(+)-Catechin and Quercetin from Pawpaw Pulp A

Home , Annona, Asimina incana, Asimina parviflora, Asimina tetramera, Asimina triloba, Atemoya

Characterization of (+)-Catechin and Quercetin from Pawpaw Pulp

A thesis presented to

the faculty of the College of Health Sciences and Professions of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Jinsoo Ahn

June 2011

This thesis titled

Characterization of (+)-Catechin and Quercetin from Pawpaw Pulp

JINSOO AHN

has been approved for

the School of Applied Health Sciences and Wellness

and the College of Health Sciences and Professions by

Robert G. Brannan

Assistant Professor of Applied Health Sciences and Wellness

Randy Leite

Interim Dean, College of Health Sciences and Professions 3

ABSTRACT

AHN, JINSOO, M.S., June 2011, Human and Consumer Sciences, Food and Nutrition

Characterization of (+)-Catechin and Quercetin from Pawpaw Pulp

Director of Thesis: Robert G. Brannan

This thesis investigates the concentration of total phenolics and total flavonoids in pulp extracts of pawpaw harvested in 2008, 2009, and 2010, and the concentration of (+)- catechin and quercetin flavonoids in 2010 pawpaw pulp extracts using high performance liquid chromatography (HPLC). Next, influence of frozen storage and air or vacuum packaging of pawpaw pulp on the concentration of (+)-catechin and quercetin flavonoids was examined. In addition, properties of pawpaw pulp such as moisture content, lipid content, percent sugar, color, and pH were measured. Total phenolics were determined using the Folin-Ciocalteu assay and reported as µmol gallic acid equivalent (GAE)/ g wet tissue. The concentration was observed in the order of 2009 sample (3.91 ± 1.61) < 2008 sample (11.19 ± 0.57) < 2010 sample (14.11 ± 1.90). Total flavonoids were determined spectrophotometrically using flavonoid rutin as a standard and reported as µmol rutin/ g wet tissue. The order of concentration was 2009 (0.46 ± 0.24) = 2010 (0.76 ± 0.40) <

2008 (2.01 ± 0.44). By using HPLC, the concentration of (+)-catechin and quercetin was determined as 0.43 ± 0.79 mg/ 100 g wet pulp and 0.46 ± 0.79 mg/ 100 g wet pulp, respectively. In both air and vacuum packaging under frozen condition, the concentration of (+)-catechin and quercetin increased during the first 4 months and decreased until the twelve-month period. In the meantime, an unknown compound was detected after quercetin was eluted. Pawpaw pulp has 75.24 % moisture, 0.45 % lipid, and 24.1 % 4 percent sugar and color was close to white, magenta, and yellow. The pH of pawpaw pulp was 4.85 which indicates slightly acidic. In conclusion, pawpaw pulp has considerable concentration of phenolic compounds which include flavonoids, and (+)-catechin and quercetin were major phenolic flavonoids in the pulp which increase their concentration during frozen storage before extraction. These results suggest that the two important flavonoids from pawpaw pulp can be essential sources for value-added functional food ingredients by optimization of storage condition of the pulp.

Approved: ______

Robert G. Brannan

Assistant Professor of Applied Health Sciences and Wellness 5

To my late father, Professor Jong-Cheol Ahn

ACKNOWLEDGMENTS

Foremost, I would like to express my sincere gratitude to my advisor Professor

Robert G. Brannan, committee chairman for his continuous support for my master’s study and research. His guidance helped me during the entire time required to accomplish the research and write this thesis. Without his advice, this study would not have been successful.

I also would like to acknowledge the members of my thesis committee, Professor

Sharon Rana and Professor Michael Held, for their insightful comments and considerations.

My special thanks go to Professor David H. Holben for all his encouragement, advice, and offering me a graduate assistantship.

I also appreciate to Dr. Jennifer Horner, the Associate Dean of Research and

Graduate Studies in the College of Health Sciences and Professions for proofreading and editing this thesis.

In addition, I thank my fellow lab colleagues, other graduate students, and all the staff members in the School of Applied Health Sciences and Wellness and in the Food and Nutrition program.

Last but not the least, I would like to thank my late father, mother, and brother for encouraging me to pursue this degree and supporting me throughout my life. 7

TABLE OF CONTENTS

Page

Abstract ...... 3 Dedication ...... 5 Acknowledgments ...... 6 List of Tables ...... 10 List of Figures ...... 11 CHAPTER 1: INTRODUCTION ...... 12 Overview and Background ...... 12 Statement of the Problem ...... 14 Research Questions ...... 15 Significance of Research Study ...... 15 Limitations ...... 17 Glossary ...... 17 CHAPTER 2: REVIEW OF CRITICAL LITERATURE ...... 19 Pawpaw ...... 19 Background ...... 19 The Description and Horticulture of Pawpaw ...... 20 Harvesting ...... 24 Postharvest Challenges ...... 26 Characteristics of Pawpaw Fruit ...... 27 Sensory Characteristics ...... 27 Nutritional Facts...... 28 Antioxidants ...... 29 Antioxidants in Fruits ...... 30 Introduction ...... 30 Oxidation...... 33 Oxidation in Biological Systems ...... 33 Oxidation in Foods ...... 34 Types of Antioxidants ...... 36 8

Flavonoids ...... 38 Introduction ...... 38 Function ...... 39 Subgroups ...... 40 Daily Intake ...... 41 Flavonoid Subclasses ...... 43 Flavonols ...... 43 Flavones ...... 44 Flavanones ...... 45 Flavan-3-ols ...... 46 Anthocyanidins ...... 47 Isoflavones ...... 48 Conclusion ...... 49 CHAPTER 3: METHODOLOGY ...... 50 Materials ...... 50 Pawpaw Sample Preparation...... 50 Pawpaw Pulp Extraction ...... 51 Measurements of Total Phenolics ...... 52 Measurements of Total Flavonoids ...... 52 Moisture, Lipid, Percent Sugar, Color, and pH ...... 53 HPLC Analysis ...... 54 Sampling Plan ...... 57 Statistical Analysis ...... 57 CHAPTER 4: RESULTS ...... 59 Measurements of Total Phenolics and Total Flavonoids ...... 59 Analysis of (+)-Catechin and Quercetin ...... 60 Evaluation of the Stability of (+)-Catechin and Quercetin in Stored Samples ...... 61 Objective Evaluation ...... 63 Summary ...... 63 CHAPTER 5: DISCUSSION AND CONCLUSION ...... 65 Measurements of Total Phenolics ...... 65 9

Measurements of Total Flavonoids ...... 69 Characterization of (+)-Catechin and Quercetin ...... 70 Evaluation of the Stability of (+)-Catechin and Quercetin in Stored Samples ...... 72 Objective Evaluation ...... 75 Conclusion ...... 76 Future Directions ...... 76 REFERENCES ...... 77

LIST OF TABLES

Page

Table 1: Total Phenolics of Common Fruits ...... 32

Table 2: Primary and Synergistic Antioxidants ...... 37

Table 3: Flavonoid Subclasses and Food Sources ...... 41

Table 4: HPLC Mobile Phase Gradient Program ...... 55

Table 5: Identification of Retention Time (Ret Time), Area, and Height of (+)- Catechin and Quercetin Peaks According to Concentration Changes ...... 57

Table 6: Comparison of Total Phenolics, Total Flavonoids, and the Ratio of Flavonoid to Phenolics in Pawpaw Pulp Harvested in 2008, 2009, and 2010……………………..…60

Table 7: Retention Times and Concentration of (+)-Catechin and Quercetin in Pawpaw Pulp Harvested in 2010...…………………………………………….………………….61

Table 8: The Concentration of (+)-Catechin (mg/100g wet pulp), Quercetin (mg/100g wet pulp), and Unknown Compound (mAU) in Pawpaw Pulp Extracts, Stored in Air or Under Vacuum for 0, 2, 4, 6, 8, 10, and 12 Months…………………………………………...62

Table 9: Moisture Content, Lipid Content, Percent Sugar, Color, and pH Values of Pawpaw ...... 63

Table 10: The Amounts of Total Phenolics in Commonly Consumed Fruits Based on Several Recent Studies; pawpaw pulp extract was prepared from pawpaws harvested in 2010………………………………………………………...... 68

Table 11: The Amounts of Flavonoids in Selected Fruits Based on Recent Studies; banana, mango, and pineapple which have similar flavor to pawpaw has been selected ...... 70

Table 12: The Amounts of (+)-Catechin and Quercetin in Selected Fruits Based on Recent Research…………………………………………………………………………72

LIST OF FIGURES

Page

Figure 1: Flowers, developing fruited clusters, and the pawpaw fruit...... 21

Figure 2: Eicosapentaenoic acid (EPA), with bis allylic methylene carbons circled .....35

Figure 3: Basic structure of flavonoids, showing C6-C3-C6 backbone ...... 38

Figure 4: Basic structure of flavonols ...... 43

Figure 5: Basic structure of flavones ...... 44

Figure 6: Basic structure of flavanones ...... 45

Figure 7: Basic structure of flavan-3-ols ...... 46

Figure 8: Basic structure of anthocyanidins...... 47

Figure 9: Basic structure of isoflavones...... 48

Figure 10: (+)-Catechin and quercetin standard curves ...... 56

CHAPTER 1: INTRODUCTION

Overview and Background

Pawpaw [Asimina triloba (L.) Dunal] is a native American tree bearing the largest tree fruits native to the United States, and was first reported in 1541 by Hernando de

Soto, the Spanish explorer, who discovered Native Americans cultivating pawpaw in the

Mississippi Valley (Layne, 1996). In the early 1900s, both blueberry and the pawpaw fruit aroused consumers’ interest as new high value fruit. However, unlike blueberry, the pawpaw fruit has not been a commercially successful farm product because of its rapid decomposition and short shelf life (Pomper & Layne, 2005). In 2000, the Ohio Pawpaw

Growers Association was founded in order to study pawpaw genetics, educate the advantages of pawpaw cultivation, and develop a pawpaw industry. This association annually supports the Ohio Pawpaw Festival near Albany, Ohio. Two international pawpaw conferences have been convened in 1994 and 2001, and the Third International

Pawpaw Conference “Pawpaw: Its Past, Present, and Future” will be held at Frankfort,

Kentucky in September, 9-10, 2011, sponsored by Kentucky State University, the Ohio

Pawpaw Growers Association, and the PawPaw Foundation.

Recently, growing interests and demands in high quality alternative fruit crops and advances in breeding techniques have facilitated the cultivation of more than forty pawpaw varieties including Shenandoah, Susquehanna, Rappahannock, Allegheny,

Potomac, and Wabash (Pomper, Layne, Peterson, & Wolfe, 2003). The pulp of the pawpaw fruit is custard-like and delicious, and its tropical taste and pleasant smell attract consumers. Various recipes are present for processing: pawpaw pies, custards, cookies, 13 cakes, ice cream, and pudding. Also, the pawpaw pulp can be a partial fat-reducing agent in baked goods (Duffrin, Holben, & Bremner, 2001).

Nevertheless, the quick ripening nature and postharvest perishability of the pawpaw fruit have been the main setbacks to bringing the fruit to both fresh and processing markets. In order to overcome these impediments, different approaches for pawpaw commercialization have been considered, such as utilizing the chemical compounds from pawpaw. For example, Annonaceous acetogenins have been extracted from the pawpaw twigs. Acetogenins are believed to have a variety of biological activities: antitumor, antimalarial, insecticidal, antifeedant, and immunosuppressive effects (Kojima & Tanaka, 2009). Because of those biological activities, acetogenins have been patented as antitumor agents and pesticides. Related to this, a head lice remover shampoo made from Annonaceous acetogenins has been patented (Pomper &

Layne, 2005).

Another approach concentrates on utilization of pawpaw as an antioxidative food additive that can preserve fat-containing foods from rancidity resulting from lipid oxidation. Antioxidants delay or stop oxidation reactions, and fruits and vegetables have considerable amounts of antioxidants (Wu et al., 2004). There are several types of antioxidants such as enzymes, vitamins, polyphenolics, carotenoids, and hormones.

Flavonoids are the most plentiful polyphenolic compounds present in plants and are believed to have antioxidant, antitumor, and anti-inflammatory activities (Taylor &

Grotewold, 2005; Williams & Grayer, 2004). It has been reported that pawpaw seeds have phenolic antioxidant compounds (Brannan & Salabak, 2009). Also, Kobayashi, 14

Wang, & Pomper (2008) showed that pawpaw pulp has phenolic antioxidants, and Harris

& Brannan (2009) demonstrated that the pulp has both phenolic and flavonoid antioxidants and antioxidant capacity. However, in order to understand the efficacy of the pawpaw pulp as an antioxidative agent, research on identification of components in the pawpaw pulp needs to be performed.

Statement of the Problem

Previous research has shown that the pawpaw pulp contains polyphenolic antioxidant compounds that are effective at inhibiting lipid oxidation. Recent studies found that the pawpaw pulp has flavonoids which are one of the most abundant polyphenolics. However, the identification of certain flavonoids in the pulp such as (+)- catechin and quercetin has never been investigated. Thus, the objective of this study is to determine total phenolic and total flavonoid contents from pawpaw pulp and to isolate and characterize (+)-catechin and quercetin from pawpaw pulp. In addition, (+)-catechin is a stereoisomer of (-)-catechin and the major form of catechin in nature, so that this study focuses on (+)-catechin.

Research Questions

Question Approach

1 What is the amount of phenolic Assess the amount of phenolic

compounds and flavonoids in compounds and flavonoids by

the pawpaw pulp extract? spectrophotometry.

