ASSESSMENT OF SELECTED ESSENTIAL MICRONUTRIENTS IN SOME INDIGENOUS IN KAKAMEGA COUNTY, KENYA

MUTEMBETE RICHARD WAWIRE RIMU (Bed. Sc.) I56/CE/11217/07

A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science in Applied Analytical Chemistry in the School of Pure and Applied Sciences of Kenyatta University

AUGUST, 2020

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DECLARATION

I hereby declare that this is my original work and has not been presented for the award of degree in any other university

Richard Wawire Rimu Mutembete Reg. No. I56/CE/11217/07)

Signature………………………………………….. Date…………………………….

This thesis has been submitted with our approval as University supervisors.

Signature………………………………………….. Date…………………………….

Prof. Ruth Wanjau Chemistry Department Kenyatta University

Signature………………………………………….. Date…………………………….

Prof. Hudson Nyambaka Chemistry Department Kenyatta University

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DEDICATION

This work is dedicated to my loving and supportive wife Rehema, sons Ronsmas and

Ryann and daughter Relyne.

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ACKNOWLEDGEMENTS

I thank God the Almighty for taking me this far. I sincerely thank my supervisors; Prof.

Ruth Wanjau and Prof. Hudson Nyambaka of Kenyatta University for their wonderful guidance throughout the research period. Their useful suggestions, comments and corrections helped me to reach the end of my research. I am grateful to National

Commission of Science and Technological Innovation (NACOSTI) for the ST&I grant that supported the study. I also appreciate the technical staff of Chemistry department of

Kenyatta University for their assistance while in the laboratory and the technical staff of

Masinde Muliro of Science and Technology for the assistance in preservation of fruits during sampling. Other gratitude goes to Lang’at of Vetcare Pharmaceutical Company at

Ruaraka, Nairobi who assisted me in HPLC analysis of , the technical staff of

Ministry of Geology and Mines in Nairobi’s industrial area who assisted in analysis of chromium, manganese and copper and a natural resourced Mr. Ceaser Wanyama who helped in botanical identification of . I also appreciate the communities of

Ingotse Boys’ High School and Friends School Keveye Girls for their understanding during my study.

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TABLE OF CONTENTS

TITLE………..……………………………...…...…….………………………………….i DECLARATION...... ii DEDICATION...... iii ACKNOWLEDGEMENTS ...... iv TABLE OF CONTENTS ...... v LIST OF FIGURES ...... ix LIST OF TABLES ...... x ABBREVIATIONS AND ACRONYMS ...... xi ABSTRACT ...... xiii CHAPTER ONE ...... 1 1 INTRODUCTION...... 1 1.1 Background information ...... 1 1.2 Problem statement and justification ...... 3 1.3 Hypotheses ...... 4 1.4 Objectives ...... 5 1.4.1 General objective ...... 5 1.4.2 Specific objectives ...... 5 1.5 Significance of study ...... 5 1.6 Scope and limitations ...... 6 CHAPTER TWO ...... 7 2 LITERATURE REVIEW ...... 7 2.1 Food and nutrition security ...... 7 2.2 Indigenous fruits studied ...... 8 2.2.1 Psidium quajava L (Guava- Mapera) ...... 8 2.2.2 peruviana L. (Cape goose - Local name, Emilwa) ...... 11 2.2.3 Rhus vulgaris M...... 13 2.3 Importance of micronutrients to health ...... 15 2.3.1 Introduction ...... 15 2.3.2 Essential trace elements ...... 16

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2.3.2.1 Chromium ...... 17 2.3.2.2 Zinc ...... 19 2.3.2.3 Iron...... 21 2.3.2.4 Manganese ...... 24 2.3.2.5 Copper ...... 28 2.3.3 Vitamins and health ...... 30 2.3.3.1 A (Retinol) ...... 32 2.3.3.2 ...... 37 2.3.3.3 Vitamin E ...... 42 2.4 Methods of analysis ...... 46 2.4.1 Introduction ...... 46 2.4.2 Theory of atomic absorption spectroscopy (AAS) ...... 47 2.4.3 Instrumentation of AAS...... 49 2.4.4 High performance liquid chromatography (HPLC) ...... 52 2.4.4.1 Theory of HPLC ...... 52 2.4.4.2 HPLC instrumentation and components...... 53 2.5 Precautions in and tocopherol analysis ...... 55 CHAPTER THREE ...... 57 3 METHODOLOGY ...... 57 3.1 Research design ...... 57 3.2 Study area ...... 57 3.3 Sampling...... 58 3.4 Sample treatment ...... 59 3.5 Chemicals, reagents and solvents ...... 59 3.6 Cleaning of glassware and sample containers ...... 60 3.7 Instrumentation...... 60 3.8 Laboratory procedure ...... 61 3.8.1 Preparation of standard solutions ...... 61 3.8.1.1 Standard solutions for AAS ...... 61 3.8.1.2 Standard solutions for HPLC ...... 62 3.8.2 Method validation ...... 63

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3.8.2.1 Calibration ...... 63 3.8.2.2 Reproducibility ...... 64 3.8.2.3 Recovery ...... 65 3.8.2.4 Detection limit ...... 65 3.8.3 Sample preparation ...... 66 3.8.3.1 Determination of β-carotene and α-tocopherol...... 66 3.8.3.2 Determination of L-ascorbic acid ...... 67 3.8.3.3 Determination of trace metallic elements ...... 67 3.9 HPLC separation and quantification ...... 68 3.10 Data analysis ...... 69 CHAPTER FOUR ...... 70 4 RESULTS AND DISCUSSION ...... 70 4.1 Introduction ...... 70 4.2 Method validation ...... 70 4.2.1 Method validation for trace elements ...... 70 4.2.2 Method validation for HPLC ...... 73 4.3 Micronutrients levels ...... 76 4.3.1 Levels of essential trace elements ...... 76 4.3.1.1 Levels of zinc ...... 78 4.3.1.2 Levels of iron ...... 79 4.3.1.3 Levels of manganese ...... 81 4.3.1.4 Levels of copper ...... 82 4.3.1.5 Levels of chromium ...... 83 4.3.2 Vitamins levels ...... 86 4.3.2.1 HPLC separation and chromatograms ...... 86 4.3.2.2 Levels of vitamin C (Ascorbic acid) ...... 91 4.3.2.3 Levels of (beta-carotene) ...... 92 CHAPTER FIVE ...... 97 5 CONCLUSIONS AND RECOMMENDATIONS ...... 97 5.1 Conclusions ...... 97 5.2 Recommendations from this study ...... 98

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5.3 Recommendations for further research ...... 99 REFERENCES ...... 100 APPENDICES ...... 110 Appendix I: Calibration curves for trace elements ...... 110 (a) Manganese ...... 110 (e) Copper ...... 112 Appendix II: Calibration curves for vitamins ...... 113 (a) Ascorbic acid ...... 113 (b) Beta-carotene ...... 113 (c) Alpha tocopherol ...... 114 Appendix III: Chromatograms of vitamins ...... 115 (a) Vitamin C in Rhus Vulgaris L ...... 115 (b) Vitamin C standard...... 116 (c) β-carotene standard ...... 117 (d) α-tocopherol standard ...... 118

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LIST OF FIGURES Figure 2.1: White guava ...... 10 Figure 2.2: Red guava variety ...... 10 Figure 2.3: Ripe guava on a branch ...... 11 Figure 2.4: Ripe Physalis peruviana in opened calyx ...... 13 Figure 2.5: Fruiting R. vulgaris Meikle ...... 14 Figure 2.6: Ripe Rhus vulgaris M. fruit ...... 15 Figure 2.7: Vitamin A structure ...... 32 Figure 2.8: Structure of ...... 33 Figure 2.9(a): Ascorbic acid structure ...... 37 Figure 2.9(b): Dehydroascorbic acid structure ...... 37 Figure 2.10: Structure of tocopherol ...... 42 Figure 2.11: Structure of tocotrienol ...... 42 Figure 2.12: Schematic diagram of equipments used for AAS...... 51 Figure 2.13: Schematic diagram of HPLC instrumentation...... 53 Figure 3.1: Population density of Kakamega County ...... 58 Figure 4.1: Calibration curve for manganese ...... 70 Figure 4.2: Calibration curve for beta-carotene ...... 73 Figure 4.3: Chromatogram of α-tocopherol and β-carotene in white sweet guava...... 87 Figure 4.4: Chromatogram of vitamin C standard ...... 88 Figure 4.5: Chromatogram of a mixture of α-tocopherol and β-carotene standards ...... 89

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LIST OF TABLES

Table 2.1: Levels of , minerals and water-soluble bioactives in P. peruviana pulp ...... 12 Table 2.2: Chromium levels in some fruits ...... 18 Table 2.3: Zinc levels in some fruit juices consumed in Saudi Arabia ...... 21 Table 2.4: Iron levels in some fruits ...... 24 Table 2.5: Manganese levels in some fruit juices consumed in Nigeria ...... 27 Table 2.6: Copper levels in some fruits and vegetables consumed in Pakistan ...... 30 Table 2.7: Beta-carotene content in some foodstuffs consumed in UK ...... 37 Table 2.8: Contents of AA, DHAA and total ascorbic acid (TAA) in fresh vegetables in Japan ...... 41 Table 2.9: Content of AA in some fruits ...... 41 Table2.10: Recommended Dietary Allowances (RDAs) for Vitamin E (Alpha-Tocopherol) ...... 46 Table 3.1: HPLC operating conditions ...... 60 Table 3.2: AAS operating conditions ...... 61 Table 4.1: AAS method validation statistics ...... 71 Table 4.2: Reproducibility analysis of trace elements in two varieties of fruits ...... 72 Table 4.3: Validation data for HPLC method ...... 74 Table 4.4: Reproducibility results of vitamins in some indigenous fruits ...... 75 Table 4.5: Levels of trace elements indigenous fruits in sub counties of Kakamega County ...... 77 Table 4.6: Levels of essential trace elements in selected indigenous fruits in Kakamega County ...... 84 Table 4.7: Amount (g) required to provide RDA/day and the % RDA contribution ...... 85 Table 4.8: Levels of vitamins in indigenous fruits in the sub counties of Kakamega County ...... 90 Table 4.10: Amount (g) required to provide RDA of vitamins/day and % RDA ...... 96

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ABBREVIATIONS AND ACRONYMS

AA Ascorbic Acid

AAS Atomic Absorption Spectrometry

AIDS Acquired Immune Deficiency Syndrome

APHA American Public Health Association

ATPASE Adenosine Triphosphate

AWWA American Water Works Association

DDR Daily Dietary Requirements

DHAA Dehydroascobic Acid

DNA Deoxyribonucleic Acid

DRIR Dietary Reference Intakes Report

EDXRF Energy Dispersive X-ray Flourescence

FAO Food Agricultural Organization

FFA Free Fatty Acids

FNB Food and Nutrition Board

GC-MS Gas Chromatography Mass Spectroscopy

HGT Hydride Generation Technique

HIV Human Immuno Deficiency Virus

ICAP Inductively Coupled Argon Plasma

ICP-AES Inductively Coupled Plasma Atomic Emission Spectroscopy

ICP-MS Inductively Coupled Plasma Mass Spectroscopy

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IDDM Insulin Dependent Diabetes Mellitus

MDGs Millennium Development Goals

NAA Neutron Activation Analysis

NGA Nutrition Goals for Asia

NIDDM Non-Insulin Dependent Diabetes Mellitus

RDA Reference Dietary Allowance

SNK Student Newman Keul

SPSS Statistical Package for Social Scientists

TAA Total Ascorbic Acid

UK United Kingdom

UNEP United Nations Environmental Programme

UNDP United Nations Development Programme

USAID Agency for International Development

USDA United States Department of Agriculture

WHO World Health Organization

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ABSTRACT

Fruits are a good source of micronutrients, yet they are less consumed. Kakamega County has various types of fruits either planted or growing wildly. Most of the fruits consumed are exotic such as avocadoes, mangoes, pawpaw, oranges, passion fruits and pineapples while the wild and indigenous fruits are usually neglected, seen as weeds and food for children and very poor rural people. In Kakamega County the indigenous fruits are well distributed among the rural areas and members of the community lack awareness of the nutritional importance of such fruits as Physalis peruviana L, Rhus vulgaris M, and Psidium guajava L and therefore they do not consume well. This has caused some members especially the poor to be nutritionally affected. These indigenous fruits are rich in essential micronutrients such as vitamins and essential trace elements that help in immune boasting, destruction of free radicals and general physical growth. The need to promote such fruits requires that the levels of micronutrients present be known. Thus this study assessed levels of some micronutrients such as trace elements (Zn, Fe, Mn, Cu, and Cr) and vitamins {L-ascorbic acid (vitamin C), α-tocopherol (vitamin E), and β-carotene (vitamin A)} in selected indigenous fruits in Kakamega County, Kenya. The vitamin content was determined using HPLC and the elements analyzed using AAS. Data was analyzed by one way ANOVA followed by SNK test where there was no significant difference at p<0.05. The mean levels of Zn, Fe, and Cu were significantly high in P. peruviana (4.54±1.41, 1.48±0.10 and 11.86±1.43 mg/100g respectively) compared with other fruits. Rhus vulgaris was significantly high in Mn (14.81±1.69 mg/100g) and Cr (0.73±0.04 mg/100g) while guava varieties were significantly high in vitamins A, E and C compared with other fruits. Among the guava varieties studied the red sweet guavas were significantly high in ascorbic acid (231.64±8.40 mg/100g), followed by red bitter guavas (205.16±4.53 mg/100g). White sweet guavas had significantly high mean level of β-carotene (3.40±0.05 mg/100g) while red bitter guavas showed significant high levels of α-tocopherol (0.54±0.00 mg/100g). The study showed that the levels of micronutrients in the fruits studied were significantly different between sub-counties and between the fruit varieties. Many of the fruits studied met and some even exceeded the RDA requirements. A fresh piece of guava of mass 200 g is sufficient to provide twice the RDA for β- carotene and greater than six times of vitamin C. A fresh piece of 200 g of white sweet and red bitter guavas can provide more than half the RDA for Mn (69.96% and 86.96% respectively) and Cr (58.02% and 63.00% respectively). The same piece of red sweet guava can provide 64.27% RDA for Cu and 50.0% for Cr. Consuming 200g of wet fresh Physalis peruviana can provide 78.77% of Cu whereas the same amount of fresh Rhus vulgaris provides 145.99% of RDA of Cr and 118.48% of Mn. 200 g of wet fresh Physalis peruviana and 200 g of wet fresh Rhus vulgaris is sufficient to provide the adult daily requirement of beta-carotene and ascorbic acid. Promotion of these indigenous fruits will provide cheap sources of essential micronutrients that can help in reducing micronutrient deficiency. The results of the study is useful to food policy makers and also used to sensitize the public on nutritional matters.

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CHAPTER ONE

1 INTRODUCTION

1.1 Background information

Africa and Asia have some known ethnic communities who still live in bushes without modern medical care facilities, clothing, and food sources but still maintain good health by eating traditional food including wild fruits, vegetables and game meat (UNEP, 1995).

People who consume a lot of fruits and vegetables are much less likely to suffer from non-communicable health related diseases compared to those who consume less (Sarma and Swaminathan, 1993). Kenya compared with other developing countries as India reports more incidences of these diseases (WHO, 2006). For incidence about 42-51% of adolescents in Kenya are anaemic as compared to Nepal with 20-28% (WHO, 2005). Iron deficiency anaemia in school attending girls in western Kenya was reported to be more prevalent than other parts of Kenya (Leenstra, 2007).

Fruits are an important part of the diet of mankind as they are rich sources of essential trace elements and vitamins which help the body to fight against diseases (Bean, 2007;

Burchi et al., 2011). The fruits are many and of varied kinds, both exotic and indigenous.

The exotic fruits such as bananas, avocadoes, oranges, mangoes, pawpaw, pineapples and passion are relatively large in size, more juicy and fleshy than indigenous fruits and, therefore, preferred by many for propagation at the expense of the latter (Burchi et al.,

2011).

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In Kakamega County bananas, mangoes, pawpaw, and avocadoes are the most common exotic fruits. The indigenous fruits commonly found in this County include Psidium guajava L (guavas), Rhus vulgaris M (known locally as owusangula) and Physalis peruviana L (gooseberry), Rhus natalensis, and Carissa edulis. These indigenous fruits are partially cultivated though considered as weeds (Kwee and Chong, 1990; Ivens, 1971) and therefore neglected in spite of being rich in essential micro-nutrients. These fruits are known to have low saturated and cholesterol and are rich in dietary fibers which may reduce the risk of coronary heart diseases (Kwee and Chong, 1990). They contain key nutrients such as vitamin C and iron among others (Kwee and Chong, 1990). Guavas, for instance, have been exploited in some parts of Kenya for their juice (UNDP, 2005). In

Western Kenya, there is no known commercial importance about guavas just as the other indigenous fruits. What is common is land clearance and harvest for firewood, charcoal, and timber, more so than harvest of fruits for medicinal and food use. This has a detrimental effect on wild medicinal and food resources (Marshall, 1997).

Kakamega County has most of its arable land under sugarcane cultivation. This, coupled with the high population of 1,660,651(USAID, 2011), poses a threat to food security in the county. This is evidenced by high food prices as compared to many other parts of the country. Given that economic status of people in this county is low (USAID, 2011), raising enough food to feed the population from the remaining small portions of land is hard and, therefore, equally difficult to meet a balanced diet. Assessment of the nutritional value of indigenous fruits will aid in their promotion, hence reduce the risks of food insecurity in the county especially among the rural community.

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1.2 Problem statement and justification

Balanced diet and physical fitness help to maintain good human health. A balanced diet includes proteins, , salts, vitamins, fats, and water. Mineral salts, especially trace elements, and vitamins are known to play a vital role in boosting body immune system which aids in management of dreaded diseases as cancer, diabetes, malaria, osteoporosis, HIV/AIDS, high blood pressure and other heart ailments (Janabai et al., 1990) which are on an increase in Kenya. This has led to enhancement of their provision through food supplements and pills. In this way, there is an increased chance of excessive intake which is unhealthy (WHO and FAO, 2002; Akan et al., 2009). The best way is to obtain them naturally by eating food rich in these nutrients as fruits especially indigenous ones.

