ANTIOXIDANT PROPERTIES OF SORGHUM, A Dissertation by JOSEPH MOBUTU AWIKA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2003, Major Subject: Food Science and TechnologyUMI Number: 3088114 UMI UMI Microform 3088114 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346, ‘Ann Arbor, MI 48106-1346ANTIOXIDANT PROPERTIES OF SORGHUM by A Dissertation JOSEPH MOBUTU AWIKA Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved as to style and content by: : Tow ‘Rodney (Chair of Committ eo Bide, Ronald Richter (Member) Lloyd’W. Rooney { (Chair of Food Science an Technology Faculty) May 2003 GLE@ (Member) er ogee Luis Cisneros-Zevallos (Member) (eave ‘Mark Hussey (Head of Department) Major Subject: Food Science and Technologyiii ABSTRACT Antioxidant Properties of Sorghum. (May 2003) Joseph Mobutu Awika, B.Sc., Egerton University; MS., Texas A&M University Chair of Advisory Committee: Dr. Lloyd W. Rooney Sorghum varieties grown in Texas between 1998 and 2002, as well as processed sorghum products, were analyzed for antioxidant potential using three methods; oxygen radical absorbance capacity (ORAC), 2,2’-azinobis (3-ethyl-benzothiaziline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). Phenolic antioxidants from these samples were separated and characterized by HPLC. Two extraction solvents, 1% HCI in methanol (Me-HCh, and 70% aqueous acetone (Ac-aq), were compared. The Me-HCl generally extracted sorghum antioxidants better than Ac-aq. This effect was more significant in black sorghums than brown sorghums. The ORAC activities (Trolox equivalents, TE) among black and brown sorghum brans were high (1,000 ~ 3,100 mol TE/g) compared to common fruits and vegetables (80-900 mol TE/g). Retention of antioxidant activity after processing was 57 — 78% for baked, and 70 100% for extruded sorghum products relative to raw samples. Antioxidant values by the ORAC method were 2-3 times higher than ABTS or DPPH values for the sorghums. However, the three methods correlated highly with one another (R’> 0.95). The ABTS method was overall the most suitable for sorghums.Brown sorghum grains and brans had high procyanidin contents (21-58 mg/g) compared to blueberry (20 mg/g), as measured by HPLC. Polymers with DP>10 were the major procyanidin constituents (66-84%) of brown sorghums. The relative ratio of oligomers (DP<10) to polymers (DP>10) increased significantly in processed products. Black sorghum brans had high anthocyanin content (4.5-11.0 mg/g) compared to commercial sources (0.8-10.0 mg/g). Luteolinidin and apigeninidin were the major anthocyanins in the black sorghums, accounting for about 50% of total anthocyanins. ‘The red sorghum bran had high levels of naringenin (1.0 mg/g) compared to the other sorghum brans (0.17-0.26 mg/g). Specialty sorghums are a rich source of different phenols with high antioxidant potential and are a commercially viable source of these compounds for foods or pharmacological applications. High retention of antioxidant activity in processed products implies the sorghums are valuable food ingredients.ACKNOWLEDGEMENTS I wish to extend big thanks to Dr. Rooney for making all this possible. Special thanks to Dr. Waniska for being very helpful through all stages of the study. Sincere gratitude goes to Dr. Richter and Dr. Cisneros for serving on my committee and general help. I especially thank Dr. Cisneros for allowing me the use of his laboratory facilities, and also his invaluable help with the initial DPPH analysis. Special thanks to Dr. Prior of the Arkansas Children’s Hospital, Little Rock, AK, and his staff, Dr. Gu and Dr. Wu for being very helpful with time and resources for normal phase HPLC and ORAC analysis. Many thanks go to Dr. Senseman, and Audie Sciumbato (Soil & Crop Sciences) for generous help with the initial HPLC analysis. 1 also thank Dr. Keeton and Bryan (Meat Science) for allowing me access to facilities in their laboratory. The CQL lab crew was all very helpful at various stages of this study. Thanks to Cassandra for helping with acquisition of most of the ‘tools’ for the work and her general advice. Many thanks to Linda Dykes for being very helpful with parts of the analysis, I salute David Acosta and Mark Baron for assisting with the dirty work! Thanks to the rest of CQL members for their general support.vi TABLE OF CONTENTS Page ABSTRACT... esse cceeceeee ACKNOWLEDGEMENTS. TABLE OF CONTENTS. wi LIST OF TABLES.. viii LIST OF FIGURES . CHAPTER I INTRODUCTION ............ I LITERATURE REVIEW........ Sorghum tannins and their antioxidant potential 3 Sorghum anthocyanins and their potential benefits 9 Extraction of phenolic compounds... wl Separation of phenolic compounds using HPLC... 15 Evaluating antioxidant activity of phenolic compounds 220 Ill MATERIALS AND METHODS... Samples... Sample extraction. Analytical procedures......... HPLC analysis. Special note. IV RESULTS AND DISCUSSION... Extraction of sorghum antioxidants Methods for measuring antioxidant activity Comparing ABTS and DPPH antioxidant methods on sorghum extracts. : Use of antioxidant activity to compare extraction efficiency of Ac-aq with Me-HCI Antioxidant properties of pure phenols HPLC separation of sorghum phenolics .vii CHAPTER Page Environmental and varietal effects on phenol and antioxidant levels of sorghums ..... _ Effect of processing on antioxidant activity of sorghum Dietary implications of the phenol and antioxidant status of the processed sorghums Comparison of sorghum products with other dietary sources Vv SUMMARY, 100 LITERATURE CITED ..... 