BINDURA UNIVERSITY OF SCIENCE EDUCATION

FACULTY OF AGRICULTURE AND ENVIRONMENTAL SCIENCE

DEPARTMENT OF ENVIRONMENTAL SCIENCE

Determination of essential and trace elements in Indigenous : Implications for food safety

TANYANYIWA ALICE B1233325

A dissertation submitted in partial fulfilment of the requirements of the Bachelor of Environmental Science Honours Degree in Safety, Health and Environmental Management March 2017 TABLE OF CONTENTS Contents Page DEDICATION ...... iv ACKNOWLEDGEMENTS ...... v ABSTRACT ...... vi LIST OF TABLES ...... viii LIST OF ACRONYMS AND ABBREVIATIONS ...... ix CHAPTER ONE ...... 1 1.1Background to the study ...... 1 1.2 Problem statement ...... 2 1.3 Justification ...... 4 1.4.1 Aim...... 4 1.4 Justification for the study ...... 4 1.5 Hypotheses ...... 4 CHAPTER TWO ...... 5 2.0 Literature review ...... 5 2.1 Introduction ...... 5 2.2 Historical use of wild edible ...... 5 2.3 Benefits of indigenous fruits to food and nutritional security ...... 5 2.4 Active chemical components of wild fruits ...... 7 2.5 Common nutritional deficiency diseases ...... 7 2.6 Essential elements, trace elements and public health ...... 8 CHARPTER THREE...... 10 3.0 Materials and methods ...... 10 3.1 Description of the study site ...... 10 Study map area ...... 11 3.2 Sampling and sample preparation ...... 12 3.4 Data analysis ...... 13 3.5 Quality assurance and quality control ...... 14 3.6 Statistical analysis ...... 14

ii

CHAPTER FOUR ...... 15 4.0 Data presentation and analysis ...... 15 4.1 Variation of essential and trace elements in wild fruits ...... 15 _Toc4789481544.2 Comparison of elemental concentrations in wild and exotic fruits ...... 17 CHAPTER FIVE ...... 19 5.0 Discussion ...... 19 5.1 Variation of essential and trace elements in wild fruits ...... 19 5.2 comparison of elemental concentrations in wild and exotic fruits ...... 21 CHAPTER SIX ...... 22 6.0 Recommendations and conclusions ...... 22 6.1 Conclusions ...... 22 6.2 Recommendations ...... 22 REFERENCES ...... 23 APPENDICES ...... 27 Appendix 1 Mean nutritional content of Exotic fruits ...... 27 Appendix 2 Preparation of Elemental 100ml/ stock solutions ...... 27 Appendix three Determination of trace and essential elements in Indigenous fruits ...28

iii

DEDICATION This project is dedicated to my family members for their support which they gave me during my project time.

iv

ACKNOWLEDGEMENTS

I would like to extend my gratitude to my supervisor Mr. A. Kanda for his expert advice and knowledge he has imparted for this project to be accomplished. The input by the Bindura University Environmental Science lab is greatly appreciated.

I would like also to express my heartfelt gratitude to my parents, brothers and sisters for their unwavering support, prayers and comfort they gave to me during the time of my research project.

Above all i give praise to the Almighty God for permitting me to reach this far.

v

ABSTRACT The current study was conducted in Bindura Mashonaland Central Province to determine the essential and trace element concentration of indigenous fruits (Uapaca kirkiana and mauritiana) consumed by Bindura residents. Samples were collected from six farmers from different places namely Masembura, Jingo and Dengu for Uapaca kirkiana , Mavhuradonha, Dande and Muzarabani for Ziziphus mauritiana . Twenty seven samples were collected once in the month of January 2017. The samples were taken to Bindura University laboratory where they were analyzed for trace elements (Cr, Cd, Fe Zn, Mn and Cu) and essential elements (Mg, K, Ca and Na) using FAAS (Model ICE 3000 Series). One-way ANOVA was used to determine whether there were any significant differences in measured mean element concentrations from the different places of origin of the fruits. Indigenous fruits were then compared with two exotic fruits C. sinensis and M domestica (Orange and Apple) to compare the dietary benefits between indigenous and exotic fruits. The nutritional information of exotic fruits was taken from U SDA nutrient for standard reference (2015). Data were analyzed for significant difference using one sample t-test. The findings of this study revealed high concentrations of essential elements with Ca being the highest in U. kirkiana (137±19.2) and Z. mauritiana (131±19.2).Concentrations of Cd and Cr were not detected. Fe concentrations were also detected with Z. mauritiana having the highest mean concentrations of (43.5±11.4).Lowest concentrations of Fe were detected in U. kirkiana (14.6±1.23) It was observed that all trace and essential elements levels in indigenous fruits varied with sampling areas. Basing on the findings it was concluded that indigenous fruits have a great potential dietary contribution to nutrition and food safety. Their nutrition value of indigenous fruits should be well communicated in order to promote their consumption both in rural and urban areas. Key words : indigenous fruits, essential elements, trace elements, food security, food safety.

