CHARACTERIZATION of SELECTED PHENOLIC COMPOUNDS in GEORGIA-GROWN MUSCADINE GRAPES by EDUARDO PASTRANA-BONILLA (Under the Direct

CHARACTERIZATION OF SELECTED PHENOLIC COMPOUNDS

IN GEORGIA-GROWN MUSCADINE GRAPES

EDUARDO PASTRANA-BONILLA

(Under the Direction of CASIMIR C. AKOH)

ABSTRACT

Muscadine grapes (Vitis rotundifolia Michx.) are indigenous to the southeastern United

States but they grow wild from Delaware to the Gulf of Mexico. The increasing interest in grapes and wine is due to their antioxidant properties and claimed health effects. This interest has expanded to the muscadines as potential nutraceutical products. Major polyphenolics in muscadine grapes were determined by HPLC. Total phenolic content of grapes and leaves was determined by the Folin-Ciocalteu reagent method. Total antioxidant capacity was assessed as TEAC (trolox equivalent antioxidant capacity), and total anthocyanin content was determined spectrophotometrically by a pH differential method. The stability of unpasteurized muscadine grape juices, as affected by temperature and addition of ascorbic acid, was examined by following the kinetics of antioxidant capacity, total phenolic content, and total and individual anthocyanins during an 8 week storage study.

Ellagic acid, myricetin, quercetin, kaempferol, resveratrol, the diglucosides of delphinidin, petunidin, malvidin, cyanindin, and peonidin, as well as petunidin monoglucoside were found in grape skins. Gallic acid, catechin, epicatechin, and

delphinidin-3,5-diglucoside were found in seeds. Phenolics in muscadine leaves were myricetin, quercetin, gallic acid, ellagic acid and kaempferol with average concentrations of 213, 15, 28, 10 and 3% higher than in fruits, respectively. Phenolics in seeds contributed 75% of the total phenolic content in grapes and accounted for 95% of the total antioxidant capacity of the fruits. Leaves had on average, 667% more antioxidant capacity than the fruits. Anthocyanins accounted for about 11.5% of the total phenolics in grapes. Delphinin-3,5-diglucoside, petunidin-3,5-diglucoside and malvidin-3,5- diglucoside were the most abundant anthocyanins in the fruits. The addition of ascorbic acid negatively affected the stability of anthocyanins in juices and was accentuated by high storage temperature. Added ascorbic acid increased the antioxidant capacity of muscadine juices but interfered with the Folin-Ciocalteu method of determination of total phenolics. The by-products of muscadine processing and production like seeds, pomace, and leaves have the potential to be used as nutraceuticals due to their high content of polyphenolics.

INDEX WORDS: Anthocyanins, antioxidant capacity, muscadine grapes, muscadine juice, nutraceuticals, phenolics, TEAC, Vitis rotundifolia Michx.

CHARACTERIZATION OF SELECTED PHENOLIC COMPOUNDS

IN GEORGIA-GROWN MUSCADINE GRAPES

EDUARDO PASTRANA-BONILLA

Ingeniero Agrícola, Universidad Surcolombiana, Neiva, Colombia, 1983

M.S., California State University, Fresno, USA, 1995

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2003

Eduardo Pastrana-Bonilla

CHARACTERIZATION OF SELECTED PHENOLIC COMPOUNDS

IN GEORGIA-GROWN MUSCADINE GRAPES

EDUARDO PASTRANA-BONILLA

Major Professor: Casimir C. Akoh

Committee: I. Jonathan Amster Ronald R. Eitenmiller William L. Kerr Philip E. Koehler

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia May 2003

DEDICATION

To my wife, Gloria, whose patience and love gave me the strength and hope to make this dream come true.

To my daughters, Maria Paula and Maria Carolina, who are my motivation.

To my parents, Carlos and Soledad, because without their nurturing love and guidance I would not be who I am. They taught me the best of life and encouraged me to pursue higher education to fulfill my personal goals. v

ACKNOWLEDGEMENTS

I am deeply thankful to all people who contributed to the completion of this dissertation. All help was fully appreciated because no assistance was little when goodwill was involved.

I want to especially thank my major professor, Dr. Casimir C. Akoh, not only for supporting me in the accomplishment of this doctoral program but also for showing me such great empathy and sensibility to understand my personal goals and needs.

I express also my deep appreciation to each professor that served on my advisory committee, Dr. Jonathan Amster, Dr. Ronald Eitenmiller, Dr. Philip Koehler and Dr.

William Kerr.

I wish to thank Dr. Subramani Sellappan and Ms. Brenda Jennings for their technical assistance.

I want to thank the Fulbright-Colciencias-IIE program for its financial and unconditional personal support.

Last but not least, thanks to the faculty and staff of the Department of Food

Science and Technology for being part of my graduate student life and for their contributions in my personal and professional development. vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... x

CHAPTER

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 4

3 PHENOLIC CONTENT AND ANTIOXIDANT CAPACITY OF

MUSCADINE GRAPES ...... 40

4 IDENTIFICATION AND QUANTIFICATION OF ANTHOCYANINS IN

MUSCADINE GRAPES BY HPLC AND HPLC-MS...... 67

5 CHANGES IN ANTHOCYANINS, TOTAL PHENOLICS AND

ANTIOXIDANT CAPACITY DURING STORAGE OF JUICES FROM

MUSCADINE GRAPES GROWN IN SOUTH GEORGIA ...... 89

6 SUMMARY AND CONCLUSIONS ...... 119

vii

LIST OF TABLES

Page

Table 3.1: Phenolics in muscadine grape parts...... 55

Table 3.2: Phenolics in muscadine grapes ...... 56

Table 3.3: Major phenolics in muscadine leaves ...... 57

Table 3.4: Total phenolics, total anthocyanins, and antioxidant capacity of muscadine grape parts...... 58

Table 3.5: Total phenolics (TPH), total anthocyanins (TAC), and Trolox equivalent antioxidant capacity (TEAC) of muscadine grapes and leaves...... 59

Table 3.6: Dry matter of muscadine grape fruits and fruit parts...... 60

Table 4.1: Anthocyanins in muscadine grape skins ...... 80

Table 4.2: Delphinidin-3,5-diglucoside in seeds and pulps...... 81

Table 4.3: Anthocyanins in muscadine grapes ...... 82

Table 4.4: Total anthocyanins in muscadine grape parts...... 83

Table 4.5: Total anthocyanin content of some berries...... 84

Table 5.1: Initial values of trolox equivalent antioxidant capacity (TEAC) and total phenolics for muscadine juices under different treatments...... 108

Table 5.2: Percentage of reduction of antioxidant capacity and total phenolics under different treatments ...... 109

Table 5.3: Initial content of total anthocyanins and individual anthocyanins in refrigerated muscadine juices as affected by ascorbic acid...... 110 viii

Table 5.4: Initial content of total anthocyanins and individual anthocyanins in muscadine juices stored at room temperature as affected by ascorbic acid ...... 111

Table 5.5: Percentage of reduction of total anthocyanins and individual anthocyanins during 8 week storage under different treatments...... 112

LIST OF FIGURES

Page

Figure 2.1: Shikimic acid pathway...... 6

Figure 2.2: Basic structure of flavonoids...... 7

Figure 2.3: Structure of naturally occurring anthocyanidins and their substitution pattern8

Figure 2.4: Effect of pH on malvidin-3-glucoside...... 9

Figure 2.5: Structures of the main phenolic compounds in muscadine grapes...... 16

Figure 3.1: Diode-array spectra of ellagic acid and trans-resveratrol for standards and a sample of the skin of the cultivar “Carlos.”...... 61

Figure 3.2: HPLC chromatograms of selected standards for muscadine skin analysis, at

260 nm (DAD1 A), 360 nm (DAD1 D), 313 nm (DAD1 E), and fluorescence detection at

Ex=330 and Em= 374 (FLD1 A) of ellagic acid (1), myricetin (2), trans-resveratrol (3), quercetin (4), and kaempferol...... 63

Figure 3.3: HPLC chromatograms of the skin of grapes of the cultivar “Carlos,” at 260 nm (DAD1 A), 360 nm (DAD1 D), 313 nm (DAD1 E), and fluorescence detection at

Ex=330 and Em= 374 (FLD1 A) of ellagic acid (1), myricetin (2), trans-resveratrol (3), quercetin (4), and kaempferol (5)...... 65 x

Figure 4.1: (A) HPLC chromatogram at 520 nm of anthocyanin monoglucosides standards: delphinidin-3-glucoside (1), cyanidin-3-glucoside (2), petunidin-3-glucoside

(3), peonidin-3-glucoside (4), malvidin-3-glucoside (5). (B) HPLC chromatogram at 520 nm of the anthocyanins found in the skin of the grapes of the cultivar, Paulk: delphinidin-

3,5-diglucoside (6), cyanidin-3,5-diglucoside (7), petunidin-3,5-diglucoside (8), malvidin-3,5-diglucoside (9), peonidin-3,5-diglucoside (10)...... 85

Figure 4.2: Integrated mass spectrum of the molecular ions found in a skin sample of the

Paulk cultivar. Petunidin-3-glucoside (A), cyanidin-3,5-diglucoside (B), peonidin-3,5- diglucoside (C), delphinidin-3,5-diglucoside (D), petunidin-3,5-diglucoside (E), and malvidin-3,5-diglucoside (F). Other peaks were not identified...... 87

Figure 5.1: Kinetics of antioxidant capacity for cultivar “Supreme.”...... 113

Figure 5.2: Kinetics of the total phenolics content for cultivar “Supreme.” ...... 115

Figure 5.3: Kinetics of the total anthocyanins for cultivar “Supreme.”...... 117 1

CHAPTER 1

INTRODUCTION

Muscadine grape (Vitis rotundifolia Michx) is an important food crop indigenous to the southeastern United States. As member of the Vitis family, it may share with other grapes a high content of phenolic compounds which have received increased attention because of their potential antioxidant activities and positive health effects on humans.

Phenolic compounds are among the most talked about dietary ingredients these days. Phenolic compounds are a class of phytochemicals found in high concentrations in wine, tea, grapes and a wide variety of other plants and have been associated with heart disease and cancer prevention (1). In general terms, phenolic compounds or polyphenolics, have a similar basic structural chemistry including at least a phenolic ring.

Over 4,000 phenolic compounds have been identified in a dozen chemical sub-categories

(2). Phenolic compounds are responsible for the brightly colored pigments of many fruits and vegetables, but also they protect plants from diseases and ultraviolet light, and help prevent damage to seeds until they germinate (3).

Numerous in vitro studies have shown that polyphenolic compounds are powerful antioxidants that can protect cell membranes and cellular DNA from the effects of free radical induced oxidative damage. Several epidemiological studies have indicated that regular consumption of foods rich in polyphenolic compounds (fruits, vegetables, nuts, whole grain cereals, red wine, green tea) is associated with reduced risk of developing cardiovascular disease and certain cancers (4,5). Experimental studies in both animals

2 and humans have shown that increasing polyphenol intake can protect LDL cholesterol from becoming oxidized (a key step in developing atherosclerosis), lower blood pressure in hypertensive subjects, reduce the tendency of the blood to clot and elevate total antioxidant capacity of the blood (6,7).

Six chapters compose this dissertation including the introduction, summary and conclusions. The second chapter presents a literature review of topics related to muscadine grapes, phenolic compounds and their claimed health effects. The third chapter presents the identification and quantification of the major phenolic compounds

(excluding anthocyanins) in the skins, pulps, seeds and leaves of ten muscadine grape cultivars. Total antioxidant capacity, total phenolic content and total anthocyanins in each fruit part and in leaves were also determined. All values were calculated on fresh weight basis but the dry weight was also determined in order to facilitate the comparison of the results to the results of other researchers.

The fourth chapter deals with the identification and quantification of the total and individual anthocyanins found in the ten studied cultivars of muscadine grapes.

Identification and quantification were performed by HPLC and the identity of the individual anthocyanins was confirmed by HPLC-MS.

The stability of juices from 10 cultivars of muscadine grape was evaluated in the fifth chapter. Changes in total and individual anthocyanins, antioxidant capacity and total phenolics were determined during 8 weeks storage at two different temperatures and two levels of added ascorbic acid.

The sixth chapter summarizes chapters 3, 4 and 5, and presents general conclusions.

LITERATURE CITED

(1) Wildman, R. E. C. Handbook of Nutraceuticals and Functional Foods; CRC

Press: Boca Raton, Fla., 2001.

(2) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. Flavonoid antioxidants: chemistry,

metabolism and structure- activity relationships. J. Nutr. Biochem. 2002, 13, 572-

584.

(3) Kahkonen, M. P.; Hopia, A. I.; Vuorela, H. J.; Rauha, J. P.; Pihlaja, K.; Kujala, T.

S.; Heinonen, M. Antioxidant activity of plant extracts containing phenolic

compounds. J. Agric. Food Chem. 1999, 47, 3954-3962.

(4) Cook, N. C.; Samman, S. Flavonoids - chemistry, metabolism, cardioprotective

effects, and dietary sources. J. Nutr. Biochem. 1995, 7, 66-76.

(5) Di Carlo, G.; Mascolo, N.; Izzo, A. A.; Capasso, F. Flavonoids: old and new

aspects of class natural therapeutic drugs. Life Sci. 1999, 65 (4), 337-353.

(6) Hollman, P. C. H.; Katan, M. B. Dietary flavonoids: intake, health effects and

bioavailability. Food Chem. Toxicol. 1999. 37, 937-942.

(7) Meyer, A. S.; Heinomen, M.; Frankel, E. N. Antioxidant interactions of catechin,

cyanidin, caffeic acid, quercetin, and ellagic acid on human LDL oxidation. Food

Chem. 1988. 61, 71-75.

CHAPTER 2

LITERATURE REVIEW

MUSCADINE GRAPES

Muscadine grape (Vitis rotundifolia Michx.) is indigenous to the southeastern

United States and was described for the first time by the Italian explorer Giovanni de

Verrazzano who while exploring the North Carolina’s Cape Fear River Valley found many vines of the “big white grape” as colonists called it (1). Later, in 1584, Sir Walter

Raleigh’s explorers wrote that the North Carolina coast was abundant in muscadine vines. Bronze muscadine grapes are also called scuppernongs, the name that comes from the surrounding area of the Scuppernong Lake where they were found by two Tyrrell county (NC) hunters in 1775 (2). The scientific name of muscadines is derived from Vitis which is the Latin name for grape and rotundifolia which refers to the roundish leaves that they have (1).

Muscadines are known as American wild grapes and grow only in the hot, humid southeastern region of the country. Unlike other grapes, muscadines are resistant to most fungal and bacterial infections due to the production of antioxidants by the plants.

Botanically, muscadines are different from other Vitis species. The leaves are small, round and hairless. Muscadines grow and ripen in clusters of berries, not in a bunch.

They can also send out air-roots from the vines to gather moisture from the surrounding atmosphere. A muscadine cluster has only a few (6 to 24) berries, which ripen very unevenly and drop off individually when ripe (1). The large berries have thick skin and a

5 thick, slippery pulp that is difficult to press (3). They need a minimum of 200-day growing season, with at least 30 inches of rain, and can be damaged by temperatures below 10°F (3). Muscadines differ from bunch grapes by two chromosomes (2n = 38 for bunch grapes and 2n = 40 for muscadines) (3). The scuppernong variety of muscadine has a tough skin and is bronze-green in color, rather than black or purplish as were its ancestors (1). Its abundant juice is sweet, with a kind of musky, fruity flavor (1). The

State of Georgia is the largest muscadine producer in the United States with about 1100 acres with an average yield of 5 tons per acre (4).

PHENOLIC COMPOUNDS

Phenolic compounds are secondary metabolites in plants that are formed via the shikimic acid pathway, also known as the phenylpropanoid pathway (Figure 2.1) (5).