103 APPENDIX A. AIS APPENDIX B.. 116 APPENDIX C... 7 VITA ... 118viii LIST OF TABLES TABLE Page 1 1 rit Vv VI vu vill Ix XI x XI xIV xv XVI ‘Tannin and phenol levels in Me-HICI and Ac-aq extracts of brown sorghums Anthocyanin levels in sorghum brans extracted with Me-HCl and Ac-aq. Antioxidant activity of sorghum grains, brans and products measured by ORAC, ABTS and DPPH.. . . A summary comparison of the different antioxidant methods .AT Comparing antioxidant activity of brown sorghums extracted for different times . Antioxidant activity of flavonoids... Antioxidant activity of phenolic acids. 53) Molar characteristics anthocyanin molecules . 61 Anthocyanin contents of black and red sorghum brans determined by HPLC. 64 Procyanidin content of brown sorghums compared to those of freeze- dried cocoa and blueberry 70 ABTS antioxidant and phenol levels among sorghums and other cereals.........75, Effect of processing on procyanidin polymer contents of Sumac (SU99) bran measured by normal phase HPLC. — Change in measurable phenolics due to extrusion .. ORAC levels per serving of bread and cookies made with sorghum brans........94 ORAC levels in sorghum brans and extracts relative to common fruits...... 97 Anthocyanin content of sorghum brans relative to common fruits and vegetables... 98ix LIST OF FIGURES FIGURE 1. Che al structure of common anthocyanidins.... 2. Typical crude anthocyanin spectra for black sorghum bran extracted with Me-HCI and Ac-ag. «.....esssssssess 3. Correlation coefficients for the three antioxidant methods used. 42 4, Comparing antioxidant DPPH methods ivities of brown sorghum brans using ABTS and 5. Comparing antioxidant activities of black sorghum brans using ABTS and DPPH methods . 43 6. Antioxidant activities of brown sorghum brans extracted in Me-HCl and Ac-aq 50 7. Antioxidant activities of black sorghum brans extracted in Me-HCI and Ac-aq 8. Comparing antiradical activity of phenolic compounds in DPPH versus ABTS-EtOH and ABTS-PBS systems. 9. Correlation coefficients between ABTS in ethanol (ABTS-EtOH) and aqueous buffer (ABTS-PBS) versus DPPH for different phenol groups....0.0...56 10, Effect of the number of OH substituents on the antioxidant activity of phenolic compounds in DPPH, and alcoholic (EtOH) and aqueous buffer (PBS) ABTS systems. 58 11. Absorption spectra for selected anthocyanin standards in pH 1.0 buffer solution seceeeeeneenene 60 12, HPLC profiles of Me-HCI anthocyanin extracts from black and red sorghum brans... 13. HPLC anthocyanin profiles of black sorghum (BKO1) bran extracts monitored at 375 nm ..... 265 14, Spectral characteristics of luteolinidin and apigeninidin peaks isolated from black sorghum (BK01) bran by HPLC 66FIGURE Page 15, Normal phase HPLC procyanidin profiles of two brown sorghum grains, Sumac (SU99), and Hi Tannin (HTO1), 69 16. HPLC profiles of Me-HCl flavonoid extracts from brown (SU99) and red (RDO) sorghum brans monitored at 280 nm .. sevens 17. Phenol levels for various sorghum brans compared to wheat bran... 18. ABTS values for various sorghum brans compared to wheat brat...uc.ssssssss000.77 19, Changes in antioxidant activity of sorghum brans after baking. 81 20. Normal phase HPLC procyanidin profiles of Sumac (809s) bran before and afier baking in cookies and bread... es 21, Change in Sumac (SU99) bran procyanidins due to processing in cookies and bread. 84 22. Change in ABTS activity of sorghums due to extrusion. 88 23. Normal phase HPLC procyanidin profiles of Hi Tannin (HTO1) grain before and after extrusion. . 89 24. Change in Hi Tannin (H'T01) grain procyanidins due to extrusion. 1CHAPTERI INTRODUCTION Interest has increased in phenolic compounds from plants due to their potential benefits as antioxidants. Sorghum, like many other cereal plants, contains phenols that are thought to provide natural defense mechanisms against insects and diseases. In sorghum, these phenols are concentrated in the outer layers of the kernel. Decortication to remove outer layers of the kernel can concentrate these compounds 3-7 fold (Hahn and Rooney 1985, Awika 2000). Such fractions possess antioxidant properties comparable to those of berries (Awika 2000) and can be used in foods to provide nutraceutical benefits. Sorghum offers a cost effective source of phenolics due to its storage stability and high grain yield. This is especially important given the rapidly increasing demand for natural sources of nutraceuticals for food. Condensed tannins are a major phenolic component of sorghums with a pigmented testa. They are concentrated in the testa and pericarp of such sorghums. The superior ability of tannins over simple phenols as antioxidants is known (Hagerman et al 1998, Bors et al 2000). Hagerman et al (1998) found tannins were 15-30 times more powerful at quenching peroxy radicals than simple phenols. Bors et al (2000) also reported an exceptionally high radical scavenging potential of condensed and hydrolysable tannins in their reaction with hydroxyl radicals. The tannin contents of sorghums correlated This dissertation follows the style and format of Cereal Chemistry.positively with their antioxidant properties as measured by the ORAC method (Awika 2000). Isolation of these polyphenols from sorghum and measuring their antioxidant ability in purified forms is necessary. This will give a better understanding of how they influence the antioxidant activity of bran fractions. Anthocyanins are becoming increasingly important not only as food colorants, but