Green Leafy Vegetables of Rural India: Ethnobotany and Contribution To

Green Leafy Vegetables of Rural India: Ethnobotany and

Contribution to Eye Health

Julie Bélanger

Degree of Doctor of Philosophy

Plant Science Department, Faculty of Agricultural and Environmental

Sciences

McGill University

Montreal, Quebec, Canada

May 2010

A thesis submitted to McGill University in partial fulfillment of the

requirements of the degree of Doctor of Philosophy

©Julie Bélanger 2010.

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DEDICATION

This dissertation is dedicated to the women of Madanapalle, who generously gave their time and shared with us their knowledge of the plants they use and value.

ABSTRACT

Recognition of the contribution of biological diversity to human health demands more scientifically sound evidence than currently exists, while the multifactorial nature of this relationship calls for innovative research frameworks. This thesis presents a multidisciplinary case study on the contribution of elements of biological diversity, namely wild and cultivated leafy vegetables, towards age-related cataract prevention in a rural developing country context. At the center of this thesis, an ethnobotanical study identified determinants of consumption of leafy vegetables and demonstrated how perceived properties and cultivation status significantly influence consumption patterns. Plant species of interest, analysed by High

Performance Liquid Chromatography, were found to exhibit high concentrations of lutein and β-carotene. Drawing on ethnobotanical and analytical data, an eye hospital-based case-control study was conducted to compare leafy vegetable consumption and diversity, along with lutein and zeaxanthin intake, in female patients identified with and without age-related cataract. Conflicting results for associations between leafy vegetable species and age-related cataract, and protective associations for elements of traditional diets, including yogurt and tea, were observed. The integration of results across isolated studies in a multidisciplinary framework further

reflected the complex biological, socio-economic and environmental components of eye health and leafy vegetable diversity, and highlighted new knowledge with important application in the eye health of populations at risk.

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RÉSUMÉ

Afin de reconnaître la contribution de la biodiversité à la santé humaine, de solides preuves scientifiques additionnelles sont requises. D’autre part, la nature multifactorielle de cette relation nécessite l’élaboration de cadres de recherche innovateurs. Ce mémoire présente une étude de cas multidisciplinaire sur la contribution d’éléments de la biodiversité, en particulier les légumes feuillus cultivés et sauvages, en relation avec la prévention de la cataracte liée à l’âge dans le contexte rural de l’Inde du Sud.

Au coeur de ce projet, une étude ethnobotanique a permis d’identifier les facteurs déterminant la consommation de légumes feuillus, et de démontrer l’influence significative des propriétés qui leur sont attribuées et de leur statut de culture sur les habitudes de consommation. Les espèces analysées par chromatographie en phase liquide à haute performance ont affiché d’importantes concentrations de lutéine et de β-carotène. Se basant sur ces données ethnobotaniques et analytiques, une étude cas témoin a été conduite dans un centre d’ophtalmologie afin de comparer la consommation de légumes feuillus, en quantité et en diversité, et de lutéine et zéaxanthine, chez des patientes diagnostiquées et des témoins sains. Des associations contradictoires concernant la consommation de légumes feuillus et le risque de cataracte ont été observées. En revanche, certains aliments traditionnels,

comme le yaourt et le thé, ont démontré une association négative avec la cataracte. L’intégration de ces études à l’intérieur d’un cadre multidisciplinaire a permis de tenir compte des relations complexes entre les composantes biologiques, socio-économiques et environnementales de la santé de l’oeil et de la diversité botanique, permettant ainsi la découverte d’importantes connaissances applicables à la prévention de la cataracte chez des populations à risque.

ACKNOWLEDGEMENTS

My first word of thank is to my supervisor, Professor Timothy Johns, for his precious advices and constant positive and encouraging support. Professor

Johns is an exceptional mentor, and his deep involvement in his research on the conservation of indigenous food and health systems and environment, is truly inspiring.

I also express profound gratitude to Dr. Shoba Katumalla without whom this complex project would never have been possible. We conducted the study in the world-class eye hospital she herself established, and she personally examined each participant of this study. Her professionalism, dedication and sincere desire to make a difference by helping people in need were for me very influential examples.

I would like to thank the numerous participants of this study who gave their time and shared their knowledge with us in the course of this study.

I am very thankful to my two research assistants, Hymavathi Kalimidi and Girija Rani, for commitment and attention to detail in their work, and above all for their friendship and introducing me to their culture. Also, I am indebted to the Siloam Eye Hospital staff for their extensive technical and logistic support throughout this adventure, especially Mr. Anand and Ms.

Salome.

The carotenoid analysis was made possible thanks to Dr. P. Sudhakar, and Dr. R. K. Reddy of the Acharya Ranga Reddy Agricultural Research

Station (Tirupati). I would like to thank Mungara Balakrishna for technical and logistic support, as well as for advices, friendship and patience. Many thanks also go to Dr. Latha, Mr. Sreenu and the other members of the Plant

Physiology Laboratory.

Thanks to the invitation of Dr. Rodriguez-Amaya and the supervision of her graduate students, I acquired skills and knowledge of the analysis of carotenoids in foods. The advices and logistic help from Dr. P. Nirmalan, Dr.

M. Aruna and Mrs. Salome Yesudas were invaluable in finding appropriate contacts and research location.

I am grateful to my advisory committee members, Dr. Jacqueline

Bede, Dr. Alan Watson, and to Dr. Grace Egeland and Dr. Grace Marquis for providing constructive criticism. Dr. Jose Correa of the McGill Statistical

Consulting Service gracefully provided advices for statistical analysis. I also express gratitude to Louise Johnson-Down for advices on dietary data and methodology to estimate intakes.

I thank many people for useful discussions, and ought to mention my colleagues Dr. Patrick Owen and Bronwen Powell. My gratitude also goes to the secretaries of the Plant Science Department, Mrs. Carolyn Bowes and

Mrs. Roslyn James.

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I acknowledge the International Development Research Center

(IDRC), Fonds Québécois de la Recherche sur la Nature et les Technologies

(FQRNT), and Natural Sciences and Engineering Research Council

(NSERC) for providing funding through graduate scholarships. I also thank

McGill University for funding through Graduate Studies Fellowship and

Recruitment Excellence Fellowship.

Last but not least, my gratitude goes to my family and friends for their continuous encouragements, and my deepest gratitude goes to my partner

François for infinite patience, support and motivation throughout my studies.

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TABLE OF CONTENTS

DEDICATION………………………………………….………………………..i

ABSTRACT……….……………………………………………………………..ii

RÉSUMÉ.………………………………………………………………………..iv

ACKNOWLEDGEMENTS.…………………………………………………...vi

TABLE OF CONTENTS ...... ix

LIST OF TABLES ...... xv

LIST OF FIGURES...... xvii

LIST OF ABBREVIATIONS ...... xviii

THESIS FORMAT ...... xx

1. Introduction ...... 1

1.1 General context ...... 1

1.2 Methodological considerations ...... 3

1.3 Thesis components ...... 5

1.4 Traditional diets, biological diversity and green leafy vegetables..... 6

1.5 Overview of cataract...... 8

1.6 Lutein and zeaxanthin ...... 10

1.7 Cataract in India...... 12

1.8 Leafy vegetables and cataract in Andhra Pradesh...... 13

1.9 Objectives...... 15

1.9.1 Broad development objectives ...... 15

1.9.2 Specific objectives...... 15

1.10 Hypotheses...... 17

Preface to Chapter 2 ...... 18

2. Biological diversity, dietary diversity and eye health in developing country populations: establishing the evidence-base.19

2.1 Abstract...... 19

2.2 Introduction ...... 20

2.3 Biological diversity, diet and health ...... 22

2.3.1 Identifying measurable outcomes ...... 22

2.3.2 Biological diversity, conservation and health ...... 24

2.4 Dietary diversity and eye health...... 26

2.4.1 Nutrition and ocular diseases...... 27

2.4.2 Vitamin A deficiency...... 28

2.4.3 Epidemiologic evidence for age-related diseases...... 28

2.4.4 Phytochemicals, minerals and eye health ...... 31

2.5 Research in a developing country context ...... 35

2.5.1 Case studies ...... 36

2.6 Research gaps and ways forward...... 38

2.6.1 Laboratory activities...... 38

2.6.2 Population-based studies...... 39

2.6.3 Community activities...... 40

2.6.4 Ecohealth determinants ...... 41

2.7 Conclusion ...... 42

Preface to Chapter 3 ...... 49

3. Perceived health benefits and cultivation status influence leafy vegetable consumption and conservation...... 50

3.1 Abstract...... 50

3.2 Introduction ...... 51

3.3 Research area ...... 54

3.4 Methods ...... 55

3.5 Results...... 57

3.5.1 Edible leafy greens in Madanapalle...... 58

3.5.2 Perceived health properties associated with leafy vegetable

consumption ...... 59

3.5.3 Sources of GLV ...... 61

3.6 Discussion ...... 61

3.6.1 Nutrition and taste...... 62

3.6.2 Perceived health properties...... 64

3.6.3 Consumption patterns of foods, folk functional foods and

food medicines ...... 65

3.6.4 Cultivation status and medicinal attributes ...... 66

3.6.5 Traditional knowledge of leafy vegetables and their

properties...... 68

3.6.6 Implications for eye health...... 69

3.6.7 Implications for agro-biodiversity conservation ...... 70

3.7 Conclusions...... 71

Preface to Chapter 4 ...... 84

4. Contribution of selected wild and cultivated leafy vegetables from South India to lutein and β-carotene intake...... 85

4.1 Abstract...... 85

4.2 Introduction ...... 86

4.3 Material and methods...... 88

4.3.1 Dietary intake ...... 88

4.3.2 Plant material ...... 89

4.3.3 Chemicals and standards...... 89

4.3.4 Carotenoid extraction...... 90

4.3.5 HPLC conditions ...... 91

4.3.6 Identification and quantification ...... 92

4.4 Results...... 93

4.4.1 Dietary intake ...... 93

4.4.2 Qualitative analysis...... 94

4.4.3 Fresh leafy vegetables...... 94

xii

4.4.4 Cooked GLV ...... 95

4.4.5 Contribution to total β-carotene and lutein intake ...... 96

4.5 Discussion ...... 97

Preface to Chapter 5 ...... 108

5. Traditional foods and age-related cataract in women of rural

South Andhra Pradesh, India...... 110

5.1 Abstract...... 110

5.2 Introduction ...... 111

5.3 Experimental methods...... 113

5.3.1 Study design ...... 113

5.3.2 Participants ...... 113

5.3.3 Dietary data collection ...... 114

5.3.4 Statistical analysis ...... 117

5.4 Results...... 118

5.4.1 General characteristics of the subjects...... 118

5.4.2 Dietary intakes...... 119

5.5 Discussion ...... 121

6. General discussion ...... 137

6.1 Verification of hypotheses...... 137

6.2 General considerations...... 140

7. Future research directions ...... 143

xiii

8. Contributions to knowledge...... 144

8.1 Overall contributions to science...... 144

8.2 Contributions to Ethnobotany and Plant Science ...... 144

8.3 Contributions to Nutrition ...... 146

8.4 Contributions to Nutritional Epidemiology and Plant-based

Nutrition ...... 146

List of References ...... 148

Appendix A...... 190

Supplementary material for Chapter 3 ...... 190

Individual consent form...... 191

Free list records of green leafy vegetables ...... 193

Seasonality, appreciation and health properties...... 194

Appendix B ...... 195

Supplementary material for Chapter 4 ...... 195

Appendix C ...... 197

Supplementary material for Chapter 5 ...... 197

Individual consent form...... 198

Individual 24 hour recall ...... 200

Individual consent form...... 202

Individual 7-day food frequency questionnaire...... 204

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LIST OF TABLES

Table 2.1: Definitions of the most common eye diseases...... 44

Table 2.2: Description of eight population-based studies evaluating the effect of diet on the risk for cataract development...... 45

Table 2.3: Dietary sources of compounds and minerals (nutrients and non- nutrients) beneficial to eye health...... 48

Table 3.1: Leafy vegetables consumed in Madanapalle, Andhra Pradesh,

India...... 73

Table 3.2: Leafy vegetable species most quoted (%) by the participants

(n=100)...... 77

Table 3.3: Health properties attributed to the consumption of selected leafy vegetables by women of the Madanapalle area...... 78

Table 4.1: Cultivation status, botanical families, scientific and Telugu names, yearly availability and dietary intake among Madanapalle women (n=100) of the selected leafy vegetable species...... 102

Table 4.2: Lutein and β-carotene content (µg/g) with standard deviation (s.d.) of fresh and cooked leafy vegetables compared with values obtained from other studies reported in µg/g fresh weight...... 104

Table 5.1: Demographic characteristics and potential risk factors for cataract in the study population: hospital-based, case‒control study on intake of green leafy vegetables and age-related cataract risk, Madanapalle, 2007-2008 .... 128

Table 5.2: Effect of food group daily intake and other dietary factors on cataract in the study population: hospital-based, case‒control study on intake of green leafy vegetables and age-related cataract risk, Madanapalle, 2007-

2008...... 131

Table 5.3: Effect of selected food items intake on cataract in the study population: hospital-based, case‒control study on intake of green leafy vegetables and age-related cataract risk, Madanapalle, 2007-2008 ...... 135

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LIST OF FIGURES

Figure 1.1: Structure of the human eye...... 10

Figure 1.2: Structure of the xanthophylls pigments lutein and zeaxanthin. 12

Figure 3.1: Location of the research site...... 79

Figure 3.2: Reported (emic) categories of perceived health properties attributed to (A) leafy vegetable consumption in general and (B) intake of specific leafy vegetables...... 80

Figure 3.4: Cultivation status of reported species, associated consumption and proportions of perceived health properties...... 82

Figure 4.1: High Performance Liquid Chromatography chromatograms of

Allmania nodiflora (L.) R. Br. Ex Wight fresh (A) and cooked (B)...... 107

Figure B.1: The 10 species selected for lutein/zeaxanthin and β-carotene analysis by High Performance Liquid Chromatography...... 196

xvii

LIST OF ABBREVIATIONS

AMD, age-related macular degeneration

CI, confidence interval

CINE, Center for Indigenous peoples’ Nutrition and Environment

DDS, dietary diversity score

FAO, Food and Agriculture Organization

FFQ, food frequency questionnaire

FVS, food variety score

GLV, green leafy vegetables

HPLC, high performance liquid chromatography

LOCS III, Lens Opacity Classification System III

M, plants managed/grown in pots, home gardens, pots and neighbourhoods

MDG, Millennium Development Goals

PSC, posterior subcapsular

OR, odds ratio

R, rainy season (June-September)

S, summer season (February-May)

SD, standard deviation

SEM, standard error of the mean

TEA, triethylamine

xviii

UN, United Nations

UV, ultra-violet

VAD, vitamin A deficiency

WHO, World Health Organization

W, winter season (October-January)

We, weed

Wi, wild

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THESIS FORMAT

This thesis is presented in manuscript-based format. Four manuscripts are included as chapters. Chapter 2, entitled “Biological diversity, dietary diversity, and eye health in developing country populations: Establishing the evidence-base”, is a literature review published in EcoHealth in 2008.

Chapter 3, entitled “Perceived health benefits and cultivation status influence leafy vegetable consumption and conservation”, has been submitted for peer- review to the journal Economic Botany. Chapter 4, entitled “Contribution of selected wild and cultivated leafy vegetables from South India to lutein and

β-carotene intake”, is presently in press in the Asia Pacific Journal of Clinical

Nutrition. Chapter 5, entitled “Traditional foods and age-related cataract in women of rural South Andhra Pradesh, India”, will be submitted for peer- reviewed publication. The four manuscripts have been reformatted for thesis consistency.

CONTRIBUTIONS OF AUTHORS

Chapter 2 is co-authored by J. Bélanger and Dr. T. Johns. J. Bélanger did the literature review research and wrote the manuscript in collaboration with Dr.

T. Johns. Both authors have participated in writing the final version of the manuscript.

Chapter 3 is co-authored by J. Bélanger, Dr. S. Katumalla and Dr. T.

Johns. J. Bélanger and Dr. T. Johns designed the project. J. Bélanger performed the research in collaboration with Dr. S. Katumalla, under the supervision of Dr. T. Johns. J. Bélanger wrote the initial manuscript in collaboration with Dr. T. Johns. All authors have revised the final version of the manuscript.

Chapter 4 is co-authored by J. Bélanger, M. Balakrishna, Dr. P. Latha,

Dr. S. Katumalla and Dr. T. Johns. J. Bélanger and Dr. T. Johns designed the project. J. Bélanger performed the research in collaboration with M.

Balakrishna under the supervision of Dr. P. Latha at the Regional

Agricultural Research Station, Acharya N. G. Ranga Agricultural University,

Tirupati, Andhra Pradesh, India, and with assistance from Dr. S. Katumalla.

J. Bélanger wrote the initial manuscript. All authors have revised the final version of the manuscript.

xxi

Chapter 5 is co-authored by J. Bélanger, Dr. S. Katumalla and Dr. T.

Johns. J. Bélanger and Dr. T. Johns designed the study. J. Bélanger performed the research in collaboration and with professional assistance of

Dr. S. Katumalla at the Siloam Eye Hospital, Madanapalle, Andhra Pradesh,

India, under the supervision of Dr. T. Johns. J. Bélanger wrote the initial manuscript. All authors have revised the final version of the manuscript.

xxii

1. Introduction

1.1 General context

The health and nutrition of expanding world populations, projected to reach over 9 billion people by 2050 (United Nations, 2008), are major upcoming challenges especially in developing countries. Research strategies include increasing crop yield, biomass and stress tolerance by means of conventional plant breeding and genetic engineering. However biotechnologies are currently developed for the needs of industrial farms (Rosegrant & Cline,

2003) and their potential impact on dietary diversity reduction raise concerns

(Johns & Eyzaguirre, 2007). The Millennium Development Goals (MDG) endeavour at halving extreme poverty by 2015, but progress and efforts are jeopardized by the ongoing global economic and food crises (United Nations,

2009). In this context, initiatives supporting sustainable agriculture and improving finances, use of available and renewable resources, diversification of systems and participatory and social processes, are relevant in both current and future food security perspectives (Pretty, 1999).

The nutrition and health objectives of the MDG rest in part on the contribution of plant diversity for food and medicine. Indeed, plant foods are sources of energy, micronutrients and nutrients essential to health, in addition to phytochemicals with further health benefits including glycaemic

control, immuno-stimulation or antioxidant activity (Johns & Eyzaguirre,

2006) and with clear applications in light of the nutrition transition underway in low-income countries where malnutrition coexists with obesity and associated non-communicable diseases (Popkin, 2004). The potential of functional plant foods with additional physiological benefits beyond that of meeting basic nutritional needs (Milner, 1998), mostly attributable to non- nutrients, is largely overlooked in local, less common and uncultivated plant foods (Johns & Eyzaguirre, 2006). Yet preventive interventions of dietary or other nature could have tremendous effects in reducing the burden of diseases; for example, in the case of cataract in India, if a national intervention could delay the development of cataract by 10 years, it would reduce by 45% the need for cataract surgery (Kupfer, 1984). Plant scientists, conducting research on the improvement of crop attributes, on the forests and agroecosystems diversity, and on the nutritional and health properties of uncultivated and underutilized plant resources, are key actors in finding solutions and initiating sustainable strategies to overcome present and upcoming health and nutrition challenges.

Other international agendas address the critical importance of the

“conservation and sustainable use of biological diversity […] for meeting the food, health and other needs of the growing world population” (COP8, 2006).

The importance of biological diversity to human nutrition and health is

increasingly recognized (Chivian & Sullivan, 2002; Grifo & Rosenthal, 1997;

Wilby, et al., 2009). Yet, the complex interrelations between diet, lifestyle, genetic and other factors, complicate in theory and in practice the demonstration of the association between health and biological diversity

(Bélanger & Johns, 2008; Johns & Eyzaguirre, 2006; Michels, 2003). To address such difficulties, multidisciplinary frameworks are promising approaches to gain further insights in this important relationship.

1.2 Methodological considerations

We propose a multidisciplinary case study on the relation between the utilization and deployment of existing plant diversity and eye health, specifically age-related cataract. The concepts of human health and biological diversity both integrate diverse biological, environmental, cultural and sociological factors and relevant issues can hardly be addressed by a single discipline. In this case study, we believe the combined contribution of the fields of plant science, ethnobotany, phytochemistry, nutrition, epidemiology and ophthalmology can together better capture the relation between plant diversity consumption and eye health. However challenges to such approaches include the transmission and integration of knowledge across disciplines and calls for integrative frameworks.

The field of ethnobotany itself is multidisciplinary in nature, as it combines botanical and anthropological data in order to highlight patterns and relations between plants and their uses by people, which would escape specialists of other disciplines. Furthermore, ethnobotanical information may be of use to a large range of social, economic, environmental and pharmacological disciplines, and more importantly to local populations. In spite of the descriptive nature of ethnobotany, the contribution of other fields of research to this field can stimulate the formulation and testing of hypotheses. Nutritional epidemiologists investigate the relationships between specific foods or food groups and the health of populations, with the assistance of health practitioners, but tend to overlook uncultivated and less common species. Ethnobotanists categorize plants of interest for the population under study and with the help of botanists identify the selected plants. Plant scientists and biochemists perform laboratory analyses to identify active compounds in the selected plants. Altogether, each field contributes to a global objective, i.e. promote the sustainable use of local biological diversity for health benefits identified by the population.

Challenges reside in collecting, treating and integrating appropriate data across fields of research in order to provide profitable and acceptable advices.

1.3 Thesis components

This thesis integrates three complementary studies on aspects of plant food consumption and age-related cataract, namely an ethnobotanical evaluation of the determinants of consumption, a laboratory analysis of compounds of interest extracted from local plant foods, and an eye hospital-based case- control study comparing dietary plant intakes of female participants identified with and without age-related cataract. At the center of this multidisciplinary framework is the ethnobotanical study, in which the choice and consumption of species by local participants is investigated. The interrelations between foods, folk functional foods and food medicines with cultivation status and consumption of species are examined. The selection of plant foods to be further analysed in the laboratory depends on the ethnobotanical findings, as is the choice of plants to be included in the dietary assessment of the case-control study. Also closely interrelated are the laboratory analyses providing results to be verified in the epidemiological investigation.

In Chapter 2 we review the existing scientific literature on the relation between botanical dietary diversity and eye health, in particular for vitamin A deficiency, age-related cataract and macular degeneration. We examine various compounds found in plant foods under evaluation for potential eye health benefits. The multidisciplinary framework for a case study on eye

health is also described. Some concepts less discussed in this published literature review, namely traditional diets, leafy vegetables, xanthophylls, eye structures and the burden of cataract in India, are presented in the following sections of the Introduction, before the Objective and Hypotheses sections.

In Chapter 3 we report the results of an ethnobotanical study conducted in

Madanapalle, rural South India. The laboratory evaluation of selected plants is presented in Chapter 4, and we outline the results of a case-control study in

Chapter 5.