2 What are the concentration of HPLC analysis for isolation and

(+)-catechin and quercetin detection of (+)-catechin and

flavonoids in the pawpaw pulp quercetin flavonoids in the

extract? pawpaw pulp extract.

3 How does frozen storage and HPLC detection of (+)-catechin

packaging condition of and quercetin flavonoids in

pawpaw pulp affect (+)- pawpaw pulp stored under

catechin and quercetin vacuum or in the presence of air

flavonoids? at 0, 2, 4, 6, 8, 10, and 12 months

of frozen storage.

Significance of the Research Study

In the food industry, synthetic antioxidants such as butylated hydroxyanisole

(BHA), propyl gallate (PG), butylated hydroxytoluene (BHT), and tert- butylhydroquinone (TBHQ) are widely used. However, health perceptions about these synthetic antioxidants have been growing since some studies have revealed that they are 16 possible carcinogens. As such, consumers’ interests have shifted from synthetic antioxidants to natural antioxidants from fruits, vegetables, beans, nuts and so on. Thus, this study on natural antioxidants in pawpaw fruit pulp will gain public attention.

Secondly, to my knowledge, there have been no published data on catechin and quercetin flavonoids in the pawpaw pulp. It is the first time that those flavonoids in the pawpaw fruit have been identified, so it may be very influential to future research projects.

Last but not least, the pawpaw is native to the eastern United States including the

Appalachian region which contains the southeastern part of Ohio. Thus, the pawpaw is a local fruit which is important to the region. Commercialization efforts by the Ohio

Pawpaw Growers’ Association are underway, but have not been met with much success.

My research will be beneficial to cultivators in Ohio.

Overall, this research can help to increase the commercialization potential of the pawpaw. For example, pomegranate, a fruit native to Iran that is widely used for food components, has been marketed by many food and dietary supplement makers due to the phenolic flavonoid content. Identification of individual flavonoids may allow the pawpaw to obtain a growing interest in terms of its health-promoting flavonoid content and the potential as a value-added functional food ingredient.

Limitations

Pawpaw can be harvested only one time from the middle of August to late

September. Pawpaws were obtained from a single tree in Athens, Ohio. There are at least forty varieties of pawpaws; however, this study will only feature one wild unidentified variety. A conversation with a grower indicated that the pawpaw fruits are likely of the

Shenandoah variety, but this has not been determined. Our instrumentation only allows identification of (+)-catechin and quercetin, so a complete flavonoid profile could not be obtained. Also, an unknown peak was identified, but concentration of the unknown compound could not be measured because a standard curve was not prepared due to lack of many different types of phenolic compounds.

Glossary

1-methylcyclopropene . A derivatives of cyclopropene and a plant growth regulator that is believed to interact with ethylene, plant hormone, receptors inhibiting ethylene perception. It is commercially used to delay the ripening of fruit and preserving flowers (Watkins, 2006).

Aminoethoxyvinylglycine (AVG) . An inhibitor of ethylene biosynthesis which can decrease the ripening of climacteric fruit (Saltveit, 2005)

Cleft graft . To cut off the limb; insert the budwood; wrap with aluminum foil to keep the heat off; cut a hole in the tip of a poly bag and carefully slip over the budwood; tie with a twisty and cut a small hole in the poly bag to drain any rain water; wait for 3 weeks and then remove coverings; cleft graft starts to force about a month later 18

Ethephon . A plant growth regulator that is the most widely used and is converted to ethylene

Gynoecium . The female part of a flower, consisting of one or more carpels.

Carpels are the part of a plant in which seeds are produced

Hardiness zone . Geographically divided region where a particular plant can grow withstanding the minimum temperature of the area

Magnoliales . An order of flowering plants

Mulch . To cover the soil or the roots of a plant in order to improve the quality of the soil or to stop weeds growing

Peduncle . A stalk bearing a flower or fruit, or a main stem that support a group or cluster of flowers

Protogynous . Having the female reproductive organs come to maturity before the male

USDA plant hardiness zone . Hardness zone which is represented by average annual minimum temperature (e.g., Ohio: 5b [minimum(-26°C)] ~ 6a[minimum(-23°C)])

Whip-and-tongue graft . To make a slanting cut on both rootstock and scion; cut the tongue 1/3 of the way from the tip on the rootstock; cut the tongue 1/3 of the way from the tip on the scion; the tongues will overlap by 1/3 to jam the graft together; slip the rootstock and scion together; slip the rootstock and scion together

CHAPTER 2: REVIEW OF CRITICAL LITERATURE

Pawpaw

Background

Pawpaw [Asimina triloba (L.) Dunal] is a small tree that is locally distributed in the eastern part of the United States.This region is composed of 26 states including the

Appalachian region of Southeastern Ohio, West Virginia, and Eastern Kentucky

(McGrath & Karahadian, 1994). It grows as an understory tree or a dense aggregation of shrub in the temperate woodlands or mesic hardwood forests that have fertile, moist soils at the bottom of rivers (Pomper & Layne, 2005). Specifically, U.S. Department of

Agriculture (USDA) plant hardiness zone 5 (average annual minimum temperature of -

29°C) through 8 (average annual minimum temperature of -7°C) is an ideal cultivation condition (Pomper, Crabtree, Layne, Peterson, Masabni, & Wolfe, 2008).

Among 10 families in the order Magnoliales that is an order of flowering plants, the pawpaw belongs to the Annonaceae , the tropical custard-apple family, which is the largest family of Magnoliales. This Annonaceae family is composed of about 2300 species and 130 genera such as genus Annona , genus Asimina, genus Guamia , genus

Monodora , genus Oxandra A. Rich, etc. For example, genus Annona is composed of

Annona cherimola Mill (cherimoya), Annona muricata L. (soursop), Annona reticulate L.

(custard apple), and Annona squamosa L. [sugar apple (sweetsop)], and genus Oxandra

A.Rich contains Oxandra laurifolia (Sw.) A. Rich (haya) .

Among the genera, only the genus Asimina grows in the temperate climate zone, southeastern United States. The other genera thrive in the tropical region (Callaway, 20

1993). The genus Asimina is composed of 9 species including Asimina angustifolia Raf,

Asimina incana (Bartram) Exell, Asimina xnashii Kral, Asimina obovata (Willd.) Nash

(bigflower pawpaw), Asimina parviflora (Michx.) Dunal, Asimina pygmea (Bartram)

Dunal (dwarf pawpaw), Asimina reticulate Shuttlw.ex Chapm, Asimina tetramera Small

(fourpetal pawpaw), and Asimina triloba (L.) Dunal (pawpaw) (Kral, 1960; Pomper &

Layne, 2005). The 9 species are native to the United States, and most of them are mainly confined to extreme southeastern region of the United States. However, A. triloba

(pawpaw) is distributed in the eastern United States (i.e., from northern Florida to southern Ontario, Canada, and as far west as eastern Nebraska, Kansas, Oklahoma, and

Texas) and A. parviflora occurs throughout the southeastern United States (Callaway

1993; Pomper & Layne, 2005).

Although the pawpaw is usually harvested in the wild, an attempt is being made to cultivate it in small orchards in several states including Alabama, California,

Maryland, Michigan, Missouri, North Carolina, Kentucky, West Virginia, and Ohio. In addition, it has also been planted in Italy, China, Japan, Israel, Belgium, Portugal, and

Romania because of its commercial potential (Duffrin & Pomper, 2006; Pomper &

Layne, 2005).

The Description and Horticulture of Pawpaw

Morphologically, pawpaw is a small tree which usually reaches 5 to 10 m in height and often found in clusters, with a straight stem and dark green leaves. It is believed that root suckering and seedlings growing near original trees cause clusters.

Leaves are long, drooping, egg-shaped or oblong in shape with the narrower end 21 connected to the stem. They may be 10 to 15 cm wide and 15 to 30 cm in length. The texture of the leaves of Asimina triloba is usually membranous which is different from leathery texture of most of the other Asimina species (Kral, 1960; Layne, 1996).

Flowers are dark maroon colored, attain 5 cm in diameter, and have up to 4 cm long robust peduncles. They become visible before leaves in the middle of spring. There are three-lobed petals in inner and outer layers of mature blossoms. As shown in Figure

1, the gynoecium of flowers is generally made up of three to seven carpels which produce three to seven fruited clusters with possible upper limit of nine fruited clusters (Pomper &

Layne, 2005). Cross pollination is accomplished by pollinators such as beetles

(Nitidulidae), flies (Diptera), and other nocturnal insects, because the flowers are strongly protogynous and unable to self-fertilized (Layne, 1996; Wilson & Schemske, 1980).

Figure 1. Flowers, developing fruited clusters, and the pawpaw fruit. 22

The pawpaw fruit is the largest tree fruit originated from North America. It is oblong-cylindrical in shape, and normally 3 to 15 cm long, 3 to 10 cm wide, and weighs

100 to 1000g (Figure 1). The fruit grows singly or in clusters like banana, and it is a pulpy berry and is sometimes called “poor man’s banana” due to its distinctive flavor and custard texture similar to a creamy blend of banana ( Musa xparadisiaca L.), mango

(Mangifera indica L.), and pineapple [ Ananas comosus (L.) Merr]. Flavors differ among

varieties and some cultivars have complex flavor profile (Jones & Layne, 1997; Pomper

& Layne, 2005).

The inedible skin turns green to brownish-black as the fruit ripens. Inside of the skin is the edible flesh (pulp), the color of which ranges from creamy white through bright yellow to orange. The seeds are bean shaped, dark brown, and flattened laterally.

They are found in two rows from 12 up to 20. The length of these seeds is up to 3 cm which is about the size of an almond. Alkaloids present in the endosperm of the seeds are emetic and cause nausea (Layne, 1996; Pomper & Layne, 2005). Racoon [ Procyon lotor

(L.) Elliot], Red foxes [Vulpes fulvus (Desmarest) Merriam], opossums (Didelphis virginiana kerr), and deer (Odocoileus virginianus) primarily consume the fallen fruits while dispersing the seeds (Pomper & Layne, 2005).

The seeds become unviable within 1 year if stored moist at warm temperature (25

°C). In addition, freezing conditions (-15°C) terminates the embryo of the seeds. On the other hand, the seeds can be preserved in ziplock bags for 2 to 3 years, retaining their viability and high germination rate in a moist, chilled (5°C) environment (Geneve,

Pomper, Kester, Egilla, Finneseth, & Crabtree, 2003). 23

Pawpaw orchards can be established in a site that has temperate climate and well- drained, deep, fertile, slightly acidic (pH 5.4-7.0) soil. Frequent flooding and waterlogged soil have a negative effect on the development of pawpaw trees. In order to increase the survival rate, preserving moisture in soil by mulching with straw is recommended (Pomper & Layne, 2005).

Although pawpaw seedlings grow a sturdy taproot, the root system of the seedlings is fragile and easily damaged by digging, and thus carefully designed containers are needed for nurseries to deliver and breed the seedlings. The size of the container is essential, so that tall containers that can provide a room for the developing taproot are necessary. For instance, Rootrainers (Spencer-Lemaire Industries Limited,

Edmonton, Alta., Canada), which are 5.1 × 6.35 × 25.4 cm (2 × 2.5 × 10 inches) with

737.4 cm 3 (45 inch 3) in volume, have been used for the propagation of seedlings. In

addition, a potting substrate that has a high aeration rate, water holding capacity, cation

exchange capacity, and a sphagnum peat moss part of more than 75% by volume is an

effective source of pawpaw container construction (Pomper, Layne, & Jones, 2003).

In the wild, pawpaws usually are present at shaded understory area. Low to

moderate shading outdoors positively affects the development of pawpaw seedlings in the

containers and increase chlorophyll a and b concentrations in leaves (Pomper & Layne,

2005). This shading regime is useful for nurseries to treat containerized pawpaw

seedlings. However, lateral root dry mass did not increase by administration of 0.7L of

Cu(OH) 2 to inside Rootrainers, unlike the effect on the other kind of trees (Pomper &

Layne, 2005). 24

There are several cloning techniques for the propagation of pawpaw, including budding and grafting such as whip-and-tongue graft and cleft graft. When the seedling rootstock is growing and has at least 0.5 cm diameter thickness (pencil thick), chip budding is the best method among them. Also, some nurseries has used whip-and-tongue, grafting scion to a developing rootstock. These grafting and budding methods are being used for the improvement of pawpaw breed (Layne, 1996; Pomper & Layne, 2005).

The expected life span of the pawpaw tree is 20 years or more. Grafted trees live less than seedling trees possibly due to the incompatibility of scion and rootstock. A low rate of tree decline occurs in some orchards. Various fungi, mainly a complex of

Mycocentrospora aiminae (Ellis et Kellerm) Deighton, Rhopaloconidium asiminae (Ellis et Morg) Petr, and Phyllosticta asiminae (Ellis et Kellerm) are responsible for a leaf spot in some pawpaw leaves and sometimes cause infections on the fruit skin (Peterson,

1991).

Harvesting

The harvest season of the pawpaw fruits is mid-August to late September when they ripen (Pomper, Crabtree, & Layne, 2008). Because the quality of ripe fruit is determined by the stage of ripeness, there has been research conducted to identify ripening indicators. As pawpaw ripens, soluble solids concentration increases up to 20% and the production of volatile compounds like ethyl and methyl esters is enhanced.