vi

LIST OF FIGURES

Figure 3. 1 showing the study area ...... 111 Figure 3. 2 showing mashonaland central districts ...... 122

vii

LIST OF TABLES

Table 4.1 Variation of essential and trace elements in indigenous fruits...... 15

Table 4.2 comparisons of elemental concentrations in exotic and indigenous fruits...... 16

viii

LIST OF ACRONYMS AND ABBREVIATIONS

WHO - World Health Organization

FAO - Food and Agriculture Organization

Ca - Calcium

Na - Sodium

Mn - Manganese

Mg - Magnesium

Zn - Zinc

Fe - Iron

Cu - Copper

Cr - Chromium

Cd - Cadmium

K - Potassium

UNSCN - United Nations System- Standing Committee

SPSS - Statistical Package for social Science SSA - Sub Saharan

ix

x

CHAPTER ONE

1.0 INTRODUCTION

1.1BACKGROUND TO THE STUDY

Existence of food security is described by FAO (1997) as when all people at all times have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preference for an active and health life. It comprises food access, food distribution, and stability of food supply and the use of food. On the other hand food insecurity is part of the continuum that includes hunger, malnutrition and famine. Food insecurity and malnutrition are affecting much of the world’s population (Godfray et al, 2010). It is unfortunate that food insecurity and malnutrition are issues that affect approximately one in seven people worldwide (Crute et al , 2010) Two million people from each country of the earth are estimated to suffer from micronutrient deficiencies that make them more susceptible to disease that can be a significant obstacle to economic growth (FAO, 2012). According to (FAO, 2015), 11.3% of the world’s population is hungry and roughly 805 million people who go undernourished on a daily basis consuming less than the recommended calories a day. Hunger kills more people each year than AIDS, malaria and tuberculosis combined (FAO, 2015). Food security issues are severe in Sub- Saharan Africa (FAO, 2011) where projected crop yield declines due to climate change, environmental degradation and reliance on manual farming and low levels of technology (Brown and Funk 2008: Muller et al .2011).

The high risk groups affected by food insecurity are children and the elderly (WHO, 2015). In 2012, 47% of the population of Sub-Saharan Africa lived on $1.90 or less per day which is a principal factor in causing widespread hunger (World Bank, Sub-Saharan Africa poverty and equity data, 2012). In Zimbabwe, malnutrition continues to be a chronic problem due to the practice of a ‘mono-diet’. Maize still accounts for half of the national caloric intake in Zimbabwe contributing to undernourishment from 30 to 39% of the total population (Zimbabwe Food Security Brief, 2014).

1

Historically wild plants and animals were the solely dietary components for hunter and gatherer and forager cultures (Bharucha et al . 2010). Today quite a number of wild plants which were used by rural and tribal populations and contributed to their livelihood and food security have long escaped recognition and scientific enquiry (Serdia et al. 2013). The distribution, conservation mode of harvest and optimal use of wild plants by local people require region specific assessment in order to intergrate them into development (Mahapatra et al . 2012). Most people in developing countries derive a significant part of their subsistence needs and income from gathered and animal products. There is need to identify alternative bio-nutritional food sources, study their nutritive values and composition in order to prioritize their edibility by indigenous people in order to improve food security (Serdia et al. 2013). It appears some plant species are being underutilised yet their potential to boost food security has not been established by scientific enquiry. Today environmental quality has been impaired by pollution. The quality of soil in which fruits grow has been contaminated by human activities. It remains unknown as to what degree has environmental pollution affected the quality of wild fruits eaten today, considering that fruits are eaten raw

Fruits are generally acceptable as a good source of nutrients and supplement for food in a world faced with the problem of food scarcity. They are known to be excellent sources of nutrients such as and minerals (Nigrea et al , 2014). FAO/WHO (2003) urge people to consume at least 400g of and vegetables every day (excluding potatoes and other starchy tubers) for the prevention of chronic diseases and alleviation of several micronutrient deficiencies especially in less developed countries. Consumption of fruits in Sub-Saharan Africa (SSA) has been estimated to fall short of the recommended daily amount (400g) (FAO/WHO. 2003). Indigenous fruit trees could serve millions of lives by providing children and adults vulnerable to malnutrition with a nutritious source of food as well as valuable vitamins and minerals. However, there is need to evaluate the nutritional values of such fruits and presence of toxic substances for food safety

1.2 PROBLEM STATEMENT

There is little information available on nutritional composition of the indigenous fruits of Zimbabwe . Most indigenous fruit trees have not been cultivated on-farm, therefore there is only little knowledge about their fruit production and fruit nutritional value (Rathore. 2009). They are

2 often undervalued and underutilised as more exotic fruits become accessible. The gap between wild or indigenous edible fruits and exotic or cultivated ones is wide and needs to be bridged by shedding more light on potential wild food biodiversity (Hegazy et al. 2013) Information on the nutrient content of many indigenous fruits are either unavailable or unreliable (Colfer et al . 2006). Despite the benefits reaped from indigenous fruit trees, their products are often undervalued or little known amongst urban and international communities.