Kakamega County community depends on maize as the staple food. Beans are also produced and consumed by many. Fruits such as bananas and avocadoes are produced but not in sufficient amount for the community. This causes a lot of fruits to be outsourced from other counties and from the neighbouring country, Uganda. As a result so many households cannot afford to consume fruits daily in required ratios. The county has realized an increase in cases of non-communicable diseases as diabetes and the communicable ones such as malaria and HIV/AIDS (USAID, 2011).

Kakamega County has many indigenous fruits such as Physalis peruviana L, Psidium guajava L, Rhus vulgaris M, Rhus natalensis, and Carissa edulis. Many of these fruits are neglected and seen as weeds (Kwee and Chong, 1990). During land clearance for

4 agricultural activities and settlement, the indigenous fruits are cut and uprooted (Ivens,

1971). In spite of the destruction, the fruits are common and well distributed especially among the rural areas. Large population of the rural do not consume these fruits. To them they regard indigenous fruits as food for children, hence occurrence of communicable diseases in the area (USAID, 2011). Studies show that indigenous fruits are rich in micronutrients (Nanyili, 1995 and Ismail et al., 2011). Levels of micronutrients in indigenous fruits such as Physalis peruviana L, Psidium guajava L, and Rhus vulgaris M have been reported elsewhere but not in Kakamega County yet their levels depend on soil type and climatic conditions. Thus the unavailability of data on levels of micronutrients of these fruits makes it difficult to promote them among the populace.

There is need therefore to assess the levels of micronutrients in these fruits in order to promote them as a measure of reducing incidences of hidden hunger. The purpose of this study, therefore, was to assess the levels of zinc, iron, chromium, manganese, and copper, and vitamins A, C, and E in the selected indigenous fruits in Kakamega County. This will help to encourage the county community to propagate the fruits for consumption and sale in order to improve the health and economic status of its people.

1.3 Hypotheses

(i) Levels of essential micronutrients in indigenous fruits including Psidium guajava

L, Rhus vulgaris M, and Physalis peruviana L of Kakamega County do not differ

significantly between sub-counties and between the fruit varieties.

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(ii) Levels of essential micronutrients in indigenous fruits including Psidium guajava

L, Rhus vulgaris M, and Physalis peruviana L of Kakamega County do not meet

the RDA values.

1.4 Objectives

1.4.1 General objective

To assess the levels of selected essential micronutrients in different varieties of Psidium guajava L, Rhus vulgaris M, and Physalis peruviana L fruits in various sub-counties of

Kakamega County, Kenya.

1.4.2 Specific objectives

(i) To determine the levels of zinc, chromium, iron, manganese, and copper in

three varieties of Psidium guajava L (red bitter, red sweet and white sweet)

and in Physalis peruviana L and Rhus vulgaris M in various sub-counties of

Kakamega County.

(ii) To determine the levels of L-ascorbic acid (vitamin C), α-tocopherol (vitamin

E) and β-carotene (vitamin A) in three varieties of Psidium guajava L (red

bitter, red sweet and white sweet) and in Physalis peruviana L and Rhus

vulgaris M various sub-counties of Kakamega County.

1.5 Significance of study

The study will provide useful information on the levels of iron, zinc, chromium, manganese, copper, beta-carotene, ascorbic acid, and alpha-tocopherol in some

6 indigenous fruits in Kakamega County. This is important to policy makers, agronomists, food and nutrition security planners, and health workers, NGOs and other organizations interested in food security and promotion of good health. This will go a long way in reducing or alleviating poverty, hunger and child mortality especially among the poor members of the community. The study also will contribute to the field of knowledge in rich, cheap and affordable micro- food sources for people and this will form basis for future research in the area. The study will help to promote the propagation of indigenous fruits which will help to improve economic status of the community and preserve the plants from extinct.

1.6 Scope and limitations

This study focused only on levels of selected essential micronutrients, iron, zinc, manganese, copper, chromium, beta-carotene, ascorbic acid, and alpha-tocopherol in

Psidium guajava L (red sweet, red bitter and white sweet varieties), Rhus vulgaris M, and

Physalis peruviana L in Kakamega County. Only one common available species of each kind of fruit was considered. Factors affecting levels of the micronutrients in fruits including seasons (climate variation), extent of ripening and soil condition were not considered.

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CHAPTER TWO

2 LITERATURE REVIEW

2.1 Food and nutrition security

One of the world’s greatest challenges is to have sufficient and healthy food for all which is part of the 1st millennium sustainable development goal (SDG) and to do so in an environmentally sustainable manner (Burchi et al., 2011). Millions of people in African countries, Kenya included, are vulnerable to hunger and malnutrition due to high level of food prices, vulnerability to man-made and natural disasters, slow rate of attaining the

MDGs, and emerging issues such as HIV/AIDS, and climate change which influence agriculture, nutrition and health sectors (Babu, 2011). Hunger continues to be a dramatic problem in developing and emerging countries with nearly one billion (925 million) hungry people (FAO, 2010) and about two billion people suffering from chronic micronutrient deficiencies also known as hidden hunger (Burchi, et al., 2011).

Food-based approaches, which include food production, dietary diversification and food fortification, are sustainable strategies for improving the micronutrient status of population. Practical sustainable actions for overcoming micronutrient deficiencies are through increased access to and consumption of adequate quantities and an appropriate variety of safe good-quality food (Thompson and Amoroso, 2011). In developing nations promotion of agricultural productivity to enhance food security is being emphasized. This is done by promotion of labour-saving technologies, fertilizer, pesticides and various types of improved seeds. This promotion is mainly focused predominantly on the

8 increased production of staple grains as maize, rice and wheat consisting mainly carbohydrates, modest amounts of protein and a few other nutrients essential to meet human nutritional requirements (Remans et al., 2011). The push to concentrate on a few staple crops may be a contributory factor to the simplified diets, which promotes under- nutrition in Kenya and other developing nations and wide spread hidden hunger.

2.2 Indigenous fruits studied

2.2.1 Psidium quajava L (Guava- Mapera)

Guava belongs to myrtaceae family which has between 110 to 130 species of trees and shrubs. Guava trees can flower and bear fruits continuously in the tropics. However, there are normally two crops a year. Fruits mature 90 to 150 days after flowering depending on variety or clone of the fruit and weather conditions (Nakasone and Paull, 1988). Guava is native to the American tropics and probably originally grew from north of Mexico and Caribbean but it is widely spread in the region that its origin cannot be clearly discerned anymore (Ahmed and Amjad, 2010). Guavas are deliberately spread but they are also dispersed by animals eating the fruits. Today, guavas are found in all subtropical and tropical parts of the world. In western Kenya guavas are considered as weeds because they invade habitats and displace native species.

Guavas are cholesterol, saturated fats and sodium free, plus low in and calories and hypoglycemia in nature (Kwee and Chong, 1990). Guavas help in reduction of cholesterol in blood and prevent it from thickening, thereby maintaining fluidity of blood

9 and prevent blood pressure. Studies have shown that food stuffs which lack fiber add to blood pressure, due to quick conversion to sugar (Ahmad and Amjad, 2010).

Guava is a great fruit because it contains key nutrients like vitamin C, carotenoids, folate, fibre, potassium, calcium and iron. Guava is very rich in vitamin C (Gatamba, et al.,

2010) and iron which prevent against cold and viral infections (Ahmad and Amjad,

2010). The edible rind of guava contains five times more vitamin C than an orange

(Kwee and Chong, 1990). Nanyili (1995) found 123.0 mg/100g of vitamin C in white guavas of Ukambani and 375.0 mg/100g in red guavas and Belitz and Grosch (1999) reported 50 mg/100g of vitamin C in oranges. Leong and Shui (2002) reported ascorbic acid of fresh guava at 270 ±18.8 mg/100g. Lim et al., (2007) reported ascorbic acid levels of 218±79 mg/100g for seeded guava and 176±54 mg/100g for seedless guava. Guava is one of the cheapest and good sources of vitamin C and pectin. Guava fruit contains 82.50 per cent water, 2.45 per cent reducing sugar, 2.23 per cent non-reducing sugar, 9.73 per cent total soluble solids, 0.48 per cent ash and 260 mg vitamin C per 100g of fruit pulp as well as good amount of iron, calcium and phosphorus (Ismail et al., 2011). These constituents may differ with the , stage of maturity, soil fertility, and season. The guava fruit is a good source of pectin (0.78%) (Ismail et al., 2014).

Siow and Hui (2013) reported 120.65 mg/100g of ascorbic acid in guava. Charoensiri, et al., (2009) reported 13.8 μg/100g beta carotene and Ramadan and Morsel (2003) reported

13.10 mg/100g of beta carotene in guavas. Ismail et al., (2011) reported 0.28 mg/100g of

10 zinc, 0.50 mg/100g of manganese and copper in guava. Janabai et al., (1990) reported

14.4 μg/100g of chromium in guava.

Guavas also help control diabetes, and reduce the risk of prostate cancer. The juice of leaves cures toothache, swollen gums and oral ulcers, heals wounds when applied externally and other bacterial infections (Ahmad and Amjad, 2010). In western Kenya people chew immature leaves of guava tree to cure stomachache. Guava helps in weight lose without compromising with intake of proteins, vitamins and fiber. Thus, guava is very filling and satisfies appetite very easily, being rich in roughage and very rich in vitamins, proteins, minerals and water, but with no cholesterol and less digestible carbohydrates (Ahmad and Amjad, 2010). Photographs of white, red and unplucked ripe guavas are shown in Plates 2.1, 2.2 and 2.3 respectively.

Figure 2.1: White guava Figure 2.2: Red guava variety

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Figure 2.3: Ripe guava on a branch

2.2.2 Physalis peruviana L. (Cape goose berry- Local name, Emilwa)

This is a and the whole plant is softly hairy. The upper leaves are often oppositely arranged, up to 4 inches long by 2 inches wide and more or less triangular in shape with entire margins or a few large shallow teeth, and petioles up to 2.5 inches long.

The flowers are about 5/8 inches across. The inflated calyx is similar in shape to and berries are golden when ripe (Ivens, 1971). Some farmers cultivate it but it is commonly found as a weed. The ripe berry is sweet to taste. The plant is fairly adaptable to a wide variety of soils.

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Puente et al., (2011) and Musinguzi et al., (2007) reported 1.24 mg/100g and 7 mg/100g of iron respectively in Physalis peruviana L. Puente et al., ( 2011) reported 22.50 g/kg P.

Peruviana pulp and skin oil and 0.88 g/kg seed oil. Ramadan and Morsel (2004) reported

43.0 mg/100g of ascorbic acid in P. Peruviana pulp. Levels of nutrients, minerals and water-soluble bioactives in P. peruviana pulp are shown in Table 2.1.

Table 2.1: Levels of nutrients, minerals and water-soluble bioactives in P. peruviana pulp

Bioactive mg/100g

Moisture 789 Protein 0.5-3 Lipid 1.5-20 196 Phosphorus 55.3 Iron 1.2 Carotene 1.6 Ascorbic acid 43.0 0.1

Source: Ramadan and Morsel (2004)

Cape gooseberry has unique storage properties, wherein the fruits can be kept for a long time. It is used in folk medicine for treating diseases such as malaria, asthma, dermatitis, diuretic, rheumatism, strengthener for optic nerve, treatment of throat infections and elimination of intestinal parasites, amoebas as well as albumin from kidneys (Ramadan and Morsel, 2004). It has an anti-ulcer activity and it is effective in reducing cholesterol

13 level (Arun and Asha, 2007). A photograph of Physalis peruviana in opened calyx is shown in Figure 2.4 below.

Figure 2.4: Ripe Physalis peruviana in opened calyx

2.2.3 Rhus vulgaris M

Rhus vulgaris M (Owusangula) is a common shrub at forest edges and in woodlands. It is used as firewood, farm tools (stem) and food (fruit). Rhus vulgaris M is a shrub or small tree that occasionally reaches 6m. Branch lets are brown and hairy. The leaves are oval to rounded, usually five centimeters long. The top of the leaf is rounded, notched or sharp.

The flowers are yellow-green, with bright-yellow stamens (Figure 2.5). They bear thin green fruits which when ripe are yellow and others yellow-red edible fruits. They are flat

14 and round with 3-5 mm across. The fruits are bitter to taste but are better eaten if heated

(Tesemma et al., 1993).

In Western Kenya, children like eating them after overfeeding. People do not like eating these fruits due to their very small size, and bitter taste. They are used to treat diarrhoea, malaria and vomiting (Nanyunja, 2003). They are known to produce very high quality charcoal, hence preferred for charcoal burning at the expense of food or medicinal use.

The photographs of fruiting and ripe Rhus vulgaris M are shown in Figures 2.5 and 2.6 respectively.

Figure 2.5: Fruiting R. vulgaris Meikle

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Figure 2.6: Ripe Rhus vulgaris M. fruit

2.3 Importance of micronutrients to health

2.3.1 Introduction

Micronutrients are nutrients (dominantly vitamins and minerals) required by humans only in small amounts throughout life in order to carry out a vast range of physiological functions (Graham et al., 2007). Micronutrients do not provide energy but help regulate

16 the production of energy from macronutrients. Graham et al., (2007) argues that at least fifty one different nutrients are needed in adequate amounts by human beings. There are nineteen essential micronutrients for physical and mental development, immune system functioning and various metabolic processes (Kennedy et al., 2007).

Recent data highlights that an estimated two billion people suffer from one or more micronutrient deficiencies demonstrating that hidden hunger is responsible in part for global malnutrition burden (Graham et al., 2007). This is quite evident in the health sector which has been faced with a number of challenges including communicable and infectious diseases, nutrition deficiency disorders and parasitic infections. Adequate intake of micronutrients can be sufficient enough to provide preventive measures either wholly or partially to these healthcare problems. Productivity of the human capacity depends on good health and nutrition. This in turn enhances economic growth, contributes to poverty reduction, hence the realization of the Kenya vision 2030 social goals. In this study eight essential micronutrients were considered. These are iron, zinc, chromium, manganese and copper as trace elements and vitamins A (β-carotene), C (L- ascorbic acid), and E (α-tocopherol).

2.3.2 Essential trace elements

Trace element is an element in a sample that has an average concentration of less than

100 ppm. They function in hormones and enzymes through complex series of chemical reactions in the body and are essential in the regulatory and maintenance of normal body functions. These body functions include growth, cells replacement, energy production,

17 reproductive functions, muscles formation and contraction and transmission of nerve impulses (WHO, 1996b).

2.3.2.1 Chromium

Chromium is a transition element that was originally discovered in 1797. Most salts of chromium are brightly coloured and are used widely as pigments (Bean, 2007). It is an important trace element that occurs in a number of valence states with 3+ and 6+ being the most common. The Cr3+ is the most stable form in biological systems in the environment and in food (Bowman and Russel, 2001; Bowen, 1994). The Cr3+ is less absorbed than Cr6+ but Cr6+ is much more toxic than the trivalent one (Mindell and

Mundis, 2004).

Chromium stimulates the enzymes involved in glucose and energy metabolism by regulation of blood sugar and therefore involved in insulin metabolism in man, hence aid in reduction of medication needs in diabetic patients (Bakhru, 2006 and Paraphona,

2004). Thus the active agent in blood sugar (glucose tolerance factor) consists of chromium chemically bound with nicotinic acid which is a member of vitamin B complex. Lack of chromium may result in full blown diabetes in man (Mindell and

Mundis, 2004). Studies have shown that levels of chromium in body tissues drop with age which likely explains why there is development of maturity onset diabetes (Al

Durtsch, 1999).

18

Chromium activates vitamin C which is beneficial to human health since it increases resistance to infection and prevents cancer. It plays a role in reduction of oxidative stress and HIV viral load and aids in iron absorption (WHO, 1996b). Refining of food and over consumption of refined carbohydrates remove the chromium naturally present in the food stuffs and in the body. Chromium sources include fresh fruits, vegetables, eggs, wheat germ, chicken meat, corn oil, brewers’ yeast, and calves’ livers (Horrobin, 1981; USDA,

1990).

Chromium deficiency diseases include diabetes, cardiovascular diseases and hypertension. High levels of chromium exceeding 800 μg/day may lead to cancer, and renal haemorrhage bronchitis (Underwood, 1977). The daily dietary requirement ranges between 50 to 200 μg (Al Durtsch, 1999). The levels of chromium have been analyzed in several fruits as presented in Table 2.2.

Table 2.2: Chromium levels in some fruits

Fruit Mean (μg/100g)

Melon 46.8 Guava 14.4

Sapota 14.4 Papaya 14.3 Banana 16.4

Source: Janabai et al., (1990)

19

2.3.2.2 Zinc

Interest in zinc as an essential trace element has been renewed since 1960’s when evidence for its need by man was shown (Bowman and Russel, 2001). It influences the activity (co-factor) of two hundred enzymes in man (Horrobin, 1981; Bowman and

Russel, 2001; NGA, 2003; Grodner et al., 2000). It is an active component of the enzyme carbonic anhydrase which functions in maintaining equilibrium between carbon dioxide and carbonic acid

CO2 + H2O H2CO3

It also activates enzymes which function in digestion of proteins by hydrolyzing specific peptide linkages.

Zinc is needed for healthy immune system, wound healing, skin and cell growth (Bean,

2007). It is a constituent of antioxidant superoxide dismutase that prevents damaging effects of oxygen free radicals on cells. Thus, zinc in diet may help in preventing most of the chronic diseases that include cancer, bronchitis, heart disease and anaemia. Food rich in zinc can enhance body immunity activities which include production of antibodies, T- cells and white body cells. In a study conducted by Bakhru (2006), it was discovered that low dosages of zinc in eighty percent regrowth of thymus glands, increase in active hormones and T-cells that fight infections. Zinc reduces incidence of opportunistic infections of diseases as pneumonia in HIV/AIDS patients by up to forty five percent

(Kabi, 2004).