also as antioxidants. There is a growing commercial need for alternative natural sources of food colorants (Boyd 2000, Francis 2000). Awika (2000) found black sorghum, (Tx430 variety) bran had significantly more anthocyanins than other sorghums, with good antioxidant properties comparable to those of berries. This was the only non-tannin sorghum with such high antioxidant activity, which suggests anthocyanins played an important part in its antioxidant activity. The potential of this sorghum for commercial exploitation cannot be overestimated. It is important to isolate and determine the antioxidant properties of the anthocyanins in more purified forms. ‘Numerous other phenolic compounds exist in sorghum brans (Hahn 1984, Gous 1989), Data is needed on the antioxidant properties of these sorghum phenols. Such phenols are known to have different antioxidant properties and their relative proportions can affect the overall antioxidant activity of bran samples. In order to determine the antioxidant activity of the sorghum phenols, itis essential to extract them in a consistently efficient manner. This will allow for accurate estimation of their activity as well as allow for comparison with other natural antioxidant sources, Several methods are available for extraction of phenols from different sources. In sorghums, acidified methanol is reported as most efficient (Hahn and Rooney 1985,Gous 1989), whereas for fruits and vegetables 70% aqueous acetone is reportedly most efficient (Kallithraka et al 1995, Garcia-Viguera et al 1998). However, the acidified ‘methanol and aqueous acetone methods are seldom compared side by side. The main goal of studying natural antioxidants is to improve human health. The most effective way to deliver the antioxidants to consumers is through foods that are consumed widely and consistently. Prior investigations in our lab have shown that specialty sorghum brans high in phenolics can produce good quality breads (Gordon 2001), and cookies (Mitre-Dieste et al 2000). However, the rate of retention of sorghum antioxidants in these products is not known. This information is important if the specialty sorghums are to be promoted as a viable source of antioxidants for human health, Numerous methods are available for estimating antioxidant activities of compounds in vitro, However, since the main goal is to improve human health, methods that would most closely predict the bioeffects of these compounds are preferable. So far ORAC is the only in vitro method that has correlated in vitro activities of various antioxidants with in vivo plasma antioxidant status (Cao et al 1996). However the method is expensive and hence not widely available. Other methods with similar or better predictive value for sorghum antioxidants should be assessed. ‘This work aims to detail the effects of sorghum phenolic components on overall antioxidant activity of sorghum brans, and how processing affects their activity. Itis a continuation of preliminary work done in our laboratory as reported by Awika (2000).Specific objectives were » 2) 3 4) To determine the most efficient method for extracting sorghum antioxidants. ‘To compare different methods for antioxidant analysis to determine the most suitable for sorghum. Isolate and identify the sorghum compounds responsible for antioxidant activity using HPLC. To determine the effects of processing on antioxidant activity of sorghum.CHAPTER IL LITERATURE REVIEW Sorghum tannins and their antioxidant potential ‘Tannins are high molecular weight polyphenols, and are classified as hydrolyzable or condensed. Hydrolyzable tannins contain a central core polyhydric alcohol such as glucose and hydroxyl groups which are esterified wholly or partially by gallic acid or hexahydroxydiphenic acid. Condensed tannins, found in sorghum, are mainly polymerized products of flavan-3-ols and/or flavan 3,4-diols. Sorghum tannins have generally been viewed as undesirable due to theit antinutritive properties, i., they complex with food macromolecules reducing their digestibility Price and Butler 1980, Salunkhe et al 1982, King-Thom et al 1998). However, current data suggest that tannins may have nutraceutical benefits due to their powerful antioxidant activity (Kinsella et al 1993, Clifford 1996, Hagerman et al 1998, Heinennonen et al 1998). Consequently research efforts are directed at understanding their precise mode of action and bioavailability in biological systems and how their consumption can be enhanced in human diets. Specialty sorghum brans rich in these ‘compounds (Awika 2000) need detailed analysis to determine how they can be used in foods. The activity of tannins as antioxidants is thought to be two-fold; i) they may chelate transition metals (Cu and Fe) making them unavailable for Fenton-type reactions (Bors et al 1990, Carbonaro et al 1996); ii) they may act as scavenger antioxidants by donating an electron to highly reactive free radicals and quenching the reactivity of the radical by6 delocalizing the unpaired electron on the phenolic ring (Husain et al 1987, Chimi et al 1991). Conflicting data exist, however, on ability of tannins to chelate transition metals. Carbonaro et al (1996) found no evidence to suggest that the ability of tannins to reduce hydroxyl radical formation was due to a chelating activity. They concluded it was purely due to a direct interaction of the polyphenol molecule with hydroxyl radicals. On the other hand, Hagerman et al (1998) found both condensed and hydrolysable tannins were