Exploring the Nutritional Potential of Carica papaya Linn Cultivars Emmy Hainida Khairul Ikram Bachelor’s Degree in Nutrition and Community Health/Master’s Degree in Nutritional Biochemistry

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 Queensland Alliance for Agriculture and Food Innovation (QAAFI)

Abstract

Carica papaya, commonly known as papaya or paw paw, is widely cultivated for its sweet, ripe edible fruits. However, papaya is also known for its health uses in some non-western cultures where green fruits and leaves are also eaten cooked as well as raw. This research project has looked at phytochemicals (carotenoids (provitamin A active compounds), polyphenols, ascorbic acid, and vitamin E) and antioxidant capacity (ORAC assay and total phenolic content) of fruit pulp, fruit peel and leaves from commercially available Australian papaya cultivars. Analysis was undertaken at different stages of fruit ripeness and leaf growth to understand the impact on nutritional quality and potential health properties. In addition, the release/bioaccessibility of the main phytochemicals was assessed as an initial measure to predict their potential bioavailability using an in vitro digestion procedure. Among cultivars, red fleshed papaya fruits exhibited the highest phytochemical values. Mid mature and/or fully ripen fruits gave the highest antioxidant capacities as well as ascorbic acid and carotenoid content. Ascorbic acid content ranged from 37.0-58.5 mg/100 g FW with large variations between cultivars and an increase as the fruits ripen. Major carotenoids found in red-fleshed papaya were β-carotene, β-cryptoxanthin, and lycopene with lycopene representing 42-58% of the total carotenoids. However, lycopene was undetectable in the yellow-fleshed cultivar. Leaves were found to exhibit the highest ORAC and total phenolic content (young and mature; ORAC: 93-185 µM trolox equivalents/g FW; total phenolic content: 148-210 mg gallic acid equivalents/100g FW) compared to the peels and pulps. Overall, the highest antioxidant capacity, ascorbic acid and polyphenols could be found in papaya leaves followed by peels and pulp. Vitamin E was below the limit of quantification in all samples. Cooking of the leaves and unripe fruit resulted in a significant reduction of ascorbic acid and antioxidant capacity. In addition, in vitro digestion clearly demonstrated that the major papaya phytochemicals are bioaccessible from the pulp as well as the leaf matrix. Heat treatment (boiling) may enhance the bioaccessibility of TPC papaya fruits and leaves. The findings of this study showed that the phytochemical composition and antioxidant capacity varied across different papaya cultivars, maturity stages and plant parts affecting the nutritional value of this important tropical fruit.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis.

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Publications included in this thesis

1. Ikram EHK, Stanley R, Netzel M, Fanning K (2015) Phytochemicals of papaya and its traditional health and culinary uses – a review. Journal of Food Composition and Analysis 41: 201-211 Incorporated with modifications as part of Chapters 2. Contributor Statement of contribution Emmy Hainida Conception and design (80%) Analysis and interpretation of the research data (70 %) Drafting and critically reviewing (70%) Roger Stanley Conception and design (20%) Analysis and interpretation of the research data (20 %) Drafting and critically reviewing (20%) Michael Netzel Conception and design (15%) Analysis and interpretation of the research data (15%) Drafting and critically reviewing (20%) Kent Fanning Conception and design (5%) Analysis and interpretation of the research data (5%) Drafting and critically reviewing (10%)

2. Ikram EH, Netzel ME, Fanning K, Stanley R (2015) Phytochemicals in the tissues of Australian-grown papaya cultivars. Acta Horticulturae (ISHS) 1106: 175-178. Incorporated with modifications as part of Chapters 3. Contributor Statement of contribution Emmy Hainida Conception and design (80%) Analysis and interpretation of the research data (70 %) Drafting and critically reviewing (70%) Michael Netzel Conception and design (20%) Analysis and interpretation of the research data (20 %) Drafting and critically reviewing (20%) Roger Stanley Conception and design (15%) Analysis and interpretation of the research data (15%) Drafting and critically reviewing (20%) Kent Fanning Conception and design (5%) Analysis and interpretation of the research data (5%) Drafting and critically reviewing (10%)

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Submitted manuscripts included in this thesis No manuscripts submitted for publication.

Other publications during candidature Abstracts

1. Ikram EHK, Stanley R, Netzel M, Fanning K (2012) Antioxidant capacity of Carica papaya Linn Cultivars. Abstract presented (Oral presentation) at QAAFI 2012 – Annual Research Meeting, Sanctuary Cove, Gold Cost, QLD, July 10-11, 2012, Abstract Collection. 2. Ikram EHK, Stanley R, Netzel M, Fanning K (2013) Maximising the potential health properties of papaya fruit and leaf eaten as a food. Abstract presented (Oral presentation) 46th Annual AIFST Convention Yesterday, Today, Tomorrow, Brisbane, QLD, Australia, July 14-16, 2013, Program & Abstracts: 42. 3. Ikram EH, Netzel M, Fanning K, Stanley R (2013) Phytochemicals in Australian papaya cultivars: the effect of maturity and cooking. Abstract presented (Oral presentation) 37th Annual Scientific Meeting of the Nutrition Society of Australia in conjunction with the Nutrition Society of New Zealand, Brisbane, QLD, Australia, December 4-6, 2013, Australasian Medical Journal 2013, 6, 11: 605. 4. Ikram EHK, Netzel M, Fanning K, Stanley R (2014) Carotenoid and polyphenol content of Carica papaya Linn cultivars. Abstract presented (Oral presentation) AIFST Food Science Summer School 2014, St. Leo’s College, The University of Queensland, St. Lucia, QLD, Australia, February 5-7, 2014, Program & Abstracts. 5. Ikram EH, Netzel M, Fanning K, Stanley R (2014) Phytochemicals in different plant parts of Australian papaya cultivars. Abstract presented (Oral presentation) 29th International Horticultural Congress, Brisbane, QLD, Australia, September 17-22, 2014, Abstract: 01452. 6. Ikram EH, Stanley R, Netzel M, Fanning K (2014) Maximising the potential health properties of papaya fruit and leaf eaten as a food. Abstract presented (poster presentation- finalist) at World Food Day Poster Competition. Global Change Institute Atrium, University of Queensland, St Lucia, Brisbane, Australia. 16 October 2014, Abstract collection. 7. Ikram EH, Stanley R, Netzel M, Fanning K (2014) Getting your fruits and greens from the one tree. Abstract presented (Poster presentation) at QAAFI Annual Research Meeting (ARM). Brisbane Convention Centre, Brisbane, Australia. 25-26 November 2014, Abstract collection. 8. Ikram EH (2016). Innovation of Nutrient dense Papaya Gummies. Gold Award. Abstract

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presented at Invention, Innovation & Design Exposition, Shah Alam, Selangor, Malaysia. 20-23 September 2016, Abstract collection. 9. Ikram EH (2018). DiPPs- Innovation of Dipping Papaya Sauce. Gold Award. Abstract presented at Invention, Innovation & Design Exposition, Shah Alam, Selangor, Malaysia. 24-28 September 2018, Abstract collection.

Contributions by others to the thesis

No contributions by others.

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Statement of parts of the thesis submitted to qualify for the award of another degree

No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects

No animal or human subjects were involved in this research.

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Acknowledgements

I would like to gratefully acknowledge excellent advice, positive criticism, encouragement, kind attention, incredible patience, insight and continuous support from my principal advisor, Dr Michael Netzel and my associate advisors, Professor Roger Stanley and Dr Kent Fanning, during the preparation and completion of this research and thesis. It has been an unforgettable and valuable experience to work with them who helped me to cope with many research and academic difficulties.

I wish to give special thanks to Ministry of Higher Education Malaysia for the scholarship and financial supports and my employer, Universiti Teknologi MARA Malaysia for giving me permission and encouragement to pursue this study.

I am pleased to acknowledge the laboratory staff and Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland for all the administrative and technical support and who have been very helpful in all laboratory works.

Most of all, I am forever indebted to my parents, Mas Zabidah Mas Haris and Khairul Ikram, my beloved husband, Mohd Syrinaz Azli, my daughter, Adelia Nashwa and my son Emil Haqqi, for their endless help, love, understanding, patience and constant encouragement when it was most required. Their presence helped make the completion of this enormous work possible.

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Financial support

The Malaysian government has sponsored my studies through a Malaysian Award Scholarship. Attendance at the Conferences and Seminar was part-funded by Ministry of Health Malaysia and University of Queensland travelling award.

Keywords

Papaya, phytochemicals, carotenoids, antioxidants, ascorbic acid, polyphenols, maturity stages, thermal treatment, in-vitro digestion, bioaccessibility.

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Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 070601, Horticultural Crop Growth and Development, 50% ANZSRC code: 060705, Plant Physiology, 30% ANZSRC code: 070605, Postharvest Horticultural Technologies, 20%

Fields of Research (FoR) Classification

FoR code: 0706, Horticultural Production, 50% FoR code: 0908, Food Sciences, 30% FoR code: 1111, Nutrition and Dietetics, 20%

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Table of Contents

Abstract ...... i

Declaration by author ...... ii

Acknowledgements ...... vii

List of Tables ...... xiii

List of Figures ...... xiv

List of Abbreviations ...... xviii

CHAPTER 1 ...... 1

1. Introduction ...... 1 1.1 Aims, objectives and hypothesis ...... 2 1.1.1 Aims ...... 2 1.1.2 Objectives...... 3 1.1.3 Hypotheses ...... 3

CHAPTER 2 ...... 4

2. Literature Review ...... 4 2.1 Interest in papaya as food and health products ...... 4 2.2 Composition of papaya ...... 6 2.3 Antioxidative capacity and polyphenols in papaya ...... 15 2.4 Health benefits of papaya ...... 24 2.4.1 Anti-inflammatory and immunomodulatory effects ...... 24 2.4.2 Other health benefits ...... 26 2.5 Health Benefits of Fermented Papaya Product (FPP) ...... 28 2.6 Toxicological effects ...... 31 2.7 In vitro digestion studies ...... 33 2.8 Papaya in Australia ...... 35

CHAPTER 3 ...... 38

3. Antioxidant capacity, Vitamins and Polyphenols content of different parts (Flesh and peel) of Papaya Linn cultivars ...... 38 3.1 Introduction ...... 38 3.2 Materials and methods ...... 40 3.2.1 Sample preparation ...... 40 3.2.2 Morphological traits and characteristic of fruits ...... 40 x

3.2.3 Color analyses ...... 41 3.2.4 Chemicals ...... 43 3.2.5 Equipment ...... 43 3.2.6 Carotenoid analysis ...... 44 3.2.7 Ascorbic acid determination ...... 44 3.2.8 Polyphenols ...... 45 3.2.9 Antioxidant capacity (ORAC assay and total phenolics) ...... 45 3.3 Statistical analysis ...... 46 3.4 Results and Discussion...... 46 3.4.1 Morphological characteristic and colour parameters of fruit...... 46 3.4.2 Carotenoids ...... 48 3.4.3 Ascorbic acid...... 52 3.4.4 Polyphenols ...... 56 3.5 ORAC and TPC ...... 64 3.5.1 Provitamin A, AA and polyphenols per serving in fruit ...... 68 3.6 Conclusions ...... 69

CHAPTER 4 ...... 70

4. Ascorbic acid, Carotenoids, Polyphenol and Antioxidant capacity of different parts of C.papaya Linn cultivars: Unripe fruits and leaves and after traditional preparation for culinary uses. 70 4.1 Introduction ...... 70 4.2 Materials and methods ...... 71 4.2.1 Sample preparation ...... 71 4.2.2 Chemicals ...... 73 4.2.3 Equipment ...... 74 4.2.4 Method ...... 74 4.3 Statistical analysis ...... 75 4.4 Results and Discussion...... 76 4.4.1 Ascorbic acid...... 76 4.4.2 Carotenoids ...... 79 4.4.3 Polyphenols ...... 83 4.4.4 ORAC and TPC ...... 88 4.5 Conclusions ...... 91

CHAPTER 5 ...... 92

5. In-vitro Bioaccessibility of phytochemical in Papaya fruits and leaves eaten as food 92 5.1 Introduction ...... 92 5.2 Materials and method ...... 92 5.2.1 Samples preparation ...... 92 5.2.2 In vitro digestion ...... 93 5.2.3 Determination of AA, total phenolics, and carotenoids ...... 94 5.3 Statistical analysis ...... 94 xi

5.4 Results and discussion ...... 96 5.4.1 Release (bioaccessibility) of AA and carotenoids in papaya fruit (%) ...... 96 5.4.2 Release (bioaccessibility) of AA and TPC in papaya leaves (%) ...... 100 5.5 Conclusion ...... 103

CHAPTER 6 ...... 104

6. General conclusions and Future directions ...... 104 6.1 Phytochemical content of Australian papaya cultivars ...... 105 6.2 Phytochemical content of unripe Fruits and Leaves and/or with cooking treatment as per traditionally prepared culinary ...... 106 6.3 Possible route of carotenoids and polyphenols after human consumption...... 106 6.4 Recommendation for Future Studies ...... 106 6.5 Proposed Publications ...... 107 REFERENCES ...... 108 APPENDICES ...... 131

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List of Tables

Table 2.1 Score range of papaya maturity according to skin color when stored under room temperature...... 7

Table 2.2 Proximate composition of papaya pulp, leaves and seeds...... 12

Table 2.3 General composition of FPP (Immun’Age®)...... 13

Table 2.4 Ascorbic acid (mg/100g FW) and provitamin A carotenoids (µg RAE/100g FW) in different types of papaya cultivars and origin...... 14

Table 2.5 Phenolic compounds in different parts of papaya...... 20

Table 2.6 Papaya varieties grown in Queensland...... 36

Table 3.1 Sample Table...... 47

Table 3.2 Morphological traits and characteristics of fruits from different papaya cultivars...... 47

Table 3.3 Skin colour parameters (L:lightness; a and b:chromaticity) of different papaya cultivars stored at 25-260C...... 48

Table 3.4 Ascorbic acid (mg/100g FW) and pro-vitamin A carotenoids (µg RAE/100g FW) in different types of papaya cultivars and origin...... 54

Table 5.1 Release (bioaccessibility) of AA and carotenoids in papaya cultivars...... 98

Table 5.2 Release (bioaccessibility) of AA and TPC in papaya leaves...... 102

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List of Figures

Figure 2.1 Oxidation mechanism of ascorbic acid. Adapted from Khairi (2007)...... 9

Figure 2.2 The sites of the gastrointestinal tract simulated by the developed in-vitro enzymatic digestion and . Adapted from Netzel et al. (2011)...... 34

Figure 3.1 Papaya commercial plantation located at Mareeba, North Queensland, Australia. Source:

Gerard Kath, Lecker Farming...... 41

Figure 3.2 Ripening stages for papaya. From left to right: Stage (1) represents papaya with yellow area on 0–25% of the skin); Stage (2) represents papaya with yellow area on 25–50% of the skin;

Stage (3) represents papaya with yellow area on 50–75% of the skin; Stage (4) represents papaya with yellow area on 75–100% of the skin...... 42

Figure 3.3 Characteristic morphological dimensions of papaya fruit (halved fruit). Adapted from

Schweiggert et al.(2011)...... 43

Figure 3.4 Carotenoid content concentrations [mg/100g FW] in papaya flesh and peels at different maturity stages and cultivars. ß-carotene content (A); ß-cryptoxanthin, (B), and Lycopene (C).

Results are given as mean±SD of 6 independent analysis. Values with different letters are significantly different at p < 0.05...... 51

Figure 3.5 Ascorbic acid [mg/100g FW] content of papaya flesh and peels at different maturity stages and cultivars. Results are given as mean±SD of 6 independent analysis. Values with different letters are significantly different at p < 0.05...... 55

Figure 3.6 Phenolic acids [mg/100g FW] content of papaya flesh at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05...... 59

Figure 3.7 Flavonoids [mg/100g FW] content of papaya flesh at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05...... 60

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Figure 3.8 Phenolic acids [mg/100g FW] content of papaya peels at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05...... 61

Figure 3.9 Flavonoids [mg/100g FW] content of papaya peels at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05...... 62

Figure 3.10 Molecular structure of secondary metabolites analyzed in papaya extract: Gallic acid

(A); Rutin (B); Quercetin (C), and Caffeic acid (D)...... 63

Figure 3.11 Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g

FW) content of Y1B papaya flesh and peel at different maturity stages. Results are given as mean±SD of 6 independent analysis...... 66

Figure 3.12 TPC of papaya flesh and peel at different maturity stages (mg GAE/100g FW). Results are given as mean±SD of 6 independent analysis. Values with different letter are significantly different at p < 0.05...... 67

Figure 4.1: The young and matured papaya leaves ...... 72

Figure 4.2: The separation between the young papaya leaves with the matured leaves ...... 72

Figure 4.3: Sample preparation of leaves and unripe fruits using traditional cooking method...... 73

Figure 4.4 Ascorbic acid (mg/100g FW) content of unripe papaya and thermal treatment (5 min of boiling) at different maturity stages and cultivars. Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05...... 78

Figure 4.5 Ascorbic acid (mg/100g FW) content of raw and thermal treated leaves (2 min of boiling) at different maturity stages and cultivars. Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05...... 78

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Figure 4.6: -carotene (mg/100g FW) content of papaya leaves (Mature and young). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05...... 81

Figure 4.7: lutein (mg/100g FW) content of papaya leaves (Mature and young). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05...... 81

Figure 4.8. Carotenoid profiles of Mature leaves (A) and shoots (B). Rt and Mass spectra were compared with that of the standard compounds 1; lutein 2: Chlorophyll b; 3: Chlorophyll a; 4: 13- cis-b-carotene; 5: all trans-b-carotene; 6:9-cis-b-carotene ...... 82

Figure 4.9: Polyphenols compounds of papaya shoots and boiled shoots (BS) (mg/100g FW).

Results are given as mean±SD of six measurements. Analysed by HPLC ...... 85

Figure 4.10 Polyphenols compounds of papaya mature leaves (ML) and boiled mature leaves

(BML) (mg/100g FW). Results are given as mean±SD of triplicate measurements. Analysis was determined by HPLC ...... 86

Figure 4.11 Analyses of polyphenols in the leaves extracts of papaya, using HPLC. Mass spectra were compared with that of the standard compounds. 1: gallic acid;2: Gallic acid; 3: Chlorogenic acid; 4: Chlorogenic derivatives; 5:Quercetin derivatives; 6: Rutin; 7: Ferulic acid; 8,12: Quercetin derivatives; 9: p-coumaric acid; 10: Caffeic acid; 11: Quercetin; 13: Apigenin; 14: Kaempferol. ... 87

Figure 4.12: Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g

FW) content of RB4 and RB2 papaya pulp, boiled and peel (n=6). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05. 89

Figure 4.13: Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g

FW) content of RB1 and Y1B papaya pulp, boiled and peel (n=6). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05. 89

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Figure 4.14: Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g

FW) content of mature leaves and shoots. Results are given as mean±SD of six measurements.

Values with different lettersare statistically significant at the level of p < 0.05...... 90

Figure 4.15: TPC of papaya flesh and leaves (mg GAE/100g FW). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05. 90

Figure 5.1: In Vitro digestion Model ...... 95

Figure 5.2 Carotenoids compounds profile comparison pre and post digestion for Red-fleshed RB4

Stage 3. Rt and Mass spectra were compared with that of the standard compounds 3: β- cryptoxanthin; 5: β-carotene; 6: lycopene ...... 99

Figure 5.3 The path of polyphenols after ingestion. Adapted from Barros and Junior (2019) ...... 101

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List of Abbreviations

% Percentage µm Micrometre Abs Absorbance C* Chroma variable of colour Cm Centimetre DW Dry weight basis FW Fresh weight basis G Gram H Hour HPLC High performance liquid chromatography Kg Kilogram L Litre L* Lightness variable of colour M Metre M Molar Mg Milligram Min Minute mL Millilitre Mm Millimetre mM Millimolar RH Relative humidity ROS Reaction oxygen species Rpm Revolutions per minute Μg Microgram μL Microliter C.papaya Carica Papaya AA Ascorbic acid Β Beta Α Alpha

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TPC Total Phenolic content FPP Fermented papaya product AOC Antioxidant Capacity DHAA dehydroascorbic acid BITC benzyl isothiocyanate AAPH fluorescein disodium salt, 2,2’-Azobis(2 methylpropionamidine) dihydrochloride BHT butylated hydroxy toluene

Lmax Maximum length

Dmin minimum diameter

Dmax maximum diameter ORAC oxygen radical absorbance capacity H-ORAC hydrophilic oxygen radical absorbance capacity L-ORAC Lipophilic oxygen radical absorbance capacity RB1 Red-fleshed hybrid 1 cultivar RB2 Red-fleshed hybrid 2 cultivar RB4 Red-fleshed hybrid 4 cultivar Y1B Yellow-fleshed hybrid 1B yellow cultivar Stage (1) Represents papaya with yellow area on 0–25% of the skin. Stage (2) Represents papaya with yellow area on 25–50% of the skin. Stage (3) Represents papaya with yellow area on 50–75% of the skin. Stage (4) Represents papaya with yellow area on 75–100% of the skin. ND Not detected NA Not available

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CHAPTER 1

1. Introduction

Carica is a genus of flowering plants and a tree-like herbaceous plant which belongs to the small family of Caricaceae (Samson, 1986). Caricaceae was thought to comprise 31 species in three genera (namely Carica, Jacaratia and Jarilla) and one genus, Cylicomorpha, from equatorial Africa (Nakasone & Paull, 1998). However, recent genetic evidence has resulted in the genus being restricted to single species of papaya, widely known as papaya or paw paw. Accordingly, the classification has been revised to comprise Cylicomorpha and five South and Central American genera (Carica, Jacaratia, Jarilla, Horovitzia and Vasconcella) in the Caricaceae (Badillo, 2002, 1971). Some of the species that were formerly known under Carica have been classified to the genus of Vasconcellea (Badillo, 2002) such as Vasconcellea cundinamarcensis formerly known as Carica vasconcellea or mountain papaya and Vasconcellea ×heilbornii formerly known as Carica pentagona or babaco. To date, only these three type of plants under the family of Caricaceae are known to be used as food or medicinal remedy. There are a number of different cultivars of papaya around the world. Examples are 'Kapoho Solo' or 'Puna Solo', 'Dwarf Solo', 'Waimanalo', 'Higgins', 'Wilder', 'Hortus Gold', 'Honey Gold', 'Bettina', 'Petersen', 'Improved Petersen', 'Hybrid No. 5', ‘Hybrid 1B’, ‘Hybrid 11B’, ‘Hybrid 13’, ‘Hybrid 14’, ‘Hybrid 29’, ‘Richer Gold’, ‘PG Lines’, ‘Petersen’, ‘Improved Petersen’, ‘Sunnybank’, ‘Airline/57’, ‘OE’, ‘Yarwun Yellow’, ‘Guinea Gold’ and ‘NT Red’ . (Agrifutures, 2019; Australian Papaya Industry Association, 2008; Morton, 1987; Nafiu, Alli-Oluwafuyi, Haleemat, Olalekan, & Rahman, 2019; Papaya Australia, 2019).

The market value of papaya fruit is very much dependent on its taste, structure and texture. Ripe fruit can be fragile, without a long shelf life and messy to eat. However it is proven to have a high ascorbic and carotenoid content (Hernández, Lobo, & González, 2006; Wall, 2006). The over ripe stage is commercially unmarketable. Unripe fruit, is also not marketable in Australia, but in Asian countries it is used in salads and cooking. Unripe fruit contains high carbohydrate and starch levels (Oloyede, 2005). Present study discussed on the effect of fruit ripening and processing (raw versus boiled) of the unripe fruit and leaves of papaya. Heat is known to improve availability of some nutrients, inactivate enzyme activities that speed up nutrient damage, destroy undesirable microorganisms and food contaminants as well as favourably change the physical attributes of food (Adams, 1991; Bakshi & Masoodi, 2010; Cortés, Esteve, Rodrigo, Torregrosa, & Frígola, 2006; Gonçalves, Pinheiro, Abreu, Brandão, & Silva, 2010). Nevertheless, loss of nutrients is also one of the undesirable changes in food processing and should be tested. 1

For the phytochemicals to fullfil their health-promoting effects, they must be bioavailable in the body once administered. The bioavailability of a nutrient is the fraction available for use by the systemic circulation. Not all phytochemicals are likely absorbable and useable in humans upon ingestion. Changes are expected to occur in the concentrations and forms as a result of the physiochemical effects of the digestion and absorption processes in the stomach and small intestine. In vitro enzymatic digestion processes are methods that are widely used to study the gastro- intestinal behaviour of food or pharmaceuticals. The in vitro enzymatic digestion models mimic conditions and luminal reactions which occur in the mouth, stomach, small intestinal and occasionally large intestinal fermentation. Although human nutritional studies are still being considered the “gold standard” for addressing diet-related questions, in vitro methods have the advantage of being more rapid, less expensive, less labour intensive without ethical restrictions. So far, there is limited and contradictory information on the bioavailability of papaya. This information is essential to have some estimation of bioavailability of papaya for human consumption. For marketing strategy, this may open the door to the aiming of potential papaya cultivars/hybrid.

1.1 Aims, objectives and hypothesis

1.1.1 Aims

The aim of this study was to carry out a phytochemical and antioxidant capacity (AOC) of commercially available Australian papaya cultivars, focusing on maturity stages and different parts of the plant (pulp, peel and leaves). Although there have been similar studies conducted on the different types of papaya cultivars mesocarp (edible portion) (Gayosso-García Sancho, Yahia, & González-Aguilar, 2011; Marelli de Souza, Silva Ferreira, Paes Chaves, & Lopes Teixeira, 2008; Salinas, Hueso, & Cuevas, 2019; R.M. Schweiggert, Steingass, Heller, Esquivel, & Carle, 2011; Wall, 2006), there is still a gap on phytochemical content of papaya flesh from cultivars planted in Australia. Moreover, the analytical attention of also focusing on other parts of the plant such as the peel and the leaves and measuring bioavailability will extend the knowledge of what is already in the literature. The study will also look into the effect of food processing (raw versus boiled) of the unripe fruit and leaves of papaya. The information gathered will then be used to understand the effect of maturity, types of cultivars, traditional cooking practices and gastrointestinal digestion processes on phytochemical (Vitamins, AOC and polyphenols) concentrations. These in turn will be used to estimate the amount of nutritional benefits that can be gained from consuming papaya. 2

1.1.2 Objectives

The objectives of the present work are to:

i. Identify and quantify the phytochemical (Vitamins and polyphenols) and AOC contents and in vitro antioxidant activities of selected commercially available Australian papaya cultivars; ii. Investigate the effect of different papaya cultivars/varieties, parts of the plant (pulp, peel and leaves) and maturity stages on phytochemical (Vitamins and polyphenols) composition and AOC; iii. Determine the effect of in vitro digestion on the stability of vitamins and polyphenols; iv. Better or improved understanding of the nutritional quality of papaya.

1.1.3 Hypotheses

The general hypotheses for this research work are that Australian papaya cultivars are a good source of phytochemicals compounds and are highly bioaccessibility in the human gastrointestinal tract.

The introductory section (Chapter 1) of this thesis provides a comprehensive overview into the PhD project with the hypothesis and aims. Literature review (Chapter 2) provides a summary of the current scientific knowledge of papaya along with plant parts and its health promoting properties.

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CHAPTER 2

2. Literature Review

2.1 Interest in papaya as food and health products

Papaya originated from lowland of eastern central America (Nakasone & Paull, 1998). The genus name Carica is derived from the Latin word which means a fig because of similarity in shape of the leaves and the fruits of papaya to those of figs. The plant can now be found and grown in all tropical countries and many sub-tropical regions of the world. Among a wide array of tropical fruits, papaya is deemed to be one of the most economical fruits, which not only is cultivated roughly in 60 countries but also is marketed worldwide (Hardur Venkatappa Annegowda & Bhat, 2016).

Among plant-derived foods, papaya is known as the ‘fruit of the angels’ or ‘the most magnificent fruit in the world’ (Singh, 2012). Almost all parts of the plant are used for either food, beauty or health products. Papaya has been cultivated for its edible fruits for hundreds of years but it is now also used for the production of jams, preserves, soft drinks, ice-cream, cocktail, crystallized fruit and canned in syrup (Ezike, Akah, Okoli, Ezeuchenne, & Ezeugwu, 2009; Nafiu et al., 2019; Villegas, 1997; Workneh, Azene, & Tesfay, 2012). The fruits are widely used as a treatment of paediatric burns in Africa (Starley, Mohammed, Schneider, & Bickler, 1999). The green mature fruit, or so called unripe fruit, is eaten as a vegetable in some Asian countries, usually after cooking or boiling. It is used as a substitute for marrow and applesauce (Villegas, 1997). Apart from that, it is traditionally used to treat skin problems and is forbidden for consumption during pregnancy (Amenta, Camarda, Di Stefano, Lentini, & Venza, 2000; Anuar, Zahari, Taib, & Rahman, 2008; Fasihuddin & Ghazally, 2003). In South-East Nigeria, extracts of the unripe fruit including the seed were taken daily for management of ulcers for which it is claimed to be highly effective (Ezike et al., 2009). In Philippines, the fruit is regarded as a medicinal fruit and fermented for use as food (Priscilla, 2008; Quisumbing, 1978). The fermented fruit has been commercialized as a health product in Japan since the 19th century (Imao, Wang, Komatsu, & Hiramatsu, 1998). To date there is one product known as Fermented Papaya Preparation (FPP) that is commercialized in the United States and some European countries.

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The leaf of the plant was reported as being consumed by people living on the Gold Coast of Australia for its purported anti-cancer activity (Harald, 2003). In addition, the leaf extracts have also been used for a long time by indigenous people as a remedy for various disorders, including cancer and infectious diseases. In Malaysia, young papaya leaf are consumed for treating diabetes and high blood pressure (Ong & Norzalina, 1999) whereas in Nigeria, the fruits and roots are used in the management of diabetes mellitus (Abo, Fred-Jaiyesimi, & Jaiyesimi, 2008). The seeds of the plant are normally discarded and not eaten due to their spicy and pungent flavour and are believed to have antifertility and contraceptive effects. However, in Nigeria, the seed is also used in the production of a fermented condiment called ‘daddawa’, which is used as food seasoning (Mansurah A. Abdulazeez & Sani, 2011; Dakare, Ameh, & Agbaji, 2011).

The market value of papaya fruit is very much dependent on its taste, structure and appearance. Ripe fruit can be fragile, with a short shelf life and messy to eat. However, ripe fruit have a high antioxidant activity due to mainly to the content of ascorbic acid and carotenoids (Hernández et al., 2006; Wall, 2006). Unripe fruit on the other hand is not marketable in countries like Brazil and Australia, but in Asian countries, it is used in salads and in cooking. Unripe fruit contains high levels of carbohydrate in the form of starch (Oloyede, 2005).

There are numerous commercially available papaya products such as beauty products, skin treatment cream and health supplements. Many studies have been published on the antioxidant and health properties of the plant. In a review by Krishna, Paridhavi, and Patel (2008), the authors briefly discussed the nutrient, uses and medicinal value of the papaya plant. However, there is a lack of information on its antioxidative value and health benefits that is based on scientific research. Moreover, de Oliveira and Vitoria (2011), in their review, focused on the loss in quality of the fruits due to physiological disorders that occur postharvest. They also briefly explored the nutritional and pharmacological characterization of the fruits.

This literature review focuses on the possible health benefits, nutritional quality and potential medical and toxicological effects as they pertain to different parts of the papaya plant and the antioxidant properties and vitamins composition during ripening in relation to the different cultivars.

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2.2 Composition of papaya

Researchers have found that differences in cultivars, growing location, sunlight exposure, agricultural practices, stage of ripeness and postharvest handling have significant effects on the chemical composition of the fruits (Hardur Venkatappa Annegowda & Bhat, 2016; De Rosso & Mercadante, 2005; Gayosso-García Sancho et al., 2011; Kimura, Rodriguezamaya, & Yokoyama, 1991; Ornelas-Paz, Yahia, & Gardea, 2008; Wall, 2006). For instance, the progression of fruit through different maturity stages results in physiological and biochemical changes that modify fruit composition and enables its consumption (Pereira et al., 2009). The loss of fruit firmness is a consequence of changes in plant cell wall constituents that lead to weak cell-to-cell links and thus lose of rigidity and firmness. The softening indicates ripening. These changes can be observed in papaya fruit tissue by means of microscopic examination (Pereira, et al., 2009). For commercial production papaya fruit maturity is often based on the changes of skin colour of the fruits (Table 2.1).

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Table 2.1 Score range of papaya maturity according to skin color when stored under room temperature.

Maturity Score range a Score range b stage 0 Fruit completely developed. 100% Fruit completely developed. 100% green green skin color. skin color.

1 Yellow color does not cover more Represents papaya with yellow area on 0– than 15% of the fruit surface. 25% of the skin.

2 1/4 mature. Fruit with up to 25% of Represents papaya with yellow area on 25– the surface yellow, surrounded by 50%. light green color.

3 1/4 mature. Fruit with up to 50% of Represents papaya with yellow area on 50– the surface yellow, surrounded by 75%. light green color.