However, those factors are not easily detectable (Archbold, Koslanund, & Pomper,

2003). Also, a study showed that green color intensity declines in some genotypes as pawpaw becomes ripe but it is not clearly distinguishable (McGrath & Karahadian, 25

1994). A color change often occurs later in ripening period. On the other hand, a decrease in fruit firmness is relatively obvious and detectable indicator of maturity. It has been reported that enzymes such as cellulase, polygalacturonase, endo-β-mannanase, and

pectin methylesterase are involved in fruit softening (Archbold et al., 2003). The ripe

fruits are touched to determine if they are ready for harvesting, and collected by hand

when they have lost firmness to some extent.

While handling pawpaws, there can be slight bruising which may lead to off-

flavors in 1 or 2 days, depending whether or not the pawpaw variety has a more leathery,

thicker skin (Peterson, 1991). Another challenge in harvesting is that the ripening period

of fruits on a given tree differs from others, partially due to the staggered bloom period in

spring time, so that harvesting takes 2 weeks or longer. Even fruits within a single cluster

grown from a certain flower frequently ripen at different times. In addition, different

varieties may have various ripening time. To solve these problems and maintain the high

quality of the fruit, multiple harvests are currently being performed. It is also

recommended to harvest fruit in the early ripening period in order to increase the storage

time (Archbold et al., 2003; Pomper & Layne, 2005).

Administration of growth regulators before ripening for ethylene-regulated fruit

species has been tested in order to shorten the duration of harvest. For instance, ethephon

for ethylene release contributes to consistent ripening and aminoethoxyvinylglycine

(AVG) for ethylene action inhibition plays a role in detaining ripening and harvest.

However, pawpaw may be very vulnerable to ethylene manipulation because it is 26 ethylene climacteric. Thus, further research on the growth regulators and their effect on pawpaw are required (Archbold et al., 2003).

Another problem in terms of harvesting is that it is often hard to spot pawpaw because they are mostly located under dense foliage and both pawpaws and leaves have the same green color. In addition, picking tools are needed, because pawpaws are hanging on trees 5 to 10 m high, and also the tree is weak and flexible so that ladders cannot be used (Peterson, 1991).

In summary, the pawpaw suffers from harvest issues such as the lack of reliable ripeness indicators, bruising while handling, variable ripening period, and difficulties in locating and picking the pawpaw fruits.

Postharvest Challenges

Postharvest issues are the major limitation to pawpaw commercialization.

Pawpaw is classified as a climacteric fruit, and it attains ethylene (C 2H4) and respiratory

climacteric peak within 3 days after harvest at ambient temperature. At the same time, the

fruit softens rapidly (Galli, Archbold, & Pomper, 2008). There are other Annonaceae , the tropical custard-apple family members, which display similar climacteric patterns or two respiratory peaks. For example, cherimoya ( Annona cherimola Mill.), sweetsop (sugar

apple, Annona squamosal L.), soursop ( Annona muricata L.), and atemoya ( Annona

×atemoya ) are climacteric (Palma, Aguilera, & Stanley, 1993).

The firmness of pawpaw fruit rapidly decreases after harvest, so that, within 5 days, it becomes too soft for handling (Galli et al., 2008). The perishability of pawpaw hinders its merchandizing in both fresh and processing markets. The shelf life of pawpaw 27 fruit that is ripened on the tree has been reported to be from 2 to 3 days at room temperature (Layne, 1996) to 5 to 7 days at room temperature (Archbold et al., 2003).

Refrigeration (4°C) can delay the loss of fruit firmness for 1 month (Pomper & Layne,

2005). The respiration peak of cold-stored pawpaw fruit is higher than other harvested fruits, and long storage may cause the pulp and skin of pawpaw fruit to turn brown or black. This could be chilling injury which has been reported in cherimoya (Galli et al.,

2008). Further research should focus on the most favorable storage temperature and period.

Alternatively, application of 1-methylcyclopropene after harvest, an inhibitor of ethylene recognition, has been successfully tried to delay the ripening process of the other climacteric species, and this treatment may be tested for pawpaw fruit (Archbold et al.,

2003). Another postharvest impediment is fruit packaging problems. For packaging, different fruit size and shape should be considered and techniques for minimize bruising are required (Pomper & Layne, 2005).

In short, the quick ripening nature of the pawpaw fruit hinders its commercialization. In order to elongate its shelf life, further research on cold storage, plant growth regulators, packaging, and minimizing bruising is required.

Characteristics of Pawpaw Fruit

Sensory Characteristics

The postharvest fruit which is ripened has a distinctive aroma that results from the high concentration of methyl and ethyl esters (Galli et al., 2008). Overripe fruit has a very strong aroma and taste which can be unpleasant to consumers. The green color of the skin 28 and creamy white or yellow color of the pulp are attractive unless there are many dark spots resulting from bruising or overripening. The custard-like texture of the flesh (pulp) is very smooth, but from 12 to 20 inedible seeds in the center of the fruit are almond size and hard. Currently, seedless pawpaw is not cloned (Pomper & Layne, 2005).

Pawpaw has been tested as a food ingredient. Pawpaw fruit was regarded as a good replacing agent for banana in recipes which require banana (Jones & Layne, 1997).

In a consumer acceptance study performed in the 2 nd Annual Pawpaw Field Day at

Kentucky State University, pawpaw ice cream received the highest acceptance percentage, making this ice cream variety promising (Templeton, Marlette, Pomper, &

Jones, 2003). Research has been conducted on the potential of pawpaw as a fat-reducing substance. Muffins made with pawpaw pulp puree were satisfactory in the same degree with the exception of appearance (color) compared to regular fat-containing muffins

(Duffrin et al., 2001). Plain shortened cake using pawpaw fruit puree was also examined for a partial fat replacement. This study showed that 25% fat replacement was equally acceptable with no replacement control to panelists in all sensory characteristics (Wiese

& Duffrin, 2003).

Nutritional Facts

Pawpaw is usually compared to banana in terms of nutritional composition, and it has been reported that pawpaw and banana have similar pattern of food energy, protein content, dietary fiber content and overall nutritive facts (Jones & Layne, 1997). It has been reported that approximately 1.0% of the weight of wet pulp and 9.0% of the weight of seed is lipid, and the ratio of neutral lipid and polar lipid is nearly 1:1. Also, the 29 amount of lipid was dependent on the maturity even though the ratio of lipid neutral and polar lipid was similar between mature and immature fruit (both pulp and seed). The immature fruit was composed of less than 25% of the lipids of mature fruit (Wood &

Peterson, 1999).

Antioxidants

There has been little information about antioxidants in pawpaw fruit, but two studies reported the antioxidant capacity of pawpaw pulp. In 2008, Kobayashi et al. reported phenolic content and antioxidant capacity in fruit of two pawpaw cultivars at different stages of ripening by using acetone extraction. Both phenolic content and antioxidant capacity tended to decrease with ripening.

In the other study, Harris and Brannan (2009) reported the level of total phenolics and flavonoids in pulp extracts from underripe, ripe, and overripe pawpaws. The study showed that when methanol extraction was used, the amount of total phenolics was not significantly different regardless of the level of ripeness. However, the level of total flavonoids was significantly higher in the pulp extract from underripe pawpaw (1.34 ±

0.14 µmol/100g pulp) than those from ripe (0.81 ± 0.17 µmol/100g pulp) and overripe

(0.71 ± 0.32 µmol/100g pulp) pawpaws. In this study, the effects of both refrigerated storage and cooking were also demonstrated. During refrigerated storage, there was a significant increase in the level of total flavonoids in overripe pulp, but both reducing potential and radical scavenging effect which represent antioxidant capacity were not significant. In the cooking treatment, a similar trend as that of the refrigerated storage was observed in overripe pulp; however, significant increases in both reducing potential 30 and radical scavenging activity were shown in underripe pulp. In summary, this result indicates the potential use of heat treatment in underripe pulp in order to increase the antioxidant capacity.

Both studies suggested that the phenolic content has an influence on antioxidant capacity and both indicated that there are differences in the relationship between ripeness and antioxidant capacity in pawpaw.

Antioxidants in Fruits

Introduction

An antioxidant is defined broadly as any substance that efficiently slows down or inhibits oxidation of oxidizable substrates, when present at low concentrations compared to the substrates (Halliwell, Aeschbach, Loliger, & Aruoma, 1995). It has been reported that antioxidants are rich in berries, berry products, other fruits, and fruit juices

(Halvorsen et al., 2006). Also, research shows that total phenolic content shows significant correlation with antioxidant capacity in grain (Hodzic, Pasalic, Memisevic,

Srabovic, Saletovic, & Poljakovic, 2009) and pawpaw (Harris & Brannan, 2009).

Phenolics are secondary metabolites synthesized in plants and usually show antioxidant properties. In this sense, measurement of total phenolics is commonly used for quantifying antioxidants. Phenolics are characterized by containing at least one aromatic ring bearing one or more hydroxyl groups, including derivatives (e.g., glycosides). Approximately 8000 phenolic structures have been introduced such as simple phenols (e.g., monophenols and diphenols), phenolic acids (e.g., hydroxycinnamic acid and hydroxybenzoic acid), flavonoids (e.g., anthocyanins, flavonols, and flavones), 31 tannins (e.g., condensed tannins and hydrolysable tannins), and stilbenes (e.g., resveratrol). Examples of hydroxycinnamic acids are p-coumaric acid, caffeic acid, ferulic acid, and chlorogenic acid. Hydroxybenzoic acid includes gallic acid which is the precursor of hydrolysable tannins. An example of condensed tannins is proanthocyanidins which are polymers of catechin and epicatechin flavonoids (Crozier,

Jaganath, & Clifford, 2009)

Table 1 compares the amount of total phenolics in common fruits. Note that pawpaws exhibit a phenolic content that is in the middle range of these fruits.

Table 1

Total Phenolics of Common Fruits Total Fruits Reference phenolic s1 Wild Blueberry 22.8 Wolfe, Kang, He, Dong, Zhang , & Liu, 2008 Plum 19.6 Chun , Kim, Smith, Schroeder , Han, & Lee, 2005 Pomegranate 18.0 Wolfe et al., 2008

Grape, red 15.5 Isabelle, Lee, Lim, Koh, Huang , & Ong, 2010 Cranberry 15. 3 Wolfe et al ., 2008

Strawberry 12.0 Chun et al ., 2005

Soursop (Annona muricata ) 11. 8 Isabelle et al ., 2010

Pawpaw (Asimina triloba ) 9.16 Harris & Brannan, 200 9

Mango 7.3 9 Isabelle et al ., 2010

Apples 6.27 Chun et al ., 2005

Banana 6.00 Chun et al ., 2005

Orange 5.74 Isabelle et al ., 2010

Persimmon 5.42 Isabelle et al ., 2010

Kiwifruit 3. 20 Isabelle et al ., 2010

Pear 2.8 5 Sun, Chu, Wu, & Liu, 2002

Pineapple 2.1 5 Sun et al ., 2002

Melon, honeydew 0. 80 Isabelle et al ., 2010

Watermelon, red 0.58 Isabelle et al ., 2010

Note . 1 Total phenolics reported as µmol gallic acid equivalents per gram tissue.

Oxidation

Oxidation in Biological Systems

Oxygen is indispensible to living organisms for aerobic metabolism and energy production in mitochondria. However, when oxygen (O2) combines with a single

·- electron, the superoxide radical (O 2 ) is made and further protonation reaction produces peroxyl radical (HO 2·) from approximately 1% of superoxide radical at physiological pH.

Metal ions including copper (Cu) and iron (Fe) catalyze this auto-oxidation. Those free

radicals, especially the peroxyl radical, are highly reactive and can damage biomolecules

such as DNA, proteins, lipids, and other small molecules through irreversible oxidation

reactions when cellular defense mechanisms are impaired, resulting in oxidative stress.

For example, superoxide can reduce the activity of several enzymes; ribonucleotide

reductase that is required in DNA synthesis, calcineurin protein which is involved in

signal transduction, NADH dehydrogenase for energy metabolism, and antioxidant

enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Oxidative

damage has been related to many chronic diseases including cancer, cardiovascular

disease, type II diabetes, and neurodegenerative disease, as well as aging (Willcox, Ash,

& Catignani, 2004). Epidemiological studies have shown the inverse relationship

between frequent intake of fruits and vegetables and the risk of chronic disease

(Lucenteforte, Garavello, Bosetti, & La Vecchia, 2009; Willcox et al., 2004).

Oxidation in Foods

Oxidation in lipid is a primary cause of oxidative deterioration in foods and has harmful effects on the overall quality of foods. Oxidative instability not only gives rise to the production of off-flavors, rancidity, discoloration, and changes in texture but also can reduce overall nutritional value and bioactivity, as well as form toxic secondary reaction products. Thus lipid oxidation is one of the greatest obstacles to food companies which produce lipid-containing food products and has a large negative impact on consumer acceptance of those items (Frankel, 1980; Shahidi & Zhong, 2010).

Lipids are naturally occurring organic compounds which are mainly composed of fatty acids esterified to glycerol and are one of the major macronutrients in foods. Lipids are generally soluble in organic solvents including chloroform, ethanol, and hexane and insoluble in water, with the exception of some monoacylglycerols that are more soluble in water than in organic solvents. Lipids include a broad group of compounds such as triacylglycerols that are energy storage molecules, phospholipids which constitute cell membranes, and plant sterols. Triacylglycerols can be further classified by physical properties: Triacylglycerols in solid state are fats, and in liquid state are oils. Also, in food processing, lipids act as a heat transfer medium, and lipids in food products contribute to appealing texture and flavor (Shahidi & Zhong, 2010). Lipids are largely found in plants (e.g., oilseeds, fruits, spices, and herbs), and animals (e.g., land animals, marine mammals, and fish).