able to chelate iron (Fe™) at a pH range of 6.4 ~ 8.4, besides reacting directly with free radicals. Similar results were reported by Moran et al (1997) for low molecular weight polyphenols at pH 5.8 and 7.4. Hagerman et al (1998) found tannins were 15-30 times more powerful at quenching peroxy radicals than simple phenolics or Trolox. ce condensed and hydrolyzable tannins are structurally different, but were equally effective as antioxidants, they concluded that high molecular weight and the proximity of many aromatic rings and hydroxyl groups are more important to free radical scavenging by tannins than specific functional groups. Ariga and Hamano (1990) found that the ability of oligomeric, flavonoids to scavenge azobis-generated peroxyl radicals was proportional to their degree of polymerization. Hagerman et al (1998) also showed that tannins, unlike simple phenols, were unable to act as prooxidants or were very weak prooxidants. Similar results were reported by Bors et al (2000), who suggested this could be because procyanidin and gallate o-quinone, unlike flavan(ol) 0-quinones which can act as prooxidants by forming reactive oxygen species through futile redox cycling, are capableof producing oligomeric compounds by various pathways. Such phenolic coupling reactions retain the number of hydroxy groups which enhance their antioxidant capacity. ‘An important factor to consider in assessing the activity of dietary components is their availability and/or activity after ingestion. Since tannins complex with proteins and other macromolecules making them resistant to digestion, they are likely to remain in the digestive tract and not be absorbed and transported to other tissues (Jimenez-Ramsye et al 1994). This way, they may serve a unique role as antioxidants by sparing other antioxidants and thus indirectly increase antioxidant level in other tissues (Hagerman et al 1998). They may also be able to protect proteins, carbohydrates and lipids in the digestive tract from oxidative damage during digestion (Marshall and Roberts 1990). To evaluate antioxidant ability of tannins when complexed with proteins, Hagerman et al (1998) used tannin-gelatin complexes as antioxidants in metmyoglobin assay. These complexes retained at least half the activity of free tannins. It is possible proteins complexed this way may be less susceptible to oxidative damage. Recent data suggest that tannins and other flavonoids are absorbed by humans in larger quantities than previously thought (Pietta 2000, Ross and Kasum 2002). Deprez et al (2001) reported that procyanidins could be absorbed through the intestinal cell monolayer, but only up to trimers. However, the interflavan bond in the procyanidins ‘was found to be unstable in acid environments, suggesting the higher molecular weight procyanidins could be broken down in the stomach (Spencer et al 2000). These authors tested depolymerization of procyanidins in simulated gastric juice (pH 2) and found that tetramers to hexamers degraded to monomers and dimers in 1.5-3.5 hr. This wouldpotentially improve the absorption of the procyanidins in the small intestine, and hence their bioavailability. ‘A major part of the ingested flavonoids is, however, apparently not absorbed directly, but is largely degraded by the intestinal microflora. The bacterial enzymes catalyze several reactions, including hydrolysis, cleavage, of the heterocyclic oxygen- containing ring, dehydroxylation, and decarboxylation (Pietta 2000). The net products are several phenolic acids which can be reabsorbed through the colon, enter circulation and offer antioxidant protection (Pietta et al 1997, Deprez et al 2000, Tapiero et al 2002) In general, tannins appear to be the most effective natural phenolic antioxidants in- vitro. Available data suggest that tannins may also produce health benefits in-vivo through direct absorption or degradation into phenolic acids in the colon, or by complexing with and protecting food molecules from oxidative damage. Since specialty sorghum brans are a rich source of these compounds (Hahn 1984, Awika 2000), they could be incorporated in diets as part of natural food ingredients. Work in our laboratory using these sorghum brans in bread and cookies has shown they produce good quality products (Mitre-Dieste et al 2000, Gordon 2001, Rudiger 2003). It is essential to assess the nutraceutical properties of such products to determine how processing and storage affect the interaction of tannin with food macromolecules and its antioxidant properties. This may help determine significance of sorghum as a source of tannins for nutraceutical applications.Sorghum anthocyanins and their potential benefits Anthocyanins are natural colorants belonging to the flavonoid family. They are widely distributed in flowers, fruits and vegetables, and are thought to play a role in plant resistance to insect attack (Strack and Wray 1993). All anthocyanins are natural glycosides and acylglycosides of anthocyanidins (Wang et al 1997), Figure 1 shows the general structure of common anthocyanidins found in nature. Most of these anthocyanidins have an OH group on the C-3 of the C ring. However, some —— Ri Ry H H ‘OH H H on ou H OCH OCHs ou H Delphinidin OH OH OH H Petunidin OH OCH OH H Peonidin H OCH; on H Luteolinidin On H H H Apigeninidin H H H H Fig 1. Chemical structure of common anthocyanidins.10 anthocyanidins, with a small distribution in nature, lack the OH group on C-3 and are normally referred to as 3-deoxyanthocyanidins. These