliferative effect without inducing apoptosis- (or necrosis-) mediated cytotoxicity in human melanoma cells (Serafino et al., 2004). Similarly, an anthocyanin rich extract derived from chokeberry, which was dominated with cyanidin-3-galactoside, was found to halt the cell cycle progression of the human colon cancer cells at the G l / G o and G 2 / M phase without inducing apoptosis (lack of cells in sub Go) (Malik et al., 2003). The cell cycle arrest occurs concomitantly with an increased expression of p 2 1 W A F 1 and p 2 7 K I P 1 , the cyclin dependent kinase inhibitors (CDKI) and a decreased expression of cyclin A and cyclin B 22 genes. Cyclins are members of cell cycle regulators which bind to cyclin dependent kinase ( C D K ) and in turn regulate cell cycle progression. A n increased expression of the C D K inhibitors p 2 1 W A F 1 and p 2 7 K l p l was found to coincide with the cell cycle arrest in the G l / G o phase, whereas a decreased expression of the cyclin A and B genes were associated with the arrest at G 2 / M phase (Malik et al., 2003). On the other hand, the main anthocyanins in Oryza sativa cv. Heugjinjibyeo, the cyanidin and malvidin, caused a cell cycle arrest at G 2 / M phase as well as apoptosis mediated cytotoxicity in human monocytic leukemia cell U937 (Hyun and Chung, 2004). Exposure of delphinidin to uterine carcinoma (HeLa S3) and colon adenocarcinoma cells (Caco2) resulted in a reduction of cells in G l phase which was accompanied by an increased fraction of cells with a hypodiploidic D N A content normally associated to apoptotic cells (Lazze et al., 2004). The mechanisms by which anthocyanins induce apoptosis have been investigated by Chang et al., (2005) and Yeh and Yen, (2005). Chang et al., (2005) experimented with anthocyanins extracted from Hibiscus, a flower extensively used in Chinese herbal medicine. Hibiscus anthocyanins, which largely consisted of delphinidin, were found to induce apoptosis mediated cytotoxicity in HL-60 . The authors proposed that Hibiscus anthocyanins induced apoptosis by stimulating the p38 MAPkinase (i.e. stress activated kinase) to phosphorylate c-Jun, which then increased the expression of Fas ligand (death receptor ligand). This FasLigand may interact with Fas death receptors and form a death inducing complex which activates the caspase 8/t-bid signaling module. The activated t-bid caused mitochondrial translocation which resulted in cytochrome c release. This in 23 turn cleaves and thus activates caspase 3, which plays a key role in inducing events that lead to apoptosis. Yeh and Yen, (2005) discovered similar activation of caspase 3 in delphinidin treated hepatoma cells (HepG2). It was concluded that delphinidin may effectively induce apoptosis in HepG2 cells through generation of oxidants thus activating the c-Jun N -terminal kinase cascade and regulation of Bcl-2 family (Yeh and Yen, 2005). Exposure of cells to delphinidin results in an increased level of c-Jun m R N A as well as increased phosphorylation of J N K , which in turn activates c-Jun signaling cascade. This may lead to an alteration of the Bax/Bcl2 ratio, the pro-apoptotic/ anti-apoptotic mitochondrial protein, which balance determines the fate of cell. It was found that delphinidin treatment upregulated the expression of Bax and downregulated the expression of Bc l2 , thus swaying the ratio more towards apoptosis (Yeh and Yen 2005). In addition, anti-tumor properties of 5 aglycones (cyanidin, delphinidin, malvidin, pelargonidin, and peonidin) and 4 associated glycosylated anthocyanins have shown growth inhibition and cytotoxicity of human gastric adenocarcinoma cells that indicated the induction of apoptosis instead of necrosis (Shih et al., 2005). Protective Effect of Anthocyanins While the antioxidant capacity o f anthocyanins has been well established, the question regarding its practical application in vivo remains unknown. Current investigations to answer this question have been centered on the manifestation of the antioxidant capacity of anthocyanins in protecting against free radical associated damages in biological systems. A strawberry extract was found to exhibit a dose dependent 24 inhibition of H202-induced cytotoxicity in P C 12 neuronal cell system and was therefore considered to have a neuroprotective effect (Heo and Lee, 2005). H u et al., (2005) demonstrated that anthocyanins extracted from Saskatoon berries or black rice scavenges H202-initiated intracellular free radicals. Cultured red blood cells as well as red blood cells withdrawn from rats displayed an increased resistance towards H2O2 induced oxidative stress after treatment or feeding with blueberry anthocyanins respectively (Youdim et al., 2000b). Cyanidin-3-glucoside in particular was effective at counteracting free radical generation as well as preventing free radical mediated D N A damage induced by ochratoxin in cultured human fibroblast cells (Russo et al., 2005). Cyanidin-3-glucoside was also found to prevent U V - A induced apoptosis in a human keratinocyte cell line (Tarozzi et al., 2005). This protective effect was attributed to the neutralization of the H202 that was released after U V A radiation. While anthocyanins may scavenge free radicals generated outside or inside the cell, it is prudent to learn i f anthocyanins can be uptaken into the cell to a degree that may exert significant protective effect against free radicals generated within the cells. Anthocyanins from elderberry extract were found to be incorporated into the plasma membrane and the cytosol, which results in increased resistance against H2O2; A A P H and FeSOVAscorbic acid induced damage (Youdim et al., 2000a). This finding was supported by Tarozzi et al., (2005) who found that cyanidin-3-glucoside treatment to human keratinocyte increased the antioxidant activity in a membrane rich fraction to a greater extent than in the cytosol (55% vs. 19%, respectively). 25 Using human umbilical vein endothelial cells and thoracic aortas from rats, Serraino et al., (2003) demonstrated the protective effect of cyanidin-3-glucoside, extracted from blackberry, against peroxynitrite-induced endothelial dysfunction and vascular failure. Blackberry anthocyanins were also found to yield a dose dependent suppression of acute inflammation response induced by the injection of carrageenan into the pleural cavity of rats (Rossi et al., 2003). Furthermore, Tsuda et al., (1999a) demonstrated the in vivo antioxidant activity of cyanidin-3-glucoside, by using hepatic ischemia/reperfusion (I/R) injury as a model of oxidative stress in rats. It was discovered that cyanidin-3-glucoside feeding suppressed the changes associated with this particular injury which involved an increase in serum concentration of thiobarbituric acid-reactive substances ( T B A R S ) (a marker for l ipid peroxidation), and activities of liver marker enzymes of injuries, as well lower concentrations of reduced liver glutathione (Tsuda et al., 1999a). Mol ler et al., (2004) however pointed out that large amount of dietary antioxidants may not necessarily be beneficial to healthy adequately nourished humans. These workers showed that supplementation of blackcurrant juice or an anthocyanin rich drink for three weeks (average 397 and 365 g/day respectively) did not decrease the already low steady state of oxidative D N A damage of mononuclear blood cells in healthy humans. This result was supported by Riso et al., (2005) who found that 600 ml of blood orange juice (which was rich in anthocyanin, vitamin C and carotenoids) per day for three weeks to sixteen healthy female, improved only the lymphocyte D N A to oxidative stress but had no influence on other biomarkers tested (e.g. plasma antioxidant status and lipid peroxidation). 26 Cell Proliferation and Death A fine balance exists with the initiation of cell proliferation and cell death. When the balance is tipped in favor of proliferation or death, the resulting events are generally attributed to various types of disease states. A n uncontrolled rate of proliferation for example can lead to the development of cancer cells. Normal cells go through several cell cycle phases ( G l , S, G2, and M ) to survive and proliferate. In order to divide, cells in the G l phase have to pass a check point to enter the S phase, where D N A synthesis occurs. Cells then go through a G2 phase where cells have to go through another check point before entering the M phase, where mitosis takes place. A t this phase, a cell divides into two cells and subsequently enters a resting stage (Go). The check points are of importance to avoid the formation of abnormal cells (King and Cidlowski , 1998). Ce l l death is an equally important mechanism in the strict regulation of cell metabolism. Apoptosis and necrosis are two most commonly known mechanisms of cell death. Apoptosis or programmed cell death causes cell shrinkage, chromatin condensation followed by D N A fragmentation, membrane blebbing and formation of apoptotic bodies. While apoptosis occurs under normal physiological condition in response to intracellular or extracellular stimulation, necrosis is an uncontrolled cell death response to an extracellular insult. Unlike apoptotic cells, necrotic cells swell, and eventually rupture resulting in low cellular organelle content (Columbano, 1995; K i n g and Cidlowski , 1995). 27 Cell Lines Five distinct human derived adherent cell lines: two breast cancer lines ( M D A - M B -453 and M C F - 7 ) , two intestinal cell lines (Caco-2 and INT-407), and one prostate cancer cells (LNCaP) , were used in this thesis. Caco-2 is a human adenocarcinoma cancer line that resembles large intestinal cells and is often used as a model to simulate the gastrointestinal tract (Popovich and Kitts, 2004; Walgren et al., 1998). Similarly, Int 407 is also used as a model of intestinal response, but is derived from normal human embryonic intestinal tissue and established via HeLa cell contamination (Henle and Deinhardt, 1957; Kitts and Nakamura, 2006). M C F 7 is a human adenocarcinoma cancer line that expresses estrogen receptor, whereas M D A - M B - 4 5 3 is a metastatic carcinoma cancer line that does not express estrogen receptor (Hall et al., 1994; Rodgers and Grant, 1998). L N C a P is a human prostate cancer cell line that expresses an androgen receptor (Romijn et al., 1988). M C F - 7 and L N C a P cell line have been used widely in breast and prostate cancer research to serve as model cell line for breast and prostate cancer respectively (Ye et al., 2002). Cytotoxicity Assays A. MTT method M T T is a relatively inexpensive, widely used assay that can be used for routine cytotoxicity evaluation. It is a cell viability assay that is based on the reduction of the yellow tetrazolium salt M T T (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) to purple formazan by metabolically active cells (Mosmann, 1983). Mitochondrial succinate dehydrogenase in live metabolizing cells transforms the yellow 28 tetrazolium salt to the insoluble purple formazan dye, whereas non viable cells do not have this property. B . B r d U While the M T T assay measures cell viability based on cell metabolism, the B r d U assay quantitates cell proliferation. B r d U (5-bromo-2-deoxyuridine) is apyrimidine analogue that can be incorporated into the D N A in place of thymidine during D N A synthesis. The BrdU-labelled D N A can be quantitated by E L I S A based chemiluminescence detection. The incorporated B r d U is detected with anti-BrdU-peroxidases, which upon reaction with substrate generates light thus allowing for quantification. It is a non-radioactive alternative to the traditional [ 3H]-thymidine incorporation method that does not compromise its sensitivity (Gratzner, 1982; M u i r et al., 1990). C . Ce l lT i t e r -Glo Similar to B r d U , CellTiter-Glo is a chemiluminescence based assay, but instead of measuring D N A proliferation, it measures the A T P content of cells. The assay is based on the ability of luciferase enzyme to convert luciferin to oxyluciferin and photonic energy (light) in the presence of A T P . Therefore the more viable cells present in the well , the higher the A T P content which is proportional to the luminescent signal emitted (Crouch etal., 1993). 29 RESEARCH HYPOTHESIS AND OBJECTIVES OVERALL HYPOTHESIS The antioxidant capacity of blackberry anthocyanins can scavenge free radicals in cultured cells thus protecting against free radical-associated oxidative stress and cytotoxicity. OVERALL OBJECTIVE To demonstrate that the antioxidant capacity of blackberry anthocyanins can protect against free radical-induced intracellular oxidation and cytotoxicity in cultured cells. 30 EXPERIMENT 1. CHARACTERIZATION, IDENTIFICATION, QUANTIFICATION AND THE ANTIOXIDANT CAPACITY OF BLACKBERY ANTHOCYANINS NULL HYPOTHESIS (H0)i: The anthocyanin from blackberry can be effectively extracted, concentrated and is mainly composed of cyanidin-3-glucoside. OBJECTIVE 1: To extract total anthocyanins from blackberry, determine a standardized crude blackberry extract, and fractionate from crude blackberry anthocyanins to obtain an anthocyanin-enriched extract. OBJECTIVE 2: To characterize chemically the properties of the anthocyanins found in blackberry using H P L C . NULL HYPOTHESIS (Ho)2: The antioxidant capacity of anthocyanin-enriched extract is higher than the crude extract OBJECTIVE: To evaluate the antioxidant capacity of the blackberry crude extract and the anthocyanin-enriched extract using the O R A C assay. EXPERIMENT 2. CYTOTOXICITY OF BLACKBERRY EXTRACT AND THE ANTHOCYANIN-ENRICHED EXTRACT NULL HYPOTHESIS (H0): Anthocyanins recovered from blackberry are not cytotoxic at physiological concentrations in different cell culture systems. OBJECTIVE 1: To determine the most appropriate cell viability assay suitable for evaluating anthocyanins cytotoxicity. OBJECTIVE 2: To establish baseline LC-50 values on five different cell lines for crude and anthocyanin-enriched extract. 31 EXPERIMENT 3. THE PROTECTIVE EFFECT OF BLACKBERRY ANTHOCYANINS AGAINST FREE RADICAL-INDUCED OXIDATION AND CYTOTOXICITY NULL HYPOTHESIS (Ho): Anthocyanins from blackberry can suppress free radical-initiated intracellular oxidation as well as protect multiple cell lines from free radical-induced cytotoxicity. OBJECTIVE 1: To monitor the manifestation of the antioxidant capacity of both blackberry crude and anthocyanin-enriched extracts in suppressing free radical-initiated intracellular oxidation in five distinct cell lines over time. OBJECTIVE 2: To quantitate the protective effect of both blackberry extracts against free radical-induced cytotoxicity. EXPERIMENT 4. POTENTIAL PROTECTIVE EFFECT OF BLACKBERRY ANTHOCYANINS AGAINST FREE RADICAL-INDUCED CYTOTOXICITY NULL HYPOTHESIS (H0): Anthocyanins neutralize free radical-induced cytotoxicity. OBJECTIVE: To probe the mechanism by which blackberry anthocyanins protect against free radical-induced cytotoxicity in a model of the gastrointestinal cells (Caco-2). 32 EXPERIMENT 1: CHARACTERIZATION, IDENTIFICATION, QUANTIFICATION AND THE ANTIOXIDANT CAPACITY OF BLACKBERY ANTHOCYANINS INTRODUCTION There is overwhelming evidence for the involvement of reactive oxygen species (ROS) in the development of various diseases. A n increased level of R O S that overloads the cellular defense system can ultimately result in oxidative damage to cellular D N A , protein and l ipid constituents, which in turn may eventually lead to cell death (Halliwell and Whiteman, 2004; Packer, 1996). The oxidative damage attributed to oxidative stress has been identified as an underlying mechanism behind the pathogenesis of more than 50 chronic disease states, such as cancer, heart disease and diabetes (Halliwell and Gutteridge, 1989). Antioxidants, which neutralize free radicals, are considered to have the potential to prevent the progression of peroxidation reactions that lead to various diseases (Wang et al., 1996). For this reason, there has been an intense interest in identifying different dietary antioxidant sources, especially those from plant and animal material (Dol l , 1990; Gey, 1990). Anthocyanins are the red to blue pigment found in berries which exhibit potent antioxidant activity (Kahkonen and Heinonen, 2003). Blackberries (Rubus fruticosus sp.) are of particular interest because of a high anthocyanin and phenolic content, which has been reported to contribute to a high antioxidant capacity (Wang and L i n , 2000). In addition to being the most commonly found anthocyanin in blackberry, cyanidin-3-glucoside has also been reported to have the highest antioxidant capacity out of 14 anthocyanins tested (Mazza and Miniat i , 1993; Wang et al., 1997). 