Antioxidant Activity of Anthocyanins In Vitro and In Vivo - American

Chapter 8

Antioxidant Activity of Anthocyanins In Vitro and In Vivo

Downloaded by COLUMBIA UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch008

Garry Duthie, Peter Gardner, Janet Kyle, and Donald McPhail Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom

Diets rich in antioxidants may decrease the risk of developing major diseases. Intakes of anthocyanins, polyphenolic secondary plant metabolites responsible for the red and purple colors of soft fruits and certain vegetables, may exceed 200mg/day but little is known about their ability to act as antioxidants. Application of electron spin resonance spectroscopy to 13 anthocyanins illustrated the importance of the degree of hydroxylation, glycosylation and pH in determining antioxidant capacity. Although consumption of anthocyanin-rich extracts decrease DNA damage and indices of lipid peroxidation in rats with a compromised antioxidant status, it is not yet clear whether anthocyanins function as antioxidants at nutritionally-relevant dietary intakes.

Introduction Several epidemiological studies suggest that diets rich in polyphenols products of the phenylpropanoid biosynthetic pathway in plants decrease the risk of developing major diseases including heart disease and certain cancers (7). Such potential health effects may be a reflection of the ability of many polyphenols to prevent the oxidation in vitro of biological molecules such as proteins, lipids and DNA (2). Such antioxidant effects are mainly due to the ease with which an Η-atom from an aromatic hydroxyl group of polyphenols can be

90

© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


91 donated to a free radical and the ability of the aromatic group to then support the unpaired electron via délocalisation around the π-electron system (3). Countries in Northern latitudes which generally have the greatest number of premature deaths from chronic diseases also tend to have a habitually low consumption of foods rich in phytochemicals with antioxidant activity (4). This irçay be due to traditional dietary patterns and the expense and lack of availability offreshfruits and vegetables. For such populations, a potentially important source of antioxidant-rich food may be locally grown soft fruits (eg. raspberries, blackberries, blueberries, cranberries and blackcurrants). These are rich in anthocyanins (Figure 1), which are glycosidic-linkedflavonoidsresponsible for the red, violet, purple and blue colors of many plants (5). Anthocyanins are also increasingly used as food colorants. Consequently, in this chapter we discuss relevant data assessing the ability of anthocyanins to act as antioxidants in chemical and biological systems.

R

1

OH 1

Pg-3-glc Mv-3-glc Pn-3-glc Pt-3-glc Cy-3-glc Dp-3-glc

R H OMe OMe OMe OH OH

2

R H OMe H OH H OH

Figure 1. The structures ofsome anthocyanin glycosides: Pg - pelargonidin; Mv - malvidin; Pn - peonidin; Pt - petunidin; Cy - cyanidin; Dp - delphinidin. The anthocyanidin aglycones have OH at the 3 position.

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Antioxidant capacity of anthocyanins in vitro The antioxidant potential of anthocyanins were determined by their abilities to reduce stable radical species by hydrogen atom or electron transfer reactions. In organic media, galvinoxyl radical (2,6-di-/er/-butyl-a-(3,5-di-ie^-butyl-4oxo-2,5-cyclohexadien-l-ylidene)-p-tolyloxy) was used. This is a resonancestabilised, sterically-protected, phenoxyl radical which dissolves in a wide range of organic solvents, including ethanol, and has a half-life of several hours. Consequently, it may be a useful determinant of relative activities in the aprotic environment of the lipid phase of foods and in cell membranes. Fremy's radical (potassium notrosodisulfonate), which is highly water soluble, can give complementary information on relative activities in the aqueous environment. Both radicals have well-defined electron spin resonance (ESR) spectra which can be monitored to assess the extent of reduction in the presence of antioxidant functionals thereby allowing the reaction stoichiometry to be determined. Furthermore, they are not sufficiently oxidising to indiscriminately abstract hydrogen atoms from a wide range of biological substrates and thus react preferentially with compounds which may fulfill an antioxidant role in-vivo. In comparison with coulorimetric methods, ESR detection has distinct advantages in terms of sensitivity, lack of interference with the highly-resolved and unique radical spectra, and the ability to work with strongly-colored or turbid solutions. Full details of the method are described in Garder et al (6).