include luteolinidin and apigeninidin (Fig. 1) (Sweeny and Iacobucci 1981, Clifford 2000). The common anthocyanins are either 3- or 3,5- glycosylated, When the number of sugar residues is higher than three, they may be attached to the basic molecule with altemating sugar and acyl linkages. ‘Anthocyanins are thought to have some therapeutic benefits including, vasoprotective and anti-inflammatory properties (Lietti et al 1976), anti-cancer and chemoprotective properties (Karaivanova et al 1990), as well as antineoplastic properties (Kamei et al 1995), Anthocyanins are considered to contribute significantly to the beneficial effects of consuming fruits and vegetables (Wang et al 1997). Most of the anthocyanins that have been identified were isolated from fruits and vegetables, Limited data exists on the types and levels of anthocyanins in cereals, probably because they have never been regarded as a commercially significant source. Nip and Bums (1969, 1971) were able to isolate and identify apigeninidin, apigeninidin- S-glucoside, luteolinidin and pelargonidin-3,5-diglucoside in red and white sorghum varieties by paper chromatography. Gous (1989) also reported luteolinidin and apigeninidin from a black sorghum variety. Cyanidin and pelargonidin were also reported in corn (Francis 1989), and sorghum (Yasumutsa et al 1965) Available quantitative data on anthocyanins vary widely from author to author, partly because of different standards used. In general, values of 1.2-23.6 mg/g forblueberries (Prior et al 1998, Mazza and Miniati 1993), blackberries 3.9-15.5 mg/g, cranberry 3.5 mg/g, raspberry 0.9-19.5 mg/g (Mazza and Miniati 1993), and red cabbage 1.6 mg/g (Timberlake and Henry 1988) have been reported (all values converted to dry ‘weight basis), Gous (1989) reported anthocyanin levels of 3.7 mg/g for a black sorghum variety. However, comparing values across authors may not be valid due to widely varying standards and extinetion coefficients, as well as extraction methods used by each author. Antioxidant properties of anthocyanins have been reported (Wang et al 1997, Satué- Gracia et al 1997, Lapidot et al 1999, Saint-Cricq de Gaulejac et al 1999). Awika (2000) also attributed high antioxidant properties of a black sorghum variety, which had a very Jow tannin level, to its anthocyanin content. Data from several authors on the antioxidant activity of anthocyanins and their aglycons are conflicting. Tsuda et al (1994) found that aglycons were better antioxidants than glucosides, whereas Wang et al (1997) reported that some glucosides were better antioxidants than aglycons. Different systems and catalyst used by different authors may be responsible for the conflicting data. For example, Lapidot et al (1999) reported that in a system catalyzed by H2O>-activated myoglobin, malvidin-3-glucoside was the best antioxidant, followed by catechin, whereas in a system catalyzed by an iron redox cycle catalyzer, resveratrol was the most efficient and catechin the least efficient among four flavonoids they tested (malvidin, catechin, resveratrol and malvidin-3-glucoside) In general, mechanistic studies of antioxidant activity of individual anthocyanin molecules have not provided meaningful information. Apparently, differenthydroxylation and glycosylation patterns modulate the antioxidant properties of anthocyanins. Saint-Cricq de Gaulejac et al (1999) reported that anthocyanins in red wine act synergistically to provide radical scavenging activity compared to pure anthocyanin molecules. This shows that measuring antioxidant activities of individual anthocyanin molecules, though insightful, may not give a good picture of the effectiveness of anthocyanins as antioxidants in their natural form. Such data would need to be compared against the crude forms of anthocyanins as they exist in nature. Antioxidant properties of sorghum anthocyanins and their individual constituents have not been reported. Such data is essential if sorghum anthocyanins are to be promoted as a viable commercial source of natural food pigments. This is becoming increasingly important with the rising demand for natural sources of food colorants (Boyd 2000), ‘A major concern over the exploitation of natural food pigment sources is the stability of anthocyanins in food systems. Anthocyanins have poor color stability relative to synthetic food colorants which presents a challenge to scientists. Use of natural anthocyanins in beverages is hampered by their sensitivity to light, pH changes, and especially their bleaching by sulfur dioxide which is a common preservative (Harborne 1988). However, the 3-deoxyanthocyanidins (which include apigeninidin and luteolinidin) were reportedly very stable in acidic solutions relative to other anthocyanidins commonly found in fruits and vegetables (Sweeny and Iacobucci 1981). The lack of oxygen at the C3 is believed to improve their stability. These 3- deoxyanthocyanidins are not widely distributed in food plants (Clifford 2000), but haveB been identified as major anthocyanins in sorghums (Nip and Burns 1969, Gous 1989). This points to the potential of sorghum anthocyanins as a viable commercial source of anthocyanins. Mechanisms for improving anthocyanin stability are known. Timberlake and Bridle (1980) reported that reacting anthocyanins with other flavonoids such as flavan-3-ols could stabilize the quinonoidal base and improve resistance to SO. Such reactions may occur during extraction of anthocyanins from samples that contain flavan-3-ols (Haslam 