33 Although the phytochemical profile of blackberry fruit appears to be well established, there are not many studies that have evaluated the anthocyanin profile of blackberry together with its associated antioxidant capacity. The purpose of this experiment was to characterize, identify and quantify the anthocyanin content in blackberry extracts using cyanidin-3-glucoside as an external standard. In addition, the antioxidant capacities of both crude and anthocyanin-enriched extracts were assessed using the O R A C assay. MATERIALS AND METHODS Materials Blackberries were supplied from Sandhu Farm, Abbotsford, B . C . Biogel-P-2 was obtained from Bio-Rad Laboratories (Richmond, Ca). Sodium acetate (CH3CO2Na.3H.2O) was purchased from Fisher Scientific (Fair Lawn, NJ) . Acetic acid (CH3CO2H), phosphoric acid ( H P L C grade), methanol ( H P L C grade) and hydrochloric acid were obtained from Fisher Scientific (Nepean, ON) . Cyanidin-3-glucoside and cyanidin-3-chloride were purchased from Polyphenol A S (Sandnes, Norway). 2, 2 '-azobis (2-amidinopropane) dihydrochloride ( A A P H ) were from Wako Chemicals U S A (Richmond, V A ) . Potassium chloride (KC1), Trolox, and fluorescein were obtained from Sigma-Aldr ich Canada Ltd. (Oakville, ON) . Extraction One hundred grams of frozen blackberry were blended with 100 ml of 80% ethanol using a Waring blender for five minutes. The slurry was transferred to an 34 Erlenmeyer flask, and extracted overnight. The extract was filtered through a Buchner funnel using Whatman number 1 in an erlenmeyer flask and rinsed with 2 x 25 ml of 80% ethanol. The filter cake was transferred to a new Erlenmeyer flask and 150 ml of 80% ethanol was added to re-extract the sample using an orbital shaker at 400 rpm (Innova 4000, New Brunswick Scientific, NJ) for an hour. The filtrate was pooled together with the first filtrate, and the filter cake was re-extracted for the third time. A l l sample extractions were performed in quadruplet. The ethanol in the pooled filtrate was removed under reduced pressure at 35°C. The residue was then freeze-dried to a powder and kept at 4°C. Enrichment of Total Anthocyanin Content with Gel Filtration Gel filtration chromatography was performed to concentrate the total anthocyanin contents of the crude extract as described by H u et al (2003). Reconstituted blackberry crude extract (300 mg/ml) was loaded onto a Biogel P2 gel filtration column (2.5 cm x 14.5 cm). Acetic acid (pH 2.5) was used to reconstitute the blackberry powder, and to elute the anthocyanin fractions (flow rate = 2 ml/min). The extract constituents were separated based on molecular weight, with higher molecular weight compounds being eluted earlier. The red colored fraction was collected and pooled together over several loadings. This anthocyanin rich fraction was then freeze-dried to powder and stored at 4°C. 35 Determination of Total Anthocyanins Content Total anthocyanin content of the blackberry extract was measured using p H differential method as described by Wrolstad and Giusti in Current Protocols in Food Analytical Chemistry (2000). A preliminary experiment was carried out to determine the maximum absorbance wavelength of blackberry extracts and the dilution factor required to adjust the absorbance reading to the acceptable range of the spectrophotometer (<1.200). Blackberry extracts, dissolved in KC1 buffer (0.025 M , p H 1) were scanned with Shimadzu UV-160 spectrophotometer (Shimadzu Corporation) for maximum absorbance wavelength (A,vjS-max)- The A,vis-max o f the extracts was determined to be 510 nm. The dilution factor was determined by adjusting the blackberry extracts to KC1 buffer (0.025 M , p H 1) ratio. Blackberry crude and anthocyanin-enriched extract were separately dissolved in potassium chloride buffer (0.025 M , p H 1) and sodium acetate ( C H 3 C 0 2 N a . 3 H 2 0 , 0.4 M , p H 4.5) with the pre-determined dilution factor. The mixtures were then allowed to equilibrate for 15 minutes at room temperature and the absorbances were read at 510 nm and 700 nm (to correct for haze) against a blank cell containing distilled and deionized water (ddHiO). The absorbance (A) o f the diluted sample was then calculated as follows: A = (Axvis-max ~ A700 nm)pH 1.0 _ (Axvis-max — A700 nm)pH 4.5 The monomeric anthocyanin pigment concentration in the original sample was calculated according the following formula: Anthocyanin content (mg/L) = A x M W x D F x 1000 e x 1 ; where M W is the molecular weight, D F is the dilution factor and 8 is the molar absorptivity. 36 The molecular weight and molar absorptivity for cyanidin-3-glucoside was used. M W = 449.2 and s = 26,900. Characterization of Blackberry Anthocyanins Characterizations of anthocyanins in blackberry extracts were performed using H P L C (High Performance Liquid Chromatography). Blackberry extracts were dissolved in a mixture of 50% acidified water (3%> phosphoric acid, H P L C grade) and 50% methanol ( H P L C grade). The reconstituted extracts were passed through a 0.45 p M Nylon membrane-filter. The H P L C analyses was performed using an Agilent 1100 series H P L C system (Agilent Technology 1100 series, Palo Alto , Ca), equipped with quaternary pumps, inline degasser, autosampler and a diode array detector. Anthocyanin and phenolic acid separation were performed using a Zorbax R X - C 1 8 column (5 pm, 4.6 mm x 250 mm). The column temperature was maintained at 30 °C. Mobile phases were: A , 100% methanol and B , acidified water (3%> phosphoric acid). The gradient condition started with 23%> A at 0 minute, increasing linearly to 24.3% A at 15 minute, then to 50% A at 20 minute and 55 % A at 25 minute. The flow rate was 1 ml/minute. The U V - V i s spectra were recorded simultaneously during analysis at 525 nm, 360 nm, 320 run, 280 nm and 255 nm with a peak scan between 220 to 700 nm (2 nm range step). Peak identification of unknown compounds of interest was performed by matching the retention time of unknown compounds to external standards (cyanidin-3-glucoside and cyanidin-3-chloride). The ChemStation software (version A . 10.02) was used to analyze the chromatograms of blackberry extracts and to calculate the proportion of cyanidin-3-glucoside relative to the total anthocyanin content. 37 Antioxidant Capacity of Blackberry Extracts The antioxidant capacities of both crude and anthocyanin-enriched extract were evaluated using the Oxygen Radical Absorption Capacity assay ( O R A C ) , as described by Kitts and H u (2005). Briefly, blackberry samples and a standard antioxidant (Trolox) were dissolved in phosphate buffer (50 m M , p H 7.0), followed by 60 n M fluorescein in a 96-well plate (Nunc, Fluorescent microplate). Plates were incubated at 37 °C for 15 min. The peroxyl radical initiator A A P H was added to a final concentration of 12 m M and fluorescence (Ex = 485nm, E m = 527nm) was continuously taken for 60 min (Fluoroskan Ascent F L , Labsystems). The data transformation and interpretation was performed according to Davalos et al. (2004). The O R A C value was expressed as pmol Trolox/g sample (Kitts and Hu, 2005). Statistics Experiments were performed in triplicate and results were expressed as mean ± standard deviation. Significant differences were detected with student t-test at p <0.05. RESULTS Determination of Total Anthocyanin Content of Blackberry Extracts The total anthocyanin content of the blackberry crude extract, as determined by p H differential method was 17.1 ± 0.9 mg/g of freeze dried powder, which was equivalent to 176.0 ± 9.5 mg/100 g of blackberry (Table 5). A small but significant (p < 0.05) batch to batch variation of total anthocyanin content was detected (Table 4). The 38 anthocyanin-enriched extract contained 371.1 ± 15.4 mg of total anthocyanin per g of freeze dried powder (Table 5). Characterization and Identification of Blackberry Anthocyanins The cyanidin-3-glucoside standard was eluted from the H P L C column at 13.6 minutes (Figure 3.A), whereas the aglycone cyanidin-3-chloride, was eluted at 22.9 minutes (Figure 3.B). A t 525 nm, the crude extract contained a single major peak that eluted at 13.7 minutes, a retention time that coincides with the cyanidin-3-glucoside standard. Minor peaks eluting at 22.5 minutes (Figure 3.C) were also seen. One of the minor peaks was identified as the aglycone. The anthocyanin-enriched extract contained a similar elution profile, with a major peak at 13.7 minutes and a minor peak at 22.5 minutes (Figure 3.D). The cyanidin-3-glucoside composed 87.5% and 90.1% of total anthocyanins in blackberry crude extract and anthocyanin-enriched extract, respectively. Antioxidant Capacity of Blackberry Extracts The antioxidant capacity of crude extract, as measured by O R A C was 69.2 ± 5 . 3 8 pmole Trolox/g of blackberry whereas the O R A C value for the anthocyanin-enriched extract was 501.6 ± 5.38 pmole Trolox Equivalent/g. 39 Table 4. Total anthocyanin content of blackberry crude extracts1 Total Anthocyanin Content (mg/100 g blackberry) Batch 1 Batch 2 Batch 3 Batch 4 166.6 ± 5 . 3 a 169.2 ± 3.0 a 180.5 ± 2.6 b 188.0 ± l . l b 'Different superscript letters indicate significant difference between treatment means (n = 3) at p < 0.05 40 Table 5. Anthocyanin profile and associated antioxidant activity of blackberry extracts1 Total Anthocyanin Content* Cyanidin-3-Glucoside T (mg/g) Crude Extract (% of Total Anthocyanin) 17.1 ± 0 . 9 87.5 O R A C (umole Trolox Equivalent/g) 673.8 ± 52.4 Anthocyanin Enriched Extract 371.1 ± 15.4 90.1 4884.7 ± 52.4 Total anthocyanin content and O R A C value are expressed per gram of freeze dried blackberry (n = 8) f Cyanidin-3-glucoside as characterized using H P L C . * Total anthocyanin content as determined by p H differential method 1 ' r 5 13.607 ~ i 1 r~ . 1 i B 10 15 20 n n n * B min 22.915 10 13.686 15 20 22.540 mm min mm Figure 3. HPLC profile (at 525 nm) of A = cyanidin-3-glucoside standard, B = cyanidin-3-chloride, C = anthocyanins from crude extract, D = anthocyanins from anthocyanin-enriched extract. 42 DISCUSSION The total anthocyanin content of blackberry crude extract, as evaluated by p H differential method (176.0 ± 9.5 mg/100 g of blackberry) in this study was within the range of the total anthocyanins reported for blackberry (83-326 mg of anthocyanins per 100 g) (Mazza and Miniat i , 1993). Differences in total anthocyanin content between four batches of extractions can be attributed to variation in extraction process or in blackberry size. The H P L C analysis of blackberry crude extract revealed the presence of one major anthocyanin, which was identified by external standard to be cyanidin-3-glucoside. This finding is consistent with the current literature information regarding blackberry anthocyanin composition. It was reported that cyanidin-3-glucoside was the one and only major anthocyanin in blackberry (Mazza and Miniat i , 1993; Siriwoharn et al., 2005). The proportion of cyanidin-3-glucoside (87.5 % - 90.1 %) to total anthocyanins in this study was within the concentration range reported by Siriwoharn et al., (73.8 % - 93.9 %) (2004; 2005), but was higher than the average (82.9%) of various blackberry selections. Gel filtration with Biogel P2 of the crude extract was shown to effectively concentrate total anthocyanins for quantitative recovery. The anthocyanin-enriched fraction obtained from gel filtration contained approximately 20 times more total anthocyanins and cyanidin-3-glucoside than the crude extract on a percent dry basis. Despite the significantly higher (p <0.05) amount of total anthocyanins in the-enriched fraction, the anthocyanin profile was similar to the crude extract. The H P L C analysis of anthocyanin-enriched extract confirmed the presence of cyanidin-3-glucoside as the only major anthocyanin (90.1% of total anthocyanins) in blackberry extracts. The aglycones of 43 both crude and anthocyanin-enriched extract were identified at a later retention time (tR = 22.5 minutes), and contributed only 5 - 6.5 % of the total anthocyanin content. In addition to cyanidin-3-glucoside and its aglycone, several minor peaks were detected slightly before or after the elution time of the aglycone (tR = 21.8 -22.9 minute). These compounds may be structurally similar to cyanidin. Some other anthocyanins which have been reported to be present in smaller amounts by other authors include cyanidin-3-rutinoside, cyanidin-3-xyloside, malvidin-3-glucoside, cyanidin-3-glucoside acylated with malonic acid, and cyanidin-3-dioxalylglucoside (Dugo et al., 2001; Fan-Chiang and Wrolstad, 2005; Pericles, 1982; Stintzing et al., 2002b). The antioxidant capacity of blackberry crude extract (69.2 ± 5 . 3 8 pmole Trolox equivalent /g) is in agreement with the finding of Moyer et al., (2002), who reported that the O R A C antioxidant capacity of blackberry, ranged from 33.3 to 78.8 pmole Trolox equivalent /g. The antioxidant capacity of the anthocyanin-enriched extract in this study (501.6 ± 5 . 3 8 pmole Trolox /g) was markedly greater (p < 0.05) than that of the blackberry crude extract. This antioxidant capacity of blackberry however did not follow a linear relationship with total anthocyanin content. For example, the concentration of total anthocyanins in the anthocyanin-enriched extract was approximately 20 times greater than the crude extract, but the antioxidant capacity ( O R A C value) of the anthocyanin-enriched extract was increased by only 7.3 times (dry basis) in comparison to the crude extract. This finding might be due to the presence of other phenolic compounds, which have antioxidant capacity and which may work additively or synergistically with anthocyanin to yield antioxidant activity in the crude extract. Some phenolic compounds such as quercetin glycosides, catechin and epicathechin, as well as 44 ellagic acid derivatives, have been reported to be present in blackberry (Siriwoharn et a l , 2004; Siriwoharn et al., 2005). These phenolic compounds in the crude extract were likely removed by gel filtration in this study and thus were not present in the anthocyanin-enriched extract. Conclusion In conclusion, anthocyanins were successfully extracted from blackberry and concentrated using Biogel P2 gel filtration. The total anthocyanin content of blackberry extract was slightly higher than that reported in the literature. The anthocyanin-enriched extract contained approximately 20 times greater amount of anthocyanins in comparison to the crude extract. Cyanidin-3-glucoside was identified to be the only predominating anthocyanin in both crude and anthocyanin-enriched blackberry extract. Lastly, the anthocyanins-enriched extract was a more effective peroxyl radical scavenger than the crude extract when expressed as equivalent dry weight basis. 45 EXPERIMENT 2: COMPARISON OF FOUR CYTOTOXIC ASSAYS TO ASSESS THE CYTOTOXICITY OF BLACKBERRY ANTHOCYANINS IN MULTIPLE CELL LINES INTRODUCTION There is a growing interest to use anthocyanins, the red to blue pigments present in berries, as a source of dietary antioxidant that can prevent the development of various free radical associated diseases and related health disorders. To date, numerous studies have characterized the antioxidant potential of anthocyanins in various in vitro chemical systems; albeit less is known about the associated potential health benefits of anthocyanins at reducing the risk of heart disease and cancer (Bomser et al., 1996; Kamei et al., 1995; Zhang et al., 2004). Blackberry (Rubus fruticosus sp) in particular has been shown to contain the greatest antioxidant capacity relative to other fruits, which can be attributed to its high anthocyanin content (Pellegrini et al., 2003; Wang and L i n , 2000). While the antioxidant capacity o f anthocyanins has been well established, less is known about the direct implications of this antioxidant capacity in preventing the progression of the free radical associated diseases. Additional studies are required to determine the potential benefits of antioxidant activities from blackberry in biological system. Cultured cell lines are a convenient and very appropriate preclinical model test system that may, to some extent, simulate in vivo condition. Even though the information regarding the effect of various anthocyanins containing extracts in some cultured cell lines in abundantly available, there remains very little known about the specific biological effect of blackberry extract and its anthocyanins-enriched fraction in different cancer cell lines. 