Lipids are vulnerable to oxidation especially when their level of unsaturation or the number of bis-allylic methylene carbons is high. An example of a compound with 35 four bis-allylic methylene carbons, eicosapentaenoic acid (EPA), which is highly susceptible to oxidation, is shown in Figure 2.

Figure 2. Eicosapentaenoic acid (EPA), with bis-allylic methylene carbons circled.

There are three steps of lipid oxidation: initiation, propagation, and termination. The initiation step begins with the presence of pro-oxidants such as transition metals and reactive oxygen species (ROS). During this initiation, free radicals are generated. The hydroxyl free radical (HO ·) is the most oxidizing radical (Buettner, 1993). The propagation step is characterized by a free radical mediated chain reaction generating reactive free radicals. In the termination stage, radical-radical collision occurs and nonradical species are produced.

In order to decrease oxidation and enhance oxidative stability in lipid-possessing food products, many strategies have been developed. Some, such as microencapsulation, have been adapted to package lipid products into capsules that protect lipid from atmospheric oxygen. However, using antioxidants for delaying or blocking oxidation is the most convenient, effective, and economical method (Shahidi & Zhong, 2010).

Types of Antioxidants

Antioxidants developed by organisms for cellular protection include: (a) antioxidant enzymes which remove initiation factors (pro-oxidants) for oxidation (e.g., superoxide dismutase, catalase, and glutathione peroxidases), (b) large molecule antioxidants (e.g., albumin, ceruloplasmin, and ferritin), (c) small molecule antioxidants which are chain-breaking, repairing oxidizing radicals (e.g., tocopherol, ascorbic acid,

[poly] phenols, carotenoids, urate, and glutathione), and (d) hormones (e.g., estrogen, angiotensin, and melatonin) (Buettner, 1993; Prior, Wu, & Schaich, 2005).

In food products, small molecule antioxidants predominate and can be subcategorized as primary antioxidants and synergistic antioxidants. Primary antioxidants (see Table 2) terminate the free radical chain reaction (propagation) and form stable radical end products. Synergistic antioxidants (see Table 2) can perform their role in several ways. Some synergists scavenge oxygen and prevent its addition to lipids.

Others are metal chelators, which bind to divalent metals such as iron and copper that can promote lipid oxidation. Other synergists, such as ascorbic acid, catalyze the reduction of primary antioxidants back into their active form after they have become oxidized. Yet others, such as polyphenols in green tea, have shown the capacity of chelating transition metals as well as quenching free radicals (Lambert & Elias, 2010; Rice-Evans & Miller

1997).

Table 2

Primary and Synergistic Antioxidants

Primary Antioxidants Synergistic Antioxidants

Phenols Sulfites

Gallates Ascorbic Acid

Flavonoids Erythorbic Acid

BHA Polyphosphates

BHT EDTA

TBHQ Organic Acids

Tocopherols Carnosine

Ethoxyquin Nitrates

Trolox-C Amino Acids

Herb/Spice Extracts Spice Extractives

Carotenoids Tea Extracts

Ascorbate Glutathione

Carnosine Selenium

An example of synergism that more fully explores the role of both primary and synergistic antioxidants is the relationship between lipid soluble α-tocopherol (primary)

and water soluble ascorbic acid (synergist), which act as cooperative chain-breaking

natural antioxidative small molecules (Buettner, 1993). First of all, α-tocopherol reacts directly with free radicals and scavenges radicals. This type of antioxidant scavenges reactive oxygen species (ROS) in the initiation step and scavenge lipid peroxyl radicals 38

(LOO ·). On the other hand, ascorbic acid works as a synergistic antioxidant. Ascorbic acid recycles α-tocopherol by donating hydrogen to α-tocopherol radicals, maintaining α- tocopherol level in the membrane (Niki, Saito, Kawakami, & Kamiya, 1984).

Flavonoids

Introduction

Flavonoids are the largest and the main polyphenolic phytochemical compounds ubiquitously found in plants. Flavonoids have a C6-C3-C6 backbone and low molecular weight (see Figure 3). They are secondary metabolites which are not essential for plant growth and development but biologically active. Flavonoids are synthesized from phenylalanine through the flavonoid branch of the phenylpropanoid and acetate-malonate metabolic pathway (Buer, Imin, & Djordjevic, 2010; Yao et al., 2004). This biosynthetic pathway is regulated by genes and enzymes in response to environmental stimuli such as ultraviolet radiation (UV) stress, phytotoxins, diseases, insects, and climate (Winkel-

Shirley, 2002). These factors may give rise to the alteration of the amount of flavonoids in plant foods.

Figure 3. Basic structure of flavonoids, showing C6-C3-C6 backbone.

Function

Flavonoids play a physiologically diverse role in plants, including pigmentation of flowers, defense against environmental cues (i.e., UV irradiation) and pathogens, transferring hormone auxin, allelopathy, and pollen viability. For example, flavonoid- deficient mutants are vulnerable to UV light with the exception in case of overproduction of sinapate esters which can make up for the deficiency in some cases (Buer et al., 2010;

Treutter, 2005). Also, it has been reported that the flavonoid-less mutants cannot generate a functional pollen tube in maize and petunia (Mo, Nagel, & Taylor, 1992). Flavonoids in legumes are involved in symbiosis with bacteria and root nodule organogenesis (Buer et al., 2010). In addition, in animals, flavonoids display antioxidant, anti-inflammatory, anti-proliferative, anti-tumor, pro-apoptotic activities (Taylor & Grotewold, 2005;

Williams, 2004). For instance, P-glycoprotein MDR1, that makes tumors resistant to antitumor drugs, can be down-regulated by flavonoids (Buer et al., 2010). More specifically, proanthocyanidins and flavan-3-ols which are subclasses of flavonoids can increase the release of endothelial nitric oxide (NO) and cause vasodilation decreasing the risk of cardiovascular disease. LDL cholesterol oxidation can be blocked by the antioxidant effect of anthocyanidins (USDA Database for Flavonoid Content of Selected Foods,

2007).

In foods, flavonoid antioxidants have been identified in a plethora of plants and incorporated into many different food products. The selection of flavonoids for foods has depended largely on their function, which is dictated by the type of flavonoids found in plants. 40

Subgroups

Flavonoids have been identified from at least 557 raw and processed fruits, vegetables, juices, herbs, tea, wine, and beer, and categorized by the USDA into the

Database for Flavonoid Content of Selected Foods (Chun, Chung, & Song, 2007).

According to this comprehensive data, USDA has classified flavonoids into 6 major subgroups based on their chemical structures (see Table 3). The subgroups are flavonols

(e.g., isorhamnetin, kaempferol, myricetin, quercetin, and rutin), flavones (e.g., apigenin, luteolin, chrysin, and diosmetin), flavanones (e.g., eriodictyol, hesperetin, naringenin, and neohesperidin), flavan-3-ols (e.g., catechins, gallocatechin, epicatechins, epigallocatechin gallate, theaflvins, and thearubigins), anthocyanidins (e.g., cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin), and isoflavones (e.g., daidzein, genistein, glycitein, biochanin A, and formononetin).

Table 3

Flavonoid Subclasses and Food Sources

% Daily intake of each Representative flavonoid from the Subclasses Flavonoids food sources representative source, (mg/day) compared to other food sources (%)

Flavonols Isorharmnetin, Tea (5.5) Tea (43.0) Kaempferol, Myricetin, Vegetables (1.6) Vegetables (12.6) Quercetin, Rutin Apple (0.6) Apple (4.3)

Flavones Apigenin, Luteolin, Grains (0.9) Grains (57.3) Chrysin, Diosmetin Vegetables (0.3) Vegetables (17.4)

Flavanones Eriodictyol, Hesperetin, Citrus juice (8.1) Citrus juice (56.3) Naringenin, Citrus fruit (3.3) Citrus fruit (23.1) Neohesperidin

Flavan-3-ols Catechins, Tea (151.6) Tea (96.9) Gallocatechin, Wines (2.1) Wines (1.4) Epicatechins, Apple (1.2) Apple (0.8) Epigallocatechin gallate, Theaflavins, Thearubigins

Anthocyanidins Cyanidin, Delphinidin, Wines (1.5) Wines (49.3) Malvidin, Pelargonidin, Melon & Melon & Peonidin, Petunidin Berries (0.7) Berries (23.5)

Isoflavones Daidzein, Genistein, Legumes (0.7) Legumes (55.6) Glycitein, Biochanin A, Fluid milk (0.2) Fluid milk (0.2) Formononetin

Daily Intake

Chun et al. (2007) reported the main dietary sources for each and total flavonoid intakes of U.S. adults aged 19+ in the National Health and Nutrition Examination Survey 42

(NHNAES) 1999-2002 based on the USDA categorization of the subclasses (see Table

3). In this research, tea was the most valuable source for flavonols and flavan-3-ols. Tea constituted 43.0% of the daily intake of flavonols and 96.9% of the daily intake of flavan-

3-ols. Also, 157.1 mg of flavonols and flavan-3-ols were taken from tea per day, which is

82.8% of total flavonoid intake daily and placed in the highest percentage.

Flavonoid structure determines its capacity as an antioxidant. For example, flavonoids with a 3’4’-catechol structure in the B-ring, such as catechin, generally exhibit enhanced antioxidant power (Heim, Tagliaferro, & Bobilya, 2002). The occurrence of a -

OH group on position 3 of the C-ring, such as catechin and quercetin, stabilizes the flavonoid radical and is a reactive site that allows conjugation. Flavonoids that exhibit

O-methylation on the B-ring have been shown to decrease antioxidative effectiveness

(Heim et al., 2002). Examples of this type of flavonoid are tamarixetin. Flavonoids that lack a 2-3 double bond coupled with a 4-carbonyl group on the C-ring usually are less potent antioxidants because the presence of these functional groups helps to stabilize flavonoid radicals due to resonance stabilization (Heim et al., 2002). Conjugation with carbohydrates such as glucose, rhamnose, and rutinose and polymerization of basic structures into larger flavonoids are thought to decrease antioxidant potency compared to aglycones. On the other hand polymerization of basic flavonoids into larger structures has been shown to increase antioxidant effectiveness (Heim et al., 2002).

Flavonoid Subclasses

Flavonols

Figure 4. Basic structure of flavonols.

The basic structure of flavonols is shown in Figure 4. Flavonols are characterized by having an OH group on position 3 in the C ring of flavones and a 2-3 unsaturated double bond in conjugation with a 4-carbonyl group (see Figure 4). The OH group gives stability of flavonols and the conjugation increase the antioxidant activity (Heim et al.,

2002). Flavonols are found in a number of plants. For instance, quercetin and kaempferol are present in fruits, vegetable, and leaves. Isorhamnetin is found in onions, and myricetin is in berries, maize and tea. Among them, quercetin is one of the most plentiful flavonoids. From 4 to 68 mg quercetin is consumed by an individual daily in the United

States, Europe, and Asia. Research has shown that quercetin has antioxidative, anti- inflammatory, vasodilating effects (Chen, Zhou, & Ji, 2010). In addition, it has been reported that flavonoid quercetin-3-O-glucoside derived from Annona squamosa leaves controls hyperglycemia and shows antioxidant activity in rat model (Panda & Kar, 2007).

Flavones

Figure 5. Basic structure of flavones.

The basic structure of flavones is shown in Figure 5. Flavones have 2- phenylchromen-4-one (2-phenyl-1-benzopyran-4-one) backbone and a 2-3 double bond conjugated with 4-carbonyl group, but unlike flavonols, they lack a 3-OH group (see

Figure 4 and 5). The absence of this 3-OH can result in a decline of free radical scavenging activity. The plant flavone apigenin is commonly contained in fruits such as oranges and grapefruit, vegetables like parsley and onions, and beverages such as chamomile tea. In recent years, cancer preventive effect of apigenin has been reported

(Shukla & Gupta, 2010). Flovone luteolin is present at low concentration in the form of glycosides in vegetables such as spinach, peppers, and celery and some spices such as parsley, sage, and thyme. Less than 1mg per day of luteolin is contained in human diet, but its anti-inflammatory effects on cardiovascular diseases and cancers, and high bioavailability of orally taken luteolin have been recently reported (Seelinger, Merfort, &

Schempp, 2008). 45

Flavanones

Figure 6. Basic structure of flavanones.

The basic structure of flavanones is shown in Figure 6. Flavanones are structurally distinguished by deficiency of a 3-OH and a 2-3 double bond and having a 4- carbonyl group. This shortage of free 3-OH can lead to the reduction of free radical scavenging ability. The flavanones hesperidin and naringenin exhibit blood sugar level lowering activity in diabetic mice (Fowler & Koffas, 2009).

Flavan-3-ols

Figure 7. Basic structure of flavan-3-ols.

The basic structure of flavan-3-ols is shown in Figure 7. Flavan-3-ols has 3’4’- catechol structure in the B-ring which strongly increases the antioxidant effectiveness.

This subclass does not contain a 4-carbonyl group and a 2-3 double bond, but has a OH group or gallate on position 3 of the C-ring. This 3-OH group makes a planar structure that increases scavenging activity. Flavan-3-ols are the most prevalent group of flavonoids in the diet and are considered as functional ingredients of beverages such as tea and red wine. For instance, about 70% of polyphenols in green tea are catechin, and epigallocatechin gallate (EGCG) is the most plentiful catechin in green tea. A 32% of antioxidant capacity of green tea is derived from EGCG (Moore, Jackson, & Minihane,

2009). It has been reported that increased green tea consumption is inversely related with total cholesterol and triacylglycerol (TAG), reducing the risk of cardiovascular disease

(Imai & Nakachi, 1995).