20

Potentially many people do not have an adequate amount of zinc but data of the prevalence of its deficiency has proven difficult to quantify and global prevalence statistics of zinc deficiency remain estimates (Hotz and Brown, 2004). Zinc deficiency results in retarded growth (dwarfism), delayed wound healing, immature gonads (testicles and ovaries), dermatitis, lose of testes and probably sickle cell diseases (Paraphona,

2004). Zinc is also associated with night blindness that does not respond to vitamin A being a component of retinene reductase in the retina (Horrobin, 1981). Since zinc is lost in sweat, the deficiency is likely in hot countries (WHO, 1998). The daily requirement for man is 15-20 mg/day (WHO, 1998).

Diseases associated with deficiency of zinc include diabetes, anaemia, prostate cancer, cardiovascular diseases and kidney diseases (Janet, 2000). Deficiency symptoms of zinc include slow hair growth, white sports on finger nails, reduced sense of smell, spotty skin and love for salt in food. Levels above 1000 mg of body zinc are toxic and effects include vomiting, nausea and impaired immune function (Mindell and Mundis, 2004). In acute intoxication renal failure occurs following haemodialysis which is characterized by nausea, fever and severe anaemia. Some of the best sources of zinc include liver, sea food, wheat germ, brewer’s yeast, pumpkin seeds, and leafy vegetables. Several studies have been conducted on analysis of zinc is food stuffs. Anke et al., (2005) reported 4.9 mg/100g of zinc in oranges, Kipkemboi (2011) reported 2.49 mg/100g in Carissa edulis,

Puente et al., (2011) reported 0.40 mg/100g in P. peruviana and Ismail et al., (2011) reported 0.28 mg/100g in guava. Some more are shown in the Table 2.3.

21

Table 2.3: Zinc levels in some fruit juices consumed in Saudi Arabia

Fruit juices Zinc (ppb)

Orange 894.8

Apple 524.00

Mango 486.57

Source: Farid and Enani (2010)

2.3.2.3 Iron

Iron is the most investigated and best understood micronutrient. Research on iron nutrition has been facilitated by the relative ease of sampling blood (of body tissues, erythrocytes contain the most iron). To a large extent iron metabolism and factors leading to iron metabolism and factors leading to iron related disorders are well defined. In recent years, concern about iron overload has also stimulated research on regulatory aspects of iron metabolism (Bowman and Russel, 2001). In solid form iron exists as a metal ion or an ion-containing compound. In aqueous form it exists as Fe2+, the ferrous form and Fe3+, the ferric form (Bowman and Russel, 2001).

Human body contains 4-5 g of iron (Underwood, 1977). Most of the body’s iron exists in complex forms bound to protein. Thus, along with protein, iron form haemoglobin, the oxygen carrying pigment in the red blood cells (Cronam, 1965; Janssen and Shinkels,

2009). Haemoglobin iron occupies 60-70% of body iron in man (Underwood, 1977).

Liver, spleen, kidney and heart are the body organs that have the highest concentration of iron. The 2001 dietary reference intakes report by the food and nutrition board gives the

22 recommended dietary allowances (RDAs) for iron as 27 mg/day for pregnant women, 15 mg/day for women aged 14-18 years, and the highest level in men being 11 mg/day for ages 7-12 months and 14-18 years (Bowman and Russel, 2001; FNB, 2001).

Pregnancy increases the requirement of iron by approximately 3.5 mg/day because of extra demand by the foetus (Williams and Caliendo, 1988). Children and adolescents need iron for maintenance of their haemoglobin concentration and to increase iron storage during their period of growth. Iron helps in energy production and cell division, immune and central nervous systems (Janssen and Shinkels, 2009). Iron boosts body immunity by being involved in formation of white blood cells in the bone marrow. Thus, iron forms a significant part of bone marrow enzymes involved in the formation of white blood cells. Iron plays a role in elimination of old blood cells and synthesis of new ones.

Eight percent of total iron intake is absorbed into the blood stream (Mindell and Mundis,

2004).

Low iron levels can cause people to develop iron deficiency anaemia which is characterized by fatigue, weakness, rapid heartbeat, fainting, susceptibility to infection and swelling of the tongue (Janssen and Shinkels, 2009; Bowman and Russel, 2001).

These signs occur for severe anaemia; otherwise iron deficiency is detected by routine physical examination (Bowman and Russel, 2001). Sometimes iron deficiency individuals will show appetite for ice, clay, paste or other non-nutritional substances.

23

Deficiency of iron in the body can impair vital functions like oxygen carrying capacity of the red blood cells, haemoglobin formation and functions of blood and enzymatic activity of respiratory function, especially the cytochrome involved in cellular respiration. Food sources rich in iron are pork, beef, liver, red meat, oysters, nuts, legumes and green leafy vegetables. In a study conducted in India millet has a high concentration of 5.48 mg/100g fresh weight (Gosh, 2007). Too much iron leads to liver, pancreas and heart damage. Too much iron in blood, a condition known as haemochromatosis, causes liver cirrhosis, arthritis, hypogonadium (underactive sex organs), diabetes and congestive heart failure

(Janssen and Shinkels, 2009). Some studies of iron levels in several food stuffs have been done in the past and some of the results are shown in Table 2.4.

24

Table 2.4: Iron levels in some fruits

Fruits Mean (mg/kg) Source

Rhus natalensis 651.67 Kipkemboi (2011)

Carissa edulis 87.10 Kipkemboi (2011)

Ficus sycomorus (figs) 75.05 Kipkemboi (2011)

Mangifera indica (mangoes) 51.64 Kipkemboi (2011)

Mangifera indica (mangoes) 45.6 Stanton (1995)

Carica papaya (pawpaw) 136.31 Kipkemboi (2011)

Mulberry 14.6 Ngigi (2014)

Citrus sinensis (orange) 75.05 Stanton (1995)

Physalis peruviana 12.4 Puente et al., (2011)

Physalis peruviana 70 Musinguzi et al., (2007)

Apples 12.4 Gosh (2007)

Mangoes 13.0 Gosh (2007)

Bananas 3.6 Gosh (2007)

2.3.2.4 Manganese

Manganese is one of the recognized essential elements for humans. It occurs in relatively constant amounts in tissues and organs of both plants and animals and is especially concentrated in the reproductive organs (Grossman and Wendell, 1985). Biological active form of manganese is Mn2+ but Mn3+ is also important. Manganese was recognized as an essential mineral element for growth and reproduction in mice and rats in 1931.

25

A few years later it was discovered that a deficiency of Mn was largely responsible for crippling disease of chickens known as perosis (Pennington and young, 1991). This elicited a lot of interest in Mn nutrition. Mn concentrarion ranges from less than 1 ppm to as much as 7000 ppm with an average of 500 to 600 ppm in soil (Pennington and Young,

1991). Normal levels in plants range from 8 to 20 μg/dm3 (Williams and Caliendo, 1988).

In human body manganese has levels relatively high in liver, bones, pancreas and kidney while muscles have very little. The skeleton accounts for about 25 percent of total body manganese (Berman, 1980). This reserve, however, is not readily used when dietary intake is low. The oral absorption of manganese into the body is naturally slow and is transported in blood serum by β-globulin and its absorption apparently occurs equally well throughout the lengthy of the small intestine (Berman, 1980). Although specific mechanisms for manganese absorption and transport have not been determined, some evidence suggests that it can share common absorption and transport pathways with iron, cobalt, calcium and magnesium. Thus an individual’s status of the four elements can affect manganese bioavailability.

Manganese, just like other essential trace elements, activates enzymes and acts as a constituent of metalloenzymes (Pond and Maner, 1984). Many enzymatic reactions associated with metabolism of organic acids, carbohydrates, nitrogen, and phosphorous are activated with manganese and they include hydrolases, kinases, decarboxylases, and transferases (Sappey, 1994). Manganese is essential for normal growth, skeletal formation and for normal reproductive functions in mammals and poultry.

26

Manganese helps in regulation of blood sugar level, hence may help prevent or cure diabetes. Previous studies showed that diabetics invariably have low manganese levels in blood; therefore, lack of it in regular diets could be linked to prevalence of diabetes in man (Williams and Caliendo, 1988). Manganese superoxide dismutase (MnSOD) is the principle antioxidant enzyme in the mitochondria. Therefore, Mn deficiency causes abnormalities in cell function and ultra-structure, particularly involving the mitochondria

(Ajit et al., 1999). Humans with convulsive disorders, including epileptics, showed whole blood manganese concentrations significantly below normal (Bratter and Schramel,

1980).

Manganese is required for utilization of fats and lipid metabolism and building of nucleic acid (carrier of genetic information). Manganese can cause the blockage of replication of

HIV inside cells; hence can prevent it from causing AIDS (Akan et al., 2009). Although the specific mechanisms for manganese absorption and transport have not been determined some evidence suggests that iron and manganese can share common absorption and transport pathways. Thus, iron status of an individual can affect manganese bioavailability.

Signs of manganese deficiency include impaired growth, hearing and reproductive function, ataxia, skeletal abnormalities, impaired glucose tolerance, altered carbohydrates and lipid metabolism, schizophrenia and ligament weakness. Its deficiency diseases include diabetes, anaemia, and cardiovascular diseases. At levels exceeding 20 mg,

27 manganese toxicity occurs and symptoms include anorexia, impulsiveness, spastic gait and Parkinson disease (WHO, 1996b; Berman, 1980).

The recommended daily allowance of manganese is not well defined, but the estimated safe and adequate intake for adults is 2 to 5 mg/day and 0.3 to 0.5 mg/day for infants and children (Mindell and Mundis, 2006; Bratter and Schranel, 1980). About 3.7 mg of manganese are ingested daily from a well-balanced diet (William and Caliendo, 1988).

Food reach in manganese include leafy green vegetables, nuts, fruits, whole grains, tea and coffee, legumes, fish and shellfish. A number of studies have been done on levels of manganese in foodstuffs. Table 2.5 shows manganese levels in some fruit juices consumed in Nigeria.

Table 2.5: Manganese levels in some fruit juices consumed in Nigeria

Fruit type Mn (ppm)

Grape 0.80

Pineapple 15.00

Apple 0.53

Orange 0.45

Lemon 0.23

Source: Akan et al. (2009)

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2.3.2.5 Copper

Copper is an essential trace element required for proper health in plants and animals in an appropriate limit. Copper absorption in human body takes place from the intestine and is taken to different other organs where it is utilized in copper dependent enzymes (Gerbasi et al., 2003). Copper absorption can be deficient in human body because of excessive iron and zinc and on the contrary excessive copper can cause iron inadequacy (Ismail et al., 2011). Trace elements are the natural constituents of the soil and due to the variation of the atmospheric conditions their uptake varies through root to the shoot. On the contrary copper contents do not mobilize and remain stagnant in roots (Ismail et al.,

2011). Normal levels in plant range from 5 to 20 ppm (Underwood, 1977).

Copper is a component of lysyl oxidase, an enzyme that participates in the synthesis of collagen and elastin connective tissues (Adams and Keen, 2005; Ismail et al., 2011).

Copper plays a vital role in lowering low density lipoprotein cholesterol and raise the high density lipoprotein cholesterol and help in prevention of oxidation of low density lipoprotein cholesterol that makes it more destructive to arteries (Bakhru, 2006). The low density lipoprotein cholesterol is associated with cholesterol deposits in blood vessels, hence harmful to the body. On the other side, high density lipoprotein cholesterol helps to remove cholesterol from circulation, thereby reducing the risk of heart disease, hence beneficial to health.

Copper is known to improve utilization of ammonia sources of nitrogen, resulting in improved growth in plants. Human body contains copper concentrations of 100 to 150

29 mg (Mindell and Mundis, 2004) with the muscle, bones and liver containing 64 mg, 23 mg and18 mg respectively (Underwood, 1977). Copper in the human body helps in the conversion of iron to haemoglobin, and makes amino acids usable, allowing it work as a pigmentation factor for hair and skin. It activates superoxide dismutase, an enzyme that is responsible for elimination of oxygen-free radicals from the body. The oxygen-free radicals are usually charged and unstable; hence have the effect of causing weakness in the nervous system, inflammation of joints, turning cells cancerous and clogging of arteries.

The recommended daily allowance for adults is in the range of 1.5 to 3.0 mg (Mindell and Mundis, 2004). Copper deficiency causes growth retardation, skin ailments, gastrointestinal disorders, skeletal defects, rheumatic arthritis, osteoporosis, heart disease, leukemia and ruptured blood vessels (WHO, 1999b). Sources of copper include sea foods, dried beans, peas, whole wheat and shrimp (Gerrior and Zizza, 1994). Copper toxicity is rare but a tolerable upper intake level for adults is about 10000 μg and between

100 and 500 μg for children (Mindell and Mundis, 2004). Copper toxicity can cause liver damage, abdominal pains and postpartum depression. Several studies of copper in food stuffs have been done. Table 2.6 shows copper levels in some fruits and vegetables consumed in Pakistan.

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Table 2.6: Copper levels in some fruits and vegetables consumed in Pakistan

Fruit Cu (mg/100g fresh weight) Guava 0.50 Mango 0.90 Banana 0.11 Okra 0.10 Cucumber 0.10 Bitter gourd 0.11 0.13 Brinjal 0.90 Chili pepper 0.17

Source: Ismail et al. (2011)

2.3.3 Vitamins and health

The term vitamin describes a group of food boasters that are peculiarly essential to life

(Bowman and Russel, 2001). The harm of a generally poor diet cannot be corrected by a vitamin pill. Thus, vitamins should be obtained from food (Bowman and Russel, 2001).

Vitamins cannot be synthesized by humans and therefore should be obtained from diet.

Many researchers believe that vitamin supplements can drastically reduce free radical damage, prevent and delay the onset of chronic degenerative diseases, and possibly extend life span (Jacob and Sotoudeh, 2002). Numerous epidemiological studies have demonstrated an association between higher intakes or higher blood concentrations of certain antioxidants and a lower incidence of certain degenerative diseases (Zhao et al.,

2004).

31

Free radicals are highly reactive molecules that react with and damage the cells throughout the body (Zhao et al., 2004). They are suspected of causing cardiovascular disease, cancer, neurological disorders, cataracts, arthritis, aging and other conditions.

They are produced continuously as a normal part of cell processes (Bean, 2007).

Antioxidants are molecules, which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged (Zhao et al., 2004). There are several enzyme systems within the body that scavenge free radicals, whereby the principle micronutrient (vitamin) antioxidants are vitamin C, vitamin E and β-carotene. The body cannot manufacture these micronutrients so they must be supplied in diet (FNB, 2001).

Intense exercise raises levels of harmful free radicals. The body generally produces higher levels of antioxidant enzymes in response to regular exercise, but additional antioxidants from food, or food supplements, are needed to help strengthen body defenses

(Bean, 2007). Many researchers believe that vitamin supplements can drastically reduce free radical damage, prevent and delay the onset of chronic degenerative diseases, and possibly extend lifespan (Zhao et al., 2004). Clinical studies have shown that supplemental levels of antioxidant vitamins reduce an individual’s risks of certain cancers and cardiovascular diseases. Evidence also suggests that vitamins C, E and β- carotene supplementation have ergogenic or performance enhancing effects (Zhao et al.,

2004).

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2.3.3.1 Vitamin A (Retinol)

Vitamin A is a pale yellow or yellow-orange fat soluble compound classified as retinoids and carotenoids (Fox and Cameron, 1984; Weissenberg et al., 1997). The term retinoids refer to compounds whose name starts with ret-. These are retinol, retinaldehyde, retinoic acid, and other similar cofounds both natural and synthetic. Retinol is the immediate precursor to the two important active metabolites, retinal and retinoic acid. The retinols are absorbed by the body 3 to 5 hours after ingestion whereas the conversion and absorption of carotenes takes 6 to 7 hours (Stevenson, 1987). Retinoids give preformed vitamin A, the kind of nutrient that the body can use right away. The structure of retinoids is shown in Figure 2.1.

R

Figure 2.7: Vitamin A structure

If: R= CH2OH, an alcohol, it is called retinol; R= CHO, an aldehyde, it forms retinal or retinaldehyde and; R= COOH, an acid, it forms retinoic acid (Combs, 1998).

Vitamin A is found in animal tissues with the most concentration in fish liver oil

(Williams, 1989). Other sources include milk and milk products as well as eggs (Fox and

Cameron, 1984). Fruits, leafy vegetables, carrots and beans are rich in vitamin A activity.

They contain pigments known as carotenoids (provitamin A) which are converted into vitamin A in the small intestine. Fruits contain various carotenoids such as lycopene,

33 alpha carotene, β-carotene and gamma carotene- beta carotene being the most common and important (Combs, 1998). β-carotene is a red solid which was first isolated in carrots hence its name. One molecule of β-carotene is twice that of vitamin A. The carotenoids have a tail- conjoined retinoid dimer as shown in Figure 2.2 (Combs, 1998).

Beta-carotene structure

15

15

Alpha-carotene

Gamma-carotene

Figure 2.8: Structure of carotenoids

Beta-carotene is found widely in plant tissues in the more stable all-trans (E) configuration. Its characteristic chromophore associated with the presence of eleven conjugated double bonds displays an absorbance maximum at an approximately 450 nm,

34 which makes it useful as a colouring agent for foods, cosmetics and pharmaceuticals, either as plant extracts and dehydrated powders, or in synthetic form (Weissenberg et al.,

1997). Beta-carotene was the first synthetic carotenoid to be marketed. It has been classified by FAO/WHO in class A under the label E160a as acceptable for use in foods

(Weissenberg et al., 1997).

Vitamin A is primarily absorbed at the upper part of the intestinal tract. It is at this point that fat-splitting enzymes and bile salts convert carotene into a usable nutrient

(Stevenson, 1987). The conversion is stimulated by thyroxine hormone obtained from the thyroid gland. Once converted to vitamin A, carotene is absorbed in the same way as the preformed vitamin A. The absorbed vitamin A is then carried through the blood stream to other body tissues (Dunne, 1990).

The conversion of β-carotenes into vitamin A is only about one-sixth as effective as retinol. Other provitamin A in fruits are about half the activity of β-carotene

(Weissenberg et al., 1997). Only about one third of the carotene in food is converted into vitamin A (Dunne, 1990). Less than a quarter of the carotene in carrots and root vegetables, and about a half of the carotene in leafy vegetables undergoes conversion

(Kutsky, 1981). Some unchanged carotene is absorbed into the circulatory system and stored in the fat tissue rather than in the liver. Unabsorbed carotene is excreted in feaces.