46 A widely used assay to assess cytotoxicity is the M T T (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) redox assay. It is a well established cell viability assay based on the reduction of the yellow tetrazolium salt to the purple formazan by viable cells (Mosmann, 1983). Antioxidants, however, can potentially interfere with the M T T assay, by reducing the yellow M T T to the purple formazan, which w i l l lead to false positive result (Bruggisser et al., 2002). Nevertheless, a slight modification to this assay was suggested to eliminate the aforementioned interference by removing the antioxidants prior to M T T addition (Bruggisser et al., 2002). Based on the potential inaccuracies at determining cytotoxicity using the M T T assay, blackberry extracts were evaluated for cytotoxicity using not only the modified M T T assay, but also through comparison, cell counting as the reference assay. In addition, the B r d U assay, which measures D N A synthesis, and the CellTiter-Glo assay, which measures cell metabolism based on the A T P content, was also evaluated against cell counting. The purpose of this experiment was to first assess the relative sensitivities of different cytotoxicity assays, with distinct end-point measures for evaluating the biological effects of the blackberry extract and the anthocyanins-enriched fraction. Evaluations were conducted in five distinct cell lines to determine i f cell type was a factor in characterizing anthocyanin effects. 47 MATERIALS AND METHODS Materials Blackberries were supplied from Sandhu Farm, Abbotsford, B . C . Biogel-P-2 was obtained from Bio-Rad Laboratories (Richmond, Ca). Acetic acid (CH3CO2H), and hydrochloric acid ( H O ) were from Fisher Scientific (Nepean, ON) . 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) was from Sigma Co. (St. Louis, M O ) . Sodium dodecyl sulfate (SDS) was from Fisher Scientific (Springfield, NJ) . B r d U (chemiluminescence) kit was purchased from Roche Diagnostics Corporation (Indianapolis, IN). CellTiter-Glo® Luminescent Cel l Viabili ty Assay kit were from Promega Corporation (Madison, WI). Adenosine triphosphate (ATP) was from Sigma-Aldr ich Canada Ltd. (Oakville, ON) . Extraction and Enrichment of Blackberry Anthocyanins Blackberry anthocyanins were extracted and concentrated to an anthocyanin-enriched extract as described in Chapter 1. Cell Culture The cell lines used in this study were: Caco-2, INT-407, M C F - 7 and M D A - M B -453, and L N C a P , all obtained from A T C C (Manassas, V A ) . Caco-2 was maintained in Dulbecco's Modified Eagle's Medium ( D M E M ) , INT407 and M C F - 7 were maintained in Min imum Essential Medium Eagle ( M E M E ) . M D A - M B - 4 5 3 and L N C a P were maintained separately in Leibovitz's L-15 and RPMI-1640, respectively. A l l media were purchased from A T C C except for D M E M (Sigma, St. Louis, M O ) , and were 48 supplemented with 10% fetal bovine serum (Gibco, Grand Island, N Y ) , penicillin (100 U) and streptomycin (100 pg/ml) (Gibco). Upon confluency, cells were subcultured according to A T C C recommendation and maintained at 37°C in a 5% CO2 humidified incubator. The only exception to this was for M D A - M B - 4 5 3 , which was maintained at 37°C in 100% air incubator. Effect of Blackberry Extracts on Cultured Cells Each cell line was seeded at 2.5 x 10 4 cells/well in a 96-well plate and left overnight to allow for attachment. Blackberry extracts were dissolved into culture medium with serial dilutions made to achieve a concentration range of 0.01 mg/ml - 10 mg/ml for final evaluation. The blackberry extracts were added to the cells and were incubated in 96 well plates for 24 hour. The test control contained untreated cells and the appropriate cell culture medium without blackberry extracts. Each treatment was done in triplicate. A t the end of the 24 hour incubation, cells were recovered for cytotoxicity testing using one of the following methods: M T T , B r d U , CellTiter-Glo or cell counting. In a separate experiment, the effect of a blackberry anthocyanin-enriched extract (2 pg/ml - 500 pg/ml) was performed on cells cultured for 24 hours and evaluated using the M T T and CellTiter-Glo assays only. Cytotoxicity Assays Cell Counting Upon 24 hour exposure to blackberry extracts, culture medium was removed and 0.25%) Tryps in-EDTA was added. Cells from different cell lines were incubated at 37°C 49 until detachment from the plates occurred. Cells were manually dispersed to attain a single cell suspension. The trypsin was neutralized by added fresh culture media to different cells, which was followed by cell counting using a haemocytometer. Viable cells were assessed by trypan blue (0.04%) exclusion dye. MTT Assay Following incubation of various cell types with blackberry extracts, culture media were replaced with fresh medium in order to prevent direct interaction between blackberry antioxidants and the M T T substrate. M T T was added to cells to a final concentration of 0.5 mg/ml and the plates were incubated in the dark for four hours. To solubilize the formazan crystal, SDS (10%) in HC1 (0. IN) was added and the plates were incubated overnight. Optical density readings were taken at 570 nm in a microplate reader (ThermoLabsystems Multiscan Spectrum, Thermolabsystem, Chantilly, V A ) . BrdU Assay The B r d U assay was performed according to the manufacturer instructions. A t the end of the 24-hour incubation period with blackberry extracts, a B r d U labeling solution was added to cells, and the 96 well plates were further incubated at 37°C for 24 hour to allow for cell uptake of BrdU. The medium and unincorporated B r d U was removed and cells were fixed with FixDenat solution, which also denatured the cellular D N A . An t i -BrdU-peroxidase was added and re-incubated for 90 minutes, followed by three washing steps. Finally the substrates of the peroxidase enzymes (H2O2) were added and the 50 reaction was measured using a luminometer (Fluoroskan Ascent F L , Labsystem, Helsinki, Finland). Interference of Anthocyanin with CellTiter-Glo Assay To evaluate potential interference of anthocyanin with CellTiter-Glo assay, the assay was performed over a concentration range of anthocyanin in the absence of cells. Briefly, blackberry crude extract was reconstituted with culture media and serial dilutions were made in a 96 well plate to achieve a final concentration range of 0.125 mg/ml - 10 mg/ml. Controls contained culture medium only without blackberry crude extract. Equal amount of A T P (4.4 m M ) was added to the different concentrations of anthocyanins as a substrate to the subsequently added luciferase enzyme, the primary component of CellTiter-Reagent. The reaction was measured using a luminometer (Fluoroskan Ascent F L , Labsystem, Helsinki, Finland). CellTiter-Glo Assay The CellTiter-Glo assay was performed according to the manufacturer instructions, with a slight modification. This modification was required since anthocyanins were found to interfere with the original CellTiter-Glo assay. To remove interference, the blackberry containing medium was replaced with fresh medium before the addition of CellTiter-Glo reagent to the cells. The plates were shaken for 2 minutes, allowed to stabilize for 15 minutes and the reaction was measured using a luminometer (Fluoroskan Ascent F L , Labsystem, Helsinki, Finland). 51 Statistics Each concentration treatment with anthocyanin was performed in triplicate. Results were expressed as mean ± standard deviation. One way A N O V A was used to analyze the LC50 data derived from cell viability curves obtained using M T T , CellTiter-Glo , B r d U and cell counting methods. Post hoc analysis of treatment means using Tukey test was performed to detect significant difference at (p < 0.05). Pearson's correlation coefficients between cell counting and alternative cytotoxicity assays were assessed using SPSS statistical program (release 11.0). RESULTS Interference of Anthocyanin with CellTiter-Glo Assay A cell free CellTiter-Glo assay was used to evaluate the potential interference of blackberry extract. Using this method, the blackberry crude extract was demonstrated to decrease the generation of luminescence in a concentration-dependent manner (p < 0.05, Figure 4). This suppression of luminescence occurred regardless of the cell culture media used to re-constitute the blackberry crude extract and maintain the cell lines. Moreover, the magnitude of interference by the blackberry constituents on the CellTiter-Glo response appeared to be dependent on the composition of the different culture media used with individual cell lines. Additional studies are required to elucidate the significance of this finding. 52 Evaluation of Cytotoxicity Assays The effect of the blackberry crude extract on multiple cell lines, evaluated using four different assay methods was expressed as concentration response curves (Figure 5A-E). Concentration response curves obtained from cell counting gave the lowest LC50 values for Int-407, L N C a P and M D A - M B - 4 5 3 . On the other hand, the L C 5 0 concentration response curve obtained using the M T T assay produced the greatest deviation from the standard cell counting method for INT-407, L N C a P and M C F 7 cells. The concentration response curves obtained from CellTiter-Glo concentration response curves were closer to cell counting for Int-407, L N C a P and M C F 7 , whereas response curves for the B r d U assay resembled the M T T assay for Caco-2, Int-407 and L N C a P , respectively. LC50 of Crude Extracts Based on Four Cytotoxicity Assays The LC50 for individual cell types in each assay derived from the concentration response curves (Figure 5A-E) , is summarized in Table 6. Significantly different (p < 0.05) LC50 were obtained for blackberry crude extract within the same cell lines depending on the cytotoxicity assay used. Cel l counting yielded a LC50 for blackberry crude extract of 3.40 ± 0 . 1 2 mg/ml for the Caco2 cell line, which was significantly (p < 0.05) different than L C 5 0 values obtained from CellTiter-Glo (6.05 ± 0.49 mg/ml) and B r d U (2.72 ± 0.38 mg/ml) assays, but similar to the M T T (3.00 ± 0.27 mg/ml) assay. A different result was obtained in the INT-407 cell line, where LC50 values from cell counting (2.39 ± 0.14 mg/ml) were not different than the CellTiter-Glo (2.48 ± 0 . 1 3 mg/ml) but significantly (p < 0.05) lower than both M T T (5.04 ± 0 . 1 0 mg/ml) and B r d U (3.46 ± 0.36 mg/ml). Similarly, there was no significant differences in LC50 values 53 obtained for the blackberry crude extract in L N C a P using cell counting (0.56 ± 0.08 mg/ml) and CellTiter-Glo (0.62 ± 0.06 mg/ml). The LC50 values from these assays using L N C a P cells however were lower (p < 0.05) than the M T T (1.04 ± 0.05mg/ml) and B r d U (0.91 ± 0.04 mg/ml) LC50, respectively. The cell counting LC50 value for the crude extract in M D A - M B - 4 5 3 (0.44 ± 0.12mg/ml) was significantly different (p< 0.05) than all other assays tested (e.g. 2.04 ± 0.11 mg/ml in M T T , 1.58 ± 0.11 mg/ml in B r d U , and 3.14 ± 0.11 mg/ml in CellTiter-Glo). Lastly, cell counting of viable M C F - 7 cells yielded an LC50 value of 6.33 ± 0.66 mg/ml, which was not significantly different from the LC50 values obtained using the CellTiter-Glo (7.18 ± 0.36 mg/ml) (p <0.05), but higher (p< 0.05) to B r d U (2.82 ± 0.35 mg/ml) and lower (p < 0.05) than M T T (9.39 ± 0.31 mg/ml) assay, respectively. LC50 of Crude Extracts for Five Cell Lines Significantly different L C 5 0 values of the blackberry crude extract was also obtained between cell lines when assessed using each assay (Table 6). Using the cell counting method, M D A - M B - 4 5 3 cells were found to have the lowest L C 5 0 value (0.43 ± 0.12 mg/ml), which was not significantly different than the L C 5 0 value for the L N C a P cells (0.57 ± 0.08 mg/ml). Significantly (p < 0.05) higher concentration of the blackberry crude extract was required to obtain LC50 values for INT-407 (2.39 ± 0 . 1 4 mg/ml), Caco-2 (3.41 ± 0.12 mg/ml) and the M C F 7 (6.33 ± 0.67 mg/ml); all L C 5 0 values were also significantly different of each others (p < 0.05). The LC50 values of the blackberry crude extract for five distinct cell lines were significantly different from each other (p < 0.05) when assessed by the modified M T T 54 assay. The order by which blackberry crude extract imposed cytotoxicity to the different cell lines was as follows: L N C a P > M D A - M B - 4 5 3 > Caco-2 > INT-407 > M C F - 7 (Table 6). Assessing cell viability using the B r d U assay gave similar results for the different cell lines exposed to the blackberry crude extract. Consistent with the order of LC50 values obtained using the modified M T T assay, L N C a P cells were the most sensitive (p < 0.05, LC50 of 0.92 ± 0.4 mg/ml) to the exposure of blackberry crude extract. This was followed by M D A - M B - 4 5 3 cells (1.59 ± 0.10 mg/ml) and Caco-2 cells (2.72 ± 0.38 mg/ml), respectively. The LC50 values for M C F - 7 cells exposed to the blackberry crude extract were however not significantly different than that obtained for Caco-2 cells. The INT-407 cells exhibited the least sensitivity (LC50 = 3.46 ± 0.36 mg/ml) to blackberry extract when measured by the B r d U assay (Table 6). LC50 values for the different cell lines when evaluated by the CellTiter-Glo assay were ranked as follows: L N C a P (0.63 ± 0.06 mg/ml) < M D A - M B - 4 5 3 (3.15 ± 0.12 mg/ml) ~ INT-407 (2.48 ± 0 . 1 3 mg/ml) < Caco-2 (6.05 ± 0.49 mg/ml) < M C F - 7 (7.18 ± 0.36 mg/ml). Overall, this relative order of cytotoxicity was comparable to that obtained using the cell counting method. Nevertheless, unlike the order of cytotoxicity that was established by cell counting, the LC50 value of the blackberry crude extract for L N C a P cells was significantly (p < 0.05) lower than that obtained for M D A - M B - 4 5 3 cells; both of which were not different from the LC50 values obtained for INT407. 55 Correlation between Cell Counting and Alternative Cytotoxicity Assays The correlation coefficient between cell counting and alternative cytotoxicity assays (e.g. CellTiter-Glo, B r d U and modified M T T assay) is summarized in Table 7. The LC50 values obtained from cell counting method correlated well (p < 0.01) to that obtained from the CellTiter-Glo and B r d U assays in all cell lines tested; the only exception being the M D A - M B - 4 5 3 cells. The correlation coefficient between the cell counting and these alternative assays (e.g. r = 0.932 - 0.999) was also higher for Caco-2, INT-407 and M C F - 7 cells, in comparison to the LC50 values from the modified M T T assay (r = 0.750 - 0.826, p < 0.05). A similar high degree of correlation (r = 0.939 -0.978, p < 0.01) between the LC50 values from cell counting method and the three different assays used were found for L N C a P cells. The LC50 values for M D A - M B - 4 5 3 from cell counting correlated best to that from the B r d U assay, followed by the CellTiter-Glo , but was weakly correlated to LC50 derived from the modified M T T assay. Cytotoxicity of Blackberry Crude Extract and Anthocyanin-Enriched Extracts The effect of crude blackberry extract on each cell line as evaluated by M T T assay and validated by CellTiter-Glo is shown in Figure 6. The prostate cancer cell (LNCaP) line was the most sensitive to the presence of blackberry extract (e.g. LC50 -1.05 ± 0.05 mg/ml by M T T and 0.62 ± 0.06 mg/ml by CellTiter-Glo), whereas the breast cancer cell (MCF7) line was the least sensitive (LC50 = 9.39 ± 0.31 mg/ml by M T T and 7.12 ± 0.36 mg/ml by CellTiter-Glo). Evaluations made with the modified M T T assay produced LC50 values of blackberry crude extract that were 3.00 ± 0.27 mg/ml for Caco-2 cells, 5.04 ± 0.10 mg/ml for INT407 cells and 2.02 ± 0 . 