1980, Dallas et al 1996, Remy et al 2000), like brown sorghums, and this may further improve stability of such sorghum anthocyanins. Acetylation also improves stability of anthocyanins (Francis 1989). Sugar attachment and methylation of one or more of the free hydroxyl groups may also improve pigment stability (Cooper-Driver 2001. Co- pigmentation is another mechanism that improves stability of anthocyanins. Co- pigments are important since under very weakly acidic conditions (pH 4-6) as is typical in plant cell vacuole, and in absence of other substances, most anthocyanins exist substantially in stable colorless forms (Cooper-Driver 2001). At this pH range anthocyanin structural types exist in equilibrium as follows: quinonoidal anhydro base <> flavylium cation <= carbinol bases Co-pigments may act either inter- or intramolecularly (Figueiredo et al 1996). They have little or no color in themselves, but probably stabilize and enhance the color of anthocyanins by intermolecular hydrophobically reinforced x-x stacking of their aromatic nuclei with those of the pigment (Brouillard et al 1991, Mistry et al 1991).14 Such a-7 interactions may stabilize the flavylium cation/anhydro base forms which prevent reaction with water to form the colorless carbinol base forms (Cooper-Driver 2001). Various molecules have been identified as good natural copigments, including hydroxycinnamoyl esters such as chlorogenic acid, galloyl esters (tannins) and flavone and flavonol glycosides (Figueiredo et al 1996) Extraction of phenolic compounds Several methods are used to isolate phenols and assess their antioxidant activity. Since most sorghum phenols are concentrated in the bran fractions (Hahn and Rooney 1985, Awika 2000), separation of these fractions through abrasive decortication is an effective way of concentrating the phenols. These fractions are also rich in dietary fiber and phytin that can provide additional benefits in foods. Extraction of phenols from fruits and vegetables for quantitative determination is usually achieved using a polar solvent like methanol, acetone, ethanol and acidic methanol, among others (Kanner et al 1994, Prior et al 1998, Velioglu et al 1998). Hahn 1984, and Gous (1989) found 1% HCI in methanol to be the most efficient solvent for extracting sorghum phenolics. On the other hand, 70% aqueous acetone is reported as the most efficient at extracting fruit phenolics (Kallithraka et al 1995, Garcia-Viguera et, al 1998), Kallithraka et al (1995) found 70% aqueous acetone to be most effective at extracting grape seed procyanidins. However, most of the solvent comparisons have not involved the acidified methanol and aqueous acetone side by side. Garcia-Viguera et al (1998) compared 1% HCI in1s ‘methanol with 70% aqueous acetone, among other solvents, for extraction of strawberry anthocyanins. They concluded that the aqueous acetone was most efficient at extracting the anthocyanins, However, they stated that their conclusion may have been due to the difficulty involved in post-extraction processing (prior to analysis) of acidified methanol and other solvents relative to aqueous acetone, rather than their actual extracting power. Also, recently Lu and Foo (2001) observed significant anthocyanin reaction with aqueous acetone to form pyranoanthocyanins during extraction. They reported that the degree of the re: jon depended largely on duration of solvent-anthocyanin interaction, as well as temperature. For example, they found that blackcurrant anthocyanin HPLC peaks almost completely disappeared after three days in 70% aqueous acetone at 40°C, with a concomitant increase in pyranocyanin peaks. Hence depending on the extraction regimen, aqueous acetone may greatly underestimate anthocyanin content of a sample. Due to the controversies, it is essential to compare the two solvents side by side on sorghum fractions to determine the most effective method for extracting sorghum phenols. This information is not only essential for consistent analysis and comparison of different samples, but also for potential extraction for commercial exploitation. Separation of phenolic compounds using HPLC High performance liquid chromatography (HPLC) has become very popular due to its versatility, precision and relatively low cost. Most frequently the method uses reversed-phase Cs or Cx columns in conjunction with an aqueous mobile phase; and methanol and acetonitrile buffers as modifiers (Escarpa and Gonzalez 2001, Chen et al 2001). However, quantitative comparison of results as reported in literature is not easy16 because the extraction and chromatographic techniques vary. There is controversy in. sample preparation methods; some authors use solid-phase extraction with Cis or strong anion-exchange (SAX) cartridges; others use liquid-liquid extraction with different organic solvents like ethyl acetate or diethyl ether; while some inject samples directly without any preparation steps (Rodriguez-Delgado et al 2001). Depending on the nature ” of the compounds, recoveries are different. Sample preparation procedures for HPLC analysis have been studied (Schieber et al 2001, Malovand et al 2001). Malovand et al (2001) concluded that liquid-liquid extraction was more efficient than solid-phase extraction for analysis of trans-resveratrol and other polyphenols in wine by HPLC. Schieber et al (2001) used a stationary phase with polar endcapping to separate phenolic acids and flavonoids of apple and pear, and obtained good resolution of flavonol glycosides. Mataix