The kind of food and the form in which food is ingested determines the body’s ability to utilize carotenes. Cooking pureeing or mushing of fruits and vegetables ruptures cell

35 membranes and makes carotenes available for absorption. Vitamin A and carotene absorption can be interfered with factors as stress, physical activity performed within four hours of consumption, intake of mineral oil, excessive consumption of alcohol and iron and use of cortisone and other drugs (Combs, 1998). Stevenson (1987) notes that, unless antioxidants are present, carotenes are rapidly destroyed by intake of polyunsaturated fatty acids. Nearly ninety percent of the body’s vitamin a is stored in the fat tissues, lungs, kidneys and retinas of eyes (Dunne, 1990). Zinc aids in mobilization of vitamin A out of the storage sites.

Vitamin A is important for immunity, cell differentiation, growth, reproduction and vision (Combs, 1998). Retinal is a necessary structural component of rhodopsin or visual purple, the light sensitive pigment within rod and cone cells of the retina. If inadequate quantities of vitamin A are present, vision is impaired. Vitamin A has shown to enhance resistance to infectious diseases in children. The immune response of children vaccinated against measles has been shown to be boosted by vitamin A (Semba, 1981). Lack of vitamin A leads to dysfunction of many epithelia- the skin becomes keratinized and scaly, and mucous secretion is suppressed. This is due to impaired transcriptional regulation due to deficits in retinoic acid signaling (Combs, 1998).

Vitamin A helps in bone remodeling. Thus, normal functioning of osteoblasts and osteoclasts is dependent upon vitamin A (Stevenson, 1987). Normal levels of vitamin A are required for sperm production, reflecting a requirement for vitamin A by spermatogenic epithelial (sertoli) cells. Similarly, normal reproductive cycles in females

36 require adequate availability of vitamin A. Adverse effects of vitamin A deficiency include night blindness and poor immune response in children, pregnant and lactating women.

Vitamin A is a powerful antioxidant. Thus, it is capable of deactivating reactive chemical species such as singlet oxygen, triplet photochemical sensitizers, and free radicals that would otherwise damage DNA inside cells and could trigger cancer-inducing mutations and adversely affect specific immune functions. A variety of ethnic vegetables (green leafy vegetables and other coloured vegetables), namely legumes/dhal, tomatoes and coriander are rich sources of carotenoids. Other sources include spinach, carrots and ghee.

The recommended dietary allowance (RDA) of vitamin A is 4000-5000 IU for adults and

1500-4000 IU for children (Institute of Medicine, 2002). These amounts increase during disease, trauma, pregnancy and lactation (Dietary Reference Intakes, 2001). Factors as smoking, polluted environment ease of absorption of vitamin A, and amount of stored vitamin A vary the requirement of the vitamin by a human body (Diplock et al., 2000).

Depletion of the stored vitamin A can be caused by diseases as pneumonia or nephritic.

Beta-carotene content in some foodstuffs consumed in UK is shown in Table 2.7.

37

Table 2.7: Beta-carotene content in some foodstuffs consumed in UK

Food μg/100g of β-carotene Source

Spinach 4066 Khokhar et al., 2012

Carrots 2324 Khokhar et al., 2012

Bitter lettuce 6.80 Weinberger and Msuya, 2004

2.3.3.2 Vitamin C

Vitamin C is a water soluble vitamin which occurs as both ascorbic acid (ascorbate, AA) and dehydroascorbic acid (DHAA) involved in many biological processes in flora and fauna (Velisek and Cejpek, 2007). The chemical structures of AA and DHAA are shown in Figure 2.3 (a) and Figure 2.3 (b) respectively.

HO OH HO OH O O O O

HO OH O O

Figure 2.9(a): Ascorbic acid structure Figure 2.9(b): Dehydroascorbic acid structure

Ascorbic acid is involved in maintaining the reduced state of metal cofactors, for example, monoxygenase (Cu+) and dioxygenase (Fe2+). In cells, AA helps in reduction of hydrogen peroxide (H2O2), which preserves cells against reactive oxygen species (Mitic et al., 2011). Synthesis of AA by the body is not possible. The only way people can obtain AA is through food but the exact requirements of vitamin C for humans are not yet

38 clear. Currently the estimated average requirement of AA is 100g/day and recommended dietary allowance is 120 g/day (Mitic et al., 2011).

Vitamin C has been widely exploited in cosmetic and pharmaceutical preparations to protect them against oxidation and to exert physiological/ biological activities (Velisek and Cejpek, 2007). Ascorbic acid is a strong reducing substance. The oxidation of ascorbic acid leads, over a radical intermediate steps, to dehydroascorbic acid, in vivo.

These forms constitute a reversible redox system. The AA is rapidly oxidized to DHAA due to the presence of two hydroxyl groups in its structure. Oxidation reactions can be induced by exposure to increased temperatures, high pH, light, presence of oxygen or metals and enzymatic action. Further oxidation generates diketogluconic acid (DKG), which has no biological function and the reaction is no longer reversible (Mitic et al.,

2011).

Vitamin C if taken orally, most of it is absorbed through the mucous membranes of the mouth, stomach and upper part of the small intestines (Weber et al., 1996). The larger the dose the less is absorbed (Naidu, 2003). Therefore, it is advisable to take vitamin C in small doses several times a day. The human body is fully saturated by 500 mg of vitamin

C, of which 30 mg are found in the adrenal glands, 200 mg in the extracellular fluids, and the rest distributed in varying concentration throughout the body cells (Stevenson, 1987).

Prolonged administration of antibiotics or cortisone, smoking, inhalation of dimethyldiphenyltricloromethane fumes of petroleum, stress, high fever and ingestion of

39 aspirin or any other pain killers and sulphur drugs increase urinary excretion of vitamin C by 2 to 3 times the normal amount (Gayla and Kirchmann, 1996).

The National Research Council recommends 60 mg of vitamin C for adults (Dietary

Reference Intakes, 2001). The requirement may vary due to differences in weight, amount of activity, rate of metabolism, ailments and age. Periods of stress caused by anxiety, infection, injury, surgery, burns or fatigue increase the body’s need for vitamin C

(Institute of Medicine, 2002). The level of ascorbic acid in blood reaches a maximum in 2 to 3 hours after ingestion of moderate quantity and then decreases as it is eliminated in urine and perspiration.

Acute vitamin C deficiency results in scurvy which is characterized by impaired wound healing, edema haemorrhage in the skin, mucous membranes, internal organs and muscles and weakening of collagenous structures in bone, cartilage, teeth and connective tissues (Semba and Tang, 1999). Mild vitamin C deficiency results in several nonspecific prescorbustic signs and symptoms including lassitude, fatigue, anorexia, muscular weakness. Mild deficiency can be caused by low dietary intake as well as a variety of factors that increase ascorbate turnover in the body such as smoking, stress, chronic diseases that include diabetes (Gayla and Kirchmann, 1996).

The AA plays an important role in hydroxylation reactions in the synthesis of collagen, therefore important for the de novo synthesis of bone, cartilage and tooth and for the healing of wounds together with proteins. Ascorbic acid promotes the reabsorption of

40 iron in the intestine. Being an antioxidant, it has a protective effect against the oxidizing effects of free radicals. It protects other substances from the oxidizing effects of oxygen.

Presence of AA is necessary for the activity of dopamine β-hydroxylase, prevention of coronary heart diseases and cancer (Mehmet and Tuba, 2010). The AA/DHAA ratio can be an indicator of the redox state of an organism. The content of vitamin C in fruits (the sum of the contents of AA plus DHAA) is used as an index of the health-related quality of fruits (Mehmet and Tuba, 2010).

Vitamin C has been found to prevent the formation of N-nitroso compounds, the cancer causing substances from nitrates and nitrites found in preserved meats and some drinking water (Du et al., 2009). In general L-ascorbic acid (AA) constitutes 90% of the total vitamin C content of vegetables and fruits (Agar, 1995). Therefore, it is important to measure ascorbic acid in fruits and vegetables for vitamin C activity (Mehmet and Tuba,

2010). Contents of AA, DHAA and total ascorbic acid (TAA) in fresh vegetables in

Japan is shown in Table 2.8 while content of AA in some fruits is shown in Table 2.9.

41

Table 2.8: Contents of AA, DHAA and total ascorbic acid (TAA) in fresh vegetables in Japan

Vegetable Edible portion (mg/100g)

AA DHAA TAA Sweet pepper 78.54 6.94 85.48 Broccoli 73.85 8.26 82.11 Pumpkin 36.92 18.99 55.91 Malabar nightshade 43.58 8.31 51.89 Asparagus bean 24.57 8.76 33.333 Tomato 14.68 2.13 16.81 Carrot 4.04 3.34 7.38

Source: Yoshihiro and Sanae (2006)

Table 2.9: Content of AA in some fruits

Fruit mg/100g Source

Cape gooseberry 46 Belitz and Grsoch (1999)

Pear 4 Belitz and Grsoch (1999)

Apple 6 Belitz and Grsoch (1999)

Peach 7 Belitz and Grsoch (1999)

Orange 50 Belitz and Grsoch (1999)

Strawberry 60 Belitz and Grsoch (1999)

Guavas 123-375 Nanyili (1995)

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2.3.3.3 Vitamin E

Vitamin E is a fat soluble vitamin which designates a family of related compounds

(tocopherol and tocotrienol) with a chromanol head and a phytyl tail. The two compounds share a common structure as shown in Figures 2.4 and 2.5.

R1

HO

R2 O

Figure 2.10: Structure of tocopherol: Source: Ruperez et al., (2001)

R1

HO

R2 O

Figure 2.11: Structure of tocotrienol: Source: Ruperez et al., (2001)

R1 R2 Compound

CH3 CH3 α

CH3 H β

H CH3 γ

H H σ

Alpha tocopherol is the most active and common of the naturally occurring vitamin E compounds (Bean, 2007). Green vegetables, seeds and nuts are rich dietary sources of

43 vitamin E (Combs, 1998). Vitamin E accumulates in adipose tissue, liver and muscle. It acts as anti-oxidant that protects against cellular damage by inhibiting peroxidation of polyunsaturated fatty acids in cell membranes. Lack of vitamin E in the body causes the body immune effectors cells to be damaged by the reactions of free radicals (Meydani et al., 1990).

Vitamin E exists in eight different forms (α-, β-, γ-,and σ-tocopherols and α-, β-, γ-, and

σ-tocotrienols ); α-tocopherol being the most active form and one of the most powerful biologically active antioxidants. Each form has a different level of potency (functional use) in the body (Ng and Ko, 2012).The only structural difference between tocopherols and tocotrienols is that tocopherols have a saturated phytyl chain, and tocotrienols have an unsaturated isoprenoid chain.

If oxidation of α-tocopherol occurs in the organism, a variety of compounds appears. In case that such oxidation of tocopherol does not exceed 20%, the main oxidation product is α-tocopherolquinone (TQ), which could be reduced by cellular mechanisms to α- tocopherolhydroquinone (THQ). Other products of tocopherol reactions with free radicals, as 5, 6-epoxy-a-tocopherolquinone (TQE1) and 2, 3-epoxy-a-tocopherolquinone

(TQE2) can also be formed (Ruperez et al., 2001).

Vitamin E has a number of health benefits for the body. It is particularly important for the protection of cell membrane as well as keeping skin, heart and circulation nerves, muscles and red blood cells healthy. One or more members of the vitamin E family may

44 also reduce cellular aging, inhibit the potentially damaging peroxynitrite radical, inhibit melanoma (skin cancer) cell growth, prevent abnormal blood clotting, synergize with vitamin A to protect the lungs against pollutants, protect nervous system and retina, lower the risk of ischemic and coronary heart disease, lower the risk of certain kinds of cancer, protect immune function and reduce the risk of Alzheimer’s disease, particularly if high doses are taken in combination with vitamin C (Meydani et al., 1990).

In the body the liver is the determinant factor of serum concentrations of vitamin E (α- tocopherol). The liver takes up the nutrient after the various forms are absorbed from the small intestine. The liver preferentially resecretes only α-tocopherol via the hepatic α- tocopherol transfer protein (Traber, 2006) and metabolizes and excrets the other vitamin

E forms (Traber, 2007). This causes blood and cellular concentrations of other forms of vitamin E to be lower than those of α-tocopherol and have been subject of less research

(Sen et al., 2006 and Dietrich et al., 2006).

Vitamin E may block the formation of nitrosamines, which are carcinogens formed in the stomach from nitrites consumed in the diet. Vitamin E deficiency is rare in humans although likely to occur under certain specific situations, when an individual cannot absorb dietary fat, was born premature, is a very low birth weight infants (birth weights less than 1500 grams or 3.5 pounds), and in individuals with rare disorders of fat metabolism. Deficiency of vitamin E in the body causes the body immune effectors cells to be damaged by the reactions of free radicals (Meydani et al., 1990). Its deficiency is usually characterized by neurological problems due to poor nerve conduction.

45

The digestive tract requires fat to absorb vitamin E. Therefore, people with fat- malabsorption disorders are more likely to become deficient than people without such disorders. Deficiency symptoms include peripheral neuropathy, ataxia, skeletal myopathy, retinopathy, and impairment of the immune response (Institute of Medicine,

2002). People with Crohn's disease, cystic fibrosis, or an inability to secrete bile from the liver into the digestive tract, for example, often pass greasy stools or have chronic diarrhoea; as a result, they sometimes require water-soluble forms of vitamin E, such as tocopheryl polyethylene glycol-1000 succinate (Traber, 2006). Table 2.10 shows the

FNB’s Recommended Dietary Allowances for α-tocopherol.

46

Table2.10: Recommended Dietary Allowances (RDAs) for Vitamin E (Alpha-

Tocopherol)

Age Males Females Pregnancy Lactation 0–6 months* 4 mg 4 mg (6 IU) (6 IU)

7–12 months* 5 mg 5 mg (7.5 IU) (7.5 IU)

1–3 years 6 mg 6 mg (9 IU) (9 IU)

4–8 years 7 mg 7 mg (10.4 IU) (10.4 IU)

9–13 years 11 mg 11 mg (16.4 IU) (16.4 IU)

14+ years 15 mg 15 mg 15 mg 19 mg (22.4 IU) (22.4 IU) (22.4 IU) (28.4 IU)

*Adequate Intake (AI)

Source: Institute of Medicine (2002)

2.4 Methods of analysis

2.4.1 Introduction

Trace elements can be analyzed by various methods including flame atomic absorption spectroscopy (AAS) (Akinyele and Shokunbi, 2015; Beyhan, et al., 2014), inductively coupled plasma atomic electron spectroscopy (ICP-AES) (Soruraddin et al., 2011), inductively coupled plasma mass spectroscopy (ICP-MS) (Tokalioglu, 2012; Soruraddin et al., 2011), neutron activation analysis (NAA) (Mohammed, 2012), flame atomic absorption spectroscopy (FAAS) (Rodriguez et al., 2015) and energy dispersive x-ray

47 fluorescence spectroscopy (EDXRF) (Kipkemboi, 2011). The AAS method was chosen for this study due to its availability, speed, simplicity in terms of sample preparation and operation, sensitivity due to low detection limit and selectivity.

Similarly vitamins can be analyzed by various methods including high performance liquid chromatography (HPLC) (Sami et al., 2014), ultra performance liquid chromatography (UPLC) (Inga and Anna, 2014), and gas chromatography mass spectrometry (GC-MS) (Ruperez et al., 2007). In this study HPLC was used because of availability and is considered a sensitive and selective method, therefore, suitable for active substance determination (Mitic et al., 2011).

2.4.2 Theory of atomic absorption spectroscopy (AAS)

The samples are converted into free atoms of the analytical elements by a flame. A beam of monochromatic radiation is passed through these atoms from a hollow cathode lamp source of characteristic wavelength specific for each element. The absorbed radiations by atoms are directly proportional to the concentration of atoms in flame. This obeys Beers’

Law, given in Equation 2.1

A = εbc = logІo/I ------(Equation 2.1)

Where: A= Absorbance b=path length (cm) through the medium c=concentration (moles/litre) of the absorbing species ε= molar absorptivity (l/mol/cm) Io=incident radiation I=attenuated radiation (Skoog et al., 2001; Mendham et al., 1999).

48

A calibration curve plotted using absorbance from known metal standards within the concentration range of the sample, help to determine the sample concentration traced from its observed absorbance by interpolation (Van Loon, 1980).

The analytical signal is obtained from the difference between the intensity of the source in the absence of the element of interest and the decreased intensity obtained when the element of interest is present in optical path. Transition from one steady state with lower energy, Eo, to another with higher energy, Ej, involves absorption of energy (photons).

The energy absorbed is given by the Equation 2.2.

Ej-Eo= Ѵojh ------(Equation 2.2)

Where: Eo is the ground steady state energy Ej is the excited state energy Ѵoj is the frequency of o-j transition h is the plank’s constant Eo is the energy at ground state Ej is energy at excited state

The emission intensities are affected by flame temperature. Thus, the flame temperature has a significant effect on the ratio between the excited and unexcited atomic particles as given by the Boltzmann Equation 2.3 (Skoog et al., 1998).

Where: Nj is number of atoms in excited state No is number of atoms in ground state Pj statistical weight of excited state Po is statistical weight of ground state K is Boltzmann’s constant (1.38x10-16 erg/deg) T is absolute temperature

49

Eo is energy at ground state Ej is energy at excited state

Sample solution is aspirated through the nebulizer into the air/acetylene flame. The nitrous oxide/acetylene flame is used for refractory metals or elements because it creates higher temperatures (Skoog et al., 1998). Electrically heated graphite is used when very high sensitivity is required. The sample solution in the nebulizer is dispersed into a fine spray of aerosols which is then led into flame where it gets evaporated into dry salt. The dry salt is then vaporized and dissociated into atoms that absorb resonance radiation from external source. The unabsorbed radiation is allowed to pass through the monochromator which isolates the existing analyte spectral lines which are detected by the detector and amplified and recorded by the signal processor and read out system.