1 2 mg/ml for M D A - M B - 4 5 3 56 cells. The LC50 values obtained from the CellTiter-Glo for these cell lines however were significantly different (p < 0.05) than that obtained from the modified M T T assay (e.g. 6.05 ± 0.49 mg/ml for Caco 2, 2.48 ± 0.13 mg/ml for INT-407 and 3.15 ± 0.12 mg/ml for M D A - M B - 4 5 3 cells). The effect of the anthocyanin-enriched extract on multiple cell lines is shown in Figure 7. The concentrations of the anthocyanin-enriched extract tested were adjusted so that the anthocyanin content would be equivalent to that present in the crude extract. For example, 10 mg/ml of crude blackberry extract contained an equivalent amount of anthocyanins to 0.5 mg/ml of the anthocyanin-enriched extract. Both M T T and CellTiter-Glo results demonstrate that at highest anthocyanin concentrations tested (e.g. 0.5 mg/ml), cell viability was not reduced beyond 50%, and thus the LC50 values were not obtained at this concentration range. In the M T T assay, 0.5 mg/ml of the anthocyanins-enriched extract produced viability changes to all cells to 103 ± 3.4 % in Caco-2, 98.9 ± 4.39% in INT-407, 68.4 ± 4.0 % in L N C a P , 67.6 ± 1.62 % in M C F - 7 , and 154.5 ± 11.5 % in M D A - M B - 4 5 3 . A similar result was obtained using the CellTiter-Glo assay (110.0 ± 5.9 % in Caco-2, 95.6 ± 0.34 % in INT-407, 85.4 ± 3.99 % in L N C a P , 94.7 ± 3 . 1 6 % in M C F 7 , and 74.6 ± 7.28 % in M D A - M B - 4 5 3 ) . 57 120 i 0.16 0.63 2.5 10 Blackberry Concentration (mg/ml) Figure 4. Interference of blackberry extract dissolved in DMEM (I), MEME (H), RPMI (H) and Leibovitz-15 (•) culture media in a cell free CellTiter-Glo assay, supplemented with 4.4 uM ATP to generate luminescence. Bars with different letters indicate significant difference (p < 0.05) between concentration means within the same culture media. 58 0 2 4 6 8 10 Concentration (mg/ml) Figure 5. Cell response curve of blackberry crude extract incubated for 24 hours on Caco2 (A) and INT-407 (B) as evaluated with MTT (•), BrdU (•), CellTiter-Glo ( A ) , and cell counting (x). 59 140 n 120 4 0 2 4 6 8 10 Concentration (mg/ml) 0 2 4 6 8 10 Concentration (mg/ml) Figure 5. Cell response curve of blackberry crude extract incubated for 24 hours on MCF-7 (C); MDA-MB-453 (D) as evaluated with MTT (•), BrdU (•), CellTiter-Glo ( A ) and cell counting (x). 60 140 - i 120 100 : > 80 J n CO > 60 a? 40 20 4 6 Concentration (mg/ml) Figure 5. Cell response curve of blackberry crude extract incubated for 24 hours LNCaP (E) as evaluated with MTT (•), BrdU (•), CellTiter-Glo ( A ) and cell counting (*). 61 Table 6. LC50 values for blackberry crude extracts on five distinct cell lines evaluated using four different assays1. L C 5 0 (mg/ml) Ce l l Counting Modified M T T B r d U CellTiter-Glo Caco-2 3.41 ± 0 . 1 2 C T 3.01 ± 0 . 2 8 c T ° 2 . 7 2 ± 0 . 3 8 c w 6 . 0 5 ± 0 . 4 9 c * INT-407 2 . 3 9 ± 0 . 1 4 b t 5 . 0 4 ± 0 . 1 0 d e 3 . 4 6 ± 0 . 3 6 d ¥ 2 . 4 8 ± 0 . 1 3 b t L N C a P 0 . 5 7 ± 0 . 0 8 a t 1 . 0 5 ± 0 . 0 5 a e 0 . 9 2 ± 0 . 0 4 a e 0 . 6 3 ± 0 . 0 6 a t M C F - 7 6 . 3 3 ± 0 . 6 7 d t 9 . 3 9 ± 0 . 3 1 e e 2.82 ± 0 .35° ¥ 7 . 1 8 ± 0 . 3 6 d t M D A M B - 4 5 3 0 . 4 3 ± 0 . 1 2 a t 2 . 0 4 ± 0 . 1 2 b e 1.59 ± 0 .10 b ¥ 3 . 1 5 ± 0 . 1 2 b § Different letters indicate significant difference (p < 0.05) between treatment means of different cell lines assessed with the same assay. Different symbols (f, 0 , ¥, §) indicate significant difference (p< 0.05) between treatment means of different assay of within same cell line. Values are expressed as mean ± standard deviation (n = 8). 62 Table 7. Correlation coefficient (r) between cell counting and alternative assays Assay Cell Lines Caco-2 Int-407 LNCaP MCF-7 MDA-MB-453 Modified MTT 0.871* 0.826* 0.939** 0.750* 0.156 BrdU 0.932** 0.990** 0.954** 0.965** 0.812 CellTiter-Glo 0.999** 0.970** 0.978** 0.939** 0.622 1 Values are expressed as mean ± standard deviation (n = 4-8) * Correlation is significant at the 0.05 level ** Correlation is significant at the 0.01 level 63 10 n Caco2 Int407 LNCaP MDAMB453 MCF-7 Figure 6. The LC50 of blackberry crude extracts on multiple cell lines as evaluated with MTT (•) and CellTiter-Glo (•) assays. * Significant difference between the LC50 values obtained from the MTT and CellTiter-Glo assays were detected at p < 0.05 64 140 120 100 !5 ro > 60 0 s -40 20 B. 0.1 0.2 0.3 Concentration (mg/ml) 0.4 0.5 Figure 7. The effect of blackberry anthocyanins-enriched extract in Caco-2 (•), INT-407 (•), LNCaP ( A ) , MCF-7 (x) and MDA-MB-453 (•), as evaluated with MTT assay (A) and CellTiter-Glo (B). 65 DISCUSSION Suppression of luminescence by blackberry crude extract occurred in a cell free CellTiter-Glo assay despite the fact that equal amount of A T P was available along with the different concentration of blackberry supplied. This result strongly suggests that blackberry anthocyanins interfere with the CellTiter-Glo assay, plausibly due to inhibition of luciferase enzyme activity. Luciferase requires A T P to generate luminescence, the end-point measure for evaluating cell viability. This experiment shows the importance of removing blackberry anthocyanins prior to the addition of CellTiter-Reagent to the cells, which i f not done wi l l result in the underestimation of cell viability. The fact that blackberry crude extract yielded notably different LC50 values in individual cells can be explained potentially on the basis that each assay had markedly different end-point measure parameters for assessing cytotoxicity. For example, the M T T assay measures the mitochondrial oxidation-reduction (Redox) activity, whereas B r d U and CellTiter-Glo assays measure D N A proliferation and cellular A T P content, respectively. Ce l l counting on the other hand, is the only direct measure of cell viability determined by the uptake of trypan blue by dead cells. In the present study, the modified M T T assay gave the greatest discrepancy from cell counting for determining anthocyanin LC50 values. Better agreement in respective LC50 values amongst different cell lines was obtained between cell counting and CellTiter-Glo. In general, however, high correlations between the different assays for measuring cell viability were also found to be specific to the individual cell line tested. For example, both cell counting and B r d U LC50 results values were comparable for both INT-407 and M D A - M B - 4 5 3 cells, whereas Caco2, L N C a P and M D A - M B - 4 5 3 cell lines 66 gave similar cytotoxicity estimates for cell counting and CellTiter-Glo methods. Significant correlations were found between cell counting and B r d U for INT-407 and M D A - M B - 4 5 3 . Similar agreement in LC50 values were obtained in Caco-2, L N C a P and M D A - M B - 4 5 3 cells when determined by cell counting and CellTiter-Glo, respectively. A t the present time, there is no explanation for these findings other than suggesting that events associated with cellular metabolism and subsequent proliferations are specific to different cell types. Even though a considerable effort was made to remove the potential interference o f anthocyanins in the M T T assay, the modified M T T assay still produced overestimated cytotoxicity results for blackberry anthocyanin. This was especially true when high concentrations of blackberry anthocyanins were tested. A clear example of this observation was demonstrated by the concentration response curves obtained for M D A -MB-453 . The crude blackberry extract appeared to reduce cell viability followed by an unexpected proliferation that occurred at higher concentration when tested using the M T T assay. This finding was also observed to a lesser extent in Caco-2 and L N C a P cell lines, but in all three cases, the observed proliferation of cells was not substantiated by CellTiter-Glo, B r d U assay or cell counting. One possible explanation for this finding has come from Youdim et al., (2000a) who reported that anthocyanins derived from elderberry are strongly incorporated into the cell membrane following high exposure to elderberry extracts. Thus, despite thorough washing of cells exposed to anthocyanins, a distinct possibility exists that uptake of pigments into the membrane predisposes a potential false positive response i f interference with the M T T substrate occurs. 67 Despite the overestimation of cytotoxicity from the blackberry extract using the M T T assay, it is important to note that this procedure is relatively rapid, inexpensive and well established in the literature. In addition, the interference of anthocyanins in the M T T assay can be considered to occur only at concentrations that far exceed physiological intake, thereby providing a strong reason to support the modified M T T assay for testing when concentrations of anthocyanin are relevant to typical dietary exposure. Notwithstanding this, employing an alternative assay to assess cytotoxicity was shown to be prudent herein to confirm results obtained from blackberry extracts in different cell lines. The CellTiter-Glo and B r d U assays produced closer estimates of cytotoxicity for the blackberry crude extract when tested on multiple cultured cells. Individual blackberry extract concentration response curves obtained from alternative assays agreed well with the viability data obtained by cell counting. The higher correlation obtained between both CellTiter-Glo and B r d U with cell counting, compared to the modified M T T assay, confirmed this finding. Although B r d U assay is a useful non-radioactive assay for estimating cytotoxicity, it does have a potential limitation with non-specific binding of the anti-BrdU-peroxidases that can only be overcome by thorough washing of cells. This source of error was found to be particularly relevant to Int-407, Caco-2 and M D A - M B - 4 5 3 cells. The CellTiter-Glo assay is rapid and less expensive than the B r d U assay. Moreover, the CellTiter-Glo assay gave the greatest precision for viability estimates especially when compared to cell counting. In addition, the absolute results that defined concentration response curves obtained from CellTiter-Glo were closer to the results 68 obtained with cell counting and better than the M T T assay. Taking these findings together, it can be concluded that CellTiter-Glo was the best predictor of LC50 values, which was evident for three out of five cell lines examined herein (e.g. L N C a P , PNT-407, and M C F - 7 ) . The LC50 values derived from the concentration response curves from each cell line exposed to the blackberry crude extract reflected different individual sensitivities of cells to the anthocyanin containing extract. L N C a P cells, for example, were consistently shown using four different cytotoxicity assays to be the most sensitive to the blackberry crude extract of the five cell lines tested. On the other hand, M C F - 7 cells, the breast cancer cell which express estrogen receptor, was consistently the least sensitive cell line to blackberry crude extract. It is plausible that anthocyanin, which was reported by Schmitt and Stopper (2001) to have estrogenic property, may have interacted with the estrogen receptors expressed by the M C F - 7 cells. This event would consequently stimulate cell proliferation despite the opposing effect of blackberry crude extract at higher concentrations. A s a result, the observation that the LC50 values for blackberry crude extract obtained from M C F - 7 were significantly greater than that obtained from M D A - M B - 4 5 3 cells may be explained on the relative estrogen dependency of each breast cancer cell line. The intestinal cell lines tested in this study (e.g. Caco-2 and INT-407) were demonstrated to have sensitivities to blackberry extract that were mid-point to that of L N C a P cells and M C F - 7 cells. The order by which all different cells were affected by the blackberry crude extract was however dependent on the assay used to evaluate cell viability. This finding strongly suggests that the relative magnitude o f change in cell viability was affected by the assay used to express the effect, in addition to cell type. 69 While the LC50 values of all cell lines ranged from 0.7 to 9 mg/ml for the blackberry crude extract, the concentrations of anthocyanins that have been reported in physiological fluids is lower than 0.7 mg/ml (Ichiyanagi et al., 2005; Kay et al., 2004). Pharmacokinetic studies have shown that anthocyanins can be detected in many organs in addition to the plasma, such as the stomach, jejunum, liver, kidney and to a smaller extent in the brain. In the jejunum, the concentration of anthocyanins was estimated to be between 67.4 pg to 269.5 pg of cyanidin-3-glucoside equivalence/ g of tissue, compared to the plasma, where cyanidin-3-glucoside ranged from 2.25 ng/ml to 1.56 pg/ml in both rat and humans (Talavera et al., 2005; Tsuda et al., 1999b). Using an average molecular mass of 447.2 g/ml for conversion, it can be calculated from other studies, that anthocyanin concentration in human plasma ranges between 2.246 ng/ml to 442.9 ng/ml (Ichiyanagi et al., 2005; Kay et al., 2004; Matsumoto et al., 2001). This range o f anthocyanins concentration is equivalent to the concentration range (0.063 pg/ml - 42.5 pg/ml) of blackberry extract used in the present study with cultured cells. Using this extrapolation to mimic the relevant concentration of cyanidin-3-glucoside to cells in culture, it is noteworthy that the blackberry crude extract had no apparent cytotoxic properties in any of the cell lines tested, regardless of the methods used to assess viability. In the case of the anthocyanin-enriched extract, LC50 values could not be obtained for all cells using both the modified M T T assay and the CellTiter-Glo. This finding strongly indicates that anthocyanin from blackberry in particular is not cytotoxic. Using a similar extrapolation to that with the blackberry crude extract, it can be estimated that the concentrations of anthocyanin found in human plasma was equivalent to 2.98 ng/ml to 70 2.03 pg/ml of anthocyanin-enriched extract. The cytotoxicity data reported in this study indicates that exposure of cells to anthocyanin-enriched extract at these concentrations are not cytotoxic. This implies that anthocyanin at concentrations relevant to typical dietary intake is not likely to impose an adverse effect on human subjects. Conclusion Due to the high probability that the modified M T T assay yielded overestimated results of cytotoxicity, it can be concluded that alternative bio-assays for measuring effects of blackberry anthocyanins on cell viability are necessary to make accurate conclusion on bioactivity. The CellTiter-Glo assay was shown to be the best alternative method to the modified M T T assay, having the advantage of correlating well with cell counting and being more rapid and economical to use. Using the modified M T T assay and confirmed by the CellTiter-Glo assay, it was also found that neither the blackberry crude extract nor an anthocyanin-enriched extract from blackberry were cytotoxic at concentrations relevant to a typical exposure in human. 