The Protective Effect of Blackberry Anthocyanins against Free Radical-Induced. Oxidation and Cytotoxicity in Multiple Cell Lines

Transcription

1 The Protective Effect of Blackberry Anthocyanins against Free Radical-Induced Oxidation and Cytotoxicity in Multiple Cell Lines By INGRID ELISIA B.Sc. The University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The FACULTY OF GRADUATE STUDIES (Food Science) THE UNIVERSITY OF BRITISH COLUMBIA November 2005 Ingrid Elisia, 2005

2 ABSTRACT 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 HPLC. Different cytotoxicity assays (MTT, 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 (MDA-MB-453 and MCF-7), two intestinal cell lines (Caco-2 and INT-407), and one prostate cancer cell line (LNCaP), using MTT and CellTiter-Glo assay. In other tests, AAPH (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 AAPH initiated intracellular oxidation was monitored with a dichlorofluorescin diacetate (DCFH-DA) probe. The protective effect of blackberry anthocyanins against AAPH-induced cytotoxicity was measured with MTT and CellTiterGlo assays. Cell cycle analysis was

3 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-initiated intracellular oxidation. This effect corresponded a protection effect against free radical-induced cytotoxicity. Cell cycle analysis with propidium iodide staining demonstrated that blackberry anthocyanins prevented cytotoxicity by scavenging the generated peroxyl radicals, thus alleviating AAPH-induced apoptosis.

4 TABLE OF CONTENTS ABSTRACT, ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ACKNOWLEDGEMENT iv vi vii vii ix INTRODUCTION 1 LITERATURE REVIEW 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 Cell Proliferation and Death 27 Cell Lines 28 iv

5 Cytotoxicity Assays 28 A. MTT assay 28 B. BrdU assay 29 C. CellTiter-Glo assay 29 RESEARCH HYPOTHESIS AND OBJECTIVES 30 EXPERIMENT 1: Characterization, identification, quantification of and the antioxidant capacity of blackberry anthocyanins 33 EXPERIMENT 2: Comparison of four cytotoxic assays to assess the cytotoxicity of blackberry anthocyanins in multiple cell lines 46 EXPERIMENT 3: The protective effect of blackberry anthocyanins against free radical-initiated intracellular oxidation and free radical-induced cytotoxicity 72 EXPERIMENT 4: A proposed protective mechanism of blackberry anthocyanins against free radical-induced cytotoxicity Ill OVERALL CONCLUSION AND FUTURE STUDIES 120 REFERENCES 122 APPENDIX A 134 v

6 LIST OF TABLES 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 of blackberry extracts 41 Table 6: LC50 of 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 of 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 MTT 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 MTT assay 97 vi

7 LIST OF FIGURES Figure 1: The flavylium cation 4 Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: Figure 7: Figure 8-12: Figure 13: Structural transformation of anthocyanins in aqueous acidic solution at room temperature 6 HPLC profile blackberry anthocyanins and cyanidin-3-glucoside standards 42 Interference of blackberry crude extracts and a cell free CellTiter- Glo assay 58 Concentration response curve of blackberry crude extract incubated for 24 hours evaluated with four different cytotoxicity assays 59 LC50 of blackberry crude extracts on five distinct cell lines evaluated using MTT and CellTiter-Glo assay 64 The effect of anthocyanins rich extract in multiple cell lines as evaluated with MTT and CellTiter-Glo assays 65 Suppression of intracellular oxidation for five distinct cell lines by blackberry anthocyanins 79 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: Figure 20: Histogram of cell cycle distribution of Caco-2 cells upon treatment with AAPH and/or an anthocyanins rich extract 118 Percentage of cells in each cell cycle phase in Caco-2 cells upon treatment with AAPH and/or anthocyanins rich extract 119

8 LIST OF ABBREVIATIONS AAPH ABTS* p-pe Caco-2 DCFH-DA DPPH inos INT-407 LDL LNCaP MCF-7 MDA-MB-453 MTT 'o 2 OH' (h, ORAC PI 2, 2' -azobis (2-amidinopropane) dihydrochloride 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) P-phycoerythrin Human intestinal adenocarcinoma cancer cell line dichlorofluorescin diacetate 1, l-diphenyl-2-picrylhydrazyl inducible nitric oxide Human embryonic intestinal cell line Low density lipoprotein Hormone sensitive prostate cancer cell line Hormone sensitive breast cancer cell line Hormone insensitive breast cancer cell line 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide singlet oxygen hydroxyl radicals superoxide radicals Oxygen radical absorbance capacity Propidium iodide R- alkyl radical R0 2 ' ROS TBARS peroxyl radicals Reactive oxygen species thiobarbituric acid-reactive substances

9 ACKNOWLEDGEMENT I would like to acknowledge first and foremost, my parents, Samuel Elisia and Utami Soebiantoro, for their full support, sacrifices and their ceaseless prayers. My 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. My special thanks for Dr. Susan Cheung for her listening ears, prayers, technical counsels and encouraging words. I am grateful to Dr. Charles Hu 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. ix