Anthocyanidins

Figure 8. Basic structure of anthocyanidins.

The basic structure of anthocyanidins is shown in Figure 8. Anthocyanidins contain a free 3-OH group that can exhibit biological activities when bound to metal ions such as Mg 2+ and Ca 2+ under alkali conditions (Naithani et al., 2008). Anthocyanidins are responsible for the red, blue, and violet colors of vegetables and fruits. Anthocyanins are glycosides that are derivatives of anthocyanidins. Individual consumption rate of anthocyanidins is estimated to be from 180 to 215mg per day in the United States, which is much higher than the other antioxidants (e.g., apigenin: 20-25 mg/day). It has been suggested that anthocyanidins have anti-oxidant and anti-inflammatory properties, and reduce the risk of cancer, diabetes, and cardiovascular disease (Wang & Stoner, 2008).

Isoflavones

Figure 9. Basic structure of isoflavones.

The basic structure of isoflavones is shown in Figure 9. Unlike flavones, the phenyl group is located position 3 of the C-ring. Isoflavones show preventive effects on heart disease, cancer, and diabetes. They are mainly contained in soybean foods, and genistein and daidzein are predominant isoflavones (Mateos-Aparicio, Redondo Cuenca,

Villanueva-Suárez, & Zapata-Revilla, 2008). It has been shown that both soy protein and soybean isoflavones decrease the atherosclerosis, and epidemiologic studies have found that higher soy consumption is negatively associated with the risk of developing cardiovascular disease (Beavers, Serra, Beavers, Cooke, & Willoughby, 2009).

Conclusion

In summary, pawpaw [ Asimina triloba (L.) Dunal] belongs to the custard-apple family and has distinctive tropical aromas and flavors. There are harvesting and postharvest issues including quick ripening nature although the pawpaw fruit has a great potential to become a mainstream fruit crop. Research showed that pawpaw can be a potential fat-reducing agent. Also, research focus on antioxidant properties of the components of the pawpaw fruit. There are several types of antioxidants and pawpaw contains polyphenolic antioxidants. Among phenolics, flavonoids are the most common type and there are six subclasses of flavonoids. The structure of flavonoids is tightly correlated to their functions.

CHAPTER 3: METHODOLOGY

Materials

All chemicals and reagents were purchased from Fisher Scientific (Waltham,

MA) or Sigma-Aldrich (St. Louis, MO). Pawpaws [Asimina triloba (L.) Dunal] were obtained locally.

Pawpaw Sample Preparation

For measurements of total phenolics, total flavonoids, and High performance liquid chromatography (HPLC) analysis of refrigerated (4 ˚C) samples in 2010, 25 local underripe pawpaw ( A. triloba ) fruits were obtained from a single pawpaw tree in Athens,

Ohio in September 2010. A conversation with a grower suggested that the pawpaw fruits are likely of the Shenandoah variety, but this has not been confirmed. Once in the laboratory, pawpaws were vacuum-packaged and refrigerated. The total weight of pawpaws was 3,343 g with an average of 133 g per fruit. After the pawpaw was peeled and the skins and seeds separated from the pulp, 1,772 g of wet pulp was obtained, for a

53% yield. The wet pulp was dried in a food dehydrator (Telewares, TFD 4096) for at least 48 hours or in an oven (70 ˚C) for 2 days for moisture determination. The dried pulp was milled into a fine powder in a grinder (Boca Raton, IDS 55).

In addition, for September 2009 sample extracts for test of total phenolics and total flavonoids, frozen stored (-18 ˚C) pulp in 2009 were dried and milled into a powder.

Percent sugar, color, and pH were measured using frozen stored pulp from 2009.

For September 2008 samples, previously reported data (n = 3) was used for total phenolics and total flavonoids. 51

For HPLC analysis of frozen pawpaw pulp stored in air or under vacuum for 0, 2,

4, 6, 8, 10, and 12 months, pawpaw pulp from a single tree was separated from the skins and seeds and then all of the pulp was pooled and divided into 100g portions. Once portioned, the pawpaw pulp was placed into polyethylene/nylon FoodSaver (Jarden

Corp., Rye, NY) 27.94-cm bags with an oxygen transmission rate of 6.7 cc/m 2/24

h/23°C/0% RH. The bags were randomly selected prior to labeling. Once the bags were

filled, they were either vacuum sealed (vacuum) or sealed without attempting to remove

air prior to sealing (air), then immediately transferred into frozen storage at -18 ºC. At 2-

month intervals, pawpaw samples were directly extracted in methanol, and then

transferred to a freezer at -40 ºC to be analyzed.

Pawpaw Pulp Extraction

For measurements of total phenolics, total flavonoids, and high performance

liquid chromatography (HPLC) analysis of refrigerated (4 ˚C) samples in 2010, the

extraction of organic compounds from the pawpaw pulp powder was performed by

mixing the powders with HPLC-grade methanol (CH 3OH). The ratio between the powder

and methanol varied in order to find the optimum ratio of the extraction. The ratio of

dried pulp powder (g) to methanol (ml) was 1:7, 1:18, and 1:36, which corresponds to

1:2, 1:5, and 1:10 on a wet pulp basis. After 2 hours of mixing, the samples were

centrifuged at 2000 x g (Beckman AccuSpin FR Refrigerated Centrifuge, Beckman, Inc.)

for 15 min at room temperature. The supernatant was filtered through a 0.45 µm

membrane. The extracted pawpaw pulp solutions were stored in a refrigerator (4°C) until

methanol was evaporated by using Rotavapor (Buchi, R-114). After methanol 52 evaporation was complete, the extracts were weighed and reconstituted in methanol which is twice their volume. These reconstituted samples were stored at 4°C.

For HPLC analysis of pawpaw pulp stored in air or under vacuum for 0, 2, 4, 6, 8,

10, and 12 months, pawpaw fruit pulp (10 g) was extracted in 50 ml of methanol using a

Waring blender followed by agitation for one hour. Methanol-extracted samples were filtered and stored in glass screw top vials in which the headspace has been flushed with

N2 gas.

Measurements of Total Phenolics

The measurement of total phenolics was conducted by using the Folin-Ciocalteu

assay according to published methods (Harris & Brannan, 2009). Folin-Ciocalteu reagent

was first diluted 10-fold with deionized water and the diluted reagent (750 µl) mixed with

diluted aliquots of the pawpaw pulp extracts (0.1 ml) and 7.5% sodium carbonate

solution (750 µl). After 120 min at room temperature in the absence of light, absorbance

was measured at 750 nm using a Spectronic Genesys 5 (Thermo Electric Corporation,

Madison, WI). Total phenolics was quantified according to a standard curve prepared

from gallic acid, and was expressed as µmol gallic acid equivalent per g wet weight of

pawpaw pulp tissue (µmol GAE/g wet pulp tissue).

Measurement of Total Flavonoids

Total flavonoids were measured spectrophotometrically (Harris & Brannan,

2009). Pawpaw extract (0.5 ml) was mixed with methanol (1.5 ml), to which 10%

aluminum chloride (AlCl 3, 0.1 ml), 1 M potassium acetate (CH 3COOK, 0.1 ml), and

deionized water (2.8 ml) were added. The solution was allowed to sit for 40 min at room 53 temperature in the absence of light, and the absorbance measured at 415 nm using a

Spectronic Genesys 5 (Thermo Electric Corporation, Madison, WI). The amount of total flavonoid was measured according to a standard curve prepared from rutin, and was displayed as µmol rutin equivalent per g pawpaw wet pulp tissue (µmol rutin/g wet pulp tissue).

Moisture, Lipid, Percent Sugar, Color, and pH

Moisture content of pawpaw pulp (~1 g) was measured as the difference in weight before and after drying in an oven (Fisher Isotemp Model 255D, Fisher Scientific) at 70

◦C. The samples were dried for two days until constant weight was achieved. Once the

samples were dry, lipid was extracted using hexane via the Soxhlet extraction process

(AOAC 920.39). Percent lipid was calculated from the weight before and after lipid

extraction. Percent sugar of wet pulp was tested using a refractometer. Wet pulp was

placed on the refractometer and percent sugar was measured. Color was measured by

three-dimensional scale (L*=0 represents black and L*=100 yields diffuse white; a*

negative values indicate green and a* positive values represent magenta; b* negative

values yields blue while b* positive values indicate yellow). The CIE L*, a*, and b*

values of the pulp was measured using a Konica BC-10 (Konica Minolta Sensing

Americas Inc., Ramsey, NJ, U.S.) that was calibrated against a standard white plate

before each use. The lightness (L ∗) and chromaticity coordinates (a ∗ and b∗) was calculated as the mean of three readings at different positions on the top surface of each patty. The pH of the pulp was determined by plunging the pH meter (Accumet AB15 54

Plus, Fisher Scientific) calibrated to pHs 4, 7, and 10 into approximately 10 g of pawpaw pulp.

HPLC Analysis

For both HPLC analysis of refrigerated (4 ˚C) pulp extracts in 2010 and HPLC analysis of extracts of frozen (-18 ˚C) pawpaw pulp stored in air or under vacuum for 0,

2, 4, 6, 8, 10, and 12 months in 2009, separation of (+)-catechin and quercetin flavonoids was achieved using HPLC (HP Agilent 1100, Hewlett Packard, Santa Clara, CA) equipped with ChemStation software, a degasser G1322A, a quaternary pump G1311A, and a variable wavelength detector G1315B. The column used was a 5 μm Hypersil ODS

with a security guard column, operated at room temperature. Table 4 shows that the

mobile phase was 2% acetic acid in water (eluent A) and 0.5% acetic acid in water and

acetonitrile (50:50, v/v; eluent B). The binary mobile phase gradient program was as

follows: 10% B to 55% B (4 min), 55% B to 100% B (3 min), 100% B to 10% B (8 min)

which was modified from one of the current methods (Schieber, Keller, & Carle, 2001).

The injection volume for all samples was 20 µl. Spectra was performed at 280 nm at a

flow rate of 1ml/min. The pressure of the column was maintained below 100 bars.

Quantification was conducted using linear standard curves of commercial (+)-

catechin and quercetin standard compounds. A standard curve represents retention times

of (+)-catechin and quercetin are 2.843 min and 2.987 min at 280 nm, respectively, when

using the HPLC gradient program of this study (see Figure 10 A). It was confirmed by

several tests. (+)-catechin alone showed a peak at 2.830 min (see Figure 10 B) and

quercetin alone displayed a peak at 2.964 min (see Figure 10 C). Also, when the 55 concentration of (+)-catechin was increased twice as much but that of quercetin remained, the retention time of (+)-catechin was almost the same (2.859 min) and the height of the (+)-catechin peak increased considerably from 1264 mAU to 1882 mAU but the quercetin peak at 2.997 min was changed only from 1203 mAU to 1145 mAU (see

Table 5). On the other hand, when only the concentration of quercetin increased twice as much, the height of quercetin peak at 2.990 min increased significantly from 1203 mAU to 1663 mAU but (+)-catechin peak at 2.854 min changed only from 1203 mAU to 1303 mAU (see Table 5). In addition, when a pawpaw sample was mixed with authentic (+)- catechin, the (+)-catechin peak of pawpaw extract around 2.840 min increased according to the concentration of authentic (+)-catechin. Similarly, commercial quercetin enhanced the quercetin peak of pawpaw extract around 2.990 min in proportion to the concentration of quercetin.

Table 4

HPLC Mobile Phase Gradient Program

Time Solvent A Solvent B Flow-rate (min) (%) (%) (ml/min)

0 90 10 1.0 4 45 55 1.0 7 0 100 1.0 15 90 10 1.0

A A

B A

Figure 10 . (+)-Catechin and quercetin standard curves.

A: (+)-catechin (5mg (+)-catechin/ 10ml methanol, i.e., 1.72 mM) and quercetin (5mg quercetin/ 10ml methanol, i.e. 1.65 mM); B: (+)-catechin (5mg (+)-catechin/ 10ml methanol, i.e., 1.72mM); C: Quercetin (5mg quercetin/ 10ml methanol, i.e., 1.65 mM).

Table 5

Identification of Retention Time (Ret Time), Area, and Height of (+)-Catechin and

Quercetin Peaks According to Concentration Changes

Ret Time Area Height mg/10ml MeOH mM (min) (mAU *s) (mAU)

(+)-catechin 5 1.72 2.843 5753 1264 quercetin 5 1.65 2.987 6968 1203

(+)-catechin 10 3.45 2.859 8505 1882 quercetin 5 1.65 2.997 7507 1145

(+)-catechin 5 1.72 2.854 5788 1303 quercetin 10 3.31 2.990 9344 1663

Sampling Plan

The measurement of total phenolics and total flavonoids was generated for 3 trials for each extraction ratio described previously (n = 30). For 2009 extracts, the number of subjects was 15 (n = 15), and for 2008 samples, the number was three (n = 3). Lipid and moisture content was reported as the mean of six pawpaw pulp samples (n = 6). Six samples were also employed to determine mean pH, color, and percent sugar values. For each extraction concentration and each sample from different duration (0, 2, 4, 6, 8, 10, and 12 month storage), at least three HPLC injections were made to determine the presence of (+)-catechin and quercetin flavonoids.