1.4 Traditional diets, biological diversity and green leafy vegetables

Traditional food systems depend on and reflect biological diversity as they typically incorporate locally available foods of plant and animal origin, are high in species variety and have rich nutrient sources (Kuhnlein & Receveur,

1996; Tontisirin, et al., 2002). However with the ongoing nutrition transition and diet simplification (Popkin, 2004), attention is given primarily to three major crops (rice, wheat and maize) in the global food supply, and the variety of fruits and vegetables in the diet is declining in many developing countries

(Johns, 2007). Successful dietary systems in transition (e.g. Mediterranean,

South Korean), showing improved longevity and decreased prevalence of cancer, diabetes, obesity and cardiovascular diseases (Lee, et al., 2002;

Trichopoulou & Lagiou, 1997; Trichopoulou, et al., 2000), have high dietary diversity, and include wild and locally grown fruit and vegetable species

(Johns, 2007; Rivera, et al., 2006; Zeghichi, et al., 2003).

Green leafy vegetables (GLV), in particular wild and weedy species, are key elements of traditional diets, as they are accessible, locally gathered or cultivated and diversified sources of nutrients and phytochemicals

(Chweya & Eyzaguirre, 1999; Grivetti & Ogle, 2000; Ogle, et al., 2001;

Tarwadi & Agte, 2003; Tontisirin, et al., 2002). GLV are sources of nutrients and micronutrients of great interest to nutritionists such as β-carotene, iron and vitamin C, which are lacking from staple foods (Kennedy, et al., 2005). In addition, GLV are primary sources of lutein and zeaxanthin (Mangels, et al.,

1993a; Sommerburg, et al., 1998), which have been identified as important eye protective agents. They are the only carotenoids found in the human lens

(Handelman, et al., 1988; Yeum, et al., 1995), where they act as antioxidants and protect the ocular structures from light damage (Junghans, et al., 2001;

Landrum & Bone, 2001; Miller, et al., 1996).

The consumption of leafy vegetables has been associated with various eye health benefits, including age-related cataract prevention (Brown, et al.,

1999; Chasan-Taber, et al., 1999; Cumming, et al., 2000; Hankinson, et al.,

1992; Lyle, Mares-Perlman, Klein, Klein, & Greger, 1999; Mares-Perlman, et al., 1995; Tavani, et al., 1996). Furthermore, increasing evidence in Western

populations supports the preventive properties of lutein and zeaxanthin intake against age-related cataract (Brown, et al., 1999; Chasan-Taber, et al.,

1999; Christen, et al., 2008; Delcourt, et al., 2006; Gale, et al., 2001; Jacques, et al., 2001; Jacques, et al., 2005; Lyle, Mares-Perlman, Klein, Klein, &

Greger, 1999; Lyle, Mares-Perlman, Klein, Klein, Palta, et al., 1999; Mares-

Perlman, et al., 1995; Moeller, et al., 2008; Olmedilla, et al., 2003; Richer, et al., 2004; Taylor, et al., 2002; Vu, et al., 2006).

1.5 Overview of cataract

The human crystalline lens is a transparent, avascular, biconvex structure suspended within the eye (Figure 1.1). It is responsible for light convergence and divergence and focuses light beams on the retina where the sight signal is sent to the brain. The lens is composed of water, lipids and proteins. These proteins, the crystallines, are organized in a pattern allowing light to pass through with minimal distortion. Cataract is the opacification of the lens, preventing light from reaching the retina in a correct focus (Taylor & Hobbs,

2001). The formation of high-molecular-weight aggregates, occurring with photo-induced oxidation of proteins, is believed to contribute to the opacification of the lens (Bron, et al., 2000). Untreated, cataract may progress over the years and lead to blindness. One or both eyes can be affected and visual symptoms include blur, glare, halos and double vision.

Epidemiologic evidence points to six categories of risk factors for age- related cataract, summarized by Taylor (1999) and reviewed in (West, 2007): daylight (UV exposure), diet (lack of vitamins, minerals), drugs (smoking, alcohol consumption, analgesic use, steroid use, hormone replacement therapy), diabetes, dehydration (caused by severe diarrhea) and other unidentified factors. Putative risks for cataract include gender, ethnicity, family history, iris color, education, employment, socioeconomic status and glaucoma. Factors like age probably represent the cumulative effects of factors not yet identified (Taylor, 1999). Oxidative damage is an important feature of age-related cataract (Spector, 1995; Truscott, 2005; Vinson, 2006) and oxidative stress and antioxidant status have been related to age-related cataract in a number of studies (Chiu & Taylor, 2007; Gale, et al., 2001; Li, et al., 2009; Tarwadi & Agte, 2004).

Figure 1.1: Structure of the human eye.

From the National Eye Institute (2009).

1.6 Lutein and zeaxanthin

Antioxidant compounds including retinol (vitamin A), ascorbic acid (vitamin

C), tocopherol (vitamin E), flavonoids and carotenoids are synthesized in plants. The later group is of particular interest and comprises provitamin A carotenoids (α and β-carotene, β-cryptoxanthin) and xanthophylls (lutein and zeaxanthin) which cannot be converted to vitamin A. Lutein and zeaxanthin are two dihydroxy-carotenoids with the ring systems being substituted at both the 3 and 3’ carbon (Figure 1.2). In plants, zeaxanthin is the less abundant of the two isomers. Structural differences are weak but have important spectroscopic implications. Zeaxanthin has 2 β rings and lutein both β and ε

rings. Due to their polarity caused by the presence of hydroxyl groups, lutein and zeaxanthin have a shorter reversed-phase HPLC retention time than non-hydroxy carotenoids. Lutein and zeaxanthin are soluble in organic solvents such as acetone, petroleum ether, ethyl acetate or ethyl ether. They are more soluble in ethanol than in methanol. In ethanol, lutein and zeaxanthin peak absorption wavelengths are 445 nm and 450 nm respectively

(Rodriguez-Amaya, 1999).

GLV, particularly spinach (Spinacia oleracea L., Amaranthaceae) and kale (Brassica oleracea L. var. acephala, Brassicaceae), contain high concentrations of lutein. Other sources such as squashes (Cucurbita spp.,

Cucurbitaceae), peas (Pisum sativum L., Fabaceae) and broccoli (Brassica oleracea L. var. botrytis, Brassicaceae) contain lutein and zeaxanthin in lower concentrations. Zeaxanthin is mostly found in corn (Zea mays L., Poaceae)

(USDA, 2005; Maiani, et al., 2009). In humans, xanthophylls originate exclusively from dietary intake and accumulate in adrenal, adipose, pancreas, kidney and breast tissues, with the highest concentrations in an ocular tissue, the macula lutea (Ribaya-Mercado, et al., 2004). Concentrations of lutein and zeaxanthin in the human lens range from 15.1 to 44.1 ng/g of wet weight

(Mares-Perlman, et al., 2002).

Figure 1.2: Structure of the xanthophylls pigments lutein and zeaxanthin.

From Krinsky, et al. 2003.

1.7 Cataract in India

Age-related cataract is responsible for more than 40% of the world’s blindness and occurs principally in developing countries (WHO, 2005). In

India, cataract is reported to cause over 60% of the country’s total blindness

(Murthy, et al., 2005) making it the leading cause for blindness before trachoma, glaucoma, macular degeneration and vitamin A deficiency (VAD).

These statistics will increase in the near future with the ongoing Indian demographic transition towards ageing (Resnikoff, et al., 2004).

In rural India, eye health services are being underused by people who could benefit from it, the principal reasons identified being fear of eye damage, costs, family responsibilities, ageism, fatalism, and an attitude of

being able to cope with blindness (Fletcher, et al., 1999). There is an alarming disparity between rural and urban prevalence of cataract, mainly caused by an unequal distribution of resources and a lack of primary eye care services (Bhaduri & Bandyopadhyay, 2004). In the state of Andhra Pradesh,

44% of blindness is attributable to cataract (Dandona, et al., 2001). In this state, the risk of cataract increases, and use of eye care resources decreases with being female, rural resident and illiterate (Nirmalan, 2003; Nirmalan, et al., 2004). Complementary to the ongoing efforts of eye health organizations to render high quality cataract surgery available to all Indians, prevention strategies, including dietary approaches, are considered and investigated.

1.8 Leafy vegetables and cataract in Andhra Pradesh

Even if the nutritional importance of wild foods to human health has been demonstrated in many studies, uncultivated foods tend to be overlooked in nutritional surveys (Grivetti & Ogle, 2000; Kennedy, et al., 2005). It appears that a significant proportion of GLV are wild plants, as observed in various surveys such as Pieroni et al. (2002), and this seems even truer in Indian traditional food systems. Supporting examples in Andhra Pradesh include

Kuhnlein and Pelto (1997), Rajyalakshmi et al. (2003) and the Center for

Indigenous Peoples’ Nutrition and Environment (CINE) (2006), who found that few leafy greens are cultivated, the majority being collected in the wild,

along roadside or semi-cultivated in home gardens. Within the context of age-related cataract research, ethnobotanical investigation of the extent and determinants of wild and cultivated leafy vegetable consumption, and consideration of such information in designing epidemiological studies, will give a more precise picture of the relation between local plant diversity consumption and health.

Proteins, lipids, carbohydrates, fibers, and most essential minerals and vitamins are routinely analyzed in nutrient databases, although more data on the carotenoid composition of foods are needed (Gopalan & Tamber, 2003;

Rodriguez-Amaya, 1999). Indeed, wild, weedy and cultivated GLV species are increasingly included in nutrient analyses; however few species have been quantified in terms of lutein and zeaxanthin content. The biochemical analysis of lutein and zeaxanthin content of local wild and cultivated GLV will improve the understanding of the causal pathway, if any, between GLV consumption and prevention of cataract.

Relatively few researchers have explored the health benefits resulting from diversifying fruit and vegetable intakes. Even if precise modes of action explaining how a diversified diet can procure health benefits have not been identified, we can assume that such a diet constitutes a ‘buffer against uncertainties posed by lack of knowledge and by environmental changes’

(Johns, 2003; Johns & Sthapit, 2004). Dietary diversity has been related to

nutrient adequacy, improved health and nutrition status, reduced mortality, preventive of some forms of cancer and lower DNA and lipid peroxidation

(Fernandez, et al., 2000; LaVecchia, et al., 1997; Ruel, 2003; Thompson, et al., 2006; Tucker, 2001). To date, the relation between plant diversity and age-related cataract has not been tested.

1.9 Objectives

1.9.1 Broad development objectives

The broad objectives of this research address issues on biodiversity and health. We aim to study the relation between the utilization and deployment of existing plant diversity and improved eye health. Another objective, in relation with the CBD Cross-cutting Initiative linking Biodiversity, Food and

Nutrition is to document the value of biodiversity, thus providing empirical evidence to support the efforts in international development and conservation initiatives.

1.9.2 Specific objectives

The specific objectives are to conduct an ethnobotanical study on the diversity and uses of GLV including their analysis for carotenoid content and to develop an eye hospital-based case-control study to examine the

contribution of GLV to eye health in rural India with regards to cataract.

Specific objectives leading to testable hypotheses are to: a) Ethnobotany of local GLV

• Document local GLV used for nutrition and investigate

determinants of consumption in relation with perceived health

properties and cultivation status.

b) Carotenoid analysis of local GLV

• Analyze lutein and zeaxanthin content of selected plants,

including the quantification of β-carotene because of its

recognized health benefits.

c) Contribution of GLV consumption to age-related cataract prevention

• Conduct an eye hospital-based case-control study on female

participants presenting and identified with age-related cataract

after comprehensive ocular examinations, and controls without

age-related cataract, using a food frequency questionnaire

(FFQ) to assess dietary intake.

1.10 Hypotheses

The research hypotheses tested in this doctoral project are as follows:

• Perceived health benefits and cultivation status influence the

consumption of GLV in the study area.

• Fresh and cooked leafy vegetables in the study area have

concentrations of lutein/zeaxanthin and β-carotene comparable with

globally available species.

• Consumption of leafy vegetables is associated with age-related

cataract prevention: controls are expected to have higher intake of

GLV (in weight) than cases.

• Diversity of leafy vegetable intake is associated with age-related

cataract prevention: controls are expected to consume a higher

number of different GLV species than cases.

• Lutein and zeaxanthin intake is associated with age-related cataract

prevention: controls are expected to have a higher intake of lutein and

zeaxanthin (in weight) than cases.

Preface to Chapter 2

We propose in this thesis a unified research framework to evaluate the contribution of plant diversity to eye health. In the following chapter, we explore the rationale for investigating biological diversity’s contribution to eye health based on the literature review of phytochemical, pharmacological and clinical data. We specifically evaluate the significance of existing evidence and, keeping in mind the multifactorial nature of disease, underlying poverty and malnutrition typically found in developing country context, the research needed to demonstrate the relationship between biological diversity, dietary diversity and eye health. Drawing from scientific literature, we present, within an ecohealth paradigm, a model case for investigating eye diseases and blindness in relation to dietary diversity.

The following chapter is a literature review published in the journal

EcoHealth: Bélanger, J. and Johns, T. 2008. Biological diversity, dietary diversity, and eye health in developing country populations: Establishing the evidence-base. EcoHealth, 5(3): 244-256. Participation of each author is described in the Contributions of Authors section. Tables are presented at the end of this chapter and references are listed in the List of References.

2. Biological diversity, dietary diversity and eye health

in developing country populations: establishing the

evidence-base.

2.1 Abstract

Human and ecosystem health converge around biological diversity issues.

Cultivated and wild plants as food and medicine make essential contributions to human health, which in turn provides rationales for conservation. While wild and cultivated plant diversity reasonably facilitates dietary diversity and positive health outcomes, the challenges of demonstrating this relationship limit its impact in concept, policy and practice. We present a rationale for testing the dietary contribution of biological diversity to improved eye health as a case study based on existing phytochemical, pharmacological and clinical knowledge. We consider the empirical evidence needed to substantiate, interpret and apply this relationship at a population and ecosystem level within a unified research framework. Epidemiological data strongly support the prevention of childhood vitamin A deficiency blindness, cataract and age- related macular degeneration by fruit and vegetable consumption.

Phytonutrients, including the carotenoids lutein and zeaxanthin, protect the eye from oxidative stress and harmful light exposure. Laboratory, community

and population level research should prioritize food composition of dietary plants from both agriculture and the wild. Intervention studies, focus groups and transmission of knowledge of local species and varieties within communities will further interpretation of epidemiological data. Population- based studies combining clinical data and measures of access and consumption of biological diversity are key to demonstrating the important relationships among biodiversity, dietary diversity and health outcomes.

2.2 Introduction

Biological diversity is defined as “the variability among living organisms from all sources including, inter alia, terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part; this includes diversity within species, between species and of ecosystems” (UNEP, 1992).

Wild and cultivated plant species make fundamental contribution to human health and nutrition by securing food supplies, meeting energy and micronutrients requirements and providing medicinal resources (Esquinas-

Alcazar, 2005; Grifo & Rosenthal, 1997; Johns & Eyzaguirre, 2006; Toledo &

Burlingame, 2006). Species and varieties have different nutrient and non- nutrient compositions (Kennedy, et al., 2005), and the consumption of a variety of foods across and within food groups, dietary diversity, has been shown to benefit human health by improving nutritional status and reducing

risks of specific diseases (Fernandez, et al., 2000; Kant, et al., 1995;

LaVecchia, et al., 1997; Ruel, 2003; Tucker, 2001). Even though the nature of the evidence is still circumstantial, the assumption that increased agricultural and forest biological diversity leads to a more varied diet, which in turn improves specific health outcomes, is reasonable and compelling.

Contemporary demographic and socioeconomic changes while exacerbating erosion of agricultural and forest biological diversity (Wilson,

1988) also simplify diets and profoundly change the nature of malnutrition and disease in developing countries (Popkin, 2004). Positive synergies for addressing conservation together with nutrition and health objectives can come from integrated interventions at the level of food systems (Johns &

Sthapit, 2004) but require innovation in policy and practice. Decision

VIII/23A of the Conference of the Parties (COP8) (Convention on

Biological Diversity, 2006) calls for a Cross-Cutting Initiative on Biodiversity for Food and Nutrition that in turn necessitates novel models of implementation. Defining the way forward in a manner garnering political and financial support within the context of the competing priorities articulated by the Millennium Development Goals (UN, 2000) and other international agendas demands a stronger evidence base than currently exists

(Johns & Maundu, 2006).

2.3 Biological diversity, diet and health

2.3.1 Identifying measurable outcomes

The dearth of empirical demonstrations of links between access and consumption of biological diversity and specific health outcomes of people subsisting within local ecosystems, or deriving benefits from biological diversity obtained through markets, in part stems from lack of research effort.

Although biological diversity presumably contributes to health through various physiological and other mechanisms (Chivian, 2002; Grifo &

Rosenthal, 1997), the multi-factorial nature of the relationship and the difficulty in establishing causal effects of diet and lifestyle that are inherently long term (Michels, 2003) presents theoretical and practical challenges.

Measurable outcomes should have a rational basis, an effect distinct enough to distinguish from multiple biological and socioeconomic factors and be approachable through practical field methods. Within a developing country context, relevance to public health and social priorities will most convince policy makers. Drawing on these criteria, population-level research reasonably focuses on consumption of biological diversity and three health outcomes:

i) those mediated through micronutrients including nutritional status

and deficiency related vision impairment;

ii) incidence of infectious disease, as mediated through the impacts of

nutritional adequacy and immunostimulatory phytochemicals on

immune function; iii) incidence of and risk factors for non-communicable disease,

specifically diabetes and age-related blindness.

Economic, socio-cultural, demographic, environmental, institutional and biological factors determine biological diversity and health relationships.

Hypotheses formulated on the relationships between biological diversity access and consumption and these three health outcomes are empirically testable. Secondarily, ancillary data can identify socio-economic and other determinants of consumption, while pharmaco-physiological approaches establish mechanisms of action. Such a combination of direct measure of the biological diversity and health relationship, supported by assessment of determining factors and physiological validation does not exist for either of these three disease categories. Some data offer insight into the contribution of dietary diversity to nutrition and health.

This review evaluates a model case for investigating one type of disease, specifically blindness and vision impairment, relative to the above criteria. Drawing on phytochemical, pharmacological and clinical data we explore the rationale for biological diversity’s contribution to eye health by

evaluating the likely robustness of the effect from literature assessing the relationship between diet and eye disease, and we elaborate the evidence needed to substantiate, interpret and apply the relationship at a population and ecosystem level. In recognition of the synergistic nature of its central issue for human and ecosystem health, this paper is directed at researchers and practitioners sharing a multidisciplinary interest in sustainable development and ecohealth, namely in public health nutrition, agriculture and biological diversity conservation.

2.3.2 Biological diversity, conservation and health

In increasingly market-oriented economies, biodiversity from wild and agricultural ecosystems provide a significant portion of the food and medicines consumed by rural and urban populations (Heywood, 1999; Johns,

2007). Moreover, traditional food systems generate economic benefit and embody socio-cultural values, identity and well being (Kuhnlein, 2004;

Padulosi, et al., 2002). Indigenous peoples’ foods form part of rich knowledge systems in drawing on local biological diversity for sustainable production and land management within distinct environments (Johns & Eyzaguirre,

2006). This knowledge of the potential utility of local plant resources for habitat management, food procurement and medicinal purposes, has been

demonstrated to improve overall child health indices in the Bolivian Amazon

(McDade, et al., 2007).

Biological diversity research and promotion programs focus at local or national levels within developing countries and in conjunction with the CBD initiative Bioversity International and FAO coordinate relevant international activities among multi-sectoral partners (COP8, Curitiba, 2006).

In addition to globally important staples (e.g. rice, wheat, maize, potatoes), traditional agriculture draws on locally-important millets and other cereals, leafy vegetables, roots and tubers, legumes and fruits (Johns,

2007). Small-scale tropical farmers supplement their diets with animal-source foods and a plethora of plant species from home gardens, field margins and natural areas (Grivetti & Ogle, 2000). Ethnobiologists document the range of plant parts obtained by agriculturalists and hunter-gatherers from forests, savannahs and other environments. Although many traditional subsistence systems depend on staples, a balanced diet is obtained by ingesting small but complementary amounts of wild animal-source foods including birds, fish, insects and mollusks and plants as sauces, condiments, snacks and beverages

(Johns & Sthapit, 2004). Traditional farmers typically maintain agrobiodiversity as multiple varieties of numerous crops (Altieri, 1999).

Urbanization, commercialization of cuisines, global trade in staple foods, poverty, cultural erosion and environmental degradation all contribute

to simplification of diets in developing regions (Popkin, 2004). Successful food systems in transition (e.g. Western Pacific and Mediterranean) draw on locally-available foods and traditional food culture (Trichopoulou & Lagiou,

1997) with potential synergies for human and ecosystem health. The case study of eye health justifies and supports a strategy for reconstructing food systems in regions that typically harbor both the richest and most vulnerable biological diversity and the greatest disease burden.

2.4 Dietary diversity and eye health

Food-based strategies for reducing micronutrient malnutrition (Tontisirin, et al., 2002) make important contributions to eye health in developing countries.

Approaches promoting dietary diversification, such as home gardening and the use of traditional foods are demonstrated to alleviate vitamin A deficiency (VAD) and consequential visual impairment (HKI/Asia-Pacific,

2001; Low, et al., 2007; Ruel & Levin, 2000). The emerging recognition that other major eye diseases are prevented by ingesting plant constituents further underlines the potential of food-based interventions to reduce global blindness.

The majority of long-term epidemiological studies has been performed with Western populations and has focused on isolated micronutrients rather than whole foods and dietary patterns. In developing

countries, fruit and vegetable intake in reducing childhood VAD blindness is extensively studied, although controversy surrounding the bioavailability of provitamin A (Castenmiller & West, 1998; de Pee, et al., 1998) usurps inordinate attention. The contribution of food systems promoting botanical dietary diversity to overall eye health deserves substantially more evaluation than it has received to date.

2.4.1 Nutrition and ocular diseases

Of some 37 million blind persons in the world, 90% live in developing countries (Ho & Schwab, 2001; Resnikoff, et al., 2004). Main causes of visual impairment in all regions, except industrialized nations where age-related macular degeneration (AMD) predominates, are cataract (47.8%), glaucoma

(12.3%), AMD (8.7%), corneal opacities (5.1%), diabetic retinopathy (4.8%) and childhood blindness (3.9%) (Resnikoff, et al., 2004) (Table 2.1). Most conditions are associated with ageing as a consequence of genetics, diabetes, obesity, malnutrition and oxidative stress; VAD is the major cause of childhood blindness (Coleman, 1999; Evans, 2001; Taylor, 1999). Other priorities (WHO, 2006) include trachoma and onchocerciasis (both caused by parasitic infection), refractive errors and low vision.

2.4.2 Vitamin A deficiency

VAD underscores the crucial role of diet in eye health (Dowling & Wald,

1958). Vitamin A (retinol) is found in foods such as eggs, liver and whole milk. Provitamin A carotenoids in yellow and orange-fleshed fruits and vegetables and dark-green leafy vegetables (GLV) are transformed into retinol in the body. In the developing country context where intake of animal-source foods is restricted, β-carotene from plant sources provides most vitamin A (Tontisirin, et al., 2002). Improvement of childhood vitamin

A status is achieved with interventions including breast-feeding promotion, infection control, food fortification, supplementation and dietary diversification (Johns & Eyzaguirre, 2006; Underwood & Arthur, 1996).