4 3/4 mature. Fruit with 50–75% of Represents papaya with yellow area on 75– the surface yellow, surrounded by 100%. light green color.

5 Mature. Fruit with 76–100% of the - surface yellow. Only area near the stem is green. aAdapted from the export program of Brazilian Papaya. Ministry of Agriculture, Livestock, and Environment (Pereira et al., 2009). bSantamaría-Basulto et al. (2009)

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Papaya fruits have a good nutritional health profile being an excellent source of provitamin A and ascorbic acid. They rank as one of the top fruit for ascorbic acid content. There is no significant variation of the ascorbic acid content of different cultivars of the fruits but there is a large variation in the provitamin A content between the red and yellow-fleshed papaya cultivars (Chandrika, Jansz, Wickramasinghe, & Warnasuriya, 2003; Wall, 2006) as well as a variation due to the level of ripeness of the fruit (Gayosso-García Sancho et al., 2011) (Table 2.4). The ripening process begins when the chlorophyll is degraded, which coincides with carotenoid synthesis and results in significant colour changes from green to yellow-orange colour. Moreover, during ripening, the content of esterified carotenoids increases, which allows esterified carotenoids to integrate more quickly into the membranes. This in turn increases the color of the fruit and its accumulation in chromoplasts (Andersson, Olsson, Johansson, & Rumpunen, 2008; Yahia & Ornelas-Paz, 2010). Many tropical fruits such as mango have similar behaviour as papaya in that the colour is conferred by the carotenoid level. Carotenoids like β-cryptoxanthin, and β-carotene are found in all types of papaya cultivars. However, contradictory results are found on lycopene availability in yellow- fleshed fruits. Lycopene in yellow-fleshed fruits was not detected or found in a low amount compared with the red-fleshed fruits. (Gayosso-García Sancho et al., 2011; Marelli de Souza et al., 2008; Wall, 2006). Colour intensity resulting from carotenoid content plays a vital role in fruit acceptability by consumers (Yahia & Ornelas-Paz, 2010). Red-fleshed cultivars such as ‘Solo’ often outweigh the yellow-fleshed fruits in consumer preference. However, the former has excellent flavor but very poor shape and appearance, whereas the latter has very good appearance (Drew, 2005).

Ascorbic acid (AA) is widely distributed in plant cells where it plays many crucial roles in growth and metabolism. As a potent antioxidant, AA has the capacity to eliminate several different reactive oxygen species, keeps the membrane-bound antioxidant a-tocopherol in the reduced state, acts as a cofactor maintaining the activity of a number of enzymes by keeping metal ions in the reduced state, appears to be the substrate for oxalate and tartrate biosynthesis and has a role in stress resistance (Arrigoni & De Tullio, 2002). AA is known as a reducing agent or an electron donor. Being an electron donor, the lose of an electron forms an intermediates species called semi dehydroascorbic acid before spontaneously converting to dehydroascorbic acid (DHAA) (Figure 2.1). Due to its equivalence in biological activity to AA, recent studies often consider the sum of DHAA and AA as vitamin C activity (Martí, Mena, Cánovas, Micol, & Saura, 2009). Papaya is a rich food source of AA with an average range of between 45-60 mg/100g, with the highest reported

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value of 154 mg/100g (Table 2.4). Most studies have shown no significant difference in the AA content of papaya between cultivars (Tripathi et al., 2010; Wall, 2006). However, the AA content measured depends on the availability of light to the crop and to individual fruits and also the methods used to determine the AA content. The preferred choice for AA determination are separation techniques: capillary electrophoresis (Y. Tang & Wu, 2005; Versari, Mattioli, Parpinello, & Galassi, 2004), gas chromatography (F. O. Silva, 2005) and liquid chromatography (Nováková, Solich, & Solichová, 2008). Determination of actual dehydroascorbic acid (DHAA), an oxidized form of AA has not always been done due to the analytical challenge (Nováková et al., 2008). Usually, DHAA is determined as the difference between the total AA after DHAA reduction and the AA content of the original sample prior DHAA reduction.

Figure 2.1 Oxidation mechanism of ascorbic acid. Adapted from Khairi (2007).

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There are very few available reports on vitamin E content of papaya. Food composition databases from countries like US, Japan and Malaysia indicate 0.3 mg/100g value of vitamin E content for papaya (Hagiwara, 2001; Tee, Noor, Azudin, & Idris, 1997; USDA, 2015). However, Charoensiri and co-workers, in their study on vitamin content of selected Thai fruits, found no detectable value of vitamin E content in papaya (var Khakdahm) fruits (Charoensiri, Kongkachuichai, Suknicom, & Sungpuag, 2009).

Papaya is one of the few examples known of a plant containing both glucosinolates and cyanogenic glucosides (Bennett, Kiddle, & Wallsgrove, 1997; Olafsdottir, Bolt Jørgensen, & Jaroszewski, 2002). Generally, plants producing glucosinolates are not cyanogenic, however, a few plant species have been positively identified as containing both glucosinolates and cyanogenic glucosides. Benzylglucosinolate was found to be detected in all of the tissues of papaya (Aruna & Subrata, 2008; Jiao, Deng, Li, Zhang, & Cai, 2010; Tripathi et al., 2010). Nonetheless, in papaya fruits, the amount detected was very low compared to other parts of the plant such as leaves and seeds (Bennett et al., 1997; MacLeod & Pieris, 1983). In addition, the leaves also contain alkaloids (including carpain and pseudocarpain), enzymes (papain, chymopapain, cystatin), tocopherol, ascorbic acid, flavonoids, tannins, nicotinic acid, saponins and phenolics (Bennett et al., 1997; Duke, 2011; Seigler, Pauli, Nahrstedt, & Leen, 2002; C. S. Tang, 1979; Tee et al., 1997). The proximate composition of papaya is shown in Table 2.2.

Papaya fruit pulp contains nonvolatile organic acids such as citric, fumaric, malic, malonic, succinic, and tartaric acids (pH 4.5–5.9). Furthermore, the level of organic acids fresh weight (FW) in ripe papaya (per 100mg) contains the following: citric acid, 335mg; l-malic acid, 209mg, succinic acid, 52mg, quinic acid, 52mg, tartaric acid, 13mg, oxalic acid 10mg, and fumaric acid, 1.1mg (Hernández, Lobo, & González, 2009; Spínola, Pinto, & Castilho, 2015).

The seeds represent a rich source of biologically active isothiocyanate (Nakamura et al., 2000; Nakamura et al., 2007). Studies have confirmed that benzyl isothiocyanate (BITC) is the predominant compound in papaya seed extracts, regardless of cultivar (Duke, 2011; Kermanshai et al., 2001; Wilson, Kwan, Kwan, & Sorger, 2002). Several epidemiological studies have indicated that the dietary consumption of isothiocyanates or isothiocyanate containing foods inversely correlates with the risk of developing cancers and potential evidence of cancer prevention in humans (Cavell, Syed Alwi, Donlevy, & Packham, 2011; Hwang & Lee, 2006; Seow et al., 2002). In contrast, there are also many studies published on the toxicological effects of the seeds and its 10

contraceptives properties, especially to men. More research is required on the health benefits and toxicology of the seeds for human consumption. The seed was also high in lipid content (Table 1.2), with oleic acid (77.7%) as the predominant fatty acid (Dakare et al., 2011).

Fermented papaya is used as a health product. A commercial Fermented Papaya Preparation (FPP) is produced as a white granular food supplement product by yeast fermentation of non-genetically modified papaya. It has been sold as a natural functional healthy food in Japan and other countries. Nutritional analysis reveals that FPP (Immun’ Age®) has amino acids and carbohydrates (Osato Research Institute, 2003) (Table 2.3). Fermented papaya seed was also shown to have essential amino acids, protein (24%) and lipids (54%). Moreover, fermentation reduced the level of antinutritional factors of the seeds. Oxalate reduced from 210.1 to 40.2 mg/100g, phytic acid from 102.0 to 68mg/100g, tannin from 15.5 to 8.3 mg/100g and typsin inhibitor from 2431.2 to 63.0 mg/100g (Dakare et al., 2011).

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Table 2.2 Proximate composition of papaya pulp, leaves and seeds.

Proximate composition Moisture Carbohydrate Proteina Lipid Fiber References (per 100 g) (per 100 g) (per 100 g) (per 100 g) (per 100 g)

Pulp Mature green fruit 88.07-88.5 14.9-17.9 0.41-1.14 0.11-0.5 1.05-1.3 Bari et al. (2006) 87.10-87.7 N.A. 0.74-0.83 <0.10-0.16 0.63 Tripathi et al. (2010)

Ripe 88.06 10.82 0.47 0.26 1.7 USDA (2015) 92.70-94.41 20.3-23.5 1.9-2.01 0.27-0.37 0.90-1.01 Bari et al. (2006) 85.00-86.60 N.A. 0.70-0.84 0.49-0.58 0.49-0.576 Tripathi et al. (2010)

Leaves 83.30 8.2 5.6 0.4 1.0 Tee et al. (1997)

Seeds 4.93-6.2 6.21-14.55 27.8-30.54 28.3-60.41 22.6-60.72 Marfo, Oke, and Afolabi (1986a, 1986b); Passera and Spettoli (1981); Dakare et al. (2011)

a The nitrogen to protein conversion factor was 6.25. N.A: Not available

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Table 2.3 General composition of FPP (Immun’Age®). Component Level per 100g FPP® Carbohydrateb 90.7g Proteina 0.3g Dietary fiber N.D. Fat N.D Vitamin B6 17μg Folic acid B9 2μg Niacin 240μg Calcium 2.5mg Iron 0.29mg Potassium 16.9mg Magnesium 4.6mg Zinc 75μg Copper 14μg

Amino acids Arginine 16mg Lysine 6mg Histidine 5mg Phenylalanine 11mg Tyrosine 9mg Leucine 18mg Isoleucine 9mg Methionine 5rng Valine 13mg Alanine 12mg Glutamic acid 37mg Serine 11mg Threonine 8mg Aspartic acid 27mg Tryptophan 2mg The nitrogen to protein conversion factor was 6.25. bThe formula used was 100−(moisture + protein + fat + ash) ND: Not detected Source: Osato Research Institute (2003)

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Table 2.4 Ascorbic acid (mg/100g FW) and provitamin A carotenoids (µg RAE/100g FW) in different types of papaya cultivars and origin.

Papaya Cultivars Source AA (mg/100g) Provitamin A References (µg RAE/100g)a Kapoho Hawaii 45.4 29.9 Wall (2006) Laei Gold Hawaii 51.3 48.2 Wall (2006) Rainbow Hawaii 51.8 50.3 Wall (2006) Rainbow Hawaii 57.4-84.9 N.A Tripathi et al. (2011) Rainbow (Hybrid line) Hawaii 43.6-75.9 N.A Tripathi et al. (2011) Sunrise and Kp hybrid Hawaii 68.3-84.9 87.6-262 Tripathi et al. (2011) Sunrise Hawaii 55.6 45.6 Wall (2006) Sun Up Hawaii 45.3 20.4 Wall (2006) Pococi Costa Rican Costa Rica N.A. 132-166 Schweiggert, Steingass, Mora, Esquivel, and Carle (2011) Red lady Florida 153.8 N.A. Mahattanatawee et al. (2006) Maradol Mexico 25.1-58.6 30.84-85.8 Sancho et al. (2011) Local 1 Yellow fleshed Sri Lanka N.A. 75.84 Chandrika et al. (2003) Local 2 Red fleshed Sri Lanka N.A. 152.92 Chandrika et al. (2003) Local 1 Bangladesh 8.09-31.0 20.8-33.8 Bari et al. (2006) Local 2 Bangladesh 7.3-29.4 16.7-32.5 Bari et al. (2006) Honey dew India N.A. 58.3 b Veda et al.(2007) Surya India N.A. 60.8 b Veda et al. (2007) Solo Malaysia 48.5 42.4 Isabelle, et al. (2010) Solo Malaysia 67.8 N.A. Leong and Shui (2002) Foot Long Malaysia 45.2 N.A. Leong and Shui (2002) Solo Brazil 101.4-112.4 27.5-40c Marelli de Souza et al.(2008) Formosa Brazil 59.9-77.8 15.8-46.7c Marelli de Souza et al.(2008) N.A. Brazil N.A. 168 c* Silva et al. (2014) BaiXinho do Santa Spain 98-154 N.A. Hernández, Lobo, and Amalia González (2006) Local Italy 88.2 N.A. Vinci et al. (1995) Oblong Red Nigeria 41.1 310.3c Nwofia, Ojimelukw, and Eji Pear Orange Nigeria 39.3 305.6c (2012) Round orange Nigeria 39.9 309.3c Nwofia et al. (2012) Oblong yellow Nigeria 36.4 258.7c Nwofia et al. (2012) Round yellow Nigeria 43.4 293.7c Nwofia et al. (2012) Nwofia et al. (2012) N.A. N.A. 60.9 47.0 USDA (2015) aProvitamin A carotenoids include ß-carotene, α-carotene, and ß-cryptoxanthin. Retinol Activity Equivalents (RAE): (µg ß-carotene/ 12) + (µg α-carotene/ 24) + (µg ß-cryptoxanthin/ 24) bRAE was calculated based on stage (3) and (4) fruits. cRAE was calculated based on ß-carotene value only. *based on dry weight N.A.: Not Available

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2.3 Antioxidative capacity and polyphenols in papaya

Plants contain a large variety of substances called phytochemicals that possess antioxidant activity. Some of the compounds that exhibit high antioxidant capacity (AOC) include vitamins and polyphenol compounds. Most studies have shown that papaya has a high AOC in comparison with other fruits (Faller & Fialho, 2010; Isabelle et al., 2010; Leong & Shui, 2002; Lim, Lim, & Tee, 2007; Mehdipour et al., 2006; Osato, Santiago, Remo, Cuadra, & Mori, 1993). However, there are some studies that detected a low AOC in comparison to other fruits (Patthamakanokporn, Puwastien, Nitithamyong, & Sirichakwal, 2008; Stangeland, Remberg, & Lye, 2009).

Due to the lack of a standard method for AOC, it is difficult to compare the results reported from different research groups. Several reviews have been published, and the opinions vary considerably (D. Huang, Ou, & Prior, 2005; Prior, Wu, & Schaich, 2005; Sánchez-Moreno, 2002). However, for botanical samples or juices, the most commonly used methods used for AOC are 2,2-di(4- tertoctylphenyl)-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and total radical-trapping antioxidant parameter (TRAP). In the case of AOC of papaya plant, DPPH seems to be the most prominent method used for majority researchers. However, the reason for this maybe because this method is easy, relatively quick and suitable for fruit and vegetables juices or extracts (Sánchez-Moreno, 2002). In contrast, Huang and colleagues (2002) in their review indicated that DPPH assay was much less chemically sound as a valid assay for antiradical activity measurement due to its reaction with other compounds. However, there are pros and cons of all these AOC assays and different methods are needed to fully evaluate and compare any one product.

Lim and co-workers (2007) observed that the papaya fruit variety ‘Solo’ was a very potent radical scavenger with a low IC50 (the amount of sample extracted into 1 ml solution necessary to decrease by 50% the initial DPPH concentration) of 3.5 mg/ml. This result was supported by other researchers, who found similar results of high radical scavenging activity. Scavenging activity of papaya fruits were found to be higher compared to other fruits like mango, tangerine, apple as well as standard vitamin E using the same method (Faller & Fialho, 2010; Mehdipour et al., 2006). On the contrary, using a ferrous ion-chelating capacity method the peel showed relatively low radical scavenging activity but much higher ability to chelate pro-oxidant metal ion (Matsusaka & Kawabata, 2010). Reports on AOC of non-edible parts of the plant such as the peel, seeds and leaves are very rare in the literature, thus the AOC of these parts could not be confirmed from reported studies. .

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Leong and Shui (2002) examined the AOC of the papaya varieties ‘Solo’ and ‘Foot long’ papaya using two methods: DPPH and ABTS free radical decolorization assay. They found that AA contributes the most to the antioxidant properties of the fruits with a contribution percentage of 48% for ‘Solo’ and 62.3% for ‘Foot long’ papaya. The result was later confirmed by Isabelle and co- workers (2010) who found that AA contributes to 97% of AOC of the fruits. However, the result was only based on the hydrophilic ORAC (H-ORAC) method which found a relatively low AOC level compared to other tropical fruits. This result was supported by Patthamakanokporn, et al. (2008) who found a relatively low AOC using the same method. On the other hand, the lipophilic antioxidant content of the fruit was found to be relatively high, probably due to the high amounts of ß-cryptoxanthin, lycopene and ß-carotene (Isabelle et al., 2010).

A low AOC was found in papaya fruits using the FRAP method (Patthamakanokporn et al., 2008; Stangeland et al., 2009). The FRAP method is an electron transfer-based antioxidant capacity assay and the mechanism is quite similar to the Folin Ciocalteu method (Singleton, Orthofer, & Lamuela- Raventós, 1999) used to determine the ‘total phenolic content’. Numerous publications have applied the total phenolic content assay using the Folin Ciocalteu reagent and an electron transfer-based antioxidant capacity assay (such as the FRAP). They have often found excellent positive correlations between the total phenolic content and AOC (D. Huang et al., 2005; Netzel, Netzel, Tian, Schwartz, & Konczak, 2006; Prior et al., 2005). This is expected because the chemistry of these assays is quite similar.

The AOC level in FPP (PS-501®) was found to be high with an IC50 value of 12.5mg/ml (Imao et al., 1998). In this study, FPP had the ability to scavenge 80% of hydroxyl radicals generated by Fenton reagents and 50% lipid soluble radicals and was dose-dependent in the range of 5-50mg/ml by DPPH method. Moreover, oral administration of the FPP for 4 weeks was found to decrease the elevated lipid peroxide levels in iron-injected cortex of rats (Imao et al., 1998). The result of this study was further confirmed by Noda and co workers (2008), using the same product (PS-501). FPP was found to have an IC50 value of 8mg/ml which inhibits hydroxy radical generation from methylguanidine, a type of neurotoxin/nephrotoxin which generates reactive oxygen species (ROS) (Noda et al., 2008). Thus, FPP may have a beneficial effect in reducing ROS as well as preventing methylguanidine related diseases (Noda et al., 2008). In contrast, Calzuola and colleagues (2006) showed that FPP (Immun’Age®) had lower AOC levels compared to wheat sprouts, white tea and Morinda citrifolia extracts. The result indicated that 1g of FPP could only reduce potassium ferricyanide reagent by 1.05±0.09 µmol compared with standard compound of vitamin C (4.8  0.7 µmol of reduced ferricyanide/mg compound), rutin (3.8  1.2 µmol of reduced ferricyanide/mg 16

compound) and quercetin (4.8  1.7 µmol of reduced ferricyanide/mg compound), respectively (I. Calzuola et al., 2006; Isabella Calzuola, Marsili, & Gianfranceschi, 2004). However, the discrepancy with the results of the AOC observed in these studies could be restricted to the method used, and the type and concentration of the sample. In addition, a study by Bolling et al. (2012) found that dilution factors may also affects the AOC of the sample. In general, for routine determination of AOC in extracts or beverages is normally diluted in a buffer or solvent in order to be within the linear standard graph. However, in their study, it is found that increased dilution factor resulted in higher AOC in pomegranate and grape juice studied. Therefore, dilution factors should be considered in AOC assays for more accurate AOC interpretation (Bolling et al., 2012).

Fruits and vegetables contain many types of phytochemicals, of which many such as vitamin C, vitamin E and carotenoids are antioxidant compounds, (Prior et al., 1998). However, polyphenol compounds, such as flavonoids, procyanidins and phenolic acids also contribute to the beneficial effects of this group of foods (Del Rio, Costa, Lean, & Crozier, 2010; Fraga, Galleano, Verstraeten, & Oteiza, 2010; Petti & Scully, 2009). Polyphenols are the most abundant bioactive compounds in our diet, and their content is much higher than that of all other classes of phytochemicals and known dietary antioxidants. Their total dietary intake could reach as high as 1 g/day, which is approximately 10 times higher than the vitamin C intake (Scalbert, Johnson, & Saltmarsh, 2005). However, the polyphenolic composition in papaya pulp had not been unambiguously determined until Gayosso- García Sancho et al., (2011), recently listed and quantified some of the major polyphenols compounds found in papaya pulp of ‘Maradol’ cultivars. They were ferulic acid, p- coumaric acid, and caffeic acid (Table 1.5). The result coincides with the first report on ‘Maradol’ cultivars which found a similar profile pattern of phenolics compounds (Rivera-Pastrana, Yahia, & Gonzalez-Aguilar, 2010) but the concentration was very different (Table 1.5). Other researchers suggested only low amounts or traces of phenolic compounds in papaya pulp or only identified compounds without quantifying (Franke, Custer, Arakaki, & Murphy, 2004; Jindal & Singh, 1975; Lako et al., 2007; Simirgiotis, Caligari, & Schmeda-Hirschmann, 2009) (Table 1.5). The low content and number of polyphenols in papaya pulp found in all these studies could explain the limited data for polyphenols in papaya pulp.

Total phenolic content in papaya pulp, determined using the standard Folin Ciocalteu’s method (Singleton et al., 1999) was found to be quite low, ranging from 0.02-2.08 (mg GAE/100g fresh weight (FW)) (Faller & Fialho, 2010; Isabelle et al., 2010). The low value of total phenolics perhaps explained the low AOC using the FRAP method as discussed earlier. Nevertheless, some

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researchers had found moderate levels of total phenolics in this fruit, with the highest value of 54 mg GAE/100g FW (Lako et al., 2007; Patthamakanokporn et al., 2008) (Table 2.5). The values of total phenolics using the Folin Ciocalteu’s reagent may possibly result from the formation of blue- molybdenum-tungsten complex from AA in the fruits rather than from the phenolics compounds. It is necessary to correct the absorbance originating from AA especially in fruits that have high AA like Papaya. The literature reports do not indicate whether correction had been made for ascorbic acid.

The content of polyphenols was found to be high in the leaves and in the peels of papaya. The quantitative analysis on phenolic compounds of papaya leaves was done by Canini et al., (2007) and Husin et al. (2019) which revealed the presence of phenolic acids (i.e. caffeic acid, p-coumaric acid and protocatechuic acid) as the main compounds. Chlorogenic acid was found in trace amounts, compared to the flavonoids and coumarin compounds. On the contrary, Miean & Mohamed (2001) detected a high level of total flavonoids (126.4 mg/100 g) in young leaves of papaya, with quercetin and kaempferol as the main compounds. Quercetin and kaempferol are compounds found abundantly in most edible plants including leafy vegetables, fruits and beverages. Phenolic acids on the other hand are present in appreciable amounts in a large number of vegetables. Both compounds demonstrate antioxidant and antioxidative properties (Lu et al., 2006; Moon, Tsushida, Nakahara, & Terao, 2001; Rusak et al., 2010; Sasaki, Toda, Kaneko, Baba, & Matsuo, 2003; Sternberg et al., 2008). In papaya peel, the polyphenols content was found to be higher than the pulp (Faller & Fialho, 2010; Matsusaka & Kawabata, 2010). Recent quantitative analysis indicated the presence of ferulic and caffeic acids as the most abundant compounds (Rivera-Pastrana et al., 2010) in the peel of the fruits. No recent data are available on the polyphenol compound in the seeds.

The quantity of polyphenols in foods can be affected by agricultural practices such as the use of synthetic fertilizers that offer more bioavailable sources of nitrogen which can accelerate plant and the production of secondary metabolites. Polyphenol content could also be affected by higher exposure of the plant to stressful environment such as the absence of synthetic pesticides. Attack by insects or fungi can induce the production of natural defence substances such as phenolic compounds (Winter & Davis, 2006; Woese, Lange, Boess, & Bogl, 1997). Both hypotheses would result in foods with higher antioxidant capacity as a consequence of the higher polyphenol content. Faller and Fialho (2010) had compared the polyphenol content of organic and conventional plant foods. They found that among fruits studied, papaya seems to be more affected by alteration in agricultural management with approximately 70% higher hydrolysable polyphenols in organic fruit compared to the conventional counterpart. This could be one of the reasons for different polyphenol 18

value found in the same type of papaya cultivars discussed earlier. Nonetheless, polyphenol composition in food can also be altered by storage, location, handling, and processing conditions (Rinaldo, Mbéguié-A-Mbéguié, & Fils-Lycaon, 2010).

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Table 2.5 Phenolic compounds in different parts of papaya. Plant parts Cultivars Source Analyzed Compound(s) Concentration References Fruits/pulp N.A. Thailand Total phenolicsa 54 (mg GAE/100 g FW) Patthamakanokporn et al. (2008)

Solo Malaysia Total phenolicsa 0.02 (mg GAE/100 g FW) Isabelle et al. (2010)

N.A. Total phenolicsa 37.9-75.7 (mg GAE/100 g FW) Annegowda et al. (2013) Total Tannins 1.25-3.36 (mg CE/100g FW) Total flavonoids 1.48-3.10 (mg CE/100g FW)

N.A. Japan Total phenolicsa 100-1200 (mg GAE/100g DW) Matsusaka and Kawabata (2010) N.A. Brazil Soluble polyphenolsa 0.11-0.98 (mg GAE/100 g FW) Faller and Fialho (2010) Hydrolysable 0.60-2.08 (mg GAE/100 g FW) polyphenols a

Total phenolicsa 1263.70 (mg GAE/100g DW) Silva et al. (2014)

Hawaiian Fiji Myricetinb 3 (mg/100g FW) Lako et al. (2007) Quercetinb 2 (mg/100g FW) Kaempferolb 2 (mg/100g FW) Morinb 2 (mg/100g FW) Fisetinb <1 (mg/100g FW) Isorhamnetinb <1 (mg/100g FW) Total anthochyaninsb 100 (mg/C-3-G100g FW) Total phenolicsa 26 (mg GAE/100 g FW)

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Table 2.5 Phenolic compounds in different parts of papaya (continuation). Plant parts Cultivars Source Analyzed Compound(s) Concentration References Fruits/pulp Hawaiian Fiji Lako et al. (2007) Caffeic acide Not quantified Jindal and Singh (1975) Gentisic acide Not quantified m-coumaric acid e Not quantified p-coumaric acid e Not quantified Salicylic acide Not quantified Quercetine Not quantified

Maradol Mexico Ferulic acid b 277.49 to 186.63 (mg/100g DW) Gayosso-García Sancho et p-coumaric acid b 229.59 to 135.64 (mg/100g DW), al. (2011) Caffeic acid b 175.51 to 112.89 (mg/100g DW)

N.A. Hawaii Apigenin b 0.01(mg/100g FW) Franke et al. (2004) Luteolinb 0.02(mg/100g FW) Kaempferol b 0.01(mg/100g FW) Myricetin b 0.03(mg/100g FW) Quercetinb 0.00(mg/100g FW)

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Table 2.5 Phenolic compounds in different parts of papaya (continuation). Plant parts Cultivars Source Analyzed Compound(s) Concentration References Peel N.A Brazil Soluble polyphenolsa 0.38-0.54 (mg GAE/100g FW) Faller and Fialho (2010) Hydrolysable 1.30-1.78 (mg GAE/100g FW) polyphenolsa

N.A Japan Total phenolicsa 132 (mg GAE/ 100g DW) Matsusaka and Kawabata (2010) Maradol Mexico Caffeoyl hexoside b Not quantified Rivera-Pastrana et al. Gallic acid- (2010) deoxyhexoside b Not quantified Protocatechuic acid- hexoside b Not quantified Caffeoyl hexose- deoxyhexose b Not quantified Myricetin b Not quantified Quercetin b Not quantified Isorhamnetinb Not quantified Kaempferol b Not quantified Caffeic acid b 4.6–6.8 (g/100g DW) Rutin b 1.0–1.6 (g/100g DW) Ferulic acid b 13.3–16.2 (g/100g DW)

Leaves N.A. Africa Protocatechuic acidc 0.11(mg/g DW) Canini et al. (2007) p-Coumaric acidc 0.33(mg/g DW) 5,7-Dimethoxycoumarinc 0.14(mg/g DW) Caffeic acidc 0.25(mg/g DW) Kaempferolc 0.03(mg/g DW) Quercetinc 0.04(mg/g DW) Chlorogenic acidc <0.001(mg/g DW)

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Table 2.5 Phenolic compounds in different parts of papaya (continuation). Plant parts Cultivars Source Analyzed Compound(s) Concentration References Leaves N.A. Malaysia Total flavonoidsb 126.4 (mg/100g DW) Miean and Mohamed Quercetinb 81.1 (mg/100g DW) (2001) Kaempherolb 45.3 (mg/100g DW)

N.A. N.A. Flavonols 0-0.02 (mg/100g FW) Duke (2011) Tannins 0.05-0.06 (mg/100g FW)*

N.A. Australia Total phenolicsa 9.43-23.06 (mg GAE/100g FW) Vuong et al. (2013) Total flavonoidsd 6.44-17.07 (mg CE/100g FW) Saponinsd 26.36-82.88 (mg Aes/g FW) proanthocyanidinsd 1.91-7.91 (mg CE/100g FW)

Seeds N.A. N.A. Tanninsf 6.35 (g/100g DW)* Marfo, Oke, and Afolabi (1986a) DW= dry weight; FW= fresh weight a Folin-Ciocalteau method b HPLC c GCMS d UV-vis spectrophotometery e paper chromatography f Vanillin-Hydrochloric Acid test GAE: Gallic Acid Equivalents C-3-G: Cyanidin-3-Glucoside CE: Catechin Equivaents Aes: Aescin equivalents N.A.: Not Available * Unit is not available

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2.4 Health benefits of papaya

2.4.1 Anti-inflammatory and immunomodulatory effects

Researchers have suggested the possibility of anti-inflammatory and immunomodulatory effects of papaya pulp, leaves and seeds. A recent published study with 15 healthy subjects suggested that ingestion of papaya fruit is inversely associated with the reduction of IFN-λ+CD4+ T cells which play a vital role in mediating inflammatory responses (Abdullah et al., 2011). The anti- inflammatory effects of papaya leaf extract has also been found to significantly reduce carrageenan- induced paw oedema, cotton pellet granuloma and formaldehyde-induced arthritis in rat models (Owoyele et al., 2008). The leaf extract was also shown to have immunomodulatory activities by enhancing Th 1 types cytokines from human lymphocytes, that relate to antitumor immunity (i.e. IL-12p40, IL-12p70, IFN-λ or TNF-α) in the human immune system (Otsuki et al., 2010). Similarly, the seed extract has also shown the ability to enhance phytohemagglutinin reactivity of lymphocytes isolated from human blood (Mojica-Henshaw, Francisco, De Guzman, & Tigno, 2003). This implies that the seed extract contains substances or components which have or display growth-promoting actions and thus act as immunomodulators. In the same manner, some bioactive fractions of the seed extract are also reported to have the ability to inhibit cell lysis as shown using an in vitro classical complement-mediated hemolytic pathway assay. This indicates a possible effect towards inhibiting inflammation (Mojica-Henshaw et al., 2003). As mentioned earlier, papaya especially the leaves contain flavonoids, saponins, tannins and glycosides that have all been associated with various degrees of anti-inflammatory activities (González Mosquera et al., 2011; Pelzer, Guardia, Juarez, & Guerreiro, 1998; Sparg, Light, & van Staden, 2004; Thomas & Filho, 1985). Therefore, the anti-inflammatory effects may be due to the activity of one or a combination of some of the identified constituents.

2.4.1.1 Anticancer effects papaya is rich in glucosinolates, isothiocyanates and BITC and thus is presumably a promising plant source for use in chemoprevention of cancer. Many scientific studies validate that BITC induces apoptosis specifically in cancer cells and protects against tumorigenesis. It has been suggested that anticarcinogenic effects of isothiocyanates are related to their capacity to induce phase II enzymes such as glutathione S-transferase, nicotinamide adenine dinucleotide phosphate and quinine reductase (Cavell et al., 2011; Nakamura et al., 2000). Moreover, plant sources of glucosinolates, have been of great interest for potential use in the chemoprevention of cancer (Aruna & Subrata, 2008; Cavell et al., 2011). The glucosinolates are known to be degraded into isothiocyanates by 24

enzymatic action of plant-specific myrosinase or intestinal flora in the human body (Aruna & Subrata, 2008). Interestingly, only recently was the effect of inhibitory activity of C. papaya leaf extract on tumor cell lines documented. The leaf extracts were shown to significantly inhibit the proliferative responses of solid tumor cell lines derived from cervical carcinoma, breast adenocarcinoma, prostate cancer, hepatocellular carcinoma, lung adenocarcinoma, pancreatic epithelioid carcinoma, and mesothelioma (Otsuki et al., 2010; Pandey et al., 2017). This studies reporting on the antitumor properties of the leaf extracts and suggested that the extract may potentially provide the means for the treatment and prevention of selected human diseases such as cancer, and may also serve as immunoadjuvants for vaccine therapy (Otsuki et al., 2010). There is no current scientific data available on the exact compound that demonstrated the anti-cancer and immunomodulatory effects, however isothiocyanates extracted from papaya fruits have been proven to act as a glutathione S-transferase inducers (Nakamura et al., 2000). Otsuki et al (2010) have done fractionation of papaya leaf and suggested that a fraction of lower than molecular weight 1000 could increase the Th 1 cytokines and exhibited inhibitory effects of proliferative response of tumor cell lines and may also have the possibility to prevent various allergic disorders. In addition, papaya fruit was also rich in lycopene content. Several in vitro studies with human cancer cells, particularly prostate cancer cell lines, have indicated that lycopene can promote apoptosis in these cells and therefore might have potential as a chemotherapeutic agent (van Breemen & Pajkovic, 2008).