2.4.3 Instrumentation of AAS

AAS has the radiation, atomizers, monochromators, detectors and read out system. There two types of radiation source: Continuous and line sources. The continuous source of radiation includes deuterium lamp, xenon lamp, mercury vapour lamp and tungsten lamp.

Continuous source gives a wide range of radiation but only a small band of radiation passed by the monochromator is absorbed and a large portion of the unabsorbed radiation falls on the detector which reduces sensitivity. Therefore, one needs a high concentration of sample.

Line source include the laser but most common is the hollow cathode lamp which is made of metallic or alloy of interest. Open end of the hollow cathode faces the anode

50 which is made of either tungsten wire or disc or ring. Window of cathode is made of borosilicate glass or quartz for UV. The lamp is filled with inert gas as argon. Sufficient potential is applied to ionize the argon gas at the anode and accelerate it towards the cathode where it knocks off (or vaporizes) metal ions at the surface of the cathode, a process known as sputtering. Further collisions of free atoms with the ionized inert gas result in the atoms being excited. The excited metal atoms return to ground state by emitting characteristic radiations.

The two types of atomizers used are flame and electrothermal (flameless) atomizers. In flame atomizers, ratio of fuel and oxidant and the type of oxidant used determine the flame temperature. A nebulizer converts a liquid sample into a fine spray of solid molecular aerosols, which is the fed into the flame. In the flame dissociation takes place leading to atomic gas.

In the electrothermal atomizer, an electric furnace is used and sample is heated in stages.

The atomizers are made of carbon or graphite rod heated in an electrical discharge. A small sample of usually order of 2-30μL is loaded and first evaporated at a low temperature and the ashed at a higher temperature. After ashing, the current is rapidly increased to several hundred amperes, causing the temperature to increase to 2000-

3000oC. This causes atomization of the sample in a few milliseconds to seconds.

Monochromators are analyzers that present monochromatic radiations onto the detector and normally disperse or separate radiations so that selected wavelength corresponding to

51 a particular energy within the sample are transmitted. The simplest of the monochromators are filters which are made of materials that absorb a certain range of wavelength and allow others to go through. Others are gratings and prisms that separate radiations. Diffraction gratings are preferred to prisms as they offer accuracy over a wide range of wavelengths.

Detectors are transducers that convert electromagnetic radiations into electrical signal which is measured and related to intensity of radiation. They include phototube, photomultiplier tube, photovoltaic cell and photodiode array detectors. These are digital and interfaced with microprocessors that allow the programming of various aspects, bringing simplicity in the operations. A schematic diagram of equipments used for AAS is shown in Figure 2.6.

Hollow Lens Burner/Nebulizer Slit Monochromator Slit Detector and Cathode readout Lamp system

Figure 2.12: Schematic diagram of equipments used for AAS. Source: (Skoog et al., 2001).

52

2.4.4 High performance liquid chromatography (HPLC)

2.4.4.1 Theory of HPLC

In HPLC analysis mutually immiscible phases, stationary and mobile, are brought in contact. A solute (sample) is introduced in the mobile phase and is carried along through the column containing uniformly distributed stationary phase. Species undergo repeated interactions between mobile and stationary phases. When in mobile phase the species are carried forward with it but remain virtually stationary during the time they spend in stationary phase. The rate of migration of each solute is therefore determined by the proportion of time it spends in the mobile phase (distribution ratio). This leads to the separation of solute into bands in the mobile phase. At the end of the process, separated components are eluted in order of increasing polarity with the mobile phase. The eluted component passes through a flow cell where it is irradiated with the monochromatic light.

The sample concentration in the flow cell is related to the fraction of the light transmitted through the cell by Beer’s law as shown by Equation 2.4.

Log Io/I = εbc ------(Equation 2.4)

Where: Io is the intensity of incident light I is the intensity of transmitted light ε is the molar absorptivity (molar extraction coefficient) of the sample b is the path length in cm c is the concentration of the sample in moles per litre

The light absorption HPLC detectors are designed to provide an output in absorbance that is linearly proportional to the concentration of the sample in the cell as shown in

Equation 2.1.

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2.4.4.2 HPLC instrumentation and components.

A schematic diagram of HPLC instrumentation is shown in Figure 2.6

Solvent reservoir

Gradient device

Pump

Pressure gauge Sample injector Column Detector Recorder

Figure 2.13: Schematic diagram of HPLC instrumentation. Source: Skoog et al.,

(2001).

Solvent reservoir contains the mobile phase from where it is pumped through the feed line. The feed line is fitted with a filter to prevent the particles from being drawn in to the pump to prevent clogging (Skoog et al., 2001). The gradient device is used for changing the mobile phase composition during the analysis. Normally the HPLC system is classified into isocratic systems where the mobile phase composition is kept constant and the gradient system where the mobile phase composition is continually changed during the analysis. Gradient elution can be able to separate complex mixtures having widely varying capacity factors (Mendham et al., 1999).

The pump is used to overcome the resistance offered by the smaller particles used to pack the columns hence higher pressure is used. The pump produces a constant reproducible supply of the mo bile phase to the column. The pressure gauge monitors the pump

54 pressure through the column and indicates any problem such as plugging or leaks, while the sample injector device, usually a loop fitted with a fixed volume sample into the column. The use of pressure increases the linear velocity (speed) giving the components less time diffuse within the column, leading to improved resolution in the resulting chromatogram. Modern HPLC systems have been improved to work at much high pressures, and therefore are able to use much smaller particle sizes in the column (> 2

μm). This can work up to 100 Mpa (15000 Ibf/in2), or about 1000 atmospheres (Skoog et al., 2001).

The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of the retardation depends on the nature of the analyte, stationary phase and mobile phase composition (Skoog et al., 2001;

Mendham et al., 1999; Fifield and Kealy, 1990). The column consists of a tube of stainless steel which has been tightly packed with small particles of the material used to effect separation. They are normally short and straight to avoid excessive pressure drops and air pockets which will lead to tailing of peaks.

The column is the core of any chromatography and primary aim of designing any chromatographic procedure must ensure that the full potential of the column is realized.

Widely used column packing materials are rigid solids based on silica matrix which can withstand high pressure. The mobile phase contains exchangeable counter ions to keep neutrality. During separation, the sample ions exchange with counter ions which is

55 mainly applicable to the analysis of fats. Exclusion/ Gel permeation chromatography has separation based upon the molecular sizes of the solute. The stationary phase consists of a gel packing with an inert porous surface such that during separation small molecules enter the network and larger ones pass through unretained. This method is mainly used for fractionation of polymers (Mendham et al., 1999).

There are several ways of detecting when a substance has passed through the column.

Commonly used HPLC detectors are UV/Visible absorption, fluorescence and refractive index detectors. The output from the detector is recorded as a chromatogram (series of peaks) - each peak representing a compound in the mixture passing through the detector and absorbing UV light when UV/Vis detector is used (Mendham et al., 1999; Fifield and

Kealy, 1990).

2.5 Precautions in carotenoid and tocopherol analysis

Light, heat and oxygen cause oxidation of carotenoids, and tocopherols. Metal salts of iron, zinc and silver and unsaturated fatty acids catalyze the oxidation by oxygen

(Nyambaka, 1998). Completion of analysis within the shortest time possible, exclusion of oxygen, protection from light, avoiding high temperatures and contact with acids and use of high analytical grade solvents help to avoid formation of artifacts and large losses

(Tonucci et al., 1995). Antioxidant addition during extraction process helps to prevent vitamin oxidation (Nyambaka, 1998). Such antioxidants include butylated hydroxytoluene (BHT), pyrogallol, ascorbic acid, ethoxyquin, sodium ascorbate, sodium sulphite, and hydroquinone, BHT being the most commonly used (Tonucci et al., 1995).

56

Most of the carotenoids are stable under alkali conditions but decompose, dehydrate or even isomerizes in the presence of acids (Tonucci et al., 1995). Effect of light is reduced using suitable blinds or tinted glass, or wraping containers or chromatography columns using aluminum foil. UV-light causes the carotenoids and tocopherol to undergo structural photo-transformations. Immediate analysis of samples helps to prevent oxidation and polymerization. If the sample is not analyzed immediately it should be stored frozen in the dark at -70oC (Furr et al., 1992). All solvents should be degassed prior to use to remove the molecular oxygen which absorbs in the UV region (Tonucci et al., 1995).

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CHAPTER THREE

3 METHODOLOGY

3.1 Research design

The study analyzed and compared the levels of some micronutrients in varieties of indigenous fruit samples of Psidium guajava L, Rhus vulgaris M, and Physalis peruviana

L from ten sub counties of Kakamega County and compared with RDA requirements.

3.2 Study area

The study area was Kakamega County which is found on latitude 1 o N and longitude 38 o

E. the county lies within altitude 1250-2000 m above sea level. The local inhabitants are mostly the Luhya tribe whose economic activity is mainly farming. It has a population of

1,660,651 (USAID, 2011). The region receives equatorial type of rainfall with long rains in the months of April to August and short rains in the months of September to November with average annual rainfall of 1250-1750 mm. December to March is usually characterized by a dry spell. Kakamega County has exotic fruits such as bananas, avocadoes, passion, mangoes, pawpaw and pineapples and indigenous fruits including guavas of different varieties, Rhus vulgaris M, Physalis peruviana L, and amarula.

Fruiting of most indigenous fruits occurs in rainy season. The map of Kakamega County and population densities of the sampling sub-counties is shown in Figure 3.1.

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Figure 3.1: Population density of Kakamega County

3.3 Sampling

Sampling of fruits was carried out during the ripe fruit season in the months of April,

August, September and October. The wild fruits were collected randomly by hand picking healthy fruits from all sides of the plant canopy. For guavas, eighteen fruits of each kind were picked from each sampling area (sub-county), six fruits from each of the three sampling points within the sampling area. For Physalis peruviana and Rhus vulgaris several were picked because they are small in size. They were packed in

59 perforated black polythene bags, labeled, put in a cool box and transported to the laboratory and preserved in deep freezer until analysis time.

3.4 Sample treatment

In the laboratory the samples were washed under tap water and then in distilled de- ionized water to remove any dust and other unwanted particles. For the analysis of trace elements guavas were cut into small pieces by use of a pre-cleaned stainless steel kitchen knife and then dried at 100 oC for twenty four hours to ensure constant weight. Physalis peruviana and Rhus vulgaris were similarly dried but without cutting into small pieces because of their small size. The dried fruits were then ground using heavy duty blender

(MRC scientific instruments) and pulverized into fine powder and put in self-sealing polythene bags, labeled and preserved. The dry samples of Physalis peruviana were stored in desiccators because of their hygroscopic nature. For analysis of vitamins fresh fruits were crushed in a mortar and using pestle to give a homogenized paste.

3.5 Chemicals, reagents and solvents

All chemicals, reagents and solvents used in this research were of analytical grade. Nitric acid and hydrochloric acid were obtained from Thomas Baker Chemicals Ltd. Mumbai

India and perchloric acid purchased from Sigma- Aldrich Laboratory Chemicals.

Chromium, iron, zinc, copper, manganese and vitamins standards were sourced from

Fluka Chemical Company, USA. Distilled de-ionized water was used throughout the study.

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3.6 Cleaning of glassware and sample containers

All sampling containers and glassware used were washed with liquid detergent and hot water. They were then rinsed several times in tap water, and then soaked in 10 % analytical grade nitric acid for 24 hours to leach out adsorbed metal ions. They were then rinsed in tap water followed by distilled deionized water. Glass wares were oven dried at about 80 oC and plastic bottles dried on open racks. All the apparatus were stored in dry and clean lockable drawers.

3.7 Instrumentation

HPLC (Shimadzu) was used to analyze β-carotene, α-tocopherol and L-ascorbic acid.

Table 3.1 shows the HPLC operating conditions.

Table 3.1: HPLC operating conditions

Operating Ascorbic acid β-carotene α-tocopherol parameters Wavelength (nm) 254 297 297

Column Reversed column Reversed column Reversed column (ODS (ODS 4.6X250mm) (ODS 4.6X250mm) 4.6X250mm) Detection limit(μg/mL) 0.5 0.17 0.09

Mobile phase ACN:n-hexane: ACN:Methanol:TEA ACN:Methanol:TEA ethanol (50:40:40) (50:50:0.01) (50:50:0.01)

Mobile flow rate 1.2 1.2 1.2 (mL/minute)

Sensitivity (aufs) 1.00 1.00 1.00

AAS (Varian spectra, 10 model) was used to analyze trace elements iron, zinc, chromium, manganese and copper. Table 3.2 shows the AAS operating conditions.

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Table 3.2: AAS operating conditions

Operating Zinc Iron Manganese Copper Chromium parameters Wavelength 213.9 248.3 279.5 324.7 357.9 (nm) Slit width 1.0 0.2 0.2 0.2 0.2 (nm) Flame type Air/acetylene Air/acetylene Air/acetylene Air/acetylene Air/acetylene Oxidant flow 1.5 1.5 1.5 1.5 1.5 rate (L/min) Sensitivity 0.009 0.062 0.024 0.04 0.055 (ppm) Detection 0.01 0.5 0.003 0.003 0.02 limit (ppm) Flame Oxidizing Oxidizing Oxidizing Oxidizing Reducing stoichiometry Lamp current 5 5 5 3 5 (mA) Optimum 0.4-1.6 2.5-10 2.0-8.0 2.0-8.0 1.0-4.0 working range (ppm)

3.8 Laboratory procedure

3.8.1 Preparation of standard solutions

3.8.1.1 Standard solutions for AAS

Stock solutions were prepared from analytical grade granulated metals of high purity

(99.9 %). Each metal was first dried at 105 oC, cooled in desiccators prior to weighing

and transferred into 250 mL volumetric flasks.

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Zinc stock solution was prepared by dissolving 0.25 g of zinc metal in 40mL HCl (1:1) and diluting it to the mark using distilled de-ionized water to give 1000 μg/mL.

Chromium stock solution was prepared by dissolving 0.25 g of the metal in 30 mL HCl

(sp gr. 1.18) with gentle heating then cooled and diluted to a litre to give 1000 μg/mL.

Iron stock solution was prepared by dissolving 0.25 g of the metal in 40 mL of HCl and diluting it to the mark using distilled de-ionized water to give 1000 μg/mL.

The stock solutions were stored in polyethylene bottles and labeled appropriately.

Working standards were freshly prepared from stock solutions each time an analysis was to be carried out using serial dilution procedure. Five working standard solutions of varied concentrations in ppm were prepared for metals analyzed.

3.8.1.2 Standard solutions for HPLC

Stock solutions of β-carotene and α-tocopherol each containing 100 μg/mL (100 ppm) were prepared by weighing 10.0 mg of the β-carotene and α-tocopherol acetate standard reagents into 100 mL volumetric flask. 5 mL of acetone was added to aid in dilution. The solution was made to the mark using n-hexane to make 100 ppm. The stock solutions, which were kept under refrigeration, could be used for a period of about 4 weeks. A working solution consisting of β-carotene and α-tocopherol mixture was prepared by taking an aliquot from each stock solution (such as 1.0 mL) into a 100 mL round bottomed flask and treating it according to extraction procedure given in the next page.

However solvent extraction was done once, while the residue after vacuum evaporation

63 was dissolved in 10 mL of ethanol. When kept under refrigeration conditions, this solution could be used for about one week. Working standards of 2, 4, 6, 8, and 10 ppm were prepared.

The solutions for the HPLC were prepared according to Skoog et al., (2001). Ethanolic potassium hydroxide was prepared by dissolving 5.60 g of potassium hydroxide dissolved in 100 mL of distilled de-ionized water contained in 1000 mL volumetric flask and then made up to the mark using ethanol and then shaken. Sodium sulphite was prepared by dissolving 12.60 g of sodium sulphite powder in 100 mL beaker using distilled de-ionized water. This was then transferred to 1000 mL volumetric flask and made up to the mark using distilled de-ionized water. Potassium hydroxide was prepared by dissolving 5.60 g of potassium hydroxide in 100 mL beaker using distilled de-ionized water. It was then transferred to 1000 mL volumetric flask and the made up to the mark.

3.8.2 Method validation

Validation of the methods to determine the reliability was done through calibration

(linearity), reproducibility, recovery and detection limit.

3.8.2.1 Calibration

The AAS instrument was calibrated using freshly prepared working standards with wavelengths set at 248.3 nm, 213.9 nm, 357.9 nm, 279.5 nm, and 324.7 nm for iron (4, 6,

8, and 10 ppm), zinc (2, 4, 6, 8, and 10 ppm), chromium (0.5, 1.0, 1.5 and 2.0 ppm), manganese (0.5, 1, 4, and 8 ppm), and copper (0.5, 2, 4 and 5 ppm) respectively. The

64 standard solutions of increasing concentrations were nebulized into the flame. Plotting the absorbance of the standards against concentrations (ppm) generated calibration curves for the elements. The process was done at the start and in the course of the sample readings.

The HPLC was calibrated using freshly prepared standards ( 2, 4, 6, 8, and 10 ppm ) with the wavelength set at 297 nm for β-carotene and α-tocopherol which were analyzed simultaneously and (100, 500, 1000 and 1500 ppm) standards for L-ascorbic acid set at wavelength 254 nm. The standard solutions of increasing concentration were injected into the HPLC column. The peak areas of the standards were plotted against concentration of standards (ppm) to generate calibration curves. Precision of the analytical methods were obtained on replication of the analysis of samples and the coefficient of variation calculated using Pearson’s coefficient of correlation.

3.8.2.2 Reproducibility

The reproducibility test for AAS was done by analyzing five sub-samples of ready samples of red bitter and red sweet guavas six times. For HPLC white sweet guavas, R. vulgaris and P. peruviana samples were used. The mean, standard error and coefficient of variation were then calculated. The analytical instrument used for sample analysis was also the one used for reproducibility test.

65

3.8.2.3 Recovery

Recovery was done to validate both the HPLC and AAS methods. Recovery data for

HPLC was obtained by adding known amounts of β-carotene, α-tocopherol and L- ascorbic acid standards to the homogenized fruit samples of white sweet guavas, R. vulgaris and P. peruviana that were ready for extraction and comparing the increased calculated vitamins content to the amounts added. The recoveries of the vitamins after liquid phase extraction were calculated by comparing observed β-carotene, α-tocopherol and L-ascorbic acid peak areas in the extract to those of non-processed standard solutions. Recovery data for AAS was obtained by analyzing the spiked sample and the percentage recovery determined. This was done by adding 10 mL of 10 ppm of a standard to equal volume of sample.