2.4.3 Epidemiologic evidence for age-related diseases

2.4.3.1 Cataract

Epidemiologic evidence suggests that both fruits and vegetables prevent cataract (Table 2.2). A first prospective cohort study showed a strong protective effect of GLV, particularly spinach (Spinacia oleracea L.), intake in women in the highest as compared to the lowest quintile of consumption

(Hankinson, et al., 1992). Similar results were reported in five other studies for spinach and/or kale (Brassica oleracea var. acephala L.) and other greens

(Brown, et al., 1999; Chasan-Taber, et al., 1999; Lyle, Mares-Perlman, Klein,

Klein, & Greger, 1999; Mares-Perlman, et al., 1995; Tavani, et al., 1996).

Moeller et al. (2004) observed a 42 % decrease of nuclear opacity in high fruit consumers as compared to a low intake group. A subsample of ascorbic acid (vitamin C) non-users showed 57% reduced odds of nuclear opacification between the quartiles consuming the highest and lowest number of different food. Christen et al. (2005) reported a 10 to 15% decrease in cataract prevalence of women in the highest fruit and vegetable intake quintile compared to the lowest. Together these results indicate that an increased consumption of fruits and GLV, particularly spinach, kale and broccoli (B. oleracea var. botrytis L.), has a protective effect against cataract, and decreases risk from 10% to 50%. Dietary diversity is generally not measured as such, but presumably candidates in the highest quintiles of fruit and vegetable intake consume greater quantity and greater variety.

2.4.3.2 Glaucoma

Herbal medicines, especially cannabinoids, ginkgo biloba and bilberry, are promoted for glaucoma treatment (West, et al., 2006). Coleman et al. (2006) report decreased odds of developing glaucoma with consumption of vegetables, particularly carrots and collard greens/kale (OR = 0.34 and OR

= 0.28, respectively) in a population-based cohort study involving 1196 elderly women.

2.4.3.3 Age-related macular degeneration

The Nurses Health Study (Cho, et al., 2004) following 77,562 women and

40,866 men for 12 to 18 years found a protective role of fruit intake on risk of neovascular AMD. Daily consumption of 3 or more fruit servings was associated with a relative risk of 0.64 compared to a daily diet including 1.5 fruits or less in both sexes (95% CI: 0.44-0.93; p-trend=0.004). In the multicenter Eye Disease Case-Control Study (Seddon, et al., 1994) vegetables, particularly GLV like spinach and collard greens (B. oleracea var. acephala L.), were strongly associated with a lower AMD risk (p- trend<0.001). An increased consumption of uncooked vegetables in an elderly non-diabetic and non-smoker Lithuanian population (170 males and

181 females) was inversely associated with AMD (Vaicaitiene, et al., 2003).

AMD prevalence was 2.0 (men) (OR=0.42; 95% CI: 0.18‒1.0; p-trend=0.05) and 2.2 (women) (OR=0.37; 95% CI: 0.15‒0.9; p-trend=0.02) times lower in subjects consuming fresh vegetables twice a week or more as compared to less than twice a week. Furthermore, vitamin A rich fruit and vegetable intake was associated with decreased risk of AMD in the first National

Health and Nutrition Examination Survey (OR = 0.59; 95% CI: 0.37-0.99; p- trend=0.047) (Goldberg, et al., 1988). Although no published study describes the relation of dietary patterns with AMD (Mares-Perlman & Moeller, 2006), the reported results justify an interest in dietary approaches to prevent AMD.

2.4.3.4 Myopia

Cordain et al. (2002) propose that high glycaemic load carbohydrate diets may produce permanent changes in the development and progression of refractive errors, especially in growth periods, resulting in juvenile-onset myopia. They hypothesized that dietary factors may interact with hormones to modify the regulation of vitreal chamber growth. Supporting observations suggest higher intake of fruit and vegetable and lower consumption of rapidly digestible refined carbohydrates may moderate development of myopia

(Johns & Sthapit, 2004).

2.4.4 Phytochemicals, minerals and eye health

Evidence of a positive effect of fruit and vegetable consumption is strongest for cataract and AMD. Although the strength of the association with other conditions needs further corroboration, research at the biochemical level evaluates the importance of isolated phytonutrients and minerals to eye health.

2.4.4.1 Oxidative stress in ocular diseases

Oxidative stress is crucial in the etiology of afflictions of the lens, retina and other ocular epithelia (Beatty, et al., 2000; Izzotti, et al., 2006; Vinson, 2006).

Eye structures are in contact with oxygen and sunlight (Taylor, 1999), with

the lens the first line of defense. Light exposure causes high oxidative stress

(Bron, et al., 2000) and provokes lipid peroxidation within the lens epithelium (Bhuyan & Bhuyan, 1984). Similarly, oxidative stress affects the retina because of its high consumption of oxygen, high proportion of polyunsaturated fatty acids, and exposure to light (Beatty, et al., 2000).

Diabetes-related hyperglycemia elevates reactive oxygen species in the retina by a number of mechanisms that are not fully understood (van Reyk, et al.,

2003). Not surprisingly antioxidant supplementation is receiving much attention from eye health researchers.

2.4.4.2 Antioxidants in the eye

Antioxidants including retinol, ascorbic acid, tocopherol (vitamin E), flavonoids and carotenoids are found in plants. The latter group includes provitamin A carotenoids (α and β-carotene, α-cryptoxanthin) and xanthophylls (lutein and zeaxanthin) which, although they cannot be converted to vitamin A, are accumulated in the eye. In mammals, carotenoids originate exclusively from the diet (Landrum & Bone, 2001; Mares-Perlman, et al., 2002). The central portion of the retina, the macula lutea, and the lens contain lutein and zeaxanthin (Yeum, et al., 1995). These xanthophylls are responsible for blue light absorption (Junghans, et al., 2001; Krinsky, 2002;

Landrum & Bone, 2001) and antioxidant protection (Khachik, et al., 2002;

Miller, et al., 1996). Vitamin A, when transformed into retinol, attaches to opsin to form rhodopsin, a protein essential to vision.

Other phytochemicals and minerals are under investigation for eye health benefits including folate, riboflavin, niacin, zinc, selenium and molybdenum (Bartlett & Eperjesi, 2004; Chiu & Taylor, 2007; Seddon, 2007), all except folate having antioxidant activity. Fruits and vegetables, GLV in particular, are excellent sources of those phytochemicals and minerals as summarized in Table 3.

2.4.4.3 Lutein and zeaxanthin in cataract and age-related macular degeneration

Clinical studies support the hypothesis that a lutein-rich diet protects against cataract and AMD as these conditions were found to be inversely associated with serum lutein levels (Jacques, 1999; Moeller, et al., 2000; Moeller, et al.,

2008). An inverse association with prevalence of nuclear cataract (OR=0.3;

95% CI: 0.1-1.2; p-trend=0.13) was seen in people 65 y of age in a study involving 400 adults aged 50‒86 (Lyle, Mares-Perlman, Klein, Klein, Palta, et al., 1999) while the risk of posterior subcapsular cataract was lowest in 372 older patients with higher serum concentrations (OR=0.5; 95% CI: 0.2‒1.0; p-trend=0.012) (Gale, et al., 2001). Vu et al. (2006) reported a significant decrease (OR: 0.64; 95% CI: 0.40-1.03; p-trend=0.018) for risk of nuclear

cataract with high intake of lutein and zeaxanthin in 1841 patients. In one small (17 subjects) randomized, double-blind, placebo controlled trial to assess the effects of carotenoid supplementation (15 mg lutein, n=5, 100 mg

α-tocopherol, n=6, three times a week for two years) compared to placebo

(n=6), only cataract patients receiving lutein experienced a significant increase in serum lutein concentration and improvements in visual acuity and glare sensitivity (Olmedilla, et al., 2003).

2.4.4.4 Dietary diversity: contribution of a wide array of phytochemicals

The limited evidence available suggests that the health benefits of diversity in diet per se (Ruel, 2003; Tucker, 2001) include, but in general exceed that of, simple nutrient adequacy. Furthermore, whole plant diets surpass supplementation strategies in providing a variety of nutrients and non- nutrients (Tontisirin, et al., 2002) with enhanced synergistic benefits. Relative to the etiology of eye disease variety of phytochemicals and minerals can play several roles that are additive or synergistic involving light, oxidative stress and inflammation et cetera in complex interaction (Mares-Perlman &

Moeller, 2006). Synergistic interactions enhance effects among antioxidants

(Jacob, 1995; Palozza & Krinsky, 1992). Diversifying phytochemical intake by increasing fruit and vegetable variety protects against cancer and cardiovascular diseases (Liu, 2003). A diet including several antioxidants - β-

carotene, vitamins C and E, and zinc - has greater lowering effect on the risk of AMD than diets counting only one antioxidant even in high amounts (van

Leeuwen, et al., 2005). In summary, plant antioxidants, alone or in synergy, protect light and oxygen exposed ocular structures.

Positive impacts on bioavailability are another readily recognizable benefit of diverse diets. For carotenoids, bioavailability depends on the nature of the plant matrix and its affinity to the compounds (Castenmiller, et al., 1999), enterocyte efficiency (Zaripheh & Erdman, 2002), carotenoid type, interaction with other carotenoids, interaction with fat and fiber, nutritional status, ageing and parasite infections (Yeum & Russell, 2002). Bioavailability of specific constituents can be optimized through food combination (food-to- food fortification) and with improved conservation methods (Ruel & Levin,

2000). In the case of provitamin A carotenoids, bioconversion and bioefficacy further modulates the effect of β-carotene on eye health.

2.5 Research in a developing country context

Ocular conditions resulting from deficiency as well as non-communicable diseases, specifically diabetic retinopathy, AMD and myopia are expected to rise in developing countries facing changing consumption patterns involving under and over-nutrition (Popkin, 2004). While caloric daily deficits of 200-

300 kcal per person persist, only three major crops (rice, wheat and maize)

provide over 50% of global caloric needs, with reduced consumption of other foods (Johns & Eyzaguirre, 2007). A decline in variety of fruit and vegetable species consumed (Fassil, et al., 2000; Johns, 2007) contributes to micronutrient deficiencies affecting some two billion people (Kennedy, et al.,

2003) and concomitant infectious and other diseases. Conversely, non- communicable diseases such as diabetes, cardiovascular diseases or obesity result from increased fat and carbohydrate intake, coupled with a reduction in food diversity (Popkin, 2004).

Poverty as the underlying cause of malnutrition is characterized by diets lacking in quantity, quality and diversity (FAO, 2000). Increasing production, access to and consumption of local and traditional food constitutes one of the main components of dietary diversification efforts supported by FAO to reduce malnutrition, hunger and disease.

2.5.1 Case studies

Dietary variety leads to greater intake of vitamin A in children (Shankar, et al., 1996). A number of studies evaluate the strength of the relationship between dietary diversity through home gardening and improved eye health outcomes. Talukder et al. (1994) reported that the variety of plants grown in a garden is associated with reduced risk of xerophthalmia in Bangladeshi children. Similar strategies provided a better intake and utilization of vitamin

A, thus preventing and curing xerophthalmia in rural Bangladesh (Bloem, et al., 1996). Such a protective effect was also observed in Nepal (Shankar, et al.,

1996), where case households (housing one or more xerophthalmic children) were over 3 times less likely to eat a variety of at least 6 different vegetables per week as compared to controls (housing non-xerophthalmic children).

A case-control study by Ojofeitimi et al. (1999) found a consistent inverse relation between plant food consumption and cataract in Nigeria.

Based on a dietary questionnaire assessing diet and lifestyle habits of patients and controls, the control group consumed more fruits and vegetables per week. Although amounts reported were below the recommended daily intake, even modest intakes of fruits and vegetable procured eye health benefits.

Presumably the fruits and vegetables consumed by Nigerians contain considerable amounts of protective eye compounds.

For instance, leafy vegetables are rich components of food biological diversity in Africa (Fassil, et al., 2000; Johns, 2003) with about a thousand edible species and varieties of greens enumerated as used traditionally. They are important sources of iron, calcium, fibre and protein, and contribute specifically to eye health via ascorbic acid (vitamin C), tocopherol (Vitamin

E), vitamin A (retinol and provitamin A), lutein, zeaxanthin, folate, riboflavin, niacin and selenium (Chweya & Eyzaguirre, 1999).

2.6 Research gaps and ways forward

The need to better demonstrate and document the association between biological diversity, dietary diversification and improved eye health requires integrative research at the laboratory, community and food system levels.

2.6.1 Laboratory activities

Few so-called underutilized species are analyzed for even basic nutrient composition, while little is known about intraspecific variation in nutrient and functional health properties. Wild foods and minor crop species, important for many rural households, need proper analytical data

(Burlingame, 2000; Grivetti & Ogle, 2000; Johns & Eyzaguirre, 2006). While protein, lipids, carbohydrates, fiber, essential minerals and vitamins are routinely included in nutrient databases, data on the carotenoid composition of foods is urgently needed (Rodriguez-Amaya, 1999). Indeed several examples of GLV and other wild plant foods have superior carotenoid concentrations to commercially available species (Betancourt-Dominguez, et al., 2006; Kobori & Amaya, 2008; Mou, 2005). Precision, accuracy and interpretability of data could be facilitated by the standardization of analytical methods.

Nutritional composition of local plant species, varieties and cultivars is important. The composition of different varieties originating from diverse

locations has proven to be surprisingly heterogeneous. Among other crops, kale, lettuce, fresh pepper (Capsicum anuum) and potatoes varieties and cultivars vary intraspecifically in concentration of carotenoids, flavonoids and mineral micronutrients (Andre, et al., 2007; Lee, et al., 1995; Mercadante &

Rodriguez-Amaya, 1991; Mou, 2005). Several traditional varieties of rice show higher values than commonly grown high-yielding varieties (Kennedy &

Burlingame, 2003). Bioavailability issues are also worth further investigation.

2.6.2 Population-based studies

Population-based studies can directly determine the contribution of local and traditional diets to eye health. The development and standardization of tools to measure dietary diversity is a considerable challenge (Ruel, 2003).

Methods to evaluate the contribution of wild species and varieties to diet also need to be elaborated (Kennedy, et al., 2005; Toledo & Burlingame, 2006) and applied in an eye research context.

Empirical data on eye health outcomes and on the use of biological diversity can be gathered using epidemiological approaches such as case- control and prospective studies, which are practical in the field if an acceptable sample size can be attained. Multidisciplinary teams would be an asset, involving clinicians measuring the prevalence of eye diseases and nutritionists assessing dietary intake of nutrients, food items and food

diversity. Since nutritional surveys have a tendency to overlook the contribution of wild foods, GLV and varieties within species (Grivetti & Ogle,

2000; Kennedy, et al., 2005), the collaboration of ethnobotanists extends knowledge on local dietary resources. The retrospective assessment of dietary exposure and the influence of socio-economic and other biological factors tend to obscure the studied effects (Michels, 2003) and calls for strategies to reduce confounding.

2.6.3 Community activities

At the community level, promotion, production and access to a variety of nutritious foods in general, and more specifically to eye-protecting dietary plants are key activities. Promotion of fruits and vegetables begins with the identification and documentation of local sources and varieties as well as perceptions regarding use, palatability, accessibility and reasons for low or high popularity. Subsequent interventions to increase consumption may involve nutrition education and transmission of knowledge on wild and cultivated foods, and of their health benefits. GLV, which are often seen as low-status, or poor people’s foods (Fassil, et al., 2000) require stronger promotion. In addition, surveys at baseline and after interventions allow testing hypotheses that increasing biological diversity, access and consumption improves health outcomes.

Increasing the production, availability and access to nutrient-rich food can be achieved by home gardening (Faber & van Jaarsveld, 2007) and improved agricultural practices, including food conservation (Ruel & Levin,

2000). Commercialization through local markets has the potential to increase the input of fruits and vegetables in daily diets. In addition to diversifying the diet, these strategies also generate household income (Johns & Sthapit, 2004).

2.6.4 Ecohealth determinants

Consumption of agricultural and wild biological diversity is determined by the interaction of manifold factors affecting accessibility, affordability and acceptability. Production and extraction of food biological diversity is mediated by environmental and agronomic factors and by farmer decision- making related to economic viability, environmental management and socio- cultural values. Market demand in conjunction with costs of production and distribution affects what farmers grow in their fields. Poverty can lead to biological diversity loss through over-exploitation of the environment as well as having direct adverse effects on human health. Documentation of the patterns of use of biological diversity and establishment of its determinants of use within a community will assist in the interpretation of epidemiological results, as well as identify key elements for designing successful program, policy and promotional interventions. Data from measures of socio-cultural,

historical, demographic, environmental, economic and market aspects can be considered in relationship to those examining dietary consumption and health indicators.

Qualitative research such as community-level focus groups can explore community understanding of these relationships, with specific attention to farmer decision-making, while seeking to identify community led solutions within an ecohealth paradigm.

2.7 Conclusion

Dietary, biochemical and physiological data attest to the contribution of plant food consumption to eye health, specifically AMD, cataract and VAD.

Indeed studies in nutritional epidemiology support a relationship between childhood blindness and VAD, and between carotenoid-containing vegetables and fruits consumption and cataract and AMD. Lutein in GLV is likely to contribute to eye health, as are carotenoids found in elements of biological diversity from both agricultural and wild areas. Synergies of different chemicals may act in ways that are not fully understood to prevent eye disease. A number of studies in developing countries have reported improved eye health outcomes with diversification of the diet.

Integrative research at the laboratory, community and food system levels will further refine the demonstration of the relationship between

biological diversity, dietary diversity and eye health. Such strategies can include the extensive compositional analysis of underutilized species, wild foods and varieties and cultivars and the examination of bioavailability issues.

Epidemiological studies coupled with measures of biological diversity consumption may empirically test the relation. Finally, the identification of community determinants of biological diversity utilization, including the commercialization of resources and the transmission of knowledge, will assist the interpretation of epidemiological results.

The case study of eye health can provide a robust and viable demonstration of the contribution of biological diversity and health in response to the CBD Cross-Cutting Initiative on Biodiversity for Food and

Nutrition and other policy objectives.

Table 2.1: Definitions of the most common eye diseases.

Disease Definition Cataract Partial or complete progressive opacification of the lens, preventing light from reaching the retina in a correct focus, thus impairing vision or causing blindness. Age-related macular AMD is a condition where light sensing cells of the macula degeneration (AMD) (central retina) malfunction because of a lack of oxygen and nutrients supply caused by the hardening of the arteries that nourish the retina. Glaucoma Glaucoma is caused by an increase in the intraocular pressure, leading to damages to the eye structures. Loss of vision or blindness can occur with the damaging of the optic nerve. Vitamin A deficiency In a progressive order, VAD cause night blindness, drying of the (VAD) related blindness conjunctiva, drying of the cornea (xerophthalmia), ulceration of the cornea (keratinomalacia) and finally blindness as inflammation or infection occurs. Superficially, the white of the eye presents foamy spots (Bitot’s spots). Corneal opacities Opacification of the cornea (clear membrane covering the outside of the eye) due to the scarring of tissues, injuries or infection, and resulting in vision loss. Diabetic retinopathy A complication of diabetes mellitus causing lesions on the retina as a result of vascular changes in the blood vessels of the retina. Myopia A refractive error caused by the abnormal length of the eyeball causing the image to come to a focus in front of the retina. The result is a poor vision of distant objects.

Table 2.2: Description of eight population-based studies evaluating the effect of diet on the risk for cataract development.

Authors No of participants Measure Outcomes Ratios Hankinson et al., 50,000 Cataract 47% lower risk when consuming spinach and other greens ≥ 5 RR: 0.53; 95% CI: 0.38-0.73; 1992 extraction times / week compared to less than once a month p-trend=0.001 Mares-Perlman et 1,919 Nuclear 31 % risk decrease in women in highest quintile of spinach OR: 0.69; 95% CI: 0.50-0.95; al., 1995 sclerosis consumption p-trend=0.03 Tavani et al., 1996 207 cataract Cataract Risk reductions for candidates with a consumption of > once a extraction patients week compared to < once a week of: and 706 controls 40% for spinach OR: 0.6; 95% CI: 0.4-0.9; p- trend<0.05 50% for cruciferous vegetables OR: 0.5; 95% CI: 0.3-0.8; p- trend<0.01 50% for tomatoes OR: 0.5; 95% CI: 0.4-0.8; p- trend<0.01 30% for peppers OR: 0.7; 95% CI: 0.4-1.1; p- trend<0.05 50% for citrus fruits OR: 0.5; 95% CI: 0.2-1.3; p- trend<0.01 50% for melon OR: 0.5; 95% CI: 0.4-0.8; p- trend<0.01 Lyle et al., 1999 246 Nuclear 40% risk decrease in persons consuming spinach and other RR: 0.6; 95% CI: 02-0.9; p- cataract leafy greens trend=0.004 Chasan-Taber et 1982 Cataract 30-38% decreased risk with consumption of spinach and kale ≥ RR: 0.62; 95% CI: 0.45-0.86; al., 1999 extraction 2 times/week compared to ≤ once a month p-trend=0.005

25% lower risk with consumption of broccoli ≥ 2 times/week RR: 0.75; 95% CI: 0.59-0.95; compared to ≤ once a month p-trend=0.03 Brown et al., 1999 36 644 Cataract 49% decrease in risk when consuming spinach ≥ 2 times/week RR: 0.51; 95% CI: 0.32-0.82; compared to ≤ once a month p-trend=0.08 23% less risk with broccoli consumption ≥ 2 times/week RR: 0.77; 95% CI: 0.61-0.97; compared to ≤ once a month p-trend=0.02 Moeller et al., 479 Nuclear 42% decrease in prevalence odds with high intake of fruit OR: 0.58; 95% CI: 0.32-1.05; 2004 opacities p-trend=0.07 275 vitamin C non- 57% decrease in prevalence odds in the highest quartile of OR: 0.43; 95% CI: 0.16-1.11; users subsample variety of food in the diet compared to the lowest quartile p-trend<0.05

Christen et al., 39 876 Cataract 10-15% risk reduction for women in the highest quintile of fruit OR: 0.87; 95% CI: 0.7-0.99; p- 2005 extraction and vegetable intake compared to the lowest trend<0.05

Table 2.3: Dietary sources of compounds and minerals (nutrients and non- nutrients) beneficial to eye health.

Compounds and Dietary sources1 minerals Ascorbic acid Fresh vegetables and fruits such as broccoli, green and red peppers, (vitamin C) collard greens, Brussels sprouts, cauliflower, lemon, cabbage, pineapples, strawberries, citrus fruits. Tocopherol (Vitamin Margarine and vegetable oil (soybean, corn, safflower, and E) cottonseed), wheat germ, whole grains, fish, peanut butter, and green leafy vegetables. Vitamin A (retinol Dark green and yellow vegetables and yellow fruits, such as broccoli, and provitamin A) spinach, turnip greens, carrots, squash, sweet potatoes, pumpkin, cantaloupe, and apricots, and animal sources such as liver, milk, butter, cheese, and whole eggs. Lutein and zeaxanthin Corn, green leafy vegetables, particularly spinach and kale, squashes, peas, broccoli. Lycopene Tomatoes, guava, rosehip, watermelon and pink grapefruit. Folate Leafy green vegetables, spinach, turnip greens, citrus fruits, dried beans and peas Riboflavin Animal products, like milk, cheese, yogurt, beef and poultry, green vegetables such as broccoli, turnip greens and spinach. Niacin Liver, meat, peanuts and other nuts, and whole grains, lean meats, liver, poultry, milk, canned salmon, green leafy vegetables. Molybdenum Legumes, such as beans, lentils, and peas. Selenium Cheese, chicken, eggs, garlic, green vegetables, liver, mackerel, mil, brazil nuts, cashew nuts, onion, salmon, shellfish, sunflower seeds, tuna, whole grains. Zinc Oysters, red meat, poultry, beans, nuts, certain seafood, whole grains, fortified breakfast cereals, and dairy products. 1From U.S. Department of Agriculture (2005) and Office of Dietary Supplements (2006).