2.4.1.2 Wound healing effects

Animal studies addressed the ability of papaya to treat skin disorders specifically wound healing properties. The extract of peels of mature green fruit (unripe) have proven to gives faster epidermal wound healing on induced wounds on mice (Anuar et al., 2008) and rats (Nayak, Pinto Pereira, & Maharaj, 2007) compared to peels of ripe fruit. This was further supported by wound healing evaluation using latex from the skin on mice burn models (Gurung & Skalko-Basnet, 2009). The latter suggested that the latex was responsible for its wound healing properties. The latex from unripe papaya fruits contains a mixture of cysteine endopeptidases such as papain, chymopapain A and B, caricain, papaya endopeptidase II, papaya endopeptidase IV, omega endopeptidase chitinase II, protease inhibitors, glutaminyl cyclase and unknown-function proteins (Azarkan, El Moussaoui, van Wuytswinkel, Dehon, & Looze, 2003; Azarkan, Wintjens, Looze, & Baeyens-Volant, 2004). Proteolytic enzymes such as papain, chymopapain and leukopapain were reported to be effective for debridement of necrotic tissues, preventing infection, promoting growth and improving the quality of the scar (Anuar et al., 2008; Starley et al., 1999). Thus, in agreement with traditional beliefs, papaya has a high potential as a treatment for wound healing. 25

2.4.1.3 Anti-ulcer effects

Papaya leaves and unripe fruits may potentially serve as a good therapeutic agent for protection against gastric ulcers. A gastric ulcer index was significantly reduced in rats pretreated with papaya extracts as compared with alcohol treated controls (Ezike et al., 2009; Indran, Mahmood, & Kuppusamy, 2008), and the standard drugs cimetidine (Ezike et al., 2009) and indomethacin (Owoyele et al., 2008), respectively. The unripe fruit was also shown to have the ability to inhibit gastrointestinal propulsion (Ezike et al., 2009). This has proven beneficial in ulcer therapy as delaying of gastrointestinal motility will increase the absorption of oral anti-ulcer drugs. Moreover, the unripe fruit was also shown to possess antimicrobial properties (Osato et al., 1993), which are probably beneficial in treating/preventing peptic ulcers by acting against Helicobacter pylori. The extracts of unripe C. papaya contain terpenoids, alkaloids, flavonoids, carbohydrates, glycosides, saponins, and steroids. Saponins are known to possess anti-ulcer activity mediated by the formation of protective mucus on the gastric mucosa and protection of the mucosa from acid. The cytoprotective and antimotility properties of the extracts may account for the anti-ulcer property of the leaves and unripe fruit.

2.4.2 Other health benefits

Antimalarial activity of papaya is mostly anecdotal but in the literature there is a report on the efficacy of a crude aqueous extract of papaya leaf on mice infested with malaria parasite (Pietretti et al., 2010). It was found that the extracts exhibited plasmodicidal activity and reduction of parasitemia (Pietretti et al., 2010; Sannella et al., 2009).

A study by Eliagita et al. (2017) reported that consuming papaya (mature ripe pulp) has a significant effect on changes in hemoglobin and hematocrit levels in pregnant women. It is suggested that consuming papaya should be one of alternative treatments for midwives to prevent anemia in pregnant women.

Diseases such as dengue result in a low thrombocyte count in the blood and need a rapid response of thrombocyte levels and plasma transfusions (World Health Organization, 1997). Papaya leaf crude formulations have been successfully employed in folk medicine in Malaysia for the treatment of dengue infections with haemorrhagic manifestations (Ching et al., 2015; Subenthiran et al.,

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2013). The formulations use suspensions of powdered leaves in palm oil as a vehicle. Papaya leaf extract in palm oil has been investigated for its effect on thrombocyte counts in mice. It showed a significantly protracted increase in platelet counts (Kathiresan, Surash, Sharif, Mas Rosemal, & Walther, 2009). The study thus suggested that the leaf may be beneficial in the release and/or production of thrombocytes. However, further studies on substance/s responsible for this effects and dose-response effects are warranted to confirm these results.

Young papaya leaf is also consumed for treating diabetes and high blood pressure (Ong & Norzalina, 1999). One of the therapeutic approaches for treating diabetes type 2 is inhibition of intestinal α-glucosidase and mild inhibition of pancreatic α-amylase. A recent published study indicated that papaya leaves contain high α-glucosidase and lower α-amylase inhibitory activities that could be considered potential dietary supplement approaches to manage early stages of hyperglycemia linked to type 2 diabetes (Loh & Hadira, 2011). Inhibition of these enzymes delays and prolongs carbohydrate digestion time, causing a reduction in the rate of glucose absorption and therefore blunting the postprandial plasma glucose rise (Rhabasa-Lhoret & Chiasson, 2004). In addition, fruits and roots are also claimed to be effective in the management of diabetes mellitus (Abo et al., 2008).

Literature also illustrate the antioxidative potential of papaya pulp juice. The in vitro and in vivo experimental studies strongly indicate that papaya leaves and pulp juice have marked antioxidant activity and are able to significantly inhibited lipid peroxidation level in blood of rats studied (Indran et al., 2008; Mehdipour et al., 2006). Moreover, the effects of consumption of papaya (400 g/day) on total antioxidant status and lipid profile (total cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol) have been studied in healthy male youths. The study showed that plasma total antioxidant capacity, glucose and glutathione reductase levels increased following treatment by papaya (Rahmat, Abu Bakar, Faezah, & Hambali, 2004). The leaf extract has also shown to increase erythrocyte glutathione peroxidise activity and thus suggest its potential to serve as an agent for protection against oxidative stress (Indran et al., 2008).

Anthelminthic properties of papaya are well published in literatures since the 19th century (Satrija, Nansen, Murtini, & He, 1995). The latex from unripe fruits contained cysteine proteinases and may possess anthelmintic properties which are used successfully against ascarids, tapeworms, whipworms and hookworms (Gillian Stepek, Behnke, Buttle, & Duce, 2004; G. Stepek, Buttle, Duce, Lowe, & Behnke, 2005). Moreover, C. papaya seeds have shown effectiveness against 27

intestinal parasites (Kermanshai et al., 2001; Okeniyi, Ogunlesi, Oyelami, & Adeyemi, 2007).

2.5 Health Benefits of Fermented Papaya Product (FPP)

Most studies on FFP to date have focused on antioxidative properties, anti-inflammatory, immunomodulatory effects and prevention of chronic and degenerative diseases such as diabetes, cirrhosis, thalassemia and aging. Recent in vivo study by Logozzi et al. (2019) reported that FPP® controlled murine melanoma tumor growth, reducing the tumor mass of about three to seven times vs. untreated mice. In this study, the optimal effect of FPP® treatment on tumor growth was at a dose of 200 mg/Kg per day for 17 days, and doubling the dose (400 mg/kg/day) did not obtain any increase in the antitumor effect of the FPP® treatment. Aruoma et al. (2010) in their commentary discussed the role of FPP in preventing some of the degenerative diseases. Supplementation with FPP has been suggested to cause a significant decrease in plasma glucose levels in both healthy subjects and type 2 diabetic patients (Danese et al., 2006). Similarly, Collard & Roy (2010) proved that FPP attenuated the gain in blood glucose in adult obese diabetic (db/db) mice. In addition, blood lipid profile (total triglycerides, total cholesterol and low density lipoprotein) levels were found to be significantly decreased whereas high-density lipoprotein level was significantly increased in FPP-supplemented mice compared to placebo-supplemented mice. They also had established its effects on diabetic wound healing after supplementation for 8 weeks before wounding. It was found that FPP increases nitric oxide production in macrophages inducible nitric oxide (iNOS) derived from wounds of FPP-supplemented mice compared with those isolated from wounds of control mice. Diabetes is characterized by a nitric oxide (NO) deficiency at the wound site, and NO is known to be involved in wound healing through multiple modes of action. The presence of iNOS in a wound setting represents a key contributor of NO.

ROS, which are generated both physiologically and pathologically, will cause damage to cellular constituents like lipid membrane and, proteins including DNA. Among many types of modifications induced by ROS, 8-hydroxy-deoxy-guanidine (8-OhdG) is an abundant stable product of oxidative DNA damage which has been indicated as an early event in hepatitis C virus infection and as a marker of liver damage as well as giving rise to carcinogenesis (Cardin et al., 2001; F. Marotta, Yoshida, Barreto, Naito, & Packer, 2007; Tarng et al., 2000). Supplementation of FPP or vitamin E in 50 patients with hepatitis C virus infection related to cirrhosis was undertaken to test a novel antioxidant or immune modulator in these patients (Marotta et al., 2007). Improvement of oxidative stress markers (i.e. reduced glutathione, glutathione peroxidase, oxidized glutathione, malondialdehyde) was obtained by both regimens (p < 0.005 patients vs control). FPP also 28

significantly decreased 8-OHdG levels in circulating leucocyte DNA and improved cytokine balance compared to vitamin E treatment. In addition, FPP was also found to decrease 8-OHdG level in circulating leucocyte DNA and enhanced DNA repair mechanisms in a group of elderly (n= 54; mean age: 72 years) (F. Marotta et al., 2006). The elderly population is more prone to depleted antioxidant defenses/malnutrition on account of poor or improper intake due to sensory impairment (Schiffman, 2009) and also from a subclinically impaired gut absorption (John E, 2007). In addition, polymorphism analysis of glutathione S-transferase M1 (GSTM1) was carried out as well in this study. At baseline assessment, GSTM1-positive smoker and the GSTM1-negative smoker showed a significantly higher level of DNA adducts and 8-OHdG concentration in leukocyte DNA. GSTM1 gene deficiency has been shown to occur in approximately half of the population of various ethnic origins (Lin et al., 1994; Nelson et al., 1995). This deficiency has been shown to increase DNA adduct formation (S. Kato, Bowman, Harrington, Blomeke, & Shields, 1995; F. Marotta et al., 2006). Supplementation with FPP showed significant improvement of the oxidative/antioxidative balance, but only in the GSTM1-negative smokers (F. Marotta et al., 2006). The significance of these findings was addressed again by Marotta and his colleagues (2010). Using 11 healthy nonsmokers, teetotaller individuals (a) detailed dietary and lifestyle questionnaire b) refrained from any multivitamin supplement or fortified food), they found that FPP significantly upregulated GSTM1 and 8-oxoguanine glycosylase gene expression. Given this promising series of results and as an open insight to nutrigenomic research, more studies of FPP are needed probably using a larger sample population, longer observation periods and observation on healthy vs clinical conditions.

The effect of FPP on the attenuation of ß-amyloid which is associated with Alzheimer’s Diseases (AD) has been assessed (Zhang, Mori, Chen, & Zhao, 2006). Researchers have indicated the association of ß-amyloid deposition on AD pathogenesis, and the possibility of free radicals as the major contributor to the neurotoxicity of AD (Reddy, 2009; Reddy & Beal, 2008; Smith et al., 2010; Vickers & Dickson, 2000). Zhang and colleagues (2006), in their study using human neuroblastoma SH-SY5Y cells, indicated that copper triggers the ß-amyloid neurotoxicity in ß- amyloid precursor protein over expressed cells, which can be taken as a model of AD. Copper is one of the free radical productions which have been associated and found within the amyloid deposits in AD brains (Hung, Bush, & Cherny, 2010; Lovell, Robertson, Teesdale, Campbell, & Markesbery, 1998). ß-amyloid and ß-amyloid precursor protein, both have a copper binding domain and can reduce Cu2+ to Cu+, leading to ROS (Hesse, Beher, Masters, & Multhaup, 1994; X. Huang et al., 1999). FPP was found to inhibit cell apoptosis through attenuation of ROS generation and thiobarbituric acid reactive substances level induced by copper and ß-amyloid (Zhang et al., 2006).

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In addition, this study also suggest that FPP inhibits the elevation of Ca2+ induced by ROS which may also trigger apoptosis (Castillo & Babson, 1998; Pinton, Giorgi, Siviero, Zecchini, & Rizzuto, 2008), and NO production which may, directly or in directly, be involved in neuronal death in AD (Brown, 2010).

ß-thalassemia is a group of genetically inherited hemoglobin (Iqbal, Kazim, & Mehboobali) disorders characterized by the inability of a person to produce hemoglobin, thus resulting in chronic anemia (Weatherall, 2010). The primary defects in thalassemia are mutations of the β-globin gene, however, many aspects of the pathophysiology of this form are mediated by oxidative stress (Rachmilewitz & Schrier, 2001; Rund & Rachmilewitz, 2005). A previous study on ß-thalassemic mice treated orally with FPP and 11 patients (eight ß-thalassemia intermedia and three ß- thalassemia major) showed that in vitro treatment of FPP reduced red blood cell sensitivity to hemolysis and phagocytosis, improved polymorphonuclear ability to generate oxidative burst, and reduced platelet tendency to undergo activation (Amer, Goldfarb, Rachmilewitz, & Fibach, 2008). Furthermore, in connection with this study, Fibach & Rachmilewitz (2010) did a study of oral treatment with FPP using two groups of patients from different countries, with ß –thalassemic major and ß–thalassemic intermedia (Israel), and E-ß–thalassemic (Singapore). They showed a significant increase of reduced glutathione (Qiu, Lu, Li, & Toivonen) content in red blood cells, externalization of phosphatidylserine, without significant changes in the hematological parameters, decreased ROS generation and membrane lipid peroxidation. The results are presented as mean fluorescence intensity, measured by flow cytometry for GSH and lipid peroxidation. Although this is not a conventional method for these parameters (Olalla-Saad, 2010) the results are consistent and encouraging. The antioxidant effect of FPP in reducing oxidative stress biomarkers in these studies is unknown, although one possible explanation could be its high content of glutamic acid, glycine and methionine, which are substrates for GSH synthesis. Besides thalassemia, oxidative stress particularly stressed red blood cells (RBC), have been observed in various congenital and hemolytic anemia including sickle cell anemia, congenital dyserythropoietic anemia, G6PD deficiency, hereditary spherocytosis (HS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as in myelodysplastic syndrome (Ghoti et al., 2011; Ghoti, Rosenbaum, Fibach, & Rachmilewitz, 2010; Mojisola, Anthony, & Alani, 2009). Ghoti and colleagues in their recent study summarized that oxidative stress plays an important role in the pathophysiology of HS which can be ameliorated by an antioxidant such as FPP. In this study, RBC from the HS patients seem to generate more ROS, membrane lipid peroxides and less reduced GSH than normal RBC. The effects of 0.1 mg/ml FPP on in vitro HS-RBC resulted in a 41% decrease in ROS and 322% increase in GSH with less

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hemolysis of RBC. Moreover, similar results for these markers were also obtained for HS patients (n=10) treated orally 3 times per day with 3g FPP for 3 months (Ghoti et al., 2011). In their previous paper, the research group had done a small study on PNH patients and proven that treatment with antioxidants (in this case FPP) may reduce the oxidative stress markers and improved clinical manifestation of PNH patients (Ghoti et al., 2010). Thus, these series of promising results suggest FPP as a potent antioxidant, which have the abilities to alleviate symptoms associated with oxidative stress in both congenital and hemolytic anemias (Amer et al., 2008; Fibach et al., 2010; Ghoti et al., 2011; Ghoti et al., 2010).

Allergic reactions are well known and have been a problem for decades. FPP has been suggested as a potential therapeutic agent for allergic inflammation on dermal and intestinal mucosa (Hiramoto, Imao, Sato, Inoue, & Mori, 2008). In this study, two types of mice models, fluorescein isothiocyanate (FITC) (Th2 type) induced ear and colon oedema, and oxazolone (Th1 type) induced ear and colon oedema was treated orally with 6 mg/day of FPP. Mice on FPP suppressed contact hypersensitivity induced by FITC or oxazolone by 50 and 37%, respectively. Furthermore, biochemical and histological analyses revealed that the FPP decreased the elevation of inflammatory cytokine levels (IFN-γ, IL-10 and TNF-α) in plasma and ear swelling and colon. Although TNF-α has been identified by its anti-tumor activity, which plays a role in many immunological and inflammatory reactions, blocking TNF-α has also been reported to inhibit skin dermatitis (Johansen et al., 2010; Olmos et al., 2007).

2.6 Toxicological effects

Research on papaya seed extract has focussed mainly on its toxicology and antifertility properties. However, recently the seeds have been emerging as a potential safe antifertility drug for men. Oral administration of the seed extract has proven it possesses male antifertility properties by showing inhibition of sperm motility and sperm count reduction in mice, rats and langur monkeys (Chinoy, D'Souza, & Padman; Nirmal K. Lohiya, Manivannan, & Garg, 2006; N.K. Lohiya et al., 2002; Nirmal K. Lohiya et al., 2005; Udoh, Udoh, & Umoh, 2005; Verma, Nambiar, & Chinoy, 2006). The spermicidal effects showing an instant fall in the sperm motility were very much dose- dependent. Most studies done using rat models with daily oral exposure of 10 up to 500 mg/kg body weight of the seed extract showed significant falls in sperm motility. It is indicated as a potentially safe male contraceptive with free general toxicity and no adverse effects to other organs as reflected in weight of vital and genital organs, hematology, serum clinical biochemistry and normal sexual behaviour (Chinoy et al.; Nirmal K. Lohiya et al., 2006; Nirmal K. Lohiya et al., 2005; Udoh et al., 31

2005; Verma et al., 2006). Solvent such as aqueous, methanol, ethanol, ethyl acetate and chloroform were used to extract the seed in all these studies. The aqueous and chloroform extracts were found to be more effective in action as contraceptive agents. For mode of action contraceptive drugs will usually inhibit sperm motility by disruption of testis epididymal epithelial cell function or directly by affecting sperm enzymes (Cooper & Yeung, 1999). Photomicrographs of testis morphology in rats treated by C. papaya seed extracts showed Leydig and Sertoli cell metaplasia and alteration of epididymis thus resulting in direct alteration of the morphology of sperm cells and causing infertility (Udoh et al., 2005). In contrast, Lohiya et al. (2005) found no significant alterations in the epididymal epithelial ultrastructure and Leydig cells and suggested a possible direct effect on sperm mediated through Sertoli cell factors. A similar effect of the seeds was also observed on the fertility of male and female Nile tilapia, respectively (S.B. Ekanem & Bassey, 2003; S.B. Ekanem & Okoronkwo, 2003). Studies done using rabbits treated orally with crude chloroform extracts of the seeds showed similar contraceptive efficacy (Nirmal K. Lohiya, Pathak, Mishra, & Manivannan, 1999), but the aqueous extract of the seeds showed no contraceptive effects (N.K. Lohiya, Pathak, Mishra, & Manivannan, 2000). The different observations of effects may be due to species specificity, relative resistance of the animals to the drug or due to other factors that affect biological activity of the plant preparations (N.K. Lohiya et al., 2000). Nevertheless, it is highly alkaloids, steroids, triterpenoids and flavonoids present in the seeds of C. papaya may be responsible for the male contraceptive effectiveness. Recently, a high dose of papaya leaf extracts (500 mg/kg body weight) was also reported to exert toxic effects on the seminiferous tubule epithelium with simultaneous reduction in reproductive potentials of the male rat (Morayo & Akinloye, 2010).

Apart from the effect on male rats, papaya seed extract also manifested antifertility, antiimplantation and abortifacient activity in female rats (Adebiyi, Ganesan Adaikan, & Prasad, 2003; Chinoy, Dilip, & Harsha, 1995). The seed was toxic to uterine tissues and resulted in spontaneous contraction. Adebiyi and co-workers (2003) suggested that BITC, could be responsible for this spontaneous contraction. BITC is the major bioactive compound in the seeds and literature has shown that it is capable of causing morphological and functional aberrations of certain cells or tissues in in vitro and in vivo studies. Uterine tissues treated with BITC give similar degenerative changes when compared to ethanolic extracted seed- treated uterus (Adebiyi, Prasad, et al., 2003). It is not only the seed of unripe or semi-ripe papaya fruits that are thought to be unsafe in pregnancy. Unripe or semi-ripe papaya flesh also contains high concentration of the latex that significantly produces marked uterine contractions (Adebiyi, Adaikan, & Prasad, 2002). However, normal consumption of ripe papaya during pregnancy should not pose any significant danger due to the fact

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that latex is only found in unripe fruits. Generally, papaya latex is found in matured unripe fruits. Ripe papaya on the other hand contains less or no latex, possibly due to cessation or breakdown in the function of the latex producing cells (OECD, 2005).

On the contrary, fermented seed was found to be safe on embryo implantation in the female uterine wall (A. M. Abdulazeez, Ameh, Ibrahim, Ayo, & Ambali, 2009; M.A. Abdulazeez, Ameh, Ibrahim, Ayo, & Ambali, 2010; Mansurah A. Abdulazeez & Sani, 2011). In this study, female rats fed with 1500 mg/kg of unfermented papaya seeds resulted in high pre-implantation losses while the same doses of fermented papaya seeds did not have such an effect. Observation of rats with lower doses (500mg/kg) of unfermented seeds also showed an increase percentage of pre-implantation loss and death of litters (A. M. Abdulazeez et al., 2009; M.A. Abdulazeez et al., 2010). The fermentation process of papaya seeds removes or dehulls the seed coat and leaves the inner part or kernel (Dakare et al., 2011). Traditionally the kernel is finally boiled and fermented in a lined jute bag containing papaya leaves (Dakare et al., 2011). The removal of the seed’s coating and boiling treatment may destroy myrosinase, an enzyme responsible for converting glucosinolates to isothiocyanate in the seed (Shapiro, Fahey, Wade, Stephenson, & Talalay, 2001; Vallejo, Tomás- Barberán, & García-Viguera, 2002). Several studies have found that inactivation of myrosinase in plants decreases the bioavailability of isothiocyanate (Conaway et al., 2000; M. Kato, Imayoshi, Iwabuchi, & Shimomura, 2011; Shapiro et al., 2001). Moreover glucosinolates and isothiocyanates may also be lost from the tissue through leaching into the cooking water. This will greatly depend on the cooking time and the amount of water used (Song & Thornalley, 2007). However more studies are required to determine the concentration of isothiocyanate in the papaya seeds after cooking treatment or fermentation, and whether the concentration of these compounds are high in the kernel or coating of the seeds. In summary, papaya seeds and unripe fruits exert toxic effects on both male and female reproductive organs. Understanding the different pharmacological properties of the plant is crucial in order to assess their safety and weigh the benefits of their use against the risks.

2.7 In vitro digestion studies

One of the principal topics concerning the beneficial effects of nutrients is their bio-availability and metabolic fate. The bio-availability of a dietary compound is dependent upon its digestive stability, its release from the food matrix which referred to as bio-accessibility, and the efficiency of its transepithelial passage. In vitro enzymatic digestion processes are methods that are widely used to study the gastro-intestinal behaviour of food or pharmaceuticals. The in vitro enzymatic digestion 33

models mimic conditions and luminal reactions which occur in the mouth, stomach, small intestinal and occasionally large intestinal fermentation (Figure 2.2-2.3). Although human nutritional studies are still being considered the “gold standard” for addressing diet related questions, in vitro methods have the advantage of being more rapid, less expensive, less labour intensive without ethical restrictions (Aref et al., 2010).

Figure 2.2 The sites of the gastrointestinal tract simulated by the developed in-vitro enzymatic digestion and . Adapted from Netzel et al. (2011).

.

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2.8 Papaya in Australia

In Australia, red or pink-fleshed cultivars are often known as ‘papaya’ to distinguish them from the yellow-fleshed fruits, known as ‘paw paw’, but both of these common names refer to the same plant species (Papaya Australia, 2019). However in most other countries, irrespective of its flesh colour, papaya is generally known as ‘papaya’. Fruit production is optimal in areas with a minimum monthly rainfall of about 100 mm, minimum relative humidity of 66% (Nakasone & Paull, 1998) and a temperature range between 21 and 33º C (Nakasone & Paull, 1998; OECD, 2003; Villegas, 1997).

In Australia, C. papaya is predominantly grown in Queensland (>95% of production) where the major production areas include Innisfail, Yarwun, Yeppoon and Gympie (Agrifutures, 2019; Garrett, 1995). In 2010, the Australian Paw Paw and papaya industry produced the biggest crop in the north and far north of Queensland (Miriam Hall (ABC), 2010). C. papaya is a new emerging industries in Australia which have been funded by the Rural Industries Research and Development Corporation (Agrifutures, 2019). The gross value of production of the selected emerging plant industries in 2011-2012 was an estimated $530 million and this was largely contributed by tropical exotic fruit (mainly papaya and lychee) (Foster & Section, 2014).

However, excess supply of fruits like papaya depresses prices and causes financial loses to the growers. This will occur especially during summer months when fruit is most plentiful. Informing consumers of the potential benefits of papaya will stimulate market demand and counter excess production. To date, there are eight types of papaya varieties most commonly grown in Queensland and available on the market (Table 2.6).

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Table 2.6 Papaya varieties grown in Queensland.

Area Varieties Type

Artherton tableland Hybrid 1 Yellow fleshed

Hybrid 11B Yellow fleshed

Hybrid 14 Yellow fleshed

Sunrise Solo Red fleshed

Coastal North Queensland Hybrid 1 Yellow fleshed

Hybrid 11B Yellow fleshed

Hybrid 13 Yellow fleshed

Hybrid 14 Yellow fleshed

Sunrise Solo Red fleshed

Central Queensland Hybrid 1 Yellow fleshed

Hybrid 11B Yellow fleshed

Hybrid 13 Yellow fleshed

Hybrid 29 Yellow fleshed

South-east Queensland PG Yellow fleshed

Ritcher Gold Yellow fleshed Source: Ross et al. (2000)

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From an industrial perspective, there are some weaknesses and strength of papaya fruit in term of sales and marketing. Strengths i. Unique, healthy product produced and available year-round. ii. Short-cycle crop for quick turnover. iii. Product has specific health/nutritional benefits. iv. Profitable industry. v. Low labour skill requirement. Weaknesses i. Inconsistent quality, taste and size of fruit. ii. Too many varieties on the market. Variability in varieties. iii. Lack of good product knowledge long the supply chain. iv. Fruit can be fragile, not long shelf life and can be messy to eat. v. Production is highly sensitive to climate variations. i. Consumption is highly sensitive to climate variations. ii. Long distance to major markets. iii. Low consumer awareness of the product.

Source: Australian Papaya Industry (2019)

The next chapter (Chapter 3) includes the methods of sampling and sample collection; preparation for 4 stages of maturity of papaya fruits, and to determine its morphological traits and color analyses for each papaya cultivars according to its maturity stages. The experiments that were undertaken will be antioxidant capacity, vitamins and polyphenols content of different parts (pulp and peel) of Papaya Linn cultivars. This chapter will fall under objective 1; Identify and quantify the phytochemical (Vitamins and polyphenols) and AOC activities of selected commercially available Australian papaya cultivars.

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CHAPTER 3

3. Antioxidant capacity, Vitamins and Polyphenols content of different parts (Flesh and peel) of Papaya Linn cultivars

3.1 Introduction

Papaya is the fourth most grown tropical fruit behind banana, mango and pineapple (FAOSTAT, 2015a). Asia including India has been the leading papaya producing region, accounting for 51% of the global production in 2011, followed by South America (20%), Africa (11%), the Caribbean (9%) and Central America (9%). In Australia, papaya is predominantly grown in Queensland (>95% of production) with 6534-7069 tonnes of fruits production between 2009 and 2011 which is less than 0.1% of world production at11.22 Mt. (FAOSTAT, 2015b).

Papaya and papaya products have a significant health profile particularly among consumers in Asia (Anuar et al., 2008; Lim, 2012; Otsuki et al., 2010). Australians however, primarily select papaya to add variety and flavour to their diet but lack the cultural background, information and knowledge to use in selecting papaya to contribute to their health. Moreover, the predominate use as a ripe fresh fruit in Western culture is different from the Asian use where immature green fruit, as well as leaves, are commonly used as food and medicinal treatments due to the phytochemical and enzymatic composition.

Papaya fruits, while usually consumed fresh some of them are processed to make dried products, juices, jams, and desserts (Nafiu et al., 2019; Rajarathnam, 2008). As the papaya processing industry increases particularly in Asia, large quantities of by-product papaya peels and seeds are produced. Usually, the peels and seeds are considered as waste, although they are occasionally used for animal feed. In most cases, they are disposed of producing phytopathogens during composting or landfill that cause ecological problems and pose risks to human health (Thomás et al., 2009).

For commercial markets, fruits are normally harvested at color break and allowed to fully ripen. Fruits with yellow area of 75-100% of the skin are considered matured, ready-to-eat fruits and commonly used as a dessert by the Australian population. The proximate composition of papaya does not show much variation from other similar eating fruits but papaya fruits are an excellent source of provitamin A and a good source of AA. Papaya is ranked as one of the top fruit sources of AA (Gebhardt & Thomas, 2002) with an average AA range of between 45-60 mg/100 g fresh weight (FW), and reported values up to 154 mg/100g FW (Table 3). Red papaya is also considered 38

a good source of lycopene and ranked number 4 of overall foods after red guava, water melon and tomatoes with average values ranging from 0.36 to 3.4 mg/100 g FW (Charoensiri et al., 2009; Gayosso-García Sancho et al., 2011; R.M. Schweiggert, Steingass, Esquivel, & Carle, 2012; R.M. Schweiggert, Steingass, Heller, et al., 2011; USDA, 2015). The analysis of other phytochemicals of papaya cultivars like polyphenols has been limited to a small number of studies. Gayosso- García Sancho et al. (2011) listed some of the major polyphenols found in papaya flesh of ‘Maradol’ cultivars, namely ferulic acid, p-coumaric acid, and caffeic acid. Other researchers reported only low amounts or traces of phenolic compounds in papaya flesh or only identified compounds such as m-coumaric acid, p-coumaric acid, quercetin, apigenin, kaempherol, myricetin and luteolin without quantifying them (Franke et al., 2004; Jindal & Singh, 1975; Lako et al., 2007).

Variation in phytochemical content reported in the literature can be due to differences in cultivar, ripeness of the fruits and growing location. However few reports link variations in phytochemical composition to the fruit ripening status. Thus, this chapter has compared and ranked the “phytochemical value” of commercially available Australian papaya cultivars according to the four internationally accepted maturity stages. This will also be the first study to compare and rank the AOC in different parts of the plant and assess the phytochemical value of papaya cultivars grown in Australia. Since papaya seed has toxicity and antifertility properties (Nirmal K. Lohiya et al., 2006; Verma et al., 2006), it was not included in the present study.

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3.2 Materials and methods

3.2.1 Sample preparation

3.2.1.1 Pulp and peels

Red fleshed papaya from hermaphrodite plants (RB1, RB3, RB4) and yellow fleshed papaya (Y1B cultivar) were supplied from a commercial plantation located at Mareeba, North Queensland, Australia (Longitude: 145.336789, Latitude: -16.956108) during summer months (November to December 2011) with mean minimum/maximum temperature of 20.2/32.0 oC (Australian Government Bureau of Meteorology, 2013) (Figure 3.1). Fruits (n=72) were selected for uniform size, picked green at commercial maturity and couriered to the laboratory facility, arriving within 2 days. Fruits were cleaned and divided into four groups of maturity stages. Stage (1) was characterized by dark green tonalities with yellow area of 0-25% and an overall whitish mesocarp with orange spots as first indication of commencing carotenoid biosynthesis. For Stage (1), fruits were processed immediately; for Stage (2), (3) and (4) (see Figure 3.2), fruits were kept at 25-26oC and allowed to fully ripen according to the selected criteria based on the maturity score used previously by Santamaría-Basulto, et al. (2009).

To prepare composite samples, individual fruit were cut longitudinally and the seeds, placenta tissue and peel were removed. Two slices of papaya flesh of equal amounts (~150 g), from each of the whole fruits (n=6) were cut into cubes and immediately snap frozen, followed by freeze-drying (type, Dynavac Linder & May Pty. Ltd., Windsor, QLD, Australia) and grinding into a fine powder using a MM400 Retsch cryomill (Retsch GmbH, Haan, Germany). Peels (5 mm thick measured from the surface of the fruit) were also snap frozen, freeze-dried and ground into a fine powder. All samples were stored at -20 oC until extraction and analysis.

3.2.2 Morphological traits and characteristic of fruits

Total fruit, peel, and flesh weights were gravimetrically assessed for each ripening stage. Maximum length (Lmax), minimum fruit diameter (Dmin) and maximum fruit diameter (Dmax) were determined (Figure 3.3).

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3.2.3 Color analyses

Changes in visual colour of fruits were assessed using a Minolta CT-310 colorimeter (Konica Minolta Inc. Mahwah, NJ, USA) by measuring Hunter L*a*b* values. Peel and flesh color was determined at 10 randomly distributed points on the exocarp and mesocarp of each fruit.