Reaction stoichiometry Using ESR, many anthocyanins were found to possess antioxidant activity similar to vitamin Ε (da-tocopherol) and its synthetic water-soluble derivative, Trolox (Figure 2). For example, one molecule of the aglycones of cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin (i.e. the anthocyanidins) is capable of reducing more than one molecule of galvinoxyl radical, stoichiometrics ranging from 1.0 to 2.2. These compare with stochiometries of 2.2 and 2.3 for da-tocopherol and Trolox, respectively. In aqueous medium, using Fremy's radical, reaction stoichiometrics ranged from 0.4 to 1.6. Structure-activity relationships were less evident than has been found previously with flavonoids where the extent of B- and C-ring hydroxylation impacted markedly on activity (6). Moreover, unlike flavonoids, substitution of the C-ring at the 3 position with a glycoside unit did not adversely affect reactivity. In fact, glycosylation generally increased antioxidant capacity although the mechanism behind this phenomenon is still unclear.



93

Figure 2. Antioxidant capacity of anthocyanins. Pg: Pelargonidin; Cy: Cyanidin; Pn: Peonidin; Dp: Delphinidin; Pt: Petunidin; Mv: Malvidin; PgGlc: Pelargonidin 3-glucoside; CyGlc: CyanidinS-glucoside; CyGal: Cyanidin 3galactoside; PnGlc: Peonidin 3-glucoside;DpGlc: Delphinidin 3-glucoside; PtGlc: Petunidin 3-glucoside; MvGlc: Malvidin 3-glucoside

Effects of pH on antioxidant capacity Unlike other products of the phenylpropanoid pathway, anthocyanins are charged species whose chemical structure changes with alterations in pH (7). At low pH, they exist predominantly as the flavylium cation. At pH 5.0, a colorless carbinol is formed and as the pH becomes alkaline, equilibrium exists between the quinoidal pseudobase form and what may be an anionic form of the flavylium structure (Figure 3). Such changes in structure can be interpolated from the differences obtained in absorption spectra between 350 and 700 ran. For example, when 0.1 mM solution of cyanidin 3-glucoside was prepared over a wide pH range using citric acid - Na HP0 buffer (pH 2.6, 5.0, 7.4) and Tris 2

4


94


HCL buffer (pH, 8.9), there was a single distinct peak at pH 2.6 and no absorption at pH 5.0 (Figure 4). The main peak in these spectra are likely to be the blue quinoidal base while die structure causing absorption between 400 and 450 nm may be an anionic form of the flavylium structure.

Figure 3: Changes in structure of delphinidin 3-glucoside due to pH (7) Clearly, as intestinal pH ranges from 2 to 8, structural changes of anthocyanins during transportation through the stomach, small intestine and colon may affect their chemical properties and thus any potential biological effects. For example, the antioxidant capacity of fresh solutions of cyanidin 3glucoside increases with pH, more Fremy's radicals being reduced in alkaline conditions (Figure 5). This suggests that the flavylium cation is the least active form. At pH 5.0, the carbinol form has a hydroxyl group attached at the 2-C position, creating a chiral center, which may be capable of donating a hydrogen atom to a radical species. In addition, the quionoidal structure formed on the Aring at alkaline pH may prime the hydroxyl group on the 5-C for hydrogen donation. Therefore, anthocyanins may be more effective as antioxidants in the small intestine and colon compared with the stomach. However, the presence of oxygen causes a decrease in antioxidant capacity at the higher pH values. This may reflect an autoxidation process, oxidation of the carbinol structure producing a colorless chalcone. The "broken" Β ring of this molecule would lead to a decrease in antioxidant potential.



95

Figure 4. Absorption spectra of cyanidin 3-glucoside at various pH values.