The shikimic acid pathway begins with the condensation of phosphoenolpyruvate from glycolysis and erythrose-4-phosphate from the pentose phosphate pathway which is catalyzed by 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase. Through a series of reactions shikimic acid is produced, which is converted to chorismate, the final precursor for the synthesis of phenylalanine, tyrosine and tryptophan. Phenylalanine and tyrosine then go on to produce alkaloids and/or phenolics, while tryptophan goes on to produce alkaloids (5).

Phenolic compounds contain at least one phenol group. Flavonoids usually have at least two rings while tannins are polymers of flavonoid units. Flavonoids are perhaps the most common in plant-based foods such as fruits, vegetables, nuts and cocoa.

Flavonoids are benzo-?-pyrone derivatives consisting of phenolic and pyrane rings.

Flavonoids have a characteristic fifteen-carbon backbone ring structure (C6-C3-C6)

(Figure 2.2) consisting of two aromatic rings (labeled A and B), generally each containing a number of substitutions that define their classification (7).

Phosphoenolpyruvate + Erythrose-4-phosphate

Shikimate

Chorismate

ALKALIODS Anthranilate Arogenate

Phenylalanine and ALKALOIDS Tryptophan Tyrosine

ALKALOIDS PHENOLICS

Figure 2.1. Shikimic acid pathway (adapted from reference (6))

Flavanols (catechin, epicatechin, epigallocatechin gallate), flavones (apigenin, rutin), flavonols (quercetin, myricetin, kaempferol), flavonones (naranginin, narangin, taxifolin, hesperidin), isoflavones (genestin, genestein, daidzin, daidzein) and anthocyanidins (cyanidin, malvidin, petunidin) are among the dietary flavonoids family.

Figure 2.2. Basic structure of flavonoids (From reference (8)).

Flavonoids may be employed by plants as visual and olfactory attractants.

Animals are used by these plants for pollination and seed dispersal (9). Simple phenolic compounds have been shown to inhibit the germination and growth of many plants.

Lignin, the most abundant organic substance in plants after cellulose, is produced in response to infection or wounding, as well as in xylem or sclerenchyma cell walls (10).

Anthocyanins are water soluble glycosylated and/or acylated flavonoid derivatives that are the source of most red, pink, purple, and blue colors in plant parts.

The aglycone is referred to as an anthocyanidin. There are 6 commonly occurring anthocyanidin structures: pelargonidin, cyanidin, peonidin, delphinidin, malvidin and petunidin (Figure 2.3) (11). However, anthocyanidins are rarely found in plants - rather they are almost always found as the more stable glycosylated derivatives. Sugars are present most commonly at the C-3 position, while a second site for glycosylation is the

C-5 position and, more rarely, the C-7 position. The sugars that are present include glucose, galactose, rhamnose, and arabinose. Sugars provide additional sites for modification as they may be acylated with acids such as p-coumaric, caffeic, ferulic, sinapic, acetic, malonic or p-hydroxybenzoic acid. Because of the diversity of glycosylation and acylation, there are at least 240 naturally occurring anthocyanins (12).

Anthocyanidin R1 R2 Name moiety moiety pelargonidin H H cyanidin OH H delphinidin OH OH

peonidin OMe H petunidin OMe OH malvidin OMe OMe

Figure 2.3. Structure of naturally occurring anthocyanidins and their substitution

pattern.

The color and stability of an anthocyanin in solution is highly dependent on the pH. They are most stable and most highly colored at low pH values but gradually lose color as the pH is increased. At around pH 4 to 5, the anthocyanin is almost colorless.

This color loss is reversible, and the red hue will return upon acidification. This characteristic limits the application of anthocyanins as a food colorant to products that have a low pH. The loss of red color with pH implies that there is equilibrium between two forms of the anthocyanin. These are the red flavylium cation and the colorless carbinol base. The flavylium cation, as the name implies, has a positive charge associated with it, while the carbinol base is a hydrated form of the anthocyanin (Figure 2.4).

Figure 2.4. Effect of pH on malvidin-3-glucoside (From reference (13))

The epidermis of the leaves of all plants contains flavonoids that protect against

UV-B radiation. These compounds absorb light in the UV-B range but allow visible light to pass through uninterrupted for photosynthesis.

Tannins are polymerized flavonoids and derive their main biochemical properties from the ability to precipitate protein at neutral pH. There are two biosynthetically distinct classes of tannins: the hydrolyzable tannins which are esters of gallic or ellagic acid and glucose; and the condensed tannins (proanthocyanidins). Hydrolyzable tannins are generally antinutritional (14).

PHENOLICS IN GRAPES

Phenolics are important to grape products because they contribute to color, flavor, mouthfeel, oxidation/antioxidant properties and related characteristics of grape products

(15,16,17). The main sources of phenolic compounds in grapes are skins and seeds (18).

The major classes of phenolics that have been identified in grapes include flavonoids such as procyanidins, anthocyanins, as well as non-flavonoid compounds (phenolic acids, stilbenoids) (18,19). Within each family of plant phenols many compounds may exist.

For instance, over 4,000 different flavonoids have been described in plants.

Due to the heterogeneity of phenolics in grape products, phenolics are commonly reported as gallic acid equivalents (GAE) (18). The concentration and type of phenolics present in grape products play an important role in controlling oxidation (18,20).

Phenolics are among the more easily oxidized compounds in grape products (18,21).

Oxidation reactions involving phenolics lead to undesirable changes in flavor and color

(18,22).

Grape cultivar affects the anthocyanin content (18,23,24,25). The effects of season and cultivar on amounts and types of phenolics in grape are significantly greater at earlier stages of ripening and decrease toward harvest (18,26).

Phytoalexins are biologically active compounds produced by plants in response to fungal infection or abiotic stress, such as heavy metal ions or UV light. In grape vines, the stress response includes the synthesis of a simple stilbene, resveratrol (trans-3,5,4’- trihydroxystilbene), and its glucoside (piceid) together with the biosynthetically related compounds viniferin and pterostilebene. These compounds have provoked an intense interest due to their antifungal properties and because they have been shown to be closely associated with grape disease resistance.

Hot press processing of grapes increases the color intensity of grape juices by increasing the extraction of anthocyanins. Heat treatment also increases the extraction of other phenolics and destroys oxidative enzymes (18,32).

METABOLISM OF PHENOLIC COMPOUNDS

The extent of intestinal absorption of phenolics is an important unsolved problem in judging their many alleged health effects. An important feature is that most phenolics are present in plants as glycosides or as esters of organic acids. Flavonoids are mostly present in foods as glycosides and they were considered non-absorbable. Only free flavonoids without a sugar molecule, the so-called aglycones, were thought to be able to pass through the gut wall (27). Hydrolysis only was thought to occur in the colon by microorganisms, which at the same time degrade flavonoids (28). However, human studies showed that glycosylation may enhance absorption depending on the type of sugar. It was found that the absorption of a flavonol glucoside was more than 50% (29).

Pharmacokinetic studies comparing various dietary flavonol glycosides and catechins showed marked differences in absorption rate and bioavailability. The flavonol quercetin was eliminated only slowly from the blood (half-life 20 h), whereas catechins were eliminated quickly (half-life 2-4 h) (30). The metabolism of flavonoids has been studied frequently in various animals, but very few data in humans are available. It is clear that flavonols are very extensively metabolized in humans; only about 1% or less of the ingested dose was excreted in urine having an intact flavonoid backbone (31). Major sites of metabolism are the small intestine wall, the liver and the colonic flora. There is evidence for methylation, sulfation and glucuronidation and glycination of hydroxyl groups. Bacterial ring fission of flavonoids occurs in the colon. The subsequent

12 degradation products, phenolic acids, can be absorbed and are found in urine of animals

(8).

MAJOR PHENOLIC COMPOUNDS FOUND IN MUSCADINE GRAPES

AND THEIR CLAIMED HEALTH EFFECTS

Anthocyanins (Figure 2.3) Recently, there has been interest in anthocyanins due to their activity as antioxidants. The antioxidant effect of cyanidin-3-glucoside on low density lipoproteins (LDL) was evaluated by Amorini et al. (33). They found that the anthocyanin inhibited malondialdehyde generation (an index of lipid peroxidation), and the inhibition was significantly higher than that obtained with equal concentrations of resveratrol and ascorbic acid. Blueberries have larger concentrations of anthocyanins than other berries (34). Anthocyanins in blueberries have been studied because their potential to prevent cancer and retard the effects of aging, particularly loss of memory and motor skills. Galli et al. (35) found that plant extracts rich in polyphenols can retard and even reverse age-related effects in brain function and in cognitive and motor performance in rats. Anthocyanins were found to show the most efficacy in penetrating the cell membrane and providing antioxidant protection. They showed that rats fed with diets containing high anthocyanin blueberry extract had smaller changes in brain neurotransmitter function than those fed other diets, resulting in improved cognitive and motor functions (35). Youdim et al. (36) examined the anti-oxidative and anti- inflammatory properties of the two main polyphenolic families in blueberries and cranberries, anthocyanins and hydroxycinnamic acids. They found that both provided equal protection to lipid peroxidation, but anthocyanins were more protective against

13 inflammatory events, despite the fact that the antioxidant capacity of hydroxycinnamic acids was twice that of anthocyanins, indicating that anthocyanins were more biologically active. Cancer research performed by Smith et al. (37) showed that the flavonoid components in wild blueberries inhibit an enzyme involved in the promotion stage of cancer. The researchers applied extracts from wild blueberries, cultivated blueberries and

European bilberries to living cells at various stages in the development of liver cancer.

Wild blueberries exhibited some of the greatest anti-cancer activity of all the berries examined (37). Katsube et al. (38) found that pure standards of the anthocyanins, delphinidin and malvidin, and the ones isolated from bilberry extracts inhibited the growth of human leukemia cells but only delphinidin was able to inhibit the growth of human colon carcinoma cells in vitro.

Quercetin (3,3’,4’,5,7-Pentahydroxyflavone) (Figure 2.5A) is found in a wide variety of plants and is the most abundant of the flavonoid molecules. It is found in many foods, including apple, onion, tea, berries, and brassica vegetables, as well as many seeds, nuts, flowers, barks, and leaves (39). It is also found in medicinal botanicals, including

Ginkgo biloba (40). It is often a major component of the medicinal activity of the plant, and has been shown in experimental studies to have numerous effects on the body (39).

Quercetin is the aglycone of a number of other flavonoids, including rutin, quercetrin, and isoquercetin. Activity comparison studies have identified other flavonoids as often having similar effects as quercetin, but quercetin usually has the greatest activity

(41). Quercetin appears to have many beneficial effects on human health, including cardiovascular protection, anti-cancer activity, anti-ulcer effects, anti-allergy activity, cataract prevention, antiviral activity, and anti-inflammatory effects. Quercetin's anti-

14 inflammatory activity appears to be due to its antioxidant and inhibitory effects on inflammation-producing enzymes (cyclooxygenase, lipoxygenase) and the subsequent inhibition of inflammatory mediators, including leukotrienes and prostaglandins (42).

Inhibition of histamine release by mast cells and basophils also contributes to quercetin's anti-inflammatory activity (43). Aldose reductase, the enzyme which catalyzes the conversion of glucose to sorbitol, is especially important in the eye, and plays a part in the formation of diabetic cataracts. Quercetin is a strong inhibitor of human lens aldose reductase (44). Quercetin exerts antiviral activity against reverse transcriptase of HIV and other retroviruses, and was shown to reduce the infectivity and cellular replication of

Herpes simplex virus type 1, polio-virus type 1, parainfluenza virus type 3, and respiratory syncytial virus (45). Quercetin is an anticarcinogen to numerous cancer cell types, including breast (46,47), leukemia (48,49), colon (50,51), ovary (52,53), squamous cell (54,55), endometrial (56), gastric (57,58), and lung (59,60).

Quercetin inhibits the formation of the inflammatory mediators known as prostaglandins and leukotrienes, as well as histamine release and is especially helpful in asthma (61). Quercetin's inhibition of xanthine oxidase decreases the formation of uric acid, and thus it may be of value in the treatment of gout (62). Quercetin has been shown to inhibit growth of Helicobacter pylori in a dose-dependent manner in vitro (63), resulting in an anti-ulcer effect. Quercetin's cardiovascular effects center on its antioxidant and anti-inflammatory activity, and its ability to inhibit platelet aggregation ex vivo (64). The Zutphen elderly study investigated dietary flavonoid intake and risk of coronary heart disease (27). The risk of heart disease mortality decreased significantly as flavonoid intake increased. Individuals in the upper 25 percent of flavonoid intake had a

15 relative risk of 0.42 compared to the lowest 25 percent in the 5-year follow-up study of men ages 65-84. Tea was used as flavonoid source and quercetin is the major flavonoid in tea (65). In a cohort of the same study, dietary flavonoids (mainly quercetin) were inversely associated with stroke incidence (66).

Few human quercetin absorption studies exist. It appears that only a small percentage of quercetin is absorbed after an oral dose, possibly only two percent, according to one study (67). A study of absorption in ileostomy patients revealed an absorption of 24 percent of the pure aglycone and 52 percent of quercetin glycosides from onions (68). Quercetin undergoes bacterial metabolism in the intestinal tract, and is converted into phenolic acids. Absorbed quercetin is transported to the liver bound to albumin, where some may be converted via methylation, hydroxylation, or conjugation

(69).

Kaempferol (3,4’,5,7-Tetrahydroxyflavone) (Figure 2.5B) has been reported to exhibit similar effects as quercetin. Most of the reports cite kaempferol and myricetin next to quercetin in health effects due to their antioxidative properties. Kaempferol can be biotransformed to quercetin in the presence of a metabolizing system in pigs and rats

(28,70). The name kaempferol was given to this flavonol in honor of Engelbert

Kaempfer, a German physician and botanist who was in Japan from 1690 until 1692 where he discovered Ginkgo trees and described the Ginkgo tree in his work

"Amoenitatum exoticarum" (71).

(A) Quercetin (B) Kaempferol

(E) Ellagic acid (F) Resveratrol

(G) Catechin (H) Epicatechin

Figure 2.5. Structures of the main phenolic compounds in muscadine grapes.

Kaempferol showed a remarkable decrease in systolic, diastolic, mean arterial blood pressure and heart rate of rats (72). In a nutritional study, the association between flavonoid intake and risk of several chronic diseases was examined. Persons with higher quercetin intake had lower mortality from ischemic heart disease and persons with higher kaempferol intake had lower incidence of cerebrovascular disease, while a trend toward a reduction in risk of type 2 diabetes was associated with higher quercetin and myricetin intakes (73). The Rotterdam study examined the association of tea and flavonoid intake with incident of myocardial infarction in the Dutch population. The intake of the dietary flavonoids quercetin, kaempferol and myricetin was significantly inversely associated with fatal myocardial infarction (74).

The anticarcinogenic effects of kaempferol are reported to be similar to that exerted by quercetin and myricetin. However, Garcia et al. (75) found no evidence of the claimed protective effect of kaempferol against bladder cancer. Additionally, some recent studies have shown a prooxidant activity of dietary flavonoids when they were metabolized by peroxidase to form phenoxyl radicals (76). The formation of reactive oxygen species, in the presence of copper ion, can damage DNA resulting in various biological processes such as mutagenesis, aging, carcinogenesis, and microbial inactivation (77).

Myricetin (3,3’,4 ,5,5’,7-Hexahydroxyflavone) (Figure 2.5C) was found in this dissertation to be the most abundant flavonol in muscadine grapes. In addition to the reported general effects of flavonols, myricetin has been found to have antimicrobial properties and effectively inhibited the growth of Gram-negative bacteria, but was ineffective against Gram-positive microorganisms (78). Morel et al. (79) and Abalea et

18 al. (80) demonstrated that in iron-treated hepatocyte cultures, myricetin was able to prevent the accumulation of oxidized bases in DNA of the cells. This action resulted from both the antioxidant activity which decreases the generation of DNA oxidation products and from an activation of repair mechanisms, eliminating oxidized products already formed in DNA. These results contrast with the one obtained by Sahu and Gray (81) who found that myricetin induced DNA degradation in vitro was enhanced in the presence of iron. The flavonols myricetin, quercetin and kaempferol found in grape juice were able to inhibit platelet aggregation and they may have some protection against the development of coronary artery disease and acute occlusive thrombosis (82). An extract of the bark of red bayberry, with myricetin as main component, was found to have in vivo anti-androgenic effect in castrated hamsters and in testosterone treated mice (83).