Figure 3.1 Papaya commercial plantation located at Mareeba, North Queensland, Australia. Source: Gerard Kath, Lecker Farming.

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Papaya hybrid RB 1

Papaya hybrid RB 2

Papaya hybrid 1B Yellow Y1B

Papaya hybrid RB4

Figure 3.2 Ripening stages for papaya. From left to right: Stage (1) represents papaya with yellow area on 0–25% of the skin); Stage (2) represents papaya with yellow area on 25–50% of the skin; Stage (3) represents papaya with yellow area on 50–75% of the skin; Stage (4) represents papaya with yellow area on 75–100% of the skin.

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Figure 3.3 Characteristic morphological dimensions of papaya fruit (halved fruit). Adapted from Schweiggert et al.(2011).

3.2.4 Chemicals

Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), fluorescein disodium salt, 2,2’- Azobis(2-methylpropionamidine) dihydrochloride (AAPH), anhydrous sodium carbonate, Folin- Ciocalteu’s phenol reagent, ascorbic acid, lycopene, β-carotene, β-cryptoxanthin and α-tocopherol were purchased from Sigma-Aldrich Chemicals Co. (St Louis, MO, USA). HPLC-grade methanol, dichloromethane and acetone were purchased from Merck (Darmstadt, Germany), hexane from Ajax (Taren Point, NSW, Australia), ethanol from LabServ (Clayton, VIC, Australia) and methyl tert-butyl from Burdick & Jackson (Muskegon, MI, USA). All chemicals used were of analytical or HPLC-grade.

3.2.5 Equipment

The HPLC system consisted of a SIL-10AD VP auto-injector (Shimadzu, Kyoto, Japan), SCL-10A VP system controller (Shimadzu), LC-10AT VP liquid chromatography (Shimadzu) and a SPD- M10A VP diode array detector (Shimadzu). A microplate fluorescence reader (Victor3V 2030 Multilable Counter, Perkin Elmer, City, MA, USA) was used for antioxidant capacity measurements.

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3.2.6 Carotenoid analysis

Carotenoid analyses were carried out without saponification as described by Fanning et al. (2010) and Schweiggert et al. (2012). Sample preparation was carried out under dim light. Aluminium covered flasks were used throughout preparation to prevent carotenoid degradation and isomerization, respectively. Flesh and peels of papaya samples were snap-frozen and cryogenically milled. A 0.3±0.01 g of the milled sample was exactly weighed into a 50 mL flask. Subsequently, 3 ml of ethanol containing 0.1% butylated hydroxytoluene (BHT) was added and mixed by vortex for 20 s. Samples were then extracted 4-5 times with 3 mL hexane until a colorless residue was obtained. Centrifugation was used to separate layers (5000×g for 3 min, at 4 ◦C). Combined hexane fractions were dried in a centrifugal evaporator at 30 ◦C and then reconstituted in 2 mL of methanol/dichloromethane (50 : 50, v/v), containing 0.1% BHT. Samples were then filtered (0.22 μm syringe filter; Grace, Sydney, NSW, Australia) and placed into HPLC vials, before storing at −80 ◦C prior to HPLC analysis. Standard solutions were prepared similarly in methanol/dichloromethane (50 : 50, v/v), containing 0.1% BHT. Thirty microlitres of each extract was injected onto a YMC C30 Carotenoid Column, 3 µm, 4.6 × 250 mm (Waters, Milford, MA, USA), with a mobile phase consisting of 92% methanol/8% 10 mmol L−1 ammonium acetate (phase A), and 100% methyl tert-butyl ether (phase B). The retinol activity equivalents (RAE) per 100 g for maturity Stage (3) and Stage (4) of all studied cultivars were calculated; ß-carotene (12 µg), or 24 µg of α-carotene or ß-cryptoxanthin is equivalent to one RAE (WHO & FAO, 2004).

3.2.7 Ascorbic acid determination

Ascorbic acid (AA) was extracted and analysed according to Sánchez-Mata et al. (2000) with slight modifications. Briefly, papaya powder samples (10-50 mg) were extracted with 3 mL of 4.5% meta- phosphoric acid for 15 min using a plate shaker at 300 rpm at 25oC. The sample was centrifuged and the supernatant was filtered through a 0.45 µm filter. The sample (20 µL) was injected onto a Phenomenex Luna C18 column protected by a Phenomenex 4.0x3.0 mm i.d. C18 ODS guard column (Phenomenex, Penant Hills, NSW, Australia) and eluted under isocratic conditions with water acidified with sulfuric acid (0.01%) to pH 2.6 in water. Detection was carried out at 245 nm at a flow rate of 1.0 mL/min. Quantification was performed using an AA standard curve with concentrations ranging from 5 to 320 mg/L. Data was expressed as milligram ascorbic acid per gram fresh weight (mg/g FW).

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3.2.8 Polyphenols

Polyphenol (free and bound) analyses were carried out as described by Vinson, Su, Zubik, and Bose (2001). Briefly, 0.5g of sample was accurately weighed in a screw-capped tube. For free phenols, 5 mL of 50% methanol/water and the sample were vortexed for one minute and heated at 90 °C for 3 h with vortexing every 30 min. After the samples were cooled, they were diluted to 10 mL with methanol and centrifuged for 5 min at 5000 rpm with a benchtop centrifuge to remove solids. Total phenols were extracted with 5 mL of 1.2 M HCl in 50% methanol/water and treated as above. The sample (30 µL) was injected into the HPLC system. Under the experimental conditions, linear calibration curves were obtained over the entire range of the standard concentration (quercetin, rutin, chlorogenic acid, quercetin -3-glucosides, gallic acid). The calibration curves demonstrated acceptable linearity with correlation coefficients r2 >0.995 except for quercetin (r2 =0.993) and chlorogenic acid (r2 =0.991). Then, the identified compounds present in the papaya extracts, before and after hydrolysis, were calculated using the corresponding calibration curves.

Identification (tentatively) of the polyphenolic compounds was carried out on a Shimadzu Nexera X2 UHPLC system (Shimadzu, Rydalmere, NSW, Australia), combined with a Shimadzu LCMS- 8050 triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source operated in negative mode. The analyses were carried out at the Department of Agriculture and Fisheries, Queensland Government, Health and Food Sciences Precinct, Coopers Plains, QLD 4108, Australia.

3.2.9 Antioxidant capacity (ORAC assay and total phenolics)

Samples were extracted according to Wu et al. (2004) using an Accelerated Solvent Extraction unit (ASE 350®, Dionex, City, CA, USA) with slight modifications. Samples (0.25 g) were mixed with diatomaceous sand (Dionex) and then transferred to a 22 mL extraction cell and were initially extracted with hexane/dichloromethane (1:1, Hex/Dc) followed by acetone/water/acetic acid (AWA) (70:29.5:0.5;v/v/v). AWA extracts were transferred to a 25 mL volumetric flask and diluted with AWA to 25 mL total volume. This solution was used to measure the hydrophilic oxygen radical absorbance capacity (H-ORAC) and total phenolic content after dilution with phosphate buffer solution (75 mM, pH 7.0). Extracts from the Hex/Dc step were evaporated and the residue was reconstituted with 10 mL of acetone. After centrifugation, the supernatant was used to measure lipophilic oxygen radical absorbance capacity (L-ORAC) following further dilution with assay buffer as necessary. ORAC assay was carried out on a Victor3V microplate fluorescence

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reader according to previous published procedures by Huang et al. (2002) and Isabelle et al. (2008). Data were expressed as micromole Trolox equivalents per gram fresh weight (µmol TE/g FW). TPC was determined using the Folin-Ciocalteau reagent as described by Isabelle et al. (2008). Data were expressed as milligram gallic acid equivalents per gram fresh weight (mg GAE/g FW).

3.3 Statistical analysis

Data were expressed as mean ± standard deviation (SD) of at least 5 independent analysis and statistically analyzed using SPSS Statistic version 25.0 for Windows (SPSS Inc, Chicago, IL, USA). Each analysis was conducted using a Multivariate ANOVA. Pearson correlation coefficients were determined and the significant difference was set at p < 0.05. The Tukey HSD and Duncan Test with a = 0.05, was used to test for differences in group means.

3.4 Results and Discussion

3.4.1 Morphological characteristic and colour parameters of fruit.

Morphological dimensions, traits and characteristic of C. papaya cultivars are shown in Table 3.1 and 3.2. Weight, Lmax, Dmax and Dmin of fruit observed were significantly different (p<0.05) between cultivars. The fruit samples of all four cultivars ranged from 619 to 820 g in weight, 16–17 cm in

Lmax, 9-12 cm and 4-6 for Dmax and Dmin among the different stages of fruit ripening, respectively (Table 3.2). All four papaya cultivars ripened fully after about nine days at 25-260C. Colour parameters showed dark green tonalities in the peel at stage (1) (lightness L=36 to 52, a= -15.02 to - 12.13 and b= 25.01-38.57 chromaticity), which successively turned into bright yellow and orange colour reaching L readings from 59-67 (Table 3.3). Progression of fruit through different maturity stages results in physiological and biochemical changes that modify fruit composition and enables its consumption (Pereira et al., 2009). Recent study showed maturity stages from ripened on-tree Maradol papayas cultivar acquire better quality parameter such as epicarp color, total soluble solids, mesocarp firmness, moisture content, pH, and titratable acidity and sugar-acid ratio than in storage (Barragan-Iglesias, Mendez-Lagunas, & Rodriguez-Ramirez, 2018). However, for commercial markets, fruits are normally harvested at color break stage (1) and allowed to fully ripen. Fruits at stage (3) and stage (4) were considered matured, edible and commercially used as deserts for the Australian population.

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Table 3.1 Sample Table. Sample Code Description RB1 Red-fleshed hybrid 1 cultivar RB2 Red-fleshed hybrid 2 cultivar RB4 Red-fleshed hybrid 4 cultivar Y1B Yellow-fleshed hybrid 1B yellow cultivar Stage (1) Represents papaya with yellow area on 0–25% of the skin. Stage (2) Represents papaya with yellow area on 25–50% of the skin. Stage (3) Represents papaya with yellow area on 50–75% of the skin. Stage (4) Represents papaya with yellow area on 75–100% of the skin.

Table 3.2 Morphological traits and characteristics of fruits from different papaya cultivars. RB1 RB2 RB4 Y1B Number of fruit 24 24 12 12 Flesh colour class Red Red Red Yellow Gender Hermaphrodite Hermaphrodite hermaphrodite Gynodioecious Type Hybrid Hybrid Hybrid Hybrid Fruit weight (g) 619.22±17.9 799.07±18.48 820.89±2.13 795.95±20.04 L max/cm 17.74±2.32 16.64±1.40 17.31±1.56 15.82±1.21 D max/cm 8.97±1.02 11.95±1.32 11.03±1.24 11.40±1.21 D min/cm 4.4±0.22 4.63±0.26 4.98±0.57 6.05±0.66 Results are given as mean±SD of triplicate measurements. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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Table 3.3 Skin colour parameters (L:lightness; a and b:chromaticity) of different papaya cultivars stored at 25-260C. Colour Maturity stages parameters Stage (1) Stage (2) Stage (3) Stage (4)

RB1 L 53.34±0.59a 60.70±0.602b 65.43±0.43 c 66.43±0.38 c A -12.77±0.77 a -12.00±0.40 a 1.085±0.86 c 11.09±0.31 d B 38.57±0.95 a 46.46±0.59 b 57.23±0.55 c 65.88±0.47 d

RB2 L 53.69±0.53 a 66.06±0.36 b 63.68±0.55 c 67.19±0.39 d A -15.02±0.61 a -0.365±0.45 b 3.05±0.66 c 12.11±0.74 d B 35.73±0.5 a 58.06±0.47 b 55.65±0.75 b 59.12±0.48 d

RB4 L 35.69±0.23 a 40.62±0.37 b 62.37±0.52 c 62.70±0.53 c A -12.35±0.14 a -5.32±0.36 b 2.46±0.72 c 11.85±0.58 d B 25.01±0.22 a 32.92±0.62 b 58.76±7.72 c 57.70±0.65 c

Y1B L 36.19±0.25 a 40.61±0.38 b 59.45±0.86 c 65.33±0.38 d A -12.13±0.17 a -1.32±0.46 b 3.57±0.68 c 14.55±0.39 d B 27.02±0.40 a 40.92±1.22 b 52.56±1.28 c 59.05±0.71 d

Results are given as mean±SD of ten measurements. Values with different letter are significantly different at p < 0.05 within the same row. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

3.4.2 Carotenoids

Mean values and standard deviation for carotenoids (-carotene, -cryptoxanthin and lycopene) are presented in Figure 3.4. The content of total carotenoids in papaya flesh at different ripeness stages expressed as mg/100 g FW increased 2-fold with the level of ripeness of the fruit. Analysis of variance showed significant differences (p<0.05) in lycopene and -carotene content between Stage (2) and Stage (4) within and between cultivars. However, Stage (3) and Stage (4) showed no significant differences in -carotene content within red-fleshed cultivars compared to yellow- fleshed cultivar (Figure 3.4).

Major carotenoids found in red-fleshed papaya were β-carotene, β-cryptoxanthin, and lycopene with lycopene representing 42-58% of the total carotenoids. In contrast, β-carotene represents 60-78% of the total carotenoids with no detectable lycopene in the yellow-fleshed cultivar. Present results coincided with those of Marelli de Souza et al. (2008) who observed similar profiles of carotenoids found in ‘Solo’ and ‘Formosa’ papaya with lycopene representing 65% of the total carotenoids.

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Similar carotenoids profile were also observed with ‘Maradol’ papaya (Rivera-Pastrana et al., 2010)(Rivera-Pastrana et al. (2010) (2008; 2010). Previous studies had reported on the differences in carotenoid profiles and organisation of carotenoids in the cell for yellow- and red-fleshed varieties of papaya (Chandrika et al., 2003; R.M. Schweiggert, Steingass, Mora, et al., 2011). Lycopene content of yellow fleshed fruit was undetectable or was more than 400 times lower than the red cultivars. This was elucidated by molecular study which observed the presence of two types of lycopene -cyclase genes in carotenoid accumulation in red and yellow-fleshed papaya fruit (Devitt, Fanning, Dietzgen, & Holton, 2009). In yellow fruit, both genes are expressed, resulting conversion of all of the available lycopene to -carotene (and subsequently to -cryptoxanthin). However, in red papaya fruit, one of the genes is nonfunctional resulting in the accumulation of both lycopene and -carotene in red-fleshed papaya fruit as maturity increases (Devitt et al., 2009). Present results for lycopene content in red fleshed papaya (0.7-1.1mg/100g FW) were close to the USDA lycopene content of papaya with values of 1.8mg/100g FW. Reported values for lycopene in papayas ranged from 0.36-3.4 mg/100g FW (Charoensiri et al., 2009; Gayosso-García Sancho et al., 2011; R.M. Schweiggert et al., 2012; R.M. Schweiggert, Steingass, Heller, et al., 2011). Lycopene does not have vitamin A activity but contributes to papaya flesh reddish color. Consumption of food products containing significant amounts of lycopene, demonstrate an inverse relationship between lycopene intake and prostate cancer risk, from prospective and retrospective epidemiological studies supported by in vitro and in vivo experiments(van Breemen & Pajkovic, 2008). Furthermore, in vitro experiments indicate other mechanisms of chemoprevention by lycopene including induction of apoptosis and antiproliferation in cancer cells (van Breemen & Pajkovic, 2008). Tomatoes are considered a good source of lycopene, with average levels ranging from 3.0 to 5.0 mg/100 FW (Perkins-Veazie, Roberts, & Collins, 2007; Thompson et al., 2000). Although lycopene content of papaya fruit in the present study was less than that of tomatoes, the levels are considered high in ranking of overall food (USDA, 2015) and papaya can also be considered as a good source of this carotenoid. In addition, recent human randomised cross-over study found that bioavailability of lycopene from papaya was approximately 2.6 times more bioavailable than that from tomatoes. Beta carotene from papayas was also found to be approximately three times higher than that from carrots and tomatoes (R.M. Schweiggert et al., 2014).

The content of total carotenoids in papaya flesh reported in this study (1.16-2.47mg/100 g FW) is within the same range as reported by others (0.92-3.27 mg/100 FW) (Bari et al., 2006; Gayosso- García Sancho et al., 2011; Veda et al., 2007; Wall, 2006). Differences between cultivars observed 49

in the present study can be attributed to papaya hybrid differences, or possibly, the differences in the age of harvested fruit. Although papaya was harvested at the stage designated as color break slight variability in maturity and ripening could potentially contribute to a wide range of differences in carotenoid levels. Similar to the present study, other studies have reported that carotenoid levels in papaya fruit were highly variable between and within cultivars. For example, variations in carotenoid levels of up to 4 to 7-fold between papaya cultivars and regions have been reported (Ikram, Stanley, Netzel, & Fanning, 2015).

-carotene levels in peels from all cultivars studied (0.45-1.88 mg/100 g FW) were found to be higher (p<0.05) compared to the flesh with values ranging from 0.45-1.88 mg/100g FW. Moreover, -cryptoxanthin values of the peels were also higher (p<0.05) in Y1B, RB2 and RB1 Stage (4) compared to the flesh (Figure 3.4). Lycopene content on the other hand, was significantly lower (p<0.05) by 25-75% in the peels compared to papaya flesh. There are no other literature reports on the content of papaya peels, previous studies on the peels of tropical fruits, e.g. mango peel, showed high carotenoid content with values ranging from 0.55-3.33 mg/100g FW (Ajila, Naidu, Bhat, & Rao, 2007; Ketsa, Phakawatmongkol, & Subhadrabhandhu, 1999). Although peel is inedible, it is fermented to make functional extracts such as “Fermented Papaya Preparation” (Patidar, Nighojkar, Kumar, & Nighojkar, 2016). As the consumption, harvest and processing increases, larger quantities of papaya peels, a so called by-product, accumulate which could potentially be utilised as a source of functional ingredients for the food industry.

Vitamin E (α-tocopherol) content was below the detection limit (<0.001µg/g) for all the samples. The vitamin E content for papaya fruits has been reported as undetectable or in low concentration (0.1-0.3mg/100g FW)(Charoensiri et al., 2009; Monge-Rojas & Campos, 2011; USDA, 2015).

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A

e h h d d d g,d d d c g,d g g g g,d g a g g b f f g f

g B g

c i,c c c c c c a,d c d a d a a a a,b b b b b f e

c c C c a c c c a a a a a d a,d d

b b b

Figure 3.4 Carotenoid content concentrations [mg/100g FW] in papaya flesh and peels at different maturity stages and cultivars. ß-carotene content (A); ß-cryptoxanthin, (B), and Lycopene (C). Results are given as mean±SD of 6 independent analysis. Values with different letters are significantly different at p < 0.05. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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3.4.3 Ascorbic acid

Ascorbic acid content of papaya flesh in the present study ranged from 37.0-61.3 mg/100g FW. Results indicated that AA increases as the fruits ripen. This can be observed by higher value for Stage (3) and Stage (4) maturities for all cultivars except RB1 cultivars (Figure 3.5). RB4 has the highest AA content followed by Y1B, RB1 and RB2, respectively. AA for cultivar RB4 and Y1B was significantly higher (p<0.05) by 25-38% than cultivar RB1 and RB2. No significant differences were found for AA at Stage (3) and Stage (4) within papaya cultivars studied except for RB1, which had significantly lower values (p<0.05) at Stage (4). Ioannidi et al. (2009) and Rubinskienė et al. (2008) reported the lowest level of AA in tomatoes and berries as fruit approach ripeness. AA for peels from all cultivars studied except for RB1 were found to be significantly lower (p<0.05) compared to the flesh with values ranging from 31-48 mg/100g FW (Figure 3.5). No significant differences were found in the AA content at different maturity stages.

The increase in AA content as fruits approached ripeness in this study was similar to work conducted by Tripathi et al. (2011) who found the highest AA content for fully ripe (off the tree) and ready-to-eat papaya fruit compared to matured green or colour break stage. During fruit ripening and softening, plants convert uronic acids to ascorbic acid as a mechanism to salvage carbon arising from the breakdown of cell walls (Davey et al., 2000). Thus, the higher concentrations of ascorbic acid observed in papaya fruits are probably associated with carbohydrate metabolism and accumulation of maximum levels of sugar.

The AA values observed in this study were almost 2 times higher than the AA from local Bangladesh papaya cultivars (Bari et al., 2006), on a par with other published AA data obtained from different geographical origins (Kelebek, Selli, Gubbuk, & Gunes, 2015; Leong & Shui, 2002; Septembre-Malaterre, Stanislas, Douraguia, & Gonthier, 2016; Tripathi et al., 2011; USDA, 2007; Wall, 2006) and 2 to 3 times lower than Spanish and Brazilian papaya cultivars. Comparison of AA in the present study with different types of papaya cultivars and origin was defined in Table 3.4. Most studies have shown no significant difference in the AA content of papaya between cultivars (Tripathi et al., 2011; Wall, 2006). However, the AA contents measured are affected by the amount and intensity of sunlight exposure to the crop and individual fruit. Longer day lengths and higher light intensities in the summer months, for example, can increase the concentrations of ascorbic acid and glucose in fruits (Lee & Kader, 2000). These present Australian papaya cultivars were collected during summer months with mean minimum/maximum temperature of 20.2/32.0 oC. This could be

52

one of the reasons for different AA values found in present cultivars and results previously published of cultivars at different weather locations (Table 3.4). In addition, AA values in the present study were also comparable to that of citrus orange cultivars ranging from 45 (Florida var), 49 (Valencias California), 59 (Navel) and 64 mg/100g FW (blood oranges). Thus, Australian cultivars can be considered as a good source of AA (FSANZ, 2017).

The AA values were found to correlate well with H-ORAC (r = 0.64). AA is a significant contributor to the hydrophilic antioxidant capacity of fruits (D. Huang et al., 2005; Prior et al., 2005). Thus, the high H-ORAC value observed in ripe papaya in the present study may be due to the high levels of AA in the fruits. Similarly, Leong and co-workers (2002) found that AA contributes the most to the antioxidant properties of the fruits with contributions of 48% for ‘Solo’ and 62.3% for ‘Foot long’ papaya. The result was later confirmed by Isabelle and co-workers (2010) who found that AA contributes to 97% of AOC of the fruits.

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Table 3.4 Ascorbic acid (mg/100g FW) and pro-vitamin A carotenoids (µg RAE/100g FW) in different types of papaya cultivars and origin. Papaya Cultivars Source AA (mg/100g) Pro-vitamin A References (µg RAE/100g)a RB4 Australia 55.3-61.3 88.3-99.8 b Present result RB2 Australia 37.0-39.7 67.1-67.8b Present result RB1 Australia 46.3-51.0 64.2-64.6 b Present result YB1 Australia 49.3-58.5 30.8-62.5 b Present result Kapoho Hawaii 45.4 29.9 Wall (2006) Laei Gold Hawaii 51.3 48.2 Wall (2006) Rainbow Hawaii 51.8 50.3 Wall (2006) Rainbow Hawaii 57.4-84.9 N.A. Tripathi et al. (2011) Rainbow (Hybrid line) Hawaii 43.6-75.9 N.A. Tripathi et al. (2011) Sunrise and Kp hybrid Hawaii 68.3-84.9 87.6-262 Tripathi et al. (2011) Sunrise Hawaii 55.6 45.6 Wall (2006) Sun Up Hawaii 45.3 20.4 Wall (2006) Pococi Costa Rican Costa Rica N.A. 132-166 Schweiggert, Steingass, Mora, Esquivel, and Carle (2011) Red lady Florida 153.8 N.A. Mahattanatawee et al. (2006) Maradol Mexico 25.1-58.6 30.84-85.8 Sancho et al. (2011) Local 1 Yellow fleshed Sri Lanka N.A. 75.84 Chandrika et al. (2003) Local 2 Red fleshed Sri Lanka N.A. 152.92 Chandrika et al. (2003) Local 1 Bangladesh 8.09-31.0 20.8-33.8 Bari et al. (2006) Local 2 Bangladesh 7.3-29.4 16.7-32.5 Bari et al. (2006) Honey dew India N.A. 58.3 b Veda et al.(2007) Surya India N.A. 60.8 b Veda et al. (2007) Solo Malaysia 48.5 42.4 Isabelle, et al. (2010) Solo Malaysia 67.8 N.A. Leong and Shui (2002) Foot Long Malaysia 45.2 N.A. Leong and Shui (2002) Solo Brazil 101.4-112.4 27.5-40c Marelli de Souza et al.(2008) Formosa Brazil 59.9-77.8 15.8-46.7c Marelli de Souza et al.(2008) N.A. Brazil N.A. 168 c* L. M. R. d. Silva et al. (2014) BaiXinho do Santa Spain 98-154 N.A. Hernández, Lobo, and Amalia González (2006) Local Italy 88.2 N.A. Vinci et al. (1995) Oblong Red Nigeria 41.1 310.3c Nwofia et al. (2012) Pear Orange Nigeria 39.3 305.6c Nwofia et al. (2012) Round orange Nigeria 39.9 309.3c Nwofia et al. (2012) Oblong yellow Nigeria 36.4 258.7c Nwofia et al. (2012) Round yellow Nigeria 43.4 293.7c Nwofia et al. (2012) N.A. N.A. 60.9 47.0 USDA (2015) aProvitamin A carotenoids include ß-carotene, α-carotene, and ß-cryptoxanthin. Retinol Activity Equivalents (RAE): (µg ß-carotene/ 12) + (µg α-carotene/ 24) + (µg ß-cryptoxanthin/ 24) bRAE was calculated based on stage (3) and (4) fruits. cRAE was calculated based on ß-carotene value only. *based on dry weight RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid. N.A.: Not Available

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a c c c c c d d d d d d,b,f d,b,f d d b f e b f f g h i i c

Figure 3.5 Ascorbic acid [mg/100g FW] content of papaya flesh and peels at different maturity stages and cultivars. Results are given as mean±SD of 6 independent analysis. Values with different letters are significantly different at p < 0.05. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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3.4.4 Polyphenols

Free and bound phenolic content of papaya flesh and peel were determined and is shown in Fig 3.6- 3.7 Tentatively, plant polyphenols may form both ester and ether linkages due to their bifunctional nature through reactions involving their carboxylic and hydroxyl groups which allows phenolic compound to form cross-links with cell wall macromolecules such as polysaccharides, proteins or fatty acids (You, Wang, & Kuo, 2001). These cross-linked fractions referred to as conjugated or bound phenolics, are not extractable in aqueous organic solvents, but may be released by acid, alkali or enzymatic treatment. Thus, in present study, acid was used to hydrolyse and extract bound phenolics.

Four major phenolic compounds in papaya flesh and peel of the studied Australian grown cultivars have been separated and tentatively identified by HPLC and LC-MS. Caffeic acid was the most abundant of phenolics acids in the flesh and peels, followed by gallic acid. In the case of flavonoids, rutin and quercetin were the major compounds detected. Total phenolic acids content in papaya flesh and peels were found to be in the range of 0.002–0.16 mg/100g FW and 0.5-4.24 mg/100g FW, respectively, whereas flavonoids ranged from 0.02-0.04mg/100g FW and 0.9-3.36 mg/100g FW, respectively. Phenolics compounds, but not the concentrations, coincide with the previous report on the identification of phenolics in papaya fruit made by Rivera-Pastrana et al. (2010) and Kelebek et al. (2015).

Gallic acid in Stage (2) to (4) for the red cultivars was in the range of 0.01-0.03 mg/100g FW (p< 0.05) which increased 20-50% during ripening. Caffeic acid had a concentration of 0.07-0.1 mg/100 g FW in Stage (2), and increased significantly (p<0.05) during ripening in Stage (4). Rutin content for papaya cultivars was in the range of 0.01-0.02 mg/100 g FW, without significant differences with respect to maturity stages. The same pattern was observed for quercetin except for RB4 with significantly higher values in Stage (4) (Fig 3.7-3.8). In contrast, the phenolic acids and flavonoids content of the peels tend to decrease significantly by 13-33% from Stage (2) to (4).

From the results, papaya have a large proportion of the phenolics as bound, ranging from 16 to 91%. Phenolic compounds occur mostly as soluble conjugates and insoluble forms, covalently bound to sugar moieties or cell wall structural components. The phenolic contents and antioxidant activities of fruits could be underestimated if the bound phenolic compounds are not considered. However, absorption mechanisms for bound phenolic compounds in the gastrointestinal tract

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greatly depend on the liberation of sugar moieties (Acosta-Estrada, Gutiérrez-Uribe, & Serna- Saldívar, 2014). Food processes such as alkaline and acid hydrolyses have potential to release phenolics associated to cell walls. In the present samples submitted to acid hydrolysis, the concentrations of caffeic acid and quercetin increased significantly and this point demonstrates that such compounds are present in papaya tissues mostly as esters and/or glycosides bound to the plant matrix. Chlorogenic acid was detected in trace amounts.

The phenolic compounds in the peels in present study was significantly (p<0.01) higher compared to the flesh. Studies performed in other fruits like mango have determined that fruit skin normally has higher concentrations of phenolic compounds than flesh. The reason for this is probably because phenolic compounds are tend to accumulate in the dermal tissues of the plant body due to their potential role in protecting against ultraviolet radiation, acting as attractants in fruit dispersal, and as defence chemicals against pathogens and predators (Toor & Savage, 2005). Present study was in agreement with study from Rivera-Prastana et al. (2010) who reported caffeic acids to be among the highest quantified compounds for peels with values ranging from 4.6–6.8 g/100g DW. However, ferulic acid were reported to be the highest compounds detected in peels (13.3–16.2 g/100g DW )(Rivera-Pastrana et al., 2010). In terms of maturity stages, the present study coincides with earlier study on mangoes which reported that extractable phenolics were higher in raw peel compared to ripe peel (Ajila & Rao, 2008) whereas in the flesh, the values increased as ripening occured (Palafox-Carlos, Yahia, & González-Aguilar, 2012). Nonetheless, an increase or no change of total or individual phenolic content during fruit ripening has also been documented in several other studies (Kim et al., 2009; Robles-Sánchez et al., 2009). Polyphenol composition of papaya cultivars has been limited to a small number of studies. Caffeic acid and quercetin compounds were found to be detected in the flesh and peels by others. Gayosso- García Sancho and co-workers (2011) in their study quantitate ferulic acid (187-277 mg/100g dry weight (DW)), p-coumaric acid (136-230 mg/100g DW), and caffeic acid (113-176 mg/100g DW) from ‘Maradol’ papaya cultivars. Other researchers reported different types of polyphenol compounds in papaya flesh such as m- coumaric acid, p-coumaric acid, quercetin, apigenin, kaempherol, myricetin and luteolin with and without quantifying the levels (Franke et al., 2004; Jindal & Singh, 1975; Lako et al., 2007). Present values for quercetin (0.01-0.03 mg/100g FW) were higher than results of <0.001 mg/100g FW found in the study by Franke and co-workers (2004)) for papaya planted in Hawaii. In contrast, Lako and co-workers (2007) found higher values of quercetin (2 mg/100g FW) for Hawaiian papaya cultivars, planted in Fiji, compared to the present study.

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The total polyphenols (HPLC) measured in the present study were significantly different (p<0.05) between papaya cultivars. The quantity of phenolics or polyphenols in foods can be affected by agricultural practices such as the use of synthetic fertilizers that offer more accessible sources of nitrogen which can accelerate plant and the production of secondary metabolites. Polyphenol content could also be affected by higher exposure of the plant to stressful environment such as the absence of synthetic pesticides. Attacks by insects or fungi can also induce the production of natural defence substances such as phenolic compounds (Winter & Davis, 2006; Woese, Lange, Boess, & Bogl, 1997). Both hypotheses would result in foods with higher antioxidant capacity as a consequence of the higher polyphenolic content. Faller and Fialho (2010) had compared the polyphenol content of organic and conventional plant foods. They found that among fruits studied, papaya seems to be more affected by alteration in agricultural management with approximately 70% higher hydrolysable polyphenols in organic fruit compared to the conventional counterpart. This could be one of the reasons for different phenolic values found in present cultivars. Nonetheless, phenolic content composition in food can also be altered by storage, location, handling, and processing conditions (Rinaldo, Mbéguié-A-Mbéguié, & Fils-Lycaon, 2010).

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b b b b b b b

a a

b b d d d d a a

e

c c c

Figure 3.6 Phenolic acids [mg/100g FW] content of papaya flesh at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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b a.b b b b b b a a

c c c

b a a

c c c

d e d d e d c

Figure 3.7 Flavonoids [mg/100g FW] content of papaya flesh at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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a a b e e f

g g c c c,d d

b b a c f h i g g

d e e

Figure 3.8 Phenolic acids [mg/100g FW] content of papaya peels at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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a b b b b f f f f

c ,c c

a a b b

c c c c c

d d d

Figure 3.9 Flavonoids [mg/100g FW] content of papaya peels at different maturity stages and cultivars. Results are given as mean±SD of 5 independent analyses. Values with different letters are significantly different at p < 0.05. RB1, RB2, RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid.

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A B

D C

D Figure 3.10 Molecular structure of secondary metabolites analyzed in papaya extract: Gallic acid (A); Rutin (B); Quercetin (C), and Caffeic acid (D).