71 EXPERIMENT 3: THE PROTECTIVE EFFECT OF BLACKBERRY ANTHOCYANINS AGAINST FREE RADICAL-INITIATED INTRACELLULAR OXIDATION AND FREE RADICAL-INDUCED CYTOTOXICITY INTRODUCTION Reactive oxygen species (ROS) are highly unstable and thus reactive molecules, which are continuously generated in cells as byproducts of ordinary metabolism (Halliwell , 1994b; Henriksen and Endresen, 1994). Excessive generation of R O S w i l l result in oxidative damage to cellular components, and i f not neutralized may lead to a condition commonly known as oxidative stress (Halliwell and Gutteridge, 1989; Wang and Jiao, 2000). The oxidative damage to cellular proteins, membrane lipids or nucleic acids w i l l eventually lead to impaired metabolism and result in adverse biochemical and physiological changes that play a key role in the progression of more than 50 diseases (Moyer et al., 2002). Dietary antioxidants, especially those from natural plant sources, are of interest to scientists who study the link between soft fruit consumption patterns and protection against free radical associated diseases. Anthocyanins, the blue-red colored pigments found in berries fruit have been reported to exhibit potent antioxidant activity (Wang and L i n , 2000). Blackberry (Rubus fruticosus) in particular, is one of the richest sources of anthocyanin and has been considered to have excellent antioxidant capacity which is almost exclusively due to the high anthocyanin content (Pellegrini et al., 2003; Wang and L i n , 2000). Blackberry is also of particular interest, since its simplicity as an anthocyanin source is seen with the only major anthocyanin being identified as cyanidin-3-glucoside (Pericles, 1982). Despite the fact that the antioxidant capacity of blackberry has been well demonstrated in various in vitro chemical systems, there is a paucity of information 72 concerning modeling antioxidant capacity in cell culture systems which are relevant to in vivo biological systems. The experiments reported in this chapter were aimed to demonstrate the effect of blackberry crude extract and a derived anthocyanin-enriched fraction, on free radical-initiated intracellular oxidation initiation and alleviation of free radical-induced cytotoxicity in multiple cell lines. M A T E R I A L S A N D M E T H O D S Mater ia ls Blackberries were supplied from Sandhu Farm, Abbotsford, B . C . Biogel P2 was obtained from Bio-Rad Laboratories (Richmond, Ca). Cyanidin-3-glucoside was purchased from Polyphenol A S (Sandnes, Norway) and 2, 2'-azobis (2-amidinopropane) dihydrochloride ( A A P H ) was obtained from Wako Chemicals U S A (Richmond, V A ) . 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) came from Sigma Co. (St.Louis, M L ) . Sodium dodecyl sulfate (SDS) was purchased from Fisher Scientific (Springfield, NJ) . CellTiter-Glo® Luminescent Ce l l Viabil i ty Assay kit were obtained from Promega Corporation (Madison, WI). 2',7'-dichlorofluorescin diacetate ( D C F H -D A ) was from Sigma-Aldrich Canada Ltd. (Oakville, ON). 73 Extraction and Enrichment of Blackberry Anthocyanins Blackberry anthocyanins were extracted and-enriched according to methods previously described in Chapter I. Cell Culture The multiple cell lines used in this chapter were cultured according to the conditions and procedures described in Chapter II. Evaluation of blackberry anthocyanins antioxidant activity against intracellular oxidation and free radical-induced cytotoxicity were performed for all individual cell lines. Cultured cells were initially seeded at a concentration of 2.5 x 10 4 cells/well in a 96 well plate and left overnight in a 37 °C, 5% CO2 incubator for attachment and attainment of growth. Intracellular oxidation The effect of blackberry extracts on AAPH-ini t ia ted intracellular oxidation was evaluated as described by H u et al., (2005) with minor modification. In this experiment, cells were seeded into 96 black well plates. Final concentrations of blackberry crude extract (8 pg/ml - 1 mg/ml) or the anthocyanin-enriched extract (0.02 pg/ml - 50 pg/ml) were added to culture media used in different cell lines. The blackberry extracts were co-incubated with the 5 p M D C F H - D A probe for three hours, followed by the addition of 1 m M A A P H to initiate intracellular oxidation. Fluorescence readings were taken from cells using a microplate reader (Fluoroskan Ascent F L , Labsystem) at zero minutes immediately upon addition of A A P H addition and at 1 to 7 hours and 24 hours, respectively, after the initial addition of A A P H . The excitation wavelength was set at 485 74 nm and emission wavelength was set at 527 nm. A negative control was constructed to consist of cells exposed to only the D C F H - D A probe. A positive control consisted of cells cultured with the D C F H - D A probe and the peroxyl radical initiator ( A A P H ) . A l l results were expressed according to the following formula, where: Fluorescence = Fluorescence ti Fluorescence to ; Where Fluorescence ti is fluorescence reading taken at time 1 to 7 hours and 24 hours Fluorescence to is initial fluorescence reading taken upon A A P H addition at 0 minute. Protection against free radical-induced cytotoxicity Experiments designed to evaluate protection against free radical-induced cytotoxicity involved exposure of different cell lines to both the blackberry crude and anthocyanin-enriched extracts at final concentrations of 31 pg/ml - 1 mg/ml and 0.8 - 25 pg/ml respectively, for three hours. Different concentrations of A A P H (5, 10 or 15 m M ) were added to cells to induce cytotoxicity and the mixture was further incubated for 24 hours at 37°C. Each treatment was done in triplicate. Cel l viability was measured using both M T T and CellTiter-Glo assay. The negative control consisted of cells cultured in media only, whereas a positive control consisted of cells treated with the A A P H without the blackberry extract. Cel l viability of treated cells was expressed as a percent o f viable cells present in the negative control. For the M T T assay, the anthocyanin containing media was replaced with fresh media followed by the addition of 0.5 mg/ml M T T reagent. Microtiter plates were incubated for four hours at 37°C in the dark, followed by the addition of SDS (10% in 75 0.01 HC1) to solubilize the formazan crystal formed. Absorbance readings were read at 570 nm using a microplate reader (Multiskan Spectrum, ThermoLabsystem, Chantilly, V A ) . The CellTiter-Glo assay was performed as described in Experiment II. The plates were shaken for two minutes at 1200 rpm and an equal volume of CellTiter-Reagent was added. The plates were left at room temperature for 15 minutes before the luminescence was read using a luminometer (Fluoroskan Ascent F L , Labsystem, Helsinki , Finland). Statistics A l l treatments were performed in triplicate. Results were expressed as mean ± standard deviation. A T-test was used to determine significant (p < 0.05) differences between control and treatment means for both M T T and CellTiter-Glo assays. RESULTS Intracellular Oxidation The effect of both the crude and anthocyanin-enriched extracts to reduce the extent o f AAPH-generated free radical in five different cell lines is shown in Figures 8-12. Exposure of different cell lines to A A P H (positive control) resulted in a time-dependent increase in free radical induced fluorescence. The presence of the D C F H - D A probe alone produced no change in fluorescence. Both the blackberry crude extract and the anthocyanin-enriched extract consistently suppressed (p < 0.05) fluorescence development in a time-dependent concentration response for all cell lines tested, over a wide concentration range of anthocyanin (Figure 8-12). Blackberry crude extract and 76 anthocyanin-enriched extract were shown to delay the generation of free radicals during the earlier stage of the intracellular oxidation (0 h - 7 h) in a concentration dependent manner (Table 8). This was evidenced by a significant (p < 0.05) increase in the T 2 5 (i.e. the time at which fluorescence intensity was 25% that generated by control cells at 7 hour) upon treatment of various cells with increasing concentration of blackberry extracts. The extent to which a concentration of blackberry extract inhibits free radical generation was dependent on the specific cell line tested. For example, 60 pg/ml of the blackberry crude extract and 6.3 pg/ml of the anthocyanin-enriched extract were sufficient to increase the T 2 5 value for all cell lines to greater than 7 hour. The only exception was with the L N C a P cells (Table 8). It is noteworthy that the concentration range of 0.1 pg/ml - 50 pg/ml of the anthocyanin-enriched extract produced a comparable delay o f free radical generation that was obtained over a range o f 0.01 mg/ml - 1 mg/ml of the blackberry crude extract (Table 8). The IC50 values obtained from the blackberry crude extract from different cell lines measured at 24 hour treatment ranged from 25.8 pg/ml to 56.1 pg/ml (Table 9). This corresponded to an IC50 range o f 2.6 pg/ml to 6.5 pg/ml for the blackberry anthocyanin-enriched extract (Table 9). In comparison, more than 0.25 mg/ml of blackberry crude extract was needed to suppress approximately 80%> of the A A P H induced free radical generation, and only 12.5 pg/ml of the blackberry anthocyanin-enriched extract was needed to produce a similar result (Figure 13). The anthocyanin-enriched extract exhibited a suppression of peroxyl radicals in cells that followed the order of L N C a P > M D A - M B - 4 5 3 > INT-407 > M C F - 7 > Caco-2. This response was different to the blackberry crude extract which ranked M D A - M B - 4 5 3 77 > INT-407 > M C F - 7 > Caco-2 > L N C a P for inhibiting intracellular free radical generation (Figure 13, Table 9). 7 0 T I 1 I 1 1 0 5 1 0 1 5 2 0 2 5 Time (hour) 140n Time (hour) Figure 8. The suppression of intracellular oxidation in Caco-2 cells cultured with A . anthocyanin-enriched extract and B . blackberry crude extract. Figure labels corresponding to the top panel (A) are represented by anthocyanin-enriched extract concentrations of: 50 pg/ml (o), 25 pg/ml (+), 12.5 pg/ml (A) , 6.2 pg/ml (x), 3.1 pg/ml (—), positive control or 0 pg/ml (•) and negative control ( A ) . The bottom panel (B) labels are represented by blackberry crude extract concentrations of: 1 mg/ml (o), 0.5 mg/ml (+), 0.25 mg/ml (A) , 0.13 mg/ml (x), 62 pg/ml ( - ) , 31 pg/ml (0), 7.8 pg/ml (•), positive control or 0 pg/ml (•) and negative control (A) .Values represent mean ± standard deviation (n = 3) 79 1201 Time (hour) 250 0 5 10 15 20 25 Time (hour) Figure 9. The suppression of intracellular oxidation in INT-407 cells cultured with A . anthocyanin-enriched extract and B . blackberry crude extract. Figure labels corresponding to the top panel (A) are represented by anthocyanin-enriched extract concentrations of: 50 pg/ml (o), 25 pg/ml (+), 12.5 pg/ml (A) , 6.2 pg/ml (x), 3.1 pg/ml (—), positive control or 0 pg/ml (•) and negative control ( A ) . The bottom panel (B) labels are represented by blackberry crude extract concentrations of: 1 mg/ml (o) , 0.5 mg/ml (+), 0.25 mg/ml (A) , 0.13 mg/ml (x), 62 pg/ml ( - ) , 31 pg/ml (0), 7.8 pg/ml (•), positive control or 0 pg/ml (•) and negative control (A) .Values represent mean ± standard deviation (n = 3) 80 3 01 Time (hour) Figure 10. The suppression of intracellular oxidation in L N C a P cells cultured with A . anthocyanin-enriched extract and B . blackberry crude extract. Figure labels corresponding to the top panel (A) are represented by anthocyanin-enriched extract concentrations of: 50 pg/ml (o), 25 pg/ml (+), 12.5 pg/ml (A), 6.2 pg/ml (x), 3.1 pg/ml (—), positive control or 0 pg/ml (•) and negative control ( A ) . The bottom panel (B) labels are represented by blackberry crude extract concentrations of: 1 mg/ml (o), 0.5 mg/ml (+), 0.25 mg/ml (A), 0.13 mg/ml (x), 62 pg/ml ( - ) , 31 pg/ml (0), 7.8 pg/ml (•), positive control or 0 pg/ml (•) and negative control (A) .Values represent mean ± standard deviation (n = 3) 81 10 15 Time (hour) 20 25 20 25 10 15 Time (hour) Figure 11. The suppression of intracellular oxidation in M C F - 7 cells cultured with A . anthocyanin-enriched extract and B . blackberry crude extract. Figure labels corresponding to the top panel (A) are represented by anthocyanin-enriched extract concentrations of: 50 pg/ml (o), 25 pg/ml (+), 12.5 pg/ml (A), 6.2 pg/ml (x), 3.1 pg/ml (—), 1.5 pg/ml (•), positive control or 0 pg/ml (•) and negative control ( A ) . The bottom panel (B) labels are represented by blackberry crude extract concentrations of: 1 mg/ml (o), 0.5 mg/ml (+), 0.25 mg/ml (A) , 0.13 mg/ml (x), 62 pg/ml (-), 31 pg/ml (0), 7.8 pg/ml (•), positive control or 0 pg/ml (•) and negative control (A) .Values represent mean ± standard deviation (n = 3) 82 10 15 Time (hour) 20 25 120 10 15 20 25 Time (hour) Figure 12. The suppression of intracellular oxidation in M D A - M B - 4 5 3 cells cultured with A . anthocyanin-enriched extract and B . blackberry crude extract. Figure labels corresponding to the top panel (A) are represented by anthocyanin-enriched extract concentrations of: 50 pg/ml (o), 25 pg/ml (+), 12.5 pg/ml ( A ) , 6.2 pg/ml (x), 3.1 pg/ml (—),1.5 pg/ml (•), positive control or 0 pg/ml (•) and negative control ( A ) . The bottom panel (B) labels are represented by blackberry crude extract concentrations of: 1 mg/ml (o), 0.5 mg/ml (+), 0.25 mg/ml ( A ) , 0.13 mg/ml (x), 62 pg/ml ( - ) , 31 pg/ml (0), 7.8 pg/ml (•), positive control or 0 pg/ml (•) and negative control (A) .Values represent mean ± standard deviation (n = 3) 83 Table 8. Concentration dependent inhibition of AAPH induced intracellular oxidation in various cell lines by blackberry extracts Concentration T25 (hour)1 Blackberry Crude Extract (mg/ml) Caco2 INT-407 LNCaP MCF-7 MDA-MB-453 0.0 2.4 ± 0 . 1 2.5 ± 0 . 1 1.6 ± 0 . 3 2.2 ± 0 . 1 2.8 ± 0 . 2 0.01 3.6 ± 0 . 1 3.9 ± 0 . 3 2.2 ± 0.2 3.4 ± 0 . 2 4.1 ± 0 . 1 0.02 5.2 ± 0.2 5.7 ± 0 . 4 2.6 ± 0 . 5 5.0 ± 0 . 2 5.0 ± 0 . 1 0.03 6.8 ± 0 . 1 6.5 ± 0.5 2.8 ± 0.2 6.7 ± 0 . 2 6.2 ± 0 . 1 0.06 >7h >7h 2.9 ± 1.3 >7h >7h 0.25 >7h >7h 6.0 ± 0 . 6 >7h >7h 1.00 >7h >7h >7h >7h >7h Negative Control >7h >7h >7h >7h - >7h Anthocyanin Enriched Extract (pg/ml) 0.0 2.6 ± 0 . 1 2.6 ± 0 . 1 2.6 ± 0 . 1 2.2 ± 0 . 1 2.8 ± 0 . 1 0.1 2.8 ± 0 . 1 3.1 ± 0 . 2 2.6 ± 0.2 2.4 ± 0 . 1 3.3 ± 0 . 1 0.4 3.4 ± 0 . 1 3.8 ± 0 . 2 3 . 0 ± 0 . 1 3.0 ± 0 . 1 3.6 ± 0 . 2 1.6 5.6 ± 0 . 4 5.7 ± 0 . 3 3.6 ± 0 . 2 5.0 ± 0 . 1 5.1 ± 0 . 2 6.3 >7h >7h 4.9 ± 0 . 5 >7h >7h 13 >7h >7h 6.6 ± 0 . 1 >7h >7h 50 >7h >7h >7h >7h >7h Negative Control >7h >7h >7h >7h >7h Lag phase is described by an arbitrary T 2 5 value.*T 25 is defined as the time required to generate 25% of the fluorescence obtained from control cells treated with 1 m M A A P H alone for 7 hour. T 2 5 is a calculated value. 120 Concentration (pg/ml) Figure 13. Percent Inhibition of Intracellular Oxidation by Blackberry Crude Extract (A) and Anthocyanin-Enriched Extract (B) in all Cell Lines at 24 hour treatment. Figure labels corresponding to both panels are represented by Caco-2 (•), INT-407 (•), LNCaP ( A ) , MCF7 (x), and MDA-MB-453 (•).Values represent mean ± standard deviation (n = 3) 85 oo Table 9 Inhibition (IC50) of AAPH-induced intracellular oxidation by blackberry extracts in different cell lines ~~ ICsodig/ml) Caco-2 INT-407 LNCaP MCF-7 MDA-MB-453 Anthocyanin Enriched Extract 6 . 5 ± 0 . 3C 4 . 1 ± 0 . 4 b 2 . 6 ± 0 . 1 a 6.1 ± 0.3C 2.8 ± 0.2 a Blackberry Crude Extract 5 5 . 1 ± 2 . 4 b 2 6 . 8 * 1 . 5 a 5 6 . 1 ± 4 . 7 b 49.7 ± 8.5 b 2 5 . 8 ± 1 . 0 a 'Different subscript letter indicates significant difference (p < 0.05) in treatment means between cell lines of the same treatment. Values are mean ± standard deviation (n = 3). Protective Effect against AAPH-Induced Cytotoxicity The protective effect o f both blackberry crude extract and anthocyanin-enriched extract against free radical-induced cytotoxicity is summarized in Tables 10-13. A A P H effectively induced (p < 0.05) cytotoxicity in all cell lines tested in a concentration dependent manner as determined using both M T T and CellTiter-Glo assays. Pre-incubating Caco-2 cells with blackberry crude extract produced a concentration-dependent (p < 0.05) protective effect after 24 hour exposure to 15 m M A A P H (Table 10, 11). For the M T T assay, a significant (p < 0.05) protective effect of blackberry crude extract occurred at a minimum concentration of 60 pg/ml. The extent of protection of AAPH-treated cells by the crude extract reached a maximum value of 67.5% viability at 1 mg/ml o f the blackberry crude extract (Table 11). Using identical culture conditions, but assessing viability by CellTiter-Glo, the minimum protective effect of 60 pg/ml anthocyanin was higher at 35.5% viability. Also increasing anthocyanin pre-incubation concentration to 0.5 mg/ml produced maximum (e.g. 41%) viability. O f interest was the finding that highest concentration (1.0 mg/ml) o f blackberry crude extract used to pre-incubate cells resulted in the lowest (p < 0.05) cell viability (36.3% viability); compared to maximum protection obtained at 0.5 mg/ml (Table 10). Maximum protection against 10 m M AAPH-induced free radicals was obtained for INT-407 cells at 0.5 mg/ml of blackberry crude extract (Table 11). A smaller yet significant (p < 0.05) degree of protection (e.g. 2-6.5%) against 5 and 15 m M A A P H also occurred at a minimum concentration of 0.13 mg/ml of the blackberry crude extract. The protective effect pattern of blackberry antioxidants in the L N C a P cell line, as evaluated by the M T T assay, was found to be unique compared to other cell lines. 