3.8.2.4 Detection limit

Concentrations of zinc, iron, manganese, copper and chromium in the samples were determined from calibration graphs. Detection limit is the concentration of the element that produces an analytical signal equal to twice the standard deviation of the background and is given by the y-intercept ± 3 standard deviation of the blank while sensitivity is a measure of the instrument’s response to the analyte (Richard et al., 2005). Elements with greater sensitivity will have the lowest concentration values in that category. The values of sensitivity in Table 3.2 above are amounts in ppm required to give an absorbance reading of 0.200.

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3.8.3 Sample preparation

3.8.3.1 Determination of β-carotene and α-tocopherol

This was done according to the procedure provided by Nyambaka (1998) with some changes. A sample of 3.00 g was weighed and crushed in a mortar and pestle. It was then transferred into 150 mL round bottomed flask and 50 mL of 0.5 M ethanolic potassium hydroxide, 5.0 mL of 10% sodium sulphite solution (w/v) and 0.30 g of ascorbic acid added. The mixture was dispersed in the solution with a glass rod. 30 mL of n-hexane was added to the mixture, shaken thoroughly for 2 minutes and allowed to separate. The hexane layer was decanted into 250 mL separating funnel and corked. The residue was similarly re-extracted with 30 mL n-hexane 5 times and the n-hexane layer decanted. The combined n-hexane layer in the separating funnel was washed with 50 mL of 1.0 M potassium hydroxide solution, followed by portions of 50 mL of distilled water until there was no colouration on phenolphthalein solution. The hexane layer was the dried by filtering over anhydrous sodium sulphate and evaporated to almost dryness in a rotatory evaporator at 40 oC. The residue was immediately dissolved in 5 mL of ethanol and 20

µL aliquot of the solution was be injected into the HPLC system. This procedure was performed in minimum light in a dark room and samples protected from light with aluminum foil. Some steps were carried out promptly and fast to avoid vitamin oxidation by air. The samples were analyzed in triplicates.

Vitamin A equivalent of beta-carotene was determined in terms of the Retinol Activity

Equivalent (RAE) as shown in Equation 3.1.

12 μg all beta-carotene= 1μg RAE ------(Equation 3.1)

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3.8.3.2 Determination of L-ascorbic acid

This was done according to the procedure outlined by Shimada and Ko (2006) with some changes. Fruits were chopped into small sections with a kitchen knife. 10 g of these small sections were homogenized with 10 mL of 10% metaphosphoric acid in a mortar and pestle. The slurry obtained was transferred to a centrifuge tube with 20 mL of 5% metaphosphoric acid and centrifuged for 20 minutes at 3000 rpm. The supernatant was diluted 20 fold with distilled water. 10 mL of diluted supernatant had its pH adjusted with

0.13 mL of 2.5 M K2HPO4 to give a final pH of 7.0. A 20 μL aliquot of this solution was injected into the HPLC system.

3.8.3.3 Determination of trace metallic elements

The dry ground samples were re-dried at 104 oC in an oven for an hour and then cooled in desiccators. Exactly 3.00 g of each sample was weighed (analytical balance, model ATX

224 S/N D307000623, Shimadzu corporation, Japan) into a pre-washed, dry pyrex beaker and 18.0 mL of concentrated analytical grade nitric acid was added. The mixture was then heated on a hot Figure at 70-80 o C for one hour until the dense brown fumes began to clear, and then allowed to cool to room temperature. Afterwards, 3.0 mL of concentrated perchloric acid was added and digestion allowed to continue on the hot mantle until the solution was clear and white fumes observed. The solution was allowed to cool and filtered (Whatman No 42 filter paper) into 100 mL volumetric flask and then diluted to the mark with distilled de-ionized water. They were then transferred into separate plastic bottles, sealed and labeled appropriately and stored at room temperature.

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The samples were analyzed of iron, zinc, chromium, manganese and copper in triplicates under the same conditions of standard and blank. For better precision, standards were measured before and after the sample solutions. The blank was measured between the standards and the samples to ensure stability of the base line.

The concentration of each element in mg/g of the dry weight from the AAS instrument was done using Equation 3.2

3.9 HPLC separation and quantification

Separation on HPLC systems was performed by isocratic elution of the mobile phases in a reversed phase column (ODS 4.6x250 mm) and mobile phase for β-carotene and α- tocopherol acetonitrile: methanol: triethylamine (50:50:0.01) and acetonitrile: n-hexane: ethanol (50:40:40) for L-ascorbic acid. The elution took 8 minutes for the carotene and tocopherol and 4 minutes for L-ascorbic acid at a mobile flow rate of 1.2 mL/minute. The vitamins were monitored using a SPD 20 UV detector at 297 nm (for β-carotene and α- tocopherol) and at 254 nm for L-ascorbic acid at a sensitivity of 1.00 absorbance units full scale (aufs). The individual analytes in the samples were identified by comparing the retention times with those of standard solutions.

Peak areas were used for quantification of vitamins expressed in μg/g or μmol/L weight of fruits using Equation 3.3

69

μ μ

Where:

Pa(s) is the peak area of analyte (mm2) Conc(std) is the concentration of standard solution (μg/g or μmol/L) Vol(s) is volume of the sample solution (ml or μL) Pa(std) is the peak area of standard (mm2) W(s) weight in grams of fruit samples used

3.10 Data analysis

The data obtained from the various determinations was statistically analyzed and results given as mean ± standard error of the nutrients in the varieties of fruits and presented in tables. One way ANOVA was employed in comparison of micronutrient concentration in various fruits in different and within sub counties at p-value <0.05 which was considered significant. A post ANOVA was employed using Student Newmann Keul (SNK) test for measurements that showed a significant difference.

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CHAPTER FOUR

4 RESULTS AND DISCUSSION

4.1 Introduction

The results obtained from the study are presented in tables and figures and then discussed. The arrangement of the results is as per the objective.

4.2 Method validation

4.2.1 Method validation for trace elements

The calibration curves for the elements were generated by plotting the absorbance of the standards against concentrations in ppm. The calibration curve of manganese is shown in

Figure 4 while the calibration curves for the other trace elements are shown in Appendix

I.

0.5 y = 0.0571x - 0.0006 R² = 0.9971 0.4

0.3

0.2

0.1

0 0 2 4 6 8 10 -0.1

Figure 4.1: Calibration curve for manganese

71

The R2 values ranged between 0.9933-0.9999, hence indicating that over 99.33% of the responses correlate to concentration (Table 4.1). The correlation coefficients found in this study are comparable with those reported elsewhere. Kipkemboi (2011) reported

0.99987, 0.99992, 0.99978, 0.99993 and 0.99986 correlation values for chromium, copper, iron, manganese and zinc respectively. Ngigi (2014) reported R2 values of zinc and iron as 0.980 and 0.996 respectively. The regression lines show a linear relationship between the analytical signals (y) and the concentration (x), with a good linearity of r2>0.99, which assures accurate measurements (Miller and Miller, 1998). Thus, the AAS instrument used in this study had reliable response and therefore valid for analysis of the samples. The regression parameters, detection limits and percentage recovery are given in

Table 4.1.

Table 4.1: AAS method validation statistics

Element Correlation Regression equation Detection Recovery

coefficient limit(ppm) (%)

Fe 0.9976 y=0.037x+0.022 0.5 99.99

Zn 0.9933 y=0.04x+0.008 0.01 99.98

Cr 0.9999 y=0.03x 0.02 96.78

Mn 0.9971 y=0.057x 0.003 98.99

Cu 0.9933 y=0.060x+0.002 0.003 99.09

Recovery data was obtained by analyzing the spiked sample to determine percentage recovery. Recovery was ≥ 96.78% which shows that the method of analysis used was reliable (Taylor et al., 2006). Onyambu (2014) reported a recovery of 97.50% (Zn),

72

98.00% (Mn) and 98.57% (Cr). The detection limit of the AAS instrument was calculated using the formula y-intercept ± 3SE (standard error) of blank. This study had detection limit ranging from 0.003 to 0.5 pmm which lies within the acceptable limits for trace elements. Kipkemboi (2009) reported detection limit of 0.0219, 0.464, 0.0431, 0.0302 and 0.0143 for chromium, copper, iron, manganese and zinc respectively.

Five sub-samples of the red bitter and red sweet guavas were pretreated, extracted and analyzed as per procedure outlined in section 3.4 and 3.8.3. The coefficient of variation was calculated and used to determine the reproducibility of the AAS method. Table 4.2 shows the reproducibility of AAS for the analysis of trace elements.

Table 4.2: Reproducibility analysis of trace elements in two varieties of fruits

Fruit Element Mean ± SE (mg/100g) Variation (%) n=5

Red bitter guava Zn 2.04 ± 0.10 9.80 Fe 0.50 ± 0.02 8.00 Mn 10.90 ± 0.42 7.71 Cu 3.00 ± 0.04 2.67 Cr 0.34 ± 0.01 5.88

Red sweet guava Zn 3.04 ± 0.10 6.62 Fe 0.62 ± 0.01 3.23 Mn 8.01 ± 0.31 7.74 Cu 1.80 ± 0.04 4.44 Cr 0.30 ± 0.01 6.67

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The coefficient of variation ranged from 6.62 % to 9.80 % for Zn, 3.23 % to 8.00 % for

Fe, 7.71 % to 7.74 % for Mn, 2.67 % to 4.44 % for Cu and 5.88 % to 6.67 % for Cr. All the values are less than ten. This shows that the AAS method used was reproducible and hence valid (Miller and Miller, 1998).

4.2.2 Method validation for HPLC

Calibration curves were generated by plotting peak area counts of the standards against their concentration in ppm. The calibration curve for beta carotene is shown in Figure 4.2 and the other vitamins are shown in appendix II.

Peak area against concentration 900000 800000 y = 51105x + 2904.4 700000 R² = 0.9997 600000 500000 400000 300000 200000 100000 0 0 5 10 15 20

Figure 4.2: Calibration curve for beta-carotene

Table 4.3 shows the data obtained for various procedures of validation for the HPLC method.

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Table 4.3: Validation data for HPLC method

Micronutrient Correlation Regression Detection Recovery

coefficient equation limit (%)

α-tocopherol 0.999 y=54052x+2964 0.09 92.0

β-carotene 0.999 y=50901x+5205 0.17 94.8

L-ascorbic acid 0.999 y=271.9x-6336 0.5 88.2

The data from the table indicates that 99.9 % of the responses correspond to the concentration. The correlation coefficients obtained in this study are comparable to those obtained in other studies. Nyambaka and Nyaga (1991) obtained r2=0.9970 for β-carotene and α-tocopherol using a HPLC system consisting of μ Bondak C18 reversed phase column and a methanol-ACN-chloroform-water in ratio 46:30:18:6 mobile phase at a wavelength of 297 nm. Nambafu (2011) obtained r2=0.999 for both β-carotene and α- tocopherol using a mobile phase consisting of methanol:DCM:water (83:15:2) containing

0.1 % BHT at a wavelength of 297nm for both β-carotene and α-tocopherol. Nderitu

(2006) reported r2=0.9987 for β-carotene using a HPLC system and mobile phase of methanol:DCM:water (76:15:2) and a detector limit of 450 nm. Nawiri (2008) reported r2=0.9981 using a HPLC system and mobile phase of methanol:DCM:water (83:15:2) and a wavelength of 450 nm.

These values indicate that there was a linear relationship between the chromatographic peak areas and the concentrations of β-carotene or α-tocopherol. The calibration lines also show that there was good response of HPLC detectors to different concentrations of

75 the analytes (Meyer, 1984). The calibration curves for the micronutrients were used to determine the concentration of the same micronutrients in the fruit samples.

The mean recoveries of β-carotene, α-tocopherol and L-ascorbic acid were 94.8 %, 92.0

% and 88.2 % respectively (Table 4.3). The results were similar to what has been reported in other studies. Nambafu (2011) reported a mean recovery of 94.8 % of β- carotene and 95.3 % of α-tocopherol in pumpkin and 98.4 % β-carotene and 97.6 % α- tocopherol in nightshade. Nyambaka (1998) reported a mean recovery of 94.3 % for β- carotene and 93.5 % for α-tocopherol. Nderitu (2006) reported a mean recovery of 94.3

% for β-carotene. Recoveries of between 90-110% indicates a satisfactory extraction procedure (Milller and Miller, 1998).

Five subsamples of three fruit varieties were pretreated, extracted and analyzed six times as per the procedure outlined in section 3.4 and 3.8.3. The coefficient of variation was used to determine the reproducibility of the methods. The results are given in Table 4.4.

Table 4.4: Reproducibility results of vitamins in some indigenous fruits

β-carotene α-tocopherol ascorbic acid Fruit Mean±S.E Variation Mean±S.E Variation Mean±S.E Variation n=6 (%) n=6 (%) n=6 (%) WS 3.33±0.10 6.06 0.17±0.00 4.71 189.01±4.4 4.66

RV 1.89±0.03 3.17 0.21±.00 9.52 41.30±1.06 5.31

PP 1.89±0.02 2.12 0.13±0.00 3.08 40.17±1.41 7.02

WS- White sweet guava RV- Rhus vulgaris M PP- Physalis peruviana L

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The coefficients of variation ranged from 2.12 % to 6.06 % for β-carotene, 3.08 % to 9.52

% for α-tocopherol, and 4.66 % to 7.02 % for ascorbic acid at 95% confidence level. All the values were less than ten, hence the AAS method used was reproducible and valid

(Miller and Miller, 1998). The results obtained are comparable to those obtained in the study by Nyambaka and Nyaga (1991) who reported a coefficient of variation range of

2.0-7.3 % for β-carotene and 2.1-6.3 % for α-tocopherol. Nambafu (2011) reported a coefficient of variation range of 2.0-3.1 % for β-carotene and 2.3-6.3 % for α-tocopherol.

4.3 Micronutrients levels

4.3.1 Levels of essential trace elements

The fruits analyzed in this study are Psidium quajava L (white sweet guavas, red sweet guavas, and red bitter guavas, Figures 2.1-2.3), Physalis peruviana L (Figure 2.4) and

Rhus vulgaris M (Figure 2.5). The results obtained for dry matter of the fruits are presented in Table 4.5 for the individual sub counties in Kakamega County. There was no significant difference in the levels of trace elements in all the varieties analyzed in this study (p ˂ 0.05) at 95 % confidence level using one way Anova apart from iron in

Kakamega North, Kakamega Central, Matete, Butere and Likuyani and chromium in

Mumias among the fruit varieties. The mean levels of nutrients in each fruit variety are discussed in the sections that follow.

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Table 4.5: Levels of trace elements in dry indigenous fruits in sub counties of Kakamega County