Preface to Chapter 3

We evaluated in Chapter 2 a model case for the study of eye health and local plant food consumption and diversity. In the suggested model, identifying the determinants of biological diversity utilization, including the valorisation of resources and the transmission of knowledge are necessary to assist the elaboration and interpretation of epidemiological studies. We investigate in this ethnobotanical study the determinants of GLV consumption by women of Madanapalle, Andhra Pradesh, India, particularly the roles perceived health properties and cultivation status play in the choice and consumption of species. This work also provides the foundation information around which are articulated the laboratory and epidemiological studies, and identifies species to be analysed for carotenoid content (Chapter 4), and to be included in an eye hospital-based case-control evaluation (Chapter 5).

This manuscript is co-authored with Dr. Shoba Katumalla and Dr.

Timothy Johns, and was submitted for peer review in the journal Economic

Botany. Participation of each author is described in the Contributions of

Authors section. Tables and figures are presented at the end of this chapter and references are listed in the List of References. Additional information is presented in Appendix A.

3. Perceived health benefits and cultivation status

influence leafy vegetable consumption and

conservation.

3.1 Abstract

Objective: The overlapping nutritional and medicinal uses of plant foods underline their importance for the health and nutrition of traditional societies and provide further support for their sustainable management. As leafy vegetables are one of the food group most attributed with health properties, we investigate the role perceived health benefits play, along with cultivation status, in the choice and consumption of local leafy vegetables, and eventually in their conservation.

Methods: An ethnobotanical study including a quantitative food frequency questionnaire was conducted with one hundred women from Madanapalle,

Andhra Pradesh, India.

Results: All informants reported consumption of leafy vegetables. Celosia argentea L. had exceptional popularity with 93% consuming. Leafy vegetables with no reported health properties were consumed more frequently than species with reported health properties (p<0.01) and food medicines were consumed less frequently on a weekly basis than functional foods (p<0.01), indicating a therapeutic versus preventive pattern of use.

Cultivation status influenced plant use with wild species less consumed and managed species most attributed with health properties.

Conclusions: Local wild and domesticated leafy vegetable diversity is used and esteemed by Madanapalle women for nutrition and health. Perceived properties, along with cultivation status, significantly influence consumption patterns. However, traditional knowledge of leafy vegetables use shows signs of erosion. Our results, combined with a biochemical and epidemiological evaluation of the potential of leafy vegetable consumption toward age- related cataract prevention, highlights the importance of plant genetic resources and their conservation to the health of rural populations.

3.2 Introduction

Ethnobotanists continue to elucidate the overlapping roles of plants used within both a nutritional and a therapeutic context to promote health and respond to disease (Etkin, 1994; Etkin, 1996; Etkin & Ross, 1982; Johns,

1990; Pieroni, 2000). The ingestion of functional phytochemicals found in traditional foods has direct implications for the well-being of indigenous and local populations (Johns, 1996; Johns & Chapman, 1995) that may in part explain the contribution of traditional diets (e.g. Mediterranean, South

Korean) to improved longevity and decreased prevalence of cancer, diabetes, obesity and cardiovascular diseases (Lee, et al., 2002; Trichopoulou, et al.,

2003; Trichopoulou, et al., 2000). Such diets are typically characterized by greater dietary diversity, and by the incorporation of wild and locally grown fruits and vegetable species (Johns, 2007; Rivera, et al., 2006; Zeghichi, et al.,

2003). Plants used deliberately for their perceived medicinal attributes may also contain phytochemicals with pharmacological and physiological activities

(McCune & Johns, 2002).

Green leafy vegetables (GLV), in particular wild and weedy species

(Bye, 1981; Stepp & Moerman, 2001), represent an important proportion of foods with medicinal value recognized in Europe (Pieroni, et al., 2002;

Rivera, et al., 2005), Africa (Chweya & Eyzaguirre, 1999; Dovie, et al., 2007;

Kimiywe, et al., 2007), South America (Hanazaki, et al., 2006) and Asia

(Jeambey, et al., 2009; Misra, et al., 2008; Ogle, et al., 2003). Limited information is available on the perceptions of medicinal properties associated with leafy vegetable consumption in South Andhra Pradesh, India. Moreover, most ethnobotanical studies on leafy vegetables concentrate on wild and weedy species and do not take into account knowledge and beliefs on cultivated and managed species (in home gardens and pots, neighborhood trees, protected in fields). We hypothesize that cultivated plants as candidates for selection for reduced toxic and palatability-mediating phytochemicals will be attributed fewer medicinal and health-promoting properties than wild and weedy species.

While the importance of biological diversity to human nutrition and health is increasingly acknowledged (Chivian & Sullivan, 2002; COP8, 2006;

Wilby, et al., 2009), empirical demonstrations of this relationship are scarce

(Johns & Eyzaguirre, 2006). The added value traditional use of genetic resources brings has significant applications in terms of biological diversity conservation (Etkin, 2002; Swaminathan, 1996). Indeed, the extent to which people relate to and esteem plant species determines how they manage them and the habitats in which they occur (Johns & Sthapit, 2004). As ethnobotanical investigations of the "perceived" food-medicinal multifunction of ingested botanicals (Johns, 1990) provide evidence on the traditional role of plant genetic resources, they offer a rationale and impetus for both sustainable use and promotion of their consumption.

This study as it supports biochemical and epidemiological evaluation of the potential of leafy vegetable consumption towards prevention of age- related cataract (Bélanger & Johns, 2008), underlines the importance of local traditional leafy vegetables to the health of rural populations. We further hypothesize that assumed health benefits, along with cultivation status, play a central role in the choice and utilization of local plant foods. By documenting traditional knowledge of South Andhra Pradesh plant foods and by establishing their importance in terms of consumption, we highlight health,

nutrition and cultivation relationships with potential impacts on local environmental and food policies.

3.3 Research area

Research took place in the municipality (mandal) of Madanapalle and surroundings, Chittoor District, state of Andhra Pradesh, India, located at

13° 33'N latitude and 78° 30'E longitude (Figure 3.1) and part of the

Rayalaseema geographic area. Madanapalle’s population is over 100, 000, with a majority of low socio-economic status. In the selected villages, the main religion is Hinduism and the main language is Telugu.

The region is hilly, being part of the Eastern Ghats, and Madanapalle stands at 695 m above sea level. The agro-ecology of the region is characteristic of the Deccan Plateau and the vegetation is mostly tropical dry deciduous (Pullaiah et al., 1998). The climate is composed of a rainy season

(June-September), winter season (October‒January) and summer ‒ dry season (February-May). Madanapalle, a drought-prone area with an average annual rainfall of 848 mm (2000-2004) (Reddy, 2007), is the center of an agricultural region growing mostly fruits and vegetables, and is known for its tomatoes. In Chittoor District, the main crops include rice (Oryza sativa L. ssp indica), sugar cane (Saccharum officinarum L.), pigeon pea (Cajanus cajan (L.) Millsp), chillies (Capsicum anuum L.), jowar (Sorghum bicolor

(L.) Moench), groundnuts (Arachis hypogaea L.) and sesame (Sesamum indicum L.) (Andhra Pradesh Government, 2002).

3.4 Methods

Data were collected from August 2007 to January 2008 during the rainy and winter seasons. Twenty-two (22) villages were selected following an opportunistic sampling provided by the community outreach program of the

Siloam Eye Center. Since most villagers do not own a vehicle and access to public transportation is limited, the Center conducts a systematic everyday door-to-door screening of all surrounding villages, in which we participated.

All participants were female since the larger study concerns the eye health of women. Moreover, women are typically at the center of wild plant food use and management in agricultural environments (Price, 2006).

Ethnobotanical information was obtained from 100 randomly selected women (age range: 17-80, mean: 42.5) using structured and semi-structured questionnaires (Cotton, 1996; Cunningham, 2001; Kuhnlein, et al., 2004;

Martin, 2004) (Appendix A). All interviews were conducted individually at the residence of the informant (Alexiades, 1996). Market surveys in the local daily market gathered further information (Martin, 2004). Ethics approval was obtained from the Human Subjects Ethics committee of McGill

University and the Human Research Project Review Board of the L.V.

Prasad Eye Institute, Hyderabad, Andhra Pradesh.

Qualitative interviews comprised a free-listing of all consumed GLV including herbs, regardless of seasonal availability, followed by a discussion of the provenance and cultivation status (wild, weedy, managed, cultivated)

(modified from Dufour & Warren, 1994) and ecological areas where gathered from, seasonality and cooking methods for all listed species.

Perceived health properties of GLV in general and for specific taxa, and the source of the knowledge were solicited. The increased or decreased use and availability of GLV over the last decades and the underlying reasons were discussed. Usual frequency of consumption on a weekly basis was recorded for of all listed GLV species (Henry & Macbeth, 2004; Kuhnlein, et al., 2004;

Ulijaszek, 2004). All interviews were conducted in Telugu.

When needed, the informant was asked to show a specimen of a species and photographs were used to confirm the identity of the plant with the informant. Plant walks and collections were performed with willing informants. Voucher specimens of plants (40) were prepared in triplicate

(Alexiades, 1996) and deposited at the Sri Krishnadevaraya University

Herbarium, Anantapur, Andhra Pradesh, under the direction of Dr T.

Pullaiah; the herbarium of a local high-school (Rishi Valley Education

Center, Madanapalle); and the McGill University Herbarium, Montreal,

Canada. Thirty-four species were formally identified.

Plant species individually mentioned by at least three distinct informants (Johns, et al., 1990) are reported. Species associated with general health benefiting properties, e.g. “good for health”, “good for the body”, were designated as folk functional foods and with specific health properties as food medicine (Pieroni & Quave, 2006) with the latter categorized in emic groups according to local conceptualization of health. Non-parametric

Mann-Withney U test was used to compare frequency of consumption for

GLV, following a non-normal distribution, with and without associated health properties. Kruskal-Wallis analysis of variance was used to detect difference in consumption and both cultivation status and nature of perceived health benefits while chi-square ( " 2 ) test compared reported attributed health properties and cultivation status. Results were analyzed ! using the JMP 7.0.2 statistical software (SAS Institute Inc, Cary, NC).

3.5 Results

From 100 interviews, 986 reports of use of leafy vegetable for food were recorded. Consumption on a monthly or weekly basis represented 801 of these reports.

3.5.1 Edible leafy greens in Madanapalle

Forty different akukuralu, leafy vegetables in Telugu, were mentioned. Of these, 34 species belonging to 20 botanical families were identified. Table 3.1 reports the 32 identified species mentioned by a minimum of three (3) informants along with Telugu name, seasonality (R: rainy season, W: winter,

S: summer), cultivation status (C: cultivated, commercially grown, M: grown in home gardens or pots, trees of the neighborhood, Wi: wild or We: weeds), ecological area where gathered from, and average weekly frequency of intake by consumers.

Table 3.2 lists the GLV most quoted by the participants. On average, women mentioned 9.85 ± 4.62 different GLV species. Celosia argentea L. was the most widely consumed leafy vegetable with 93% of respondents reporting consumption. Coriandrum sativum L. and Murraya koenigii (L.)

Spreng. were the species most frequently consumed weekly. Leaves of these species are used as herbs for flavoring a wide range of dishes and they are commonly used in small quantities.

Only the leaves were used for cooking. All species but two were consumed cooked, with only Mentha spicata L. and C. sativum occasionally used fresh for garnishing. Recipes that include leafy vegetables are pappu

(lentil curry), talimpu (fry), pachadi (chutney), pickles, pulusu kuura (gravy cooked with tamarind or lime juice), sambar (vegetables boiled with variable

amounts of cooked toor dal ‒ pigeon pea, tamarind and spices) and rasam

(thin soup made of coriander, tamarind, pepper and spices).

The majority of reported leafy greens are herbaceous species, with the exception of 4 trees, namely Sesbania grandiflora (L.) Poir., Tamarindus indica L., Moringa oleifera Lam. and M. koenigii. Most species are available either all year long or during both the rainy and winter seasons (from June to

January) (Table 3.1). T. indica is available only during the summer season when the young leaves are collected.

3.5.2 Perceived health properties associated with leafy vegetable consumption

During the interviews, the participants were blinded to the fact that we were working on a project on eye health in order to avoid biases in their answers.

A majority of interviewees (67%) reported health properties associated with consuming leafy vegetables in general (Figure 3.2A), with general health properties (“good for health”), blood tonic, good for eye health and body strengthener most mentioned.

When asked for known health properties of specific plant foods, 81% of respondents reported at least one species with health properties. Good for health, blood tonic, joint pain reliever and good for eye health were the properties most attributed to consumption of specific GLV (Figure 3.2B). M.

oleifera had the highest numbers of uses reported by at least 3 informants (5), and the highest number of respondents reporting health properties (38)

(Table 3.3).

Of a total of 801 species-consumption reports, 65.5% were not associated with health properties. Leafy vegetables were less frequently consumed, on a weekly basis, when attributed with health properties

(p<0.01). There were more reports of food medicine consumption than functional foods; however, food medicines were less frequently consumed on a weekly basis than functional foods (p<0.01) (Figure 3.3). Participants reporting perceived food medicine properties associated with specific leafy vegetable were significantly older than those reporting no health properties or functional food attributes (median 45 years old (interquartile range 35-55),

40 (29.5-53), 44 (24-52), respectively; p<0.01).

Up to 45% of the respondents reported they were aware of the health benefits attributed to the consumption of GLV through their doctor. The older generation (parents, neighbors) was cited as transmitting this knowledge to 25% of the respondents. Exposure to television, radio, commercial advertisement, personal experience, family members and education was mentioned by smaller numbers of respondents.

3.5.3 Sources of GLV

Among the plants consumed at least once a month, the largest number

(37%) are considered weeds in fields and along roadsides or wastelands

(Figure 3.4A). Another 30% are cultivated in small-scale farms and sold at the daily or weekly market, or produced by the consumer. Managed plants in home gardens, pots or neighborhood account for 21%. Only 12% of reported plants are found as wild plants around hills, ponds, wetlands and forests. On a weekly basis, wild leaves are significantly less consumed than weedy, cultivated and managed species (p<0.01) (Figure 3.4B). A higher proportion of respondents report health properties for managed leafy vegetables than for cultivated and weedy species (p<0.01) (Figure 3.4C).

3.6 Discussion

Leafy vegetables are one of the most diverse groups of food and are found in a variety of environments (Chweya & Eyzaguirre, 1999; Johns, 2007; Rivera, et al., 2006; Zeghichi, et al., 2003). In this study, most of the thirty-two (32) species (Table 3.1) used by the women of Madanapalle are common leafy vegetable species found in other parts of Andhra Pradesh (CINE, 2006;

Gopalan, et al., 2004; Rajyalakshmi, et al., 2001; Reddy, et al., 2007; Reddy &

Yesudas, 2006). C. argentea, an annual herb mostly found as weed in fields and wastelands (Pullaiah, et al., 1998), had an exceptional popularity

unreported previously. Its extended availability during both rainy and winter season and its ubiquity in a broad range of habitats may in part explain this very high popularity.

In the Nutritive Value of India Foods guide (Gopalan, et al., 2004),

105 GLV are reported for all states of India. In their study in the Medak district, researchers of the Center for Indigenous Peoples’ Nutrition and

Environment (CINE) enumerated 55 GLV consumed by the Dalit community (CINE, 2006). Rajyalakshmi et al. (2001) and Reddy et al. (2007) have listed 70 and 54 leafy vegetables, respectively, consumed by tribals in different districts of Andhra Pradesh. Although we report a lower diversity than studies in other areas in Andhra Pradesh, the number of informants and our methodology might explain in part this difference as we report only leafy vegetables consumed by at least three participants (Johns, et al., 1990).

Notwithstanding, Madanapalle women on average make use of this diversity on a weekly basis with the reported mean consumption of 9.85 different species.

3.6.1 Nutrition and taste

Leafy vegetables, both from wild or cultivated sources, are often perceived as being ‘low-status’ food (Fassil, et al., 2000). Nonetheless, a substantial evidence-base supports their use to alleviate and combat micronutrient

deficiencies (Tontisirin, et al., 2002). Nutritionally, edible greens are rich sources of micronutrients and minerals such as folate, iron, calcium, fibre, protein, ascorbic acid (vitamin C), tocopherol (vitamin E), provitamin A carotenoids, lutein, zeaxanthin, folate, riboflavin, niacin, and selenium

(Chweya & Eyzaguirre, 1999; Grivetti & Ogle, 2000). At least 13% of the respondents reported consuming more leafy vegetables today than a decade earlier because they were now aware of the nutritional benefits of GLV. In addition, GLV are low cost or free sources of nutrients and are found year round (Devadas Rajammal, et al., 1996).

Wild species, in particular, contain a variety of chemical compounds responsible for their different tastes (pungent, astringent, sour or bitter).

People respond individually to taste stimuli because of genetics and physiology, and at the community level specific tastes are culturally preferred

(Johns, 1994). In South Andhra Pradesh, a very common sour pickle is prepared with Hibiscus cannabinus L. leaves (gongura). With the occasional exception of M. spicata and C. sativum, GLV reported are cooked before consumption. Indeed, leafy vegetables are often potentially noxious, containing compounds such as phytic, oxalic and/or tannic acids, trypsin and chemotrypsin inhibitors (Mosha & Gaga, 1999). Domestic cooking effectively reduces the content of these antinutrients and inhibitors (Yadav & Sehgal,

2002).

3.6.2 Perceived health properties

Madanapalle respondents consume leaf species as vegetables for dietary reasons, and at times for general or specific health properties. The medicinal activities attributed to GLV in general and to species in particular, mainly blood tonic, ophthalmic and joint pain reliever, are consistent with local health priorities for women, which include anemia, night blindness and pain due to intensive fieldwork. GLV in general contain pro-vitamin A carotenoids, folic acid and iron which are responsible for the ophthalmic and blood tonic properties reported by the participants.

M. oleifera (syn. pterygosperma), the drumstick or horseradish tree, had the highest number of health reports, including folk functional food properties (good for health) and food medicine properties (laxative, joint pain, eye health, blood tonic, other). Indeed the leaves, fruits, flowers and immature pods are widely consumed as vegetables in India and are considered highly nutritious (Makkar & Becker, 1997; Morton, 1991). In addition, the leaves have shown antioxidant activity in vitro (Siddhuraju &

Becker, 2003; Tarwadi & Agte, 2003). The blood pressure regulation activity of M. oleifera leaves and its active compounds have been tested and confirmed in animal trials (reviewed in Anwar, et al., 2007). Leaves cooked and eaten as vegetables are used in India to improve eyesight (Viswanathan

& Singh, 1996) and to relieve eye infection (Anwar, et al., 2007; Muthu, et al.,

2006). Other valued GLV species for health properties are Alternanthera sessilis (L.) R. Br. Ex DC and Trigonella foenum-graecum L. (Table 3.3).

3.6.3 Consumption patterns of foods, folk functional foods and food medicines

Plants may be ingested both for food or medicine without any interrelation between the two uses, for health benefits that may provide an additional physiological benefit beyond that of meeting basic nutritional needs (i.e. folk functional foods), or consumed in a food context for specific medicinal action

(i.e. medicinal foods or food medicines) (Pieroni & Quave, 2006). In the

Madanapalle area, foods, folk functional foods and food medicines follow different consumption patterns. A majority of leaves are consumed as vegetables for taste and nutrition. These leafy vegetables are consumed more frequently, on a weekly basis, than species with reported food medicine activity. The lower frequency of the latter fits a pattern of therapeutic use on a need basis in response to specific ailments (as above). Conversely, although folk functional foods are consumed as frequently as the non-medicinal leaves, their reported additional health benefiting properties fit a preventive pattern of consumption for strengthening the organism instead of responding to illness (Pieroni & Quave, 2006). Therefore, the perception of health

properties associated with leafy vegetable consumption, and the nature of these properties, influence their use pattern.

3.6.4 Cultivation status and medicinal attributes

An exhaustive definition of what is ‘wild’ and ‘cultivated’ in terms referring to a continuum of human-plant interactions is discussed by Etkin (1994). For the purpose of simplification, we follow descriptors modified from Dufour and Warren (1994): cultivated (C) plants have been introduced in human agroecosystems and are nurtured in a prepared seed bed, mostly for commercial purposes; managed (M) plants are being protected from human actions that might harm it and planted in areas other than prepared seed beds, here in home gardens, pots or trees in the neighborhood; weeds (We) are plants found mostly in disturbed areas such as fields and roadside, often in competition with cultivated plants; wild (Wi) plants are any plant used but neither managed nor cultivated and found primarily in undisturbed areas.

Not surprisingly, a majority of leafy vegetables are weeds, followed by cultivated, managed and wild species. Weeds are indeed free and available in a broad range of habitats, especially in the vicinity of fields and roadsides.

Wild species, on the opposite, are found in remote areas and their collection demands additional efforts. Consequently, their weekly consumption is less frequent than weedy, cultivated and managed species.

Most ethnobotanical studies on the nutritional and/or medicinal role of edible leaves concentrate on gathered wild and weedy species (Bye, 1981;

Nebel, et al., 2006; Pieroni, 2000; Pieroni, et al., 2002; Pieroni & Quave,

2006; Stepp & Moerman, 2001; Vieyra-Odilon & Vibrans, 2001). In many cases selection for cultivation aims at reducing naturally occurring antinutrients and bitter tasting phytochemicals, such as phenolics or saponins, which are often bioactive compounds (Johns, 1990, 1994). In this study, we have taken into account the managed and cultivated species in order to recognize the complex reality of the wild-cultivated axis, to include all possible sources of leafy vegetables and to allow further comparison and analysis. Almost a third of the reported species are cultivated, and 21 % is grown and managed in home gardens, pots and neighborhood.

Managed species are attributed with more health properties than cultivated and weedy species. One explanation may reside in the fact that neighborhood trees such as T. indica, S. grandiflora, M. koenigii and M. oleifera belong to this cultivation category and their leaves are considered to be both highly nutritious and health benefiting when consumed as vegetables.

Such life forms typically accumulate compounds in order to deter herbivory, play allelopathic activity or protect against UV light, among other reasons, which may lead to phytochemically enhanced leaves (McCune & Johns,

2007). Other frequently managed species include Brassica juncea (L.) Czern.

& Coss., Basella rubra L., M. spicata and Mirabilis jalapa L. Wild species, including A. sessilis, Cassia italica (Mill.) Spreng., Hygrophila auriculata

(Schum.) Heine and Trianthema portulacastrum L., are also perceived to have health properties. In accordance with the theory of selection by cultivation for reduced bitter tasting compounds (Johns, 1990), cultivated species, in comparison with wild and managed leaves, are less associated with health benefits. However, in this study weedy species are also less associated with health properties than wild and managed species.