1.3 JUSTIFICATION FOR THE STUDY

The determination of essential and elements and trace elements in wild fruits may help provide information to choose alternative cheaper and accessible sources of such essential elements which may in turn provide remedies to malnutrition and food insecurity. People may not utilise wild fruits due to lack of knowledge of their nutritive values. However these wild plant foods do not require formal cultivation and have been used in the past without treatment. Commercially available essential elements, mineral and fortified foods have been made a solution to micronutrient deficiencies in regular diets in order to help manage diet-related diseases by providing deficient micronutrients ( Muller et al . 2011) However, they are inaccessible and expensive, especially the most vulnerable in society. Gaining scientific knowledge by the local populations that readily available wild fruits contain essential elements for health living may encourage their consumption even by the most vulnerable groups of society.

The current study will document concentrations of selected essential (Ca, Na, K, and Mg) and trace elements (Zn, Fe, Cu, Mn, Cr and Cd) in fruits of wild plants U. Kirkiana and Z. Mauritiana . Information gathered may be used to sensitise people on nutritive value of wild plant food sources and their role in human health. Since most of the people in Zimbabwe cannot afford food supplements and balanced diets (Zimbabwe Food Security Brief, 2014). Research findings will be used to encourage people to forage for wild plant products. The study on wild fruits and other wild edible plants is also intended to promote preservation of these wild plant species presently under threat by human activities such as land clearing for agriculture, firewood for energy and building infrastructure. The economic advantages of these fruits through local trade may be boosted.

3

1.4.1 AIM

To evaluate the nutritive value in Uapaca Kirkiana and Ziziphus mauritiana bought at an open local market.

1.4.2 SPECIFIC OBJECTIVES

(a) To determine the levels of exposure to trace elements in indigenous fruits of Uapaca Kirkiana and Ziziphus mauritiana

(c) To determine the potential dietary contribution of the selected indigenous fruits to food safety and human health.

1.5 HYPOTHESES

H0. There is no significant difference in nutritional values between wild fruits Ziziphus mauritiana and Uacapa Kirkiana fruits and most cultivated fruits M.domestika and Citrus Cinensis

H0. There are no significant toxic metal concentrations in wild fruits of Ziziphus mauritiana and Uacapa Kirkiana

4

CHAPTER TWO

2.0LITERATURE REVIEW

2.1 INTRODUCTION

This chapter explores literature on the issues related to historical use of wild edible plants, benefits of indigenous fruits to food and nutrition security and other health benefits of indigenous fruits

2.2 HISTORICAL USE OF WILD EDIBLE PLANTS

Wild edible plants or indigenous plants refer to plants growing without intended cultivation (Lui et al . 2003). They include predominantly native and naturalised species (Kalle et al , 2012). Wild edible plants have been defined by FAO (2011) as “plants that grow spontaneously in self- maintaining populations in natural and semi-natural ecosystems and can exist independently of direct human action”. The evidence of man’s dependency on wild plants for his survival can be demonstrated by ethno-botanical findings from prehistoric archeological sites (Sawian et al , 2007). Wild fruits and vegetables have been used since ancient times by native people all over the world. Before domestication of agriculture, humans depended on wild plants for their daily uses (Maanda et al . 2010). They lived a nomadic life without permanent households but depended on the availability of natural resources.

Wild plants formed most of our ancestor’s diet in a multitude of ways including bread ingredients, vegetables, fruits, spices, snacks or beverages (Tardio et al, 2011). In many tropical countries, rural people still follow the traditional harvesting of a wide range of roots, tubers and fruits from the forest because of their cultural uses, as food supplement labelled as famine or hunger food. This shows that wild plants play a significant role in food and nutrient security (Laddha et al . 2014). Wild plants did not only provide edible food but also have their importance in providing fuel, fodder and medicine.