Myricetin was found to be less effective than quercetin and kaempferol in growth retardation of androgen-independent human prostatic tumor cells. Myricetin was the most effective flavonoid in reducing lipid oxidation of refined, bleached and deodorized marine oils and its effect was similar to that of tertiary-butylhydroquinone (TBHQ) (84).

Myricetin was able to stimulate lipogenesis and enhance insulin-stimulated lipogenesis in isolated rat adipocytes, showing a protective antidiabetic effect (85).

Gallic acid (3,4,5-Trihydroxybenzoic acid) (Figure 2.5D) is a plant phenolic found in green tea and grape seeds. In screening for anti-cancer agents, gallic acid was found to show cytotoxicity against all cancer cells that were examined (86,87,88,89).

Additionally, gallic acid did not harm healthy cells, but was able to distinguish between normal cells and cancer cells. A study of structurally related compounds suggested that the cytotoxicity shown by gallic acid was not a common feature in phenolic compounds,

19 but was a fairly specific characteristic of gallic acid. Three adjacent phenolic hydroxyl groups of gallic acid were responsible for the cytotoxicity, and the carboxyl group was not responsible, but seemed to be implicated in distinguishing between normal cells and cancer cells (90). Frankel et al. (91) found that gallic acid has higher antioxidant activity compared to other polyphenols found in red wine. Additionally, gallic acid has antibacterial (92,93,94), anti-inflammatory (95), and anti-asthmatic (96,97) properties.

Ellagic acid (4,4’,5,5’,6,6’-Hexahydroxydiphenic acid 2,6,2’,6’-dilactone)

(Figure 2.5E) has not been reported in grape species other than muscadine (18,98).

Ellagic acid is also found in blackberry, strawberry, raspberry, bayberry, fueijoa, pineapple and pomegranate (99). Hot press process increases the extraction of ellagic acid when compared to other methods of juice and must preparation (18). Research in animal and laboratory models has found that ellagic acid inhibits the growth of tumors caused by certain carcinogens (100,101). Akagi et al. (102) analyzed the effect of ellagic acid on different kinds of cancer in rats and found that ellagic acid had a moderate inhibition effect on small intestinal cancer and weak inhibition effect on lung cancer, with no effect on stomach, liver, large intestinal and kidney cancer. Small intestinal cancer is not frequent in humans, so ellagic acid may not be very promising as a chemopreventor of colon cancer. Studies in humans are underway to determine the effect of long-term daily consumption of raspberries on cell activity in the human colon. The Hollings

Cancer Institute of the Medical University of South Carolina has examined the ability of ellagic acid to prevent colon and cervical cancers from developing (103). Ellagic acid is abuntantly present in whisky. Iino et al. (104) found that whisky, due to the antioxidant activity of ellagic acid, is less irritant to the rat gastric mucosa than ethanol.

Ellagic acid has been found to cause apoptosis in cancer cells in the laboratory

(105,106,107). An animal study found that ellagic acid protected mice against chromosome damage from radiation therapy (108). Two separate studies of ellagic acid indicated that it was effective at inhibiting tumor growth from esophageal cancer cells in mice (109,110). Ellagic acid prevents the binding of carcinogens to DNA, and strengthens connective tissue, which may keep cancer cells from spreading

(109,111,112). Ellagic acid has also been said to reduce heart disease (113,114), birth defects (115), and lung fibrosis in rats (116).

Resveratrol (3,4’,5-Trihydroxy-trans-stilbene) (Figure 2.5F) is a phytoalexin found in grapes and other food products. It is formed as a response to the phytopathogenic fungus Botrytis cirinea. It has been shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis.

Resveratrol was found to act as an antioxidant, antimutagen, and to induce phase II drug- metabolizing enzymes (anti-initiation activity) (117); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity) (118); and it induced human promyelocytic leukemia cell differentiation

(antiprogression activity) (119,120). In addition, it inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture (121) and inhibited tumorigenesis in a mouse skin cancer model (122). Resveratrol was also found to have cardioprotective effects and it was related directly to the so called “French

Paradox.” Resveratrol and other polyphenolics in wine, provide cardioprotection by their ability to function as in vivo antioxidants while its alcoholic component by itself imparts cardioprotection by adapting the hearts to oxidative stress (123). The action of resveratrol

21 as cardioprotective agent may be attributed to its peroxyl radical scavenging activity

(124). In addition, resveratrol was effective in protecting LDL against oxidation by decreasing the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin

(125).

(+)-Catechin ((+)-trans-3,3',4',5,7-Pentahydroxyflavane) (Figure 2.5G) and

(-)-epichatechin (cis-3,3',4',5,7-Pentahydroxyflavane) (Figure 2.5H) possess numerous biological activities including antimutagenic, antibacterial, hypocholesterolemic, antioxidant, antitumor and cancer preventive activities. A study on the survival of the fly

Drosophila melanogaster under a pesticide as source of the superoxide anion, found that the survival ratio of flies fed on a diet enriched with the superoxide scavenging antioxidants cathechin and epicathechin ranged from 77 to 87% (126). The bark of

Saraka asoca, a commonly used ingredient in the traditional Indian ayurvedic medicine, was found to inhibit the growth of skin cancer cells in mice; the active ingredient responsible for the antitumour/anticarcinogenic activity of the plant preparation was epichatechin (127). An experiment with an extract of green tea whose major compound was identified as epicatechin, showed that green tea inhibited the mutagenicity and/or chromosomal damage caused by different carcinogens in both bacterial and mammalian cells (128). The Saudi Coronary Artery Disease study found that those who drink 6 or more cups of tea per day had a significantly lower prevalence of coronary heart disease than the none tea drinkers (129). Epicatechin was reported to have insulin mimetic action with protective effects on erythrocytes in a manner similar to insulin (130). Catechins are also reported to inhibit alpha-amylase, thereby preventing the digestion and absorption of carbohydrates that would otherwise contribute to increased blood sugar levels (131).

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CHAPTER 3

PHENOLIC CONTENT AND ANTIOXIDANT CAPACITY OF MUSCADINE

GRAPES

______Eduardo Pastrana-Bonilla, Casimir C. Akoh, Subramani Sellappan and Gerard Krewer. Submitted to the Journal of Agricultural and Food Chemistry, January 23, 2003.

ABSTRACT

Fruits of ten cultivars of muscadine grapes (five bronze-skin and five purple-skin) were separated into skin, seed and pulp. Each fruit part, as well as leaves from the corresponding varieties, were extracted for HPLC analysis of major phenolics. Total phenolics were determined colorimetrically using Folin-Ciocalteu reagent. Total anthocyanins were determined by a pH-differential method, using a UV-visible spectrophotometer. Antioxidant capacity was determined by the TEAC (Trolox equivalent antioxidant capacity) assay. Gallic acid, (+)catechin and epicatechin were the major phenolics is seeds with average values of 6.56, 789 and 1234 mg/100 g fresh weight (FW), respectively. In muscadine skins ellagic acid, myricetin, quercetin, kaempferol and trans-resveratrol were the major phenolics with respective average values of 16.5, 8.34, 1.74, 0.64 and 0.14 mg/100 g FW. Contrary to other previous reports, ellagic acid and not resveratrol was the major phenolic in muscadine grapes. HPLC solvent system in addition to fluorescence detection allowed separation and confirmation of ellagic acid from resveratrol. This paper includes the phenolic content and antioxidant capacity of muscadine leaves, which have not been previously reported. Major phenolics in muscadine leaves were myricetin, ellagic acid, kaempferol, quercetin and gallic acid with average concentrations of 157.6, 66.9, 8.9, 9.8 and 8.6 mg/100 g FW, respectively.

Average total phenolics were 21.78, 3.75, 0.24, and 368.8 mg/g gallic acid equivalent

(G.A.E.) in seed, skin, pulp, and leaves, respectively. Total anthocyanins contents were

1.34 and 118 mg/100 g FW, in skins of bronze and purple grapes respectively; 4.4 and

1.0 mg/100 g FW in seeds and pulps, in that order. Antioxidant capacity values were, on

42 average, 1.0, 11.4, 285 and 238.9 µM TEAC/g FW for pulps, skins, seeds, and leaves, respectively.

Keywords: Vitis rotundifolia, muscadine grapes, phenolics, anthocyanins, antioxidant capacity

INTRODUCTION

Muscadine grapes (Vitis rotundifolia Michx.) are indigenous to the southeastern

United States. Muscadines are vigorous vines that may grow up to 100 ft in the wild.

They differ botanically from other grapes and are placed in a separate sub-genus,

Muscadinia. Muscadine fruits are round, 1 to 1 ½ inch in diameter with thick, tough skin and may have up to 5 seeds. The Georgia Agricultural Experiment Station and the U.S.

Department of Agriculture have introduced a number of improved varieties that currently are standard cultivars (1). Plants contain a large variety of phytonutrients, many having antioxidant properties. Antioxidant compounds include vitamins, phenolic compunds, and carotenoids. Among the phenolic compounds, flavones, isoflavones, flavonones, flavonols, anthocyanins and catechins are the most important, and exhibit substantial antioxidant activity (2,3), that may prevent the incidence of cardiovascular disease (4).

Phenolics are secondary plant metabolites and are widely found in majority of fruits, vegetables, and teas (5). Even though plants are the basis of most traditional medicinal therapy (6), the positive effect of antioxidants found in fruits and vegetables was demonstrated, among others, by Ames et al. (7) and Hertog et al. (8,9,10,11), and from that point a good number of papers have shown the impact of plant antioxidants on human health. Just in recent years, many studies have demonstrated that free radicals are

43 the leading cause of degenerative diseases like several forms of cancer, cardiovascular disease, and neurological diseases (12). Plant antioxidants work as singlet and triplet oxygen quenchers, free radical scavengers, peroxide decomposers, and enzyme inhibitors

(13). Many of their protective biological effects are derived from their antioxidant functions (14). There is interest in knowing the phenolic content of fruits in order to envision their potential use as nutraceuticals or functional foods.

There are few research papers on phenolic compounds content of muscadine grapes. Ector et al. (15), Meepagala et al. (16), Goldy et al. (17), and lately Talcott and

Lee (18), are some of the reports on the phenolic content of muscadine grapes. Studies from our laboratory represent one of the few attempts to measure the polyphenolic content of muscadines and their antioxidant capacity. The objective of this study is to determine the major phenolic compounds found in grapes and leaves, their total polyphenolic content, as well as the antioxidant capacity of the muscadine fruits and leaves.

MATERIALS AND METHODS

Chemicals. Pure standards of ((+) catechin, (-) epicatechin, gallic acid, ellagic acid, myricetin, quercetin, kaempferol and trans-resveratrol, were purchased from Fluka

(Milwaukee, WI) and Sigma (St. Louis, MO). Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), and ABTS (2,2’-azinobis(3-ethylbenzothiazoline

6-sulfonic acid) diammonium salt were purchased from Fluka (Milwaukee, WI).

Acetonitrile, methanol, and water (HPLC grade) were purchased from Fisher Scientific

(Norcross, GA)

Samples. Fruits and leaves from ten muscadine grape cultivars, five bronze

(Carlos, Early Fry, Fry, Summit and Late Fry), and five purple (Paulk, Cowart, Supreme,

Ison and Noble), grown in South Georgia and provided by Mr. Jacob Paulk (Paulk

Vineyards, Wray, GA) were studied. Fruits were separated into skins, seeds and pulps, and extracted, in triplicate, for the corresponding analysis as described below.

Major phenolics. Each sample was mashed, using mortar and pestle, to a very fine paste and diluted with 80% methanol in 6 N HCl. The samples were vortexed for 1 minute and then placed in a water-bath shaker set at 60 ºC and 200 rpm for 2 h for acid hydrolysis of flavonoid glycosides to aglycones. Finally, the samples were vortexed for 1 minute to ensure total extraction. The extracted samples were filtered through a 0.2- micron syringe nylon filter, and injected into a Hewlett-Packard (Avondale, PA) HP 1090

HPLC system with diode array and fluorescence detectors. The mobile phase was: solvent A: water, solvent B: methanol/acetic acid/water, (10:2:88, v/v/v), and solvent C: acetonitrile; in a gradient suitable for phenolic separation as follows: at 0 min 100% solvent B, at 5 min 90% solvent B and 10% solvent C, and at 25 min 30% solvent B and

70% solvent C, with 5 min post-run with 100% solvent A. Flow rate: 1 mL/min. Column:

Beckman Ultrasphere C18 ODS 4.6 x 250 mm. Column temperature : 40 ºC.

Total phenolics. Samples of fruit parts and leaves were extracted in 2% HCl in methanol for 24 h in the dark, at room temperature, and extracts diluted to an appropriate concentration for analysis. Total phenolics were measured by the Folin-Ciocalteu reagent method (19). 200 µL of sample extract was introduced in a test tube, 1.0 mL of Folin

Ciocalteu reagent and 0.8 mL of sodium carbonate (7.5%) were added, the test tube content mixed and allowed to stand for 30 min. Absorption at 765 nm was measured in a

Shimadzu 300 UV-Vis spectrophotometer (Shimadzu UV-1601, Norcross, GA). The total phenolic content was expressed as gallic acid equivalent (GAE), in milligrams per gram of sample, using a standard curve generated with 100, 200, 300, and 400 mg/L of gallic acid.

Total anthocyanins. Grape parts (skin, seed or pulp) were extracted in 2% HCl in methanol for 24 h, following the method described by Revilla et al. (20) as the one that gives the highest extraction of anthocyanins in grapes, and extracts diluted to an appropriate concentration with potassium chloride buffer, pH 1, until the absorbance of the sample was within the linear range of the Shimadzu 300 UV-Vis spectrophotometer.

The spectrophotometer was zeroed with distilled water. Two dilutions of each sample were prepared, one with potassium chloride buffer, pH 1, and the other with sodium acetate buffer, pH 4.5, and the dilutions allowed to equilibrate for 15 min. The absorbance was measured at 520 and at 700 nm (to correct for haze) against a blank cell filled with distilled water, following the pH differential method described by Giusti and

Wrolstad (21).

Antioxidant capacity. Determined as TEAC (Trolox equivalent antioxidant capacity), following a slight modification to the method described by Re et al. (22).

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a vitamin E analog, was used as an antioxidant standard. ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) diammonium salt was dissolved in water to a concentration of 7 mM and allowed to react with a 2.45 mM potassium persulfate solution, for 16 h in the dark. This reaction will form ABTS radical cations (ABTS*+). The ABTS*+ solution was diluted in ethanol to an absorbance of 0.70 (+/- 0.02) at 734 nm. 1.980 µL of diluted ABTS*+

46 solution was placed into a quartz cuvette and after exactly one min, 20 µL of antioxidant compound or Trolox standard was added and mixed. The absorbance reading was recorded up to 6 more minutes. The percentage inhibition of absorbance at 734 nm was calculated and plotted as a function of concentration of antioxidants and of Trolox for the standard reference data. The ratio between the area under the curve for the reaction of the specific antioxidant and that for Trolox gave the relative antioxidant capacity. For calibration, Trolox standards of 0, 300, 600, 900, 1200, and 1500 µM were prepared to obtain final concentrations in the cuvette of 0, 3, 6, 9, 12, and 15 µM, respectively. Data from the spectrophotometer were saved as Excel file (Microsoft Corporation). The area under the curve was calculated using the software TableCurve 2D V5.0 (SPSS Inc.

Chicago, IL).

Dry weight determination. Sample dry weight was determined following the guidelines of the official method AOAC 967.03, 1990 (23). For each cultivar, 500 g of fruits were separated into pulps, seeds and skins. Each fruit part fraction was weighed and the composition of the fruit (by percentage of its fruit parts) was determined.