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3.5 ORAC and TPC

The AOC of flesh and peels varied according to maturity stages of different papaya cultivars as presented in Figure Among the 4 cultivars studied, RB4 and RB1 flesh contained the highest H- ORAC and L-ORAC ranging from 11-43 and 5-9 uM TE/g FW, respectively (Figure 3.11). H- ORAC in papaya peels in the present study were 2-12 fold higher compared to the flesh. The present results showed no significant differences for red-fleshed cultivars, except RB2 at Stage (2) and (3) fruits, compared to Stage (4) which dropped to less than half the content (p<0.05). In contrast to the red-fleshed cultivars, AOC for YB1 increased significantly (p<0.05) by 19-27% (from Stage 2 to Stage 4) as fruit ripened. No significant differences in AOC for peel were observed at any stage of maturity.

The AOC of fruit could be affected by the types of cultivar and maturity. This could be seen by the large range (up to 100% of the mean value) in the previous results reported (Hdider, Ilahy, Tlili, Lenucci, & Dalessandro, 2013; Wu et al., 2004). Hence, variation of AOC within the same fruit species in the present study are expected. In each cultivar, the hydrophilic antioxidant fraction was the main contributor for AOC for papaya fruits, flesh and peels, with levels being significantly greater (p<0.05) than lipophilic fraction. This was similarly reported by Wu et al. (2004) on H- ORAC compared to L-ORAC values of common fruits studied.

Previous studies found relatively low H-ORAC values (2.7-3.0 uM TE/g FW) for different papaya cultivars (Haytowitz & Bhagwat, 2010; Isabelle et al., 2010; Patthamakanokporn et al., 2008) compared to the present results for RB4, RB1 and YB1 flesh fruit (Figure 3.11). No literature data was found for L-ORAC for papaya for comparison, however fruits and vegetables in general have low lipophilic antioxidant values, often being less than 5 uM TE/g FW (Haytowitz & Bhagwat, 2010). The H- and L-ORAC values for papaya peels could not be traced in the literature for comparison.

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The TPC in papaya flesh and peels for RB1, RB2, RB4 and Y1B at different maturity stages is shown in Figure 3.12. TPC for Australian cultivars RB1, RB2 and Y1B was found to be in the range of previous publications with the highest reported value of 2.08 mg GAE/100g FW (Lako et al., 2007; Patthamakanokporn et al., 2008). However others had found significant high and low TPC in papaya fruit of 33.4-41.3 mg GAE/100g FW and 0.02 mg GAE/100g FW, respectively (Isabelle et al., 2010; Septembre-Malaterre et al., 2016). TPC of the peel was significantly higher (p<0.05) compared to the flesh in all cultivars studied with values ranging from 9.7-19.8 mg GAE/100g FW.

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c a a a a f f f f f

b b b b b b b g g e g g g g

Papaya cultivars at different maturity stages

Figure 3.11 Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g FW) content of Y1B papaya flesh and peel at different maturity stages. Results are given as mean±SD of 6 independent analysis. Values with different letter are significantly different at p < 0.05.

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c b

b b b b b b b b b f

a a a e g g g g g g h h

Figure 3.12 TPC of papaya flesh and peel at different maturity stages (mg GAE/100g FW). Results are given as mean±SD of 6 independent analysis. Values with different letter are significantly different at p < 0.05.

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3.5.1 Provitamin A, AA and polyphenols per serving in fruit

To evaluate the provitamin A, AA and polyphenols consumed, serving size must be taken into consideration. The defined serving size for papaya varies worldwide from 140 g-150 g per serving. In the present study, serving size for papaya was assigned based upon the USDA National Nutrient Database for Standard Reference, release 28 (2015), which defines a serving size as one cup of medium fruit (145 g) containing only the edible portion. However, the serving size was used for comparative purpose and might not reflect the actual consumption by the Australian population.

Dietary need for vitamin A can be supplied by provitamin A carotenoids in fruits. For comparison, RB4, RB2, RB1 and Y1B give a total value ranged from 88.3-99.8, 67.1-67.8, 64.2-64.6 and 30.8- 62.5 μg RAE/100 g FW, respectively. Thus, red and yellow-fleshed papaya would give approximately 93-145 and 45-90 μg RAE per serving size of papaya. Since 700 μg RE/day for women and 900 μg RAE/day for men (19–70 years) are Recommended Dietary Intake (RDI) values according to NHMRC and MoH (2006), 3-4 servings of red-fleshed papaya would cover half of the recommended daily requirements.

Values of provitamin A in the present study were in agreement with other published data obtained from different geographical origins (Chandrika et al., 2003; Tripathi et al., 2011; Veda et al., 2007; Wall, 2006). Comparison of provitamin A in present study with different types of papaya cultivars and origin is shown in Table 2.

AA value for papaya per serving for all cultivars at Stage (3) and Stage (4) in present study ranged from 54-89 mg per serving of fruits. This value is sufficient to give 100% RDI for vitamin C of 45 mg/day for the Australian population (NHMRC & MoH, 2006).

Polyphenols for papaya per serving ranged from 0.03-0.28 mg with 0.03-0.06 mg of flavonoids per serving of fruit. There are no current recommendations for daily polyphenol or flavonoid intake at this time. Although the papaya is not likely to become a fruit that contributes a significant amount of phenolic acids and flavonoids to the Australian diet, papaya fruit can be considered a good valuable contributor to healthy diet like apples, oranges and grapes (Brannan, Peters, & Talcott, 2015; Neveu et al., 2010).

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3.6 Conclusions

Overall, findings of this study suggest that phytochemical composition and AOC significantly varied across different papaya cultivars, maturity stages and plant parts. Stage (3) and (4) Australian papaya fruits will provide a valuable source of dietary provitamin A and vitamin C. One serving of Australian papaya per day would give 100% of the RDI for vitamin C and 3-4 servings per day of red-fleshed Australian papaya would cover 50% of the RDI for vitamin A, respectively. To summarize, this study has generated novel and significant data for epidemiological research and providing support for dietary guidelines and efforts to promote papaya consumption for public health benefits.

The next chapter (Chapter 4) includes the methods of sampling and sample collection and preparation of thermal treatment (for pulp and leaves) . The experiments that were undertaken will be antioxidant capacity (ORAC and TPC), vitamins and polyphenols content of different parts (pulp, peel and leaves) of papaya Linn cultivars. This chapter will fall under objective 2; Investigate the effect of different papaya cultivars/varieties, parts of the plant (pulp, peel and leaves) and maturity stages on phytochemical (Vitamins and polyphenols) composition and AOC.

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CHAPTER 4

4. Ascorbic acid, Carotenoids, Polyphenol and Antioxidant capacity of different parts of C.papaya Linn cultivars: Unripe fruits and leaves and after traditional preparation for culinary uses.

4.1 Introduction

Numerous studies on the nutritional value of Western foods have been published, whereas studies on so called ‘inedible’ (plant) food consumed in Asian countries are lacking (Ikram et al., 2009). Papaya is one of highly popular plant among Asian population (Ikram et al., 2015). In Malaysia, Indonesia and Thailand, the fruit is consumed on a daily basis as a part of the normal basal diet. Young and mature papaya leaves are cooked as main dishes and served with rice. While the young leaves are usually eaten after blanching they may also be served as salad (Aliah & Ikram, 2018) and often with ‘sambal’, a type of sauce typically made from a variety of chili peppers. Apart from its use as a food, the leaf is also consumed after boiling and eaten as a functional “vegetable” to treat diabetes and high blood pressure (Manish, Liswaria, Kosheelah, & Puvaneswaran, 2012; Ong & Norzalina, 1999).

According to scientific literature, majority of Asian and African countries depends on traditional medicine for their primary health care due to economic and geographical constraints (Ekor, 2014; Oyebode, Kandala, Chilton, & Lilford, 2016; Payyappallimana, 2010). Medicinal plants have been used widely to treat a variety of vector ailments such as malaria (Kadir, Yaakob, & Mohamed Zulkifli, 2013). The demand for plant-based medicines is growing as they are generally considered to be safer, non-toxic and less harmful than synthetic drugs (Kadir et al., 2013). Additionally, crude formulations of papaya leaf, using suspensions of powdered leaves in palm oil and juice have been successfully employed in folk medicine in continental Malaysia for the treatment of dengue infections with haemorrhagic manifestations (Kathiresan et al., 2009; Subenthiran et al., 2013). Similarly, unripe fruits are also used as a vegetable such as making pickles, cooked or eaten as salad.

Phytochemicals are plant bioactive compounds mainly found in fresh vegetables, algae and fruits. The terms are broadly classified as phenolics, carotenoids, alkaloids, and nitrogen- containing/organosulfur compounds (Chen et al., 2017; Luo et al., 2017; Zhao et al., 2019). To date, numerous studies have been focused on the effect of cooking methods on dietary phytochemicals (Bellili, Khenouce, Benmeziane, & Madani, 2019; Ramírez-Jiménez, Rangel-Hernández, Morales- 70

Sánchez, Loarca-Piña, & Gaytán-Martínez, 2019; Zhao et al., 2019). On the other hand, the benefits of food processing such as cooking cannot be ignored including the improvement of food safety, the release of natural phytochemicals with functional properties and enhancement of nutritional value.

The rationale for this study is to investigate whether Papaya unripe fruits and leaves may possess antioxidative properties with cooking treatment as per traditionally prepared culinary. Moreover, a preliminary survey on preferences and intake of papaya unripe fruits, leaves and flowers among Malaysian survey population (n:167) was conducted prior to present study. Unripe fruits or papaya Stage (1) scored the highest liking rate (64.8%), whereas young papaya leaves/shoots and flowers scored the least; 23% and 18%, respectively (Aliah & Ikram, 2018). Hence, by conducting this research, new product and findings can be established and incorporated with the previous finding regarding papaya to increase the knowledge about the benefits of this plant.

4.2 Materials and methods

4.2.1 Sample preparation

4.2.1.1 Unripe pulp and peels

Red fleshed papaya fruits from hermaphrodite plants RB4 and yellow fleshed papaya Y1B cultivars (n: 24) were supplied from a commercial plantation located at Mareeba, North Queensland, Australia (Longitude: 145.336789, Latitude: -16.956108). They were harvested during summer months (November to December 2011) with mean minimum/maximum temperature of 20.2/32oC (Australian Government Bureau of Meteorology, 2013). Fruits were selected for uniform size and picked green at commercial maturity and couriered to the Coopers Plains laboratory facility, arriving within 2 days. Latex on fruits skin was immediately cleaned once arrived by wiping with a clean damp cloth. To prepare composite samples, individual fruit was cut longitudinally and the seeds, placenta tissue and peel were removed. Two slices of papaya flesh of equal amounts (~150 g), from each of the whole fruits (n:6) were cut into cubes. The samples were then divided into two groups. One group of samples were given heat treatment by cooking in boiling water (100oC) for 5 min (according to traditional food preparation). The ratio of water to sample was 1:10. The treated sample was then immediately soaked in ice water for approximately 1 min to avoid further heating (Figure 4.3). Treated and non-treated samples was immediately snap-frozen, followed by freeze- drying (Dynavac Linder & May Pty. Ltd., Windsor, Brisbane, Australia) and grinding into a fine powder using a Retsch MM400 cryomill (Retsch GmbH, Haan, Germany). Samples were kept in an 71

air-tight container and stored in a freezer (-20 oC) until further analysis.

4.2.1.2 Leaves

Leaves (young and mature) from RB4 and Y1B cultivars were supplied from the same commercial plantation, Mareeba, North Queensland, Australia (Figure 4.1-4.2). Mature, fully green and young leaves of C. papaya were washed and shredded into small pieces less than 20mm using scissors. The samples (mature and young leaves) were randomly divided into two groups. One group of samples were immediately snap frozen and freeze-dried using a bench-top freeze-dryer dryer (Dynavac Linder and May Pty. Ltd., Brisbane, QLD, Australia), whereas the treated leaves were boiled for 2 min (1:20, w/v water) before lyophilisation (Figure 4.3). All samples were freeze-dried and ground into a fine powder using a cryomill (Retsch GmbH MM400, Retsch-Alee 1-5, Haan, Germany). Samples were kept in an air-tight container and stored in a freezer (-20 oC) until further analysis.

Figure 4.1: The young and matured papaya leaves

Figure 4.2: The separation between the young papaya leaves with the matured leaves 72

Leaves (young and mature) Unripe fruits (stage 1)

Boiling for 2 min (1:20, w/v water) Boiling for 5 min (1:10 w/v water)

The treated samples were immediately soaked in ice water for approximately 1 min to avoid further heating

Snap frozen by liquid nitrogen, followed by freeze-drying and grinding using cryomill

Kept in air-tight container and stored at -20 oC

Figure 4.3: Sample preparation of leaves and unripe fruits using traditional cooking method.

4.2.2 Chemicals

Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), fluorescein disodium salt, 2,2’- Azobis(2-methylpropionamidine) dihydrochloride (AAPH), anhydrous sodium carbonate, Folin- Ciocalteu’s phenol reagent, ascorbic acid, lycopene, β-carotene, β-cryptoxanthin, α-tocopherol, Chlorophyll a and b, polyphenols were purchased from Sigma-Aldrich Chemicals Co. (St Louis, MO, USA). HPLC-grade methanol, dichloromethane and acetone were purchased from Merck (Darmstadt, Germany), hexane from Ajax (Taren Point, NSW, Australia), ethanol from LabServ (Clayton, VIC, Australia) and methyl tert-butyl from Burdick & Jackson (Muskegon,MI, USA). All

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chemicals used were of analytical or HPLC-grade.

4.2.3 Equipment

The HPLC system consisted of a SIL-10AD VP auto-injector (Shimadzu, Kyoto, Japan), SCL-10A VP system controller (Shimadzu), LC-10AT VP liquid chromatography (Shimadzu) and a SPD- M10A VP diode array detector (Shimadzu). A microplate fluorescence reader (Victor3V 2030 Multilable Counter, Perkin Elmer, City, MA, USA) was used for antioxidant capacity measurements.

4.2.4 Method

Please refer methods 3.2.6 to 3.2.9.

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4.3 Statistical analysis

Data were expressed as mean±standard deviation of at least 6 measurements. Data were statistically analyzed using statistical software, IBM SPSS Statistic version 25.0 for windows (SPSS Inc, Chicago, IL, USA). Each analysis was conducted using a Multivariate ANOVA. Pearson correlation coefficients, were determined and the significant difference was set at p < 0.05. The Tukey HSD and Duncan Test with a = 0.05, was used to test for differences in groups means.

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4.4 Results and Discussion

In general, cooking methods such as boiling, steaming, and microwaving are typical/popular food preparation methods in Western countries and society were based on the dietary habit in western society, whereas stir-frying is a popular and common preparation methods for dishes in China and some Asian countries (Aliah & Ikram, 2018; Liu, Perera, & Suresh, 2007). In present study, cooking preparation instructions for unripe papaya and leaves were chosen and conducted according to methods used by elders of rural communities in Malaysia (Aliah & Ikram, 2018). The ‘boiling method’ was preferred and used to reflect the common household practice in these communities.

4.4.1 Ascorbic acid

AA content of unripe papaya flesh in the present study ranged from 36.3-48.4 mg/100g FW (Figure 4.4). AA content in papaya leaves varied greatly depended on the maturity of the leaves (Figure 4.5). Considering heating processes, boiling determined a significant loss of the total AA content of pulp and leaves when compared with the raw ones. The values decreased more than half of the content (50-82% ) after 2 and 5 min of boiling, respectively compared with the uncooked samples. Overall, the ranking of AA was mature leaves > shoots > pulp (Figure 4.4-4.5).

The AA content of mature leaves and papaya shoots in the present study ranged from 85.45-90.6 mg/100g FW. For comparison, this value was in agreement with Andarwulan et al. (2012) who found high value of 117.15 mg/100g FW of AA in papaya leaves grown in Indonesia. Majority of Asian vegetables (Andarwulan et al., 2012) had ascorbic acid content greater than typical western vegetables such as spinach (28 mg/100 g), green leaf lettuce (9.2 mg/ 100 g), and iceberg lettuce (2.8 mg/100 g) (USDA-ARS, 2011).

To date, many studies have been conducted to investigate the impact of preparation and cooking methods on the stability of nutrients in food. The results of these studies vary widely leading the consumer to question the best ways of preparing and cooking foods in order to maintain the nutritional qualities, especially in legumes and vegetables (Fabbri & Crosby, 2016). The available literature provides no information about heat-treatment of papaya pulp and leaves. A study by Lafarga, Viñas, Bobo, Simó, and Aguiló-Aguayo (2018) found that thermal processing significantly reduced the vitamin C content of all Brassica vegetables in their study. Moreover, Cuastumal, Ledesma, and Ordonez (2016) found that boiling for 10 minutes reduces 51.73% of ascorbic acid 76

content in green pepper. In their study, AA tends to decrease by 52-80% regardless of the cooking methods (boiling, microwaving and conventional oven). Xu et al. (2014) also reported that boiling and stir frying of red cabbage caused significant reduction of AA. All of the samples studied showed a reduction in AA content regardless of the cooking methods (boiling, steaming, microwaving and conventional stir-frying). However, Gliszczynska-Swiglo et al. (2006), found that steamed broccoli showed no significant changes in AA level compared to boiled broccoli. Moreover, Akdaş and Bakkalbaşı (2017) found that water soluble phytochemicals were significantly decreased by boiling and stir-frying, while steaming gave the lowest degradation ratio. Thus, using minimal cooking water like steaming and cooking for shorter time periods may result in better AA retention.

In the present study, we found that household cooking practices by boiling vegetables may cause significant loss of AA, which related to high temperatures and cooking time. Thermal treatment known to be able to accelerate the oxidation of AA and also as water soluble compound, boiling might cause great losses of AA by leaching into surrounding water. However, the content of AA in papaya pulp and leaves can also be significantly reduced during sample preparation, processing and storage due to its solubility in water and its sensitivity to high temperatures and oxidation conditions (Gamboa-Santos et al., 2013).

Maturity is one of the major factors that determine the compositional quality of fruits and vegetables. In present study, AA of mature leaves was at par with the young leaves (Figure 4.5). A study by Omary et al. (2012), AA content decreased with plant maturity. On the other hand, Drews et al. (2000) observed an opposite tendency for AA levels in garden and iceberg lettuce with plant maturity.

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a a c

c

b d d e

Different maturity stages and cultivars

Figure 4.4 Ascorbic acid (mg/100g FW) content of unripe papaya and thermal treatment (5 min of boiling) at different maturity stages and cultivars. Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

a c

b

d

Different maturity stages (leaves)

Figure 4.5 Ascorbic acid (mg/100g FW) content of raw and thermal treated leaves (2 min of boiling) at different maturity stages and cultivars. Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

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4.4.2 Carotenoids

Leafy vegetables contain several types of photosynthetic pigments, that are chlorophylls and carotenoids with varying bioaccessibility. (Kimura and Rodriguez-Amaya, 2002) (Chandrika, Basnayake, Athukorala, Colombagama, & Goonetilleke, 2010). The composition of these pigments produces specific colouration of the food, which is one of the assessed visual quality attributes (Xue and Yang, 2009). Chlorophyll and carotenoid concentration correlate to the physiological status as well as health benefits of the plant.

It is estimated that 85% of total vitamin A activity from leafy green carotenoids is from b-carotene (Ball, 2000). Leafy green carotenoids are composed of b-carotene, lutein, neoxanthin, zeaxanthin, and violaxanthin, with varying bioaccessibility (Chandrika, Basnayake, Athukorala, Colombagama, & Goonetilleke, 2010). It is estimated that 85% of total vitamin A activity from leafy green carotenoids is from b-carotene (Ball, 2000). In present study, major carotenoids found in the leaves were β-carotene and lutein. Both chlorophyll a and b were observed with two isomers of β - carotene: eluting at 28 min is all trans β -carotene, closely followed by the isomer cis β -carotene (Figure 4.8).

-carotene levels in mature leaves studied were found to be higher (p<0.05) compared to the young leaves with values of 4.2 mg/100g FW and 3 mg/100g. Present results for lutein content for papaya leaves ranged from 0.2-1.3 mg/100g. Results are in agreement with study done by Nam, Jang, and Rhee (2018) with -carotene levels of 3.9 mg/ 100g FW in leaves of papaya tree grown in Korea. Bhaskarachary et al. (2008) demonstrated similar domination of -carotene in 17 species of leafy vegetables. In contrast Andarwulan et al. (2012) found lower level of -carotene in papaya leaves grown in Indonesia with values 0.13 mg/100g FW. For comparison, green leaf lettuce (4.4 mg/100 g), and spinach (5.6 mg/100 g) (USDA, 2018) have equivalent or more -carotene than papaya leaves in the present study. The content of −carotene in papaya leaves reported in this study agree in general with other green vegetables in other published reports ranging from 2.6-7.4 mg/100 FW (Arscott, Howe, Davis, & Tanumihardjo, 2010; Hart & Scott, 1995; Maurizi et al., 2015; Schoefs, 2002; Žnidarčič, Ban, & Šircelj, 2011).

Carotenoid levels in the leaves depend on several factors, including species, variety, cultivar,

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production practice, maturity, as well as environmental growth factors such as light, temperature, and soil properties (Van den Berg et al., 2000). Present results were in agreement with previous studies where lutein and β -carotene have been widely reported as being two of the major carotenoids found in vegetables (Lakshminarayana et al. 2005; Calvo, 2005).

Boiling is a common domestic cooking method in Asian countries. As shown in Figure 4.6-4.7, boiling for 2 minutes did give a significant impact on single carotenoids of papaya leaves. Boiling of mature and young leaves resulted in slight (10-12%) but significant increase of -carotene concentration. Similar observation was observed by other research studies on boiling of vegetable at 2-5 minutes (F.-J. Kao, Chiu, & Chiang, 2014; F. J. Kao, Chiu, Tsou, & Chiang, 2012; Miglio, Chiavaro, Visconti, Fogliano, & Pellegrini, 2008). Increase in concentration may be the result of disruption of the carotenoid protein complexes facilitating the extraction efficiency for measurement and the inactivation of carotenoid-oxidizing enzymes preventing from degradation (Bernhardt & Schlich, 2006). Results suggested that boiling green leafy vegetables could promote the release of carotenoids from the food matrix, leading to better extractability and higher concentrations in cooked samples. In contrast, boiling is also reported to affect β-carotene content of some green vegetables (Traoré et al., 2017).

Lutein content was found to be more stable (p > 0.05) after boiling treatment (Figure 4.7). Kao et al. (2012) observed that sweet potato leaves boiled in water retained the majority of total carotenoids. In their study, among single carotenoids, boiling caused no significant effect on lutein and zeaxanthin (p > 0.05), while deep and stir- frying decreased the most of the carotenoids content. In addition, Liu et al. (2007) found that no lutein content was detected in the water after boiling vegetables in it, suggesting that no carotenoids leached out water during the boiling of the vegetables. However, in present study we did not measure carotenoid content in the water.

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a b

d c

Different maturity stages (leaves)

Figure 4.6: -carotene (mg/100g FW) content of papaya leaves (Mature and young). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

ML: Mature Leaves

a b

c c

Different maturity stages (leaves)

Figure 4.7: lutein (mg/100g FW) content of papaya leaves (Mature and young). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

ML: Mature Leaves

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mAU 450nm,4nm 400 3 350 A

300

250 200 5 150 1 2 100 4

50 6

0

-50

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min

mAU 450nm,4nm 70 5 B 60

50

40

30 1 20 6 10 2 3 4

0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 min

Figure 4.8. Carotenoid profiles of Mature leaves (A) and shoots (B). Rt and Mass spectra were compared with that of the standard compounds 1; lutein 2: Chlorophyll b; 3: Chlorophyll a; 4: 13- cis-b-carotene; 5: all trans-b-carotene; 6:9-cis-b-carotene

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4.4.3 Polyphenols

The flavonoid content of some western foods has been reported and archived in the USDA flavonoid (Release 3.3) database (USDA, 2018). However, less is known about the flavonoid content of foods from developing nations. It has been reported that plant derived antioxidants have multiple biological effects and has been the niche area of interest. A number of underutilized leafy vegetables including papaya leaves are used for both food and traditional medicine (Aliah & Ikram, 2018; Andarwulan, Batari, Sandrasari, Bolling, & Wijaya, 2010). For food, these plants are eaten raw or boiled, yet little is known about their constituents that may contribute to their functionality

Polyphenols content of papaya leaves were determined and is shown in Fig 4.9-4.11. Thirteen major peaks of phenolic compounds have been separated and tentatively identified from Australian grown papaya cultivars (Figure 4.9). Caffeic acid was the most abundant of phenolics acids in the leaves, followed by gallic acid. In the case of flavonoids, quercetin was the major compounds detected. Generally, the highest content of total polyphenolics was determined in mature leaves extracts.

As shown in Figure 4.8-4.9, the contents of flavonoids quercetin, kaempferol, apigenin and rutin of young and mature papaya leaves ranged from 5.63-76.03 mg/100g FW. Results shows that quercetin and kaempferol constituted 63-81% of the sum of flavonoids in papaya leaves. Studies on underutilized leafy vegetables showed that quercetin and kaempferol constituted more than 60% of the sum of vegetable flavonoids (Andarwulan et al., 2012). Similarly, several other studies have performed identification of the flavonoid constituents in papaya leaves and found that quercetin 3- (2G-rhamnosyrutinoside), quercetin 3-rutinoside, myricetin 3-rhamnoside, kaempferol 3-rutinoside, quercetin and kaempferol have been identified in the leaves (Husin et al., 2019; Nugroho, Heryani, Choi, & Park, 2017).

Results showed that caffeic acid, gallic acids and chlorogenic acid are the major phenolics acid in papaya leaves (Figure 4.8-4.9). Caffeic acid and chlorogenic acid is present in a number of vegetables including lettuce (3.78 mg/100 g FW), broccoli (1.78 mg/100 g FW), and endive (101 mg/100 g FW) (Neveu et al., 2010).Andarwulan et al. (2012) reported that chlorogenic acid was the most abundant phenolic acid in 18 of the 24 vegetables studied, while caffeic acid was the second most abundant phenolic acid among the vegetables studied. Caffeic acid is present in fresh herbs such as rosemary, sage, thyme, and oregeno from 2.08 to 11.7 mg/100 g (Neveu et al., 2010). Ferulic acid has previously been identified in vegetables from 0.21 to 3.01 mg/100 g FW, and is

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also found in beans and dried herbs (Neveu et al., 2010). Thus, consumption of papaya leaves may increase dietary chlorogenic acid and flavonoids. Animal studies suggest that chlorogenic acid improve antioxidant status and increase phase 2 detoxification enzymes. Dietary chlorogenic acid improved hepatic glutathione redox status in rats subjected to azoxymethane-induced colon cancer (Park, Davis, Liang, Rosenberg, & Bruno, 2010). Ferulic acid supplementation increased intestinal glutathione expression of glutathione-S-transferase and quinone reductase in rats (Bolling et al., 2011).

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f

g

i

j b c a a a

Polyphenolscontents a a a a a e d e h

Polyphenols compounds

Figure 4.9: Polyphenols compounds of papaya shoots and boiled shoots (BS) (mg/100g FW). Results are given as mean±SD of six measurements. Analysed by HPLC Quercetin derivatives was calculated based on Quercetin-3-glucoside standard.

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i

g g g

olyphenolscontents c P d a f b h f c c f f f c g g g

Polyphenols compounds

Figure 4.10 Polyphenols compounds of papaya mature leaves (ML) and boiled mature leaves (BML) (mg/100g FW). Results are given as mean±SD of triplicate measurements. Analysis was determined by HPLC Quercetin derivatives was calculated based on Quercetin-3-glucoside standard.

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Retention time (minutes)

Figure 4.11 Analyses of polyphenols in the leaves extracts of papaya, using HPLC. Mass spectra were compared with that of the standard compounds. 1: gallic acid;2: Gallic acid; 3: Chlorogenic acid; 4: Chlorogenic derivatives; 5:Quercetin derivatives; 6: Rutin; 7: Ferulic acid; 8,12: Quercetin derivatives; 9: p-coumaric acid; 10: Caffeic acid; 11: Quercetin; 13: Apigenin; 14: Kaempferol. Peak 4, 5, 8 and 12 corresponds to a compound with mass spectrum equal to chlorogenic acid and quercetin, but with different rt (most likely isomers).

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4.4.4 ORAC and TPC

The ORAC in vitro AOC of unripe pulp, peels and leaves were shown in Figure 4.11-4.13. Results of AOC was in the order of leaves>peel>pulp. The leaves and young leaves showed high levels of total ORAC of 65-192 TE/g FW, respectively compared to other parts of the plant. The AOC of the pulp and leaves decreased to less than half the content (p<0.05) after boiling treatment (Figure 4.11). Present result for leaves was higher compared to previous values of 529.45 µg TE/g DW for dried crude papaya leaf powder (Vuong et al., 2013). In their optimization study, papaya leaves were brewed (70oC, 20 min, water-to-leaf ratio of 100:7.5 mL/g) before centrifuged, partial drying at 75oC for 3-4 hours and finally freeze-dried to obtain the crude extract. Hence, the results of AOC on different preparation, extraction conditions and method used vary considerably with the present experimental approach (Huang et al., 2005).

ANOVA analysis revealed that there was a significant difference (p < 0.05) between total phenolic content of the pulp, peel and leaves. The untreated leaves and shoots showed a high phenolic content ranging from 148.2-210 mg GAE/100g FW. Results of high total phenolics contents was in agreement with previous study by Asghar et al. (2016) which reported 54.28-65.12 mg GAE/g DW in papaya leaves. Results suggested that cooking treatment of the leaves resulted in significant (p<0.05) reduction of phenolic content (Figure 4.11). Numerous studies have been focused on the effect of cooking methods on dietary phytochemicals (Bellili et al., 2019; Ramírez-Jiménez et al., 2019; Zhao et al., 2019) as it affects the stability of compounds due to chemical and enzymatic decomposition, losses by volatilization or thermal decomposition (Adams, 1991; Bakshi & Masoodi, 2010; Cortés et al., 2006; Gonçalves et al., 2010). These factors also could be the main reason in the reduction of phenolic content in the leaves studies.

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c

a

b

f

d e

Papaya cultivars

Figure 4.12: Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g FW) content of RB4 and RB2 papaya pulp, boiled and peel (n=6). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

c c

d

a a a a b ,

e

Papaya cultivars

Figure 4.13: Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g FW) content of RB1 and Y1B papaya pulp, boiled and peel (n=6). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

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a a

b b

c

Papaya cultivars

Figure 4.14: Total ORAC (Sum of H-ORAC and L-ORAC) expressed as Trolox Equivalents (TE/g FW) content of mature leaves and shoots. Results are given as mean±SD of six measurements. Values with different lettersare statistically significant at the level of p < 0.05.

d

f

e

g

a b a a,c

Papaya cultivars

Figure 4.15: TPC of papaya flesh and leaves (mg GAE/100g FW). Results are given as mean±SD of six measurements. Values with different letters are statistically significant at the level of p < 0.05.

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From our findings, the phenolic content of the peel was high compared to the flesh. Phenolic compounds tend to accumulate in the dermal tissues of the plant body due to their potential role in protecting against ultraviolet radiations, acting as attractants in fruit dispersal, and as defence chemicals against pathogens and predators (Toor & Savage, 2005). This might be the reason of high AOC and TPC in the peels compared to the flesh.

4.5 Conclusions

Overall, the findings of this study suggested that phytochemical content and AOC were in the rank leaves>shoots>peels>pulp. Boiling method have a significant impact on AA, AOC and polyphenols. In contrast, boiling could retain or promote the release of carotenoids from the food matrix. The phytochemical concentration of papaya in present study is unlikely to fully explain any associated health effects because of the large differences in the bioavailability and biological activity of individual compounds. Nonetheless, the data from this study would be valuable for epidemiological research and providing support for dietary guidelines and efforts to promote their consumption should be continued for public health benefits. Furthermore, the number of consumers following a vegetarian diet and the demand for vegan food have notably increased in many countries, and it is likely that this trend will continue to grow.

The next experiments chapter (Chapter 5) will evaluate and discuss In-vitro bioaccessibility of phytochemical in Papaya fruits and leaves eaten as food. The chapter includes the methods of sampling and sample collection and preparation of thermal treatment (for pulp and leaves) . The experiments that were undertaken will be AA, Carotenoids and TPC content of different parts (pulp, and leaves) of papaya Linn cultivars. This chapter will fall under objective 3. Determine the effect of in vitro digestion on the stability of vitamins and polyphenols.