10 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). ROS, such as superoxide radicals (O2'"), hydroxyl radicals (OH"), hydrogen peroxide (H2O2) and peroxyl (R0 2 ') radicals are constantly formed as byproducts of normal metabolism and are removed by cellular antioxidant defenses system (Halliwell 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; Poli 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 (Doll, 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

11 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

12 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

13 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 Miniati, 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). R4 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), will govern which anthocyanin is produced. All these variables increase the number of individual compounds that belong to the anthocyanin family (Galvano et al., 2004). It has been 4

14 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 ' 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 ph 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 ph, 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 ph to the colorless hemiketals at intermediate ph (e.g. 4.5) has been used as a tool for measuring total anthocyanin content. The ph differential method determines total anthocyanin content based on colour loss with increasing ph. 5

15 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. + H + OR" Flavylium Cation (AH+) Reddish at ph = 1 OR" Quinonoidal Base (A) Bluish at ph = 7 OH O OH R 2 OR" Carbinol Base (B) Colorless at ph = 4.5 OR" OR' Chalcone (C) Colorless at ph = 4.5 Figure 2. Structural Transformation of Anthocyanins in Aqueous Acidic Solution at Room Temperature. R 1 and R 2 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 ph, elevated temperature and hydration 6

16 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). ROS 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. ROS, such as those listed above are continuously generated as byproducts of cell metabolism. The high reactivity of ROS with nearby cellular components results in oxidative damage to DNA, 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 ROS produced (Yuan and Kitts, 1996). 7

17 A moderate concentration of ROS is however also necessary for life, despite its potential damaging effect, since ROS 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. UV 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 will 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 Cell injuries caused by oxidative stress arise from interrelated damage to several key cellular components: the DNA, protein and lipids. Free radicals attack DNA and cause DNA strand breakage, base lesions, DNA-protein cross-links, and DNA adducts with reactive aldehydes derived from lipid oxidation (Cadet et al., 1994). These oxidative DNA lesions have been linked to DNA 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 lipid 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

18 Stadtman, 2001). Lipid peroxidation results in the production of hydroperoxides, which readily transform to various secondary oxidation products, such as reactive aldehydes (e.g. malondialdehyde), alkanes, lipid 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. Lipid 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 will be oxidized to form peroxyl radical (LOO), which readily interacts with nearby lipid molecules causing the formation of more radicals. The chain reaction will not cease until the terminating reactions take place. This usually involves the combination of free radicals to form non reactive species. The lipid peroxidation mechanism can be briefly described by the following equations (Equation 1-7) (Nawar, 1996; Pryor, 1994). Initiation + LH RH + L«(Equation 1) Propagation L' LOO' LOO LH LOO* LOOH + L' LO» + HO«(Equation 2) (Equation 3) (Equation 4) Termination 2 LOO«L' + LOO' 2 L- Non-radical products (Equation 5) Non-radical products (Equation 6) Non-radical products (Equation 7) 9

19 -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 (Xin et al., 1996). Antioxidants Dietary consumption of 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. Non 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, DNA 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

20 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 ORAC (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, ORAC measures the peroxyl radical scavenging activity of an antioxidant(s) molecule. The original ORAC assay employed P-phycoerythrin (P-PE), a fluorescing protein which decays upon exposure to AAPH (2,2' -azobis (2-amidinopropane) dihydrochloride ), thus generating a free radical (Cao et al., 1993). AAPH is a water soluble free radical initiator that undergoes spontaneous thermal decomposition at 37 C (Peyrat-Maillard et al., 2003; Yoshida et al., 2004). AAPH dissociates to form two carbon centered radicals which quickly combine with oxygen to form peroxyl radical and initiate lipid oxidation (equation 8-9) (Niki, 1990). R-N=N-R -> (l-e) R : R + 2eR» + N 2 (Equation 8) R« > 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 -PE conformation which results in a decrease in fluorescence intensity. Antioxidants that scavenge the peroxyl radicals thus 11

21 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 (AUC) 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 ROS 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 will 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 LDL 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