and Ludue de Castro (2001) used a method based on coupling of continuous liquid-solid extraction, evaporation, HPLC individual separation and photometric detection to separate and determine anthocyanins in wine, The method was more sensitive than the batch manual method, with a wider determination range and better linearity of calibration curves. Chen et al (2001) used an acid-catalyzed hydrolysis process to liberate flavonoids and phenolic acids from their bound form and help overcome low resolution between flavonoids and phenolic acids. They were able to simultaneously determine flavonoids and phenolic acids in cranberry juice using this method. Putman and Butler (1989) developed a HPLC procedure to fractionate high molecular weight procyanidins (condensed tannins) from sorghum. They used a short7 reversed-phase C18 column to elute procyanidins through a combination of linear and step gradient coupled with a fast flow rate. Solvents used were acetic acid, methanol, and water. They included phytic acid and EDTA (ethylene diaminetetraacetic acid) in solvents to counter the effect of trace metals that may reside in the column. Through this method, they were able to identify three procyanidin polymers; B-2 (epicatechin dimer), B-3 (catechin dimer), and B-9 (epicatechin trimer), and isolate several other higher molecular weight procyanidins. Using a diode array detector they were able to show that peak separation was due to procyanidin chain length, with the lower molecular weight polymers eluting first. The monomers of catechin and epicatechin were not retained by the column (eluted with solvent front). Rigaud et al (1993) developed a normal phase HPLC method coupled with UV detection to separate procyanidins on a molecular weight basis. They were able to separate procyanidins up to tetramers for grape seed and up to pentamers for cacao beans. Oligomers and polymers beyond tetramers tended to fuse into broad unresolved humps, making the method unsuitable for quantification. Improvements of Rigaud et al (1993) method that coupled the normal phase HPLC with fluorescence detection was used to successfully separate oligomers up to pentamers from cocoa beans on a molecular weight basis (Adamson et al 1999, Hammerstone et al 1999). The authors proposed the method as a more effective tool for quantification of procyanidins over the less specific spectrophotometric methods like the Vanillin-HCI. However, since they were unable to resolve polymers with DP>10, the method underestimated procyanidin content,18 Gu et al (2002) further optimized the method used by Adamson et al (1999) and Hammerstone et al (1999) for berries, cocoa, and sorghum procyanidins. They were able to resolve the polymers with DP>10 as a single peak that could be quantified, in addition to the monomers and DP 2 ~ DP 10 oligomers. They found the high molecular weight polymers were the major procyanidins in brown sorghum bran, cocoa, cranberries and blueberries. This method allowed for a more effective quantification of procyanidins by HPLC than the previous methods. Using reversed phase HPLC-ESI MS, the authors identified epicatechin as the polymer extension unit and catechin as the major chain terminating unit (89%) in the sorghum bran procyanidins. This agreed with data obtained by Gupta and Haslam (1978), and Gujer et al (1986). HPLC presents a powerful tool for quantification of procyanidins in sorghums due to its specificity and ability to detect contribution of different chain length procyanidins to the total procyanidin content of a sample. This is important in health and food applications since polymer chain length affects organoleptic, antioxidant and other health properties of procyanidins (Rigaud et al 1993, Tebib et al 1997, Lotito et al 2000). Several phenolic acids have been isolated from sorghum using HPLC. Hahn (1984) used a multistep gradient with acetic acid-water, and butanol-methanol as solvents; and a non-polar C18 column to separate phenolic acids in sorghum. He was able to isolate and identify gallic, protocatechuic, gentinsic, p-hydroxybenzoic, chlorogenic, caffeic, syringic, p-coumaric, sinapic and ferulic acids. To obtain crude phenolic acids extracts, he used aqueous acetone and ethyl acetate for free phenolic acids, and base (NaOH) hydrolysis to extract bound and esterified phenols. Waniska et al (1989) and Gous19 (1989) used similar methods to isolate phenolic acids from sorghum. They additionally identified salicylic, vanillic, sinapic and cinnamic acids from their sorghum samples. However, most phenolic acids in sorghum are bound to cellular matrix and are only extractable in minor quantities without acid/alkaline hydrolysis. Their contribution to antioxidant activity of sorghum extracts may thus be minimal. Schieber et al (2001) used a recently available column (Aqua 5 um C18) coupled with a diode array detector to separate polyphenols in apple juice and pomace. The column was developed for the separation of very polar analytes which were usually not sufficiently retained on common reversed phases. Using acetic acid in water, and acetonitrile as solvents with simultaneous monitoring at 280, 320 and 370 nm, they were able to isolate and identify 26 phenolic compounds including procyanidins, catechin, flavonols and phenolic acids. Similar work using reversed-phases have consistently given fewer peak resolutions (Lopez et al 2001, Martinez-Ortega et al 2001, Mateos et al 2001, Ferrara et al 2001). Coupling of HPLC and UV-visible spectroscopy (HPLC/photodiode array detection) has been the most common method to identify and quantify anthocyanins (Anderson 1985, Dallas et al 1996, Garcia-Viguera et al 1997, Wang et al 2000). Wang, et al (2000) identified 13 anthocyanins in highbush blueberries using a reversed phase column with a photodiode array detector. They used aqueous formic acid and 100% methanol as solvents. Revilla et al (2001) on the other hand used acetonitrile in water at pH 1.3, and perchloric acid as solvents for determination of anthocyanins in red grapes and red wine, They were able to obtain 15 peaks with 9 identified anthocyanins. Use of20 formic acid and acetonitrile as solvents also gave a good resolution and identification of 15 anthocyanins and anthocyanin esters in red table grapes (Carrefto et al 1997). Various solvent systems are apparently effective for separation of anthocyanins in HPLC systems, However, quantitative comparison of results may be a problem Evaluating antioxidant activity of phenolic compounds Numerous methods are used to evaluate antioxidant activities of phenolic compounds in foods or biological systems. Most antioxidant activity assays consist of accelerating oxidation in a lipid system, usually by heat and excess oxygen supply, then monitoring oxygen consumption, substrate loss or product formation (Bondet et al 1997, Fukumoto and Mazza 2000). Because many factors affect oxidation, including temperature, oxygen pressure, metal catalyst, composition and form of fat, results vary depending on oxidation condition used (Frankel 1993). Assays measuring substrates or products can also give varying results depending on their specificity. Moreover, such methods are not always representative of the natural evolution of lipids in foods (Frankel 1993), Also, for many antioxidants, the risk of degradation during testing is high (Bondet et al 1997), Osawa and Shibamoto (1992) developed a HPLC method to measure malonaldehyde formed in lipid emulsion systems oxidized by FexCly/H20>, Malonaldehyde was derivatized by reaction with urea under acidic conditions to form 2- hydroxypytimidine which could be measured by HPLC. This method was used by Tsuda et al (1994) to measure malonaldehyde formed in various lipid systems, and by Fukumoto and Mazza (2000) to measure antioxidant and prooxidant activity of phenolic24 compounds. However, in the latter case, sensitivity of HPLC method was poor relative to free radical methods. Beta-carotene bleaching is a quick and simple method to measure antioxidant activity, Antioxidant activity is measured by the ability of a compound to minimize loss of B-carotene during the coupled oxidation of linoleic acid and i-carotene in an emulsified aqueous system. The reaction is usually initiated using heat (50°C) (Fukumoto and Mazza 2000). The method has good sensitivity, but is non-specific and is subject (o interference from oxidizing and reducing agents in crude extracts, and the linoleic acid used is also not representative of typical food lipids (Frankel 1993). ‘Two free radicals that are commonly used to assess antioxidant activity are 2,2 azinobis (3-ethyl-benzothiaziline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1- picrylhydrazyl (DPPH"), The ABTS assay measures the relative ability of antioxidant to scavenge the ABTS" generated in aqueous phase, compared with a Trolox (vitamin E water-soluble analog) standard. The ABTS" is generated by reacting a strong oxidizing agent (c.g., potassium permanganate or potassium persulfate) with the ABTS salt. The reduction of the blue-green ABTS” radical by hydrogen-donating antioxidant is measured by the suppression of its characteristic long wave absorption spectrum. Antioxidant reduces absorbance of the radical cation (ABTS") to an extent and on a 1e-scale dependent on the antioxidant capacity of the substance being investigated (Miller and Rice-Evans 1997), Results are usually expressed as Trolox equivalent antioxidant capacity (TEAC). The method is rapid and can be used over a wide range ofpH (Amao ct al 1999, Lemanska et al 2001), and in both aqueous and organic solvent systems. It also has good repeatability, and hence is widely reported. The method however, has not been correlated with biological effects, and hence its actual relevance to in vivo antioxidant effects of vi ‘The DPPH is a stable free radical with an absorption band at 515 nm. It loses this absorption when reduced by an antioxidant; (DPPH* + A > DPPHH + A*) or a free radical species; (DPPH’ + R* > DPPH-R). ‘The DPPH’ method is widely used to determine antiradical/antioxidant activity of purified phenolic compounds as well as natural phenol extracts (Brand-Williams et al 1995, Sripriya et al 1996, Bondet et al 1997, Mahinda and Shahidi 2000, Peyrat-Maillard et al 2000, Fukumoto and Mazza 2000). Brand-Williams et al (1995) observed three reaction kinetics for reaction of DPPH" with different phenolic compounds, The first kinetic reaction proceeded quickly, reaching a steady state immediately; the second kinetic reaction was a bit slower, reaching a steady state in 30 min. The third kinetic reaction involved longer periods of time, reaching a steady state in 1-6 hr, which suggests a complex reaction mechanism (Bondet et al 1997). Most of the phenols they tested followed the third kinetics. This suggests that antioxidant activity using DPPH” should be evaluated over time. The method also has good repeatability and is widely reported. However, like ABTS, its relevance to biological systems is unknown.