Figure 5. The effect ofpH on antioxidant activity of cyanidin 3 glucoside


96

Stability and partitioning characteristics of anthocyanins


Synthetic phenolics are widely used by the food industry to delay rancidity and extend the shelf life of food products. However, concern about the safety of synthetic antioxidants has led to increasing interest in naturally-occurring alternatives as these may not only retard rancidity but may also have added health benefits. Ease of use of natural polyphenols in food formulation will depend on a number of factors including their relative stability and their ability to partition into the lipid phases of food matrices.

Stability of solvated anthocyanins As stability is a major consideration for the inclusion of antioxidants in food processing, we assessed how storage in ethanol over 7 days at 25°C affected concentrations of a range of anthocyanins. Stability was estimated using an adaptation of the method of Ribreau-Gaynor and Stonestreet (8) in which absorbances at 520 and 700 nm are measured at pH 0.6 and 3.5. Concentration was calculatedfromthe expression: Atotal = [A52O - A oo] H 0.6 ~ [A52O ~ A7 o]pH 3.5 7

p

0

Results (Table I) indicate that delphinidin degrades rapidly with time with little remaining after 7 days. Petunidin and malvidin also showed significant loss over this period of 23 and 13%, respectively. However, the compounds were relatively stable as glycosides.

Theoretical octanol-water partitioning coefficients of anthocyanins Octanol - water partitioning coefficients (LogP values, where Ρ is the ratio of the concentration of a compound in the octanol phase over that in the aqueous phase) have been widely used to model a chemical's lipophilicity. These values are employed by the pharmaceutical industry as one of many QSAR predictors relating to bioactivity and bioavailability. A number of algorithms have been developed to allow theoretical predictions of LogP using fragmentai, atom, neural-network and quantum based approaches of which thefragmentaimethods appear most robust. Consequently, we have used the ClogP algorithm (9) to predict the partitioning coefficients of six anthocyanins in their flavylium form, their 3-glucoside derivatives and the pH-dependent equilibria structures of malvidin.


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Table I. Stability of solvated anthocyanins upon storage Anthocyanin Pelargonidin Cyanidin Peonidin Delphinidin Petunidin Malvidin Pelargonidin 3-glucoside Cyanidin3-glucoside Cyanidin 3-galactoside Peonidin 3-glucoside Delphinidin 3-glucoside Petunidin 3-glucoside Malvidin 3-glucoside

4 days -3.88 -0.23 -4.26 -75.15 -11.50 -16.54 -8.08 0.48 -5.85 -0.47 -2.20 0.23 -1.29

7 days -6.64 -3.02 -6.50 -94.48 -12.78 -22.90 -4.37 0.32 -2.44 -2.60 -4.16 -0.70 -4.18

NOTE: Data as percentage change

Table II. ClogP values offlavyliumaglycones and glycosides Compound Pelargonidin Peonidin Malvidin Cyanidin Petunidin Delphinidin Pelargonidin 3-glucoside Peonidin 3-glucoside Malvidin 3-glucoside Cyanidin3-glucoside Petunidin 3-glucoside Delphinidin 3-glucoside

ClogP value 2.16 2.02 1.80 1.56 1.47 0.897 0.783 0.636 0.415 0.187 0.090 -0.479

NOTE*. A value of zero is a prediction of50:50 octanol - water partitioning.



98 The ClogP algorithm predicts that all of the flavylium aglycones will preferentially partition within the lipid environment (Table II). The values follow the expected trend towards decreasing hydrophobicity as the B-ring becomes more hydroxylated as exemplified by the series: Pelargonidin, cyanidin and delphinidin. Methoxylation has a marginal impact on lowering hydrophobicity (pelargonidin, peonidin and malvidin or cyanidin and petunidin). Accounting for the log scale, delphinidin, although lipophilic, is predicted to have a significant presence (13%) in the aqueous phase. Introduction of the 3-glucoside unit is predicted to dramatically decrease hydrophobicity. Pelargonidin, the most lipophilic anthocyanin, when glycosylated becomes less hydrophobic than all other aglycones. Petunidin-3glucoside is predicted to partition almost equally between both phases, whilst delphinidin-3-glucoside acquires significant lipophobic character with almost 90% distributed in the aqueous phase. The potential effects of pH on partitioning are predicted by calculations on the various forms of malvidin that have been reported (7) to exist in a series of pH-dependent equlibria (Figure 6). The flavylium cation, which dominates at low pH, is highly lipophilic with only 1.6% predicted to be present in the aqueous phase. However, structures existing in the physiological pH range are much less hydrophobic with ClogP values approaching 0.5, which equates to approximately 30% presence in the aqueous phase. Although the ClogP algorithm has a high predictive accuracy with R = 0.936 when compared against experimental values from a large drug dataset, it must be emphasised that i f structural attributes are present within the anthocyanins which are not well-defined within the algorithm's training set of over 1000 compounds, then the predictive accuracy may be lower.