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CHAPTER 5

5. In-vitro Bioaccessibility of phytochemical in Papaya fruits and leaves eaten as food

5.1 Introduction

Fruits and vegetables are often considered as typical everyday healthy foods. Daily consumption of at least 400 g of fruits and vegetables (equivalent to five daily servings) decreases the risk of chronic diseases such as cardiovascular diseases and diabetes type 2. Therefore, increasing the consumption of fruits and vegetables is a core strategy for improved public health worldwide. In Australia, consuming two servings of fruits and five servings of vegetables per day is currently a national health initiative (Chapman et al., 2016). In addition to being healthy, fruits contain compounds such as phytochemicals and fibre that may prevent and delay the development of some diseases (Boehm et al., 2018). To further understand the possible beneficial effects of papaya on human health it is essential to determine the metabolic fate of its bioactive compounds. As part of the diet, food is ingested as complex mixtures which undergo a digestion process in the gut. It is important to determine how this digestion process affects nutrients and other bioactive compounds and their stability as this, in turn, will affect their bioaccessibility, intestinal absorption and potential beneficial effects. Bioaccessibility and bioavailability are critical ‘steps’ in regard to potential health benefits in vivo. In the present study, the bioaccessibility of key-phytochemicals (AA, carotenoids and TPC) in Papaya fruit and leaves was assessed using a validated in vitro digestion model. Bioaccessibility can be used as an initial measure to predict the potential intestinal absorption of nutrients and non-nutritive compounds such as phytochemicals.

5.2 Materials and method

5.2.1 Samples preparation

5.2.1.1 Fruits

Red fleshed Papaya fruits from hermaphrodite plants RB4 and yellow fleshed Papaya Y1B cultivars (n:24) were supplied from a commercial plantation located at Mareeba, North Queensland, Australia (Longitude: 145.336789, Latitude: -16.956108). For the in vitro digestion procedure, fruits were selected for uniform size and picked green at commercial maturity (stage 1) and couriered to the Coopers Plains laboratory facility, arriving within 2 days. Fruits were immediately cleaned. To prepare samples, individual fruit was cut longitudinally and the seeds, placenta tissue

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and peel were removed. Two slices of Papaya flesh of equal amounts (~150 g), from each of the whole fruits (n:6) were cut into cubes and/or were given heat treatment by cooking in boiling water (100oC) for 5 min (according to traditional food preparation). The ratio of water to sample was 1:10. The treated sample was then immediately soaked in ice water for approximately 1 min to avoid further heating. Two slices of Papaya flesh of equal amounts (~150 g), from each of the whole fruits (n=8) were cut into cubes and).

5.2.1.2 Leaves (shoots and mature)

Leaves (mature and shoots) from Y1B and RB4 cultivars were supplied from the same commercial plantation, Mareeba, North Queensland, Australia. Mature, fully green and young leaves of C. papaya were washed and shredded into small pieces less than 30mm using scissors and immediately snap frozen, followed by freeze-drying (type, Dynavac Linder & May Pty. Ltd., Windsor, QLD, Australia) and grinding into a fine powder using a MM400 Retsch cryomill (Retsch GmbH, Haan, Germany

5.2.2 In vitro digestion

In vitro digestion procedure, adopted from Netzel et al. (2011) with slight modifications, was undertaken using fruits (red-fleshed RB4 and yellow-fleshed Y1B maturity Stage (3)), and leaves (young and mature leaves). Briefly, ~5 g of was weighed into 10 individual 50 mL tubes with the addition of 5ml corn oil. Five independent digestion trials were carried out whereas five tubes (one per trial) were removed after gastric digestion and the other five (one per trial) after gastric and small intestinal digestion to measure the impact of the two digestion compartments on the bioaccessibility/release of the hydrophilic/lipophilic phytochemicals. For the digestion procedure, 6 M HCl was added, with thorough mixing, until the samples reached pH 2 and then 250 μL of pepsin solution (40 mg/mL pepsin from porcine gastric mucosa (P7000, Sigma-Aldrich) dissolved in 0.1 M HCl). The tubes were incubated for 1 h at 37 °C in a shaking water bath (85 rev/min). After 1 h the gastric samples were removed and centrifuged (10 min, 5000 rpm), and the supernatants were stored at −84 °C until analysis. 0.1 M NaHCO3 with calcium (824 mg CaCl2*2H2O in 500 mL 0.1 M NaHCO3) was added until the pH reached 5.7. The samples were mixed well and incubated for a further 30 min at 37 °C in a shaking water bath (85 rev/min). The pH was increased from 2 to 5.7 to simulate the slow increase of the pH between the gastric and small intestinal digestion. After removing the small intestinal samples from the water bath, 1 M NaOH was added until pH of 7.0

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was reached, with thorough mixing throughout. One ml of pancreatin-bile solution (2 mg/ml pancreatin from porcine pancreas (P1750, Sigma-Aldrich), 12 mg/ml porcine bile extract (B8631, Sigma-Aldrich) in 0.1 M NaHCO3) was then added and mixed well. Samples were incubated for another 2 h at 37 °C in a shaking water bath (85 rev/min). After removing from the water bath the samples were centrifuged (10 min, 5000 rpm), and supernatants were then transferred into 15 mL tubes. Samples which were designated for polyphenol analysis were mixed with 500 μL formic acid (90%). All samples were then stored at −84 °C until extraction and analysis of AA, carotenoids and polyphenols.

5.2.3 Determination of AA, total phenolics, and carotenoids

Total phenolic content (Folin-Ciocalteu method) was determined as previously described Netzel et al. (2012), and results were expressed as mg of gallic acid equivalents per 100 g (mg GAE/100 g). AA and carotenoids were analyzed by HPLC as detailed previously in method 3.2.6 and 3.27.

5.3 Statistical analysis

All experiments were repeated twice and in triplicate each time. Data were statistically analyzed using statistical software, IBM SPSS Statistic version 25.0 for windows (SPSS Inc, Chicago, IL, USA). Each analysis was conducted using a Multivariate ANOVA. Pearson correlation coefficients, were determined and the significant difference was set at p < 0.05. The Tukey HSD and Duncan Test with a = 0.05, was used to test for differences in groups means.

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0.1 M NaHCO3, 12 mM CaCl2 centrifugecentrifuge + blend samples +water + corn oil + gastric solution + Pancreatin Bile solution separate supernatant and pellet

rotate rotate

2 h at 2 h at 37 C 37 C +

pH 2 pH 7

empty step 1: step 2: step 3: Supernatant pepelletllet test tube “mouth” “stomach”“stomach” “small intestine”intestine”

ananalysisalysis of compound

Figure 5.1: In Vitro digestion Model

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5.4 Results and discussion

5.4.1 Release (bioaccessibility) of AA and carotenoids in papaya fruit (%)

Bioavailability is the proportion of the nutrient that is digested, absorbed, and metabolized through normal pathways (McGhie and Walton, 2007). The amount and bioavailability are key to elucidating the importance of diet in health. Bioaccessibility is defined as the amount of a food constituent, present in the gut as a result of its release from the solid food matrix, which might be able to pass through the intestinal barrier.

The relative release (released amount vs. total amount) of carotenoids in red and yellow fleshed RB4 and Y1B Stage (3) in present study was 11-25% (Table 5.1). Results are consistent with literature evidence for the limited bioavailability of these molecules. According to (Epriliati, D’Arcy, & Gidley, 2009), the release of carotenoids in papaya (Red-fleshed, cultivars unknown, Australian grown) tissues was found to be the lowest (5-12.5%) if compared to mangoes and tomatoes. Moreover, Schweiggert et al. (2012) also reported a lower release of carotenoids (5.3%) of Red-fleshed pulp (cultivars unknown) grown in Germany. The gastric digestion was minimal, consistent with the very low aqueous solubility of these compounds. In comparison to previous study, present result of known papaya cultivars exhibited higher release of lipophilic carotenoids from the pulp with addition of oil/lipids to the pulp/fruit which can significantly increase the released fraction of carotenoids (Lemmens, Van Buggenhout, Oey, Van Loey, & Hendrickx, 2009; Netzel et al., 2011; Parada & Aguilera, 2007).

The second phase of the in vitro digestion process, which was designed to mimic intestinal digestion, resulted in the release of lower amount (11-19%) β-carotene and β-cryptoxanthin except for lycopene. Moreover, lycopene was not detected in red-fleshed Stage (1) maturity stages. Results were in agreement with a study by Capella et al. (2015) and Bunioswka et al. (2017) which showed reduction of carotenoid release from papaya fruit mixtures in intestinal digestion if compared with gastric digestion. However, studies of fruit juices including papaya juice was reported to have higher recovery of carotenoids after gastric conditions (48-56%) (Buniowska et al., 2017; Rodríguez-Roque, Rojas-Graü, Elez-Martínez, & Martín-Belloso, 2014). The present results further showed that β-carotene and β-cryptoxanthin was better releases than lycopene. One explanation for this is maybe β-carotene and β-cryptoxanthin are less tight binding to the papaya matrix than lycopene, as type of physical deposition form of carotenoids may play a major role (Kirkhus et al., 2019; Ralf M Schweiggert, Mezger, Schimpf, Steingass, & Carle, 2012).

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The present results showed that papaya fruit could be a complementary source of AA, with the release of 64-77% during gastric digestion. However, loses of >85% can be observed in second phase of the in-vitro digestion. This was in agreement with previous studies by Pérez-Vicente, Gil- Izquierdo, and García-Viguera (2002) with AA release of 74% during gastric digestion and loses of >80% in Gastric and small intestinal digestion in pomegranate fruit juice. These losses could be partly explained if the different pH values of the media and the presence of oxygen are taken into consideration. Ascorbic acid is a moderately strong reducing agent and acidic.

As discussed earlier in Chapter 1 and 4, unripe papaya fruits are cooked and eaten as food in some Asian countries. Thus, limited studies have been done on the bioaccessibility of thermal treatment of papaya fruits maturity Stage (1). Results showed relative release of 23% and 11% of β-carotene and β-cryptoxanthin in gastric and small intestinal digestion for boiled RB4 Stage (1), respectively (Table 5.1). There are significant different (p<0.05) in-term of % release in gastric digesta and gastric and small intestinal digestion. This may be due to the breakdown of cell matrix in papaya fruit. Thermal processes can improve or release natural phytochemicals such as carotenoids by the disruption of cell walls, breakdown of complex molecular structures, and dissociation of molecular linkages between food components (Hidalgo & Zamora, 2017; Kirkhus et al., 2019). Moreover, a study by Netzel et al. (2011) found that simulated gastric digestion of blanched (10 min at 80oC) and cooked (10 min at 100oC) carrot puree samples resulted in the release of small amounts of total carotenes: 7.2% for blanched carrot puree, and 7.8% for cooked samples. However, larger release can be seen in the second phase of the in vitro digestion process, which was designed to mimic intestinal digestion; 31.6 for blanched carrot puree and 37.7% for cooked carrot puree, respectively. Our results indicate that the intestinal digestion phase is of major importance for the release and bioaccessibility of β-carotene and β-cryptoxanthin in unripe papaya tissues.

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Table 5.1 Release (bioaccessibility) of AA and carotenoids in papaya cultivars.

Papaya cultivar AA and carotenoids content Released (%)b Gastric digestion (60 min) Gastric and small intestinal digestion (180 min) RB4 Stage (3) β-carotene (mg/100g) 0.65±0.02 0.12±0.01 (18%) 0.10±0.01 (15%) β-cryptoxanthin (mg/100g) 0.36±0.01 0.09±0.01 (25%) 0.07±0.02 (19%)* Lycopene (mg/100g) 0.85±0.02 0.12±0.02 (14%) 0.10±0.01 (12%) Ascorbic Acid (mg/100g) 58.11±0.32 42.83±0.21 (73%) 6.43±0.14 (11%)*

Y1B Stage (3) β-carotene (mg/100g) 0.28±0.02 0.05±0.01 (18%) 0.04±0.01 (14%) β-cryptoxanthin (mg/100g) 0.18±0.01 0.02±0.01 (11%) 0.02±0.00 (11%)* Lycopene (mg/100g) ND ND ND Ascorbic Acid (mg/100g) 50.57±0.26 32.44±0.12 (64%) 2.32±0.11 (5%)*

RB4 Stage (1)a β-carotene (mg/100g) 0.13±0.02 0.02±0.01 (15%) 0.03±0.01 (23%) β-cryptoxanthin (mg/100g) 0.09±0.01 ND 0.01±0.01 (11%) Lycopene (mg/100g) ND ND ND Ascorbic Acid (mg/100g) 38.9±0.06 30.2±0.11 (77%) 6.2±0.22 (15%)

Values are means ± standard deviation, n=5 independent trials; RB4: Red-fleshed hybrid; Y1B: Yellow-fleshed hybrid; Stage (3): Represents papaya with yellow area on 50–75% of the skin; Stage (1): Represents Papaya with yellow area on 0–25% of the skin.%: All data are rounded;ND: Not detected. a 5 min Boiling treatment. bRelative release of AA and carotenoids (release amount vs applied dose), * Significant differences (p < 0.05) RB4 vs Y1B

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mAU 472nm,4nm 400 3 6

350

300

250 2 5 200 1

150 4 100

50

0 Carotenoids compounds pre-digestion 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

mAU 472nm,4nm120 6 110 100 90 80

70 60 50 40 3 30 1 2 5 Carotenoids compounds post-pancreatic 20 4 10 digestion 0 -10 -20 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

Figure 5.2 Carotenoids compounds profile comparison pre and post digestion for Red-fleshed RB4 Stage 3. Rt and Mass spectra were compared with that of the standard compounds 3: β- cryptoxanthin; 5: β-carotene; 6: lycopene

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5.4.2 Release (bioaccessibility) of AA and TPC in papaya leaves (%)

There are several data in the literature regarding the carotenoids and polyphenol content in fruit and vegetables extracts, but there is a lack of data regarding the polyphenols that are released after in vitro digestion for papaya leaves. Figure 5.3 shows the path of polyphenols after ingestion mimicking different phases of polyphenols in digestion model. The digestion process directly affects the composition of the extracts depending on the simulation of the physiological conditions and the sequence of events within the gastrointestinal tract.

Present results showed that papaya leaves could also be a complementary source of AA, with release of 42-53% during gastric digestion. However, loses of >90% can observed in second phase of the in-vitro digestion (Table 5.2).

The TPC exhibited different results in the leaves extracts after in vitro digestion. These results indicated that the release or bioaccessibility of papaya leaves is 27-45% of its TPC during the gastric digestion and intestinal conditions (Table 5.2). There is no significant different on bioaccessibility with regards to the maturity level of the leaves studied. However, present results demonstrate significant (p<0.05) higher release of TPC in thermal treatment leaves (boiled mature and young leaves).

When phenolic compounds are consumed in our diet, they are released from the cell matrix under mechanical action of mastication. Polyphenols are more weakly to the cell wall structure and those contained in vacuoles and the linked are more closely to the cell wall, especially in skin cells (Tagliazucchi et al., 2010). The gastrointestinal tract can be considered as an efficient extractor, where part of the phytochemicals contained in food matrices is extracted and becomes available for uptake in the intestine (Bouayed et al., 2011). Polyphenols can be unstable to physiologic pH conditions, suffering irreversible structural changes (Friedman & Jürgens, 2000; Krook & Hagerman, 2012). Reduction in TPC release can be linked to the instability of phenolic compounds in high pH, as they speculated, larger molecules may be more stable, but when hydrolyzed to smaller molecules, such as gallic acid, were not stable at high pH. Thus, this explain how simple total polyphenols in papaya fruit, might be unstable to alkaline conditions above pH 7 (Barros & Junior, 2019).

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Figure 5.3 The path of polyphenols after ingestion. Adapted from Barros and Junior (2019)

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Table 5.2 Release (bioaccessibility) of AA and TPC in papaya leaves.

Papaya Leaves AA and TPC content Released (%)b Gastric digestion (60 min) Gastric and small intestinal digestion (180 min) Mature leaves Ascorbic Acid (mg/100g) 83.34±0.32 35.04±0.2 (42%) 1.8±0.03 (2%) TPC (mg GAE/100g) 89.11±0.4 28.51±1.5 (32%) 24.45±2.1 (27%)*

Boiled mature Ascorbic Acid (mg/100g) 32.53±0.01 17.31±0.09 (53%) 0.28±0.02 (1%) Leavesa TPC (mg GAE/100g) 41.32±0.02 17.91±0.06 (43%) 17.1±0.01 (41%)*

Young Ascorbic Acid (mg/100g) 85.61±0.02 36.51±0.02 (42%) 1.87±0.02 (2%) leaves/shoots TPC (mg GAE/100g) 87.43±0.01 32.04±0.04 (37%) 28.63±0.03 (32%)*

Boiled young Ascorbic Acid (mg/100g) 28.5±0.01 13.78±0.02 (48%) 1.5±0.02 (5%) leaves/shootsa TPC (mg GAE/100g) 47.48±0.38 21.55±0.24(45%) 18.34±0.02 (38%)* Values are means ± standard deviation, n=5 independent trials; %: All data are rounded. a 2 min boiling treatment (1:20, w/v water). bRelative release of AA and carotenoids (release amount vs applied dose) * Significant differences (p < 0.05) (untreated vs treated)

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5.5 Conclusion

Present results showed that papaya fruits and leaves can be a complementary source of AA. Heat treatment (boiling) may enhance the bioaccessibility of TPC papaya fruits and leaves. However, several limitations of these in vitro digestion methods should be critically considered when interpreting the results. To date, no in vitro model is capable of covering all aspects of in vivo digestion and absorption, distribution, metabolism. Metabolites generated in vivo are of particular interest in terms of their potential biological significance in the prevention of chronic diseases as well as the maintenance of a “healthy gut”. Although human nutritional studies are still being considered the “gold standard” for addressing diet-related questions, in vitro methods have the advantage of being more reproducible, choice of controlled conditions and easy sampling. In vitro models are also suitable for mechanistic studies and hypothesis building. However, future human studies/clinical trials are still the best method on bioavailability of papaya fruits and leaves eaten as food.

The next chapter (Chapter 6) is overall conclusion (Chapters 3, 4, and 5) of the project along with future recommendations.

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CHAPTER 6

6. General conclusions and Future directions

Plant foods such as papaya are known to have numerous nutritional and health benefits. The nutritional and health benefits of plant foods have been the focal point of much scientific investigations due to their relation with the prevention of chronic and degenerative diseases. These nutritional benefits arise from a combination of different cultivars, geographical areas, maturity stages, plant parts and food preparation techniques as well as bioaccessibility and bioavailability of its nutrients/bioactive compounds after consumption. Thus, without a thorough studies on the nutritional properties of plant-based foods such as papaya plant, the individual nutrients in plant foods cannot be fully addressed. The aims of this study were therefore to investigate the a) Phytochemical content of commercially available red fleshed papaya from hermaphrodite plants (RB1, RB3, RB4) and yellow fleshed papaya (Y1B cultivar) with four different maturity stages as well other plant parts such as peels and leaves (Chapter 3 and Chapter 4) b) Effects of food processing on papaya plant as incorporated in Asian culinary use of unripe fruits and leaves of papaya with its traditional processing techniques, c) examined the digestion part under in vitro digestion model, involving the mouth, stomach and small intestine (Chapter 5) d) Suggestions of portion or serving size for the Australian papaya fruits. Together these approaches enable us to understand the phytochemical content of different types of Australian papaya cultivars that has never been discussed. Moreover, current study also incorporated new findings (available literature provides no information) on phytochemical content of unripe fruits and leaves and/or with cooking treatment as per Asian traditionally prepared culinary.

In Australia, the market value of papaya fruit is very much dependent on its taste, structure and texture. The plant is predominantly grown in Queensland. Papaya fruits is not popular compared to other fruits and excess supply of fruits during summer months when fruit is plentiful can causes financial loses to growers. Moreover, the unripe and over ripe stage is commercially unmarketable. We conduct this research to evaluate nutritional benefits as well as to inform consumers of the potential benefits of different types of Australian grown papaya cultivars and to stimulate market demand of the fruits and future functional food. Functional are define as food that, by virtue of the presence of physiologically active components, provide a health benefit beyond basic nutrition”(Birch & Bonwick, 2019) . According to this statement, a plethora of natural foods 104

are considered as functional, as well as formulated products containing bioactive ingredients.

6.1 Phytochemical content of Australian papaya cultivars

Phytochemical composition and AOC significantly varied across different papaya cultivars, maturity stages and plant parts. Major carotenoids found in red-fleshed papaya were β-carotene, β- cryptoxanthin, and lycopene with lycopene representing 42-58% of the total carotenoids. β-carotene represents 60-78% of the total carotenoids with no detectable lycopene in the yellow-fleshed cultivar. The content of total carotenoids in Papaya flesh at different ripeness stages expressed as mg/100 g FW increased 2-fold with the level of ripeness of the fruit. We found a significant differences in lycopene and -carotene content between Stage (2) and Stage (4) within and between cultivars. However, Stage (3) and Stage (4) showed no significant differences in -carotene content within red-fleshed cultivars compared to yellow- fleshed cultivar.

Ascorbic acid content of papaya flesh in the present study ranged from 37.0-61.3 mg/100g FW. We confirm that AA increases as the fruits ripen. Red fleshed papaya from hermaphrodite plants RB4 had the highest level of AA. Interestingly, the AA values observed in this study were almost 2 times higher than the AA from local Bangladesh papaya cultivars, and on a par with other published AA data obtained from different geographical origins worldwide. One serving of Australian papaya per day would give 100% of the RDI for vitamin C and 3-4 servings per day of red-fleshed Australian Papaya would cover 50% of the RDI for vitamin A, respectively.

In terms of phenolics acid, Caffeic acid was the most abundant phenolics acids in the flesh and peels, followed by gallic acid. In the case of flavonoids, rutin and quercetin were the major compounds detected. From the results, papaya have a large proportion of the phenolics as bound, ranging from 16 to 91%. Phenolic compounds occur mostly as soluble conjugates and insoluble forms, covalently bound to sugar moieties or cell wall structural components. The phenolic contents and antioxidant activities of fruits could be underestimated if the bound phenolic compounds are not considered. However, absorption mechanisms for bound phenolic compounds in the gastrointestinal tract greatly depend on the liberation of sugar moieties. Food processes such as alkaline and acid hydrolyses have the potential to release phenolics associated to cell walls. We found that samples submitted to acid hydrolysis, the concentrations of caffeic acid and quercetin increased significantly and this point demonstrates that such compounds are present in Papaya tissues mostly as esters and/or glycosides bound to the plant matrix. Chlorogenic acid was detected in trace amounts. 105

6.2 Phytochemical content of unripe Fruits and Leaves and/or with cooking treatment as per traditionally prepared culinary

Fruits and vegetables are consumed not only fresh, but also processed into products. Previous studies have reported that food processing could modulate its phytochemical content. In this study, we found that food processing, particularly thermal processing could have a significant impact on AA, AOC and TPC. In contrast, boiling could retain or promote the release of carotenoids from the food matrix. Overall, the findings of this study suggested that phytochemical composition and AOC was in the rank leaves>shoots>peels>pulp. The phytochemical concentration of papaya in present study is unlikely to fully explain any associated health effects because of the large differences in the bioavailability and biological activity of individual compounds. Nonetheless, the data from this study would be valuable for epidemiological research and providing support for dietary guidelines and efforts to promote their consumption should be continued for public health benefits. Furthermore, the number of consumers following a vegetarian diet and the demand for vegan food have notably increased in many countries, and it is likely that this trend will continue to grow.

6.3 Possible route of carotenoids and polyphenols after human consumption.

The phytochemical concentration (AA, carotenoids and polyphenols) of papaya in the present study is unlikely to fully explain any associated health effects because of the large differences in the bioavailability and biological activity of individual compounds. Nonetheless, the data from this study would be valuable for epidemiological research on papaya sites and providing support for dietary guidelines and efforts to promote their consumption should be continued for public health benefits. The findings from this study should be shared with stakeholders to develop a guideline for use of locally available fruits such as papaya in improving the health of populations beyond Australia.

6.4 Recommendation for Future Studies

Whilst the work on papaya plants provided insights on phytochemicals content within parts of plant, level of maturity stages, the interaction of phytochemicals during cooking and their effect on in-vitro digestibility, there are still area needs to be addressed. Although this study has clearly established the effect of plant maturity and food processing, further work should focus on establishing focused on human tissue research to establish the metabolism of phytochemicals such as polyphenol especially in the leaves for appropriate human doses. There are also gaps between in vitro findings and realistic in vivo environments. Hence, the actual effects under in vivo conditions 106

using human need to be determined. Moreover, studies on the porosity of cell walls and the impact of processing on nutrient release and bio-availability from papaya cell matrix are still required.

Based on the results of the present study, the following recommendations are suggested for future studies:

1. Further study should be focused on human tissue research to establish the metabolism of polyphenols especially in the leaves measured in this thesis through the use of appropriate human doses. 2. There is an existing “gap” between in vitro assays and animal models vs. human studies/clinical trials to determine bioavailability, metabolism and bioactivity. Future data analysis of the relevance and significance of the results obtained by in vitro experiments and in vivo animal study should be performed. Further, human trials are the best way to determine the real bioavailability of Papaya tissues.

6.5 Proposed Publications

1. Antioxidant capacity, ascorbic acid, carotenoids, lycopene and vitamin E composition of papaya (Carica papaya Linn) cultivars grown in Australia.

2. Chemical composition and antioxidant capacity of raw and traditionally cooked Carica papaya Linn pulp and leaves.

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REFERENCES

Abdulazeez, A. M., Ameh, D. A., Ibrahim, S., Ayo, J., & Ambali, S. F. (2009). Effect of fermented and unfermented seed extracts of Carica papaya on pre-implantation embryo development in female Wistar rats (Rattus norvegicus). Scientific Research and Essays, 4(10), 1080-1084.

Abdulazeez, M. A., Ameh, A. D., Ibrahim, S., Ayo, J. O., & Ambali, S. F. (2010). Effect of fermented seed extract of Carica papaya on litters of female wistar rats (Rattus norvegicus). African Journal of Biotechnology, 8(5).

Abdulazeez, M. A., & Sani, I. (2011). Use of Fermented Papaya (Carica papaya) Seeds as a Food Condiment, and Effects on Pre- and Post-implantation Embryo Development. In Nuts and Seeds in Health and Disease Prevention (pp. 855-863). San Diego: Academic Press.

Abdullah, M., Chai, P. S., Loh, C. Y., Chong, M. Y., Quay, H. W., Vidyadaran, S., . . . Seow, H. F. (2011). Carica papaya increases regulatory T cells and reduces IFN + CD4+ T cells in healthy human subjects. Molecular nutrition & food research, 55, 803–806.

Abo, K. A., Fred-Jaiyesimi, A. A., & Jaiyesimi, A. E. A. (2008). Ethnobotanical studies of medicinal plants used in the management of diabetes mellitus in South Western Nigeria. Journal of Ethnopharmacology, 115(1), 67-71.

Acosta-Estrada, B. A., Gutiérrez-Uribe, J. A., & Serna-Saldívar, S. O. (2014). Bound phenolics in foods, a review. Food Chemistry, 152, 46-55.

Adams, J. (1991). Review: enzyme inactivation during heat processing of food‐stuffs. International Journal of Food Science and Technology, 26(1), 1-20.

Adebiyi, A., Adaikan, G. P., & Prasad, R. N. (2002). Papaya (Carica papaya) consumption is unsafe in pregnancy: fact or fable? Scientific evaluation of a common belief in some parts of Asia using a rat model. British Journal of Nutrition, 88(2), 199-203.

Adebiyi, A., Ganesan Adaikan, P., & Prasad, R. N. (2003). Tocolytic and toxic activity of papaya seed extract on isolated rat uterus. Life Sciences, 74(5), 581-592.

Adebiyi, A., Prasad, R., & Adaikan, G. P. (2003). Benzyl isothiocyanate induced functional aberration of isolated uterine strips. The Toxicologist, 72 (S-1), 25.

Agrifutures. (2019). Paw paw/papaya. Retrieved from https://www.agrifutures.com.au/product/emerging-animal-and-plant-industries-their-value- to-australia/

Ajila, C., Naidu, K., Bhat, S., & Rao, U. (2007). Bioactive compounds and antioxidant potential of mango peel extract. Food Chemistry, 105(3), 982-988.

Ajila, C., & Rao, U. P. (2008). Protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by Mangifera indica L. peel extract. Food and Chemical Toxicology, 46(1), 303-309.

108

Akdaş, Z. Z., & Bakkalbaşı, E. (2017). Influence of different cooking methods on color, bioactive compounds, and antioxidant activity of kale. International Journal of Food Properties, 20(4), 877-887.

Aliah, N. F., & Ikram, E. H. K. (2018). Indigenous and Produce Vegetable Consumption in Selangor, Malaysia, Singapore.

Amenta, R., Camarda, L., Di Stefano, V., Lentini, F., & Venza, F. (2000). Traditional medicine as a source of new therapeutic agents against psoriasis. Fitoterapia, 71, S13-S20.

Amer, J., Goldfarb, A., Rachmilewitz, E. A., & Fibach, E. (2008). Fermented papaya preparation as redox regulator in blood cells of beta-thalassemic mice and patients. Phytotherapy Research, 22(6), 820-828.

Andarwulan, N., Batari, R., Sandrasari, D. A., Bolling, B., & Wijaya, H. J. F. c. (2010). Flavonoid content and antioxidant activity of vegetables from Indonesia. 121(4), 1231-1235.

Andarwulan, N., Kurniasih, D., Apriady, R. A., Rahmat, H., Roto, A. V., & Bolling, B. W. J. J. o. F. F. (2012). Polyphenols, carotenoids, and ascorbic acid in underutilized medicinal vegetables. 4(1), 339-347.

Andersson, S. C., Olsson, M. E., Johansson, E., & Rumpunen, K. (2008). Carotenoids in Sea Buckthorn (Hippophae rhamnoides L.) Berries during Ripening and Use of Pheophytin a as a Maturity Marker. Journal of Agricultural and Food Chemistry, 57(1), 250-258.

Annegowda, H. V., & Bhat, R. (2016). Chapter 21 - Composition of Papaya Fruit and Papaya Cultivars. In M. S. J. Simmonds & V. R. Preedy (Eds.), Nutritional Composition of Fruit Cultivars (pp. 497-516). San Diego: Academic Press.

Annegowda, H. V., Bhat, R., Yeong, K. J., Liong, M.-T., Karim, A. A., & Mansor, S. M. (2013). Influence of Drying Treatments on Polyphenolic Contents and Antioxidant Properties of Raw and Ripe Papaya (Carica papaya L.). International Journal of Food Properties, 17(2), 283-292.

Anuar, N. S., Zahari, S. S., Taib, I. A., & Rahman, M. T. (2008). Effect of green and ripe Carica papaya epicarp extracts on wound healing and during pregnancy. Food and Chemical Toxicology, 46(7), 2384-2389.

Aref, H. L., Salah, K. B., Chaumont, J. P., Fekih, A., Aouni, M., & Said, K. (2010). In vitro antimicrobial activity of four Ficus carica latex fractions against resistant human pathogens (antimicrobial activity of Ficus carica latex). Pak J Pharm Sci, 23(1), 53-58.

Arrigoni, O., & De Tullio, M. C. (2002). Ascorbic acid: much more than just an antioxidant. Biochimica et Biophysica Acta (BBA)-General Subjects, 1569(1-3), 1-9.

Arscott, S. A., Howe, J. A., Davis, C. R., & Tanumihardjo, S. A. (2010). Carotenoid profiles in provitamin A-containing fruits and vegetables affect the bioefficacy in Mongolian gerbils. Exp Biol Med (Maywood), 235(7), 839-848.

Aruna, B., & Subrata, H. (2008). Dietary isothiocyanate mediated apoptosis of human cancer cells is associated with Bcl-xL phosphorylation. International Journal Of Oncology, 33, 657-663. 109

Aruoma, O. I., Hayashi, Y., Marotta, F., Mantello, P., Rachmilewitz, E., & Montagnier, L. (2010). Applications and bioefficacy of the functional food supplement fermented papaya preparation. Toxicology, 278(1), 6-16.

Asghar, N., Naqvi, S., Hussain, Z., Rasool, N., Khan, Z., Shahzad, S., . . . Jaafar, H. (2016). Compositional difference in antioxidant and antibacterial activity of all parts of the Carica papaya using different solvents. Chemistry Central Journal, 10(1), 1-11.

Australian Government Bureau of Meteorology. (2013). Summary statistics Mareeba QWRC. Retrieved from http://www.bom.gov.au/climate/averages/tables/cw_031066.shtml

Australian Papaya Industry Association. (2008). Australian Papaya Industry Strategic Plan - 2008- 2012. In (pp. 1-28): Australian Papaya Industry Association Ltd.

Azarkan, M., El Moussaoui, A., van Wuytswinkel, D., Dehon, G., & Looze, Y. (2003). Fractionation and purification of the enzymes stored in the latex of Carica papaya. Journal of Chromatography B, 790(1-2), 229-238.

Azarkan, M., Wintjens, R., Looze, Y., & Baeyens-Volant, D. (2004). Detection of three wound- induced proteins in papaya latex. Phytochemistry, 65(5), 525-534.

Badillo, V. M. (2002). Carica L. vs. Vasconcella St. Hil. (Caricaceae) con la Rehabilitacion de este Ultimo. Ernstia 10, 74-79.

Badillo, V. M. (Ed.) (1971). Monografia de la familie Caricaceae. Associacion de Profesores: Universidad Central de Venezuela. Maracay, Venezuela.

Bakshi, P., & Masoodi, F. (2010). Effect of pre-storage heat treatment on enzymological changes in peach. Journal of Food Science and Technology, 47(4), 461-464.