3.6.5 Traditional knowledge of leafy vegetables and their properties

Traditional knowledge is a cumulative body of experiences, practices and beliefs, evolving by adaptive processes and handed down through generations by cultural transmission (Berkes, et al., 2000). Doctors, elders, family members, neighbors and teachers are the main persons responsible for sharing the knowledge about GLV species and their gathering, preparation and associated health benefits in the study area. Interestingly, older participants significantly consume food medicines more frequently, indicating they retain more of the traditional knowledge on the use of leafy vegetables for relieving various ailments. This may also indicate that their therapeutic needs increase with age. With respondents reporting changes in the availability of gathered GLV species due to changing climate and water

availability, the adaptation and continued transmission of knowledge is likely to have an influence on the future use and conservation of the local diversity.

Rapid erosion warrants recording of traditional knowledge of wild and cultivated diversity (Johns, 2007).

3.6.6 Implications for eye health

Consumption of GLV is believed to contribute to lower risk of developing age-related diseases, namely macular degeneration and cataract, by providing high amounts of the xanthophyll lutein (Sommerburg, et al., 1998) and potentially other phytochemicals and minerals (Chiu & Taylor, 2007). Many epidemiological studies involving nutrition surveys tend to overlook the contribution of non-cultivated foods (Grivetti & Ogle, 2000; Johns, 2003). In the Madanapalle situation, women consume a high proportion of both cultivated and weedy GLV. They also frequently consume leaves for nutrition and disease prevention purposes.

To test an association between the xanthophyll intake and cataract prevalence, 10 species, selected for their local popularity and lack of previous analysis, were analyzed by High Performance Liquid Chromatography

(HPLC) (Bélanger et al., in press)(Chapter 4). High content of lutein and β- carotene, comparable with kale and spinach (USDA, 2005), were found in H. cannabinus, Amaranthus viridis L., and Chenopodium album L., three

species commonly found in and around fields as weeds, in home gardens, and sometimes sold at the market (Bélanger et al., in press). Spinach and kale are the leafy vegetable species most frequently associated with cataract protection in population-based studies conducted in the Western world

(Brown, et al., 1999; Hankinson, et al., 1992; Lyle, Mares-Perlman, Klein,

Klein, & Greger, 1999; Mares-Perlman, et al., 1995; Moeller, et al., 2000;

Moeller, et al., 2008). Given the prevalence of cataract in the study area, these findings are of importance for elaborating strategies for prevention.

3.6.7 Implications for agro-biodiversity conservation

Wild, weedy, managed and cultivated leafy vegetable diversity that is used and valorized by Madanapalle women for nutrition, health, medicine and taste should be part of local agricultural or food security planning. In Sub-

Saharan Africa, Bioversity International (formerly IPGRI) and partners have led successful interventions reviving the use and value of traditional leafy vegetables, increasing their cultivation, protection and conservation, and facilitating urban access and household income through improved marketing.

As noted by O’niango et al. (2006), adding value by coupling these resources with the market and with health benefits increases likelihood of enhancing biological diversity. Research and interventions are also ongoing in other countries to promote the cultivation, management and use of traditional and

underutilized leaf vegetables (Dovie, et al., 2007; Gautam, et al., 2005; Keller, et al., 2005). Complementary with cropping systems, home gardens, pots and common village areas play an important role in agrobiodiversity conservation strategies in providing refuge for underutilized species (Engels, 2001; Ogle, et al., 2003; Padulosi, et al., 2002). Another area for conservation of biological diversity concerns the protection and selective gathering of uncultivated species in field boundaries, habitats where the highest diversity of leafy vegetables is found (Etkin, 2002). Even without explicitly aiming at conserving biological diversity, local management strategies play an important role in the use and conservation of leafy vegetable diversity.

3.7 Conclusions

Perceived health properties, either as folk functional foods or food medicines influence leafy vegetable consumption among women in Madanapalle, South

Andhra Pradesh. In addition, cultivation status relates to both health perceptions and frequency of consumption, with managed species highly valued for health benefits beyond nutrition. However reported changes in the availability of leafy vegetables due to climatic factors and water scarcity further emphasize the importance of recording traditional knowledge of their use and associated health properties. Plant genetic resources play an important role in the health and nutrition of this population, and this reality

has application in terms of conservation of biological diversity, and to the development of strategies to reduce the burden of cataract blindness.

This ethnobotanical work, along with laboratory analyses and a population-based study combining clinical data on cataract and measures of access and consumption of leafy vegetable diversity, provides an example of an integrative strategy demonstrating the important relationships among biological diversity, dietary diversity and health outcomes.

Table 3.1: Leafy vegetables consumed in Madanapalle, Andhra Pradesh, India.

Latin name, botanical family, common Telugu name, cultivation status, ecological area where gathered from, seasonality and mean frequency of weekly consumption, among users.

Latin name Botanical family Telugu name Cultivation Ecological area Seasonalityb Mean weekly statusa frequency (s.d.)c Allmania nodiflora (L.) R. Br. Ex Amaranthaceae Errabadhaku Wi, We Groundnut, tomato, green R; W 1.86 (1.18) Wight chillies and other fields, ponds Alternanthera sessilis (L.) R. Br. Amaranthaceae Ponnagantaku Wi, We Ponds and fields R; W 1.29 (0.85) Ex DC Amaranthus caudatus L. Amaranthaceae Koyagura C, We Tomato, green chillies, R; W; S 1.80 (0.97) groundnut and other fields. Amaranthus cruentus L. Amaranthaceae Thotaku C, M Tomato, other fields R; W; S 1.61 (0.94) Amaranthus tricolor L. Amaranthaceae Sirraku C, We Tomato, groundnut, ragi and R; W; S 1.27 (0.70) other fields Amaranthus viridis L. Amaranthaceae Dantaku C, We Tomato, groundnut, green R; W; S 1.78 (1.32) chillies and other fields Amaranthus viridis L. Amaranthaceae Kodijuttaku We Tomato, groundnut and other R; W; S 1.22 (0.70) fields. Basella rubra L. Basellaceae Bachalaku M, We Neighborhood, fields R; W 1.25 (1.03) Boerhavia diffusa L. Nyctaginaceae Atikimavidaku We Tomato, groundnut and other R; W 1.22 (0.96) fields Brassica juncea (L.) Czern. & Brassicaceae Avalaku, Sassaku C, M, We Groundnut fields, home gardens R; W 1.25 (0.79) Coss. Cassia italica (Mill.) Spreng. Caesalpinaceae Nelathangedu Wi Hills R; W 0.80 (0.74) Celosia argentea L. Amaranthaceae Gurugaku, Wi, We Groundnut, tomato, ragi, any R; W 1.79 (1.17)

Sannagurugaku field, roadside. Chenopodium album L. Chenopodiaceae Chakrantaku C, We Tomato, green chillies an other R; W; S 1.54 (0.72) fields Cleome gynandra L. Cleomaceae Gabbettaku We Tomato, groundnut, other fields R; W 0.85 (1.16) and anywhere. Coriandrum sativum L. Apiaceae Kothimeera C Home gardens R; W; S 5.42 (2.49) Cucurbita maxima Duchesne Cucurbitaceae Gummadaku Wi, We Roadside, fields, anywhere. R; W 2.45 (2.65) Digera muricata (L.) Mart. Amaranthaceae Chenchulaku We Groundnut, tomato and other R; W 1.11 (0.64) fields Hibiscus cannabinus L. Malvaceae Gongura C, M, We Groundnut, tomato, green R; W; S 1.09 (0.80) chillies and other fields. Hibiscus sabdariffa L. Malvaceae Bendlaku Wi Near forests R; W; S 0.58 (0.38) Hygrophila auriculata (Schum.) Acanthaceae Gorimitaku Wi Ponds. R; W; S 0.63 (0.41) Heine Mentha spicata L. Lamiaceae Pudina C, M Pots, home gardens R; W; S 2.18 (1.89) Mirabilis jalapa L. Nyctaginaceae Suryakantamaku M Home gardens, neighborhood R; W; S 0.55 (0.41) Moringa oleifera Lam. Moringaceae Munagaku C, M Neighborhood, home gardens R; W; S 1.07 (0.71) and fields. Murraya koenigii (L.) Spreng. Rutaceae Karivepaku C, M Home gardens R; W; S 5.55 (2.46) Portulaca oleracea L. Portulacaceae Pappaku, Pavillaku We Groundnut and other fields R; W; S 1.42 (1.10) Portulaca pilosa L. Portulacaceae Chavatapayilaku Wi Near ponds and sandy lands R; W 1.00 (0.00) Portulaca quadrifida L. Portulacaceae Esukapayilaku We Fields and sandy waste lands W 1.5 (1.57)

Rumex vesicarius L. Polygonaceae Chukkaku C, We Not gathered R; W; S 1.81 (3.56) Sesbania grandiflora (L.) Poir. Fabaceae Avisaku M Home gardens, neighborhood R; W; S 0.86 (0.56) Spinacia oleracea L. Amaranthaceae Palakura C Commercially grown R; W; S 1.21 (0.58) Tamarindus indica L. Caesalpinaceae Chintaku C, M Neighborhood, home gardens S 2.52 (1.79) Trianthema portulacastrum L. Aizoaceae Gadaraku Wi Near forests R; W 0.50 (0.39) Trigonella foenum-graecum L. Fabaceae Menthaku C Commercially grown R; W; S 1.38 (0.85) a Cultivation status. Wi : wild, We: weed, C: cultivated, M: managed/grown in home gardens, pots and neighborhood. b Seasonality. R: rainy season (June-September), W: winter season (October‒January), S: summer season (February‒May). According to our observations and Pullaiah et al. (1998). c Mean weekly frequency: average number of times per week the species is consumed, among users.

Table 3.2: Leafy vegetable species most quoted (%) by the participants

(n=100).

Species % quotation Species % quotation Celosia argentea 93 Mentha spicata 24 Allmania nodiflora 72 Murraya koenigii 22 Amaranthus caudatus 64 Boerhavia diffusa 17 Trigonella foenum- 64 Amaranthus viridis 15 graecum (kodijuttaku) Moringa oleifera 60 Sesbania grandiflora 15 Alternanthera sessilis 56 Cleome gynandra 13 Rumex vesicarius 47 Trianthema portulacastrum 11 Amaranthus tricolor 46 Brassica juncea 10 Coriandrum sativum 36 Basella rubra 10 Hibiscus cannabinus 36 Portulaca oleracea 7 Amaranthus viridis 34 Cucurbita maxima 6 Spinacia oleracea 34 Hygrophila auriculata 6 Digera muricata 33 Mirabilis jalapa 6 Tamarindus indica 33 Cassia italica 5 Amaranthus cruentus 26 Hibiscus sp. 5 Chenopodium album 26

Table 3.3: Health properties attributed to the consumption of selected leafy vegetables by women of the Madanapalle area.

Species Perceived health properties (n reports) Total (n reports) Amaranthus Good for health (3), laxative (3), blood tonic (4), other (5) 15 caudatus Aamaranthus Good for health (4), blood tonic (4), other (1) 9 cruentus Allmania nodiflora Good for health (5), other (6) 11 Alternanthera Good for health (9), eye health (6), appetizer (3), blood 30 sessilis tonic (3), other (9) Amaranthus tricolor Good for health (3), blood tonic (4), other (3) 10 Amaranthus viridis Blood tonic (3), other (8) 11 Brassica juncea Good for health (3), relieves joint pain (5), other (2) 10 Basella rubra Blood tonic (3), other (5) 8 Celosia argentea Good for health (9), increase bile juice (3), blood tonic (6), 24 other (6) Cleome gynandra Relieves joint pain (11) 11 Coriandrum sativum Good for health (8), other (4) 12 Digera muricata Good for health (7), other (6) 13 Hibiscus cannabinus Blood tonic (3), other (4) 7 Murraya koneigii Good for health (8), other (3) 11 Moringa oleifera Good for health (5), laxative (4), joint pain (10), eye health 38 (3), blood tonic (10), other (6) Rumex vesicarius Good for health (7), other (5) 12 Sesbania grandiflora Joint pain (5), other (5) 10 Spinacia oleracea Good for health (5), blood tonic (3), other (5) 13 Trigonella foenum- Good for health (8), blood tonic (4), other (13) 25 graecum Tamarindus indica Coolness (5), other (5) 10

Figure 3.1: Location of the research site.

Left: map of the South Indian state of Andhra Pradesh, with Chittoor District, identified at the bottom. Right: Madanapalle revenue division comprising Madanapalle Mandal, the study area.

Figure 3.2: Reported (emic) categories of perceived health properties attributed to (A) leafy vegetable consumption in general and (B) intake of specific leafy vegetables.

a Mann-Whitney U test

Figure 3.3: Medians of weekly frequency of intake (serv./pers./week) of leafy vegetables associated or not with general and specific health properties among interviewed women in Madanapalle (n=100).

Figure 3.4: Cultivation status of reported species, associated consumption and proportions of perceived health properties.

(A) number (n) of food-use reports per cultivation status category, (B) mean frequency of consumption on a weekly basis and associated standard error of the mean (SEM), and (C) proportion of participants reporting health properties (%) per cultivation status. Different letters indicate statistically significant differences, using Kruskal-Wallis analysis of variance and chi- square (χ2) test.

Preface to Chapter 4

In Chapter 3 we established the background information on leafy vegetable consumption, valorisation and cultivation in Madanapalle. Drawing on these results, we selected 10 GLV species locally consumed to be included in the following analysis of lutein/zeaxanthin and β-carotene in fresh and cooked samples by High Performance Liquid Chromatography. We selected these compounds for further evaluation on the basis of supporting epidemiological reports of a protective effect of lutein/zeaxanthin on the onset and progression of age-related cataract. The results of this analysis will be included in the lutein/zeaxanthin database to estimate and compare intakes between cases presenting cataract and controls (Chapter 5).

The following chapter entitled ‘Contribution of selected wild and cultivated leafy vegetables from South India to lutein and β-carotene intake’ and co-authored by J. Bélanger, M. Balakrishna, P. Latha, S. Katumalla and

T. Johns, is an article in press in the Asia Pacific Journal of Clinical Nutrition,

Participation of each author is described in the Contributions of Authors section. Tables and figures are presented at the end of this chapter and references are listed in the List of References section. Additional information to this chapter is presented in Appendix B.

4. Contribution of selected wild and cultivated leafy

vegetables from South India to lutein and β-carotene

intake.

4.1 Abstract

Carotenoids, especially lutein and β-carotene, offer benefits to human health in general and to eye health in particular. However, more data on the contribution of plant foods to carotenoid intake is of importance for developing strategies for promoting eye health in regions where cataract is highly prevalent such as in South India. The most frequently consumed 5 uncultivated and 5 commercially grown South Andhra Pradesh leafy vegetables were selected based on interviews with 100 local women. The lutein and β-carotene content of fresh and cooked samples was determined using reversed-phase High Performance Liquid Chromatography. Lutein values ranged from 53 to 143 μg/g and 58 to 175 μg/g in fresh and cooked samples, respectively. β-carotene content was found to range from 45 to 119

μg/g in fresh samples and from 40 to 159 μg/g in cooked samples. No significant difference was observed between the carotenoid content of wild and commercially grown species. According to their reported frequency of consumption, the 10 species considered in this study contribute 40% of the recommended daily intake of β-carotene. This is the first report of lutein

content in fresh samples of Celosia argentea L., Rumex vesicarius L., Digera muricata (L.) Mart., Amaranthus cruentus L. and in cooked samples of all species included in this study.

4.2 Introduction

Benefits imparted by β-carotene in orange and dark green fruits and vegetables in preventing and treating xerophthalmia have long been established (Dowling & Wald, 1958). Plant foods, especially green leafy vegetables (GLV), provide other carotenoids with promise in eye health

(Bartlett & Eperjesi, 2004; Seddon, 2007). The xanthophyll lutein, found mostly in leafy vegetables, has been identified as an important protective agent in several in vitro assays, epidemiologic studies and intervention trials examining plant food consumption and prevention of age-related cataract and macular degeneration (Chiu & Taylor, 2007). Antioxidant activity and absorbance of damaging blue and UV light constitute likely mechanisms of action (Krinsky, et al., 2003).

GLV are the major sources of lutein and, in developing countries where access to animal food is restricted, contribute substantially to fighting retinol deficiencies by being rich sources of the provitamin A β-carotene, notwithstanding bioavailability issues (de Pee, et al., 1998). As leafy vegetables are widely available and easy to gather from the wild or in agro-

ecosystems, or may be cultivated at low cost, their consumption and conservation is being promoted for increased health benefits (Johns, 2007).

However, epidemiologic studies on eye health and nutrition tend to overlook the contribution to diet of wild or less common species (Bélanger & Johns,

2008).

This work is part of a multidisciplinary project to document the importance of GLV consumption to the prevention of cataract in women living in Madanapalle, Andhra Pradesh, involving an ethnobotanical survey and hospital-based case-control study. The objective of this analysis is to quantify carotenoids in common, local cultivated and wild GLV, and to estimate the contribution of these vegetables to daily lutein intake in local women. Because of its recognized importance for eye health, β-carotene content is also examined.

In accordance with local culinary habits in which leafy vegetables are mostly consumed cooked, carotenoid values are reported for both fresh and cooked leafy vegetables. The effect of cooking on the retention of carotenoids is well documented and is not tested in this study (Calvo, 2005;

Liu, 2003; Maiani, et al., 2009; Rajyalakshmi, et al., 2003).

4.3 Material and methods

4.3.1 Dietary intake

Ten (10) local species of GLV were selected based on their frequency of consumption determined in ethnobotanical interviews and food frequency questionnaires conducted with 100 women randomly selected from 20 villages in the surroundings of the Madanapalle sub-district (Mandal) from

September 2007 to December 2007. Villages were selected following an opportunistic sampling provided by the community outreach program of the

Siloam Eye Center. Dietary intake was determined by administering a food frequency questionnaire among participants. Data are expressed as servings per person per week. With 78 24-hour recalls, one average portion was estimated to contains 20 g of fresh leaves, taking into account the variability between different recipes. Subject informed consent was sought before the interview and the patient anonymity was respected. Ethics approval was obtained from the Human Subjects Ethics committee of McGill University and the Human Research Project Review Board of the L.V. Prasad Eye

Institute, Hyderabad, Andhra Pradesh, to which the Siloam Eye Center is affiliated.

4.3.2 Plant material

The ten plant species with highest availability, frequency of consumption and unknown carotenoid content are listed in Table 4.1 (Appendix B). Five leafy vegetables among those are uncultivated, and the remaining are cultivated in small-scale farms. For each species, approximately 200 to 500 g of plant material was collected in three different locations (different gathering areas or cultivated in fields from different regions). Each sample was analyzed separately upon arrival in the laboratory. Plants were rinsed with distilled water, dried with absorbing paper and the composite sample of leaves without stems was divided into two portions, one to be analyzed fresh and the other after boiling for 5 minutes and draining the water. Before extraction each sample was homogenized in a household blender. Voucher specimens of each species were pressed and dried and identification was confirmed, with the collaboration of Dr. T. Pullaiah (Sri Krishnadevaraya University

Herbarium, Anantapur).

4.3.3 Chemicals and standards

HPLC grade acetone, acetonitrile, methanol and ethyl acetate plus diethyl ether, petroleum ether, sodium sulfate and sodium chloride were purchased from Merck Ltd (Mumbai, India). Acetone and triethylamine (TEA) were purchased from S.D. Fine Chemicals Ltd (Mumbai, India) and SRL

(Mumbai, India), respectively. Lutein and β-carotene standards were obtained from Sigma-Aldricht (Mississauga, Canada) and zeaxanthin was obtained from Extrasynthese (Lyon, France).

4.3.4 Carotenoid extraction

The extraction procedure is described in Rodriguez-Amaya et al. (2002;

1999). In brief, approximately 3 g of fresh or cooked homogenized sample was weighed and ground with mortar and pestle in Celite and acetone.

Acetone was filtered through a sintered disk glass funnel mounted on a suction flask with the solid residue reground in acetone until complete discoloration of the material (usually 2-3 repetitions). The acetone fraction was then partitioned to 50 ml of diethyl ether and petroleum ether [1:1 v:v] and washed 5 times with distilled water, sometimes with addition of 2-3 g of sodium chloride if an emulsion formed. After removing all water, the remaining diethyl ether and petroleum ether fractions were dried over a sodium sulfate bed and evaporated with a rotary evaporator (Superfit

Continental Pvt Ltd, Mumbai, India). The last 1-2 ml of solvent was evaporated under nitrogen gas and the extracted sample was stored at -20°C until HPLC analysis for a maximum duration of 1 week. The samples were not saponified as this treatment may alter the quantity of carotenoids and is not necessary in this case where the carotenoids of interest are separated

from the chlorophylls (Rodriguez-Amaya, 1999). All manipulations were conducted under dim laboratory light and glass material was covered with foil.

4.3.5 HPLC conditions

Separation was performed on a Shimadzu model HPLC coupled with a tertiary pump LC-10AT, a SPD-10A UV-VIS detector and a column thermostat. The integration system was Class-VP version 7. The reversed- phase Phenomenex C18 5µ [250 x 4.60 mm i.d.] column was kept at 25°C. The solvent composition was modified according to Kimura and Rodriguez-

Amaya (2002) for a tertiary pump system: solvent A contained acetonitrile and 0.05% TEA and solvent B methanol: ethyl acetate [1:1]. The selected flow rate was 1.0 ml/min. The initial proportion of solvent A and B was 95:5 increasing to 60:40 in 15 min following a concave gradient and the proportion was maintained until the end of the run (60 min). Re-equilibration took 15 min. Immediately before injection the sample was rediluted in 10 ml HPLC grade acetone, 1.5 ml were filtered through a 0.22μ PTFE Millipore filter to a

HPLC vial and 10 μl were injected in the system. Detection was performed at

450 nm.

4.3.6 Identification and quantification

Identification of lutein and β-carotene was carried out by comparison of the

HPLC retention times with corresponding standards and co-chromatography with added standards. As major carotenoid patterns are highly constant in leafy vegetables these procedures are sufficient to confirm the identification of the compounds for this validated analytical method (Britton, 1991). Our method did not separate zeaxanthin from lutein, so values are reported together. However, it should be noted that GLV contain only trace amounts of zeaxanthin (Sommerburg, et al., 1998). Values are reported for lutein and all-trans β-carotene.

Standard curves were constructed for external quantification using lutein isolated by open-column chromatography from groundnut leaves and commercial β-carotene standard purchased from Sigma (Kimura &

Rodriguez-Amaya, 2002). Purity, verified with HPLC for the isolated lutein and the commercial β-carotene, was 93% and 97% respectively. The concentrations of the standards were determined spectrophotometrically, using the following absorption coefficient values: β-carotene, 2592 in petroleum ether; lutein, 2550 in ethanol. Concentrations were corrected accordingly. The curves were constructed in triplicate at 3 and 4 different concentrations for β-carotene and lutein, respectively. The curves were linear, passed through the origin and their correlation coefficient were higher than

0.98. The means of the three locations are reported in Table 4.2 with associated standard deviation in μg/g of fresh or cooked sample. One-way

ANOVA analyses were used to test for difference between contents in non- cultivated and in cultivated GLV. Wilcoxon rank-sum test was used to compare consumption of cultivated and non-cultivated GLV (Wilcoxon,

1945). Statistical analyses were conducted using R statistical software version

2.9.0.