Nutritional relevance of anthocyanins Epidemiological studies inversely relating dietary intakes of plant polyphenols with the incidence of heart disease and cancer (10) may indicate a putative role for anthocyanins in the prevention of chronic diseases. To date, formulation of databases to allow estimates of dietary polyphenol intakes have mainly focussed on flavonols and flavones rather than anthocyanins per se. There is therefore a need for dietary compositional information to facilitate epidemiological and human intervention studies assessing the role of anthocyanins in health.

Anthocyanins in foods In the US, average daily intake of anthocyanins may be around 200mg/day (5), which is comparable to intakes of recognized nutritional antioxidants such as


99


OMe

CLogP » 0.825621

Figure 6. Relationship between ClogP and the various structures which malvidin is reported to form in the literature (7) as a function ofpH. The flavylium cation represents the majorform at low pH.

vitamin C, vitamin Ε and certain carotenoids. However, accurate determination of dietary intake of anthocyanins is problematical due to variations in analytical methodology. In addition, the concentration of anthocyanins in foods and beverages can be influenced by several factors including species, variety, light, degree of ripeness, processing and storage. In general, greatest concentrations of anthocyanins are found in soft fruit, significant amounts also being present in some vegetables and beverages (Table III). Red wine is also a rich source of anthocyanins. However, during the maturation process, there is a color change (520nm towards 420nm) reflecting the progressive displacement of the grape


100 Table III. Anthocyanin types and contents in selected foods


Food

1

Red apple Bilberry Blackberrys Blueberry

Content (g/kg) 0.1-0.2 3.7 0.3-1.1 0.8-2.8

Cherry Cranberry Current, black Elderberry

2.4 0.1-3.6 1.3-4.0 2.0-10.0

Red grapes Lychee Orange, blood Peach Plum Raspberry Strawberry Red cabbage

0.7-1.1 0.5 2 0-0.1 0.02-0.3 0.3-1.2 0.1-3.8 0.7-0.9

Red onion

0.2

Red radish

1.5 (skin)

Black beans Cocoa beans Purple basil

2.1 0-1.0 0.2

Red wine

0.01-0.5

Blackcurrant juice 0.02-0.1

2

Anthocyanin

3

Glycosides

Glu; gala; arab Cy Mv; Dp; Cy; Pt; PnGlu; gala; arab Glu; gala; rut; arab; xyl Cy;Pg Mv; Cy; Dp; Pn; PtArab; gala; glu; 6-ace-3-gly, sam; soph Glu; rut Cy;Pn Gala; glu; arab Pn;Cy Glu; rut Cy;Dp Sam; glu; sam-5-glu, 3,5Cy diglu, /7-cou-glu-5-glu Mn; Dp; Pt; Pn; CyGlu; glu-ace; glu-p-cou; rut Glu, rut, ace-glu Cy,Mv Glu; mal-glu Cy;Dp Glu; rut Cy Glu; rut; 3-ace-glu; gal Cy Cy;Dp;Mv; Pg 3-glu; soph, rut; 3-glu-rut Rut; 3-ace-gly; glu Pg;Cy 3,5-diglu; soph-5-glu Cy acylated with p-cou, fer, sin Mal-glu; di-mal-lam; malCy;Pn lam; glu; mal-3-glu; arab 3,5-diglu; soph-5-glu Pg;Cy acylated with p-cou, fer, caf Glu Dp; Pt; Mv Arab; gala; arab-glu Cy Glu; 3,5-glu; p-cou; mal; pCy,Pn cou-glu-5-glu glu; ace-glu; p-cou-glu; 3,5Mv; Dp; Pt; Pn; glu Cy Glu; rut Cy