87 Maximum protection (p< 0.05) against 10 m M A A P H (e.g. 43.2%) occurred at the minimum blackberry crude extract concentration tested, with no further protection observed at higher concentrations, when M T T assay was used to assess cell viability (Table 11). Moreover, pre-incubation of L N C a P cells with blackberry crude extract over a concentration range of 0.03 mg/ml - 1 mg/ml prior to a challenge with 5 m M A A P H resulted in a concentration dependent decrease in viability (p< 0.05). On the contrary, cells pre-treated with identical blackberry crude extract concentrations but exposed to 15 m M A A P H responded with a significant (p < 0.05) but small increase in viability (2%> -7.5%). A significant protection (p < 0.05) of M C F - 7 cells against 15 m M A A P H was afforded by the blackberry crude extract at a minimum concentration o f 30 pg/ml when assessed by the M T T assay (Table 11). Maximum (e.g. 28%) protection was obtained at 1 mg/ml of the blackberry crude extract. Exposure of M C F - 7 cells to lower concentrations of A A P H (e.g. 5 and 10 mM) , however, produced a relatively low cytotoxicity (e.g. 20%); the extent of which was not altered by pre-incubation with blackberry crude extract at the concentration range tested (Table 11). The protective effect of the blackberry crude extract against AAPH-induced cytotoxicity for INT-407, L N C a P and M C F - 7 was alternatively evaluated using the CellTiter-Glo assay. Pre-incubation of cells with blackberry crude extract (0.03 mg/ml -1 mg/ml) was shown to be ineffective at reducing AAPH-induced cytotoxicity in these the three cell lines, as measured using the ATP-based CellTiter-Glo assay (Table 10). For M D A - M B - 4 5 3 cells, the cytotoxic effect of 10 and 15 m M A A P H was significantly (p < 0.05) reduced when cells were exposed to a minimum concentration of 88 0.03 mg/ml of the blackberry crude extracts, and when evaluated by the M T T assay. Increasing the concentration of the blackberry crude extract resulted in a concentration dependent increase in protection, with a maximum protection afforded by 0.13 mg/ml -0.25 mg/ml of the blackberry crude extract. When M D A - M B - 4 5 3 cells were challenged with 10 m M A A P H , pre-treatment with 0.25 mg/ml o f the blackberry crude extract effectively prevented cytotoxicity by increasing cell viability from 74.5% to 98.4% (p < 0.05). Similarly, a maximum 18% increase in viability was obtained in M D A - M B - 4 5 3 cells when pre-incubated with 0.13- 0.25 mg/ml of the blackberry crude extract prior to the 15 m M A A P H challenge (Table 11). In comparison, assessing cell viability using CellTiter-Glo assay showed a significant (p < 0.05) protective effect against 15 m M A A P H at pre-treatment concentration of 0.25 mg/ml blackberry crude extract (Table 10). The protective effect against AAPH-induced cytotoxicity was also evaluated for the anthocyanin-enriched extract using both M T T and CellTiter-Glo viability assays. In general, the anthocyanin-enriched extract was found to have a variable but significant (p<0.05) protective effect against AAPH-induced cytotoxicity in all cell lines tested. Maximum protection against Caco-2 cells cytotoxicity resulting from exposure to 5 and 10 m M of A A P H was obtained with exposure to 6.25 pg/ml anthocyanin-enriched extract, as assessed by the M T T assay (p< 0.05; Table 13). The degree o f protection afforded by 6.25 pg/ml of the anthocyanin-enriched extract was however greater for cells challenged with 10 m M A A P H (23%) than with 5 m M A A P H (12%). Using the CellTiter-Glo assay, protection of cells against 10 m M A A P H was obtained only at 25 pg/ml of the anthocyanin-enriched extract (Table 12). On the other hand, a significant (p<0.05) protection which followed a concentration-dependent, pattern was afforded by 89 the anthocyanin-enriched extract for Caco-2 cells challenged with 15 m M A A P H . Maximum protection in this experiment was 15% when treated with 12.5 pg/ml of the anthocyanin-enriched extract (p < 0.05). For INT-407 cells challenged with 15 m M A A P H , the anthocyanin-enriched extract tested over a range of 0.78 pg/ml - 6.25 pg/ml significantly (p< 0.05) increased viability by 5 % - 8%>, as evaluated using the CellTiterGlo assay (Table 12). A t lower concentrations of A A P H (e.g. 5 and 10 m M A A P H ) used to challenge.cells, no protection was afforded by the anthocyanin rich extract, as assessed by CellTiter-Glo assay. In contrast, the M T T assay revealed a significant protection of the anthocyanin-enriched extract over the full concentration range of 0.78 pg/ml and 6.25 pg/ml of the extract against 10 m M A A P H (p < 0.05, Table 13). For L N C a P cells, the minimal concentration (0.78 pg/ml) of the anthocyanin rich extract gave a significant (p < 0.05) protective effect (e.g. 9.4%) against 15 m M A A P H , as determined by the CellTiter-Glo assay (Table 12). Increasing the concentration of the anthocyanin-enriched extract to 25 pg/ml produced a comparable protective effect against 15 m M A A P H to that obtained at 0.78 pg/ml (e.g. 10.4%). Evaluating the protective effect of blackberry anthocyanins using the M T T assay indicates a greater protection than that showed by the CellTiter-Glo (Table 12,13). For example, using the M T T assay, a significant protection against 5 m M A A P H was obtained over a range of 0.78 pg/ml -3.13 pg/ml of anthocyanin-enriched extract (8% - 18%, p < 0.05). This result was not duplicated when cells were exposed to 5 and 10 m M A A P H when assessed by CellTiter-Glo assay. In general, the extent by which the anthocyanin-enriched extract protected L N C a P cells against 15 m M A A P H was greater when evaluated using M T T assay (18%) 90 than the CellTiter-Glo (9.4%). This pattern of protective effects was similar to that obtained with 0.03 mg/ml - 1 mg/ml of the blackberry crude extract against 10 m M A A P H as assessed with the M T T assay for the L N C a P cells (Table 13). The anthocyanin-enriched extract protected M C F - 7 cells from AAPH-induced cytotoxicity in a manner that was similar to the blackberry crude extract. Pre-incubating cells with anthocyanin-enriched extract over a range o f 1.56 pg/ml - 25 pg/ml, equivalent to 0.03 mg/ml - 1 mg/ml of the crude extract, produced a significant (p < 0.05) concentration-dependent increase in cell viability (Table 12, 13). A maximum protection (e.g. 12%) was afforded by 25 pg/ml o f the anthocyanin-enriched extract or 1 mg/ml of the blackberry crude extract. N o significant protection was afforded by both the blackberry crude extract and the anthocyanin-enriched extract when cells were challenged with low concentrations of A A P H (e.g. 5 and 10 m M A A P H ) . Evaluation of cell viability using the CellTiter-Glo indicated that pre-incubation with anthocyanin-enriched extract over a concentration range of 12.5 pg/ml - 25 pg/ml protected against cytotoxicity when challenged with 5 m M A A P H (Table 12). A similar result was obtained when cells challenged with 10 m M A A P H were pre-treated with 25 pg/ml of the anthocyanin-enriched extract. The best combination of A A P H concentration used for challenging M C F - 7 cells to oxidative stress and protection afforded by blackberry extracts occurred with 15 m M A A P H and a wide concentration range of 0.78 pg/ml - 25 pg/ml of anthocyanin-enriched extract (p < 0.05). The maximum protection occurred at 12.5 pg/ml preincubation concentration. The anthocyanin-enriched extract was also effective at protecting M D A - M B - 4 5 3 cells from peroxyl radical-induced cytotoxicity, when assessed using both the M T T and 91 CellTiter-Glo assays (Table 12 and 13). The minimum concentration of anthocyanin-enriched extract found to protect against 10 m M A A P H (25.7% protection) and 15 m M A A P H (28.3%) was 0.78 pg/ml when assessed using the CellTiter-Glo assay (p < 0.05; Table 12). Pre-incubation with higher concentration of the extract also produced an increased degree of protection against AAPH-induced cytotoxicity, with a maximum protective effect obtained at 12.5 pg/ml for cells exposed to 10 m M A A P H (30%>) and 15 m M A A P H (33%; p < 0.05). A different indication of cell protection from peroxyl radical exposure by blackberry anthocyanins was obtained using the M T T assay (Table 13). For example, pre-exposure of cells to 0.78 ug/ml anthocyanin-enriched extract was only effective at significantly (p< 0.05) protecting cells exposed to 10 m M A A P H . The minimum concentration o f the anthocyanin-enriched extract to exert a significant protective effect against 5 m M and 10 m M A A P H was 1.56 pg/ml and 6.25 pg/ml, respectively. Maximum protection against 5 m M A A P H (15%>) and 10 m M A A P H (25%) was obtained at 12.5 pg/ml of the anthocyanin-enriched extract (p < 0.05). A similar magnitude of protection (21%) was obtained when M D A - M B - 4 5 3 cells were pre-incubated with 25 pg/ml of the anthocyanin-enriched extract to following exposure to 15 m M A A P H (p < 0.05). Amongst the different A A P H concentrations used to challenge cells from peroxyl radical-induced oxidative stress, blackberry extracts were shown to have the greatest protective effect against 15 m M A A P H in various cell lines (Figure 14-18). M T T assay results showed that the blackberry crude extract exerted a variable degree of protection against 15 m M AAPH-induced cytotoxicity in all cell lines tested (Figure 14D-18D). This protection however was only confirmed to occur in Caco-2 cells and M D A - M B - 4 5 3 92 cells when cell viability was assessed by CellTiter-Glo (Figure 14C-18C). Pre-incubation of various cells with anthocyanin-enriched extract at a concentration that was 20 times lower than the blackberry crude extract produced a significant protective effect in three out of five cell lines tested (e.g. L N C a P , M C F - 7 and M D A - M B - 4 5 3 ) , as assessed by the M T T assay (Figure 14B-18B). Using the CellTiter-Glo assay, a minimum of 0.8 pg/ml -1.6 pg/ml of the anthocyanin-enriched extract gave a significant (p < 0.05) protection against 15 m M A A P H in all cell lines tested (Figure 14A-18A) 93 Table 10. Protective effect of blackberry crude extract against AAPH-induced cytotoxicity for various cell lines as evaluated by CellTiter-Glo1 Concentrations of Crude Extract (mg/ml) Cell lines AAPH 0 0.03 0.06 0.13 0.25 0.50 1.00 Caco-2 0 mM 10 mM 15 mM 100 57.62 13.60 91.98* n/d n/d 87.68* 57.47 35.55* 85.14* 56.07 37.41* 84.04* 54.80 39.11* 82.22* 53.75 41.05* 79.05* 48.46 36.33* Int 407 0 mM 10 mM 15 mM 100 84.42 18.18 89.46* 80.54 18.91 89.02* 77.50 19.19 86.32* 77.28 17.58 87.11* 77.24 15.00 84.57* 70.61* 11.73* 77.81* 27.19* 0.58* LNCaP 0 mM 10 mM 15 mM 100 74.46 39.75 88.63* 72.72 41.57 89.90* 63.02* 38.51 88.77* 57.14* 35.26 83.43* 50.47* 36.85 66.56* 15.29* 3.65* 30.53* 0.33* 0.16* MCF-7 0 mM 10 mM 15 mM 100 89.32 81.71 99.21 89.24 79.78 98.60 89.71 81.72 99.49 85.03 79.68 97.95 83.45 76.08 90.33 76.22* 72.19* 92.51* 75.22* 69.96* MDA-MB-453 0 mM 10 mM 15 mM 100 54.40 21.20 101.21 61.30 35.48 101.04 61.02 32.97 96.85 60.23 32.67 96.15 59.46 43.14* 93.04* 34.28* 32.34 88.37* 6.38* 2.43* Note: Cel l viability was assessed upon 3 h pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. 1 Data are expressed as mean values of percent cell viability, n = 3 * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with anthocyanin containing extract and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 94 Table 11. Protective effect of blackberry crude extract against AAPH-induced cytotoxicity for various cell lines as evaluated by MTT assay1 Concentrations of Crude Extract (mg/ml) Cell lines AAPH 0 0.03 0.06 0.13 0.25 0.50 1.00 Caco-2 0 mM 5 mM 10 mM 15 mM 100 93.26 89.93 15.52 87.55 105.82 100.27* 18.99 102.39 110.92 101.76 24.16* 102.42 111.88 98.65 33.92* 106.19 108.04 101.24* 38.28* 109.61* 99.69 95.10 57.19* 103.7 94.29 97.17 67.54* Int 407 0 mM 5 mM 10 mM 15 mM 100 61.67 17.10 7.80 95.98* 58.46 18.15 7.85 98.78 59.01 30.21 8.75* 101.40* 68.11* 36.96* 8.30* 99.77 64.42 42.61* 9.45* 104.01* 61.72 43.31* 10.8* 112.72* 54.11 28.66* 8.75* LNCaP 0 mM 5 mM 10 mM 15 mM 100 119.14 45.77 12.45 100.36 112.3* 88.99* 17.68* 100.57 .109.54* 76.54*" 19.91* 98.38 107.54* 74.16* 19.51* 98.15 90.18* 62.26* 18.16* 90.95 38.31* 20.86* 13.25 51.36* 18.16* 15.46* 14.51* MCF-7 0 mM 5 mM 10 mM 15 mM 100 97.95 80.02 21.60 101.28 90.24* 75.49 26.42* .96.22* 96.02 80.79 35.09* 92.44* 97.95 81.56 37.50* 92.99* 95.35 79.15 31.81* 95.36* 91.49 80.89 32.39* 99.63 93.61 76.45 50.04* MDA-MB-453 0 mM 5 mM 10 mM 15 mM 100 106.13 73.28 42.18 98.42 96.92* 80.49* 50.76* 108.38 104.01 84.10* 60.59* 114.94* 111.23 92.32* 60.84* 117.99* 112.60 98.41* 59.09* 107.28* 72.9* 61.71 34.96 72.18* 43.29* 26.62* 24.01* Note: Ce l l viability was assessed upon 3 h pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3 * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with anthocyanin containing extract and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 95 Table 12. Protective effect of anthocyanin-enriched extract against AAPH-induced cytotoxicity for various cell lines as evaluated by CellTiter-Glo1 Concentrations of Anthocyanin-Enriched Extract (pg/ml) Cell lines AAPH 0 0.78 1.56 3.13 6.25 12.50 25.00 Caco-2 0 mM 5 mM 10 mM 15 mM 100 67.70 47.55 0.98 122.29* 69.30 51.85 5.90 98.11 67.68 53.76 11.11* 97.31 68.46 53.71 12.18* 92.41* 69.00 55.25 15.97* 95.03* 69.89 56.55 16.05* 96.49 68.67 62.09* 14.78* Int 407 0 mM 5 mM 10 mM 15 mM 100 86.68 72.30 22.95 97.87 85.68 73.04 28.05* 89.15* 84.81 72.02 28.66* 89.03* 84.37 71.11 29.78* 89.07* 87.26 73.78 30.67* 91.92* 86.44 72.96 24.55 91.77* 86.62 72.84 22.10 LNCaP 0 mM 5 mM 10 mM 15 mM 100 90.12 74.78 37.40 95.67 86.23 73.83 46.80* 94.51 84.44 66.78 40.44 99.56* 83.49 68.58 41.99 95.75* 85.90 67.96 41.66 96.94* 84.38 67.25 44.86* 95.14 86.85 74.39 47.75* MCF-7 0 mM 5 mM 10 mM 15 mM 100 79.24 85.54 48.25 97.21 81.54 87.88 62.96* 96.49 81.23 83.15 64.20* 96.57 82.66 85.76 63.16* 105.5 83.75 86.10 64.83* 102.7 85.86* 87.25 65.80* 100.4 86.27* 87.92* 60.69* MDA-MB-453 0 mM 5 mM 10 mM 15 mM 100 100.71 56.20 17.28 101.50 101.65 81.91* 45.53* 100.36 101.12 79.06* 48.32* 99.14 103.06 84.95* 49.14* 103.95 102.09 84.05* 49.92* 100.43 104.85 86.06* 50.46* 106.53 101.06 85.94* 46.82* Note: Cel l viability was assessed upon 3 h pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n - 3 * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with anthocyanin containing extract and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 96 Table 13. Protective effect of anthocyanin-enriched extract against AAPH-induced cytotoxicity for various cell lines as evaluated by MTT assay1 Concentrations of Anthocyanin-Enriched Extract (pg/ml) Cell lines AAPH 0 0.78 1.56 3.13 6.25 12.50 25.00 Caco-2 0 mM 5 mM 10 mM 15 mM 100 92.89 80.87 68.81 n/d 93.47 86.10 72.20 97.36* 90.51 90.00 72.67 105.82* 100.41 94.16* 71.02 108.69* 104.34* 103.65* 74.92 109.04* 103.76* 97.34* 73.54 114.32* 100.55 93.73* 67.96 Int 407 0 mM 5 mM 10 mM 15 mM 100 92.63 53.12 13.62 n/d 83.74 66.55* 14.44 100.12 90.73 57.22 12.24 104.35* 85.12 58.60 13.95 103.05 84.40 66.38* 15.60 100.69 85.78 66.38 12.08 97.37* 80.61 62.56 10.62 LNCaP 0 mM 5 mM 10 mM 15 mM 100 80.85 63.93 29.05 n/d 88.01* 60.04 47.35* 106.67* 94.21* 61.95 46.79* 104.73 98.62* 61.19 45.71* 105.98* 85.60 67.12 43.30* 102.89 84.63 62.71 40.81* 108.16 85.08* 53.38 38.99* MCF-7 0 mM 5 mM 10 mM 15 mM 100 87.24 74.99 18.13 n/d 81.84 70.63 22.25 100.69 84.08 70.38 24.80* 101.11 81.05 71.17 26.00* 101.68 86.51 73.48 25.95* 100.77 85.48 70.63 27.10* 102.03 84.39 74.02 30.25* MDA-MB-453 0 mM 5 mM 10 mM 15 mM 100 86.63 71.13 48.75 n/d 88.90 79.31* 54.47 106.69* 92.10* 81.77* 54.96 104.95* 99.05* 82.39 52.20 102.91 99.7* 90.25* 55.58* 114.78* 101.9* 95.48* 67.32* 112.44* 95.5* 91.11* 69.66* Note: Cel l viability was assessed upon 3 h pre-incubation of various cells to different concentrations o f anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3 * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with anthocyanin containing extract and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 97 25 8 20 £ 15 o 3? t : 10 .2 5 > 90 | 70 5 6 0 o 50 g. 40 I" 30 £ 20 5 10 0 0.0 0.8 1.6 3.1 6.3 Concentration (Ma/ml) 12.5 25.0 £ 40 £ 30 = 15 n .2 10 0.00 D 0.00 0.06 0.13 0.25 Concentration (mg/ml) 0.50 1.00 • I 0.03 0.06 0.13 0.25 0.50 1.00 Concentration (mg/ml) oo Concentration (ng/ml) Figure 14. Protective effect of anthocyanin enriched extract (A, C) and blackberry crude extract (B, D) against AAPH-induced cytotoxicity for Caco-2 cells as evaluated using CellTiter-Glo assay (A,B) and M T T assay (C,D). Ce l l viability was assessed upon 3 h pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3. * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with blackberry extracts and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 0.0 0.8 1.6 3.1 6.3 12.5 25.0 0.00 0.03 0.06 0.13 0.25 0.50 1.00 Concentration (vg/ml) Concentration (mg/ml) Figure 15. Protective effect of anthocyanin enriched extract (A, C) and blackberry crude extract (B, D) against AAPH- induced cytotoxicity for INT-407 cells as evaluated using CellTiter-Glo assay (A,B) and M T T assay (C,D). Cel l viability was assessed upon 3 h pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3. * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with blackberry extracts and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 0.0 0.8 1.6 3.1 6.3 12.5 25.0 0.00 0.03 0.06 0.13 0.25 0.50 1.00 Concentration {M g/ml) Concentration (mg/ml) Figure 16. Protective effect of anthocyanin enriched extract (A, C) and blackberry crude extract (B, D) against AAPH-induced cytotoxicity for L N C a P cells as evaluated using CellTiter-Glo assay (A ,B) and M T T assay (C,D). C e l l viability was assessed upon 3 pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3. * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with blackberry extracts and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 80 -=. 