4.4 Results

4.4.1 Dietary intake

Table 4.1 shows mean dietary intake of the selected leafy vegetables among the women recruited for the study. Celosia argentea L. and Allmania nodiflora (L.) R. Br. Ex Wight were the two species most frequently consumed. In total the weekly average number of GLV servings per person is

6.81 ± 3.91, comprised of 4.59 ± 3.00 servings/person/week of uncultivated species and 2.22 ± 2.14 of cultivated ones. A Wilcoxon rank-sum test indicates a difference between the number of servings of cultivated and uncultivated GLV (p < 0.01).

4.4.2 Qualitative analysis

According to the standard co-chromatography and retention times, lutein

(β,ε-carotene-3,3’-diol; tR =9.6 min) and β-carotene (β,β-carotene; tR =37.5 min) were identified. A typical GLV carotenoid chromatogram is presented in Figures 4.1A and 4.1B (fresh and cooked A. nodiflora). The elution patterns of all GLV were very similar, with the exception of Hibiscus cannabinus L. and Rumex vesicarius L., which showed different patterns when fresh and cooked.

4.4.3 Fresh leafy vegetables

The concentrations obtained for A. nodiflora, Alternanthera sessilis (L.) R.

Br. Ex DC, Amaranthus cruentus L., Amaranthus tricolor L., Amaranthus viridis L., C. argentea, Chenopodium album L., Digera muricata (L.) Mart.,

H. cannabinus and R. vesicarius ranged from 53 to 143 μg/g for lutein and from 45 to 119 μg/g for β-carotene (Table 4.2).

Our results for A. viridis compare well with Kobori and Rodriguez-

Amaya (2008) and Liu and colleagues (2007) who report 119 and 147 μg/g respectively. For the same species, Tee and Lim (1991) report 42 μg/g after saponification and both Raju et al. (2007) and Lakshminarayana et al. (2007), from the same laboratory, reported 904 µg/g on a dry weight basis which does not allow comparison (Lakshminarayana, et al., 2007; Raju, et al., 2007; Tee

& Lim, 1991). Kumar et al. (2010) reported 1850 µg/g for C. album, also on a dry weight basis. Wills and Rangga (1996) and Kidmose et al. (2006) reported 29 and 23 μg/g for A. tricolor (syn. mangostanus and gangeticus).

Previous HPLC analyses yielded values for β-carotene content of 57,

86, 11 and 12 μg/g for A. sessilis, A. tricolor, A. viridis and C. argentea

(Bhaskarachary, et al., 1995). Our results also compare well with Kobori and

Rodriguez-Amaya (2008) who reported 114 μg/g in A. viridis and

Rajyalakshmi et al. (2003) with values ranging from 56 to 90 μg/g (Table 4.2).

Wills and Rangga (1996) found a lower value of 20 μg/g in A. tricolor. A number of factors influence the carotenoid concentration among species, including timing of collection, seasonality, climate, growing conditions, geographic location, varieties (genetic variation) and cultivars, and may explain the variation among results (Calvo, 2005; Kimura & Rodriguez-

Amaya, 2002; Liu, et al., 2007; Maiani, et al., 2009; Rodriguez-Amaya, 1999).

4.4.4 Cooked GLV

The means obtained for A. nodiflora, A. sessilis, A. cruentus, A. tricolor, A. viridis, C. argentea, C. album, D. muricata, H. cannabinus and R. vesicarius range from 58 to 175 μg/g for lutein and 40 to 159 µg/g of β-carotene in the cooked samples (Table 4.2).

Although no lutein values for cooked samples have been reported for the species of interest in this study, our results compare well with the USDA nutrient database (USDA, 2005) for boiled kale (Brassica oleracea var. acephala DC.), spinach (Spinacia oleracea L.), turnip (Brassica rapa subsp. rapa L.), collards (Brassica oleracea var. viridis L.), mustard (Brassica juncea

(L.) Czern.) and dandelion (Taraxacum officinale G.H. Weber ex Wiggers) greens, with reported lutein values of 182, 113, 84, 77, 60 and 47 μg/g (cooked weight basis). During the cooking process, at least two mechanisms contribute to the decrease, increase or lack of modification in the concentration of carotenoids, namely the disruption of the food matrix and the consequent release of water and/or carotenoids and the degradation of the heat-labile carotenoids (Calvo, 2005).

Values ranging from 27 to 67 µg/g for β-carotene reported for cooked species by Rajyalakshmi et al. (2003) are different than ours because they are expressed on fresh weight instead of cooked weight basis.

4.4.5 Contribution to total β-carotene and lutein intake

Daily intakes of β-carotene and lutein were calculated on the basis of 20 g fresh leaves portions and reported frequencies (Table 4.1). The selected species contribute to 1489 μg/day of β-carotene (980 μg from non-culivated

GLV) and 1788 μg/day of lutein (1186 μg from non-cultivated GLV). One-

way ANOVA analyses of the results obtained between cultivated and non- cultivated GLV showed no significant differences (p>0.05).

4.5 Discussion

Cataract is a condition affecting the transparency of the crystalline lens, is the main cause of blindness in India and affects particularly women from rural areas (Dandona, et al., 2001). In mammalian systems carotenoids originate exclusively from the diet. Lutein and zeaxanthin are the only carotenoids found in the human lens where their concentrations range between 15.1 to

44.1 ng/g of wet weight (Mares-Perlman, et al., 2002; Yeum, et al., 1999). In both the lens and the macula lutea the two xanthophylls are responsible for blue light absorption and antioxidant protection which are the proposed mechanisms for their protective role against cataract (Krinsky, et al., 2003).

A number of studies in Western countries have demonstrated an inverse relationship between leafy vegetable consumption, especially lutein- rich species like spinach and kale (USDA, 2005; Sommerburg, et al., 1998) and the risk of developing cataract (Brown, et al., 1999; Hankinson, et al.,

1992; Lyle, Mares-Perlman, Klein, Klein, & Greger, 1999; Mares-Perlman, et al., 1995; Moeller, et al., 2000; Moeller, et al., 2008). However, no such studies have previously been conducted in India. To demonstrate the preventive effect of leafy vegetable consumption in countries where cataract

is of high prevalence and occurs early in life, more data is needed on the carotenoid content of local foods. Increasing intake of leafy vegetables and associated carotenoids may constitute efficient strategies for promoting eye health and reducing the burden of cataract. In this study, we have determined the lutein and β-carotene concentrations of commonly consumed leafy vegetables of South Andhra Pradesh thus providing useful data to further evaluate the contribution of wild and cultivated plant foods to cataract prevention.

Nutritional surveys tend to overlook the contribution of wild foods,

GLV and varieties within species (Grivetti & Ogle, 2000; Kennedy, et al.,

2005), which may represent important sources of nutrients, including lutein and β-carotene. In our study, we found the carotenoid profile and contents of uncultivated leaves to be similar to the cultivated ones, confirming their important contribution to health and nutrition and supporting their inclusion in nutritional and epidemiological surveys. Wild A. viridis and commercially available H. cannabinus had similar content of lutein and β-carotene when fresh and cooked, and both compare well with globally available raw spinach

(122 and 56 μg/g lutein and β-carotene, respectively) (USDA, 2005). In addition, within the selected species the uncultivated leaves were more frequently consumed on a weekly basis than the commercially grown ones

(Table 4.1), thus contributing further to carotenoid intake.

On a daily basis, the reported consumption of the 10 selected GLV species provides 1489 μg of β-carotene, contributing over 40% of the daily intake recommended by WHO/FAO (2004). To date, there is no recommendation for lutein intake. The reported intake attributable solely to the selected species in this study is 1788 μg/day. In the United States, various studies with women populations reported daily lutein intakes of 1832, 1300,

1860, 4404 and 1232 µg respectively (Burke, et al., 2005; Chug-Ahuja, et al.,

1993; Michaud, et al., 1998; Nebeling, et al., 1997; Slattery, et al., 2000).

Johnson-Down et al. (2002) estimated that Canadian women (18-65 years old) consumed 1382 μg/day of lutein. Lutein intake in Europe was found to be 3250, 2500, 1590,1560 and 2010 μg/day in Spain, France, United Kingdom,

Ireland and the Netherlands, respectively (male and female intakes not statistically different and reported together) (O'Neill, et al., 2001). Recently,

Hamulka et al. (2009) found a population of Polish women to consume 2160

μg/day of lutein. In Asia, Zhang et al. (2007) estimated the lutein consumption of a Chinese women population to be 1810 μg/day. To our knowledge, there is no published data on the lutein intake of Indian populations. However when compared with reported daily intakes in various countries, the 10 species of interest in this studies contribute an important portion of the lutein consumed by the women of Madanapalle.

Olmedilla et al. (2003) reported improved visual acuity and glare sensivity in patients with cataract with approximately 7 mg of lutein supplementation/day. A portion of 100 g of either fresh A. viridis and H. cannabinus provides 14 mg of lutein. Moreover, 100 g of cooked C. album or

A. tricolor will each contribute 18 mg of lutein with substantially increased bioavailability due to the disruption of the food matrix resulting from the cooking process (Liu, et al., 2007; van het Hof, et al., 1999).

In conclusion, we have determined high content of lutein and β- carotene in cultivated and wild leafy vegetable species commonly consumed by the women in the Madanapalle mandal. To our knowledge, this study is the first to report lutein values for fresh C. argentea, R. vesicarius, D. muricata and A. cruentus and for all cooked species, and β-carotene values for cooked A. cruentus, C. album and R. vesicarius. The reported concentrations can be used directly to estimate the contribution of either fresh or cooked species to lutein and β-carotene intake. One drawback of this work is the single time collection during the peak availability period. Based on these results, the selected cultivated and wild leafy vegetables are equally important sources of β-carotene and lutein. In a country such as India where cataract incidence is very high and occurs early in life, identifying strategies to help reduce or delay the burden of cataract is of primary importance.

Increasing consumption and use of local GLV might be a valuable strategy.

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These data will be used in a population-based study to further evaluate the potential of leafy vegetables to contribute to cataract prevention in women from Madanapalle. This work considering the importance of local plant foods to health contributes to the initiative of the Convention on Biological

Diversity Decision VIII/23A to provide the evidence base for the implementation of global biodiversity conservation policy.

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Table 4.1: Cultivation status, botanical families, scientific and Telugu names, yearly availability and dietary intake among

Madanapalle women (n=100) of the selected leafy vegetable species.

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Cultivation status Botanical family Scientific name Telugu name English name Availabilitya Dietary intake (serv/pers/wk) Uncultivated Amaranthaceae Allmania nodiflora (L.) R. Br. Errabadaku NA Jul. ‒ Mar. 1.28 (1.30) Alternanthera sessilis (L.) R. Br. Ponnaganti aku Sessile joyweed Aug.-Jan. 0.64 (0.89) Amaranthus viridis L. Dantu aku Slender amaranth All year 0.77 (1.28) Celosia argentea L. Gurugu aku Silver cock's comb Jul.-Dec. 1.58 (1.27) Digera muricata (L.) Mart. Chenchali aku False amaranth All year 0.32 (0.61) Cultivated Amaranthus cruentus L. Thota aku Red amaranth All year 0.41 (0.84) Amaranthus tricolor L. Sirri aku Joseph's-coat All year 0.54 (0.78) Chenopodiaceae Chenopodium album L. Chakranta aku Lambsquarter All year 0.33 (0.68) Malvaceae Hibiscus cannabinus L. Gongura Brown Indian hemp All year 0.35 (0.67) Polygonaceae Rumex vesicarius L. Chukka aku Bladder dock All year 0.60 (0.79) a) According to Pullaiah et al. (1998) and our observations. b) Intake is expressed as mean servings/person/week with standard deviation.

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Table 4.2: Lutein and β-carotene content (µg/g) with standard deviation

(s.d.) of fresh and cooked leafy vegetables compared with values obtained from other studies reported in µg/g fresh weight.

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Fresh (µg/g fresh weight)a (s.d.) Cooked (µg/g cooked weight)a (s.d.) Leafy vegetables Lutein β-carotene Lutein β-carotene Uncultivated Allmania nodiflora This study 67 (14) 45 (10) 58 (15) 40 (10) Rajyalakshmi et al. (2003) - 56 - 27c Alternanthera sessilis This study 104 (25) 92 (26) 123 (46) 101 (31) Bhaskarachary et al. (1995) - 57 (16) - - Rajyalakshmi et al. (2003) - 83 - 36c Amaranthus viridis This study 140 (15) 119 (10) 151 (63) 124 (47) Kobori and Rodriguez-Amaya (2008) 119 (21) 114 (22) - - Tee and Lim (1991) 42 32 - - Bhaskarachary et al. (1995) - 11 (4) - - Rajyalakshmi et al. (2003) - 72 - 36c Celosia argentea This study 81 (11) 69 (8) 109 (18) 96 (2) Bhaskarachary et al. (1995) - 12 (2) - - Rajyalakshmi et al. (2003) - 60 - 34c Digera muricata This study 85 (17) 81 (19) 114 (18) 99 (16) Rajyalakshmi et al. (2003) - 90 - 67c

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Cultivated Amaranthus cruentus This study 92 (16) 76 (9) 143 (22) 115 (27) Amaranthus tricolor This study 103 (18) 96 (16) 175 (77) 153 (61) Tee and Lim (1991) 20 51 - - Wills and Rangga (1996) 29 20 - - Kidmose et al. (2006) 23 (6) 18 (6) - - Bhaskarachary et al. (1995) - 86 (30) - - Liu et al. (2007) 147b (7) - - - Rajyalakshmi et al. (2003) - 74 - - Isabelle et al. (2010) 23.55 - - - Chenopodium album This study 107 (18) 93 (9) 175 (70) 159 (50) Hibiscus cannabinus This study 143 (52) 107 (33) 82.36 121 (39) Rajyalakshmi et al. (2003) - 83 - 42c Rumex vesicarius This study 53 (1) 45 (1) 127 (14) 139 (31) Bhaskarachary et al. (1995) - 26 (3) - - a) Values are reported as the mean of samples from three different locations with standard deviation. b) Lutein only. c) Fresh weight basis.

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Figure 4.1: High Performance Liquid Chromatography chromatograms of

Allmania nodiflora (L.) R. Br. Ex Wight fresh (A) and cooked (B).

Chromatographic conditions are described in the text. Peak identification: 1: neoxanthin, 2: violaxanthin, 3: lutein, 4: chlorophyll b, 5: chlorophyll a, 6: β- carotene.

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Preface to Chapter 5

In Chapter 3 we identified the species of leafy vegetables consumed by female participants of an ethnobotanical study, and determined their availability, methods of preparations and cultivation status. Based on these results, we selected 10 GLV species for further analysis and found high contents of lutein/zeaxanthin and β-carotene (Chapter 4). In both ethnobotanical and laboratory studies, we have found evidence of the contribution elements of biological diversity, namely wild and cultivated leafy vegetables, make to human health in this study area.

Chapter 5 presents the results of a third component of this multidisciplinary work and evaluates within a female population the effect of leafy vegetable consumption and diversity, along with lutein/zeaxanthin intakes, to the prevention of age-related cataract. Potential risk factors are included to evaluate confounding, and other elements of the traditional diet are also examined. Broader patterns and findings emerging from these results and their integration in the light of the other studies are discussed further in Chapter 6.

The following chapter is a manuscript to be submitted for peer- reviewed publication. Participation of each author is described in the

Contributions of Authors section. Tables are presented at the end of this

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chapter and references are listed in the List of References. Additional information to this chapter is presented in Appendix C.

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5. Traditional foods and age-related cataract in women

of rural South Andhra Pradesh, India.

5.1 Abstract

Objective: To examine the possible relationship between elements of traditional diets, including leafy vegetables, lutein/zeaxanthin intake and dietary diversity within leafy vegetable intake, in a female population of rural southern India.

Design: We conducted an eye hospital-based case-control study where cases and controls were interviewed in person. A food-frequency questionnaire was used to record food intake.

Setting: Madanapalle, Andhra Pradesh, India, from September 2007 to

February 2008.

Subjects: Unmatched cases (n=200) and controls (n=258) were included in the analysis. Definite cataract was defined as nuclear opalescence >3.0 and/or cortical cataract >3.0 and/or PSC >2.0.

Results: Unconditional multiple logistic regression showed that age-related cataract was negatively associated with dairy food consumption, yogurt and tea intake (OR: 0.32, 95% CI: 0.11-0.95; OR: 0.52, 95% CI: 0.27-0.97 and

OR: 0.25, 95% CI: 0.10-0.65, respectively) after adjusting for age, energy, literacy, being vegetarian, use of tobacco products, number of children and

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cooking fuel type. The consumption of greens chutney, vegetables in general and carrots were associated with cataract prevention in age and energy adjusted models. Leafy vegetable intake in general, lutein/zeaxanthin and dietary diversity were not related to cataract.

Conclusions: For rural women of Madanapalle our results support a protective role of dairy foods, yogurt and tea against age-related cataract.

The use of wood as cooking fuel is a strong risk factor for cataract in this region.

5.2 Introduction

Age-related cataract is responsible for more than 40% of the world’s blindness and occurs principally in developing countries (WHO, 2005). In the state of Andhra Pradesh, 44% of blindness is attributable to cataract

(Dandona, et al., 2001). Risk of cataract increases, and use of eye care resources decreases with being female, rural resident and illiterate (Nirmalan,

Padmavathi, et al., 2003; Nirmalan, et al., 2004). To complement the ongoing efforts to increase access to high quality surgery, finding alternatives promoting prevention and delaying of cataract onset may have important impacts on the quality of life of populations at risk. Dietary approaches investigating the protective role of traditional diets, typically characterized by a greater dietary diversity and by the incorporation of wild and locally grown

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plant species (Johns, 2007; Kuhnlein & Receveur, 1996), have shown positive results in reducing vitamin A deficiencies in a rural South Indian population

(Schmid, et al., 2007; Schmid, et al., 2006), and are yet to be employed in the context of age-related cataract research (Bélanger & Johns, 2008).

Green leafy vegetables (GLV) are important elements of traditional diets and their consumption has been reported to contribute to lowering the risk of age-related cataract in occidental studies (Brown, et al., 1999; Chasan-

Taber, et al., 1999; Cumming, et al., 2000; Hankinson, et al., 1992; Lyle,

Mares-Perlman, Klein, Klein, & Greger, 1999; Mares-Perlman, et al., 1995;

Tavani, et al., 1996) and more recently in India (Tarwadi, et al., 2008).

Oxidative damage being an important feature of age-related cataract

(Spector, 1995; Truscott, 2005; Vinson, 2006), the xanthophylls lutein and zeaxanthin primarily found in GLV have been associated in India with preventive properties (Dherani, et al., 2008; Tarwadi, et al., 2008) although with conflicting results outside India (Brown, et al., 1999; Chasan-Taber, et al., 1999; Christen, et al., 2008; Delcourt, et al., 2006; Gale, et al., 2001;

Jacques, et al., 2001; Jacques, et al., 2005; Lyle, Mares-Perlman, Klein, Klein,

& Greger, 1999; Lyle, Mares-Perlman, Klein, Klein, Palta, et al., 1999;

Mares-Perlman, et al., 1995; Maresperlman, et al., 1995; Moeller, et al., 2008;

Olmedilla, et al., 2003; Richer, et al., 2004; Taylor, et al., 2002; Vu, et al.,

2006). We previously reported high consumption, diversity and

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lutein/zeaxanthin content of wild and cultivated GLV in the study area

(Chapter 3 and Chapter 4). The present eye hospital-based case-control study further examines the possible relationship between dietary factors, including leafy vegetables and lutein/zeaxanthin intake and dietary diversity, and age-related cataract in a female population of rural southern India.

5.3 Experimental methods

5.3.1 Study design

The present eye hospital-based case-control study was conducted at the

Siloam Eye Hospital, Madanapalle, Chittoor District, affiliated with the L.V.

Prasad Eye Institute, Hyderabad, Andhra Pradesh, India, between

September 2007 and January 2008 during the rainy and winter seasons. The study area is a rural drought-prone region, with a population mainly of low socio-economic status. Ethics approval was obtained from the Human

Subjects Ethics committee of McGill University and the Human Research

Project Review Board of the L.V. Prasad Eye Institute, Hyderabad, Andhra

Pradesh.

5.3.2 Participants

Cases and controls were identified at the Siloam Eye Hospital within current patients or accompanying persons. The participants resided in Madanapalle

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or surroundings. Cases were consenting female patients of 30 years old and above diagnosed with current, definite age-related cataract according to the

Lens Opacity Classification System III (LOCS III) as identified with a slit- lamp biomicroscope examination (Chylack, et al., 1993). Cases were selected when presenting a LOCS III nuclear opalescence of 3.0 and/or cortical of 3.0 and/or posterior subcapsular (PSC) of 0.2 or mixed cataract (any combination of these) in at least one eye (Nirmalan, Krishnadas, et al., 2003).

Controls were included when identified with LOCS III nuclear score less than 2, and PSC and cortical 0. Candidates were tested for the following exclusion criteria: diabetes, intraocular pressure of 21mm Hg, glaucoma, and congenital or traumatic cataract. Each selected participant was invited to complete a 7-day food frequency questionnaire administered by trained

Indian research assistants fluent in Telugu, Hindi, Urdu and English.

Participation was on a voluntary basis and written consent was obtained before interviews. A total of 479 candidates participated to the study.

5.3.3 Dietary data collection

Interviews were conducted in the participants’ language, and included information on the following demographic characteristics and potential risk factors exposure: age, occupation, literacy, religion, vegetarianism, land holding, type of house, number of children, home garden or horticultural

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land ownership, daily time spent outdoors, use of protective eye or headwear and main cooking fuels (Leske, et al., 1991; Mohan, et al., 1989; Nirmalan, et al., 2004; Pokhrel, et al., 2005; Ughade, et al., 1997).

A 7-day food frequency questionnaire (FFQ) has been chosen as the instrument for measuring the dietary intake (Appendix C). It is inexpensive and brief to administer comparatively to multiple 24-hour recalls. According to Resnicow et al. (2000), the correlations of fruit and vegetable servings with specific and total serum carotenoid levels are generally higher for a seven day food frequency questionnaire than with the other dietary assessment tools

(monthly FFQ, diet history). Items to be included in the FFQ were identified with 78 24-hour food recalls (Appendix C) conducted on a selected subsample of households in the study site at the onset of the project to estimate usual dietary intakes and portion sizes in the female population. The person who cooks, generally the wife or a female sibling, was asked to describe each food and drink she cooked the previous day, meal by meal. She was asked to report quantities of each ingredient using standardized cups and spoons. The usual portion (g) of the respondent was calculated as follow: each raw ingredient (in g) of a recipe / (number of people eating x number of meals).

The portion was then adjusted, if necessary, when the respondent’s portion was smaller/bigger than the others’ portions. In other cases, the usual portion

(g) of the respondent was calculated as follow: each raw ingredient (in g) of a

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recipe x the cooked volume of the respondent portion / total cooked volume of the dish. These data in combination with the results of an ethnobotanical study on the wild, weedy and cultivated leafy vegetables consumed in the area provided a basis for constructing the FFQ (Chapter 3).

The FFQ comprised 81 items grouped in 9 food groups, grains and cereals, pulses and nuts, leafy vegetables, root vegetables, other vegetables, fruits, meats and eggs, dairy foods and sweets. The number of servings per day and the number of days per week consuming the item were recorded, along with recipe and source (purchased, produced, gathered) (for GLV species). The study was conducted during the availability seasons for GLV

(rainy and winter). Specific intakes were calculated by multiplying reported frequency and average portion size as identified with the 24-hour recalls

(expressed as weights in grams) and reported on a daily basis.