Bari, L., Hassan, P., Absar, N., Haque, M. E., Khuda, M. I. I. E., Pervin, M. M., . . . Hossain, M. I. (2006). Nutritional analysis of two local varieties of papaya (Carica papaya L.) at different maturation stages. Pakistan Journal of Biological Sciences, 9, 137-140.

Barragan-Iglesias, J., Mendez-Lagunas, L. L., & Rodriguez-Ramirez, J. (2018). Ripeness indexes and physicochemical changes of papaya (Carica papaya L. cv. Maradol) during ripening on- tree. Scientia Horticulturae, 236, 272-278.

Barros, H. D. d. F. Q., & Junior, M. R. M. (2019). Phenolic Compound Bioavailability Using In Vitro and In Vivo Models. In Bioactive Compounds (pp. 113-126): Elsevier.

Bellili, S., Khenouce, L., Benmeziane, F., & Madani, K. J. J. o. F. T. (2019). Effect Of Domestic Cooking On Physicochemical Parameters, Phytochemicals And Antioxidant Properties of Algerian Tomato. 6(1), 1-17.

Bennett, R. N., Kiddle, G., & Wallsgrove, R. M. (1997). Biosynthesis of benzylglucosinolate, cyanogenic glucosides and phenylpropanoids in Carica papaya. Phytochemistry, 45(1), 59- 66.

110

Bernhardt, S., & Schlich, E. (2006). Impact of different cooking methods on food quality: Retention of lipophilic vitamins in fresh and frozen vegetables. Journal of Food Engineering, 77(2), 327-333.

Birch, C. S., & Bonwick, G. A. (2019). Ensuring the future of functional foods. International Journal of Food Science & Technology, 54(5), 1467-1485.

Boehm, J. K., Soo, J., Zevon, E. S., Chen, Y., Kim, E. S., & Kubzansky, L. D. J. H. P. (2018). Longitudinal associations between psychological well-being and the consumption of fruits and vegetables. 37(10), 959.

Bolling, B. W., Chen, Y. Y., Kamil, A. G., & Oliver Chen, C. (2012). Assay Dilution Factors Confound Measures of Total Antioxidant Capacity in Polyphenol‐Rich Juices. Journal of Food Science.

Brannan, R. G., Peters, T., & Talcott, S. T. (2015). Phytochemical analysis of ten varieties of pawpaw (Asimina triloba [L.] Dunal) fruit pulp. Food Chemistry, 168, 656-661.

Brown, G. C. (2010). Nitric oxide and neuronal death. Nitric Oxide, 23(3), 153-165.

Buniowska, M., Carbonell-Capella, J. M., Frigola, A., & Esteve, M. J. (2017). Bioaccessibility of bioactive compounds after non-thermal processing of an exotic fruit juice blend sweetened with Stevia rebaudiana. Food Chemistry, 221, 1834-1842.

Calzuola, I., Gianfranceschi, G. L., & Marsili, V. (2006). Comparative activity of antioxidants from wheat sprouts, Morinda citrifolia, fermented papaya and white tea. International Journal of Food Sciences and Nutrition, 57(3-4), 168-177.

Calzuola, I., Marsili, V., & Gianfranceschi, G. L. (2004). Synthesis of Antioxidants in Wheat Sprouts. Journal of Agricultural and Food Chemistry, 52(16), 5201-5206.

Canini, A., Alesiani, D., D'Arcangelo, G., & Tagliatesta, P. (2007). Gas chromatography-mass spectrometry analysis of phenolic compounds from Carica papaya L. leaf. Journal of Food Composition and Analysis, 20(7), 584-590.

Carbonell-Capella, J. M., Buniowska, M., Esteve, M. J., & Frigola, A. (2015). Effect of Stevia rebaudiana addition on bioaccessibility of bioactive compounds and antioxidant activity of beverages based on exotic fruits mixed with oat following simulated human digestion. Food Chemistry, 184, 122-130.

Cardin, R., Saccoccio, G., Masutti, F., Bellentani, S., Farinati, F., & Tiribelli, C. (2001). DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study. Journal of Hepatology, 34(4), 587-592.

Castillo, M., & Babson, J. (1998). Ca2+-dependent mechanisms of cell injury in cultured cortical neurons. Neuroscience, 86(4), 1133-1144.

Cavell, B. E., Syed Alwi, S. S., Donlevy, A., & Packham, G. (2011). Anti-angiogenic effects of dietary isothiocyanates: Mechanisms of action and implications for human health. Biochemical Pharmacology, 81(3), 327-336.

111

Chandrika, U. G., Jansz, E. R., Wickramasinghe, S. M. D. N., & Warnasuriya, N. D. (2003). Carotenoids in yellow- and red-fleshed papaya (Carica papaya L). Journal of the Science of Food and Agriculture, 83(12), 1279-1282.

Chapman, K., Havill, M., Watson, W. L., Wellard, L., Hughes, C., Bauman, A., & Allman- Farinelli, M. J. A. (2016). Time to address continued poor vegetable intake in Australia for prevention of chronic disease. 107, 295-302.

Charoensiri, R., Kongkachuichai, R., Suknicom, S., & Sungpuag, P. (2009). Beta-carotene, lycopene, and alpha-tocopherol contents of selected Thai fruits. Food Chemistry, 113(1), 202-207.

Chen, X.-X., Lam, K. H., Chen, Q.-X., Leung, G. P.-H., Tang, S. C. W., Sze, S. C.-W., . . . Zhang, Z.-J. (2017). Ficus virens proanthocyanidins induced apoptosis in breast cancer cells concomitantly ameliorated 5-fluorouracil induced intestinal mucositis in rats. Food and Chemical Toxicology, 110, 49-61.

Ching, S., Ramachandran, V., Gew, L. T., Lim, S. M. S., Sulaiman, W. A. W., Foo, Y. L., . . . Veettil, S. K. (2015). Complementary alternative medicine use among patients with dengue fever in the hospital setting: a cross-sectional study in Malaysia. BMC complementary and alternative medicine, 16(1), 37.

Chinoy, N. J., D'Souza, J. M., & Padman, P. Effects of crude aqueous extract of Carica papaya seeds in male albino mice. Reproductive Toxicology, 8(1), 75-79.

Chinoy, N. J., Dilip, T., & Harsha, J. (1995). Effect of Carica papaya seed extract on female rat ovaries and uteri. Phytotherapy Research, 9(3), 169-175.

Collard, E., & Roy, S. (2010). Improved Function of Diabetic Wound-Site Macrophages and Accelerated Wound Closure in Response to Oral Supplementation of a Fermented Papaya Preparation. Antioxidants & Redox Signaling, 13(5), 599-606.

Conaway, C. C., Getahun, S. M., Liebes, L. L., Pusateri, D. J., Topham, D. K. W., Botero-Omary, M., & Chung, F. L. (2000). Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli. Nutrition and cancer, 38(2), 168-178.

Cooper, T. G., & Yeung, C. H. (1999). Approaches to post-testicular contraception. Asian J Androl, 1, 29-36.

Cortés, C., Esteve, M., Rodrigo, D., Torregrosa, F., & Frígola, A. (2006). Changes of colour and carotenoids contents during high intensity pulsed electric field treatment in orange juices. Food and Chemical Toxicology, 44(11), 1932-1939.

Cuastumal, H. G., Ledesma, M. A., & Ordonez, L. E. (2016). Vitamin C and surface color in tomato and green pepper: effect of heat treatments. Entre Ciencia E Ingenieria(20), 32-36.

Dakare, M. A., Ameh, D. A., & Agbaji, A. S. (2011). Biochemical assessment of daddawa food seasoning produced by fermentation of pawpaw (Carica papaya) seeds. Pakistan Journal of Nutrition, 10(3), 220-223.

112

Danese, C., Esposito, D., D'Alfonso, V., Cirene, M., Ambrosino, M., & Colotto, M. (2006). Plasma glucose level decreases as collateral effect of fermented papaya preparation use. Clin Ter, 157(3), 195-198.

Davey, M. W., Montagu, M. V., Inzé, D., Sanmartin, M., Kanellis, A., Smirnoff, N., . . . Fletcher, J. (2000). Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. Journal of the Science of Food and Agriculture, 80(7), 825-860. de Oliveira, J. G., & Vitoria, A. P. (2011). Papaya: Nutritional and pharmacological characterization, and quality loss due to physiological disorders. An overview. Food Research International.

De Rosso, V. V., & Mercadante, A. Z. (2005). Carotenoid composition of two Brazilian genotypes of acerola (Malpighia punicifolia L.) from two harvests. Food Research International, 38(8- 9), 1073-1077.

Del Rio, D., Costa, L. G., Lean, M. E. J., & Crozier, A. (2010). Polyphenols and health: What compounds are involved? Nutrition, Metabolism and Cardiovascular Diseases, 20(1), 1-6.

Devitt, L. C., Fanning, K., Dietzgen, R. G., & Holton, T. A. (2009). Isolation and functional characterization of a lycopene b-cyclase gene that controls fruit colour of papaya (Carica papaya L.). Journal of Experimental Botany Advance, 1-7.

Drew, R. A. (2005). development of new papaya varieties for southeast and central queensland. (FR 02024).

Duke, J. (2011). Phytochemical and Ethnobotanical Databases. Retrieved 5 February 2011 http://sun.ars-grin.gov:8080/npgspub/xsql/duke/plantdisp.xsql?taxon=209

Ekanem, S. B., & Bassey, P. O. (2003). Effect of paw-paw seeds (Carica papaya) as antifertility agent in female Nile Tilapia (Oreochromis niloticus). . J Aquaculture Tropics, 18, 181-188.

Ekanem, S. B., & Okoronkwo, T. E. (2003). Paw-paw seeds as fertility control agent on male Nile Tilapia. NAGA World Fish Centre Quarterly, 26, 8-10.

Ekor, M. J. F. i. p. (2014). The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. 4, 177.

Eliagita, C., Kuntjoro, T., Sumarni, S., Suwondo, A., Hadisaputro, S., Eliagita, C., & Mulyantoro, D. K. (2017). Effect of consuming papaya (carica papaya linn.) on the level of hemoglobin and hematocrit in pregnant women with anemia. Belitung Nursing Journal, 3(2), 120-125.

Epriliati, I., D’Arcy, B., & Gidley, M. (2009). Nutriomic Analysis of Fresh and Processed Fruit Products. 1. During in Vitro Digestions. Journal of Agricultural and Food Chemistry, 57(8), 3363-3376.

Ezike, A. C., Akah, P. A., Okoli, C. O., Ezeuchenne, N. A., & Ezeugwu, S. (2009). Carica papaya (Paw-Paw) Unripe Fruit May Be Beneficial in Ulcer. Journal of Medicinal Food, 12(6), 1268-1273.

113

Fabbri, A. D. T., & Crosby, G. A. (2016). A review of the impact of preparation and cooking on the nutritional quality of vegetables and legumes. International Journal of Gastronomy and Food Science.

Faller, A. L. K., & Fialho, E. (2010). Polyphenol content and antioxidant capacity in organic and conventional plant foods. Journal of Food Composition and Analysis, 23(6), 561-568.

Fanning, K. J., Martin, I., Wong, L., Keating, V., Pun, S., & O'Hare, T. (2010). Screening sweetcorn for enhanced zeaxanthin concentration. Journal of the Science of Food and Agriculture, 90(1), 91-96.

FAOSTAT. (2015a). Detailed Trade Data: Export Quantity Retrieved from http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor

FAOSTAT. (2015b). Production crops. Retrieved from http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor

Fasihuddin, A., & Ghazally, I. (2003). Medicinal Plants Used by Kadazan Dusun Communities Around Crocker Range. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC).

Fibach, E., & Rachmilewitz, E. A. (2010). The role of antioxidants and iron chelators in the treatment of oxidative stress in thalassemia. Annals of the New York Academy of Sciences 1202, 10-16. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20712766.

Fibach, E., Tan, E. S., Jamuar, S., Ng, I., Amer, J., & Rachmilewitz, E. A. (2010). Amelioration of oxidative stress in red blood cells from patients with beta-thalassemia major and intermedia and E-beta-thalassemia following administration of a fermented papaya preparation. Phytotherapy Research, 24(9), 1334-1338.

Foster, M., & Section, A. C. (2014). Emerging animal and plant Industries-their value to Australia. Retrieved from Australia.

Fraga, C. G., Galleano, M., Verstraeten, S. V., & Oteiza, P. I. (2010). Basic biochemical mechanisms behind the health benefits of polyphenols. Molecular Aspects of Medicine, 31(6), 435-445.

Franke, A. A., Custer, L. J., Arakaki, C., & Murphy, S. P. (2004). Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Journal of Food Composition and Analysis, 17(1), 1-35.

Australia New Zealand Food Standards Code – Schedule 4 – Nutrition, health and related claims, 1.2.7—12(1) C.F.R. (2017).

Garrett, A. (1995). The Pollination Biology of Papaw (Carica papaya L.) in Central Queensland. (PhD Thesis PhD Thesis), Central Queensland University, Rockhampton.

Gayosso-García Sancho, L. E., Yahia, E. M., & González-Aguilar, G. A. (2011). Identification and quantification of phenols, carotenoids, and vitamin C from papaya (Carica papaya L., cv. Maradol) fruit determined by HPLC-DAD-MS/MS-ESI. Food Research International, 44(5), 1284-1291. 114

Gebhardt, S. E., & Thomas, R. G. (2002). Nutritive value of foods. Beltsville, Md. US Department of Agriculture, Agricultural Research Service, Nutrient Data Laboratory. Home and Garden, Bulletin(72), 95.

Ghoti, H., Fibach, E., Dana, M., Abu Shaban, M., Jeadi, H., Braester, A., . . . Rachmilewitz, E. (2011). Oxidative stress contributes to hemolysis in patients with hereditary spherocytosis and can be ameliorated by fermented papaya preparation. Annals of Hematology, 90(5), 509- 513.

Ghoti, H., Rosenbaum, H., Fibach, E., & Rachmilewitz, E. A. (2010). Decreased hemolysis following administration of antioxidant-fermented papaya preparation (FPP) to a patient with PNH. Annals of Hematology, 89(4), 429-430.

Gliszczyńska-Świgło, A., Ciska, E., Pawlak-Lemańska, K., Chmielewski, J., Borkowski, T., & Tyrakowska, B. (2006). Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing. Food Additives & Contaminants, 23(11), 1088-1098.

Gonçalves, E., Pinheiro, J., Abreu, M., Brandão, T., & Silva, C. L. M. (2010). Carrot (Daucus carota L.) peroxidase inactivation, phenolic content and physical changes kinetics due to blanching. Journal of Food Engineering, 97(4), 574-581.

González Mosquera, D. M., Ortega, Y. H., Kilonda, A., Dehaen, W., Pieters, L., & Apers, S. (2011). Evaluation of the in vivo anti-inflammatory activity of a flavonoid glycoside from Boldoa purpurascens. Phytochemistry Letters.

Gurung, S., & Skalko-Basnet, N. (2009). Wound healing properties of Carica papaya latex: In vivo evaluation in mice burn model. Journal of Ethnopharmacology, 121(2), 338-341.

Hagiwara, K. (2001). Standard Tables of Food Composition in Japan Fifth Revised Edition. Journal for the Integrated Study of Dietary Habits, 12(2), 86-89.

Harald, W. T. (2003). Papaya the Medicine Tree: Third Edition (3 ed.): Harald, W.Tietze publishing.

Hart, D. J., & Scott, K. J. (1995). Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chemistry, 54(1), 101-111.

Haytowitz, D., & Bhagwat, S. (2010). USDA Database for the Oxygen Radical Capacity (Orac) of Selected Foods, Release 2.

Hdider, C., Ilahy, R., Tlili, I., Lenucci, M. S., & Dalessandro, G. (2013). Effect of the stage of maturity on the antioxidant content and antioxidant activity of high-pigment tomato cultivars grown in Italy. Food, 7(1), 1-7.

Hernández, Y., Lobo, M. G., & González, M. (2006). Determination of vitamin C in tropical fruits: A comparative evaluation of methods. Food Chemistry, 96(4), 654-664.

115

Hernández, Y., Lobo, M. G., & González, M. (2009). Factors affecting sample extraction in the liquid chromatographic determination of organic acids in papaya and pineapple. Food Chemistry, 114(2), 734-741.

Hesse, L., Beher, D., Masters, C. L., & Multhaup, G. (1994). The [beta] A4 amyloid precursor protein binding to copper. FEBS letters, 349(1), 109-116.

Hidalgo, F. J., & Zamora, R. (2017). Food processing antioxidants. In Advances in food and nutrition research (Vol. 81, pp. 31-64): Elsevier.

Hiramoto, K., Imao, M., Sato, E. F., Inoue, M., & Mori, A. (2008). Effect of fermented papaya preparation on dermal and intestinal mucosal immunity and allergic inflammations. Journal of the Science of Food and Agriculture, 88(7), 1151-1157.

Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., & Prior, R. L. (2002). High-Throughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. Journal of Agricultural and Food Chemistry, 50(16), 4437-4444.

Huang, D., Ou, B., & Prior, R. L. (2005). The Chemistry behind Antioxidant Capacity Assays. Journal of Agricultural and Food Chemistry, 53(6), 1841-1856.

Huang, X., Atwood, C. S., Hartshorn, M. A., Multhaup, G., Goldstein, L. E., Scarpa, R. C., . . . Moir, R. D. (1999). The Aβ peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry, 38(24), 7609-7616.

Hung, Y. H., Bush, A. I., & Cherny, R. A. (2010). Copper in the brain and Alzheimer’s disease. Journal of Biological Inorganic Chemistry, 15(1), 61-76.

Husin, F., Ya'akob, H., Rashid, S. N. A., Shahar, S., Soib, H. H. J. B., & Biotechnology, A. (2019). Cytotoxicity study and antioxidant activity of crude extracts and SPE fractions from Carica papaya leaves. 101130.

Hwang, E. S., & Lee, H. J. (2006). Phenylethyl isothiocyanate and its N-acetylcysteine conjugate suppress the metastasis of SK-Hep1 human hepatoma cells. The Journal of Nutritional Biochemistry, 17 837-846.

Ikram, E. H. K., Eng, K. H., Jalil, A. M. M., Ismail, A., Idris, S., Azlan, A., . . . Mokhtar, R. A. M. (2009). Antioxidant capacity and total phenolic content of Malaysian underutilized fruits. Journal of Food Composition and Analysis, 22(5), 388-393.

Ikram, E. H. K., Stanley, R., Netzel, M., & Fanning, K. (2015). Phytochemicals of papaya and its traditional health and culinary uses–A review. Journal of Food Composition and Analysis, 41, 201-211.

Imao, K., Wang, H., Komatsu, M., & Hiramatsu, M. (1998). Free radical scavenging activity of fermented papaya preparation and its effect on lipid peroxide level and superoxide dismutase activity in iron-induced epileptic foci of rats. Biochemistry and Molecular Biology International, 45(1), 11-23.

116

Indran, M., Mahmood, A. A., & Kuppusamy, U. R. (2008). Protective effect of carica papaya L leaf extract against alcohol induced acute gastric damage and blood oxidative stress in rats. West Indian Medical Journal, 57(4), 323-326.

Ioannidi, E., Kalamaki, M. S., Engineer, C., Pateraki, I., Alexandrou, D., Mellidou, I., . . . Kanellis, A. K. (2009). Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. Journal of Experimental Botany, 60(2), 663.

Iqbal, M. P., Kazim, S. F., & Mehboobali, N. (2006). Ascorbic acid contents of Pakistani fruits and vegetables. Pak J Pharm Sci, 19(4), 282-285.

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(1), 77-84.

Isabelle, M., Lee, B. L., Ong, C. N., Liu, X., & Huang, D. (2008). Peroxyl Radical Scavenging Capacity, Polyphenolics, and Lipophilic Antioxidant Profiles of Mulberry Fruits Cultivated in Southern China. Journal of Agricultural and Food Chemistry, 56(20), 9410-9416.

Jiao, Z., Deng, J., Li, G., Zhang, Z., & Cai, Z. (2010). Study on the compositional differences between transgenic and non-transgenic papaya (Carica papaya L.). Journal of Food Composition and Analysis, 23(6), 640-647.

Jindal, K. K., & Singh, R. N. (1975). Phenolic Content in Male and Female Carica papaya: A Possible Physiological Marker for Sex Identification of Vegetative Seedlings. Physiologia Plantarum, 33(1), 104-107.

Johansen, C., Vinter, H., Soegaard‐Madsen, L., Olsen, L., Steiniche, T., Iversen, L., & Kragballe, K. (2010). Preferential inhibition of the mRNA expression of p38 mitogen‐activated protein kinase regulated cytokines in psoriatic skin by anti‐TNFα therapy. British Journal of Dermatology, 163(6), 1194-1204.

John E, M. (2007). The Aging Gut: Physiology. Clinics in Geriatric Medicine, 23(4), 757-767.

Kao, F.-J., Chiu, Y.-S., & Chiang, W.-D. (2014). Effect of water cooking on antioxidant capacity of carotenoid-rich vegetables in Taiwan. journal of food and drug analysis, 22(2), 202-209.

Kao, F. J., Chiu, Y. S., Tsou, M. J., & Chiang, W. D. (2012). Effects of Chinese domestic cooking methods on the carotenoid composition of vegetables in Taiwan. LWT - Food Science and Technology, 46(2), 485-492.

Kathiresan, S., Surash, R., Sharif, M. M., Mas Rosemal, M. H. H., & Walther, H. W. (2009). Thrombocyte counts in mice after the administration of papaya leaf suspension. The Middle European Journal of Medicine, 121(Suppl 3), 19-22.

Kato, M., Imayoshi, Y., Iwabuchi, H., & Shimomura, K. (2011). Kinetic Changes in Glucosinolate- Derived Volatiles by Heat-Treatment and Myrosinase Activity in Nakajimana (Brassica rapa L. cv. nakajimana). Journal of Agricultural and Food Chemistry.

117

Kato, S., Bowman, E. D., Harrington, A. M., Blomeke, B., & Shields, P. G. (1995). Human lung carcinogen—DNA adduct levels mediated by genetic polymorphisms in vivo. Journal of the National Cancer Institute, 87(12), 902.

Kelebek, H., Selli, S., Gubbuk, H., & Gunes, E. (2015). Comparative evaluation of volatiles, phenolics, sugars, organic acids and antioxidant properties of Sel-42 and Tainung papaya varieties. Food chemistry, 173, 912-919.

Kermanshai, R., McCarry, B. E., Rosenfeld, J., Summers, P. S., Weretilnyk, E. A., & Sorger, G. J. (2001). Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts. Phytochemistry, 57(3), 427-435. Ketsa, S., Phakawatmongkol, W., & Subhadrabhandhu, S. (1999). Peel enzymatic activity and colour changes in ripening mango fruit. Journal of Plant Physiology, 154(3), 363-366.

Khairi, M. S. (2007). Analysis of preservatives in pharmaceutical products. Retrieved from http://www.pharmainfo.net/reviews/analysis-preservatives-pharmaceutical-products

Kimura, M., Rodriguezamaya, D. B., & Yokoyama, S. M. (1991). Cultivar Differences And Geographic Effects On The Carotenoid Composition And Vitamin-A Value Of Papaya. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie, 24(5), 415-418.

Kirkhus, B., Afseth, N. K., Borge, G. I. A., Grimsby, S., Steppeler, C., Krona, A., & Langton, M. J. F. c. (2019). Increased release of carotenoids and delayed in vitro lipid digestion of high pressure homogenized tomato and pepper emulsions. 285, 282-289.

Krishna, K., Paridhavi, M., & Patel, J. A. (2008). Review on nutritional, medicinal and pharmacological properties of papaya (Carica papaya Linn.). Natural Product Radiance, 7(4), 364-373.

Lafarga, T., Viñas, I., Bobo, G., Simó, J., & Aguiló-Aguayo, I. (2018). Effect of steaming and sous vide processing on the total phenolic content, vitamin C and antioxidant potential of the genus Brassica. Innovative Food Science & Emerging Technologies, 47, 412-420.

Lako, J., Trenerry, V. C., Wahlqvist, M., Wattanapenpaiboon, N., Sotheeswaran, S., & Premier, R. (2007). Phytochemical flavonols, carotenoids and the antioxidant properties of a wide selection of Fijian fruit, vegetables and other readily available foods. Food Chemistry, 101(4), 1727-1741.

Lee, S. K., & Kader, A. A. (2000). Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest biology and technology, 20(3), 207-220.

Leong, L. P., & Shui, G. (2002). An investigation of antioxidant capacity of fruits in Singapore markets. Food Chemistry, 76(1), 69-75.

Lim, Y. Y., Lim, T. T., & Tee, J. J. (2007). Antioxidant properties of several tropical fruits: A comparative study. Food Chemistry, 103(3), 1003-1008.

Lin, H. J., Han, C. Y., Bernstein, D. A., Hsiao, W., Lin, B. K., & Hardy, S. (1994). Ethnic distribution of the glutathione transferase Mu 1-1 (GSTM1) null genotype in 1473 individuals and application to bladder cancer susceptibifity. Carcinogenesis, 15(5), 1077.

118

Liu, Y., Perera, C. O., & Suresh, V. (2007). Comparison of three chosen vegetables with others from South East Asia for their lutein and zeaxanthin content. Food Chemistry, 101(4), 1533- 1539.

Logozzi, M., Mizzoni, D., Di Raimo, R., Macchia, D., Spada, M., & Fais, S. J. C. (2019). Oral Administration of Fermented Papaya (FPP®) Controls the Growth of a Murine Melanoma through the In Vivo Induction of a Natural Antioxidant Response. 11(1), 118.

Loh, S. P., & Hadira, O. (2011). In vitro inhibitory potential of selected Malaysian plants against key enzymes involved in hyperglycemia and hypertension. Malaysian Journal of Nutrition, 17(1), 77-86.

Lohiya, N. K., Manivannan, B., & Garg, S. (2006). Toxicological investigations on the methanol sub-fraction of the seeds of Carica papaya as a male contraceptive in albino rats. Reproductive Toxicology, 22(3), 461-468.

Lohiya, N. K., Manivannan, B., Mishra, P. K., Pathak, N., Sriram, S., Bhande, S. S., & al., E. (2002). The chloroform extract of the seeds of Carica papaya by oral administration induces long-term reversible azoospermia in langur monkey Presbytis entellus entellus. Asian J Androl 4, 17-26.

Lohiya, N. K., Mishra, P. K., Pathak, N., Manivannan, B., Bhande, S. S., Panneerdoss, S., & Sriram, S. (2005). Efficacy trial on the purified compounds of the seeds of Carica papaya for male contraception in albino rat. Reproductive Toxicology, 20(1), 135-148.

Lohiya, N. K., Pathak, N., Mishra, P. K., & Manivannan, B. (1999). Reversible contraception with chloroform extract of Carica papaya linn. seeds in male rabbits. Reproductive Toxicology, 13(1), 59-66.

Lohiya, N. K., Pathak, N., Mishra, P. K., & Manivannan, B. (2000). Contraceptive evaluation and toxicological study of aqueous extract of the seeds of Carica papaya in male rabbits. Journal of Ethnopharmacology, 70, 17-27.

Lovell, M., Robertson, J., Teesdale, W., Campbell, J., & Markesbery, W. (1998). Copper, iron and zinc in Alzheimer's disease senile plaques. Journal of the Neurological Sciences, 158(1), 47- 52.

Lu, J., Zheng, Y.-l., Luo, L., Wu, D.-m., Sun, D.-x., & Feng, Y.-j. (2006). Quercetin reverses d- galactose induced neurotoxicity in mouse brain. Behavioural Brain Research, 171(2), 251- 260.

Luo, X., Wang, Q., Zheng, B., Lin, L., Chen, B., Zheng, Y., & Xiao, J. (2017). Hydration properties and binding capacities of dietary fibers from bamboo shoot shell and its hypolipidemic effects in mice. Food and Chemical Toxicology, 109, 1003-1009.

MacLeod, A., & Pieris, N. M. (1983). Volatile components of papaya (Carica papaya L.) with particular reference to glucosinolate products. Journal Agricultural of Food Chemistry, 31, 1005-1008.

119

Mahattanatawee, K., Manthey, J. A., Luzio, G., Talcott, S. T., Goodner, K., & Baldwin, E. A. (2006). Total antioxidant activity and fiber content of select Florida-grown tropical fruits. Journal of Agricultural and Food Chemistry, 54(19), 7355-7363.

Marelli de Souza, L., Silva Ferreira, K., Paes Chaves, J. B., & Lopes Teixeira, S. (2008). L- Ascorbic acid, β-Caroteno and lycopene content in papaya fruits (Carica papaya) without physiological skin freckles. Science Agriculture, 3(65), 246−250.

Marfo, E. K., Oke, O. L., & Afolabi, O. A. (1986a). Chemical composition of papaya (Carica papaya) seeds. Food Chemistry, 22(4), 259-266.

Marfo, E. K., Oke, O. L., & Afolabi, O. A. (1986b). Some studies on the proteins of Carica papaya seeds. Food Chemistry, 22(4), 267-277.

Marotta, F., Koike, K., Lorenzetti, A., Jain, S., Signorelli, P., Metugriachuk, Y., . . . Locorotondo, N. (2010). Regulating Redox Balance Gene Expression in Healthy Individuals by Nutraceuticals: A Pilot Study. Rejuvenation Research, 13(2-3), 175-178.

Marotta, F., Weksler, M., Naito, Y., Yoshida, C., Yoshioka, M., & Marandola, P. (2006). Nutraceutical supplementation: effect of a fermented papaya preparation on redox status and DNA damage in healthy elderly individuals and relationship with GSTM1 genotype: a randomized, placebo-controlled, cross-over study. Annals of the New York Academy of Sciences, 1067, 400-407.

Marotta, F., Yoshida, C., Barreto, R., Naito, Y., & Packer, L. (2007). Oxidative-inflammatory damage in cirrhosis: effect of vitamin E and a fermented papaya preparation. J Gastroenterol Hepatol, 22(5), 697-703.

Martí, N., Mena, P., Cánovas, J. A., Micol, V., & Saura, D. (2009). Vitamin C and the role of citrus juices as functional food. Natural product communications, 4(5), 677.

Matsusaka, Y., & Kawabata, J. (2010). Evaluation of Antioxidant Capacity of Non-Edible Parts of Some Selected Tropical Fruits. Food Science and Technology Research, 16(5), 467-472.

Maurizi, A., De Michele, A., Ranfa, A., Ricci, A., Roscini, V., Coli, R., . . . Burini, G. (2015). Bioactive compounds and antioxidant characterization of three edible wild plants traditionally consumed in the Umbria Region (Central Italy): Bunias erucago L. (corn rocket), Lactuca perennis L. (mountain lettuce) and Papaver rhoeas L. (poppy). Journal of Applied Botany and Food Quality, 88, 109-114.

Mehdipour, S., Yasa, N., Dehghan, G., Khorasani, R., Mohammadirad, A., Rahimi, R., & Abdollahi, M. (2006). Antioxidant potentials of Iranian Carica papaya juice in vitro and in vivo are comparable to alpha-tocopherol. Phytotherapy Research, 20(7), 591-594.

Miean, K. H., & Mohamed, S. (2001). Flavonoid (Myricetin, Quercetin, Kaempferol, Luteolin, and Apigenin) Content of Edible Tropical Plants. Journal of Agricultural and Food Chemistry, 49(6), 3106-3112.

Miglio, C., Chiavaro, E., Visconti, A., Fogliano, V., & Pellegrini, N. (2008). Effects of Different Cooking Methods on Nutritional and Physicochemical Characteristics of Selected Vegetables. Journal of Agricultural and Food Chemistry, 56(1), 139-147. 120

Miriam Hall (ABC). (2010). Bumper papaya crop in 2010. ABC Rural Report.

Mojica-Henshaw, M. P., Francisco, A. D., De Guzman, F., & Tigno, X. T. (2003). Possible immunomodulatory actions of Carica papaya seed extract. Clin Hemorheol Microcirc, 29(3- 4), 219-229.

Mojisola, C.-O. C., Anthony, E. A., & Alani, D. M. (2009). Antisickling properties of the fermented mixture of Carica papaya Linn and Sorghum bicolor (L.) Moench. African Journal of Pharmacy and Pharmacology, 3(4), 140-143.

Monge-Rojas, R., & Campos, H. (2011). Tocopherol and carotenoid content of foods commonly consumed in Costa Rica. Journal of Food Composition and Analysis, 24(2), 202-216.

Moon, J.-H., Tsushida, T., Nakahara, K., & Terao, J. (2001). Identification of quercetin 3-O-β-D- glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin. Free Radical Biology and Medicine, 30(11), 1274-1285.

Morayo, O., & Akinloye, O. (2010). Evaluation of andrological indices and testicular histology following chronic administration of aqueous extract of carica papaya leaf in wistar rat. African Journal of Pharmacy and Pharmacology, 4, 252-255.

Morton, J. (Ed.) (1987). Fruits of warm climates.

Nafiu, A. B., Alli-Oluwafuyi, A.-m., Haleemat, A., Olalekan, I. S., & Rahman, M. T. (2019). Papaya (Carica papaya L., Pawpaw). In Nonvitamin and Nonmineral Nutritional Supplements (pp. 335-359): Elsevier.