Statistical analysis

For total phenolics and total flavonoids, means were generated from three 2008 samples

(n = 3), fifteen 2009 samples (n = 15), and thirty 2010 samples (n = 30). For (+)-catechin 58 and quercetin, means were made from three 2010 samples (n = 3). SPSS Statistics 17.0

(Chicago, IL) was used to analyze data by using the general linear model. The level of significance for all tests was set at p< 0.05. Means separations or determinations of homogenous subsets were achieved according to Duncan’s post hoc multiple-range test.

CHAPTER 4: RESULTS

The objectives of this study were to assess the concentration of total phenolics and total flavonoid compounds and to isolate (+)-catechin and quercetin flavonoids in pawpaw pulp extracts using HPLC. Also, the stability of those flavonoids was analyzed by monitoring pawpaw pulp packaged in for 0, 2, 4, 6, 8, 10, or 12 months. In addition, objective evaluations for moisture, lipid, pH, % sugar, and color were performed.

Measurements of Total Phenolics and Total Flavonoids

As shown in Table 6, statistically significant differences were observed for total phenolics from pawpaw pulp harvested in 2008, 2009, and 2010 in the order of 2009 <

2008 < 2010. Table 6 also shows that total flavonoids from pawpaw pulp harvested in

2009 and 2010 were significantly lower than total flavonoids from pawpaws harvested from 2008. Thus the order was 2009 ≈ 2010 < 2008. The percentage of total phenolics identified as total flavonoids were approximated as 18% (2008), 12% (2009), and 5%

(2010).

Table 6

Comparison of Total Phenolics, Total Flavonoids, and the Ratio of Flavonoid to

Phenolics in Pawpaw Pulp Harvested in 2008, 2009, and 2010

2008 2009 2010

Total phenolics 1 11.19 ± 0.57 B 3.91 ± 1.61 C 14.11 ± 1.90 A

2 Z Z Total flavonoids 2.01 ± 0.44 Y 0.46 ± 0.24 0.76 ± 0.40

Total flavonoids 18 % 12 % 5 % Total phenolics

Note . 1µmol Gallic Acid Equivalents (GAE)/g wet tissue; 2µmol rutin equivalents/g wet tissue; Different superscripts within a row show significant differences (p < 0.05). Each value of total phenolics and flavonoids in the table is represented as mean ± SD.

Analysis of (+)-Catechin and Quercetin

Isolation of (+)-catechin and quercetin flavonoids was conducted using reversed

phase HPLC. The retention times of authentic (+)-catechin and quercetin from the

standard chromatogram were 2.83~2.85 and 2.96~2.99, respectively (see Figure 10).

Table 7 display the retention times of (+)-catechin and quercetin (2.85~2.86 min and

2.97~2.98 min, respectively) which are very close to the retention time from the standard

chromatogram, and there was an unknown compound that eluted at 3.60~3.63 min.

Consistent with previous results made by Schieber et al (2001), the retention time of

quercetin was longer than that of (+)-catechin. Phenolic compounds in the pawpaw pulp 61 extract were (+)-catechin and quercetin. Table 7 also shows that the amount of quercetin was similar to that of (+)-catechin in the pawpaw pulp extracts. The identification and quantification of the unknown compound will need to be investigated.

Table 7

Retention Times and Concentration of (+) -Catechin and Quercetin in Pawpaw Pulp

Harvested in 2010

Concentration Compound Retention time mAU 1 (mg/ 100 g wet (min) pulp)

(+)-Catechin 2.85~2.86 275.66 ± 57.49 1.28 ± 0.85

Quercetin 2.97~2.98 304.57 ± 63.83 1.37 ± 1.00

Unknown 3.60~3.63 153.21 ± 33.24 -

Note . 1mAU=milli-absorbance unit; Each value of concentration in the table is represented as mean ± SD.

Evaluation of the Stability of (+)-Catechin and Quercetin in Stored Samples

Methanolic pawpaw pulp extracts were stored in air or under vacuum for 0, 2, 4,

6, 8, 10, or 12 months. As shown in Table 7, in both air and vacuum storage samples, the concentration of both (+)-catechin and quercetin increased during the first 4 months.

After 4 month storage, a decrease in the concentration of both (+)-catechin and quercetin was seen until the 12-month period. 62

Table 8

The Concentration of (+) -Catechin (mg/100g wet pulp), Quercetin (mg/100g wet pulp), and Unknown Compound (mAU) in Pawpaw Pulp Harvested in 2008 and Stored in Air or

Under Vacuum for 0, 2, 4, 6, 8, 10, and 12 Months

Air stored Vacuum stored

(+)- (+)- Month Quercetin 2 Unknown 3 Quercetin Unknown Catechin 1 Catechin

0 2.42 ± 3.13 ± 78.68 ± 1.97 ± 0.91 ± 45.82 ± 3.75 1.81 59.52 3.41 1.57 59.72

2 N.A. 4 N.A. N.A. 6.30 ± 3.21 ± 98.89± 5.15 5.57 72.43

4 10.04 ± 5.83 ± 242.92 ± 12.31 ± 4.25 ± 157.19± 1.54 3.73 39.70 2.39 7.36 148.52

6 9.52 ± 6.42 ± 157.48± N.D. 5 N.D. 10.64± 0.37 1.99 128.41 5.86

8 8.78 ± 3.58 ± 194.95± 0.18 ± N.D. 25.43± 0.20 1.84 167.74 0.30 26.53

10 8.28 ± 1.58 ± 146.77± 0.14 ± 0.82 ± 92.12± 4.09 2.73 121.67 0.25 1.43 70.89

12 0.46 ± N.D. 40.44± 0.06 ± N.D. 38.84± 0.79 37.09 0.10 38.74 Note . 1,2 represented as mg/100g wet pulp; 3 represented as mAU; 4 N.A.- not analyzed; 5 N.D.- not detected. Each value in the table is represented as mean ± SD.

Objective Evaluation

Objective analyses for moisture content, lipid content, percent sugar, color, and pH were performed and are shown in Table 9.

Table 9

Moisture Content, Lipid Content, Percent Sugar, Color, and pH Values of Pawpaw

Properties Pawpaw

Moisture 75.24 ± 2.07 (% wet weight)

Lipid 0.45 ± 0.38 (% wet weight)

Percent Sugar 24.10 ± 1.15 (% wet weight)

Color L* (Lightness) 54.17 ± 5.28 a* (Redness) 10.23 ± 1.86 b* (Yellowness) 27.20 ± 1.59

pH 4.85 ± 0.01

Summary

The amounts of total phenolics were significantly different among 2008, 2009, and 2010 pawpaw pulp extracts (2009 < 2008 < 2010). On the other hand, total flavonoids contents were in the order of 2009 ≈ 2010 < 2008. By using reversed phase

HPLC, (+)-catechin, quercetin, and an unknown compound were mainly detected. During 64 the first 4-month period, both air and vacuum stored samples have shown the increase in the concentration of both (+)-catechin and quercetin. Then the concentration decreased until 12 months. Objective evaluations for pawpaw pulp (analysis of moisture content, lipid content, percent sugar, color, and pH) were performed.

CHAPTER 5: DISCUSSION AND CONCLUSION

Measurements of Total Phenolics

The results of this study show that pawpaw pulp phenolics are variable from year to year. Some of this variation could be due to methodological differences among extractions, suggesting that standardization of extraction conditions for pawpaw may help further research. Also, although this research used pawpaws from a single tree in Athens,

Ohio, even slight differences in individual pawpaws could affect the amount of phenolics available for use as a functional food. Table 10 compares seventeen common fruits consumed in the United States in terms of their amounts of total phenolic compounds.

Berries tend to have high amount of total phenolics compared to other fruits including the results for pawpaw from this research. Therefore, it is not surprising that the amount of total phenolics in wild blueberry is the highest (22.80 ± 0.53 µmol GAE/ g tissue) followed by blackberry (21.90 ± 0.32 µmol GAE/ g tissue). Table 10 shows pawpaw also has considerable amount of total phenolics (14.11 ± 1.90 µmol GAE/ g tissue). Total phenolics in pawpaw compared favorably to that of pomegranate (17.97 ± 0.74 µmol

GAE/ g tissue) and cranberry (15.25 ± 0.27 µmol GAE/ g tissue).

According to Wolfe et al., 2008, the top ten fruit phenolics contributors to the

American diet are apple (33.1%), orange (14.0%), grape (12.8%), strawberry (9.8%), plum (7.3%), banana (4.3%), cranberry (3.4%), pear (2.9%), pineapple (2.6%), and peach

(2.1%) based on consumption data from the USDA Food Availability (Per Capita) Data for 2005. Consumption of fruits which have high amount of phenolics such as wild blueberry, blackberry, and pawpaw will help to increase the consumption of phenolics. 66

Many studies (Johnson, Bomser, Scheerens, & Giusti, 2011; Wu et al., 2004) has shown that diets rich in fruits and vegetables reduce the risk of major chronic diseases including cancer, cardiovascular disease, and diabetes, and it is believed that these prevention of diseases are largely attributed to phenolics or phenolic compounds in fruits and vegetables. For example, phenolic-rich black raspberry extracts inhibited the proliferation of HT-29 colon cancer cells in a dose dependent manner (from 33 to 82 % at

0.6 mg extract/mL medium and from 71 to 118% at 1.2 mg extract/ml medium), and the inhibitory effects were also dependent on production site, cultivar, and the level of maturity (Johnson et al., 2011). In another example, Kwon, Apostolidis, and Shetty

(2008) showed that phenolic antioxidant-enriched extracts of pulp of white eggplants had

α-glucosidase inhibitory activity, which leads to controlling glucose absorption and reducing hyperglycemia. This can be an effective method for management of type 2 diabetes with less side effects.

Phenolics are non-nutritive secondary metabolites derived from phenylalanine or tyrosine and are widely distributed in plants and plant foods. In plants, phenolic compounds keep species from harm such as microbial infections, UV radiation, physical damages, and drought. For instance, grape-derived resveratrol stops fungi from growing

(Szajdek & Borowska, 2008). Also, natural phenolics from fruit extracts show a higher antioxidant capacity than pure phenols and pharmaceutical antioxidant supplements

(Vinson, Su, Zubik, & Bose, 2001). It is well known that berry fruits are good sources of antioxidant. Phenolic acids from berry fruits inhibit LDL oxidation in vitro, preventing

the generation of reactive oxidized LDL which can cause tissue damage. 67

Proanthocyanidin (condensed tannin) and ellagitannins (hydrolysable tannin) can much more effectively scavenge free radicals than low molecular weight polyphenol, vitamin

C, and vitamin E (Szajdek & Borowska, 2008).

It is unknown if the phenolic profile of pawpaw will be beneficial to health.

Recently, there is a growing interest on phenolic compounds in fruits and vegetables.

Thus, it will be very important to characterize phenolics in pawpaw and find out its potential health benefits.

Table 10

The Amounts of Total Phenolics in Commonly Consumed Fruits Based on Several Recent Studies; pawpaw pulp extract was prepared from pawpaws harvested in 2010

Fruits Total phenolics1 Reference

Wild Blueberry 22.80 ± 0.53 Wolfe et al., 2008

Blackberry 21.90 ± 0.32 Wolfe et al., 2008

Plum 19.60 ± 0.67 Chun et al., 2005

Pomegranate 17.97 ± 0.74 Wolfe et al., 2008

Cranberry 15.25 ± 0.27 Wolfe et al., 2008

Pawpaw ( Asimina triloba ) 14.11 ± 1.90 Current Study

Strawberry 11.96 ± 0.14 Chun et al., 2005

Grapefruit 8.60 ± 0.41 Chun et al., 2005

Apples 6.29 ± 0.07 Chun et al., 2005

Banana 6.00 ± 0.36 Chun et al., 2005

Lemon 3.52 ± 0.18 Sun et al., 2002

Mango 3.43 ± 0.22 Wolfe et al., 2008

Orange 3.02 ± 0.05 Sun et al., 2002

Pear 2.85 ± 0.13 Sun et al., 2002

Pineapple 2.15 ± 0.05 Sun et al., 2002

Avocado 1.27 ± 0.04 Wolfe et al., 2008

Honeydew 0.82 ± 0.05 Wolfe et al., 2008

Watermelon 0.75 ± 0.00 Wolfe et al., 2008 Note . 1 Total phenolics reported as µmol gallic acid equivalents (GAE) per gram tissue. 69

Measurements of Total Flavonoids

As shown in Table 6, the amount of flavonoids also varied from year to year. The exact amount of flavonoids can be dependent on extraction solvent. All the results in

Table 6 and 11 were made by using methanol extraction. However, acetone extracts showed higher flavonoid content in banana compared to methanol extracts (Saravanan &

Aradhya, 2011). Table 11 shows the concentration of total flavonoids in selected fruits.

Flavonoids are the main constituent of phenolic compounds and exist in plants and plant foods. There are six subgroups (flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones) based on their structure. Biological roles of flavonoids include anti-inflammatory, anti-microbioal, anti-cancer, anti-proliferative activities.

Studies have shown that flavonoids can modify enzyme systems in our body (Hollman &

Katan, 1999). The systems are related to cell division, cell proliferation, detoxification, and inflammatory and immune response. For example, flavonols decreased the risk of cardiovascular disease in prospective cohort studies (Hollman & Katan, 1999).