Energy intakes were computed using the Nutritive Value of Indian

Foods (Gopalan, et al., 2004), based on individual ingredients of each recipe and calculated portions. Lutein and zeaxanthin intakes were calculated together using our results from previous analysis of local leafy vegetables

(Bélanger, et al., in press) (Chapter 4), and completed with USDA databases,

Su et al., Kidmose et al., Tee and Lim, Kopsell et al., and Hart and Scott

(USDA, 2005; Hart & Scott, 1995; Kidmose, et al., 2006; Kopsell, et al., 2007;

Su, et al., 2002; Tee & Lim, 1991) (content reported for lutein and

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zeaxanthin together). A Food Variety Score (FVS) was computed for GLV items by adding the number of different species consumed (>0.1g) over a 7- day period. A dietary diversity score (DDS) was calculated for the number of food groups consumed during the 7-day period (Hatloy, et al., 1998). All calculations were computed using custom Perl (version 5.8.6) scripts and the

R statistical software version (2.9.0).

5.3.4 Statistical analysis

Demographic characteristics and potential risk factors for cataract were compared using Mann-Whitney U test for non-normally distributed continuous variables and chi-square ( " 2) test for categorical variables. The associations of demographic characteristics and potential risk factors with ! cataract were measured fitting unadjusted and age-adjusted unconditional logistic regressions. The dietary factors variables were categorized into quintiles according to the population distribution. The lowest quintile of intake was used as the reference category. Odds ratios (OR) and 95% confidence intervals (CI) were calculated adjusting for age and energy intake in a first model (model I). The potential confounders literacy, fuel type, being vegetarian, use of tobacco products, number of children (transformed into a quadratic term since the assumption of linearity in the logit was not respected) and use of eyewear were added to age and energy intake in a

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second model (model II). Linear trend across categories was computed introducing the observed median of each quintile as a continuous variable in the model. The null hypothesis of no linear trend across quintile was tested using Wald " 2 test, as was the statistical significance of continuous variables and use/non-use categories. Assumption of linearity in the logit was verified ! for continuous variables (Hosmer & Lemeshow, 2000). We considered as statistically significant a p value of 0.05. The SAS statistical software 9.2 (SAS

Institute, Cary, USA) was used for all statistical analyses.

5.4 Results

5.4.1 General characteristics of the subjects

A total of 479 women were interviewed; 16 questionnaires were not completed or contained missing data, and thus 463 candidates were considered in the analysis. We identified 5 suspect reports of dietary intakes after analysis of the residuals, and the corresponding candidates were removed from the analysis, resulting in 200 cases and 258 controls.

Demographic characteristics and potential risk factors for cataract are presented in Table 5.1. In this population cases were significantly older than controls and after adjusting for age, odds for cataract decreased with being literate (OR: 0.50, 95% CI: 0.29-0.86, p=0.0122), being lacto-ovo-vegetarian versus non-vegetarian (OR: 0.35, 95% CI:0.15-0.77, p=0.0098), using gas

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instead of wood as cooking fuel (OR: 0.32, 95% CI: 0.18-0.57, p=0.0001) and wearing protective eyewear (OR: 0.44, 95% CI:0.25-0.77, p=0.0043). The other potential risk factors (energy intake, time spent outdoors, number of children, occupation, use of headwear), after adjusting for age, showed no relation with cataract.

5.4.2 Dietary intakes

In Table 5.2 we report the odds ratios of cataract associated with food groups, lutein/zeaxanthin, diversity in the GLV intake (FVS) and dietary diversity score (DDS) adjusted in model I for age and energy, and further adjusted for literacy, eyewear, dietary patterns, cooking fuel type and use of tobacco products in model II. Odds ratio for cataract associated with specific food items are presented in Table 5.3. Participants with the highest consumption of dairy products, including milk, buttermilk and yogurt, had lower odds for cataract, and the relation was maintained after adjusting for all other factors

(OR: 0.32, 95% CI: 0.11-0.95; p for trend=0.0159). Consumption of all vegetable groups (leafy, roots and other) showed an inverse association with cataract in model I (OR: 0.23, 95% CI: 0.08-0.72; p for trend=0.0286).

However, the association was lost when adjusting for other potential confounders. A borderline positive association was observed for the variety in the GLV food group (FVS) when introduced as a continuous variable in the

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in the fully adjusted model (OR: 1.16, 95% CI: 0.99-1.346, p value= 0.0630).

We found little evidence of association with cataract in the other food groups, including GLV intake, DDS, and lutein/zeaxanthin.

Among specific food items, consuming >200 ml of tea was inversely associated with cataract in both models (model II: OR: 0.25, 95% CI: 0.10-

0.65, p for trend=0.02). Participants consuming yogurt were less likely to have cataract in both age and energy intake and further adjusted models

(model II: OR: 0.52, 95% CI: 0.27-0.97, p= 0.0394). In fully adjusted models, consuming drumstick leaves (Moringa oleifera Lam.), sirriaku (Amaranthus tricolor L.) and lambsquarter (Chenopodium album L.) was positively associated with cataract (OR: 2.94, 95% CI: 1.23-7.05, p=0.0156, OR: 2.89,

95% CI: 1.11-7.50, p=0.0293 and OR: 5.32, 95% CI: 0.99-28.34, p=0.0504, respectively). In age and energy intake adjusted models, consumption of greens chutney and carrots was inversely associated with cataract (OR: 0.34,

95% CI: 0.13-0.89, p=0.0276 and OR: 0.44, 95% CI: 0.25-0.80, p=0.0069), however the associations were lost in further adjusted models. The analysis of coriander (Coriandrum sativum L.) and curry (Murraya koneigii (L.)

Spreng.) leaves as continuous variables per 10g units showed borderline associations in age and energy intake adjusted models (OR: 0.85, 95% CI:

0.71-1.00, p=0.0529 and OR: 0.84, 95% CI: 0.69-1.01, p=0.0634). Other food items did not show significant relationship with age-related cataract.

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5.5 Discussion

Traditional food systems characteristically incorporate locally available foods of plant and animal origin, are high in species variety and have rich nutrient sources (Kuhnlein & Receveur, 1996; Tontisirin, et al., 2002). Wild and cultivated leafy vegetables in particular are free, locally gathered or cultivated and diversified sources of nutrients and antioxidants of potential interest to eye health, including lutein and zeaxanthin of which they are primary sources (Mangels, et al., 1993b; Sommerburg, et al., 1998). In this population we found associations between age-related cataract and elements of traditional diets of the women from rural Madanapalle, South Andhra

Pradesh, namely with tea, yogurt and dairy foods in fully adjusted models.

However, both positive and negative associations were observed between leafy vegetables and age-related cataract.

Among women of this study population, drinking >200 ml of black tea daily reduced the odds of cataract by 75% when comparing highest to lowest quintile of intake (OR: 0.25, 95% CI: 0.10-0.65, p for trend=0.02). Our findings are in agreement with animal studies (Thiagarajan, et al., 2001), along with a Canadian case-control study (Robertson, et al., 1989), in which drinking five or more cups of tea per day reduced odds of cataract by 61%

(OR = 0.39, p=0.02). However Indian and Italian based studies did not support a protective role for tea consumption (Tarwadi, et al., 2008; Tavani,

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et al., 1996). In this study population, we found dairy food group consumption to reduce significantly the odds of cataract dairy products (OR:

0.32, 95% CI: 0.11-0.95; p for trend=0.0159), which supports Pohkrel et al.

(2005) reduced odds of cataract with drinking a glass of milk daily. Within dairy foods, patients in the highest quintile of yogurt intake were less likely to have cataract, and the relation was maintained after all adjustments (OR:

0.52, 95% CI: 0.27-0.97, p=0.0394). This is a first report of a protective role of yogurt against age-related cataract, in contrast with other Indian studies reporting either positive or no association (Chatterjee, et al., 1982; Tarwadi, et al., 2008).

In our study cases and controls consumed elevated quantities of GLV

(58 ± 36 g and 72 ± 38 g daily respectively), however no significant difference was observed between cases and controls after adjusting for age and energy intake and for further potential confounders (p=0.8834).

Tarwadi et al. (2008) found higher intake of GLV in Indian controls compared to patients (p < 0.001), although male cases accounted for lower intakes. Female cases and controls did not differ significantly in their GLV intake. In Western female populations, reduced odds for cataract were significantly associated with intake of leafy vegetables (Chasan-Taber, et al.,

1999; Hankinson, et al., 1992; Mares-Perlman, et al., 1995; Tavani, et al.,

1996), and borderline significant associations in protective directions were

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recently reported (Christen, et al., 2008; Moeller, et al., 2008). Considerable disparities in the lifestyles of Indian and Western populations may in part explain these different results.

Our findings did not support a protective role of lutein/zeaxanthin consumption, nor dietary diversity in leafy vegetable intake.

Lutein/zeaxanthin intakes were high in both groups (3.56 ± 2.02 mg in cases and 3.77 ± 1.83 mg in controls). By comparison, U.S. women consume between 1.23 and 1.86 mg of lutein daily (Burke, et al., 2005; Chug-Ahuja, et al., 1993; Michaud, et al., 1998; Nebeling, et al., 1997; Slattery, et al., 2000) and Canadian women 1.38 mg daily (Johnson-Down, et al., 2002). Tarwadi et al. (2008) reported lower lutein/zeaxanthin in the highest quintile of intake relative to the lowest quintile (p=0.03), but the relation did not remain statistically significant after adjusting for other nutrients. On the opposite,

Dherani et al. (2008), found an inverse relation in serum zeaxanthin and cataract in models adjusted for age, sex, smoking, BMI, and average systolic blood pressure, when comparing highest tertile to the lowest (OR: 0.66, 95%

CI: 0.45‒0.96, p=0.04). They also found lower odds ratio in adjusted analysis when doubling the natural log of the serum lutein level (OR: 0.73, 95% CI:

0.60‒0.89, p=0.01).

A further analysis of individual GLV species and preparations revealed different trends toward both protection and risk increase. Curry and

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coriander leaves are consumed on a daily basis by 95% and 88% of the respondents, respectively, and are included in most vegetable and meat preparations for flavoring and garnishing. Coriander is one of few leaves mostly eaten uncooked (Chapter 3). Continuous analysis of these plant species showed a borderline significant inverse association with increasing intakes in age and energy adjusted models (OR: 0.85, 95% CI: 0.71-1.00, p=0.0529 and OR: 0.84, 95% CI: 0.69-1.01, p=0.0634 for each 10g increase of coriander and curry intake, respectively). The association was lost when adjusting with other risk factors in model II. Similarly, consuming greens chutney reduced the odds of cataract after adjusting for age and energy intake (OR: 0.34, 95% CI: 0.13-0.89, p=0.0276), but the association did not remain in further adjusted models.

On the contrary, participants consuming drumstick, sirriaku and lambsquarter leaves, which are generally uncultivated, were more likely to have age-related cataract in fully adjusted models (OR: 2.94, 95% CI: 1.23-

7.05, p=0.0156, OR: 2.89, 95% CI: 1.11-7.50, p=0.0293 and OR: 5.32, 95%

CI: 0.99-28.34, p=0.0504, respectively). Large CI indicates imprecision and may be due to the small numbers of participants consuming drumstick (12%), sirriaku (11%) and lambsquarters (5%). With no known direct relation between the consumption of any leafy vegetable species and the increased age-related cataract, the observed risk increase could reflect other aspects of

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diet or lifestyle that differ in participants that are unknown and unmeasured.

One plausible explanation lies in the socioeconomic status of women collecting uncultivated plant species for food, since these 3 species are mostly gathered in and around fields and neighborhood. Socio-economic status refers not only to financial conditions but also unhygienic living conditions, poor sanitation, and other potential risk factors. We adjusted for literacy, but recognize that this would have only partly controlled for the complexities of socioeconomic class. In addition, our results of an ethnobotanical study conducted with local women (Bélanger et al., submitted) (Chapter 3) indicate that drumstick is sometimes consumed as a leafy vegetable to prevent or treat eye diseases in general, which could explain in part the higher consumption of these leaves by cases in this study. Other potential factors not accounted for include exposure to pesticides, for which no risk evaluation exists. To date, no studies on the effect of wild vs. cultivated foods on cataract have been conducted. Still, interventions increasing the consumption of traditional foods, including leafy vegetables in Andhra Pradesh, ameliorated the nutritional status of Dalit mothers and decreased vitamin A deficiencies

(Schmid, et al., 2007; Schmid, et al., 2006).

Combining leafy, root and other vegetables, a reduction of the odds for cataract was observed in age and energy intake adjusted models (OR:

0.23, 95% CI: 0.08-0.72; p for trend=0.0286). Similarly, carrot consumption

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had significant protective odds ratios in the age and energy intake adjusted model (OR: 0.44, 95% CI: 0.25-0.80, p=0.0069). When adding the potential confounders included in model II one at a time, the type of cooking fuel used was responsible for the loss of statistical significance in all cases. Indeed, the type of fuel did have a strong significant effect on cataract in the study population (OR: 0.32, 95% CI: 0.18-0.57, p=0.0001), in accordance with previous Indian findings (Mohan, et al., 1989; Pokhrel, et al., 2005; Saha, et al., 2005; Sreenivas, et al., 1999; Ughade, et al., 1998). It would thus be recommended to include this important risk factor in nutritional studies on cataract in regions where wood and other cheap fuels are used for cooking.

By including this risk factor, as opposed to Tarwadi et al. (2008), chances of observing statistically significant associations that are due to other confounding factors are reduced.

Possible limitations to our eye hospital-based case-control study warrant attention. The two major weaknesses of case-control studies are the biases both in selecting the cases and controls and in retrospectively measuring the dietary exposure (Hulley, et al., 2007). In addition, unknown confounders can be responsible for the observed effects. The overwhelming influences of underlying socioeconomic, genetic or environmental factors not accounted for may contribute to the observed (or lack of) association between variables of interest and age-related cataract. The size of our sample

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may not have been large enough to detect potential small but significant protective effect of the variables of interest. Likewise, we made multiple statistical comparisons and so some findings could be due to chance.

In summary we found protective associations with consumption of tea, dairy foods and yogurt with age-related cataract in a rural women population in South Andhra Pradesh. Although some species were moderately associated with reduced and increased risks, we found little evidence of a relation between leafy vegetables, dietary diversity or lutein/zeaxanthin intake and age-related cataract. In agreement with previous studies, the choice of cooking fuel was found to have a strong influence on development of age-related cataract.

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Table 5.1: Demographic characteristics and potential risk factors for cataract in the study population: hospital-based, case‒ control study on intake of green leafy vegetables and age-related cataract risk, Madanapalle, 2007-2008

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Characteristic Case (n 200) Control (n p value Crude OR (95% CI) Age-adjusted OR (95% CI) p value 258) (age-adjusted) Age (years) Mean 59.1 42.0 †0.0001 1.22 (1.18-1.26) - - SD 9.13 8.43 - - Daily energy intake (kcal) Mean 1990 2128 †0.0082 0.72 (0.54-0.96)* 0.75 ( 0.48-1.17)* 0.1976 SD 674 636 Literacy (%) Yes 28.5 55.4 ‡0.0001 0.25 (0.17-0.37) 0.50 (0.29-0.86) 0.0122 Parity (no. of children) Mean 3.53 2.31 †0.0001 2.03 (1.43-2.86)** 0.94 (0.58-1.52)** 0.8063 SD 2.35 1.09 Use of tobacco products (%) Yes 55.0 27.1 ‡0.0001 3.28 (2.22-4.85) 1.53 (0.89-2.65) 0.1276 Religion (%) Hindu 80 82.2 ‡0.7433 1.00 1.00 Muslim 16.5 13.9 1.22 (0.73-2.03) 1.83 (0.90-3.76) 0.0975 Christian 3.5 3.9 0.93 (0.35-2.49) 0.30 (0.07-1.25) 0.0979 Dietary patterns (%) Vegetarian 12.5 16.7 ‡0.2136 0.71 (0.42-1.22) 0.35 (0.15-0.77) 0.0098 Housing (n respondents)

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Modern 76.0 90.3 ‡0.0001 0.34 (0.20-0.57) 0.48 (0.23-1.01) 0.0537 Land holding (%) Yes 44.0 44.6 ‡0.9024 0.97 (0.67-1.42) 0.76 (0.44-1.29) 0.3068 Home garden (%) Yes 4.0 9.3 ‡0.0273 0.41 (0.18-0.93) 0.33 (0.10-1.06) 0.0616 Land for horticulture (%) Yes 9.5 10.85 ‡0.6361 0.86 (0.47-1.60) 1.42 (0.56-3.58) 0.4601 Occupation (%) Agriculture vs. other 73.5 76.4 ‡0.4831 0.86 (0.56-1.31) 0.69 (0.37-1.29) 0.2470 Daily hours outdoor Mean 2.36 2.21 †0.9056 1.01 (0.96-1.06) 1.05 (0.97-1.13) 0.2424 SD 3.69 3.60 Use of eyewear (%) Yes 45.0 41.5 ‡0.4495 1.16 (0.80-1.67) 0.44 (0.25-0.77) 0.0043 Use of headwear (%) Yes 27.5 24.0 ‡0.3985 1.20 (0.79-1.83) 1.25 (0.67-2.31) 0.4877 Cooking fuel (%) Wood 58.5 27.1 ‡0.0001 1.00 1.00 Gas 27.5 63.2 0.20 (0.13-0.31) 0.31 (0.17-0.56) 0.0001 Other 14.0 9.7 0.67 (0.36-1.24) 1.37 (0.58-3.24) 0.4761 †Mann-Whitney and ‡ " 2 tests. * Odds ratio for each unit increase (unit = 1000 kcal). ** A quadratic term was created with the number of children variable since the assumption of linearity in the logit was not respected. !

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Food intake Q1 Q2 Q3 Q4 Q5 p trend Grains and cereals (g) <102 102-141 142-199 200-377 >377 OR† 1.00 0.71 (0.30-1.68) 0.70 (0.30-1.64) 1.12 (0.47-2.62) 1.65 (0.68-3.98) 0.0684 OR‡ 1.00 0.70 (0.28-1.75) 0.72 (0.29-1.74) 0.89 (0.36-2.19) 0.75 (0.28-2.03) 0.8099 Pulses and nuts (g) <72 72-122 123-174 175-239 >239 OR† 1.00 0.54 (0.23-1.28) 0.68 (0.28-1.68) 0.71 (0.27-1.82) 0.57 (0.20-1.62) 0.5301 OR‡ 1.00 0.58 (0.23-1.44) 0.67 (0.26-1.71) 0.85 (0.31-2.35) 0.65 (0.22-1.94) 0.7387 GLV (g) <30 30-50 51-80 81-96 >96 OR† 1.00 1.28 (0.54-3.05) 0.69 (0.29-1.63) 0.66 (0.28-1.55) 0.62 (0.24-1.61) 0.1276 OR‡ 1.00 1.45 (0.59-3.60) 1.10 (0.44-2.76) 1.10 (0.42-2.86) 1.28 (0.44-3.78) 0.8834 Root vegetables (g) <4 4-14 15-22 23-45 >45 OR† 1.00 0.43 (0.17-1.08) 1.42 (0.66-3.05) 0.75 (0.32-1.77) 0.77 (0.31-1.91) 0.6336 OR‡ 1.00 0.50 (0.19-1.36) 1.96 (0.86-4.48) 1.24 (0.49-3.13) 1.41 (0.55-4.13) 0.3842 Other vegetables (g) <146 146-191 192-256 257-344 >344 OR† 1.00 1.05 (0.45-2.44) 0.60 (0.24-1.49) 0.62 (0.25-1.55) 0.37 (0.13-1.04) 0.0407 OR‡ 1.00 0.89 (0.36-2.21) 0.54 (0.21-1.41) 0.92 (0.35-2.45) 0.57 (0.18-1.76) 0.5234 Fruits (g) <15 15-50 51-113 114-232 >232 OR† 1.00 0.79 (0.34-1.85) 1.51 (0.64-3.56) 0.64 (0.26-1.54) 0.70 (0.25-1.95) 0.3283 OR‡ 1.00 1.49 (0.59-3.80) 2.81 (1.07-7.37) 1.34 (0.50-3.58) 1.83 (0.58-5.76) 0.7476 Meats and eggs (g) <6 6-14 15-28 29-65 >65

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OR† 1.00 1.29 (0.49-3.37) 2.05 (0.91-4.62) 0.82 (0.39-1.75) 1.30 (0.59-2.86) 0.7596 OR‡ 1.00 0.88 (0.29-2.63) 1.40 (0.57-3.48) 0.71 (0.29-1.74) 1.12 (0.44-2.81) 0.8945 Dairy foods (g) <114 114-257 258-471 472-600 >600 OR† 1.00 0.72 (0.31-1.71) 0.34 (0.14-0.85) 0.26 (0.10-0.65) 0.27 (0.10-0.69) 0.0007 OR‡ 1.00 0.86 (0.34-2.16) 0.45 (0.17-1.19) 0.39 (0.14-1.11) 0.32 (0.11-0.95) 0.0159 Sweets* (g) 0 >0 OR† 1.00 1.17 (0.65-2.09) - - - 0.6109 OR‡ 1.00 1.33 (0.71-2.46) - - - 0.3716 Lutein+zeaxanthin (mg) <2.04 2.04-2.92 2.93-3.77 3.78-5.15 >5.16 OR† 1.00 0.49 (0.20-1.18) 0.54 (0.32-1.26) 0.96 (0.40-2.33) 0.65 (0.24-1.75) 0.7944 OR‡ 1.00 0.51 (0.20-1.30) 0.73 (0.30-1.81) 1.53 (0.58-4.07) 0.93 (0.31-2.79) 0.5891 GLV diversity (FVS) <3 3 4 5-6 >6 OR† 1.00 0.99 (0.43-2.27) 0.74 (0.31-1.74) 1.60 (0.66-3.87) 1.12 (0.38-3.30) 0.5802 OR‡ 1.00 1.28 (0.53-3.06) 1.15 (0.45-2.95) 2.72 (1.02-7.22) 1.68 (0.51-5.53) 0.2334 Total vegetables (g) <206 206-271 272-362 363-461 >461 OR† 1.00 0.47 (0.19-1.10) 0.47 (0.19-1.18) 0.44 (0.17-1.16 0.23 (0.08-0.72) 0.0286 OR‡ 1.00 0.61 (0.25-1.51) 0.58 (0.22-1.51) 0.93 (0.33-2.64) 0.58 (0.17-1.98) 0.7499 DDS (score) >8 8 <8 OR† 1.00 1.30 (0.67-2.52) 1.14 (0.54-2.41) - - 0.7117 OR‡ 1.00 1.62 (0.78-3.35) 1.54 (0.64-3.75) - - 0.3115

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†Adjusted for age and energy intake ‡Further adjusted for dietary patterns, use of eyewear and cooking fuel type, literacy, use of tobacco products and number of children (quadratic term). * Because of low proportion of participants consuming and non-linearity of the logit, categories based on presence or absence of consumption during the 7-day period were compared using chi-square tests.