Approximately 10 g of each fruit part was placed into an aluminum pan (in triplicate) and dried for 16 h in an oven set at 105 °C. After the drying time, the pans were removed from the oven, allowed to cool in a desiccator, weighed, and dry weight determined as gram of dry matter per gram of sample.

Statistics. The statistical analysis was carried out using the Microsoft Excel software package (Microsoft Corporation).

RESULTS AND DISSCUSSION

Major phenolics in muscadine grape skins were identified by their retention time and characteristic spectra. Quantification was made by calibration curves of external standards built for each of the analyzed compounds (+) catechin, (-) epicatechin, gallic acid, ellagic acid, trans-resveratrol, and the aglycones of myricetin, quercetin, and kaempferol. Ellagic acid, resveratrol, and the aglycones of myricetin, quercetin, and kaempferol were found in muscadine skins, while (+) catechin, (-) epicatechin and gallic acid were found in seeds. In order to check the performance of the extraction method some skin and seed samples were spiked with selected phenolics and analyzed for recovery. Recoveries of ellagic acid and resveratrol were, respectively, 95.8% and 98.7% when skin samples were spiked with known amounts of the compounds, and recovery of gallic acid was 83.5% when seed samples were spiked. Ellagic acid was the most abundant phenolic compound in muscadine grape skins with concentrations from 6.2 +/-

0.3 to 22.2 +/- 1.6 mg/100 g FW; myricetin had concentrations from 1.8 +/- 0.1 to 19.6

+/- 2.3 mg/100 g FW; quercetin varied from 0.5 +/- 0.0 to 3.9 +/- 0.2 mg/100 g FW; kaempferol with concentrations from 0.2 +/- 0.0 to 3.0 +/- 0.2 mg/100 g FW; and, trans- resveratrol had the lowest concentrations of the detected phenolics ranging from below detection limit in two varieties to 0.2 +/- 0.1 mg/100 g of FW (Tables 3.1 and 3.2). Our result for resveratrol contradicts previous reports (15) that indicated high concentrations.

These researchers apparently were not able to separate ellagic acid from resveratrol with

UV detection alone. We were able to separate the two compounds, identify and quantify them with UV detection, and confirm the resveratrol identity and amount with fluorescence detection. Fluorescence detector was set to wavelengths of 330 nm and 374

48 nm (24,25), for excitation and emission, respectively. Figure 3.1 shows the spectra of ellagic acid and trans-resveratrol for standards and a sample of the skin of the muscadine grape cultivar “Carlos.” The HPLC chromatogram of selected standards for muscadine skin analysis, showing diode-array at different wavelengths and fluorescence detection are shown in Figure 3.2. Figure 3.3 shows the HPLC chromatogram of a sample of the skin of the muscadine grape cultivar “Carlos.” Major phenolics in muscadine seeds

(Tables 3.1 and 3.2) were (-) epicatechin, (+) catechin and gallic acid. (-) Epicatechin ranged from 450.1 +/- 25.3 to 1897.6 +/- 54.4 mg/100 g FW; (+) catechin had concentrations between 319.6 +/- 17.5 to 1424.7 +/- 46.5 mg/100 g FW; and gallic acid varied from 3.0 +/- 0.2 to 11.53 +/- 0.5 mg/100 g FW. Major phenolics in muscadine leaves (Table 3.3) were myricetin, ellagic acid, kaempferol, quercetin and gallic acid.

Myricetin varied from 107.7 ± 3.5 to 216.4 ± 4.9 mg/100 g FW, which is, on average, 50 times the concentration of the compound in the whole muscadine grapes and 18 times the concentration in skins. Ellagic acid ranged between 44.8 ± 2.8 and 80.0 ± 5.9 mg/100 g

FW, which is 10 times the concentration of ellagic acid in the fruits and 4 times the concentration in skins. Kaempferol was found in concentrations between 5.7 ± 0.2 and

11.5 ± 1.0 mg/100g FW, corresponding to 32 times the concentration of the phenolic compound in the grapes and 14 times the concentration in skins. The flavonol quercetin ranged from 6.3 ± 0.3 to 21.6 ± 1.5 mg/100 g FW, corresponding to 13 times the concentration in the whole grapes and 6 times the concentration in skins. Gallic acid varied from 6.1 ± 0.2 to 18.7 ± 2.8 mg/100 g FW, which is, on average, 29 times the concentration of gallic acid in grapes and about the same concentration in seeds. No phenolics (from the ones that we analyzed) were detected in grape pulps.

Total phenolics in muscadine grape parts were, on average, 5 times more concentrated in seeds than in the skins and 80 times more than in the pulps (Table 3.4).

This result may be due to the high concentration of catechins in seeds and the very low presence of major phenolics in pulps. The relatively high value for total phenolics in skins in comparison to the sum of individual phenolics found in them indicates that some other phenolics are present in the skin but not identified in this study. The whole muscadine fruits had, on average, 50% less total phenolics than the leaves (Table 3.5), even though the skins and seeds had high contents of phenolics. However, the high relative weight of the pulp to the skins and seeds, and their very low content of phenolics contributed to a low phenolics value in the whole fruit. Nevertheless, it is important to note that in muscadine grapes processing, like in juice, wine or jelly production, skins and seeds are discarded as waste. Therefore, the nutraceutical industry may use the muscadine seeds and skins as potential sources of phenolics, and for the farmers or processors they may provide an additional source of income. Leaves are allowed to remain in the field after harvesting the fruits, and finally will fall to the ground during the

Fall season. They could also be collected and used for extraction of polyphenolics, or processed for use as functional foods.

The analysis of total anthocyanins showed that bronze skin muscadine grapes had very low anthocyanins content in skins and seeds, and no anthocyanins in pulps. The seeds of this group of fruits had higher relative anthocyanin content. For purple skin muscadine grapes, the skins showed higher anthocyanins content, ranging from 65.5 to

174.5 mg/100 g FW expressed as cyanidin-3-glucoside. This corresponds to 65 times more anthocyanins than in the skins of bronze grapes. The total anthocyanins content in

50 the seeds of purple grapes was just 1.3 times higher than the one for bronze grape seeds, and the pulps of purple grapes had, on average, 2.0 mg/100 g FW. This could be due to some migration of the pigments from the skin to the pulp, or some tinting from ruptured skin cells during the process of separation of the fruit into its parts. Tables 3.4 and 3.5 show the results for total anthocyanins. No total anthocyanins analysis was performed on the leaves once they were collected at the time of harvest for the fruits, and at that physiological stage it is possible to assume that muscadine leave cells do not have such kind of pigments.

Tables 3.4 and 3.5 also show the antioxidant capacity data. The average values were 12.8, 281.2, 2.42, 15.3 and 236.1 µM Trolox equivalent/g FW, in skins, seeds, pulps, whole grape and leaves, respectively. This means that seeds had 22, 116, and 18 times more antioxidant capacity than skins, pulps, and the whole grape, respectively.

Additionally, seeds had, on average, 20% more antioxidant capacity than leaves.

However, taking into account that seeds had about 6 times more total phenolics than leaves, and that the ratio of antioxidant capacity/total phenolics was about 6 times higher for leaves than seeds, it is presumable that major phenolics in leaves have higher antioxidant capacity than the ones found in seeds, or that other antioxidant compounds different from phenolics, may be present in higher concentrations in the leaves than in seeds. A comparison of our results to that reported by Wang and Lin (13) indicated that antioxidant capacity of muscadine leaves was at least twice the value for the leaves of some berry plants, such as blackberry, raspberry or strawberry.

Table 3.6 shows the results for dry weight determination of muscadine grapes and the corresponding dry weight of the whole fruit calculated taking into account the

51 percentage of each fruit part in the whole fruit. This was provided as additional information to simplify the comparison of our results to those of other fruits whose results were reported on dry weight basis. Both leaf and skin of muscadine grape are a good source of ellagic acid and other phenolics. Muscadine seeds have elevated content of phenolics and have very high antioxidant capacity.

ACKNOWLEDGMENT

We thank Mr. Jacob Paulk for his valuable help by providing us with the muscadine fruits and leaves samples. We also thank Fulbright-IIE-Colciencias and the

Universidad Surcolombiana for the financial support to the graduate student, Eduardo

Pastrana-Bonilla. Research funded by the State of Georgia’s Traditional Industries

Program for Food Processing research grant.

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Table 3.1. Phenolics in Muscadine Grape Parts (mg/100 g FW of fruit part)1

Skins Seeds Cultivar Ellagic acid Myricetin Quercetin Kaempferol Resveratrol (-)Epicatechin (+)Catechin Gallic acid

Carlos 19.7 ± 2.0 19.6 ± 2.3 1.3 ± 0.2 0.4 ± 0.0 0.2 ± 0.0 1189 ± 52 1425 ± 30 9.4 ± 0.4 Early fry 19.7 ± 1.0 16.4 ± 1.1 1.6 ± 0.1 0.3 ± 0.0 0.1 ± 0.0 1603 ± 89 941 ± 80 3.3 ± 0.2 Fry 13.1 ± 0.8 4.1 ± 0.0 2.5 ± 0.1 0.8 ± 0.0 0.2 ± 0.1 1851 ± 55 356 ± 54 4.5 ± 0.3 Summit 11.7 ± 1.1 9.2 ± 0.7 3.8 ± 0.2 3.0 ± 0.2 0.2 ± 0.1 450 ± 81 349 ± 67 5.0 ± 0.4 Late fry 21.1 ± 2.0 12.1 ± 1.0 0.9 ± 0.1 0.2 ± 0.0 nd 1898 ± 60 511 ± 38 9.5 ± 1.0 Paulk 14.7 ± 1.1 1.8 ± 0.1 1.7 ± 0.5 0.4 ± 0.0 nd 1672 ± 48 320 ± 13 9.9 ± 1.2 Cowart 21.6 ± 1.7 6.4 ± 0.6 0.9 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 1181 ± 28 347 ± 27 5.0 ± 0.1 Supreme 6.2 ± 0.3 2.0 ± 0.4 3.0 ± 0.6 0.2 ± 0.0 0.2 ± 0.0 1554 ±180 461 ± 13 2.2 ± 0.5 Ison 22.2 ± 1.6 7.1 ± 0.6 1.4 ± 0.2 0.6 ± 0.1 0.2 ± 0.0 873 ± 5 542 ± 80 8.8 ± 1.1 Noble 14.6 ± 0.2 4.8 ± 0.1 0.5 ± 0.0 0.3 ± 0.0 0.1 ± 0.0 724 ± 61 333 ± 33 11.5 ± 1.1

1Values are average and standard deviation of triplicates; nd = not detected

Table 3.2. Phenolics in Muscadine Grapes (mg/100 FW of whole fruit)1

Ellagic Cultivar % Skin % Seeds % Pulp acid Myricetin Quercetin Kaempferol Resveratrol (-)Epicatechin (+)Catechin Gallic acid

Carlos 32.3 6.0 61.6 6.4 6.3 0.4 0.1 0.1 71.8 86.1 0.6 Early fry 35.7 2.0 62.3 7.0 5.8 0.6 0.1 0.1 32.4 19.0 0.1 Fry 43.3 1.8 54.9 5.7 1.8 1.1 0.4 0.1 33.1 6.4 0.1 Summit 45.8 1.5 52.7 5.4 4.2 1.8 1.4 0.1 6.9 5.4 0.1 Late fry 46.7 3.9 49.4 9.9 5.6 0.4 0.1 nd 74.0 19.9 0.4 Paulk 40.7 1.8 57.5 6.0 0.7 0.7 0.2 nd 30.4 5.8 0.2 Cowart 34.2 5.1 60.7 7.4 2.2 0.3 0.1 0.1 60.3 17.7 0.3 Supreme 47.8 1.1 51.1 3.0 1.0 1.4 0.1 0.1 17.1 5.1 nd Ison 39.1 3.5 57.3 8.7 2.8 0.5 0.2 0.1 30.9 19.2 0.3 Noble 46.2 9.2 44.6 6.8 2.2 0.2 0.2 0.1 66.6 30.7 1.1

1Values are average of triplicates

Table 3.3. Major Phenolics in Muscadine Leaves (mg/100 g FW)1

Cultivar Ellagic acid Kaempferol Myricetin Quercetin Gallic acid

Carlos 80.0 ± 5.9 8.7 ± 1.4 145.8 ± 4.2 8.0 ± 1.0 7.6 ± 0.1 Early Fry 79.0 ± 4.6 10.6 ± 1.3 216.4 ± 4.9 21.6 ± 1.5 9.3 ± 0.2 Fry 76.7 ± 4.4 8.8 ± 0.4 140.9 ± 3.3 7.9 ± 0.3 6.1 ± 0.2 Summit 55.6 ± 2.7 5.7 ± 0.2 107.7 ± 3.5 6.3 ± 0.3 6.9 ± 0.3 Late Fry 66.1 ± 3.2 10.2 ± 1.1 162.1 ± 4.6 8.1 ± 0.5 8.3 ± 0.3 Paulk 65.5 ± 5.0 10.0 ± 1.0 166.2 ± 5.3 8.4 ± 0.7 6.4 ± 0.9 Cowart 74.7 ± 3.7 7.9 ± 0.3 157.1 ± 4.8 11.8 ± 1.0 7.8 ± 0.7 Supreme 59.1 ± 2.8 7.2 ± 0.3 133.8 ± 5.7 9.9 ± 0.9 7.7 ± 0.6 Ison 65.1± 4.0 11.5 ± 1.0 178.5 ± 6.3 8.0 ± 0.7 7.1 ± 0.7 Noble 44.8 ± 2.8 8.6 ± 0.7 167.3 ± 4.9 7.9 ± 0.6 18.7 ± 2.8

1Values are average and standard deviations of triplicates

Table 3.4. Total Phenolics, Total Anthocyanins, and Antioxidant Capacity of Muscadine Grape Parts

Total phenolics (G.A.E. Total anthocyanins (mg/100 g 1 mg/100 g FW) FW as cyanidin-3-glucoside) TEAC (µM/g FW)

Cultivar Seed Skin Pulp Skin Seed Pulp Skin Seed Pulp Carlos 1920 546 25.1 2.6 1.2 nd 14.9 204.6 3.4 Early Fry 2367 303 21.3 2.5 8.7 nd 13.9 277.8 2.0 Fry 2356 332 23.8 0.8 4.6 nd 11.1 234.2 2.9 Summit 3259 541 22.3 2.8 3.1 nd 12.4 245.4 3.0 Late Fry 1986 349 24.0 2.0 3.7 nd 13.4 218.9 2.4 Paulk 1649 364 30.0 177.0 4.1 4.7 12.1 307.9 2.2 Cowart 2303 262 11.6 107.8 4.6 1.1 12.4 325.5 2.7 Supreme 1536 330 20.1 135.5 7.5 0.7 12.2 478.6 1.6 Ison 1726 365 26.0 174.5 4.6 1.9 13.3 284.8 2.1 Noble 2685 355 33.4 65.5 2.2 2.2 12.4 234.7 2.1

1Trolox equivalent antioxidant capacity, TEAC; values are average of triplicates. nd = not detected

Table 3.5. Total Phenolics (TPH), Total Anthocyanins (TAC), and Trolox Equivalent Antioxidant Capacity (TEAC) of Muscadine Grapes and Leaves1

TPH TAC TEAC G.A.E mg/100 g FW mg/100 g FW µM/g FW

Whole Cultivar fruit Leaves Whole fruit Whole fruit Leaves Carlos 307.9 350.1 0.9 18.2 229.8 Early Fry 169.1 437.0 1.1 11.2 251.0 Fry 199.0 340.4 0.4 9.8 239.8 Summit 309.7 282.1 1.3 10.2 222.0 Late Fry 252.3 355.0 1.1 15.4 235.0 Paulk 195.2 356.5 74.8 11.2 247.8 Cowart 214.2 359.3 37.8 21.7 164.0 Supreme 184.7 317.7 65.2 11.5 304.0 Ison 218.9 370.4 69.5 15.9 283.0 Noble 425.7 347.3 31.5 27.8 184.8

1Values are average of triplicates

Table 3.6. Dry Matter of Muscadine Grape Fruits and Fruit Parts (g/g FW)1

Cultivar Skin Seed Pulp Whole fruit

Carlos 0.179 0.532 0.137 0.174 Cowart 0.149 0.531 0.121 0.139 Early fry 0.159 0.562 0.149 0.161 Fry 0.139 0.523 0.144 0.148 Ison 0.182 0.559 0.157 0.184 Late fry 0.161 0.516 0.152 0.162 Noble 0.135 0.596 0.122 0.151 Paulk 0.163 0.578 0.146 0.159 Summit 0.165 0.571 0.166 0.180 Supreme 0.169 0.514 0.137 0.186

1Values are average of triplicates

Figure 3.1. Diode-array spectra of ellagic acid (left) and trans-resveratrol (right) for standards and a sample of the skin of the cultivar “Carlos.”