70 | 60 o 0 50 •s ^ 40 if 3 0 1 20 > 10 35 =• 30 S 25 •5 20 4 15 10 > 5 0.0 0.8 1.6 3.1 6.3 Concentration (Mg/ml) 12.5 25.0 0.00 0.03 0.06 0.13 0.25 . 0.50 1.00 Concentration (mg/ml) 0.0 0.8 1.6 3.1 6.3 12.5 25.0 Concentration (Mg/ml) 0.00 0.03 0.06 0.13 0.25 Concentration (mg/ml) 0.50 1.00 Figure 17. Protective effect of anthocyanin enriched extract (A, C) and blackberry crude extract (B, D) against AAPH- induced cytotoxicity for M C F - 7 cells as evaluated using CellTiter-Glo assay (A,B) and M T T assay (C,D). Cel l viability was assessed upon 3 pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3. * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with blackberry extracts and cells treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). 0.0 0.8 1.6 3.1 6.3 12.5 25.0 0.00 0.03 0.06 0.13 0.25 0.50 1.00 Concentration (ug/lml) Concentration (mg/ml) Figure 18. Protective effect of anthocyanin enriched extract (A, C) and blackberry crude extract (B, D) against AAPH-induced cytotoxicity for M D A - M B - 4 5 3 cells as evaluated using CellTiter-Glo assay (A,B) and M T T assay (C,D). Ce l l viability was assessed upon 3 h pre-incubation of various cells to different concentrations of anthocyanin containing extract followed by 24 h exposure to A A P H peroxyl radical generator. Data are expressed as mean values of percent cell viability, n = 3. * indicates significant difference (p < 0.05) between percent viability value of cells pre-treated with blackberry extracts and cells ^ treated with A A P H only (i.e. 0 mg/ml of the anthocyanin containing extract). o to DISCUSSION The blackberry crude and anthocyanin-enriched extracts both suppressed generation of intracellular peroxyl free radicals-induced by A A P H and in all cell lines tested. The affinity of blackberry anthocyanins to suppress intracellular free radicals induced by A A P H was based on the cellular uptake of a non-fluorescent probe ( D C F H -D A ) and subsequent hydrolysis by intracellular esterase. In this particular antioxidant assay, peroxyl radicals generated from A A P H oxidize the non-fluorescent substrate (dichlorofluorescin, D C F H ) to a fluorescent product (dicholorofluorescein, D C F ) . The presence of antioxidants within the cell act to quench the free radical, which in turn reduces fluorescence intensity, indicating modulation of intracellular oxidation. A concentration dependent suppression of intracellular oxidation over 24 hour period by blackberry extracts can be attributed in part to the antioxidant activity of blackberry anthocyanins which quenched A A P H generated free radicals during the initial stage of intracellular oxidation. This was clearly demonstrated by the time-dependent delay in free radical generation up to seven hours, which occurred concomitantly with an increase in blackberry extracts concentrations. While both blackberry crude and anthocyanin-enriched extracts effectively suppressed intracellular oxidation, there was greater potency from the anthocyanin-enriched extract to inhibit free radical generation. This result can be attributed to the approximately 20 times greater amount of anthocyanin that was present in the anthocyanin-enriched extract. The antioxidant capacity of this anthocyanin-enriched extract was shown earlier in Experiment I to be greater than the crude extract using O R A C assay. Thus, antioxidant activity of anthocyanin in the concentrated extract 103 accounts for significant peroxyl radical scavenging activity. Moreover, the greater antioxidant activity of the anthocyanin-enriched extract reported in Experiment I using the O R A C test was confirmed in this experiment with the cell-based intracellular assay. This result extents the characterization of anthocyanin antioxidant potential by strongly suggesting that they are involved in reducing intracellular oxidation. Calculated IC50 values for each cell line pre-incubated with blackberry extract prior to the challenge with peroxyl radical induction of intracellular oxidation, provided a quantitative assessment of the protection afforded by the antioxidant properties of anthocyanins in cells exposed to oxidative stress. The IC50 values from experiments conducted with blackberry crude or anthocyanin-enriched extracts were cell line specific in magnitude. The relative differences between anthocyanin-induced protections against intracellular oxidation may reflect the degree to which the blackberry crude or the anthocyanin-enriched extracts were taken up by individual cells to neutralize oxidative stress. For example, the fact that blackberry anthocyanins suppressed intracellular oxidation indicates that blackberry anthocyanin, in particular cyanidin-3-glucoside, was incorporated into cells to an extent that was required to reduce the presence of AAPH-ini t ia ted free radicals. This interpretation of finding is supported by Youdim et al., (2000a), who reported that elderberry anthocyanins, which are made up of cyanidin derived anthocyanins, were incorporated in the cytoplasm as well as the cell membrane of cultured endothelial cells. Tarozzi et al., (2005) also recently demonstrated that cyanidin-3-glucoside treatment to human keratinocyte increased the antioxidant activity of the membrane rich fraction (55%) and the cytosol (19%). Youdim et al., (2000a) also showed that exposure of 104 endothelial cells to elderberry extracts resulted in an increased resistance against H2O2 and AAPH-induced oxidative stress. A similar protective effect of anthocyanins against H2O2 induced-oxidative stress has been reported by H u et al., (2005) using Saskatoon berry and black rice in cultured RAW264.7 cells (Hu et al., 2003). Blueberry anthocyanins have also been reported to increase the resistance of cultured red blood cells to H2O2 when recovered from rats fed on a blueberry diet (Youdim et al., 2000b). The protective effect of blackberry anthocyanins in the present study was also evaluated for protection against AAPH-induced cytotoxicity. Upon exposure to heat, A A P H dissociates and interacts with oxygen to form peroxyl radicals, which in turn initiate l ipid peroxidation through a cascade of reactions (Nik i , 1990; Yoshida et al., 2004). L ip id peroxidation is a major factor for inducing oxidative stress that results in cellular damage (Park et al., 2002; Zhang et al., 1997). Products of membrane l ipid peroxidation include damaged membrane phospholipids, proteins and cellular D N A (Park et al., 2002; Paulet et al., 1994). Excessive exposure to free radicals, i f not counter-balanced by endogenous defenses (i.e. cellular antioxidant enzyme and low molecular weight non-enzymatic antioxidants, and cellular repair) w i l l result in the accumulation of oxidative damage that ultimately leads to cellular dysfunction and cell death (Poli et al., 2004). Park et al., (2002) demonstrated the diminishing viability of cultured U937 cells, that accompanied D N A damage which was monitored by increases in 8-OH-dG adduct and the inability of D N A to re-nature when exposed to A A P H . Other studies have reported that A A P H cleaves single stranded D N A and that lipid peroxidation products, 105 namely malondialdehyde and unsaturated aldehydes, form adducts with D N A that result in mutagenecity (Park et al., 2002; Plumb et al., 1997; Russo et al., 2005). The blackberry crude and anthocyanin-enriched extracts used in the present study effectively reduced AAPH-induced cytotoxicity. The degree of protection obtained, however, was found to be dependent on several factors, such as the concentration of A A P H used to induce cytotoxicity, the concentration of blackberry anthocyanin used to pre-incubate different cells, the cell type, and the cell viability assay used to measure cytotoxicity. Exposure of five distinct cell lines to A A P H resulted in characteristic concentration-dependent decreases in cell viability as measured using both the M T T and CellTiter-Glo assays. Although both assay methods are based on changes in cell metabolism following exposure to A A P H , the absolute cell viability values for each cell line was not equivalent and varied depending on the cell line tested. Protection against AAPH-induced cytotoxicity by blackberry crude and anthocyanin-enriched extracts was also obtained over a defined concentration range and was shown to be dependent on the A A P H exposure level and the method used to measure cell viability. For example, although exposing different cells to only 5 m M A A P H in turn resulted in a 38% reduction in cell viability, the level of protection afforded to cells by pre-incubating with blackberry extracts was relatively small and variable. Increasing the A A P H exposure of cells to 10 and 15 m M A A P H produced a markedly higher decline in cell viability (e.g. up to 99 %) which enabled a higher degree of protection by blackberry extracts to be shown. Thus optimum test conditions were required to show a potential protective effect of blackberry extract and involved not only the amount of A A P H exposed to cells, but 106 also the optimal concentration of blackberry extract used to pre-incubate cells. This combination of prooxidant-antioxidant balance varied for each cell line tested. Taken together, these findings indicate that supplementation of cells with blackberry anthocyanins provided a beneficial effect against AAPH-induced oxidative stress when exposure to free radicals was generated at a relatively high level. In contrast, exposure to lower levels of peroxyl radical-induced oxidative stress was insufficient to show protection in all cell lines by the blackberry anthocyanins. The reduced cell viability observed at high concentrations of blackberry crude extract may be explained in two ways. First, high concentrations of blackberry extract were previously found to directly result in cytotoxicity in Experiment II. Secondly, constituents of blackberry crude extract may possess pro-oxidant activity which was exacerbated in the presence of A A P H at the given concentration for specific cell lines. The anthocyanin-enriched extract of blackberry was more effective than the crude extract at inhibiting AAPH-mediated cytotoxicity. In fact, more cell lines were protected by pre-incubation with anthocyanin-enriched extract than that crude extract when challenged with A A P H . While blackberry crude extracts exhibited a certain degree of protection in all cell lines when examined using the M T T assay, the protective effect of the crude extract was confirmed for only 2 cell lines by the CellTiter-Glo assays. The protective effect of anthocyanin-enriched extract however, was confirmed by both the M T T and CellTiter-Glo assays to occur to a certain extent in all cell lines, with exception to INT-407. It should be noted the concentration of anthocyanin rich extract required to provide protection against AAPH-induced cytotoxicity was relatively lower (e.g. 0.8 pg/ml 0 25 pg/ml) than the crude extract (e.g. 31 pg/ml - 0.25 mg/ml). The anthocyanin-107 enriched extract was determined in Experiment II to contain a 20 times higher concentration of anthocyanins, and had a greater corresponding O R A C antioxidant capacity than the crude extract. The protective effect of blackberry extracts against peroxyl radical-induced cytotoxicity was more evident when evaluated using the M T T assay rather than the CellTiter-Glo assay. Both blackberry crude and anthocyanin-enriched extracts reduced AAPH-induced cytotoxicity to a similar extent in all cell lines when measured by M T T assay. On the other hand, the CellTiter-Glo indicated that only Caco-2 and M D A - M B -453 cell lines were protected by the crude blackberry extract and all but the INT-407 were protected by the anthocyanin-enriched extract. This discrepancy can be explained in part by the potential of the M T T assay to overestimate cell viability, which was attributed to interference with redox reactions shown in Experiment II. Blackberry anthocyanins were effective at protecting against free radical-induced cytotoxicity at different levels between cell lines. This cell-specific difference may be explained on the basis that crude extracts, or the anthocyanin-enriched extract, were taken up by individual cells at different rates. Taking the results of M T T and CellTiter-Glo together, it was evident that the anthocyanin-enriched extract protected Caco-2 and M D A - M B - 4 5 3 to a greater extent than the found in other cell lines. Zhang et al., (1997) reported that AAPH-ini t ia ted oxidative damage occurs at the membrane level in cultured red blood cells. Protection against free radicals generated by A A P H may therefore occur at the membrane level. The fact that cyanidin-3-glcuoside was found to increase the antioxidant activity of the membrane rich fraction to a greater extent than the cytosol of human keratinocytes cell line, indicates that the cyanidin-3-glucoside from blackberry 108 was most likely incorporated in the membrane, thus affording protection against A A P H -induced cytotoxicity (Tarozzi et al., 2005). Other studies have supported the protective effect of anthocyanins against free radical-induced cytotoxicity (Heo and Lee, 2005). In this study, a strawberry extract exhibited a concentration-dependent prevention of H 2 0 2 - i n d u c e d cytotoxicity in P C 12 neuronal cells. Cyanidin-3-glucoside in particular was shown to be effective at counteracting damage from free radical generation that consisted of reducing free radical-induced D N A damage in cultured human fibroblast cells (Russo et al., 2005). In addition to the aforementioned in vitro studies, very few animal studies have provided in vivo evidence that establishes the link between the anthocyanin consumption and the prevention of free radical associated disease. Boysenberry anthocyanins, for example, were shown to increase plasma antioxidant status while decreasing some biomarkers of oxidative damage in rats (McGhie et al., 2005). Moreover, Ramirez et al., (2005) and Cho et al. (2003), reported memory enhancing effects of blueberry and sweet potato anthocyanins, which may be associated with its antioxidant property. Blueberry anthocyanins were also found to decrease the vulnerability of rats to the enhanced oxidative stress involved in aging (Ramirez et al., 2005). The minimum concentration found to exert a protective effect in many of the cell lines in the present study was 30 pg/ml for the crude extract, and 0.78 pg/ml for the anthocyanin-enriched extract. The cyanidin-3-glucoside content of these extracts was equivalent to 1.08 pg/ml and 0.59 pg/ml for the crude and anthocyanin-enriched extract, respectively. These concentrations are well within the range of circulating anthocyanins levels reported in plasma of rats fed on an anthocyanins containing diet. The range o f 109 2.25 ng/ml to 1.53 pg/ml is also close to the range of anthocyanins measured in human plasma (e.g. 2.25 ng/ml to 0.44 pg/ml)(Ichiyanagi et al., 2005; Kay et al., 2004). C O N C L U S I O N In this study, the antioxidant capacity of blackberry anthocyanins was demonstrated to be effective at suppressing AAPH-ini t ia ted intracellular oxidation. The blackberry anthocyanins were also shown to protect various cell lines against free radical-induced cytotoxicity. The protective effect of blackberry anthocyanins against peroxyl radical-induced cytotoxicity was, however dependent on the cell lines tested, the severity of the oxidative challenge, and the concentrations of the blackberry extracts used to pre-treat the cells. Moreover, the end-point measures specific to different cell viability assays gave a measure of variability for this protective effect. The anthocyanin-enriched extract was demonstrated consistently to be more effective than the crude extract at suppressing the intracellular oxidation as well as free radical-induced cytotoxicity. The protective effect of both blackberry crude and anthocyanin-enriched extract occurred at concentrations that are relevant to a typical intake of anthocyanin rich fruits in human. 