Mean ± SE (mg/100g) n=30 Element Fruit Kak. Central Kak. North Kak. East Matete Likuyani Butere Mumias Matungu Kak. South Khwisero p-value WS 1.01±0.66cA 1.26±0.68abA 3.45±0.56bC 2.02±1.04bB 4.98±0.50cD 3.50±1.60cC 4.88±0.20cD 5.09±1.90cE 3.99±1.01bC 2.48±0.12bB RS 0.15±0.24abA 1.64±0.51aB 5.29±1.10dE 1.64±0.04aB 4.79±1.83bD 1.69±0.18bB 1.97±0.07bB 5.49±0.43cE 4.91±0.97dD 2.65±0.98bC Zn RB 0.82±0.13bA 0.95±0.55aA 1.31±0.65aB 1.23±0.26aA 2.21±2.08abC 3.96±0.28cD 4.87±0.20cE 3.08±1.10aD 0.97±0.04aA 1.00±0.21aB PP 4.60±3.30dC 4.54±2.40bB 4.87±2.22cE 4.87±2.22cE 4.50±1.30abB 4.55±1.55dB 4.23±0.52cA 4.62±0.59bB 4.46±0.58cB 4.22±1.37cA RV 0.92±0.29bB 1.40±0.62bC 1.46±0.11aC 1.02±0.37aA 1.49±1.29aC 0.88±0.24aA 0.67±0.34aA 2.01±1.27aD 1.41±0.20aC 0.97±0.55aB <0.001 p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 WS 0.48±0.17A 0.87±0.07A 0.59±0.07aA 0.82±0.14A 2.56±1.77B 0.73±0.12A 0.68±0.07aA 0.66±0.09bA 0.92±0.08bA 0.45±0.13aA RS 2.26±1.54C 0.38±0.12A 0.32±0.04aA 1.32±0.21B 0.58±0.14A 0.47±0.10A 0.46±0.10aA 0.34±0.08aA 0.61±0.05aA 0.44±0.04aA Fe RB 0.53±0.07A 1.25±0.19B 0.42±0.16aA 0.47±0.04A 0.50±0.03A 0.38±0.08A 0.46±0.02aA 0.35±0.08aA 0.50±0.05aA 0.64±0.10aA PP 1.29±0.03A 1.23±0.16A 1.48±0.09bA 1.11±0.07A 1.36±0.05A 3.15±1.84B 1.20±0.00bA 1.23±0.02cA 1.25±0.04cA 1.47±0.07cA RV 1.71±0.13B 0.99±0.41A 1.79±0.09bB 1.08±0.34A 1.45±0.02B 1.72±0.07B 1.75±0.07cB 1.51±0.04dB 1.06±0.09bcA 0.92±0.08bA <0.001 p-value 0.089 0.099 <0.001 0.076 0.080 0.069 <0.001 <0.001 <0.001 <0.001 WS 10.07±0.21cD 7.18±2.10cA 7.21±1.98bA 7.43±2.00bA 8.04±0.31B 9.59±0.70cC 8.40±0.05bB 8.45±0.12bB 8.07±0.54cB 9.29±0.08dC RS 0.67±0.16aA 0.58±0.59aA 0.73±0.31aA 6.5.46±0.16aD 15.34±1.22E 1.67±0.16aB 1.59±0.59aB 2.50±0.14aC 6.57±0.06aD 5.76±0.23aD Mn RB 7.34±0.17bB 5.33±2.01bA 15.38±1.50cC 16.60±1.31cD 8.93±1.82B 8.31±0.13cB 16.22±0.13dD 16.98±0.73dD 4.37±0.05dA 9.28±0.04cB PP 7.73±0.21bB 8.07±1.02cC 9.25±0.09bD 8.70±0.07bC 8.11±0.04C 6.15±0.49bB 8.28±0.18bC 7.51±0.18bB 4.76±0.18bA 4.48±0.05bA dC dE G dB cA cD eC eB RV 11.60±3.24dB 12.59±1.36 14.62±0.24dD 24.36±0.15 26.45±9.47 11.63±0.77 7.95±0.24 14.30±0.35 12.70±0.57 11.92±0.51 <0.001 p-value 0.044 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 WS 1.49±0.30aA 1.92±0.42bA 1.86±0.21aA 3.50±0.33bcB 1.82±0.04aA 1.48±0.02aA 2.09±0.26bB 1.74±0.09aA 1.33±0.09aA 1.37±0.07aA RS 3.11±0.02bB 1.24±0.08aA 11.02±0.04dC 2.56±0.07aA 11.03±0.04dB 3.11±0.02bB 1.15±0.02aA 2.49±0.10bA 10.25±0.11dC 2.33±0.03bA Cu RB 3.25±0.05bA 3.24±0.05cA 3.11±0.02cA 2.82±0.09abA 3.08±0.03cA 3.25±0.06cA 3.24±0.05dA 4.21±0.03cB 3.17±0.07cA 3.20±0.06bA PP 14.62±0.94cC 13.28±0.05dB 13.39±0.01eB 3.39±0.03bcA 13.29±0.01eB 13.38±0.01dB 14.51±0.24eC 15.37±0.02dD 15.43±0.00eD 14.08±0.65cB RV 3.54±0.00bB 2.37±0.18bA 2.74±0.00bA 3.66±0.20cB 2.70±0.01bA 3.13±0.01bB 2.68±0.01cA 2.68±0.01bA 2.58±0.01bA 2.70±0.02bA <0.001 p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 WS 0.12±0.00aA 0.11±0.07aA 0.41±0.80cD 0.35±0.03cC 0.28±0.01bB 0.24±0.00aB 0.27±0.00B 0.24±0.00aB 0.24±0.01aB 0.61±0.10dE RS 0.27±0.05bC 0.26±0.01bB 0.25±0.01aB 0.26±0.07aB 0.20±0.01aA 0.25±0.00aB 0.21±0.03A 0.26±0.03bB 0.26±0.03bB 0.30±0.01aD Cr RB 0.32±0.00cC 0.30±0.02cB 0.24±0.02aA 0.28±0.09bcB 0.37±0.02cD 0.32±0.02bC 0.33±0.00C 0.26±0.03bA 0.36±0.03cdC 0.36±0.01bD PP 0.36±0.02eC 0.33±0.01dA 0.37±0.02bE 0.34±0.03cA 0.36±0.01cD 0.36±0.01dD 0.39±0.13C 0.34±0.02cA 0.34±0.01cB 0.38±0.01cD RV 0.34±0.01dA 0.37±0.02eC 0.38±0.02bD 0.37±0.20dC 0.38±0.05dD 0.34±0.03cB 0.48±0.04E 0.34±0.02cA 0.33±0.02cA 0.35±0.07bB <0.001 p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.059 <0.001 <0.001 <0.001 Key: WS – White sweet guava RS – Red sweet guava RB – Red bitter guava PP – Physalis peruviana RV – Rhus vulgaris Mean value followed by the same small letter(s) within the same column for each element are not significantly different and mean value followed by the same capital letter within the same row are not significantly different (One way ANOVA, SNK test, α=0.05)

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4.3.1.1 Levels of zinc

Mean levels of zinc were significantly higher in Likuyani for white sweet guavas

(37.06±0.5 mg/100g), in Matungu (53.49±0.43 mg/100g) for red sweet guavas, Mumias

(24.87±0.20 mg/100g) for red bitter guavas, kakamega East (40.87±4.22 mg/100g) for

Physalis peruviana and Matungu (13.65±2.27 mg/100g) for Rhus vulgaris. Thus, red sweet guavas of Matungu recorded the highest levels of zinc and red sweet guavas of

Kakamega Central (1.15±0.24 mg/100g) the least amount compared with other fruit varieties in all sub-counties in Kakamega County.

There was no significant difference in the levels of zinc in white sweet guavas of

Kakamega Central and Kakamega North, Kakamega East, Matete and Khwisero and

Butere, Mumias and Kakamega South, in R. vulgaris of Kakamega North, Kakamega

East, Kakamega South and Likuyani and of Kakamega Central, Matete and Mumias and in P. peruviana of Kakamega North and Kakamega East, Kakamega Central, Matete,

Likuyani, Matungu and Butere and of Mumias, Kakamega South and Khwisero. The mean levels of zinc in red sweet guavas of Kakamega North and Khwisero and Butere,

Mumias and Matete, and Kakamega East and Matungu were not significantlly different.

Red bitter guavas of Kakamega North, Kakamega Central, Matete, and Kakamega South showed no significant difference in the mean levels of zinc. There was no significant difference in the mean levels of zinc.

Zinc levels found in this study in all the ten sub counties are greater than what was reported elsewhere by Ismail et al. (2011) in guava (0.28 mg/100g), Puente et al, (2011)

79 in P. peruviana L (0.40 mg/100g), Kipkemboi (2011) Carissa edulis (2.49 mg/100g).

Anke et al. (2005) reported a level of 4.9 mg/100g of Zn in oranges which is higher than zinc in white sweet guavas in Kakamega Central and Kakamega North, in Rhus vulgaris of Kakamega Central, Matete, Mumias and Khwisero, in red bitter guavas in Kakamega

Central, Matete and Kakamega South, and in red sweet guavas in Kakamega Central but lower than what was found in other sub counties. The variation could be due to the differences in sample varieties, soil fertility, climatic conditions or geographical site of production (Ihekoronye, 1992). Another reason of variation could be due to use of different methods of analysis.

4.3.1.2 Levels of iron

Kakamega North, Kakamega Central, Butere and Kakamega East recorded significantly higher levels of iron in white sweet guavas (2.56±1.77 mg/100g), red sweet guavas

(2.26±1.54 mg/100g), red bitter guavas (1.25±0.19 mg/100g), P. peruviana (3.15±1.84 mg/100g) and R. vulgaris (1.79±0.09 mg/100g) respectively. Mean levels of iron recorded least in red sweet guavas of Kakamega East (0.32±0.04 mg/100g) and highest in

P. peruviana of Butere (3.15±1.84 mg/100g) in comparison with other fruits studied.

Iron levels in white sweet guavas were not significantly different in all the sub-counties of Kakamega County apart from Likuyani, in red sweet guavas of all the sub-counties of

Kakamega County apart from Kakamega Central and Matete, in red bitter guavas of all the sub-counties apart from Kakamega North. Kakamega Central, Kakamega East,

Likuyani, Butere, Mumias and Matungu and in Kakamega North, Matete, Kakamega

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South and Khwisero showed no significant difference in iron levels in R. vulgaris and all the sub counties apart from Butere in P. peruviana. There was a significant difference in the levels of iron among the fruits analyzed in Kakamega Central, Kakamega North,

Matete, Likuyani and Butere.

The levels of iron in white sweet guavas found in this study are lower than what was reported elsewhere in Ficus sycomorus (75.05 mg/kg) (Kipkemboi, 2011) but are comparable with what was reported in mulberry (1.46 mg/100g) (Ngigi, 2014) and bananas (0.36 mg/100g) (Gosh, 2007). Iron levels in red sweet and red bitter guavas found in this study can be compared favourably with what was reported in mulberry (1.46 mg/100g) (Ngigi, 2014) bananas (0.36 mg/100g) (Gosh, 2007). Stanton (1995) reported higher levels of 45.6 mg/kg and 75.05 mg/kg of iron in mangoes and oranges respectively.

The levels of iron in Rhus vulgaris and P. peruviana found in this study can be compared favourably with what was reported elsewhere in mulberry (1.46 mg/100g) (Ngigi, 2014),

P. peruviana (1.24 mg/100g) (Puente et al., 2011), and mulberry (1.46 mg/100g) (Ngigi,

2014). The levels are higher than what was reported in bananas (0.36 mg/100g) (Gosh,

2007) but lower than what was reported by Musinguzi et al. (2007) in P. peruviana (7 mg/100g) and Stanton (1995) in mangoes (45.6 mg/kg) and in oranges (75.05 mg/kg).

Ramadan and Morsel (2004) reported 1.2 mg/100g of iron in P. peruviana pulp.

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4.3.1.3 Levels of manganese

There was no significant difference in the levels of manganese in white sweet guavas of

Kakamega North, Kakamega East, Likuyani, Matete, Mumias Matungu, and Kakamega

South, in red sweet guavas of Kakamega North, Kakamega East Kakamega Central,

Khwisero, Mumias and Butere and of Matungu and Kakamega South, in red bitter guavas of Kakamega Central, Butere and khwisero, of Kakamega North and Likuyani and of

Mumias, Matungu and Kakamega South, in R. vulgaris of Kakamega Central, Butere and

Khwisero, and of Kakamega South and Matungu, and in P. peruviana of Kakamega

Central and Matete, Kakamega North, Likuyani and Mumias, and Butere, Matungu and

Kakamega South.

Likuyani recorded significantly highest mean levels of manganese in R. vulgaris

(26.45±9.47 mg/100g) and Kakamega North the lowest in red sweet guavas (0.58±0.59 mg/100g) with respect to the fruit varieties studied. There was no significant difference in the levels of manganese in white sweet guavas of Kakamega North, Kakamega East and

Matete, and of Likuyani, Mumias, Matungu and Kakamega South, in red sweet guavas of

Kakamega North, Kakamega Central, Kakamega East, Khwisero, Mumias and Butere, and of Matungu and Kakamega South, in red bitter guavas of Kakamega Central, Butere and Khwisero, of Kakamega North and Likuyani, and of Mumias, Matungu and

Kakamega South. R. vulgaris of Kakamega Central, Butere and Khwisero, and of

Kakamega South and Matungu and P. peruviana of Kakamega Central and Matete, of

Kakamega North, Likuyani and Mumias, and of Butere, Matungu and Kakamega South also showed no significant difference in the levels of manganese.

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There was no significant difference in the levels of manganese in Likuyani among the fruit varieties studied at 95% confidence level. The levels of manganese in this study are favourably comparable to those reported elsewhere for Ficus sycomorus (17.021 mg/100g) (Kipkemboi, 2011) but higher than what were reported by Akan et al. (2009) in grapes (0.8 mg/kg) pineapples (15.0 mg/kg) and oranges (0.45 mg/kg).

4.3.1.4 Levels of copper

Mean levels of copper recorded highest in P. peruviana of Kakamega South (15.43±0.00 mg/100g) and lowest in red sweet guavas of Kakamega North (1.24±0.08 mg/100g). The levels of copper in white sweet guavas Kakamega Central and Butere, of Kakamega

North, Kakamega East and Likuyani, and of Kakamega South and Khwisero were not significantly different. There no significant difference in the levels of copper in red sweet guavas of Kakamega Central, Matete, Butere, Matungu and Khwisero, of Kakamega

East and Likuyani, in red bitter guavas of all the sub-counties apart from Matete and

Matungu and in P. peruviana of Kakamega North, Kakamega East, Likuyani, Butere and

Khwisero. R. vulgaris showed no significant difference in the mean levels of all the sub- counties apart from Kakamega Central and Matete. The levels of copper in all the fruit varieties found in this study are higher than what was reported elsewhere for guava (0.50 mg/100g), and mango (0.90 mg/100g) (Ismail et al., 2011), chili pepper (0.17 mg/100g), banana (0.11 mg/100g) and cucumber (0.10 mg/100g) (Ismail et al., 2011).

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4.3.1.5 Levels of chromium

White sweet guavas of Khwisero and Kakamega North recorded highest (0.61±0.10 mg/100g) and lowest (0.11±0.07 mg/100g) copper mean levels respectively. There was no significant difference in the levels of chromium in white sweet guavas of Kakamega

North and Kakamega Central, of Likuyani, Mumias, Matungu, Kakamega South and

Butere, in red sweet guavas of Kakamega North, Kakamega East, Matete, Butere, matungu and Kakamega South, in red bitter guavas of Kakamega North and Matete, of

Kakamega Central, Butere and Mumias, of Khwisero, Kakamega South and Likuyani. R. vulgaris of Kakamega Central, Butere, Matungu, Kakamega South and Khwisero and P. peruviana of Kakamega North, Matete and Matungu, of Kakamega Central and Mumias and of Likuyani, Butere and Khwisero showed no significant difference in the mean levels of chromium. The levels of chromium found in this study are higher than what was reported elsewhere for guava (14.4 µg/100g), melon (46.8 µg/100g) and banana (16.4

µg/100g) (Janabai et al., 1990). The mean levels of the trace elements for all sub counties in Kakamega County were determined (Table 4.6). The values were used to indicate the fruits that can provide high levels of trace elements.

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Table 4.6: Levels of essential trace elements in selected indigenous fruits in Kakamega County

Mean ± SE (mg/100g) Parameter Variety n=30 Range (mg/100g) p-value Zn WS 3.27±0.82c 1.01-7.06 RS 3.02±0.63c 1.15-5.29 RB 2.04±0.10b 1.12-4.87 PP 4.54±1.41d 2.86-7.54 RV 1.22±0.53a 0.97-3.65 <0.001 Fe WS 0.88±0.01a 0.45-2.56 RS 0.72±0.18a 0.32-2.26 RB 0.55±0.02a 0.35-1.25 PP 1.48±0.18b 0.48-4.25 RV 1.40±0.10b 0.11-3.15 <0.001 Mn WS 8.37±0.80b 2.18-15.07 RS 4.09±0.36a 2.58-6.224 RB 10.87±0.79c 6.93-17.98 PP 7.30±0.25b 4.48-14.25 RV 14.81±1.69d 7.95-21.25 <0.001 Cu WS 1.71±0.08a 1.33-2.09 RS 4.82±1.25c 1.15-11.03 RB 3.40±0.13c 3.08-4.23 PP 11.86±1.43d 9.23-15.43 RV 2.78±0.10b 2.37-3.54 <0.001 Cr WS 0.29±0.01a 0.11-0.61 RS 0.25±0.01a 0.20-0.30 RB 0.31±0.01b 0.24-0.36 PP 0.36±0.05b 0.33-0.39 RV 0.37±0.03c 0.33-4.08 <0.001

Key: WS – White sweet guava RS – Red sweet guava RB – Red bitter guava PP – Physalis peruviana RV – Rhus vulgaris

Mean value followed by the same small letter(s) within the same column are not significantly different (One way ANOVA, SNK test, σ=0.05)

White sweet and red sweet guavas showed no significant difference in the levels of zinc, iron and chromium, Physalis peruviana and Rhus vulgaris and white sweet, red sweet and red bitter guavas showed no significant difference in iron levels, whereas red bitter guavas and Physalis peruviana showed no significant difference in chromium levels. The levels of the essential trace elements found in Kakamega County are comparable and

85 even others higher than what was reported in other studies as has been discussed in the previous sections on individual nutrient. Table 4.7 shows amount (g) of fruit variety required to provide RDA of trace elements per day and the percentage RDA contribution by 200 g of wet fruit.

Table 4.7: Amount (g) required to provide RDA/day and the % RDA contribution by 200 g of wet fruit

Parameter Variety RDA Amount (g) to provide RDA level/ % RDA contribution (mg) day (DW): (RDA/MeanX100) by 200g wet fruit piece Zn WS 20.0 611.62 6.54 RS 662.25 6.04 RB 980.39 4.08 PP 440.53 4.54 RV 1639.34 2.44 Fe WS 15.0 1704.55 1.74 RS 2083.33 1.92 RB 2727.27 1.47 PP 1013.51 1.97 RV 1071.51 3.73 Mn WS 5.0 59.74 69.96 RS 122.25 32.72 RB 46.00 86.96 PP 68.49 29.20 RV 33.76 118.48 Cu WS 3.0 175.41 22.80 RS 62.24 64.27 RB 88.24 45.33 PP 25.30 78.77 RV 107.91 37.07 Cr WS 0.2 68.97 58.00 RS 80.00 50.00 RB 64.52 63.00 PP 55.56 36.00 RV 54.05 74.01

The average weight of a piece of fresh guava used in this study was 200 g and is taken as the reference measure in this study. Table 4.7 shows that an average amount of 200 g of

R. vulgaris is sufficient to provide Mn and Cr more than the RDA per day for a healthy adult human being.

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4.3.2 Vitamins levels

4.3.2.1 HPLC separation and chromatograms

The relative elution time for ascorbic acid was 3.651 minutes using a mobile phase of acetonitrile:n-hexane:ethanol (50:40:40) at 254 nm and that of β-carotene and α- tocopherol were 4.311 minutes and 3.110 minutes respectively using acetonitrile: methanol: triethylamine (50:50:0.01) mobile phase at 297 nm. The mobile flow rate for separation was 1.2 mL/minute.

The individual analytes in the samples were identified by comparing the retention times with those of standard solutions. Peak areas were used for quantification of vitamins expressed in μg/g or μmol/L weight of fruits (Equation 3.2). The chromatogram of α- tocopherol and β-carotene in white sweet guava, vitamin C standard and a mixture of standards of α-tocopherol and β-carotene are shown in Figures 4.3-4.45 and other chromatograms are shown in appendices.

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Figure 4.3: Chromatogram of α-tocopherol and β-carotene in white sweet guava

Column: ODS 4.6 X 250 mm Mobile Phase: Acetonitrile: Methanol: triethylamine(50:50:0.01) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 297 nm

Elution time for the vitamins was 3.110 minutes (α-tocopherol), 4.311minutes (β- carotene), 5.389 minutes (unidentified peak).