Figure 3.2. HPLC chromatograms of selected standards for muscadine skin analysis, at 260 nm (DAD1 A), 360 nm (DAD1 D), 313 nm (DAD1 E), and fluorescence detection at Ex=330 and Em= 374 (FLD1 A) of ellagic acid (1), myricetin (2), trans- resveratrol (3), quercetin (4), and kaempferol (5).

Figure 3.3. HPLC chromatograms of the skin of grapes of the cultivar “Carlos,” at 260 nm (DAD1 A), 360 nm (DAD1 D), 313 nm (DAD1 E), and fluorescence detection at Ex=330 and Em= 374 (FLD1 A) of ellagic acid (1), myricetin (2), trans-resveratrol (3), quercetin (4), and kaempferol (5).

CHAPTER 4

IDENTIFICATION AND QUANTIFICATION OF ANTHOCYANINS IN

MUSCADINE GRAPES BY HPLC AND HPLC-MS

______Eduardo Pastrana-Bonilla, Casimir C. Akoh and Gerard Krewer. Submitted to the Journal of Agricultural and Food Chemistry, February 18, 2003.

ABSTRACT

Total anthocyanin content and individual anthocyanin profile of ten cultivars (5 purple skin and 5 bronze skin) of muscadine grapes, grown in South Georgia, were assessed. Fruits were separated into skins, seeds and pulps for analysis. Total anthocyanin content was determined by a pH differential method and it varied from 31 to 75 mg/100g

FW in the purple grapes and from 0.4 to 1.3 in the bronze grapes. Individual anthocyanins were analyzed by HPLC and their identity confirmed by HPLC-MS.

Delphinidin-3,5-diglucoside was the most abundant (about 46% of the total anthocyanin content) and was found in the skins of all fruits, the seeds of 9 cultivars, and the pulps of

3. Petunidin-3,5-diglucoside (~23%) and malvidin-3,5-diglucoside (~20%) were the next in concentration but only found in the skins of 8 and 5 of the cultivars, respectively.

Cyanidin-3,5-diglucoside (~6%), peonidin-3,5-diglucoside (~3%) and petunidin-3- monoglucoside (~1%) were also found in the skins of the purple grapes. This is the first report of petunidin-3-monoglucoside in muscadine grapes.

The total anthocyanin content and the sum of the individual anthocyanins had a high correlation (R = 0.98). The average anthocyanin content of muscadine grapes was lower than published values for red European or other American red grapes, and other common berries. However, the purple muscadine grapes have anthocyanins levels that may be considered important from the nutraceutical point of view.

Keywords: Vitis rotundifolia, muscadine grapes, anthocyanins, anthocyanin diglucosides, polyphenols, food analysis.

INTRODUCTION

Muscadine grapes (Vitis rotundifolia Michx.) are indigenous to the southeastern

United States. Muscadines are vigorous vines that may grow up to 100 ft in the wild.

They differ botanically from other grapes and are placed in a separate sub-genus,

Muscadinia. Muscadine fruits are round, 1 to 1½ inch in diameter with thick, tough skin and may have up to 5 seeds.

Anthocyanins are water soluble, glycosylated derivatives based on the cyanidin aglycon (1,2) and are part of the flavonoid family. These pigments are responsible for the red, purple and blue colors of most fruits and flowers (3). Anthocyanins have the potential to be used as natural food colorants. However, enzymes, pH, temperature, and oxygen affect their color quality (2). Co-pigmentation with other phenolic compounds may increase their stability (4). Anthocyanins are part of the human diet and they occur in many fruits and vegetables (5). The average daily intake of anthocyanins in the United

States was estimated at 215 mg during the summer and 180 mg during the winter (6). The beneficial antioxidant activity and therefore, positive health effects of anthocyanins are a significant added value for their use as food colorants. The antioxidant function of anthocyanins seems to be related to their hydrogen donation capacity, metal chelation and protein binding (7). Anthocyanins have been found to be powerful antioxidants in comparison to other common antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and a-tocopherol (8,9). Anthocyanin-rich fruit extracts have been used in traditional medicine as anti-inflammatory agents (10), for the treatment and prevention of vascular diseases due to cholesterol-induced atherosclerosis, and as

70 anti-carcinogenic agents (11). Anthocyanins have also been reported to have antiulcer activity and to provide protection against UV radiation (12). Possible mechanisms for the anti-inflammatory activity of anthocyanins include inhibition of arachidonic acid metabolism and the prostaglandin synthase cyclooxygenase activity (13). Anthocyanins in red wine may have antiatherogenic effects in conjunction with other polyphenols found in the wine (14). Kamei et al. (15) studied the in vitro anticarcinogenic effect of anthocyanins on tumor cells. Glycosides of the aglycons cyanidin and delphinidin have been found to be the most abundant anthocyanins in plants (16). Cyanidin lowered serum thiobarbituric acid-reactive substance (TBARS) concentration and increased the oxidation resistance of the serum to lipid peroxidation in rats (17). Delphinidin has been reported to inhibit the growth of human tumor cell line by shutting off the epidermal growth-factor receptor downstream signaling cascade (17). Mazza and Miniati (12) and

Talcott and Lee (18) reported the presence of anthocyanidin diglucosides as the anthocyanins present in muscadine grapes. The objective of this paper was to identify and quantify the anthocyanins present in 10 cultivars of muscadine grapes grown in South

Georgia.

MATERIALS AND METHODS

Chemicals. Standards (with more than 97% purity) of malvidin-3-O-ß- glucopyranoside (mv-3-gl), delphinidin-3-O-ß-glucopyranoside (dp-3-glc), petunidin-3-

O-ß-glucopyranoside (pt-3-glc), peonidin-3-O-ß-glucopyranoside (pn-3-glc), and cyanidin-3-O-ß-glucopyranoside (cy-3-glc) were purchased from Polyphenols

Laboratories AS (Sandnes, Norway). Potassium chloride and sodium acetate were

71 purchased from J.T. Baker Chemical Company (Phillipsburg, NJ). Acetonitrile, methanol, O-phosphoric acid (85% purity, HPLC grade), formic acid and water (HPLC grade) were purchased from Fisher Scientific (Norcross, GA).

Standards preparation. Stock standard solutions (100 µg/mL) of the anthocyanins mv-3-gl, dp-3-glc, pt-3-glc, pn-3-glc, and cy-3-glc were prepared with 2%

HCl in methanol and stored for a week at –86°C. Each week new stock solutions were prepared to ensure freshness of the standards. Working standard solutions of 100, 75, 50, and 25 µg/mL were prepared in order to build the calibration curve for each compound using the software TableCurve 2D V5.0 (SPSS Inc., Chicago, IL). Randomly selected working standards were prepared daily in order to check the performance of the method and for possible degradation of the stock solutions. No degradation of the stock solutions was detected during the week that they were stored.

Samples. Fruits from selected ten muscadine grape cultivars, five bronze (Carlos,

Early Fry, Fry, Summit and Late Fry), and five purple (Paulk, Cowart, Supreme, Ison and

Noble), grown in South Georgia and provided by Mr. Jacob Paulk (Paulk Vineyards,

Wray, GA) were studied. Fruits were separated into skins, seeds and pulps, and extracted, in triplicate, for the corresponding analysis as described below.

Individual anthocyanins. One gram of each sample was mashed using mortar and pestle to a very fine paste and diluted with 2% HCl in methanol. The samples were vortexed for 1 minute and then placed in a water-bath shaker set at 25 ºC and 200 rpm for

24 h. Finally, the samples were vortexed for 1 minute to maximize the extraction. The extracted samples were filtered through a 0.45-µm syringe polypropylene filter and 20 µL aliquot injected into a Hewlett-Packard (Avondale, PA) HP 1100 HPLC system with

72 diode array detector. The mobile phase was: solvent A: O-phosphoric acid/methanol/water (5:10:85, v/v/v), and solvent B: acetonitrile. The gradient for anthocyanin separation is as follows: at 0 min 100% solvent A, at 5 min 90% solvent A and 10% solvent B, and at 25 min 50% solvent A and 50% solvent B, with 5 min post-run with HPLC-grade water. Flow rate: 0.5 mL/min. Column: Beckman Ultrasphere C18

ODS 4.6 x 250 mm. Temperature: 40ºC. Anthocyanin-3,5-diglucosides were identified and quantified by the chromatographic characteristics of their corresponding anthocyanin-3-monoglucosides, and their identity was verified by mass spectrometric analysis. The mass spectrometric analysis was performed under the same chromatographic conditions described for the HPLC analysis (except that O-phosphoric acid was replaced with formic acid), using a LC-MS system from Thermo Separations

Products HPLC instrument (San Jose, California) coupled to a Perkin Elmer Sciex API I plus quadrupole mass spectrometer (Shelton, Connecticut).

Total anthocyanins. Grape parts (skin, seed or pulp) were extracted in 2% HCl in methanol, for 24 h at room temperature in the dark, and diluted to an appropriate concentration with potassium chloride buffer, pH 1, until the absorbance of the sample was within the linear range of a Shimadzu 300 UV-Vis spectrophotometer (Rydalmere,

Australia). The spectrophotometer was zeroed with distilled water. Two dilutions of each sample were prepared, one with potassium chloride buffer, pH 1, and the other with sodium acetate buffer, pH 4.5. The dilutions were allowed to equilibrate for 15 min. The absorbance was measured at 520 and at 700 nm (to correct for haze) against a blank cell filled with distilled water, following the pH differential method described by Giusti and

Wrolstad (19).

Statistics. The statistical analysis was carried out using the Microsoft Excel software package (Microsoft Corporation, Mountain View, CA). The analysis was repeated using three samples and standard deviation recorded. Regression and area under the curve analyses were performed using TableCurve 2D V5.0 (SPSS Inc., Chicago, IL).

RESULTS AND DISSCUSSION

Anthocyanins were identified by their retention times and characteristic spectra.

Quantification was made using the calibration curves of external standards built for each of the standard compounds (mv-3-gl, dp-3-glc, pt-3-glc, pn-3-glc, and cy-3-glc), and their corresponding anthocyanidins, after acid hydrolysis. We found, based on HPLC-MS analysis and in agreement with the findings of Talcott and Lee (18), that anthocyanin-3,5- diglucosides correlated well to their corresponding anthocynidin. Table 4.1 shows that delphinidin-3,5-diglucoside (dp-3,5-di-glc) was the most abundant anthocyanin found in muscadine skins ranging from 1.1 to 2.8 mg/100 g fresh weight (FW) in the case of the bronze muscadine skins, and from 23.0 to 95.3 mg/100g FW for the purple muscadine grape skins. The second most abundant anthocyanin in skins was petunidin-3,5- diglucoside (pt-3,5-di-glc) which was present in all purple skins varying from 19.5 to

52.6 mg/100g FW, and was detected in three of the five bronze skins ranging from 0.9 to

1.3 mg/100g FW. Malvidin-3,5-diglucoside (mv-3,5-di-glc), cyanidin-3,5-diglucoside

(cy-3,5-di-glc), and peonidin-3,5-diglucoside (pn-3,5-di-glc) were not detected in the skins of the bronze fruits, but they were detected in the purple muscadines ranging from

15.8 to 45.8 mg/100g FW, from 5.9 to 13.8 mg/100g FW, and from 3.8 to 7.3 mg/100g

FW, respectively. In addition, petunidin-3-glucoside was the only monoglucoside

74 detected in the skins of the purple grapes and ranged from 1.3 to 2.3 mg/100 g FW.

Petunidin-3-monoglucoside has not been previously reported in muscadine grapes.

Delphinidin-3,5-diglucoside was the only anthocyanin detected in the seeds as well as in the pulps (Table 4.2). It was found in the seeds of four of the five bronze fruits and ranged between 3.1 to 5.8 mg/100g FW, and also detected in all the seeds of the 5 purple fruits with concentration varying from 2.4 to 6.4 mg/100g FW. Delphinidin-3,5- diglucoside was not detected in the pulps of the bronze fruits, but detected in 3 of the 5 pulps of purple fruits with concentrations ranging from 1.7 to 4.2 mg/100g FW. Table 4.3 shows the calculated individual anthocyanin content in muscadine grapes. The calculation was based on the anthocyanin content in each fruit part multiplied by the weight fraction of the fruit part to the weight of the whole fruit (data not shown). The anthocyanin profile in the whole grapes followed the same trend as the skins because skins are a major component of the fruit (41% of the whole fruit, on average). Skins account for the majority of the pigment content in the grape. The total anthocyanin content of muscadine grape parts (Table 4.4) varied from 0.4 to 1.3 mg/100g FW for the bronze skin grapes and from 31.1 to 74.5 mg/100g FW in the case of the purple grapes.

On average, 92.4% of the total anthocyanin content was found in the skins of the grapes,

6.1% in the seed and 1.5% in the pulps. The low percentage of anthocyanins in pulps may be due to some transfer of anthocyanins from the skin at the moment of the separation because most pulps were basically colorless. Anthocyanin content in muscadine grapes was found to be lower compared to other grapes or berries; however, this comparison is not reliable due to the different methods that each author reported for extraction and

75 analysis. Table 4.5 shows some values for anthocyanin content found in the literature for other small fruits.

Our results are in agreement with those published by Mazza and Miniati (12) and

Goldy et al. (20) who reported the same order in the concentration of anthocyanin- diglucosides in muscadine grapes with delphinidin-3,5-diglucoside as the major anthocyanin. However, there was no agreement in the proportions of the individual anthocyanins, which is not surprising because agro-ecological and varietal factors may affect the morphological characteristics and the chemical composition of agricultural products. In addition to the 5 diglucosides reported by Mazza and Miniati, we were able to identify a monoglucoside (petunidin-3-glucoside). Figure 4.1 shows the HPLC chromatograms for A: monoglucosides standards, and B: muscadine grape skin sample.

As indicated above, quantification of anthocyanin diglucosides was based on the calibration curves built for the monoglucosides standards and their corresponding anthocyanidins, following the method described by Talcott and Lee (18). Figure 4.2 shows the mass spectra for a grape skin sample showing the different molecular ions for the anthocyanins present. The anthocyanin profile of muscadine grapes differ greatly from the European grapes (Vitis vinifera). The anthocyanin profile is more complex in the case of the Vitis vinifera grapes which have more than 20 different anthocyanins with the major one being malvidin-3-glucoside (21). All anthocyanins in Vitis vinifera are monoglucosides, some are acylated, with no diglucosides present (12,22). In contrast, muscadine grapes (Vitis rotundifolia) have mainly anthocyanin diglucosides, none of the anthocyanins is acylated and malvidin is a minor pigment in this kind of grapes. The differences in the pattern of anthocyanins may be due to the different evolutionary paths

76 taken by the two species. In addition, European grapes have a long history of artificial breeding aimed at the improvement of wine quality and at the search for new tastes, colors and aromas. In contrast, muscadine grapes have remained close to their natural state, subjected to natural selection, and for just a few decades subjected to selective breeding.