Nakamura, Y., Morimitsub, Y., Tomoko, U., Hajime, O., Akira, M., Yuko, N., . . . Koji, U. (2000). A glutathione S-transferase inducer from papaya: rapid screening,identi®cation and structure-activity relationship of isothiocyanates. Cancer Letters, 157, 193-200.

Nakamura, Y., Yoshimoto, M., Murata, Y., Shimoishi, Y., Asai, Y., Park, E. Y., . . . Nakamura, Y. (2007). Papaya Seed Represents a Rich Source of Biologically Active Isothiocyanate. Journal of Agricultural and Food Chemistry, 55(11), 4407-4413.

Nakasone, H. Y., & Paull, R. E. (Eds.). (1998). Tropical fruits. : CAB International, Wallingford.

Nam, J. S., Jang, H. L., & Rhee, Y. H. (2018). Nutritional compositions in roots, twigs, leaves, fruit pulp, and seeds from pawpaw (Asimina triloba L. Dunal) grown in Korea. Journal of Applied Botany and Food Quality, 91, 47-55.

Nayak, S. B., Pinto Pereira, L., & Maharaj, D. (2007). Wound healing activity of Carica papaya L. in expeimentally induced diabetic rats. Indian Journal of Experimental Biology, 45, 739- 743.

Nelson, H. H., Wiencke, J. K., Christiani, D. C., Cheng, T. J., Zuo, Z.-F., Schwartz, B. S., . . . Kelsey, K. T. (1995). Ethnic differences in the prevalence of the homozygous deleted genotype of glutathione S-transferase theta. Carcinogenesis, 16(5), 1243-1246.

121

Netzel, M., Fanning, K., Netzel, G., Zabaras, D., Karagianis, G., Treloar, T., . . . Stanley, R. J. J. o. F. B. (2012). Urinary excretion of antioxidants in healthy humans following Queen Garnet plum juice ingestion: a new plum variety rich in antioxidant compounds. 36(2), 159-170.

Netzel, M., Netzel, G., Tian, Q., Schwartz, S., & Konczak, I. (2006). Sources of Antioxidant Activity in Australian Native Fruits. Identification and Quantification of Anthocyanins. Journal of Agricultural and Food Chemistry, 54(26), 9820-9826.

Netzel, M., Netzel, G., Zabaras, D., Lundin, L., Day, L., Addepalli, R., . . . Seymour, R. J. F. R. I. (2011). Release and absorption of carotenes from processed carrots (Daucus carota) using in vitro digestion coupled with a Caco-2 cell trans-well culture model. 44(4), 868-874.

Neveu, V., Perez-Jimenez, J., Vos, F., Crespy, V., du Chaffaut, L., Mennen, L., . . . Wishart, D. (2010). Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database, 2010, 024.

Noda, Y., Murakami, S., Mankura, M., & Mori, A. (2008). Inhibitory effect of fermented papaya preparation on hydroxyl radical generation from methylguanidine. J Clin Biochem Nutr, 43(3), 185-190.

Nugroho, A., Heryani, H., Choi, J. S., & Park, H.-J. (2017). Identification and quantification of flavonoids in Carica papaya leaf and peroxynitrite-scavenging activity. Asian Pacific Journal of Tropical Biomedicine, 7(3), 208-213.

Nwofia, G. E., Ojimelukw, P., & Eji, C. (2012). Chemical composition of leaves, fruit pulp and seeds in some Carica papaya (L) morphotypes. International Journal of Medicinal and Aromatic Plants 2, 200-206.

OECD. (2003). Draft Consensus Document on the Biology of Carica papaya (L.) (Papaya). . (Report No. 5 February 2003). France: Organisation for Economic Co-operation and Development

OECD. (2005). Consensus Document on the Biology of Papaya (Carica papaya) Series on Harmonisation of Regulatory Oversight in Biotechnology No. 33.

Okeniyi, J. A. O., Ogunlesi, T. A., Oyelami, O. A., & Adeyemi, L. A. (2007). Effectiveness of Dried Carica papaya Seeds Against Human Intestinal Parasitosis: A Pilot Study. Journal of Medicinal Food, 10(1), 194-196.

Olafsdottir, E. S., Bolt Jørgensen, L., & Jaroszewski, J. W. (2002). Cyanogenesis in glucosinolate- producing plants: Carica papaya and Carica quercifolia. Phytochemistry, 60(3), 269-273. Olalla-Saad, S. T. (2010). Fermented papaya preparation for beta-thalassemia? Expert Review of Hematology, 3(3), 265-268.

Olmos, A., Giner, R., Recio, M., Rios, J., Cerdá‐Nicolás, J., & Manez, S. (2007). Effects of plant alkylphenols on cytokine production, tyrosine nitration and inflammatory damage in the efferent phase of contact hypersensitivity. British journal of pharmacology, 152(3), 366- 373.

Oloyede, O. I. (2005). Chemical profile of unripe pulp of Carica papaya. . Pak. J. Nutr., 4, 379-381.

122

Ong, H. C., & Norzalina, J. (1999). Malay herbal medicine in Gemencheh, Negri Sembilan, Malaysia. Fitoterapia, 70(1), 10-14.

Ornelas-Paz, J. d. J., Yahia, E. M., & Gardea, A. A. (2008). Changes in external and internal color during postharvest ripening of [`]Manila' and [`]Ataulfo' mango fruit and relationship with carotenoid content determined by liquid chromatography-APcI+-time-of-flight mass spectrometry. Postharvest Biology and Technology, 50(2-3), 145-152.

Osato, J. A., Santiago, L. A., Remo, G. M., Cuadra, M. S., & Mori, A. (1993). Antimicrobial and antioxidant activities of unripe papaya. Life Sci, 53(17), 1383-1389.

Osato Research Institute. (2003). Immun’Age FPP (Fermented Papaya Preparation) : A Nutritional Approach to Aging In (pp. 2-30). Japan: Osato Research Institute.

Otsuki, N., Dang, N. H., Kumagai, E., Kondo, A., Iwata, S., & Morimoto, C. (2010). Aqueous extract of Carica papaya leaves exhibits anti-tumor activity and immunomodulatory effects. Journal of Ethnopharmacology, 127(3), 760-767.

Owoyele, Bamidele, V., Funmilayo, Adeoye, A., Adebukola, Olubori, M., . . . Ayodele, O. (2008). Anti-inflammatory activities of ethanolic extract of Carica papaya leaves. Inflammopharmacology, 16(4), 168-173.

Oyebode, O., Kandala, N.-B., Chilton, P. J., & Lilford, R. J. (2016). Use of traditional medicine in middle-income countries: a WHO-SAGE study. Health policy and planning, 31(8), 984- 991.

Palafox-Carlos, H., Yahia, E., & González-Aguilar, G. (2012). Identification and quantification of major phenolic compounds from mango (Mangifera indica, cv. Ataulfo) fruit by HPLC– DAD–MS/MS-ESI and their individual contribution to the antioxidant activity during ripening. Food Chemistry, 135(1), 105-111.

Pandey, S., Walpole, C., Cabot, P. J., Shaw, P. N., Batra, J., & Hewavitharana, A. K. (2017). Selective anti-proliferative activities of Carica papaya leaf juice extracts against prostate cancer. Biomedicine & Pharmacotherapy, 89, 515-523.

Papaya Australia. (2019). Homepage for papaya Australia. Retrieved from http://www.australianpapaya.com.au/home

Passera, C., & Spettoli, P. (1981). Chemical composition of papaya seeds. Plant Foods for Human Nutrition (Formerly Qualitas Plantarum), 31(1), 77-83.

Patidar, M. K., Nighojkar, S., Kumar, A., & Nighojkar, A. (2016). Papaya peel valorization for production of acidic pectin methylesterase by Aspergillus tubingensis and its application for fruit juice clarification. Biocatalysis and Agricultural Biotechnology, 6, 58-67.

Patthamakanokporn, O., Puwastien, P., Nitithamyong, A., & Sirichakwal, P. P. (2008). Changes of antioxidant activity and total phenolic compounds during storage of selected fruits. Journal of Food Composition and Analysis, 21(3), 241-248.

Payyappallimana, U. (2010). Role of Traditional Medicine in Primary Health Care : An Overview of Perspectives and Challenging (Vol. 14). 123

Pelzer, L. E., Guardia, T., Juarez, A. O., & Guerreiro, E. (1998). Acute and chronic antiinflammatory effects of plant flavonoids. Il Farmaco, 53(6), 421-424.

Pereira, T., de Almeida, P. S. G., de Azevedo, I. G., da Cunha, M., de Oliveira, J. G., da Silva, M. G., & Vargas, H. (2009). Gas diffusion in [`]Golden' papaya fruit at different maturity stages. Postharvest Biology and Technology, 54(3), 123-130.

Pérez-Vicente, A., Gil-Izquierdo, A., & García-Viguera, C. (2002). In vitro gastrointestinal digestion study of pomegranate juice phenolic compounds, anthocyanins, and vitamin C. Journal of agricultural and food chemistry, 50(8), 2308-2312.

Perkins-Veazie, P., Roberts, W., & Collins, J. K. (2007). Lycopene content among organically produced tomatoes. Journal of Vegetable Science, 12(4), 93-106.

Petti, S., & Scully, C. (2009). Polyphenols, oral health and disease: A review. Journal of dentistry, 37(6), 413-423.

Pietretti, A., Karioti, A., Sannella, A., Orsini, S., Scalone, A., Gradoni, L., . . . Bilia, A. (2010). Antiplasmodial in vivo activity of Carica papaya leaf decoction. Planta Med 76.

Pinton, P., Giorgi, C., Siviero, R., Zecchini, E., & Rizzuto, R. (2008). Calcium and apoptosis: ER- mitochondria Ca2+ transfer in the control of apoptosis. Oncogene, 27(50), 6407.

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.

Priscilla, C. S. (2008). Philippine fermented foods:principles and technology. Quezon City: The University of the Philippine Press.

Qiu, S., Lu, C., Li, X., & Toivonen, P. M. A. (2009). Effect of 1-MCP on quality and antioxidant capacity of in vitro digests from [`]Sunrise' apples stored at different temperatures. Food Research International, 42(3), 337-342.

Quisumbing, E. (1978). Medicinal Plants of the Philippines. Manila: Katha Publishing.

Rachmilewitz, E., & Schrier, S. (2001). Pathophysiology of thalassemia. Disorders of hemoglobin: genetics, pathophysiology and clinical management. MH Steinberg, BG Forget, DR Higgs, and RL Nagel, editors. Cambridge University Press. Cambridge, United Kingdom, 233–251.

Rahmat, A., Abu Bakar, M. F., Faezah, N., & Hambali, Z. (2004). The effects of consumption of guava (Psidium guajava) or papaya (Carica papaya) on total antioxidant and lipid profile in normal male youth Asia Pac J Clin Nutr (13 ), S106.

Rajarathnam, S. (2008). Perspectives of Processing Papaya (Carica papaya) Fruit: National and International Strategies. Paper presented at the II International Symposium on Papaya 851.

Ramírez-Jiménez, A. K., Rangel-Hernández, J., Morales-Sánchez, E., Loarca-Piña, G., & Gaytán- Martínez, M. J. F. c. (2019). Changes on the phytochemicals profile of instant corn flours obtained by traditional nixtamalization and ohmic heating process. 276, 57-62.

124

Reddy, P. H. (2009). Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease. Experimental Neurology, 218(2), 286-292.

Reddy, P. H., & Beal, M. F. (2008). Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer's disease. Trends in molecular medicine, 14(2), 45-53.

Rhabasa-Lhoret, R., & Chiasson, J. L. (2004). Alpha-glucosidase inhibitors. In R. A. Defronzo, E. Ferrannini, H. Keen, & P. Zimmet (Eds.), International textbook of diabetes mellitus (Vol. 1)(3rd ed.). UK: John Wiley.

Rinaldo, D., Mbéguié-A-Mbéguié, D., & Fils-Lycaon, B. (2010). Advances on polyphenols and their metabolism in sub-tropical and tropical fruits. Trends in Food Science & Technology, 21(12), 599-606.

Rivera-Pastrana, D. M., Yahia, E. M., & Gonzalez-Aguilar, G. (2010). Phenolic and carotenoid profiles of papaya fruit (Carica papaya L.) and their contents under low temperature storage. Journal of the Science of Food and Agriculture, 90, 2358−2365.

Rodríguez-Roque, M. J., Rojas-Graü, M. A., Elez-Martínez, P., & Martín-Belloso, O. (2014). In vitro bioaccessibility of health-related compounds as affected by the formulation of fruit juice-and milk-based beverages. Food Research International, 62, 771-778.

Ross, P., O'Hare, P., MacLeod, N., Kernot, I., Evans, D., Grice, K., . . . Astridge, D. (2000). Paw Paw Information Kit.

Rubinskienė, M., Viškelis, P., Stanys, V., Šikšnianas, T., & Sasnauskas, A. (2008). Quality changes in black currant berries during ripening. Sodininkystė ir daržininkystė, 27(2), 235-243.

Rund, D., & Rachmilewitz, E. (2005). -Thalassemia. New England Journal of Medicine, 353(11), 1135-1146.

Rusak, G., Piantanida, I., Mašić, L., Kapuralin, K., Durgo, K., & Kopjar, N. (2010). Spectrophotometric analysis of flavonoid-DNA interactions and DNA damaging/protecting and cytotoxic potential of flavonoids in human peripheral blood lymphocytes. Chemico- Biological Interactions, 188(1), 181-189.

Salinas, I., Hueso, J. J., & Cuevas, J. (2019). Fruit growth model, thermal requirements and fruit size determinants in papaya cultivars grown under subtropical conditions. Scientia Horticulturae, 246, 1022-1027.

Samson, J. A. (1986). Tropical Fruits. 2nd edn: Longman Scientific and Technical.

Sánchez-Mata, M. C., Cámara-Hurtado, M., Díez-Marqués, C., & Torija-Isasa, M. E. (2000). Comparison of high-performance liquid chromatography and spectrofluorimetry for vitamin C analysis of green beans (Phaseolus vulgaris L.). European Food Research and Technology, 210(3), 220-225.

Sánchez-Moreno, C. (2002). Review: Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Science and Technology International, 8(3), 121-137.

125

Sannella, A., Karioti, A., Vincieri, F., Messori, L., Maiori, G., Severini, C., & Bilia, A. (2009). Antiplasmodial activity of papaya leaf decoction and its synergistic effects in combination with artemisinin. Planta Med 75.

Santamaría-Basulto, F., Sauri-Duch, E., Espadas y Gil, F., Díaz- Plaza, R., Larqué-Saavedra, A., & Santamaría, J. (2009). Postharvest ripening and maturity indices for Maradol papaya. Interciencia, 34, 583−588.

Sasaki, N., Toda, T., Kaneko, T., Baba, N., & Matsuo, M. (2003). Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells. Chemico-Biological Interactions, 145(1), 101-116.

Satrija, F., Nansen, P., Murtini, S., & He, S. (1995). Anthelmintic activity of papaya latex against patent Heligmosomoides polygyrus infections in mice. Journal of Ethnopharmacology, 48(3), 161-164.

Scalbert, A., Johnson, I. T., & Saltmarsh, M. (2005). Polyphenols: antioxidants and beyond. The American Journal of Clinical Nutrition, 81(1), 215S-217S.

Schiffman, S. (2009). Sensory Impairment: Taste and Smell Impairments with Aging: Handbook of Clinical Nutrition and Aging. In (pp. 1-21): Humana Press.

Schoefs, B. t. (2002). Chlorophyll and carotenoid analysis in food products. Properties of the pigments and methods of analysis. Trends in Food Science & Technology, 13(11), 361-371.

Schweiggert, R. M., Kopec, R. E., Villalobos-Gutierrez, M. G., Högel, J., Quesada, S., Esquivel, P., . . . Carle, R. (2014). Carotenoids are more bioavailable from papaya than from tomato and carrot in humans: a randomised cross-over study. British Journal of Nutrition, 111(03), 490- 498.

Schweiggert, R. M., Mezger, D., Schimpf, F., Steingass, C. B., & Carle, R. J. F. C. (2012). Influence of chromoplast morphology on carotenoid bioaccessibility of carrot, mango, papaya, and tomato. 135(4), 2736-2742.

Schweiggert, R. M., Steingass, C. B., Esquivel, P., & Carle, R. (2012). Chemical and morphological characterization of Costa Rican papaya (Carica papaya L.) hybrids and lines with particular focus on their genuine carotenoid profiles. Journal of Agricultural and Food Chemistry.

Schweiggert, R. M., Steingass, C. B., Heller, A., Esquivel, P., & Carle, R. (2011). Characterization of chromoplasts and carotenoids of red-and yellow-fleshed papaya (Carica papaya L.). Planta, 1-14.

Schweiggert, R. M., Steingass, C. B., Mora, E., Esquivel, P., & Carle, R. (2011). Carotenogenesis and physico-chemical characteristics during maturation of red fleshed papaya fruit (Carica papaya L.). Food Research International, 44(5), 1373-1380.

Seigler, D. S., Pauli, G. F., Nahrstedt, A., & Leen, R. (2002). Cyanogenic allosides and glucosides from Passiflora edulis and Carica papaya. Phytochemistry, 60(8), 873-882.

126

Seow, A., Yuan, J. M., Sun, C. L., van den Berg, D., Lee, H., & Yu, M. C. (2002). Dietary isothiocyanates, glutathione S-transferase polymorphisms and colorectal cancer risk in the Singapore Chinese Health Study. Carcinogenesis, 23, 2055-2061.

Septembre-Malaterre, A., Stanislas, G., Douraguia, E., & Gonthier, M.-P. (2016). Evaluation of nutritional and antioxidant properties of the tropical fruits banana, litchi, mango, papaya, passion fruit and pineapple cultivated in Réunion French Island. Food Chemistry, 212, 225- 233.

Shapiro, T. A., Fahey, J. W., Wade, K. L., Stephenson, K. K., & Talalay, P. (2001). Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. Cancer Epidemiology Biomarkers and Prevention, 10(5), 501-508.

Silva, F. O. (2005). Total ascorbic acid determination in fresh squeezed orange juice by gas chromatography. Food Control, 16(1), 55-58.

Silva, L. M. R. d., Figueiredo, E. A. T. d., Ricardo, N. M. P. S., Vieira, I. G. P., Figueiredo, R. W. d., Brasil, I. M., & Gomes, C. L. (2014). Quantification of bioactive compounds in pulps and by-products of tropical fruits from Brazil. Food Chemistry, 143(0), 398-404.

Simirgiotis, M. J., Caligari, P. D. S., & Schmeda-Hirschmann, G. (2009). Identification of phenolic compounds from the fruits of the mountain papaya Vasconcellea pubescens A. DC. grown in Chile by liquid chromatography-UV detection-mass spectrometry. Food Chemistry, 115(2), 775-784.

Singh, C. V. (2012). Papaya - The Fruit of The Angles: Health Benefits & Medicinal Properties.

Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In P. Lester (Ed.), Methods in Enzymology (Vol. Volume 299, pp. 152-178): Academic Press.

Smith, M. A., Zhu, X., Tabaton, M., Liu, G., McKeel, J., D.W., Cohen, M. L., . . . Hayashi, T. (2010). Increased iron and free radical generation in preclinical Alzheimer disease and mild cognitive impairment. Journal of Alzheimer's Disease, 19(1), 363-372.

Song, L., & Thornalley, P. J. (2007). Effect of storage, processing and cooking on glucosinolate content of Brassica vegetables. Food and Chemical Toxicology, 45(2), 216-224.

Sparg, S. G., Light, M. E., & van Staden, J. (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94(2-3), 219-243.

Spínola, V., Pinto, J., & Castilho, P. C. (2015). Identification and quantification of phenolic compounds of selected fruits from Madeira Island by HPLC-DAD–ESI-MSn and screening for their antioxidant activity. Food Chemistry, 173, 14-30.

Stangeland, T., Remberg, S. F., & Lye, K. A. (2009). Total antioxidant activity in 35 Ugandan fruits and vegetables. Food Chemistry, 113(1), 85-91.

Starley, I. F., Mohammed, P., Schneider, G., & Bickler, S. W. (1999). The treatment of paediatric burns using topical papaya. Burns, 25(7), 636-639.

127

Stepek, G., Behnke, J. M., Buttle, D. J., & Duce, I. R. (2004). Natural plant cysteine proteinases as anthelmintics? Trends in Parasitology, 20(7), 322-327.

Stepek, G., Buttle, D. J., Duce, I. R., Lowe, A., & Behnke, J. M. (2005). Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro. Parasitology, 130(Pt 2), 203-211.

Sternberg, Z., Chadha, K., Lieberman, A., Hojnacki, D., Drake, A., Zamboni, P., . . . Munschauer, F. (2008). Quercetin and interferon-β modulate immune response(s) in peripheral blood mononuclear cells isolated from multiple sclerosis patients. Journal of Neuroimmunology, 205(1–2), 142-147.

Subenthiran, S., Choon, T. C., Cheong, K. C., Thayan, R., Teck, M. B., Muniandy, P. K., . . . Ismail, Z. (2013). Carica papaya leaves juice significantly accelerates the rate of increase in platelet count among patients with dengue fever and dengue haemorrhagic fever. Evidence- Based Complementary and Alternative Medicine, 2013.

Tang, C. S. (1979). New macrocyclic DELTA 1-piperideine alkaloids from papaya leaves: dehydrocarpain I and II. Phytochemistry, 18, 651-652.

Tang, Y., & Wu, M. (2005). A quick method for the simultaneous determination of ascorbic acid and sorbic acid in fruit juices by capillary zone electrophoresis. Talanta, 65(3), 794-798.

Tarng, D.-C., Huang, T.-P., Wei, Y.-H., Liu, T.-Y., Chen, H.-W., Wen Chen, T., & Yang, W.-C. (2000). 8-Hydroxy-2′-Deoxyguanosine of Leukocyte DNA as a Marker of Oxidative Stress in Chronic Hemodialysis Patients. American Journal of Kidney Diseases, 36(5), 934-944.

Tee, E. S., Noor, M. I., Azudin, M. N., & Idris, K. (1997). Malaysian Food Composition Database Programme Kuala Lumpur: Institute of Medical Research.

Thomás, G.-E., Rodolfo, H.-G., Juan, M.-D., Georgina, S.-F., Luis, C.-G., Ingrid, R.-B., & Santiago, G.-T. (2009). Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest by-products. Process Biochemistry, 44(1), 77-82.

Thomas, M. L. R. M. G., & Filho, J. M. B. (1985). Anti-inflammatory actions of tannins isolated from the bark of Anacardwm occidentale L. Journal of Ethnopharmacology, 13(3), 289-300.

Thompson, K., Marshall, M., Sims, C., Wei, C., Sargent, S., & Scott, J. (2000). Cultivar, maturity, and heat treatment on lycopene content in tomatoes. Journal of Food Science, 65(5), 791- 795.

Toor, R. K., & Savage, G. P. (2005). Antioxidant activity in different fractions of tomatoes. Food Research International, 38(5), 487-494.

Traoré, K., Parkouda, C., Savadogo, A., Ba/Hama, F., Kamga, R., & Traoré, Y. (2017). Effect of processing methods on the nutritional content of three traditional vegetables leaves: Amaranth, black nightshade and jute mallow. Food Science & Nutrition.

Tripathi, S., Suzuki, J. Y., Carr, J. B., McQuate, G. T., Ferreira, S. A., Manshardt, R. M., . . . Gonsalves, D. (2010). Nutritional composition of Rainbow papaya; the first commercialized transgenic fruit crop. Journal of Food Composition and Analysis. 128

Tripathi, S., Suzuki, J. Y., Carr, J. B., McQuate, G. T., Ferreira, S. A., Manshardt, R. M., . . . Gonsalves, D. (2011). Nutritional composition of Rainbow papaya, the first commercialized transgenic fruit crop. Journal of food composition and Analysis, 24(2), 140-147.

Udoh, F. V., Udoh, P. B., & Umoh, E. E. (2005). Activity of Alkaloid Extract of Carica papaya. Seeds on Reproductive Functions in Male Wistar Rats. Pharmaceutical Biology, 43(6), 563- 567.

USDA. (2007). USDA Database for the Flavonoid Content of Selected Foods. Release 2.1. Retrieved from http://www.ars.usda.gov/nutrientdata

USDA. (2015). USDA National Nutrient Database for Standard Reference. Retrieved from http://ndb.nal.usda.gov/

Vallejo, F., Tomás-Barberán, F., & García-Viguera, C. (2002). Glucosinolates and vitamin C content in edible parts of broccoli florets after domestic cooking. European Food Research and Technology, 215(4), 310-316. van Breemen, R. B., & Pajkovic, N. (2008). Multitargeted therapy of cancer by lycopene. Cancer Lett, 269(2), 339-351.

Veda, S., Platel, K., & Srinivasan, K. (2007). Varietal Differences in the Bioaccessibility of β- Carotene from Mango (Mangifera indica) and Papaya (Carica papaya) Fruits. Journal of Agricultural and Food Chemistry, 55(19), 7931-7935.

Verma, R. J., Nambiar, D., & Chinoy, N. J. (2006). Toxicological effects of Carica papaya seed extract on spermatozoa of mice. Journal of Applied Toxicology, 26(6), 533-535.

Versari, A., Mattioli, A., Parpinello, G. P., & Galassi, S. (2004). Rapid analysis of ascorbic and isoascorbic acids in fruit juice by capillary electrophoresis. Food Control, 15, 355-358. Vickers, J. C., & Dickson, T. C. (2000). The cause of neuronal degeneration in Alzheimer's disease. Progress in Neurobiology, 60(2), 139-165.

Villegas, V. N. (1997). Carica papaya L. . In E. W. M. Verheij & R. E. Coronel (Eds.), Plant Resources of South East Asia: Edible Fruits and Nuts (Vol. 2): Wageningen University, Wageningen, The Netherlands.

Vinci, G., Botrè, F., Mele, G., & Ruggieri, G. (1995). Ascorbic acid in exotic fruits: a liquid chromatographic investigation. Food Chemistry, 53(2), 211-214.

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(11), 5315-5321.

Vuong, Q. V., Hirun, S., Roach, P. D., Bowyer, M. C., Phillips, P. A., & Scarlett, C. J. (2013). Effect of extraction conditions on total phenolic compounds and antioxidant activities of Carica papaya leaf aqueous extracts. Journal of Herbal Medicine, 3(3), 104-111.

Wall, M. M. (2006). Ascorbic acid, vitamin A, and mineral composition of banana (Musa sp.) and papaya (Carica papaya) cultivars grown in Hawaii. Journal of Food Composition and Analysis, 19(5), 434-445.

129

Weatherall, D. J. (2010). The thalassemias. In E. Beutler, M. A. Lichtman, B. S. Coller, T. J. Kipps, & U. Seligsohn (Eds.), Williams Hematology (6th Edition). NY USA: McGraw-Hill.

WHO, & FAO. (2004). Vitamin and mineral requirements in human nutrition (2nd ed.).

Winter, C. K., & Davis, S. F. (2006). Organic foods. Journal of Food Science, 71, 117-112. Woese, K., Lange, D., Boess, C., & Bogl, K. W. (1997). A comparison of organically and conventionally grown foods - results of a review of the relevant literature. Journal of the Science of Food and Agriculture, 74, 281-293.

Workneh, T., Azene, M., & Tesfay, S. (2012). A review on the integrated agro-technology of papaya fruit. African Journal of Biotechnology, 11(85), 15098-15110.

World Health Organization. (1997). Dengue haemorrhagic fever:diagnosis, treatment, prevention and control. 2nd edn. . Geneva.

Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Ronald, L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agricultural and Food Chemistry, 52(12), 4026-4037.

Xu, F., Zheng, Y., Yang, Z., Cao, S., Shao, X., & Wang, H. (2014). Domestic cooking methods affect the nutritional quality of red cabbage. Food Chemistry, 161(0), 162-167.

Yahia, M. E., & Ornelas-Paz, J. J. (2010). Chemistry, stability and biological actions of carotenoids. In L. A. de la Rosa, E. Alvarez-Parrilla, & G. A. Gonzalez-Aguilar (Eds.), Fruit and vegetable phytochemicals (pp. 177−222): USA: Wiley-Blackwell.

You, B.-Y., Wang, Y.-H., & Kuo, M.-L. (2001). Role of reactive oxygen species in cupric 8- quinolinoxide-induced genotoxic effect. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 491(1-2), 45-56.

Zhang, J., Mori, A., Chen, Q., & Zhao, B. (2006). Fermented papaya preparation attenuates beta- amyloid precursor protein: beta-amyloid-mediated copper neurotoxicity in beta-amyloid precursor protein and beta-amyloid precursor protein Swedish mutation overexpressing SH- SY5Y cells. Neuroscience, 143(1), 63-72.

Zhao, C., Liu, Y., Lai, S., Cao, H., Guan, Y., San Cheang, W., . . . Xiao, J. (2019). Effects of domestic cooking process on the chemical and biological properties of dietary phytochemicals. Trends in Food Science & Technology, 85, 55-66.

Žnidarčič, D., Ban, D., & Šircelj, H. (2011). Carotenoid and chlorophyll composition of commonly consumed leafy vegetables in Mediterranean countries. Food Chemistry, 129(3), 1164-1168.

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APPENDICES

Appendix 1.

Representative chromatograms of papaya leaves measured individually.

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β-Carotene standard curve.

β-Cryptoxanthin standard curve.

Lutein standard curve.

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AA standard curve.

Trolox equivalents standard curve.

Gallic acid standard curve.

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Samples of papaya from a commercial plantation located at Mareeba, North Queensland, Australia (Longitude: 145.336789, Latitude: -16.956108) during summer months (November to December 2011) with mean minimum/maximum temperature of 20.2/32.0 oC (Australian Government Bureau of Meteorology, 2013).

Fruits were kept at 25-26oC and allowed to fully ripen according to the selected criteria based on the maturity score describe in Table 3.3.

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AUTHOR’S PROFILE

EMMY HAINIDA KHAIRUL IKRAM

Name : Emmy Hainida Khairul Ikram Telephone No. (Office) +603-32584378 Cell-phone : +6017-4242559 Email [email protected] Professional Qualification BSc (Nutrition and community Health) MSc (Nutritional Biochemistry)

RESEARCH FUNDINGS Total Funds Begin Research Project End Year Year Principal Researcher, Antioxidant capacity and phenolic content of raw and blanched selected 1 RM20,000 Dec 2008 Feb 2010 Malaysian Underutilized vegetables.

Principal Researcher, Sensory evaluation of Carica 2 Papaya products. RM30,000 Sept 2016 Sept 2018

Co-Researcher, Nutritional properties of Berembang 3 Leaves. RM50,000 Dec 2018 Nov 2021

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PUBLICATIONS 1. Kong, K. W., Ikram, E. H. K., Othman, A., Amin, I., & Tan, C. P. (2010). Effect of steam blanching on lycopene and total phenolics in pink guava puree industry by- products. International Food Research Journal, 17(2), 461-468. 2. Emmy Hainida, Khoo Hock Eng, Abbe Maleyki Mhd Jalil, Amin Ismail, Salma Idris, Azrina Azlan, Halimatul Saadiah Mohd Nazri, Norzatol Akma Mat Diton, and Ruzaidi Azli Mohd Mokhtar. 2009. Antioxidant Capacity and Total phenolic content of Malaysian underutilized fruits. Journal of Food Composition and Analysis, 22:388-393 3. Emmy Hainida, Amin I., Normah H., N. Mohd.-Esa and Ainul Z.A.B. 2008. Nutritional composition of roselle (Hibiscus sabdariffa L.) seeds powder and its effect on lipid profiles of hypercholesterolemia rats. Journal of the science of food and agriculture, 88:1043-1050 4. Emmy Hainida K.I., Amin I., Normah H., and N. Mohd.-Esa. 2008. Nutritional and amino acid contents of differently treated roselle (Hibiscus sabdariffa L.) seeds. Food chemistry, 111:906-911 5. Amin Ismail, Emmy Hainida Khairul Ikram and Halimatul Saadiah Mohd Nazri. 2008. Roselle (Hibiscus sabdariffa L.) Seeds - Nutritional Composition, Protein Quality and Health Benefits. FOOD 2(1): 1-16 (Print ISSN 1749-7140). 6. Lee wee Yee, Emmy Hainida Khairul Ikram, Abbe Maleyki Mhd Jalil, Amin Ismail. 2007. Antioxidant capacity and phenolic content of selected commercially available cruciferous vegetables. Malaysian Journal of Nutrition 139(1):71-80. 7. Amin Ismail and Emmy Hainida Khairul Ikram. 2006. Antioxidant Capacity and Phenolic Content of Amaranthus spp. Grown in Malaysia. In Floriculture, Ornamental and Plant Biotechnology Volume IV. Global Science Books, UK. 8. Amin, I., Norazaidah, Y. and Emmy Hainida, K.I. 2006. Antioxidant activity and phenolic content of raw and blanched Amaranthus species, Food Chemistry., Vol 94, pp 47-52. 9. Amin Ismail and Emmy Hainida Khairul Ikram. 2004. Effects of cooking practices (boiling and frying) on the protein and amino acids contents of four selected fishes, Nutrition and Food Sciences, Vol. 34 No.2, pp.54-59.

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