NOTE: Datafromseveral sources crude total anthocyanin content Cy - cyanidin; Dp - delphinidin, Mv - malvidin; Pn - peonidin; Pg - pelaronidin Glu - glucoside; gala - galactoside; arab - arabinoside; rut - rutinoside; xyl - xyloside; rham - rhamnoside; soph - sophoroside; mal - malonyl; lam - laminaribioside; samb sambubioside; p-cou - p-coumaroyl; ace - acetyl; caf - caffeoyl; fer - feruloyl; sin sinapic 1

2

3



101 anthocyanins by more stable polymeric pigments which are formed by a variety of reactions including co-pigmentation (77). Although anthocyanins have marked antioxidant activity in chemical systems, they have to be absorbed from the gut if they are to exert a similar effect in systemic cells and tissues. As yet, little is known about the bioavailabilty of anthocyanins although it will likely be affected by numerous factors including molecular structure, the amount consumed, the food matrix, degree of bioconversion in the gut and tissues, the nutrient status of the host and genetic factors. Direct intestinal absorption of anthocyanins in the intact glycoside form and subsequent biotransformation to methylated derivatives in the liver has been observed in animal models suggesting that some may be bioavailable (72). In addition, we have observed that anthocyanin rich-extracts may moderate oxidation in vivo (13). Rats were maintained on vitamin Edeficient diets for 12 weeks in order to enhance susceptibility to oxidative damage and then repleted with rations containing a highly purified anthocyaninrich extract at a concentration of lg/kg diet. The extract consisted of the 3glucopyranoside forms of delphinidin, cyanidin, petunidin, peonidin and malvidin. Consumption of the anthocyanin-repleted diet significantly decreased the vitamin Ε deficiency-enhanced hydroperoxides and 8-Oxo-deoxyguanosine concentrations in liver. As these are indices of lipid peroxidation and DNA damage, respectively, dietary consumption of anthocyanin-rich foods may have nutritional benefits. However, it should be noted that in such studies the animals are fed quantities of the compounds, which markedly exceed what may be achievable from diet alone. The nutritional relevance of these studies is therefore uncertain. Clarification of the absorption, bioavailability and metabolism of the anthocyanins in our diet will be an important research area in the future.

Acknowlegements Financial support is gratefully acknowleged from the Scottish Executive Environment and Rural Affairs Department (SEERAD) and the European Union Framework V Programme.

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5. Clifford, M.N. J. Sci. Food Agric. 2000, 80, 1063-1072. 6. Gardner, P.T.; McPhail, D.B.; Duthie G.G. J. Sci. Food Agric. 1997, 76, 257-262. 7. Brouillard, R.; Lang, J. Canad. J. Chem. 1990, 68, 755-761. 8. Ribreau-Gaynor, P.; Stonestreet, E. Bull. Soc. Chim. Fr. 1965, 9, 26492653. 9. Leo, Α.;Hansch,C.;Elkins, D. Chem. Rev. 1971, 71, No. 6. 10. Bravo, L. Nutr. Rev. 1998, 56, 317-333. 11. Mazza, G. Crit. Rev. Food Sci. Nut. 1995, 35, 341-371. 12. Miyazawa, T.; Nakagawa, K.; Kudo, M.; Kayo, M.; Someyo, K. J. Agric. Food Chem. 1999, 47, 1083-1091. 13. Ramirez-Tortosa, C.; Andersen, Ø.; Gardner, PT.; Wood, S.G.; Morrice, P.C.; Duthie, S.J.; Collins, A.R.; Duthie, G.G. Free Rad. Biol. Med., 2001, 31, 1033-1037.


Antioxidant Activity of Anthocyanins In Vitro and In Vivo - American

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