ACKNOWLEDGMENT

We thank Mr. Jacob Paulk for providing us with the muscadine fruits. We also thank Fulbright-IIE-Colciencias and the Universidad Surcolombiana for the financial support to the graduate student, Eduardo Pastrana-Bonilla. Research funded by the State of Georgia’s Traditional Industries Program for Food Processing research grant.

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pigments. J. Food Sci. 1977, 42, 488.

Table 4.1. Anthocyanins in Muscadine Grape Skins (mg/100 g FW)1

Cultivar Dp-3,5-di-glc pt-3,5-di-glc mv-3,5-di-glc cy-3,5-di-glc pn-3,5-di-glc pt-3-glc

Carlos 1.3 ± 0.1 1.3 ± 0.1 nd nd nd nd Early fry 1.4 ± 0.1 1.2 ± 0.1 nd nd nd nd Fry 1.1 ± 0.1 nd nd nd nd nd Summit 2.8 ± 0.1 nd nd nd nd nd Late fry 1.2 ± 0.1 0.9 ± 0.1 nd nd nd nd Paulk 95.3 ± 4.9 29.8 ± 1.9 45.8 ± 2.8 13.8 ± 0.2 6.3 ± 0.1 2.3 ± 0.1 Cowart 54.9 ± 4.0 28.8 ± 1.9 15.8 ± 0.7 6.0 ± 0.8 3.7 ± 0.2 1.4 ± 0.1 Supreme 74.9 ± 3.5 33.5 ± 2.1 20.3 ± 1.1 11.5 ± 0.9 3.9 ± 0.1 1.5 ± 0.1 Ison 85.1 ± 4.0 52.6 ± 2.9 31.5 ± 1.2 9.3 ± 0.9 7.3 ± 0.2 2.3 ± 0.2 Noble 23.0 ± 2.0 19.5 ± 1.0 31.9 ± 1.5 5.9 ± 0.5 3.8 ± 0.1 1.3 ± 0.1

1Values are average and standard deviation of triplicates; nd = not detected 81

Table 4.2. Delphinidin-3,5-diglucoside in Seeds and Pulps (mg/100 g FW)1

Cultivar Seeds Pulps

Carlos nd nd Early fry 5.8 ± 0.5 nd Fry 4.2 ± 0.7 nd Summit 3.1 ± 0.3 nd Late fry 3.3 ± 0.2 nd Paulk 4.1 ± 0.3 4.2 ± 0.1 Cowart 4.3 ± 0.2 nd Supreme 6.4 ± 0.4 nd Ison 4.0 ± 0.2 1.7 ± 0.1 Noble 2.5 ± 0.1 2.2 ± 0.1

1Values are average and standard deviation of triplicates; nd = not detected

Table 4.3. Anthocyanins in Muscadine Grapes (mg/100 g FW)1

Cultivar dp-3,5-di-glc pt-3,5-di-glc mv-3,5-di-glc cy-3,5-di-glc pn-3,5-di-glc pt-3-glc Total

Carlos 0.4 0.4 nd nd nd nd 0.8 Early fry 0.6 0.4 nd nd nd nd 1.0 Fry 0.6 nd nd nd nd nd 0.6 Summit 1.3 nd nd nd nd nd 1.3 Late fry 0.7 0.4 nd nd nd nd 1.1 Paulk 41.3 12.1 18.2 5.6 2.6 2.3 82.1 Cowart 19.0 9.8 5.4 2.0 1.3 1.4 38.9 Supreme 35.9 16.0 9.7 5.5 1.8 1.5 70.4 Ison 34.4 20.6 12.3 3.7 2.8 2.3 76.1 Noble 11.8 9.0 14.8 2.7 1.7 1.3 41.3

1Values are calculated based on the weight of each part to the whole fruit; nd = not detected 83

Table 4.4. Total Anthocyanins in Muscadine Grape Parts and Whole Fruit (mg/100 g FW as cyanidin-3-glucoside) 1

Cultivar skin seed pulp whole fruit

Carlos 2.5 1.3 nd 0.9 Early Fry 2.5 8.5 nd 1.1 Fry 0.7 4.1 nd 0.4 Summit 2.9 3.5 nd 1.3 Late Fry 2.0 4.0 nd 1.1 Paulk 174.5 4.0 4.2 74.5 Cowart 101.5 4.4 0.9 37.5 Supreme 143.2 7.8 0.8 65.6 Ison 170.2 4.1 1.7 69.4 Noble 67.8 2.1 2.1 31.1

1Values are average of triplicates; nd = not detected

Table 4.5. Total Anthocyanin Content of Some Berries (mg/100g FW)

Fruit Anthocyanin content Reference Blackberries (Rubus spp.) 83 - 326 (23) Blackberries (Rubus spp.) Georgia-grown 111 - 123 (5) Blueberries (Vaccinium spp.) 25 - 495 (12) Blueberries (Vaccinium spp.) Georgia-grown 16 - 197 (5) Cranberries (Vaccinum macrocarpon Ait.) 78 (12) Black currant (Ribes nigrun L.) 250 (12) Black raspberries (Rubus occidentalis L.) 214 - 428 (23) Red grapes (Vitis vinifera) 30 - 750 (12) Purple muscadine grapes, whole fruit (Vitis rotundifolia Michx) 31-75 Current report

Figure 4.1. (A) HPLC chromatogram at 520 nm of anthocyanin monoglucosides standards: delphinidin-3-glucoside (1), cyanidin-3-glucoside (2), petunidin-3-glucoside

(3), peonidin-3-glucoside (4), malvidin-3-glucoside (5). (B) HPLC chromatogram at 520 nm of the anthocyanins found in the skin of the grapes of the cultivar, Paulk: delphinidin-

3,5-diglucoside (6), cyanidin-3,5-diglucoside (7), petunidin-3,5-diglucoside (8), malvidin-3,5-diglucoside (9), peonidin-3,5-diglucoside (10).

Figure 4.2. Integrated mass spectrum of the molecular ions found in a skin sample of the

CHAPTER 5

CHANGES IN ANTHOCYANINS, TOTAL PHENOLICS AND ANTIOXIDANT

CAPACITY DURING STORAGE OF JUICES FROM MUSCADINE GRAPES

GROWN IN SOUTH GEORGIA

______Eduardo Pastrana-Bonilla, Casimir C. Akoh and Gerard Krewer. Submitted to the Journal of Agricultural and Food Chemistry, March 26, 2003.

ABSTRACT

Grape juice consumption has been attributed to a series of health benefits due to its polyphenolic content. Muscadine grapes belong to the Vitis family. Juices from 10 cultivars of Georgia-grown muscadine grapes were analyzed weekly for changes in antioxidant capacity, total phenolics, total anthocyanins and individual anthocyanins during an 8 week storage. Juices were stored at two levels of temperature (refrigeration and room temperature) and two levels of added ascorbic acid (0 and 0.15%). Both ascorbic acid and temperature impacted the studied variables. Ascorbic acid increased the antioxidant capacity values but interfered with the Folin-Ciocalteu method in the assessment of total phenolics. Addition of ascorbic acid produced a negative effect on the color stability of the juices. High temperature produced a deleterious effect on the evaluated responses. Petunidin-3-glucoside was the most stable anthocyanin followed by malvidin-3,5-diglucoside. Delphinidin-3,5-diglucoside and petunidin-3,5-diglucoside were the most abundant anthocyanins but the least stable pigments.

Keywords: Anthocyanins, antioxidant capacity, muscadine grape juice, total phenolics,

Vitis rotundifolia

INTRODUCTION

Fruit juices are widely consumed in the United States. In 1996, fruit juice consumption accounted for about 47% of all fruit consumed in the country, with an average consumption of 8.6 gallons per person (1). Grape juice consumption was 4.1 pounds per person (fresh-weight equivalent) in 1995/96 (2). However, muscadine grape consumption does not appear in any statistics due to the low volume produced. Some muscadine growers offer muscadine juice at prices around 5 dollars per 750 mL bottle.

The juice of muscadine grapes is appreciated by some people in southern United States due to its unique aroma (3). Other products can be made from muscadine juice like wines, jellies, jams, pie fillings, candy, fruit, butter, and fruit rollups.

Polyphenols in grape juice and wine has been attributed to prevention of atherosclerosis and coronary heart disease (4). Wang et al. (4) investigated the effect of the grape polyphenol, resveratrol, on platelet aggregation in vitro and in vivo, and found that resveratrol at 10 - 1000 µmol/L, significantly inhibited platelet aggregation in vitro induced by collagen, thrombin, and ADP in healthy subjects. Freedman et al. (5,6) found that selected flavonoids found in purple grape juice, decreased platelet aggregation, increased platelet-derived nitric oxide release, and decreased superoxide production after in vitro incubation and oral consumption. Vinson et al. (7) in an in vivo study with hamsters, found that grape juice was more effective than red wine or de-alcoholized red wine, at the same polyphenol dose, in inhibiting atherosclerosis. Chou et al. (8) reported that moderate daily consumption of purple grape juice improved flow-mediated vasodilatation of the brachial artery in patients with atherosclerotic vascular disease.

Chen et al. (9) found that grape juice may be useful in breast cancer prevention by

92 inhibiting in situ aromatase/estrogen biosynthesis. Durak et al. (10) concluded that red wine, white wine and grape juice all have high antioxidant potential to protect cellular structures against peroxidation reactions due to their high polyphenolic content. Kang et al. (11) found that grape juice consumption has a protective effect on DNA damage in human peripheral lymphocytes. O’Byrne et al. (12) compared the in vivo antioxidant efficacy of concord grape juice with that of a-tocopherol in healthy adults and found that the consumption of 10 mL of concord grape juice per kilogram of body weight per day increased serum antioxidant capacity and protected LDL against oxidation to an extent similar to that obtained with 400 IU a-tocopherol/d. The juice decreased native plasma protein oxidation significantly more than a-tocopherol, and they concluded that grape juice flavonoids were potent antioxidants that may protect against oxidative stress and may reduce the risk of free radical damage and chronic diseases (13). When the polyphenolic concentration was standardized, the antioxidant activity of grape juices toward LDL oxidation was similar to that of several California red wines. However, for normal products with undiluted total phenolic concentration, red grape juices had comparable activity to that of the red wines, while the white grape juices were less active

(14), indicating that polyphenolic concentration is a definitive factor for the oxidative inhibitory effect (15). Although flavonoids inhibit LDL oxidation, flavonoids in low concentration can promote LDL oxidation if the LDL is already mildly oxidized (16).

Leake (15) explained that the mechanism of this prooxidant activity may involve the reduction of Cu2+ to Cu+, followed by the rapid breakdown of preexisting lipid hydroperoxides in the mildly oxidized LDL by Cu+.

Flavonoids have been reported to exhibit a wide range of biological effects, including anti-ischemic, antiplatelet, antineoplastic, antiinflammatory, antiallergic, antilipoperoxidant and gastro-protective actions. Furthermore, flavonoids are potent antioxidants, free radical scavengers, metal chelators, and inhibit lipid peroxidation (16).

However, not all the effects of grape juice consumption are positive. Boato et al. (17) found that red grape juice, due to its high content of phenolic compounds bind and prevent absorption of soluble Fe, had profound inhibitory effect on iron bioavailability, while other fruit juices such as white grape, pear, apple, grapefruit, and orange juice do not exhibit such inhibitory effect. Additionally, some authors have suggested that there is no association between flavonoid intake and coronary heart disease (18) or thrombotic stroke (19).

Muscadine juices have not been extensively studied perhaps due to their very low participation in the market and localized production limited mostly to the southeastern

United States. Just recently, some studies on polyphenolic content of muscadine grape juices were published (20,21).

The objective of this study was to assess the total antioxidant capacity, the total polyphenolic content, total and individual anthocyanin content of muscadine grape juices from 10 Georgia-grown cultivars, and to determine the stability of the selected factors during an 8 week storage under different conditions.

MATERIALS AND METHODS

Chemicals. Standards of malvidin-3-O-ß-glucopyranoside (mv-3-gl), delphinidin-3-O-ß-glucopyranoside (dp-3-glc), petunidin-3-O-ß-glucopyranoside (pt-3-

94 glc), peonidin-3-O-ß-glucopyranoside (pn-3-glc), and cyanidin-3-O-ß-glucopyranoside

(cy-3-glc) were purchased from Polyphenols Laboratories AS (Sandnes, Norway). Trolox

(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and ABTS (2,2’-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt were purchased from Fluka

(Milwaukee, WI). Gallic acid, potassium persulfate, and Folin-Ciocalteu’s phenol reagent were purchased from Sigma (St. Louis, MO). Ascorbic acid was purchased from BASF

(Parsippany, NJ). Hydrochloric acid, sodium carbonate, potassium chloride and sodium acetate were purchased from J.T. Baker Chemical Co (Phillipsburg, NJ). Acetonitrile, methanol, formic acid, and water (HPLC grade) were purchased from Fisher Scientific

(Norcross, GA).

Samples. Juices from ten muscadine grape cultivars, five bronze (Carlos, Early

Fry, Fry, Summit and Late Fry), and five purple (Paulk, Cowart, Supreme, Ison and

Noble), grown in South Georgia and provided by Mr. Jacob Paulk (Paulk Vineyards,

Wray, GA.), were prepared. Fruit samples from the 10 cultivars were selected after removing under and overriped fruits and then washed. Juices were extracted by heating approximately 200 g of fruits in an equal amount of water at 85°C for 10 minutes, then strained through four layers of cheese cloth and pressed for juice extraction. 15 mL of each juice was bottled in a 20 mL scintillation vial and capped. The experimental design was an optimal design following a linear mixed model (22) with total phenolics, total anthocyanins, antioxidant capacity and 6 individual anthocyanins as dependent variables, diagonal structure of covariance with 4 classes for analysis: cultivar (10 levels due to the number of cultivars studied), temperature of storage (2 levels; refrigerated at 4°C and room temperature, 25°C), ascorbic acid added (two levels; no ascorbic acid and 0.15%

95 ascorbic acid), and time (9 levels; week 0 to 8), with two replicates for a total of 726 observations per variable analyzed. Hot-press method of juice preparation was chosen due to the low extraction of anthocyanins by cold-pressing the fruits. Blanco et al. (23) noted that the hot press process increased the color intensity of grape juices and the extraction of other phenolics, while destroying oxidative enzymes.

Analyses for antioxidant capacity, total phenolics, total anthocyanins, and individual anthocyanins were performed weekly. Antioxidant capacity was determined as

TEAC (trolox equivalent antioxidant capacity), following a modification to the method described by Re et al. (24). Trolox, a vitamin E analog, was used as an antioxidant standard. ABTS diammonium salt was dissolved in water to a concentration of 7 mM and allowed to react with a 2.45 mM potassium persulfate solution for 16 h in the dark. This reaction forms ABTS radical cations (ABTS*+). The ABTS*+ solution was diluted in ethanol to an absorbance of 0.70 (+/- 0.02) at 734 nm. 1.980 µL of diluted ABTS*+ solution was placed into a quartz cuvette and after one min, 20 µL of antioxidant compound or trolox standard was added and mixed. The absorbance reading was recorded by a computer connected to the spectrophotometer up to 6 additional minutes.

The recorded data was plotted and the area under the curve calculated using the

TableCurve 2D V5.0 software (SPSS Inc. Chicago, IL). The ratio between the area under the curve for the reaction of the grape juice sample and that for trolox gave the relative antioxidant capacity. For calibration and calculation of antioxidant capacity of the muscadine grape juices, trolox standards with final concentrations in the cuvette of 0, 3,

6, 9, 12, and 15 µM, were prepared.

Total phenolics were measured by the Folin-Ciocalteu reagent method (25,26).

200 µL of fruit juice was introduced into a test tube, 1.0 mL of Folin-Ciocalteu reagent and 0.8 mL of sodium carbonate (7.5%) were added, the test tube content mixed and allowed to stand for 30 min. Absorption at 765 nm was measured in a Shimadzu 300 UV-

Vis spectrophotometer (Shimadzu UV-1601, Norcross, GA). The total phenolic content was expressed as gallic acid equivalent (GAE), in milligrams per gram of sample, using a standard curve generated with 100, 200, 300, and 400 mg/L of gallic acid.