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Food intake OR† OR‡ Tea (ml) <200 1.00 1.00 200 0.69 (0.38-1.24) 0.81 (0.43-1.56) >200 0.25 (0.11-0.59) 0.25 (0.10-0.65) p trend 0.0038 0.0200 Yogourt Consuming vs not 0.39 (0.22-0.70) 0.52 (0.27-0.97) p value* 0.0016 0.0394 Greens chutney Consuming 0.34 (0.13-0.89) 0.48 (0.17-1.37) p value* 0.0276 0.1714 Carrot Consuming 0.44 (0.25-0.80) 0.73 (0.38-1.41) p value* 0.0069 0.3497 Drumstick leaves Consuming 2.63 (1.13-6.10) 2.94 (1.23-7.05) p value* 0.0242 0.0156 Sirri aku (A. tricolor) Consuming 2.01 (0.85-4.78) 2.89 (1.11-7.50) p value* 0.1142 0.0294 Lambsquarter (C. album) Consuming 6.75 (1.50-30.37) 5.32 (0.99-28.34) p value* 0.0128 0.0504 Coriander (10g)** Continuous OR 0.845 (0.713-1.002) 0.966 (0.793-1.178) p value* 0.0529 0.7360 Curry leaves (10g)** Continuous OR 0.836 (0.691-1.010) 0.932 (0.755-1.150) p value* 0.0634 0.5103 †Adjusted for age and energy intake ‡Further adjusted for dietary patterns, use of eyewear and cooking fuel type, literacy, use of tobacco products and number of children (quadratic term). * p values are taken from the corresponding chi-square tests. ** Odds ratios are calculated for each 10g unit increase.

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6. General discussion

The general objective of this doctoral research was to investigate, using a multidisciplinary research approach, the contribution of plant diversity to human health, specifically of wild and cultivated leafy vegetables towards age-related cataract prevention. The components of this thesis focused on the determinants of leafy vegetable consumption by women in Madanapalle, rural South Andhra Pradesh, India, on the carotenoid content of local species, and on protective aspects of GLV consumption towards age-related cataract, as evaluated in an eye hospital-based case-control study.

6.1 Verification of hypotheses

We tested the first hypothesis that perceived health benefits, along with cultivation status, influence the choice and consumption of leafy vegetables in the study area. We found significant differences in the frequency of consumption of species according to whether or not they were considered good for general health (folk functional foods) or for specific conditions

(food medicines). Differences were also found in the frequency of consumption and cultivation status of GLV species. In this ethnobotanical study, we thus confirmed our hypothesis and collected the background

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information needed to insure the cohesion of the other components of this work.

We report in Chapter 4 the results of a High Performance Liquid

Chromatography analysis of 10 selected local leafy vegetable species for lutein/zeaxanthin and β-carotene content. The hypothesis that fresh and cooked leafy vegetables, in the study area, would exhibit concentrations of lutein/zeaxanthin and β-carotene comparable with common cultivated species was confirmed for most of the species. No significant difference was observed between the carotenoid content of wild and commercially grown species. These findings support the general idea that uncommon and wild vegetables are valuable sources of micronutrients and other phytochemicals

(Gopalan & Tamber, 2003; Grivetti & Ogle, 2000; Heywood, 1999), and their inclusion in nutritional epidemiology studies to evaluate their contribution to health.

In Chapter 5 we tested associations between various elements of traditional diets and age-related cataract in female participants, using as background the information on GLV species, cooking and gathering as presented in Chapter 3, and lutein/zeaxanthin content as determined in

Chapter 4. Our third hypothesis of the negative association between leafy vegetable intake and age-related cataract remained unconfirmed in the study

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population. However, conflicting results were obtained when comparing the intakes of individual GLV species and preparations.

We questioned whether the diversity of GLV intake was associated with age-related cataract, with controls expected to consume a higher number of different GLV species than cases. We did not observe differences in the diversity score between cases and controls. We further hypothesized the lutein/zeaxanthin intakes (mg) to be inversely related to age-related cataract. In this study, we did not find statistically significant associations between lutein/zeaxanthin and age-related cataract. Adjusting for the type of fuel used for cooking changed the estimates of effect, in agreement with previous reports (Mohan, et al., 1989; Pokhrel, et al., 2005; Saha, et al., 2005;

Sreenivas, et al., 1999; Ughade, et al., 1998). This important risk factor therefore has to be included in studies related to cataract. In the point of view of biological diversity, decreasing age-related cataract prevalence could represent an important incentive to reduce the harvest and use of local woody plants as cooking fuel, thus promoting their conservation.

The retrospective nature of the exposure recorded, the sample size, the overwhelming effect of cooking fuel type or other unidentified potential confounders not accounted for in a developing country research context may in part explain the lack of association observed. Indeed, the Madanapalle population is subject to difficult drought conditions, heavy agricultural

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workload, extended sun exposure, malnutrition and precarious living conditions among other potential factors.

6.2 General considerations

In the multidisciplinary framework of this research project, we combined clinical data and measures of access and consumption of GLV in laboratory, community, and population research in relation to cataract. The common contributions of the disciplines involved, namely ethnobotany, nutrition, phytochemistry and epidemiology, participated to overcome limitations of individual fields alone. At the center of this work, the ethnobotanical study identified and described determinants of GLV consumption, thus providing a foundation for the elaboration of complementary studies. With the documentation of plant uses and patterns, hypotheses emerged on the activity of plant foods and their potential benefits to eye health. The analytical evaluation of compounds of interest, lutein and zeaxanthin, provided a quantifiable basis for their evaluation in a retrospective population-based study. Drawing on both ethnobotanical and analytical data, the epidemiological study evaluated plant foods normally overlooked in nutritional surveys, nonetheless showing important content of potential bioactive compounds. The integration of complementary studies allowed a better global understanding of a complex multidisciplinary problem.

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Interesting findings arose from the integration of data, for example,

Moringa oleifera Lam., found mostly in neighbourhoods, was reported as a highly valued species consumed as a vegetable for general and specific health properties, including ophthalmic attributes. In the case-control study, consumption of this species was positively associated with age-related cataract; however, this unexpected association is suspected to reflect other factors such as socio-economic status. Alternately, its perceived eye health properties may encourage women with cataract to consume it more frequently. Nonetheless, its β-carotene content (66.3 ± 7.8 µg/g) (Kidmose, et al., 2006) coupled with high popularity, make this widely available GLV a contributor of interest for the alleviation of vitamin A deficiency and related eye conditions. Likewise, its blood tonic and other attributed health properties are also confirmed in in vitro studies.

In the case-control study, however, observed trends for individual

GLV species and preparations encourage further studies and calls for innovative research strategies, specifically designed for the local socioeconomic, health and environmental conditions. Madanapalle GLV species contained excellent sources of lutein/zeaxanthin and β-carotene, carotenoids with promise in cataract and age-related macular degeneration, but also other conditions, such as cardiovascular diseases, skin and other cancers

(Maiani, et al., 2009; Ribaya-Mercado & Blumberg, 2004) and women were

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found to consume high quantities of lutein/zeaxanthin. The overall design of this thesis project enabled an exploratory analysis of the multiple components involved in complex health and biological diversity relations.

This approach appraises complexity and addresses important health and environment issues specifically adapted to local context.

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7. Future research directions

Interesting directions for future research arising from the present studies include:

1. Expanded ethnobotanical investigation to measure the sustainability

and ecological impacts of wild and uncultivated plant collections.

2. Intervention studies supporting home garden implementation in

willing households, measuring baseline and post-intervention

nutritional and health status, to determine their impact on health,

improve accessibility of local fruits and vegetables and contribute to

the conservation of genetic resources.

3. Evaluation of the effect of altitude and climate effects on carotenoid

content.

4. Re-evaluation of the observed borderline associations with cataract of

specific plant species and preparations with larger sample size

matched for the type of cooking fuel used.

5. Further evaluation of the mechanisms of action of tea, dairy foods and

yogurt.

6. Application of multidisciplinary frameworks to demonstrate other

relations between biological diversity and human health.

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8. Contributions to knowledge

8.1 Overall contributions to science

The overall multidisciplinary design of this work, by generating results reflecting the complex biological, socio-economic and environmental components of age-related cataract and leafy vegetable diversity, presents a model in which deeper knowledge emerges from the association of its isolated parts. Each study contributes to its respective field, and additional understanding arises from their integration in a unified framework.

8.2 Contributions to Ethnobotany and Plant Science

1. This study is the first ethnobotanical evaluation of wild and

cultivated leafy vegetables in the Madanapalle area, and it

provides useful data on the GLV species and their determinants of

consumption.

2. We have demonstrated the role cultivation status play on the

collection and frequency of leafy vegetable consumption, with wild

species less consumed than cultivated, managed or weedy species.

3. Our findings on the foods, folk functional foods and food

medicines confirms the schematic representation of the relation

between plants foods and medicines proposed by Pieroni & Quave

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(Pieroni & Quave, 2006). We further support this model with

consumption frequency data and extend it to cultivated and

managed leafy vegetable species.

4. The attribution of additional health benefits beyond nutrition and

the nature of these benefits influence the consumption patterns of

leafy vegetables in the study area.

5. This is the first report of exceptional popularity of the weedy

species Celosia argentea L.

6. This is the first report of lutein content in fresh samples of Celosia

argentea L., Rumex vesicarius L., Digera muricata (L.) Mart.,

Amaranthus cruentus L. and in cooked samples of all species

included in this study, and β-carotene values for cooked A.

cruentus, C. album and R. vesicarius.

7. The majority of selected cultivated and wild species exhibited

similar lutein/zeaxanthin and β-carotene content when compared

to globally available spinach. These findings support the

promotion of local uncultivated leafy vegetables for nutrition and

health.

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8.3 Contributions to Nutrition

1. We provide a first estimation of the lutein intake by Madanapalle

women, as contributed by the usual consumption of the 10 selected

species, based on reported frequency of intake and measured

concentrations. According to their reported frequency of

consumption and quantification of β-carotene, we estimate the 10

species considered in this study contribute 40% of the

recommended daily intake of β-carotene.

8.4 Contributions to Nutritional Epidemiology and Plant-based

Nutrition

1. We have conducted the first study on the relation between

elements of traditional diets, including leafy vegetable

consumption, and age-related cataract in South Andhra Pradesh.

2. This is the first report of a protective role of yogurt and tea against

age-related cataract, in contrast with other Indian studies reporting

either positive or no association (Chatterjee, et al., 1982; Tarwadi,

et al., 2008).

3. The overwhelming effect of the type of fuel used for cooking in

this rural area supports its systematic inclusion as a confounder in

other studies conducted in areas where such type of fuel is used.

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4. This is the first study testing the association between variety in

GLV intake and age-related cataract in India.

147

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Appendix A

Supplementary material for Chapter 3

190

Individual consent form

Ethnobotanical study on the use of green leafy vegetables.

Location of research Area deserved by the Siloam Eye Center, Madanapalle

Purpose of research This study’s goal is to investigate the role of foods in the prevention of cataracts. For this, we want to know what are the green leafy vegetables that are known and consumed in the region.

Benefits of research This work will permit further research on the relation between food consumption and eye health.

At the end of the study, the investigators of the project (Ms Julie Belanger or Dr K. Shoba Naveen) will give a full report to the community.

Procedure of research If you agree to participate in this interview, it will take about 20 to 30 minutes of your time to answer questions about the leafy vegetables you know. All information will be confidential and never publicly attached to your name. Research data will be coded, stored in locked cabinet and limited personnel will have access to the data. Results may be published in scientific journals.

Risks/discomforts of research No known risk is associated with this procedure. However, if you have further questions, you are welcome to contact the investigators (phone and address below).

Alternatives of research You have the choice to participate or not to this study. If you accept, at any time you can refuse to answer any or all of the questions and leave. I will answer any questions you may have about this study, or will refer you to my supervisors.

Student investigator 1. Julie Belanger, PhD degree candidate, Plant Science Department, McGill University. Contact in Canada: (514) 278-7944 21111 Lakeshore Road, Sainte-Anne-de-Bellevue Montreal, Quebec, Canada, H9X 3V9 [email protected]

191

Principal investigator 2. Dr K. Shoba Naveen, Siloam Eye Center, Madanapalle, Chittoor District, Andhra Pradesh. Contact: 919849148777 [email protected]

Supervisor 3. Dr Timothy Johns, Supervisor, McGill University. Contact: (514) 398-7847 21111 Lakeshore Road, Sainte-Anne-de-Bellevue Montreal, Quebec, Canada, H9X 3V9, [email protected]

Do we have your permission to begin?  Yes  No

Respondent’s signature:

Respondent’s name:

Identification number :

Interviewer’s initials, once a copy of the consent form has been offered to the respondent ______(initials). This acknowledges that the consent form has been read to the respondent in language that the respondent understands, and the respondent has been provided with a written copy if he requests so.

This form is to be kept attached to the questionnaire.

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Free list records of green leafy vegetables

Name: ______Community: ______Age:____ veg ( ) non-veg ( ) Interviewer: ______Today’s Date____(dd/mm/yy) Please list any leafy vegetable you can think of.

No Local name Source If gathered: Special health Cons./ (pu, pr, ga) ecological properties Week space1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 1. Ecological spaces : around fields, roadside ditches, forest, marsh, other.

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Seasonality, appreciation and health properties

No Jan Feb Ma Ap May Jn Jl Aug Sep Oct Nov Dec

Compared to 10-20 years ago :

1. a) Are you eating more or less GLV? Why?

b) Are people in general eating more or less GLV? Why?

2. Do you eat the same plants as before? Why?

3. Is it more or less difficult to collect plants? Why?

Are GLV good for health? How/why? What does it do to your body?

If yes (good for health) how do you know this?

Are there situations where GLV shoud be avoided or promoted (pregnancy, sickness…)?

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Appendix B

Supplementary material for Chapter 4

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Figure B.1: The 10 species selected for lutein/zeaxanthin and β-carotene analysis by High Performance Liquid Chromatography.

1: Allmania nodiflora (L.) R. Br. (Errabadaku), 2: Alternanthera sessilis (L.)

R. Br. (Ponnaganti aku), 3: Amaranthus cruentus L. (Thota aku), 4:

Amaranthus tricolor L. (Sirri aku), 5: Amaranthus viridis L. (Dantu aku), 6:

Celosia argentea L. (Gurugu aku), 7: Digera muricata (L.) Mart (Chenchali aku), 8: Chenopodium album L. (Chakranta aku), 9: Hibiscus cannabinus L.

(Gongura), 10: Rumex vesicarius L. (Chukka aku)

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Appendix C

Supplementary material for Chapter 5

197

Individual consent form

Protocol for assessment of usual dietary intake and standard portion sizes.

Location of research Area deserved by the Siloam Eye Center, Madanapalle

Purpose of research This study’s goal is to determine the usual food items and portion sizes that are consumed in the area.

Benefits of research This work will permit further research on the relation between food consumption and eye health.

At the end of the study, the investigators of the project (Ms Julie Belanger or Dr K. Shoba Naveen) will give a full report to the community.

Procedure of research If you agree to participate in this interview, it will take about 20 to 30 minutes of your time to answer questions about the food you ate yesterday. All information will be confidential and never publicly attached to your name. Research data will be coded, stored in locked cabinet and limited personnel will have access to the data.

Risks/discomforts of research No known risk is associated with this procedure. However, if you have further questions, you are welcome to contact the investigators (phone and address below).

Principal investigator 2. Dr K. Shoba Naveen, Siloam Eye Center, Madanapalle, Chittoor District, Andhra Pradesh. Contact: 919849148777 [email protected] 198

Supervisor 3. Dr Timothy Johns, Supervisor, McGill University. Contact: (514) 398-7847 21111 Lakeshore Road, Sainte-Anne-de-Bellevue Montreal, Quebec, Canada, H9X 3V9 [email protected]

Do we have your permission to begin?  Yes  No

Respondent’s signature:

Respondent’s name:

Identification number :

This form is to be kept attached to the questionnaire.

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Individual 24 hour recall Name: ______Age:______Community: ______Today’s Date_____(dd/mm/yy) Interviewer: ______Day Recalled_____(dd/mm/yy)

ID Time of Name of food Qantity Comments no. day (sc, mc, bc, tsp, (b,l,d,s) tbsp)

Ex. B Chutney 2 tbsp

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ID Name of food Ingredients Quantity No. no. (sc, mc, bc, tsp, people tbsp) and meals (p,m)

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Individual consent form

Protocol for assesment of dietary intake in cataracteous and non- cataracteous individuals.

Location of research Siloam Eye Center, Madanapalle

Purpose of research This study’s goal is to investigate the role of foods in the prevention of cataracts.

Benefits of research This work will help identify food items that help prevent the development of cataract.

At the end of the study, the investigators of the project (Ms Julie Belanger or Dr K. Shoba Naveen) will give a full report to the community. The researchers will be available to discuss results from individuals, if they wish. Results may be published in a scientific journal to describe the role of specific food items in the prevention of cataract.

Procedure of research If you agree to participate in this interview, it will take about 30 to 45 minutes of your time to answer questions about the foods you eat. We will also ask you questions for classification purposes such as occupation status, literacy, land holding, type of house, number of children, land utilized for horticulture and home garden, time spent outdoors, use of protective eye or headwear and household cooking practices (stove type, main cooking fuels). All information will be confidential and never publicly attached to your name. Research data will be coded, stored in locked cabinet and limited personel will have access to the data. We are aiming at collecting dietary data from approximately 400 participants.

Risks/discomforts of research No known risk is associated with this procedure. However, if you have further questions, you are welcome to contact the investigators (phone and address below).

Alternatives of research You have the choice to participate or not to this study. If you do not join, your care at Siloam Eye Hospital will not be affected. If you accept, at any time you can refuse to answer any or all of the questions and leave. I will answer any questions you may have about this study, or will refer you to my supervisors.

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Principal investigator 2. Dr K. Shoba Naveen, Siloam Eye Center, Madanapalle, Chittoor District, Andhra Pradesh. Contact: 919849148777, [email protected]

Supervisor 3. Dr Timothy Johns, Supervisor, McGill University. Contact: (514) 398-7847 21111 Lakeshore Road, Sainte-Anne-de-Bellevue Montreal, Quebec, Canada, H9X 3V9, [email protected]

Do we have your permission to begin?  Yes  No

Respondent’s signature:

Respondent’s name:

Identification number :

Interviewer’s initials, once a copy of the consent form has been given to the respondent ______(initials). This acknowledges that the consent form has been read to the respondent in language that the respondent understands, and the respondent has been provided with a written copy.

This form is to be kept attached to the questionnaire.

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Individual 7-day food frequency questionnaire

No.

Community: ______

Interviewer: G ( ) H ( )

Age:______

Eye health status: cataract ( ) non-cataract ( )

Today’s date ______(dd/mm/yy)

For enumeration purposes :

Occupation:______

Literate ( ) Illiterate ( )

Religion : hindu ( ) muslim ( ) christian ( )

Veg ( ) Non-veg ( )

Land holding : ______

Type of house : ______

Number of children : ______

Presence of a home garden ( ) or land utilized for horticulture ( )

Time spent outdoors daily :______

Use of protective eye ( ) or headwear ( ) ______

Use of tobacco products : yes ( ) no ( )

Household cooking practices :

Indoor ( ) Outdoor ( )

main cooking fuels: ______

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1 FOOD FREQUENCY QUESTIONNAIRE : 7-DAY

Sl. Local name Frequency Preparation Source3 No. (English/Other) No. of days No. of times/d w/ milk ( ) 1. Tea w/ sugar ( ) Grains and cereals

2. Rice, boiled

3. Sangati

4. Pongali

5. Dosa

6. Idli

Rice with Which veg : 7. vegetables

57. Upma

58. Chapatti

59. Puri

60. Appadum

Pulses and nuts

8. Sambar Semi-solid ( ) 9. Pappu Liquid ( ) 47. Groundnut chutney Which 73. Bajji vegetable :

74. Vada

78. Groundnuts

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Akukuralu

(Leafy vegetables) Thota koora C ( ) 10. (Amaranthus P ( ) cruentus) Chukka koora C ( ) 11. (Rumex vesicarius) P ( ) Palak koora C ( ) 12. (Spinach) P ( ) Ponnaganti koora C ( ) 13. (Alternanthera P ( ) sessilis) Menthikoora C ( ) 14. (Fenugreek leaves) P ( ) Pappu koora C ( ) 15. (Portulaca P ( ) oleracea) Munaga akulu C ( ) 16. (Drumstick leaves) P ( ) Gongura C ( ) 17. (Hibiscus P ( ) cannabinus) Kasaku : b ( ) n ( ) C ( ) 18. (Solanum nigrum) P ( ) Errabadhaku C ( ) 19. (Allmania P ( ) nodiflora) Sirraku C ( ) 20. (Amaranthus P ( ) tricolor) Gurugaku s ( ) C ( ) 21. (Celosia argentea) P ( ) Chakravarti aku C ( ) 22. (Chenopodium P ( ) album) Avise C ( ) 23. (Sesbania P ( ) grandiflora) Adavi pulla koora C ( ) 24. (Oxalis P ( ) corniculata) Mulla thotakoora C ( ) 25. (Amaranthus P ( ) spinosus) Mullangi akulu C ( ) 26. (Radish leaves) P ( )

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Kothimiri C ( ) 27. (Coriander leaves) P ( ) Karivepaku C ( ) 28. (Curry leaves) P ( ) Pudina C ( ) 29. (Mint Leaves) P ( ) Other : C ( ) 30. P ( ) Other : C ( ) 31. P ( ) Other : C ( ) 32. P ( ) Which greens : 45. Greens chutney

70. Gongura pickle. Thamalapaku 80. (Piper betel) Root vegetables Pu ( ) 33. Carrots Ta ( ) Pu ( ) 34. Beet roots Ta ( ) Urla gadda Pu ( ) 35. (Potato) Ta ( ) Mulinge Pu ( ) 42. (Radish) Ta ( ) Other vegetables Pu ( ) 36. Beans Ta ( ) Birakaya Pu ( ) 37. (Ridge gourd) Ta ( ) Vankaya Pu ( ) 38. (Brinjal) Ta ( ) Kakarakaya Pu ( ) 39. (Bitter gourd) Ta ( ) Benda kaya Pu ( ) 40. (Ladies finger) Ta ( ) Munaga kaya Pu ( ) 41. (Drumstick) Ta ( ) Pu ( ) 43. Tomato Ta ( )

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44.a Corn Other: Pu ( ) 44.b Ta ( ) 46. Tomato chutney

69. Tomato pickle

75. Rasam Pamdu

(Fruits) 48. Coconut chutney

49. Apple Ariti pandu 50. (Banana) Draksa 51. (Grapes) Dhanimakaya 52. (Pomagrenate) Jamakaya 53. (Guava) Seethaphalam 54. (Custard apple) Chinikaya 55. (sweet lime) Other: 56.

68. Mango pickle

71. Tamarind pickle

72. Lime pickle

Meats and eggs Chepa Fresh ( ) 61. (Fish) Dry ( ) Mamsamu 62. (Meat) Kodi 63. (Poultry)

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Fried ( ) Guddu 64. Curry ( ) (Eggs) Boiled ( ) Dairy foods Palu 65. (Milk) Perugu/dahi 66. (Yogourt) Majiga 67. (Buttermilk) Sweets

76. Kajikayalu

77. Payasam

79. Other sweets

209