Table 11

The Amounts of Flavonoids in Selected Fruits Based on Recent Studies; Banana, Mango, and Pineapple Which Have Similar Flavor to Pawpaw Has Been Selected

Flavonoids 1 Reference

Banana, giant cavendish (Musa 10.70 ± 1.70 1 Saravanan & Aradhya acuminata ) (2011)

Pawpaw ( Asimina triloba ) 3.99 ± 2.54 2 Current Study

Mango (Mangifera pajang ) 7.00 ± 0.00 3 Abu Bakar, Mohamed, Rahmat, & Fry (2009)

Strawberry (Fragaria ananassa ) 14.6 ± 3.0 4 Lin & Tang (2007)

Note. 1 Total flavonoids expressed as mg catechin equivalents per g dry extract of pseudostem. 2 Total flavonoids reported as mg rutin per 100g of wet pulp. 3 Total flavonoids expressed as mg rutin per 100g of dry sample of flesh. 4 Total flavonoids displayed as mg quercetin equivalents per 100g of fresh matter.

Characterization of (+)-Catechin and Quercetin

Reversed phase HPLC was used for the isolation of (+)-catechin and quercetin.

The mobile phase was based on the previously reported method (Schieber et al., 2001), but the gradient program was modified as described in Table 4, which produced clear (+)- catechin and quercetin peaks at 2.83 min and 2.98 min, respectively. A peak at 3.62 min was also detected. In reversed phase HPLC, nonpolar compounds stay in the hydrophobic column longer than polar compounds. Thus, the results suggest that quercetin is more 71 nonpolar compound than (+)-catechin because it stayed in the column longer than (+)- catechin. Also, the unknown peak is likely to be more nonpolarity than quercetin because the peak stayed longer than quercetin. According to HPLC results of Schieber et al. ( ≈of

phloretin is 274.26 g/mol and detected after quercetin. Therefore, it seems that polarity is

probably the major factor for the retention time.

The amount of (+)-catechin was 1.28 ± 0.85 mg/ 100 g wet pulp and that of

quercetin was 1.37 ± 1.00 mg/ 100g wet pulp (Table 7). Both (+)-catechin and quercetin

are flavonoids, and the sum of the amount (≈ 2.65 mg/100g wet pulp) does not exceed the

amount of total flavonoids (3.99 ± 2.54 mg/ 100 g wet pulp), so the results seem

consistent. Also, these results show that approximately 66.42 % of total flavonoids in the

pawpaw pulp is attributed to (+)-catechin (32.08 %) and quercetin (34.34%). As shown in

Table 12, banana and mango are sources of (+)-catechin, however, banana, mango, and

pineapples are not the sources of quercetin. According to USDA database for the

Flavonoid Content of Selected Foods (2007), raw apples with skin ( Malus domestica ) have 4.27 mg quercetin/ 100 g edible portion, and apples are one of the most important dietary sources of quercetin. Pawpaws have 0.46 mg quercetin/ 100 g wet pulp (see Table

12). Therefore, apples have approximately ten times more quercetin than pawpaws but pawpaws can still be the source of quercetin.

Catechin plays a significant role in preventing cancer cell growth. Especially green tea catechins target receptor tyrosine kinases which are important for cell proliferation. For example, epigallocatechin-3-gallate (EGCG) from green tea blocks cell proliferation and causes apoptosis of many cancer cells (Shimizu, Adachi, Masuda, 72

Kozawa, & Moriwaki, 2011). Quercetin is one of the most studied flavonoids. It is found in many plants such as blueberries, grapes, onions and broccoli and potentially delays aging process (Cherniack, 2010). In addition, quercetin induces the apoptosis of human cancer cells in vitro (Cherniack, 2010).

Table 12

The Amounts of (+)-Catechin and Quercetin in Selected Fruits Based on Recent Research

(+)-Catechin 1 Quercetin 2 Reference

Banana, raw 6.1 ± 0.53 0.00 USDA 3 (Musa X paradisiaca )

Pawpaw 1.28 ± 0.85 1.37 ± 1.00 Current Study (Asimina triloba )

Mango, raw 1.72 ± 0.00 0.00 USDA (Mangifera indica )

Pineapple, raw, all varieties 0.00 0.00 USDA (Ananus comosus )

Note. 1, 2 mg/ 100g edible portion (or wet pulp). 3 USDA-USDA Database for the Flavonoid Content of Selected Foods (2007).

Evaluation of the Stability of (+)-Catechin and Quercetin in Stored Samples

Pawpaw pulp extracts stored in air or under vacuum for 0, 2, 4, 6, 8, 10, and 12

months were tested for the stability of catechin and quercetin in the extract. In the stored

samples, the concentration of quercetin was less than that of catechin (see Table 8). It 73 was possibly due to a pH change during the storage. The optimum pH for detecting quercetin was pH 1.5 which was described by Schieber et al. (2001). On the other hand, pH 7.0 is optimum for (+)-catechin. Thus a pH increase may inhibit detection of quercetin.

Those pawpaw pulp sample extracts were frozen at -18˚C. According to Harris and Brannan (2009), frozen storage (-18˚C) for 300 days before extraction increased total phenolics fourfold and total flavonoids sixfold. It is suggested that hydrolysis of phenolic or flavonoid polymers caused the increase of the concentration of total phenolics and flavonoids. In this study, the concentration of total phenolics and flavonoids of 2009 samples in air or vacuum storage at -18 ˚C before extraction increased compared to that of 2010 pawpaw pulp extracts. It is probably due to hydrolysis of long chain phenolics or flavonoids during frozen storage.

Interestingly, a study showed that flavonoid anthocyanin, which is responsible for pigmentation and is very unstable during storage, can be retained significantly by adding sugar during frozen storage (-18 ˚C) of blackberries. Especially, addition of glucose among sugar (glucose, fructose, and sucrose) displayed the highest retention of anthocyanin (Kopjar, Tiban, Pilizota, & Babic, 2009). It was suggested that sugar addition decreases water activity, increasing the stability of anthocyanin. These findings can be a basis for developing new strategy of improving the efficiency of frozen storage by replacing fructose and sucrose with glucose in fruit preserves.

In order to slow down the loss of firmness of pawpaw fruits, refrigeration (4 ˚C) of the fruits is recommended and it does not cause the chilling injury of the fruits, unlike 74 cherimoya which gets chilling injury at below 7˚ C (Archbold et al., 2003). In addition, as mentioned above, frozen storage of pawpaw pulp before extraction can increase the concentration of total phenolic and flavonoids.

In Table 8, standard deviations of the concentrations of (+)-catechin and quercetin in air stored samples are variable. The unknown compound was expressed as mAU because the standard curve for the unknown compound could not be prepared at this time.

Some standard deviations of mAU of the unknown compound in air stored sample are quite large, but the actual values would be smaller than values of (+)-catechin and quercetin if they were converted into mg/100g wet pulp. On the other hand, standard deviations were larger than mean values in some cases, especially for the concentrations of (+)-catechin and quercetin in vacuum stored samples. It is suspected that retention time drift caused irregularity of the concentrations of (+)-catechin and quercetin, creating larger standard deviations. Retention time drift may result from changes in mobile phase composition, mobile phase flow rate, and stationary phase column temperature. During the experiment, mobile phase was changed a few times. Also, HPLC was operated for long periods of time, with vacuum stored sample tested after air stored samples. Perhaps when the vacuum stored samples were analyzed, the operation of the HPLC caused slightly higher column temperatures, resulting in slight changes in mobile phase flow rate and retention time drift. There also could be a slight alteration of the pressure inside the column, even if most of the time it was maintained below 100 bars. However, as shown in Table 5, the (+)-catechin and quercetin peaks were clearly identified by authentic 75 standards, so in spite of these standard deviation variations, these peaks represent anything other than these two flavonoids.

Objective Evaluation

Compared to mango which has approximately 75~85% moisture content, pawpaw contains somewhat less moisture in the ripe pulp (approximately 75%). Lipid content of pawpaw pulp was very low (0.45%). Similarly, McGrath & Karahadian (1994) previously reported that ripe pawpaw has 77% moisture and 0.47% lipid.

The pH of the ripe pulp in this study was 4.85, so it was slightly acidic. The low acid contents contribute to less sour taste, leading to stronger sweet flavor of pawpaw pulp. To the contrary, high acidic contents can result in more sour taste. Spore formation by foodborne pathogen Clostridium botulinum is generally inhibited below pH 4.6

(Raatjes & Smelt, 1979). In case of raw pawpaw pulp, C.botulinum can generate spores which make toxin because the pulp is above pH 4.6. Thus, lowering pH of the pulp would be needed when it is processed.

In addition, pawpaw in this study was shown to have 24% sugar, more than banana (12%), mango (14%), and pineapple (10%). McGrath and Karahadian (1994) showed that pawpaw fruits that initially displayed 8% sugar increased to

23% sugar by 28 days of sampling. During the 28-day period, the amount of percent sugar increased in one cultivar (cv. 1-59). Then it decreased a little after 28 days but it was still more than 20%. In the current study, a sample stored in 2009 in the freezer (-

18˚C) was used, so that the high amount of percent sugar could be largely due to long storage. 76

Conclusion

The results display that pawpaw pulp contains significant phenolic compounds and flavonoids. Flavonoids found in this study were (+)-catechin and quercetin flavonoids. Because they are very important phenolics and flavonoids in human diet, these findings will lead to pawpaw research on individual flavonoids. After 6 months of air and vacuum storage, the amount of (+)-catechin and quercetin flavonoids decreased.

Pawpaw has considerable amounts of moisture and sugar, but the amount of lipid is less than 1%.

Future Directions

Results from this study show the need for more research. An unknown peak was observed at 3.60~3.63 min and identification of the peak would be beneficial. There are also several smaller peaks which are needed to be identified. Complete separation of peaks by adapting different gradient programs would allow for more precise quantification. In terms of sample preparation, pawpaw skin could be a source of phenolic flavonoid compounds. The skin can be dried and grinded into powder before methanol extraction is conducted. Also, different types of cultivars could be tested. In addition, further research on optimization of storage condition for increasing the concentration of flavonoids would be needed. Lastly, the synergistic effect of the combination of flavonoid antioxidants on antioxidant capacity could be investigated, because it has been reported that combinations of antioxidants can increase in vitro antioxidant power (Parker, Miller, Myers, Miguez, & Engeseth, 2010).

REFERENCES

Archbold, D. D., Koslanund, R., & Pomper, K. W. (2003). Ripening and postharvest

storage of pawpaw. Horttechnology, 13 (3), 439-441.

Abu Bakar, M. F., Mohamed, M., Rahmat, A., & Fry, J. (2009). Phytochemicals and

antioxidant activity of different parts of bambangan ( Mangifera pajang ) and tarap

(Artocarpus odoratissimus ). Food Chemistry, 113, 479-483.

AOAC Official Methods of Analysis , 17th ed.; method 920.39. Crude Fat or Ether Extract:

Animal Foods; AOAC: Arlington, VA, 2002.

Beavers, K. M., Serra, M. C., Beavers, D. P., Cooke, M. B., & Willoughby, D. S. (2009).

Soymilk supplementation does not alter plasma markers of inflammation and

oxidative stress in postmenopausal women. Nutrition Research, 29 (9), 616-622.

Brannan, R. G., & Salabak, D. E. (2009). Ability of methanolic seed extracts of pawpaw

(Asimina triloba ) to inhibit n-3 fatty acid oxidation initiated by peroxyl radicals

and reactive oxygen, nitrogen, and sulfur. Food Chemistry, 114, 453-458.

Buer, C. S., Imin, N., & Djordjevic, M. A. (2010). Flavonoids: New roles for old

molecules. Journal of Integrative Plant Biology, 52 (1), 98-111.

Buettner, G. R. (1993). The pecking order of free radicals and antioxidants: Lipid

peroxidation, alpha-tocopherol, and ascorbate. Archives of Biochemistry and

Biophysics, 300 (2), 535-543. 78

Callaway, M. B. (1993). Pawpaw (Asimina triloba ): A "tropical" fruit for temperate

climates. In: J. Janick and J.E. Simon (eds.), New crops (pp. 505-515). New York:

Wiley.

Chen, C., Zhou, J., & Ji, C. (2010). Quercetin: A potential drug to reverse multidrug

resistance. Life Sciences, 87, 333-338.

Cherniack, E. P. (2010). The potential influence of plant polyphenols on the aging

process. Forschende Komplementarmedizin (Research in Complementary

Medicine), 17, 181-187.

Chun, O. K., Kim, D. O., Smith, N., Schroeder, D., Han, J. T., & Lee, C. Y. (2005). Daily

consumption of phenolics and total antioxidant capacity from fruit and vegetables

in the American diet. Journal of the Science of Food and Agriculture, 85, 1715-

1724.

Chun, O. K., Chung, S. J., & Song, W. O. (2007). Estimated dietary flavonoid intake and

major food sources of U.S. adults. The Journal of Nutrition, 137 (5), 1244-1252.

Crozier, A., Jaganath, I. B., & Clifford, M. N. (2009). Dietary phenolics: Chemistry,

bioavailability and effects on health. Natural Product Reports, 26, 1001-1043.

Duffrin, M. W., Holben, D. H., & Bremner, M. J. (2001). Consumer acceptance of

pawpaw ( Asimina triloba ) fruit puree as a fat-reducing agent in muffins,

compared to muffins made with applesauce and fat. Family and Consumer

Sciences Research Journal, 29 (3), 281-287. 79

Duffrin, M. W., & Pomper, K. W. (2006). Development of flavor descriptors for pawpaw

fruit puree: A step toward the establishment of a native tree fruit industry. Family

and Consumer Sciences Research Journal, 35 (2), 118.

Frankel, E. N. (1980). Lipid oxidation. Progress in Lipid Research, 19 (1-2), 1-22.