Total anthocyanin content was determined by a pH differential spectrophotometric method (27). The spectrophotometer was zeroed with distilled water.

Two dilutions of each juice sample were prepared, one with potassium chloride buffer, pH 1, and the other with sodium acetate buffer, pH 4.5, and the dilutions allowed to equilibrate for 15 min. The dilution factor varied from 5 to 40 depending on the color intensity of the juice. Lower dilution was needed in the case of bronze fruits and higher dilution for purple grape juices. The absorbance was measured at 520 and at 700 nm (to correct for haze) against a blank cell filled with distilled water.

Individual anthocyanins were analyzed and quantified by an HPLC method and the identity of the compounds validated by mass spectrometry. The juices were filtered through a 0.45-µm syringe polypropylene filter and a 20 µL aliquot injected into a

Hewlett-Packard (Avondale, PA) HP 1090 HPLC system with diode array detector. The mobile phase was: solvent A: formic acid/methanol/water (5:10:85, v/v/v), and solvent B: acetonitrile. The gradient for anthocyanin separation was as follows: at 0 min 100% solvent A, at 5 min 90% solvent A and 10% solvent B, and at 25 min 50% solvent A and

50% solvent B, with 5 min post-run with HPLC-grade water. Flow rate: 0.5 mL/min.

Column: Beckman Ultrasphere C18 ODS 4.6 x 250 mm. Temperature: 40ºC.

Anthocyanin-3,5-diglucosides were identified and quantified by the chromatographic characteristics of their corresponding anthocyanin-3-monoglucosides, and their identity was verified by mass spectrometric analysis using a LC-MS system from Thermo

Separations Products HPLC instrument (San Jose, California) coupled to a Perkin Elmer

Sciex API I plus quadrupole mass spectrometer (Shelton, Connecticut).

Statistics. The statistical analysis was carried out using SAS release 8.2 (SAS Institute

Inc. Cary, NC) and the PROC MIXED for the calculation of ANOVA’s. SAS was also used for the calculation of correlations, means and standard deviations.

RESULTS AND DISSCUSSION

Due to the large set of data involved we attempted to simplify their presentation by reporting only the initial content of the different parameters studied and the percentage of reduction of the corresponding response at the end of the 8 weeks study. However, statistical analysis was applied to the whole set of data and the results of such analysis discussed here.

Antioxidant capacity, total phenolics and anthocyanin content were affected by the addition of ascorbic acid. Antioxidant capacity and total phenolics had higher values when ascorbic acid was added. Higher antioxidant capacity may be due to the antioxidant properties of ascorbic acid.

The initial antioxidant capacity varied from 25.5 to 47.7 µM trolox/mL for juices stored at 4°C with 0.15% ascorbic acid added, 18.7 to 32.2 µM trolox/mL for refrigerated juices with no ascorbic acid added, 21.3 to 49.8 µM trolox/mL for juices stored at room

98 temperature with ascorbic acid added, and 14.5 to 34.2 µM trolox/mL in the case of samples with no ascorbic acid and stored at room temperature (Table 5.1). On the average, ascorbic acid increased the antioxidant capacity by 17 µM trolox/mL. Ascorbic acid is an antioxidant compound and had a profound effect on the TEAC values. At the end of the 8 weeks study (Table 5.2), the antioxidant capacity decreased from 8.2 to

17.9% for juices with ascorbic acid added and stored under refrigeration, 11.8 to 20.4% in the case of samples with no ascorbic acid added and stored at 4°C, 14.1 to 25.5% for juices stored at room temperature with ascorbic acid added, and 17.8 to 28.9% for samples stored at 25°C with no ascorbic acid added. The kinetic trend of antioxidant capacity changes during the study is shown in Figure 5.1 for the muscadine Supreme cultivar. At refrigeration temperature the reduction in antioxidant capacity was significantly lower than at room temperature. There was a significant difference (a=0.01) in antioxidant capacity among cultivars, levels of ascorbic acid and temperature with time. The interactions of all independent variables showed a significant effect on antioxidant capacity (a=0.05). Antioxidant capacity did not correlate with total anthocyanins (R=0.08) indicating that the antioxidant properties of muscadine juices are mainly due to the presence of other phenolics that exist in higher concentrations or that are more reactive than anthocyanins.

The total phenolic content was, on average, higher for the purple skin cultivars

(Paulk, Cowart, Supreme, Ison and Noble), but there was no significant difference

(a=0.05) in phenolic content between the two types of muscadine grapes. The initial total phenolic content (Table 5.1) varied from 767 to 1227 Gallic Acid Equivalent (G.A.E.) mg/L of juice, in the case of juices stored at 4°C with ascorbic acid added, and from 410

99 to 854 G.A.E. mg/L for juices stored at refrigeration temperature with no ascorbic acid added. Juices stored at room temperature (25°C) varied from 759 to 1212 G.A.E. mg/L with ascorbic acid added and, from 416 to 844 G.A.E. mg/L for samples with no ascorbic acid added. Ascorbic acid increased the total phenolic values by an average of 360

G.A.E. mg/L. This indicates that there is interference by ascorbic acid in the total phenolics determination using the Folin-Ciocalteu method. Table 5.2 shows that by the end of the 8 week study the total phenolic content was reduced by 6.4 to 24.5% in the case of refrigerated samples with ascorbic acid added, 20.8 to 31.7% for refrigerated samples with no ascorbic acid added, 14.2 to 31.9% for juices stored at room temperature with ascorbic acid added, and 28.1 to 38.5% in the case of juices stored at room temperature with no ascorbic acid added. This result shows that samples with ascorbic acid added had less reduction in total phenolic content, even though ascorbic acid may react with anthocyanins producing a degradation of the pigments. The stabilizing effect of ascorbic acid on total phenolics was enhanced by refrigeration temperature. The trend in the reduction of total phenolics is almost linear during the 8 weeks period with a slight increase in the slope beginning in the 7th week. Figure 5.2 shows the kinetics of the total polyphenolic content for cultivar Supreme. The statistical analysis showed that there was a significant difference in total phenolic content within cultivars and during the 8 week storage time, as well as a significant effect of ascorbic acid and temperature of storage on the stability of total phenolics (a=0.01). The correlation analysis showed that the overall average of total phenolics had a correlation coefficient (R) of 0.41 and 0.38 when analyzed against antioxidant capacity and total anthocyanin content, respectively.

100

Anthocyanin content was the most affected factor during the study. Tables 5.3 and

5.4 show the initial content of total and individual anthocyanins under the different treatment combinations. Cultivars with bronze skin (Carlos, Early Fry, Fry, Summit and

Late Fry) had total anthocyanin content ranging between 2.0 and 2.5 mg/L with low variability among them. Lowest values were found in juices with ascorbic acid added; however, there was no significant difference (a=0.01) in anthocyanin content in bronze skin grapes under the different treatments studied. In juices made from bronze skin grapes only delphinidin-3,5-diglucoside, petunidin-3,5-diglucoside and malvidin-3,5- diglucoside had concentrations high enough to be detected by the HPLC analysis.

Anthocyanins in bronze skin grapes degraded rapidly to the point that they were not detected on the 3rd week. The changes in anthocyanin content in purple grapes were noticeable even to the human eye. Color degradation was observed with a tendency of the juices to turn brown. Ascorbic acid had a degrading effect on anthocyanin stability.

Anthocyanins degraded in greater proportion when ascorbic acid was present. This result can be explained by the finding of García-Viguera and Bridle (28) who demonstrated that ascorbic acid has a degradation effect on anthocyanins through an oxidation reaction in which ascorbic acid acts as an activator of molecular oxygen producing free radicals, which cleave the anthocyanin ring structure. In the case of juices made from purple grapes, initial total anthocyanin content varied from 134 to 350 mg/L for samples with ascorbic acid added, and from 142 to 369 mg/L for juices with no ascorbic acid added

(Table 5.3). Figure 5.3 shows an example of the kinetic trend of the total anthocyanins degradation for the cultivar Supreme as affected by different treatments. This figure shows the detrimental effect of ascorbic acid on the total anthocyanin content. At

101 refrigeration temperature the anthocyanin content remained almost constant during the first 6 weeks, thereafter it began to drop rapidly to 60 mg/L. Different effect was found for samples stored at room temperature which had a faster decline in the anthocyanin content (20 mg/L) during the first 4 weeks of storage. This may be due to the quicker and faster reaction between ascorbic acid and anthocyanins favored by the relatively high temperature.

Delphinidin-3,5-diglucoside was the most abundant anthocyanin in the grapes accounting for about 45% of the total anthocyanin content, but it was the least stable of all the pigments. Degradation at the end of the 8 weeks storage study ranged from 73 to

83% for refrigerated samples, and from 79 to greater than 99% in the room temperature samples (Table 5.5). The higher degradation occurred when ascorbic acid was added to the juices. Petunidin-3,5-diglucoside was the second individual anthocyanin in concentration and in loss of stability. About 24% of the total anthocyanin content was derived from this phenolic, and degradation from 62 to 73% in refrigerated samples and from 68 to 99% in the room temperature juices. Malvidin-3,5-diglucoside was the third anthocyanin in concentration but it was the second most stable. It accounted for about

20% of the total anthocyanins and degraded from 13 to 41% in refrigerated samples, and from 29 to 99% in the 25°C juices. Cyanidin-3,5-diglucoside and peonidin-3,5- diglucoside accounted for 6 and 3% of the total anthocyanin content, respectively, and had similar rate of degradation going from 36 to 58% for refrigerated samples and from

50 to 99% in the room temperature juices. The lowest in concentration of all anthocyanins detected was petunidin-3-glucoside which represented about 1% of the total anthocyanin content but the most stable. On the average, petunidin-3-glucoside degraded

102 just 2% in the case of the refrigerated samples with no ascorbic acid added, 4% in refrigerated juices with ascorbic acid added, 82% in the room temperature juices with ascorbic acid, and 8% in samples with no ascorbic acid but stored at room temperature.

Petunidin-3-glucoside was very stable during the eight weeks of study, except when ascorbic acid was added and stored at room temperature.

García-Viguera and Bridle (28), in agreement with our findings, reported that, in an 18 days study, diglucoside anthocyanin solutions stored at 20°C and containing ascorbic acid degraded rapidly with a clear browning effect. Recently Talcott et al. (21) reported the detrimental effect of ascorbic acid on the color of purple muscadine juices.

They found that refrigerated muscadine juice samples analyzed after 72 h of processing had a reduction in total anthocyanin content of 3-5% when ascorbic acid was added to the juices. They did not run a longer storage analysis but their results might follow the trend found in this study. Additionally, they performed a comparison of the effect of different processing methods for the muscadine juice, and found that high temperature juice preparation enhanced the degradation of anthocyanins, confirming our findings. Our results of the stability of individual anthocyanins agree with Goldy et al. (29) who highlighted that delphinidin-3,5-diglucoside and malvidin-3,5-diglucoside were pigments of great importance in muscadine grapes because of the instability of the former and the stability of the later. However, the average concentration of the anthocyanin diglucosides in the muscadine grapes analyzed by Goldy et al. was different from the initial individual anthocyanins found in the current study. The different techniques used for the determination and quantification and the different agro-ecological conditions of the muscadine crops may have produced the difference in the results.

103

There is no previous report on the kinetics of antioxidant capacity and total phenolics in stored muscadine juices. Our findings show a statistically significant

(p<0.05) deteriorative process for these factors during storage, thereby reducing the potential beneficial effects of this food on human health. The reduction in phenolics and antioxidant capacity were accompanied by a rapid decrease in anthocyanin content and excessive browning of the purple juices, a factor that may affect the organoleptic quality of the juices.

ACKNOWLEDGMENT

Research funded by the State of Georgia’s Traditional Industries Program for

Food Processing research grant. We thank Mr. Jacob Paulk for providing us with the muscadine fruits. We also thank Fulbright-IIE-Colciencias and the Universidad

Surcolombiana for the financial support to the graduate student, Eduardo Pastrana-

Bonilla.

104

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Table 5.1. Initial Values of Trolox Equivalent Antioxidant Capacity (TEAC) and Total Phenolics for Muscadine Juices Under Different Treatments1

Antioxidant Capacity (µM trolox /mL) Total Phenolics (G.A.E., mg/L)

Refrigerated Room Temperature Refrigerated Room Temperature Cultivar Ascorbic No Ascorbic Ascorbic No Ascorbic Ascorbic No Ascorbic Ascorbic No Ascorbic Acid Acid Acid Acid Acid Acid Acid Acid Carlos 36.4 ± 0.6 28.4 ± 0.4 35.9 ± 0.3 34.2 ± 0.7 1182.7 ± 16.9 828.5 ± 5.8 1191.4 ± 8.5 843.8 ± 23.4 Early Fry 38.0 ± 0.2 29.6 ± 0.4 24.4 ± 0.3 29.2 ± 0.1 767.5 ± 5.4 663.4 ± 9.5 758.8 ± 0.1 684.4 ± 14.3 Fry 47.3 ± 1.0 30.4 ± 0.3 49.8 ± 0.4 34.2 ± 0.1 779.8 ± 3.4 422.0 ± 6.0 833.9 ± 11.7 430.6 ± 9.0 Summit 40.0 ± 0.3 21.0 ± 0.2 33.3 ± 0.2 19.2 ± 0.1 887.7 ± 6.3 409.6 ± 2.9 888.0 ± 0.1 416.4 ± 8.7 Late Fry 35.4 ± 0.3 18.7± 0.3 35.7 ± 0.8 20.9 ± 0.1 1181.3 ± 16.9 802.7 ± 5.7 1209.4 ± 16.9 783.8 ± 9.6 Paulk 47.7 ± 0.7 32.2 ± 0.1 40.2 ± 0.1 21.2 ± 0.1 1101.4 ± 15.4 839.8 ± 1.5 1104.3 ± 15.5 833.5 ± 11.9 Cowart 25.5 ± 0.2 18.7 ± 0.1 23.4 ± 0.3 14.5 ± 0.2 834.0 ± 17.4 560.3 ± 8.0 818.9 ± 11.7 570.2 ± 8.0 Supreme 36.4 ± 0.8 19.8 ± 0.1 27.6 ± 0.4 17.8 ± 0.1 1018.1 ± 7.2 779.8 ± 5.3 1010.2 ± 1.3 770.9 ± 5.3 Ison 28.4 ± 0.4 20.2 ± 0.8 21.3 ± 0.2 15.3 ± 0.1 1226.7 ± 25.6 853.8 ± 17.8 1210.7 ± 1.9 843.3 ± 5.9 Noble 35.6 ± 0.0 26.4 ± 0.2 33.0 ± 0.5 26.1 ± 0.6 1208.7 ± 16.9 849.6 ± 6.0 1212.2 ± 17.0 844.0 ± 1.0

1Values are average and standard deviation of duplicates

Table 5.2. Percentage of Reduction of Antioxidant Capacity and Total Phenolics Under Different Treatments

Antioxidant Capacity Total Phenolics Cultivar Refrigerated Room Temperature Refrigerated Room Temperature Ascorbic No Ascorbic Ascorbic No Ascorbic Ascorbic No Ascorbic Ascorbic No Ascorbic Acid Acid Acid Acid Acid Acid Acid Acid Carlos 10.9 11.9 14.1 17.8 24.5 31.7 30.3 38.5 Early Fry 10.0 13.8 18.3 22.1 6.4 27.7 14.2 33.1 Fry 16.9 19.8 23.4 25.7 12.7 24.6 20.8 31.3 Summit 17.9 20.4 20.2 23.0 13.0 20.8 27.3 37.8 Late Fry 8.3 11.8 15.5 18.9 21.5 26.8 31.9 36.6 Paulk 13.9 15.2 25.5 28.9 16.7 23.1 30.1 37.6 Cowart 11.1 13.0 18.7 23.7 17.3 24.4 29.6 32.7 Supreme 12.7 17.7 22.2 25.0 20.2 21.5 26.2 33.4 Ison 14.6 15.8 23.6 26.7 18.8 23.4 27.5 29.4 Noble 8.2 12.7 19.2 23.4 12.2 22.0 16.0 28.1