110 EXPERIMENT 4: A PROPOSED PROTECTIVE MECHANISM OF BLACKBERRY ANTHOCYANINS AGAINST FREE RADICAL-INDUCED CYTOTOXICITY INTRODUCTION There is overwhelming evidence that reactive oxygen species (ROS), due to its highly unstable and thus reactive nature, are involved in the pathogenesis of various diseases, such as heart disease, diabetes, cancer (Gey, 1993). The consumption of antioxidants, however, is considered to neutralize the harmful effects of R O S , and therefore reduce the risk of developing the free-radical associated diseases (Wang et al., 1997). Anthocyanins are the red to blue pigments found in various berries and are of particular interest because they are potent antioxidants (Kahkonen and Heinonen, 2003). Increasing dietary intake of anthocyanin rich sources may thus provide health benefits by affording protection against free radical associated damage of cell lipids, proteins or nucleic acids. Blackberry is a rich source of anthocyanins and has strong in vitro antioxidant capacity as shown in Experiment 2 and 3 and by others (Jiao and Wang, 2000; Pellegrini et al., 2003; 2004). There is a lack of evidence that specifically relates the antioxidant capacity to the prevention of free radical-associated disease. In the previous chapter, blackberry anthocyanins were shown to be effective at suppressing free radical-initiated intracellular oxidation and free radical-induced cytotoxicity. The antioxidant capacity of blackberry anthocyanins was demonstrated to exert a protective effect against free radical-induced damage in different cell lines. A greater understanding of the free radical-induced cytotoxicity such as apoptosis and necrosis cell death and the protective mechanism of blackberry anthocyanins against these ROS-induced events is lacking. I l l Since the intestine is the first line of defense for many food based constituents to either induce free radical reactions or alternatively prevent free radical formation, the purpose of this study was to determine a cellular mechanism in intestinal cells whereby blackberry anthocyanins could potentially reduce AAPH-induced cytotoxicity. MATERIALS AND METHODS Materials Blackberries were supplied from Sandhu Farm, Abbotsford, B . C . Biogel P2 was obtained from Bio-Rad Laboratories (Richmond, Ca). Acetic acid (CH3CO2H) was from Fisher Scientific (Nepean, ON) and 2, 2'-azobis (2-amidinopropane) dihydrochloride ( A A P H ) were from Wako Chemicals U S A (Richmond, V A ) . Propidium iodide, RNase A , and was from Sigma-Aldrich Canada Ltd. (Oakville, ON) . Extraction and Enrichment of Blackberry Anthocyanins Blackberry anthocyanins were extracted and concentrated using the methods described in Experiment 1. The major blackberry anthocyanin, cyanidin-3-glucoside was identified and quantified using H P L C as described in Experiment 1. Cell Culture Caco-2 cells were cultured and maintained as described in the methods section in Experiment 2. 112 C e l l Cycle Analysis The effect of A A P H and anthocyanin-enriched extract on Caco-2 cell cycle progression was analyzed based on the method described by Popovich and Kitts (2004). Caco-2 cells were seeded at a density of 1.56 x 10 5 cells/well in 24- well plates and incubated at 37°C in a 5% CO2 humidified incubator overnight to allow for attachment. The anthocyanin-enriched extract from blackberry was added to cells at a final concentration of 100 pg/ml and cells were returned to the incubator for three hours at 37° C. After pre-incubation of cells with anthocyanin, A A P H (20 m M ) was added to induce cytotoxicity. The mixture was further co-incubated with the cells for 24 hours. Controls consisted of untreated cells and culture medium only. The effect of 20 m M A A P H and 100 pg/ml anthocyanin-enriched extract on the cell cycle progression of Caco-2 cells were also tested individually using the same experimental design. The concentration of anthocyanin-enriched extract and A A P H used in this study was based on a preliminary experiments which demonstrate that pre-incubation of 100 pg/ml of anthocyanin-enriched extract prior to exposure of cells to 20 m M A A P H , produced the clearest indication of a protective mechanism for blackberry anthocyanin against AAPH-induced cytotoxicity. Caco2 cells were recovered after 24 hour incubation by adding of 200 pi trypsin (0.25%) and E D T A (0.03%>) followed by manual agitation to disperse cells. Cells were removed and combined with floating cells, and the suspension was centrifuged for 10 minute a 400 x g. The cell supernatant was discarded, and the pellet was washed twice with P B S by re-centrifugation at 400 x g. Cel l pellets were fixed by the addition of 1 ml of ice-cold 70%> ethanol and subsequently stored at 4°C overnight. Ethanol was removed by centrifugation (2000 x g, 5 minute), followed by the addition of 300 pi of propidium 113 iodide staining solution (50 pg/ml propidium iodide and 100 pg/ml R N A s e A in PBS) . Stained cells were incubated for 1 hour at room temperature before analysis with FACscan flow cytometry (Becton-Dickinson, Mountain View, Ca l i f ) . F low cytometry data were analyzed with W i n M D I software (La Jolla, Ca l i f ) . R E S U L T Treatment of cells with 20 m M A A P H was effective at perturbing the cell cycle progression of Caco-2 cells (Figure 19). More specifically, A A P H treatment resulted in a reduced proportion of cells in the G l phase (26%), and an increased proportion of cells in the G 2 / M phase (18%), as well as in the sub-Gl region (6%) (Figure 20). Exposing cells to 20 m M A A P H effectively arrested Caco2 cells at the G 2 / M stage, thus reducing the amount of cells found in G l phase. The increased proportion of cells found in sub G l indicates the presence of cells with hypo-diploidic D N A , which is commonly associated with apoptotic cells. Therefore, AAPH-induced cytotoxicity was most likely mediated by induction of apoptosis. One hundred pg/ml of anthocyanin-enriched extract was found to have no influence on Caco-2 cell cycle pattern (Fig 19.A, 19C). Moreover, a comparable proportion of cells were found in each cell cycle phase of both anthocyanin treated and control Caco-2 cells (Figure 20). Exposure of Caco-2 cells to 100 pg/ml of anthocyanin-enriched extract prior to exposure to 20 m M A A P H ; however, reduced the cytotoxic effect of A A P H . This followed a comparable cell cycle pattern of treated cells compared to the control (Fig 19.D). Specifically, the anthocyanin-enriched extract decreased the 114 proportion of cells in sub G l phase as well as normalizing the proportion of cells in other cell cycle phases that resembled the control (Figure 20). DISCUSSION The current finding is in agreement with other reports using mature human monocyte derived macrophages, which demonstrated AAPH-mediated cytotoxicity occurrence via an apoptosis pathway (Asmis and Wintergerst, 1998). In another study, Zimowska et al., (1997) reported that AAPH-induced apoptosis in leukaemic cells (LI 210) which was accompanied by the peroxidation of cellular lipids. The occurrence of apoptosis was further confirmed by Ishisaka et al., (2001) who demonstrated that apoptosis was mediated by the activation of caspase 3 through lysosomal cystein protease in the HL-60 cell line. The anthocyanin-enriched extract prevented the growth arrest at G 2 / M phase as well as the formation of apoptotic cells. Knowing the affinity of anthocyanin to scavenge A A P H generated peroxyl free radical suggests that this mechanism also prevented AAPH-induced apoptosis of Caco-2 cells. The protective effect of blackberry anthocyanins was attributed to cyanidin-3-glucoside, the predominate anthocyanin found in blackberry. These finding supports results from Tarozzi et al., (2005) who also showed that cyanidin-3-glucoside prevented U V - A induced apoptosis in human keratinocyte via neutralization of the H2O2 released upon U V - A radiation. It is important to note that the anthocyanin-enriched extract alone had no direct effect on the cell cycle progression of Caco-2 at the concentration tested in this study. The protective effect of the anthocyanin-enriched extract therefore occurred only as a 115 result of the antioxidant capacity of anthocyanins to mitigate the effect of AAPH-induced apoptosis on cell cycle progression. r While exposure to blackberry anthocyanins alone was found to have no effect on the cell cycle progression of Caco-2, various other sources of anthocyanins have been reported to elicit chemopreventive or anti-cancer effects on various cell lines. For example, bilberry and Hibiscus anthocyanins were shown in separate studies to induce apoptosis in H L 60 leukemia cell lines(Chang et al., 2005; Katsube et al., 2003) . In addition to apoptosis-mediated cytotoxicity, exposure o f anthocyanins from Oryza sativa cv. Heugjinjibyeo to human monocytic leukemia cell (U937) was accompanied by an arrest in cell cycle at the G 2 / M phase (Hyun and Chung, 2004). Differences in cell responses to various anthocyanin containing extracts could be due to the unique anthocyanin profile o f each anthocyanin source. Moreover, individual anthocyanin structure has distinctive effects on cell survival and proliferation (Katsube et al., 2003; Lazze et al., 2004; Zhang et al., 2005). For example, in HL-29 cancer cells, the rank by which anthocyanidin induced cytotoxicity was: delphinidin ~ malvidin > cyanidin> peonidin > pelargonidin (Cooke et al., 2005). In addition, aglycones were reported to be less cytotoxic than associated glycosides (Zhang et al., 2005). Blackberry is unique from the stand point that the predominating anthocyanin is cyanidin-3-glucoside (Pericles, 1982). In Experiment 1, cyanidin-3-glucoside comprised 90 % of the total anthocyanin content. Other anthocyanin containing fruit or vegetable extracts may contain more than one anthocyanin, each of which potentially possesses different anti-proliferation or apoptosis inducing effects. The main anthocyanin in Hibiscus for example, is delphinidin, whereas the main anthocyanins of Oryza sativa cv. 116 Heugjinjibyeo, is cyanidin and malvidin (Chang et al., 2005; Hyun and Chung, 2004). Thus it is possible that cyanidin-3-glucoside is less cytotoxic than the aglycone, thus having no effect on altering cell cycle progression of Caco-2 upon treatment with blackberry extracts. C O N C L U S I O N In conclusion, while AAPH-generated free radicals induced apoptosis-mediated cytotoxicity, pre-treatment of Caco-2 cells with an anthocyanin-enriched extract containing 90% cyanidin-3-glucoside prior to A A P H exposure reduced cytotoxicity. This protective effect from blackberry anthocyanin in general and cyanidin-3-glucoside in particular, was attributed to the antioxidant activity of its constituents, which effectively scavenged AAPH-generated peroxyl radicals. 117 Figure 19. Histogram of cell cycle distribution of treated Caco-2 cells. A = control, B = AAPH (20 mM), C = Anthocyanins-Enriched Extract (100 fig/ml), D = Anthocyanins-Enriched Extract (100 ug/ml) followed by 20 mM AAPH. 118 Control AEE AAPH AAPH-AEE Figure 2 0 . A representative percentage of Caco-2 cells in each cell cycle phase for individual treatments: control, anthocyanins-enriched extract (AEE, 1 0 0 pg/ml), or AAPH ( 2 0 mM), or AAPH ( 2 0 mM) followed by anthocyanins-enriched extract ( 1 0 0 pg/ml). Bars of different colors represent cells in SubGl (I), S (I), G 2 / M (S) and Gl (•) phases. 119 OVERALL CONCLUSION AND FUTURE STUDIES This study demonstrates that blackberry is a rich source of anthocyanins and in particular, cyanidin-3-glucoside. Blackberry also exhibits considerable antioxidant activity, which was evaluated in this study using both cell free and intracellular methods. Gel filtration of the blackberry crude extract produced an anthocyanin-enriched extract which was characterized as containing 20 times greater concentration of total anthocyanins. The antioxidant activity of the anthocyanin-enriched extract was equally greater than the blackberry crude extract. Characterization of blackberry anthocyanins using H P L C demonstrated that cyanidin-3-glucoside was the main anthocyanin (>88%) in blackberry fruit. Amongst four distinct cytotoxicity assays examined (e.g. cell counting, M T T assay, B r d U assay and CellTiter-Glo assay), CellTiter-Glo was determined to be the best alternative assay to the commonly used M T T assay due to higher precision and greater accuracy in estimating cell viability. The M T T assay and the CellTiter-Glo assay gave similar conclusions that both the blackberry crude extract and the anthocyanin-enriched extracts were not cytotoxic to any of the cell lines tested at typical human daily intake levels. The blackberry extract and anthocyanin-enriched extract exhibited a protective effect against free radical-initiated intracellular oxidation and free radical-induced cytotoxicity. The suppression of AAPH-induced intracellular oxidation and A A P H mediated cytotoxicity occurred in all different cell lines but was dependent on the concentration of anthocyanin used to treat the cells. The protective effect of blackberry extracts also varied in magnitude for various cell types and was dependent on the severity 120 of oxidative damage imposed to the cells and the assay used to assess final cell viability. The anthocyanin-enriched extract, in particular, was shown to be more effective than the blackberry crude extract at suppressing intracellular oxidation and free radical-induced cytotoxicity. Using Caco-2 intestinal cells as the model test system, the anthocyanin-enriched extract effectively prevented AAPH-induced apoptosis. This protective effect was attributed to the high antioxidant activity of the anthocyanins contained in the extract and in particular cyanidin-3-glucoside affinity to quench A A P H generated free radicals. This study therefore elucidates a potential mechanism by which the antioxidant activity of blackberry anthocyanins may protect against free radical-induced cell injury. Future studies wi l l focus on the mechanism by which anthocyanins prevent free radical mediated apoptosis, possibly through modulation of specific cell signaling pathways involved in cell death. 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