Fowler, Z. L., & Koffas, M. (2009). Biosynthesis and biotechnological production of

flavanones: Current state and perspectives. Applied Microbiology and

Biotechnology, 83 (5), 799-808.

Galli, F., Archbold, D. D., & Pomper, K. W. (2008). Loss of ripening capacity of

pawpaw fruit with extended cold storage. Journal of Agricultural and Food

Chemistry, 56, 10683-10688.

Geneve, R. L., Pomper, K. W., Kester, S. T., Egilla, J. N., Finneseth, C. L. H., Crabtree,

S. B., et al. (2003). Propagation of pawpaw. Horttechnology, 13 (3), 428-433.

Halliwell, B., Aeschbach, R., Loliger, J., & Aruoma, O. (1995). The characterization of

antioxidants. Food and Chemical Toxicology, 33 (7), 601-617.

Halvorsen, B. L., Carlsen, M. H., Phillips, K. M., Bohn, S. K., Holte, K., Jacobs, D. R.,

Jr., et al. (2006). Content of redox-active compounds (ie, antioxidants) in foods

consumed in the United States. American Journal of Clinical Nutrition, 84, 95-

135.

Harris, G. G., & Brannan, R. G. (2009). A preliminary evaluation of antioxidant

compounds, reducing potential, and radical scavenging of pawpaw ( Asimina

triloba ) fruit pulp from different stages of ripeness. LWT-Food Science and

Technology, 42, 275-279. 80

Heim, K. E., Tagliaferro, A. R., & Bobilya, D. J. (2002). Flavonoid antioxidants:

Chemistry, metabolism and structure-activity relationships. The Journal of

Nutritional Biochemistry, 13 (10), 572-584.

Hodzic, Z., Pasalic, H., Memisevic, A., Srabovic, M., Saletovic, M., & Poljakovic, M.

(2009). The influence of total phenols content on antioxidant capacity in the

whole grain extracts. European Journal of Scientific Research, 28 (3), 471-477.

Hollman, P. C. H., & Katan, M. B. (1999). Dietary flavonoids: Intake, health effects and

bioavailability. Food and Chemical Toxicology, 37, 937-942.

Imai, K., & Nakachi, K. (1995). Cross sectional study of effects of drinking green tea on

cardiovascular and liver diseases. BMJ, 310 , 693-696.

Isabelle M, Lee B. L., Lim M. T., Koh W-P., Huang D., & Ong C. N. (2010). Antioxidant

activity and profiles of common fruits in Singapore. Food Chemistry, 123, 77-84.

Johnson, J. L., Bomser, J. A., Scheerens, J. C., & Giusti, M. M. (2011). Effect of black

raspberry ( Rubus occidentalis L.) extract variation conditioned by cultivar,

production site, and fruit maturity stage on colon cancer cell proliferation. Journal

of Agricultural and Food Chemistry, 59, 1638-1645.

Jones, S. C., & Layne, D. R. (1997). Cooking with pawpaws (Bulletin #PIB-001).

Frankfort: Kentucky State University Cooperative Extension Program.

Kobayashi, H., Wang, C., & Pomper, K. W. (2008). Phenolic content and antioxidant

capacity of pawpaw fruit ( Asimina triloba L.) at different ripening stages.

HortScience, 43 (1), 268-270. 81

Kral, R. (1960). A revision of Asimina and Deeringothanmnus (Annonaceae). Brittonia

12 , 233-278.

Kojima, N., & Tanaka, T. (2009). Medicinal chemistry of Annonaceous acetogenins:

design, synthesis, and biological evaluation of novel analogues. Molecules, 14 (9),

3621-3661.

Kopjar, M., Tiban, N. N., Pilizota, V., & Babic, J. (2009). Stability of anthocyanins,

phenols and free radical scavenging activity through sugar addition during frozen

storage of blackberries. Journal of Food Processing and Preservation , 33 , 1-11.

Kwon, Y-. I., Apostolidis, E., & Shetty, K. (2008). In vitro studies of eggplant (Solanum

melongena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes

and hypertension. Bioresource Technology, 99, 2981-2988.

Lambert, J., & Elias, R. (2010). The antioxidant and pro-oxidant activities of green tea

polyphenols: A role in cancer prevention. Archives of Biochemistry and

Biophysics, 501, 65-72.

Layne, D. R. (1996). The pawpaw [ Asimina triloba (l) dunal ]: A new fruit crop for

Kentucky and the United States. Hortscience, 31 (5), 777-784.

Lin, J.-Y. & Tang, C.-Y. (2007). Determination of total phenolic and flavonoid contents

in selected fruits and vegetables, as well as their stimulatory effects on mouse

splenocyte proliferation. Food Chemistry, 101, 140-147.

Lucenteforte, E., Garavello, W., Bosetti, C., & La Vecchia, C. (2009). Dietary factors and

oral and pharyngeal cancer risk. Oral Oncology, 45, 461-467. 82

Mateos-Aparicio, I., Redondo Cuenca, A., Villanueva-Suárez, M. J., & Zapata-Revilla,

M. A. (2008). Soybean, a promising health source. Nutricion Hospitalaria, 23 (4),

305-312.

McGrath, M., & Karahadian, C. (1994). Evaluation of physical, chemical, and sensory

properties of pawpaw fruit ( Asimina triloba ) as indicators of ripeness. Journal of

Agricultural and Food Chemistry, 42, 968-974.

Mo, Y., Nagel, C., & Taylor, L. P. (1992). Biochemical complementation of chalcone

synthase mutants defines a role for flavonols in functional pollen. Proceedings of

the National Academy of Sciences, 89, 7213-7217.

Moore, R. J., Jackson, K. G., & Minihane, A. M. (2009) Green tea ( Camellia sinensis )

catechins and vascular function. The British Journal of Nutrition, 102(12), 1790-

1802.

Naithani, R., Huma, L. C., Holland, L. E., Shukla, D., McCormick, D. L., Mehta, R. G.,

et al. (2008). Antiviral activity of phytochemicals. Mini-Reviews in Medicinal

Chemistry, 8, 1106-1133.

Niki, E., Saito, T., Kawakami, A., & Kamiya, Y. (1984). Inhibition of oxidation of

methyl linoleate in solution by vitamin E and vitamin C. The Journal of

Biological Chemistry, 259 (7), 4177-4182.

Palma, T., Aguilera, J. M., & Stanley, D. W. (1993). A review of postharvest events in

cherimoya. Postharvest Biology and Technology, 2, 187-208. 83

Panda, S., & Kar, A. (2007). Antidiabetic and antioxidative effects of Annona squamosa

leaves are possibly mediated through quercetin-3-O-glucoside. Biofactors, 31 (3-

4), 201-210.

Parker, T. L., Miller, S. A., Myers, L. E., Miguez, F. E., & Engeseth, N. J. (2010).

Evaluation of synergistic antioxidant potential of complex mixtures using oxygen

radical absorbance capacity (ORAC) and electron paramagnetic resonance (EPR).

Journal of Agricultural and Food Chemistry, 58 , 209-217.

Peterson, R. N. (1991). Pawpaw (Asimina ). Acta Horticulturae, 290 , 567-600.

Peterson, R. N. (2003). Pawpaw variety development: A history and future prospects.

HortTechnology, 13, 449-454.

Pomper, K. W., Crabtree, S. B, & Layne, D. R. (2008). Flowering and fruiting

characteristics of eight pawpaw [ Asimina triloba (L.) Dunal] selections in

Kentucky. Journal of the American Pomological Society, 62 (3), 89-97.

Pomper, K. W., Crabtree, S., Layne, D., Peterson, N., Masabni, J., & Wolfe, D. (2008).

The Kentucky pawpaw regional variety trial. Journal of the American

Pomological Society, 62 (2), 58-69.

Pomper, K. W., & Layne, D. R. (2005). The north American pawpaw: Botany and

horticulture. Horticultural Review, 31, 349-382.

Pomper, K. W., Layne, D. R., & Jones, S. C. (2003). Container production of pawpaw

seedlings. HortTechnology, 13, 434-438.

Pomper, K. W., Layne, D. R., Peterson, R. N., & Wolfe, D. (2003). The pawpaw regional

variety trial: Background and early data. Horttechnology, 13 (3), 412-417. 84

Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination

of antioxidant capacity and phenolics in foods and dietary supplements. Journal

of Agricultural and Food Chemistry, 53 (10), 4290-4302.

Raatjes, G. J., & Smelt, J. P. (1979). Clostridium botulinum can grow and form toxin at

pH values lower than 4.6. Nature, 281 (5730), 398-399.

Rice-Evans, C., & Miller, N. (1997). Measurement of the antioxidant status of dietary

constituents, low density lipoproteins and plasma. Prostaglandins, Leukotrienes,

and Essential Fatty Acids, 57, 499-505.

Saltveit, M. E. (2005). Aminoethoxyvinylglycine (AVG) reduces ethylene and protein

biosynthesis in excised discs of mature-green tomato pericarp tissue. Postharvest

Biology and Technology, 35, 183-190.

Saravanan, K., & Aradhya, S. M. (2011). Polyphenols of pseudostem of different banana

cultivars and their antioxidant activities. Journal of Agricultural and Food

Chemistry, 59, 3613-3623.

Schieber, A., Keller, P., & Carle, R. (2001). Determination of phenolic acids and

flavonoids of apple and pear by high-performance liquid chromatography.

Journal of Chromatography A, 910 , 265-273.

Seelinger, G., Merfort, I., & Schempp, C. M. (2008). Anti-oxidant, anti-inflammatory and

anti-allergic activities of luteolin. Planta Medica, 74 (14), 1667-1677.

Shahidi, F., & Zhong, Y. (2010). Lipid oxidation and improving the oxidative stability.

Chemical Society Reviews, 39 (11), 4067-4079. 85

Shimizu, M., Adachi, S., Masuda, M., Kozawa, O., & Moriwaki, H. (2011). Cancer

chemoprevention with green tea catechins by targeting receptor tyrosine kinases.

Molecular Nutrition & Food Research, 55, 1-12.

Shukla, S., & Gupta, S. (2010). Apigenin: A promising molecule for cancer prevention

Pharmaceutical Research, 27, 962–978.

Sun, J., Chu, Y.-F., Wu, X., & Liu, R. H. (2002). Antioxidant and antiproliferative

activities of common fruits. Journal of Agricultural and Food Chemistry, 50,

7449-7454.

Szajdek, A., & Borowska, E. J. (2008). Bioactive compounds and health-promoting

properties of berry fruits: A review. Plant Foods for Human Nutrition, 63, 147-

156.

Taylor, L. P., & Grotewold, E. (2005). Flavonoids as developmental regulators. Current

Opinion in Plant Biology, 8, 317-323.

Templeton, S. B., Marlette, M., Pomper, K. W., & Jones, S. C. (2003). Favorable taste

ratings for several pawpaw products. Horttechnology, 13 (3), 445-448.

Treutter, D. (2005). Significance of flavonoids in plant resistance and enhancement of

their biosynthesis. Plant Biology (Stuttgart, Germany ), 7(6), 581-591.

U.S. Department of Agriculture. (2007). USDA Database for the Flavonoid Content

of Selected Foods . Beltsville, MD: U.S. Department of Agriculture, Agricultural

Research Service, Beltsville Human Nutrition Research Center, and Nutrient Data

Laboratory. 86

Vinson, J. A., Su, X., Zubik, L., & Bose, P. (2001). Phenol antioxidant quantity and

quality in foods: Fruits. Journal of Agricultural and Food Chemistry, 49, 5351-

5321.

Wang, L. S., & Stoner, G. D. (2008). Anthocyanins and their role in cancer prevention.

Cancer Letters, 269 (2), 281-290.

Watkins, C. B. (2006). The use of 1-methylcyclopropene (1-MCP) on fruits and

vegetables. Biotechnology Advances, 24, 389-409.

Wiese, T. D., & Duffrin, M. W. (2003). Effects of substituting pawpaw fruit puree for fat

on the sensory properties of a plain shortened cake. Horttechnology, 13 (3), 442-

444.

Willcox, J. K., Ash, S. L., & Catignani, G. L. (2004). Antioxidants and prevention of

chronic disease. Critical Reviews in Food Science and Nutrition, 44 (4), 275-295.

Williams, C. A., & Grayer, R. J. (2004). Anthocyanins and other flavonoids. Natural

Product Reports, 21, 539-573.

Wilson, M. F., & Schemske, D. W. (1980). Pollinator limitation, fruit production, and

floral display in pawpaw ( Asimina-triloba ). Bulletin of The Torrey Botanical

Club, 107 (3), 401-408.

Winkel-Shirley, B. (2002). Biosynthesis of flavonoids and effects of stress. Current

Opinion in Plant Biology, 5(3), 218-223.

Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L.

(2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the

United States. Journal of Agricultural and Food Chemistry, 52 (12), 4026-4037. 87

Wolfe, K. L., Kang, X., He, X., Dong, M., Zhang, Q., & Liu, R. H. (2008). Cellular

antioxidant activity of common fruits. Journal of Agricultural and Food

Chemistry, 56 (18), 8418-8426.

Wood, R., & Peterson, S. (1999). Lipids of the pawpaw fruit: Asimina triloba . Lipids,

34 (10), 1099-1106.

Yao, L. H., Jiang, Y. M., Shi, J., Tomas-Barberan, F. A., Datta, N., Singanusong, R., et al.

(2004). Flavonoids in food and their health benefits. Plant Foods for Human

Nutrition, 59, 113-122.