Table 5.3. Initial Content of Total Anthocyanins and Individual Anthocyanins in Refrigerated Muscadine Juices as Affected by Ascorbic Acid1

Cultivar Anthocyanins (mg/L) Total Individual Anthocyanins Ascorbic Acid Anthocyanins dp-3,5-di-glc pt-3,5-di-glc mv-3,5-di-glc cy-3,5-di-glc pn-3,5-di-glc pt-3-glu

Carlos yes 2.1 ± 0.1 1.0 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Early Fry yes 2.2 ± 0.0 1.1 ± 0.0 0.5 ± 0.0 0.5 ± 0.1 n.d. n.d. n.d. Fry yes 2.1 ± 0.0 0.9 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 n.d. n.d. n.d. Summit yes 2.2 ± 0.0 1.0 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Late Fry yes 2.3 ± 0.1 1.0 ± 0.4 0.6 ± 0.0 0.4 ± 0.0 n.d. n.d. n.d. Paulk yes 349.8 ± 2.5 153.9 ± 10.5 85.1 ± 2.4 70.4 ± 0.5 21.5 ± 0.6 10.6 ± 0.1 3.4 ± 0.1 Cowart yes 133.9 ± 0.1 58.2 ± 1.7 31.2 ± 0.5 26.4 ± 1.0 8.0 ± 0.3 3.9 ± 0.1 1.4 ± 0.0 Supreme yes 147.3 ± 2.3 66.6 ± 0.5 35.6 ± 1.1 30.6 ± 1.6 9.6 ± 0.2 4.9 ± 0.1 1.4 ± 0.0 Ison yes 257.8 ± 3.8 113.4 ± 4.3 65.9 ± 0.5 51.9 ± 0.4 16.2 ± 0.6 7.8 ± 0.1 2.6 ± 0.0 Noble yes 187.6 ± 3.0 79.4 ± 3.2 45.3 ± 0.7 36.5 ± 0.9 12.4 ± 0.4 5.5 ± 0.1 2.0 ± 0.0

Carlos no 2.4 ± 0.0 1.1 ± 0.0 0.6 ± 0.0 0.5 ± 0.1 n.d. n.d. n.d. Early Fry no 2.4 ± 0.0 1.1 ± 0.0 0.6 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Fry no 2.2 ± 0.0 1.0 ± 0.0 0.5 ± 0.0 0.4 ± 0.1 n.d. n.d. n.d. Summit no 2.4 ± 0.0 1.0 ± 0.0 0.6 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Late Fry no 2.4 ± 0.0 1.1 ± 0.0 0.6 ± 0.1 0.5 ± 0.0 n.d. n.d. n.d. Paulk no 368.5 ± 5.2 168.7 ± 5.9 88.0 ± 3.1 74.4 ± 2.1 21.5 ± 0.9 10.5 ± 0.2 3.5 ± 0.1 Cowart no 141.7 ± 3.0 64.2 ± 2.7 33.1 ± 1.9 27.5 ± 0.0 8.4 ± 0.4 4.2 ± 0.1 1.4 ± 0.0 Supreme no 165.0 ± 1.2 74.8 ± 1.1 38.6 ± 1.1 32.3 ± 0.7 9.5 ± 0.1 4.9 ± 0.1 1.5 ± 0.1 Ison no 277.5 ± 3.3 121.9 ± 1.0 68.6 ± 1.1 55.0 ± 1.6 16.9 ± 0.1 8.3 ± 0.4 2.5 ± 0.1 Noble no 199.9 ± 1.4 91.0 ± 4.6 48.2 ± 2.7 39.6 ± 0.3 12.2 ± 0.4 5.8 ± 0.1 2.1 ± 0.0

1Values are average and standard deviation of duplicates; n.d.= not detected

Table 5.4. Initial Content of Total Anthocyanins and Individual Anthocyanins in Muscadine Juices Stored at Room Temperature as Affected by Ascorbic Acid1

Cultivar Anthocyanins (mg/L) Total Individual Anthocyanins Ascorbic Anthocyanins Acid (mg/L) dp-3,5-di-glc pt-3,5-di-glc mv-3,5-di-glc cy-3,5-di-glc pn-3,5-di-glc pt-3-glu

Carlos Yes 2.0 ± 0.0 1.1± 0.2 0.5 ± 0.0 0.4 ± 0.1 n.d. n.d. n.d. Early Fry Yes 2.3 ± 0.0 1.1± 0.1 0.5 ± 0.0 0.4 ± 0.0 n.d. n.d. n.d. Fry Yes 2.0 ± 0.0 0.9 ± 0.1 0.5 ± 0.0 0.4 ± 0.1 n.d. n.d. n.d. Summit Yes 2.2 ± 0.0 1.0 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 n.d. n.d. n.d. Late Fry Yes 2.3 ± 0.1 1.0 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Paulk Yes 356.3 ± 5.3 164.5 ± 2.4 78.1 ± 4.4 67.9 ± 1.6 21.0 ± 0.1 10.6 ± 0.1 3.6 ± 0.1 Cowart Yes 167.0 ± 1.0 111.4 ± 0.4 26.4 ± 1.0 13.3 ± 0.1 4.0 ± 0.3 2.1 ± 0.1 1.5 ± 0.0 Supreme Yes 145.2 ± 2.4 67.5 ± 3.3 35.4 ± 1.1 32.5 ± 0.7 9.4± 0.2 4.2 ± 0.1 1.6 ± 0.0 Ison Yes 247.8 ± 1.9 114.4 ± 4.4 58.6 ± 1.8 49.2 ± 1.0 14.4± 0.1 8.1 ± 0.4 2.6 ± 0.1 Noble Yes 184.0 ± 2.9 85.3± 3.4 44.5 ± 0.7 38.0 ± 2.0 11.6 ± 0.1 5.9 ± 0.0 1.9 ± 0.0

Carlos No 2.4± 0.0 1.1 ± 0.1 0.6 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Early Fry No 2.5± 0.1 1.1 ± 0.1 0.6 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Fry No 2.2± 0.0 1.0 ± 0.1 0.5 ± 0.1 0.4 ± 0.0 n.d. n.d. n.d. Summit No 2.3 ± 0.0 1.1 ± 0.1 0.6 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Late Fry No 2.4 ± 0.0 1.1 ± 0.1 0.6 ± 0.0 0.5 ± 0.0 n.d. n.d. n.d. Paulk No 370.3 ± 7.7 170.4 ± 1.2 87.6 ± 1.2 71.2 ± 0.5 21.6 ± 0.8 10.8 ± 0.6 3.6 ± 0.1 Cowart No 141.70± 3.0 62.3 ± 0.9 33.8 ± 1.0 28.3 ± 1.0 8.5 ± 0.1 4.2 ± 0.2 1.4 ± 0.0 Supreme No 165.6 ± 1.2 73.9 ± 0.5 39.3 ± 1.4 30.8 ± 2.1 9.5 ± 0.2 4.9 ± 0.1 1.7 ± 0.0 Ison No 268.3 ± 3.8 125.3 ± 6.2 62.8 ± 0.5 52.6 ± 0.1 15.5 ± 0.1 8.1 ± 0.3 2.6 ± 0.1 Noble No 201.1± 1.4 92.9 ± 1.3 48.5 ± 2.0 39.2 ± 1.1 11.9 ± 0.3 5.9 ± 0.0 2.0 ± 0.0

1Values are average and standard deviation of duplicates; n.d.= not detected

Table 5.5. Percentage of Reduction of Total Anthocyanins and Individual Anthocyanins During 8 Week Storage Under Different Treatments

Cultivar Treatment Percentage of Reduction Individual Anthocyanins Ascorbic Total pt-3,5-di- mv-3,5-di- cy-3,5-di- pn-3,5-di- Storage Acid Anthocyanins dp-3,5-di-glc glc glc glc glc pt-3-glu

Paulk Refrig. yes 60.2 77.4 66.2 29.3 44.8 45.9 3.1 Cowart Refrig. yes 67.6 81.7 72.1 41.3 55.5 56.1 5.8 Supreme Refrig. yes 64.8 80.4 71.2 34.5 51.1 54.0 3.0 Ison Refrig. yes 63.7 79.1 70.6 35.4 51.8 52.3 2.2 Noble Refrig. yes 68.2 82.9 73.3 38.5 56.4 57.9 5.4

Paulk Refrig. no 58.1 76.7 65.8 24.7 43.2 41.5 1.8 Cowart Refrig. no 56.1 74.3 63.0 15.8 39.4 39.1 2.5 Supreme Refrig. no 54.7 73.9 62.0 12.7 36.4 39.5 1.6 Ison Refrig. no 55.6 73.1 65.4 17.1 39.6 41.7 1.4 Noble Refrig. no 55.7 75.1 63.8 17.6 44.7 41.5 2.8

Paulk Room yes 97.3 98.3 97.7 94.8 96.4 96.2 86.6 Cowart Room yes 93.4 96.4 94.6 88.3 91.4 91.7 66.9 Supreme Room yes 99.3 99.6 99.4 98.6 99.1 99.1 86.2 Ison Room yes 96.3 98.0 97.0 93.1 95.3 95.3 81.2 Noble Room yes 97.4 98.5 97.9 95.2 96.7 96.3 87.6

Paulk Room no 91.7 95.4 92.8 84.3 88.6 88.7 8.9 Cowart Room no 70.0 82.3 74.4 46.7 60.0 60.0 5.6 Supreme Room no 93.9 96.4 94.9 88.1 91.3 91.6 7.1 Ison Room no 77.0 87.5 80.1 57.0 66.9 70.1 9.1 Noble Room no 62.4 78.7 67.5 28.6 49.6 49.6 8.4 113

Figure 5.1. Kinetics of antioxidant capacity for cultivar “Supreme.”

10 Antioxidant Capacity (µM trolox/mL).

0 0 1 2 3 4 5 6 7 8 Time (weeks) Refrigerated, ascorbic acid added Room temperature, ascorbic acid added Refrigerated, no ascorbic acid added Room temperature, no ascorbic acid added

115

Figure 5.2. Kinetics of the total phenolics content for cultivar “Supreme.”

1200

1000

800

600

400

200 Total phenolics (mg/L G.A.E.)

0 0 1 2 3 4 5 6 7 8 Time (weeks)

Refrigerated, ascorbic acid added Room temperature, ascorbic acid added Refrigerated, no ascorbic acid added Room temperature, no ascorbic acid added

117

Figure 5.3. Kinetics of the total anthocyanin content for cultivar “Supreme.”

180

160

140

120

100

40 Total Anthocyanins (mg/L)

0 0 1 2 3 4 5 6 7 8 Time (Weeks) Refrigerated, ascorbic acid added Room temperature, ascorbic acid added Refrigerated, no ascorbic acid added Room temperature, no ascorbic acid added

119

CHAPTER 6

SUMMARY AND CONCLUSIONS

Muscadine grapes have good concentrations of important flavonoids and phenolic acids known to have potential beneficial effects on human health. The major phenolics in the skins of muscadine grape cultivars were ellagic acid (6-22 mg/100g FW), followed in most cases by the flavonols myricetin and quercetin (2-19 and 0.48-3.9 mg/100g FW, respectively). Kaempferol was also found but in lower concentration ranging from 0.15 to

3.0 mg/100g FW. The stilbenoid, resveratrol was present in most cultivars, but in a very low concentration (less than 0.25 mg/100g FW of skins). This result contradicts previous reports of high concentrations of resveratrol in muscadine grapes.

Major phenolics in grape seeds were identified as epicatechin, catechin, and gallic acid, ranging from 450 to 1900 mg/100g FW, 420 to 1410 mg/100g FW and below detection limit to 11 mg/100 g FW, respectively. Phenolics in leaves were myricetin, ellagic acid, kaempferol, quercetin and gallic acid, with concentrations ranging from 108 to 216 mg/100g FW, 45 to 80 mg/100g FW, 7 to 12 mg/100g FW, 6 to 22 mg/100g FW, and 6 to 19 mg/100 g FW, respectively.

Anthocyanin content of muscadine grapes was almost confined to the skins of purple grapes. The seeds of almost all cultivars, independent of the color of the skin, contain some anthocyanin and the pulps were almost free of this group of colored phenolics. Five of the 6 anthocyanins detected in muscadine grapes were anthocyanin diglucosides, with delphinidin-3,5-diglucoside as the most abundant, accounting for 46%

120 of the total anthocyanin content. Petunidin-3,5,-diglucoside, malvidin-3,5,-diglucoside, cyanidin-3,5-diglucoside and peonidin-3,5-diglucoside followed in concentration accounting for 23, 20, 6, and 3% of the total anthocyanin value. Petunidin-3-glucoside was the only monoglucoside detected with a contribution of about 1% of the total anthocyanins. The stability study showed that anthocyanins were degraded faster in the precense of added ascorbic acid. Petunidin-3-glucoside and malvidin-3,5-diglucoside were the most stable anthocyanins.

The total phenolics content of muscadine grapes was highest in the seeds (from

1650 to 3540 GAE mg/100g FW), intermediate values in skins (260-540 GAE mg/100g

FW), and lower values for pulps (less than 35 GAE mg/100g FW, in all cultivars). Total phenolic content in leaves was, on average, 42% higher than in the whole fruits, and antioxidant capacity was, on average, 1,543% higher in leaves than in the whole fruits.

Total phenolics highly correlated with the concentration of major phenolics.

Seeds have higher antioxidant capacity and their values have high correlation

(r=0.90, p<0.05) with the total phenolics content. Skins have an intermediate antioxidant capacity, and pulps have the lowest values, but there were lower correlations (r=0.56, p<0.05 and r=0.75, p<0.05, respectively) with the total phenolic content, indicating that the antioxidant capacity of skins and pulps were more closely related to the presence of other antioxidants.

Ascorbic acid increased the total phenolic and antioxidant capacity values when added to muscadine juices and imparted some stability to the measured parameters. High temperature accelerated the degradation of anthocyanins, total phenolics and antioxidant capacity, even when ascorbic acid was added to the juices.

121

This is the first report on the phenolic profile of muscadine leaves, and the first time that petunidin-3-glucoside is found in muscadine grapes. Seeds and skins that are most of the times waste in muscadine wine or juice production, and leaves that are not used at the end of the fruit season, contain important beneficial phenolics and have high antioxidant capacity. Potential exists for using them as nutraceutical products, through appropriate processing and packaging that preserve their phenolic content.

SUGGESTIONS FOR FUTURE WORK

The high concentration of important polyphenolics in muscadine leaves was one of the major findings of this study. The presence of myricetin and ellagic acid in concentrations well above their reported values in fruits and vegetables, make muscadine leaves a promising source of such compounds as nutraceuticals. The determination of the temporal fluctuation of these selected compounds during the growth season is suggested in order to determine the best time to collect the leaves to maximize their phenolic content, as well as the determination of a suitable method of processing and/or extraction that minimize lost of phenolics and ensure their stability when marketed as a nutracetutical.

Due to their promising use as a chemopreventive product, in vitro and/or in vivo studies of the effect of the extracts or capsules of muscadine leaves on different types of cancer cells is suggested. In a similar way, a study of supplementation of muscadine leave capsules to diabetic animals and/or persons can be performed to evaluate their potential as anti-diabetic agent, similar to the studies currently in progress with raspberries as ellagic acid source.

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Muscadine skins are a byproduct of juice and wine production. Through previous adequate processing, they may be used as flavoring agent for other fruits jellies or jams with the added value as a source of antioxidant compounds. The effect of processing muscadine skins on their polyphenolic content is also suggested as future work.

The potential use of muscadine seed extracts, because of their high content of catechin, epicatechin, and gallic acid, as antioxidants in food systems is suggested.

The most negative effect on muscadine juices was the rapid degradation of anthocyanins. The development of a method of copigmentation of anthocyanin diglucosides in purple muscadine grape juices is also suggested as future work.