22 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 Fe 2+ /kg Fresh Weight} (mmol Trolox/kg Fresh Weight) 1 Blackberry Blackberry Blackberry Redcurrant Olive (black) Raspberry Raspberry Olive (green) Olive (black) Olive (black) Redcurrant Redcurrant Strawberry (wild) Raspberry Strawberry (wild) Olive (green) Strawberry (wild) Cultivated strawberry Cultivated strawberry Blueberry 9.30 Olive (green) Orange Cultivated strawberry 8.56 Pineapple Blueberry Plum (red) 8.09 Orange Pineapple Pineapple 5.92 Blueberry Plum (red) Orange 5.65 Plum (red) Grape (black) Cherry 4.17 Tangerine Grapefruit (yellow) Grapefruit (yellow) 4.04 Grape (black) Tangerine 9.60 Pear 3.87 Clementine Clementine 8.88 Tangerine 2.76 Grapefruit (yellow) Cherry 8.10 Clementine 2.74 Cherry Kiwi fruit 7.41 Grape (black) 2.50 Grape (white) Prickly pear 6.97 Kiwi fruit 2.30 Fig Peach (yellow) 6.57 Apricot 2.29 Kiwi fruit Fig 5.82 Apple (red Delicious) 2.23 Pear Melon (cantaloupe) 5.73 Prickly pear 2.06 Peach (yellow) Pear 5.00 Fig 2.06 Apple (red Delicious) Apricot 4.02 Loquat 1.73 Prickly pear Apple (red Delicious) 3.84 Grape (white) 1.59 Apricot Grape (white) 3.25 Apple (yellow Golden) 1.54 Apple (yellow Golden) Apple (yellow Golden) 3.23 Peach (yellow) 1.49 Melon (cantaloupe) Loquat 2.70 Melon (honeydew) 1.12 Loquat Banana 2.28 Banana 1.05 Watermelon Melon (honeydew) 2.27 Melon (cantaloupe) 0.95 Melon (honeydew) 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 DPPH* and ABTS* radicals (Hu et al., 2005). 13

23 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 ORAC, and anthocyanin content in several varieties of Vaccinium species. Hu et al., (2003) demonstrated that the anthocyanin rich fraction of black rice extract significantly prevented supercoiled DNA strand scission induced by peroxyl or hydroxyl radicals. They were also successful at showing the suppression of human LDL 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 of 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 Miller, 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

24 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 DNA, which protects both the DNA 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 of 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 th century. The juice was recommended for mouth and eye infections. Blackberry contains approximately mg of anthocyanins per 100 g (Table 3) (Mazza and Miniati, 1993). 15

25 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 Blackberries Black chokeberries 560 Black currants Black raspberries Blueberries Cherries Cranberries Elderberry 450 Grapes Kiwi 100 Plum 2-25 Red cabbage 25 Red onions 7-21 Red radishes Red raspberries Strawberries 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 Miniati, 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

26 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 ORAC assay in comparison to black or red raspberry and strawberry (Wang and Lin, 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 ORAC 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 LDL 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 DNA and cyanidin was also observed by Sarma and Sharma (1999), who reasoned that co-pigmentation protected both DNA and the 17

27 anthocyanin from hydroxyl radical attacks. Exposure of the DNA, 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 LDL 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; Wu 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, will result in the formation of 4'-0-methyl delphinidin-3- glucoside (methylation of the 4'-OH on the delphinidin B ring) in rats (Ichiyanagi et al., 2004). 18

28 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 of the methylated form, but not glucuronidated form of cyanidin-3-glucoside in rats, and found neither the methylated nor glucoronidated metabolites in human. Wu 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

29 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. Hu et al., (2003) demonstrated that anthocyanins from black rice were effective at suppressing DNA strand scission induced by ROS as well as inducible nitric oxide (inos) production, generally associated with inflammatory response. Wang and Mazza, 20

30 (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; Xia 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 lipid 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 regeneration 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 HL60 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-: malvidinglycosides, respectively, delphinidin and malvidin aglycone and its glycosides exhibited the greatest apoptosis induction in HL60 cells. The growth inhibitory effects of delphinidin-glycosides on both cell lines however were lower than those of the 21

31 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 HNDF) to an extent less than that observed in cancerous cells (HCF116 human colon carcinoma cells). Malik 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 Gl/Go and G2/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 p21 W A F 1 and p27 KIP1, the cyclin dependent kinase inhibitors (CDKI) and a decreased expression of cyclin A and cyclin B 22

32 genes. Cyclins are members of cell cycle regulators which bind to cyclin dependent kinase (CDK) and in turn regulate cell cycle progression. An increased expression of the CDK inhibitors p21 W A F 1 and p27 Klpl was found to coincide with the cell cycle arrest in the Gl/Go phase, whereas a decreased expression of the cyclin A and B genes were associated with the arrest at G2/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 G2/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 Gl phase which was accompanied by an increased fraction of cells with a hypodiploidic DNA 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

33 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 mrna as well as increased phosphorylation of JNK, 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 Bcl2, 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 of 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

34 inhibition of H 2 02-induced cytotoxicity in PC 12 neuronal cell system and was therefore considered to have a neuroprotective effect (Heo and Lee, 2005). Hu 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 DNA damage induced by ochratoxin in cultured human fibroblast cells (Russo et al., 2005). Cyanidin-3-glucoside was also found to prevent UV-A induced apoptosis in a human keratinocyte cell line (Tarozzi et al., 2005). This protective effect was attributed to the neutralization of the H 2 02 that was released after UV A radiation. While anthocyanins may scavenge free radicals generated outside or inside the cell, it is prudent to learn if 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; AAPH 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