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Figure 4.4: Chromatogram of vitamin C standard Column: ODS 4.6 X 250mm Mobile Phase: Acetonitrile: n-hexane: ethanol (50:40:40) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 254 nm

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Figure 4.5: Chromatogram of a mixture of α-tocopherol and β-carotene standards

Column: ODS 4.6 X 250mm Mobile Phase: Acetonitrile: Methanol: triethylamine (50:50:0.01) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 297 nm

Table 4.8 shows the levels of ascorbic acid, β-carotene, and α-tocopherol in some selected fresh indigenous fruits in the ten sub counties of Kakamega County.

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Table 4.8: Levels of vitamins in indigenous fruits in the sub counties of Kakamega County

Vita Fruit Mean ± SE(mg/100g) n=30 min Kak. Central Kak. North Kak. East Matete Likuyani Butere Mumias Matungu Kak. South Khwisero p- value AA WS 228.21±4.76bE 238.45±3.85bF 186.29±6.38bA 199.47±3.57bD 226.68±10.34bE 189.01±4.42bC 180.42±7.36bAB 175.20±3.31bA 179.74±16.22A 186.06±2.28bBC <0.001 RS 260.59±5.90cD 200.47±3.88cC 234.07±3.27cB 239.91±7.11cB 240.46±4.73cC 233.37±7.04cB 223.72±1.83cA 223.86±3.61cA 231.10±3.60A 228.89±4.0cA RB 240.21±7.58cE 214.97±3.82cD 198.22±0.75cB 213.42±7.13cC 209.49±6.35cC 200.29±6.36cB 199.98±0.92cA 180.77±2.52cA 196.44±0.70B 197.82±3.84cB PP 42.25±0.33aD 41.05±0.28aC 41.80±0.11aC 39.42±0.94aB 38.44±1.11aA 39.62±0.33aB 37.54±0.07aA 41.05±0.44aC 39.74±1.22B 40.82±0.56aC RV 42.51±0.03aD 42.81±0.48Ad 41.31±0.13aC 41.95±0.09aCD 41.92±0.43aCD 42.33±1.08Ac 39.83±0.96aA 41.18±0.39aC 39.53±1.22A 40.08±1.04aB p- <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 value BC WS 3.51±0.00cD 3.42±0.02dC 3.37±0.03dB 3.42±0.04dC 3.37±0.04dB 3.37±0.00dB 3.33±0.02d 3.33±0.05dA 3.38±0.03dB 3.45±0.01dC <0.001 RS 3.24±0.00cD 3.20±0.01cC 3.14±0.01cB 3.14±0.01cB 3.13±0.00cB 3.11±0.02cB 3.09±0.02c 3.07±0.02cA 3.07±0.01cA 3.05±0.02cA RB 2.87±0.00bD 2.75±0.02bC 2.73±0.00bB 2.78±0.02bD 2.69±0.00bA 2.74±0.01bC 2.71±0.01b 2.67±0.01bA 2.73±0.00bB 2.69±0.00bA PP 1.91±0.00aA 1.91±0.00aA 1.92±0.00aB 1.92±0.00aB 1.92±0.00aB 1.92±0.00aB 1.92±0.00a 1.92±0.00aB 1.92±0.00aB 1.91±0.00aA RV 1.91±0.00aC 1.91±0.07Ac 1.89±0.00Aa 1.90±0.00aB 1.90±0.00Ab 1.89±0.00Aa 1.89±0.00a 1.89±0.00Aa 1.89±0.00Aa 1.89±0.00aA p- <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 value AT WS 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA 0.17±0.00bA <0.001 RS 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00dA 0.50±0.00cA RB 0.54±0.00eB 0.54±0.00eB 0.54±0.00eB 0.54±0.00eB 0.54±0.00eB 0.53±0.00eA 0.53±0.00eA 0.54±0.00eB 0.53±0.00eA 0.53±0.00dA PP 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA 0.13±0.00aA RV 0.21±0.00cC 0.21±0.00cC 0.20±0.00cB 0.21±0.00cC 0.21±0.00cC 0.21±0.01cC 0.20±0.00Cb 0.20±0.00bB 0.19±0.00cA 0.20±0.00cB p- <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 value

Key: WS – White sweet guava RS – Red sweet guava RB – Red bitter guava PP – Physalis peruviana RV – Rhus vulgaris Kak. – Kakamega AA- Ascorbic acid BC- Beta-carotene AT- Alpha-tocopherol

Mean value followed by the same small letter(s) within the same column and mean value followed by the same capital letter(s) within the same row are not significantly different for each fruit (One way ANOVA, SNK test, σ =0.05)

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There was a significant difference in the levels of vitamins in all the varieties analyzed in this study since the p-values were all ˂ 0.05 at 95 % confidence level using one way

Anova. The mean levels of vitamins in each fresh fruit variety are discussed in the sections that follow.

4.3.2.2 Levels of vitamin C (Ascorbic acid)

Mean levels of ascorbic acid were recorded significantly lowest in R. vulgaris of

Kakamega South (39.53±1.22 mg/100g) and highest in red sweet guavas of Kakamega

Central (260.59±5.90 mg/100g). There was no significant difference in the mean levels of ascorbic acid in white sweet guavas of Kakamega East, Kakamega South, Matungu and

Mumias, of Mumias and Khwisero and of Kakamega central and Khwisero, in red sweet guavas of Mumias, Matungu, Kakamega South and Khwisero, of Kakamega East, Matete and Butere and of Kakamega South and Likuyani and in red bitter guavas of Kakamega

East, Butere, Kakamega South and Khwisero and of Matete and Likuyani.

P. peruviana of Kakamega North, Kakamega East, Matungu and Khwisero, and of

Matete, Butere and Kakamega South and R. vulgaris of Mumias and Kakamega South, of

Kakamega East, matete, Likuyani, Butere and Matungu and of Kakamega North, Butere and Mumias, and of Kakamega Central, Matete and Likuyani showed no significant difference in the levels of ascorbic acid. All the fruit varieties studied for ascorbic acid in

Kakamega South showed a significant difference (p>0.05).

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The levels of AA in the guava varieties in this study compare favourably with what was reported elsewhere for wet white guavas (123 mg/100g) (Nanyili, 1995), 218±79 mg/100g for seeded guava and 176±54 mg/100g for seedless guava (Lim et al., 2007),

270 ±18.8 mg/100g for fresh guava (Leong and Shui, 2002) and 120.65 mg/100g for guava (Siow and Hui, 2013) but lower than what was reported for red guavas (375.0 mg/100g) (Nanyili, 1995) and higher than what was reported in oranges of 50 mg/100g

(Belitz and Grosch, 1999 and Nambafu, 2011), raw banana (8.2 mg/100g), ripe banana

(51.0 mg/100g) and pawpaw (52.0 mg/100g) (Nambafu, 2011).

P. peruviana and R. vulgaris in this study have levels that compare favourably with what was reported elsewhere for P. peruviana pulp (43.0 mg/100g) (Ramadan and Morsel,

2004), oranges (50.00 mg/100g), ripe banana (51.0 mg/100g) and pawpaw (52.0 mg/100g) (Nambafu, 2011). Lower levels of AA in avocado (17.85 mg/100g) and raw banana (8.2 mg/100g) were rported by Nambafu (2011). Musinguzi et al. (2007) reported a higher level of 114 mg/100g in P. peruviana and a lower level of 3.24 mg/100g in

Carissa edulis.

4.3.2.3 Levels of vitamin A (beta-carotene)

Kakamega Central recorded significantly highest levels of ß-carotene in white sweet guavas (3.51±0.00 mg/100g) and Kakamega East, Butere, Mumias, Matungu, Kakamega

South and Khwisero the lowest (1.89±0.00 mg/100g). White sweet guavas of Mumias and Matungu, of Kakamega East, Likuyani, Butere and Kakamega South and of

Kakamega North, Matete and Khwisero, red sweet guavas of Matungu, Kakamega South

93 and Khwisero, and of Kakamega East, Matete, Likuyani, Butere and Mumias, and red bitter guavas of Likuyani and Khwisero, of Kakamega East, Mumias and Kakamega

South and of Kakamega North and Butere showed no significant difference in the ß- carotene mean levels.

There was no significant difference in the ß-carotene mean levels in P. peruviana of all the sub-counties apart from Kakamega Central, Kakamega North and Khwisero and in R. vulgaris of all the sub-counties apart from Kakamega Central, Matete and Likuyani.

Among all the fruit varieties studied in each sub-county, there was no significant difference. The levels of beta carotene found in this study is lower than what was reported by Ramadan and Morsel (2003) of 13.10 mg/100g of beta carotene in guavas but higher than what was reported by Charoensiri et al. (2009) of 13.8 μg/100g beta carotene in guavas. The levels are comparable to what was reported in P. peruviana (1.6 mg/100g)

(Ramadan and Morsel, 2004).

4.3.2.4 Levels of vitamin E (alpha-tocopherol)

Mean levels of α-tocopherol were significantly highest in red bitter guavas of Kakamega

North, Kakamega East, Kakamega Central, Matete, Likuyani and Matungu (0.54±0.00 mg/100g). There was no significant difference in the levels of α-tocopherol in P. peruviana, white sweet and red sweet guavas of all the sub-counties. Red bitter guavas of all the sub-counties apart from Butere, Mumias, Kakamega South and Khwisero and R. vulgaris of Kakamega Central, Kakamega North, Matete, Likuyani and Butee and of

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Kakamega East, Mumias, Matungu and Khwisero showed no significant difference in the

α-tocopherol mean level.

The levels of alpha tocopherol found in this study are higher than what was reported elsewhere for seed oil of P. Peruviana (0.88 mg/ kg) (Puente et al., 2011) but lower than what was reported in P. Peruviana pulp and skin oil (22.50 mg/kg) (Puente et al., 2011).

Table 4.9 shows mean levels of ascorbic acid, β-carotene, and α-tocopherol in some selected fresh indigenous fruits in Kakamega County. The mean levels of the vitamins for all sub counties in Kakamega County were determined (Table 4.9). The values were used to indicate the fruits that can provide high levels of vitamins in Kakamega County.

Table 4.9: Levels of vitamins in some selected fresh indigenous fruits in Kakamega County

Mean ± SE (mg/100g) Vitamin Variety n=30 Range p-value Ascorbic WS 198.95±6.99b 175.20-238.45 acid RS 231.64±8.40d 185.72-240.10 RB 205.16±4.53c 181.98-228.21 PP 40.17±0.45a 37.54-42.25 a RV 41.35±0.35 39.53-42.81 <0.001 c Beta WS 3.40±0.05 3.33-3.51 c carotene RS 3.12±0.02 3.05-3.24 b RB 2.74±0.02 2.67-2.87 a PP 1.92±0.00 1.91-1.92 a RV 1.90±0.00 1.89-1.91 <0.001 Alpha- WS 0.17±0.00a 0.17-0.17 Tocopherol RS 0.50±0.00c 0.50-0.50 RB 0.54±0.00c 0.53-0.54 PP 0.13±0.00a 0.13-0.13 RV 0.20±0.00b 0.20-0.21 <0.001

Key: WS – White sweet guava RS – Red sweet guava RB – Red bitter guava PP – Physalis peruviana RV – Rhus vulgaris AA- Ascorbic acid BC- Betacarotene AT- Alpha-tocopherol

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Mean value followed by the same small letter(s) within the same column are not significantly different (One way ANOVA, SNK test, σ=0.05)

Table 4.9 shows there was there was a significant difference in the levels of the vitamins in all the fruits studied apart from ascorbic acid in P. peruviana and R. vulgaris, beta carotene in white sweet and red sweet guavas and in red bitter guavas, P. peruviana and

R. vulgaris, and alpha tocopherol in white sweet guavas and P. peruviana and in red sweet and red bitter guavas. The levels of micronutrients in Kakamega County are comparable to what was reported in other studies with some variations as has been discussed in the previous sections above. The variation could be due to the fact that micronutrient content varies with sample varieties, maturity stage, soil fertility, climatic conditions or geographical site of production, harvesting and post harvesting handling, processing and storage conditions (Ihekoronye, 1992). Another reason of variation could be due to use of different methods of analysis. Table 4.10 shows amount (g) of fruit variety required to provide RDA of selected vitamins per day and the percentage RDA contribution by 200 g of wet fruit.

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Table 4.10: Amount (g) required to provide RDA of vitamins/day and % RDA contribution by 200 g of wet fruit

Vitamin Fruit RDA Amount (g) % RDA Variety required to provide contribution by RDA value per day 200g wet fruit

Ascorbic WS 60 mg 30.16 663.13 acid RS 25.90 772.20 RB 29.25 683.76 PP 149.37 133.90 RV 145.10 137.84 Beta WS 4000IU (2.4 mg) 70.56 283.32 carotene RS 76.92 266.95 RB 87.59 228.34 PP 125.00 160.00 RV 126.32 158.32 Alpha- WS 15 mg 8823.53 2.27 Tocopherol RS 3000 6.67 RB 2777.78 7.20 PP 11538.46 1.73 RV 7500 2.67

Note: 1IU vitamin A=0.6 μg beta carotene

Table 4.10 shows that an average piece of guava of mass 200 g, just as an equivalent amount of the other fruit varieties is sufficient to provide ascorbic acid and beta carotene more than the RDA per day for a healthy adult human being. The same amount of the fruits can be a good supplement for alpha tocopherol.

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CHAPTER FIVE

5 CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

(i) All the indigenous fruits analyzed in this study recorded significantly high

levels of Zn, Fe, Cr, Mn, and Cu. This is shown by the RDA contribution per

day if on average 200 g of fresh fruit is consumed. Red bitter guavas of

Matete, Mumias and Matungu and R. vulgaris of Kakamega East, Kakamega

North, Matete, Likuyani, Matungu and Kakamega South can provide more

than 100% of the RDA levels of manganese per day. Red sweet guavas of

Kakamega East, Likuyani and Kakamega South, P. peruviana of all the sub-

counties apart from Matete can provide the RDA levels per day of copper. All

the fruits from almost all the sub-counties can contribute more than 50% of

the RDA levels of chromium per day. All the fruits analyzed in this study

recorded significantly high levels of zinc and iron as they contribute

significantly to the RDA value per day if about 200 g of the fruits are

consumed.

(ii) All guava varieties analyzed recorded very high levels of ascorbic acid. A

piece of 200 g can provide more than six times the RDA values per day for

AA and more than two times beta carotene and a significant contribution of

alpha tocopherol. A quantity of 200 g of P. peruviana and R. vulgaris in all

the sub-counties can provide over 100% of the RDA values per day of AA and

beta carotene and a significant contribution of alpha tocopherol.

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(iii) There was a significant difference in the mean levels of iron between fruit

varieties in sub-counties of Kakamega North., Kakamega Central., Matete,

Likuyani, Butere and Khwisero and of Mn and Cr between fruit varieties in

Likuyani and Mumias sub-counties respectively at a confidence level of 95%.

5.2 Recommendations from this study

(i) There is need to promote indigenous fruits of P. quajava, R. vulgaris and P.

peruviana because they have high levels of trace elements (Zn, Fe, Cr, Mn

and Cu) and vitamins A, C and E that can meet or contribute significantly to

the RDA levels. These fruits can be either eaten raw or be used for preparation

of juices for the sick, children and elderly.

(ii) Farmers should be advised to grow indigenous fruits in their home gardens

and farms to ensure that as many people as possible can access and consume a

lot of these micronutrient-rich fruits.

(iii) There is need to consume indigenous fruits in mixed or varied kinds as they

complement one another in provision of essential micronutrients.

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5.3 Recommendations for further research

(i) Studies on effects of seasonal variation, soil conditions and pesticides on the

levels of essential micronutrients in fruits covered in this study need to be

conducted.

(ii) Studies on levels of essential micronutrients in the leaves, roots and barks of

plants of the fruit varieties studied should be done. These is because the leaves

of these plants are used as vegetables and both leaves, roots and barks are

used as herbal medicine by people in Kakamega County.

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APPENDICES

Appendix I: Calibration curves for trace elements

(a) Manganese

0.5 y = 0.0571x - 0.0006 R² = 0.9971 0.4

0.3

0.2

0.1

0 0 2 4 6 8 10 -0.1

(b) Zinc Absorbance against 0.45 y = 0.0399x + 0.0085 0.4 R² = 0.9933 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 8 10 12

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(c) Iron

Absorbance against concentration 0.45 0.4 y = 0.0387x + 0.0105 R² = 0.9976 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 8 10 12

(d) Chromium

0.08 0.07 y = 0.0336x - 0.0004 R² = 0.9999 0.06 0.05 0.04 0.03 0.02 0.01 0 0 0.5 1 1.5 2 2.5 -0.01

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(e) Copper

0.45 y = 0.0399x + 0.0085 0.4 R² = 0.9933 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 8 10 12

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Appendix II: Calibration curves for vitamins

(a) Ascorbic acid

Peak area against concentration

450000 400000 y = 269.48x - 3535.1 R² = 0.9997 350000 300000 250000 200000 150000 100000 50000 0 -50000 0 500 1000 1500 2000

(b) Beta-carotene

Peak area against concentration 900000 800000 y = 51105x + 2904.4 700000 R² = 0.9997 600000 500000 400000 300000 200000 100000 0 0 5 10 15 20

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(c) Alpha tocopherol

Peak area against concentration 900000 y = 54168x + 1654 800000 R² = 0.9998 700000 600000 500000 400000 300000 200000 100000 0 0 5 10 15 20

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Appendix III: Chromatograms of vitamins

(a) Vitamin C in Rhus Vulgaris L

Column: ODS 4.6 X 250mm Mobile Phase: Acetonitrile: n-hexane: ethanol (50:40:40) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 254 nm 3.532 minutes (Vitamin C) The other peaks were unidentified

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(b) Vitamin C standard

Column: ODS 4.6 X 250mm Mobile Phase: Acetonitrile: n-hexane: ethanol (50:40:40) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 254 nm

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(c) β-carotene standard

Column: ODS 4.6 X 250mm Mobile Phase: Acetonitrile: Methanol: triethylamine(50:50:0.01) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 297 nm

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(d) α-tocopherol standard

Column: ODS 4.6 X 250mm Mobile Phase: Acetonitrile: Methanol: triethylamine (50:50:0.01) Flow rate: 1.2 mL/minute Sensitivity: 1.00 absorbance units full scale (aufs) Detector: SPD 20UV at 297 nm