2.3 BENEFITS OF INDIGENOUS FRUITS TO FOOD AND NUTRITIONAL SECURITY

Food and nutrition security are key issues for human wellbeing while indigenous fruits are underutilised in many countries (Feyssa et al , 2015). Agricultural production in African countries is under pressure to produce greater quantities of food resources. The over-reliance on a few major

5 staple crops has inherent agronomic, ecological, nutritional and economic risks and is probably unsustainable in the long run (FAO. 2011). According to Fentahun and Hager (2009) wild edible fruits contribute to nutrition and health security of rural people as they contain proteins, vitamins and minerals. (UNSCN. 2010) reported that about 30% of the inhabitants of Sub-Saharan Africa, mostly women and children suffer from malnutrition yet indigenous fruits can offer micronutrients such as vitamins and minerals necessary to support human healthy growth and activity. Many traditional or indigenous fruits and vegetables are characterised by a high nutritional value compared with global cultivated ones and are major sources of essential vitamins, micronutrients, proteins and other phytonutrients (Hegazy et al . 2013). They have the potential to play a role in strategies to attain nutritional security (Adepoju. 2013: Hegazy et al . 2013).

According to Adepoju (2009), fruits are acceptable sources of nutrients and food supplements in a world faced with problems of food scarcity. In a research carried out in Nigeria on nutrient composition and antinutritional factors of the fresh wild fruits, results showed that fruits had potential in meeting some or all of micronutrients needs of consumers (Mahapatra et al . 2013). Besides mineral elements, the fruits analysed showed other nutrients such as crude proteins, , crude fiber and crude lipids (Adepoju, 2009). Nazarudeen (2010) also states that wild or indigenous fruits are beneficial in terms of minerals and other nutrients. A study carried out in India on nutritional composition of some lesser known fruits used by the ethnic communities and local folks of Kerala showed that some of the wild fruits though not tasty and desirable unlike the popular cultivated ones, were more nutritional containing high protein, and minerals.

A comparative study between wild fruits and exotic fruits in India showed that the fruit’s nutrient values compared well with their cultivated counterparts (Mahapatra et al . 2012). The study also explored that indigenous fruits qualify as high mineral and nutrient providers as compared to their cultivated counterparts such as banana and guavas. Most related studies on wild fruits revealed the presence of high concentrations of Ca, K, and Mg which are essential for making good of worn out cells, building of red blood cells and maintaining the body mechanisms WHO (1996) The absence of these elements in diet might result in stunted growth, poor bone development and a weak body (Effiong and Udo. 2010). Other benefits of wild crops include commercial, medicinal and cultural value crops. (Neudeck et al . 2012: Teketay et al . 2010)

6

2.4 ACTIVE CHEMICAL COMPONENTS OF WILD FRUITS

Wild fruits possess various bioactivities and health benefits such as free radicals scavenging, antioxidant, anti-inflammatory, antimicrobial and anti-cancer activity (Zhang et al . 2016). Therefore wild fruits have the potential to be used as functional foods or pharmaceuticals to prevent and treat several chronic diseases. Studies have shown the presence of antioxidant activity (Lui et al. 2016). Antioxidants protect the cells against the effects of free radicals that are normally produced as a byproduct of cellular metabolism and are capable of killing bacteria, damaging biomolecules, provoking immune responses, activating oncogenes, causing atherogenesis and enhancing the aging process (Li et al. , 2014; Tapan et al. , 2012). Wild fruits have exhibited potential antibacterial, antifungal and antiviral activities (Lui et al. 2016; Ya Li et al . 2016). According to Zhang et al . (2016) several kinds of wild fruits possess anti-inflammatory activities and a relationship between fruit consumption and a reduced risk of cancer has been found. Several wild fruits have been proven to possess anti-cancer activities against breast, colon, prostrate and cervical cancer cells (Ya Li et al . 2016)

2.5 COMMON NUTRITIONAL DEFICIENCY DISEASES

Diet-related diseases such as cardiovascular diseases, strokes, diabetes and some forms of cancer have emerged as public health problems in developing countries (WHO. 2006). Deficiency of essential elements is responsible for diabetes, heart diseases anemia and cancers which continue to kill people (WHO 2011). The prevalence of diet- related non-communicable diseases worldwide is increasing at an alarming stage (WHO 2006). Research indicate that there is frequent consumption of foods containing artificial flavours, colours and other artificial additives which can diminish the effectiveness of the immune system and therefore leading to increased incidence of opportunistic infections (Bakru, 2006). In most developed countries, food choices are dominated by fast food restaurants that mainly serve fat fried meat, excess dairy products and . However such foods have been linked to many life threatening diseases (Cherop. 2009)

Indigenous foods like fruits and vegetables contain most of the micro-nutrients, natural drugs and fiber that are necessary for disease prevention, body management and its normal functioning. (Nazarudeen et al . 2010)The consumption of whole grains leads to significant decrease in the metabolic syndrome, heart diseases and lower glucose levels. Refined grains are associated with higher fasting glucose levels and increased risk of metabolic syndrome which is characterised by 7 risk factors which include elevated blood pressure, abdominal obesity and blood chemistry imbalance. (American clinical nutrition. 2006) Therefore wholesome natural food which includes plenty of vegetables and fruits is ideal for health maintenance because of their therapeutic characteristics.

Mineral deficiencies are a major public health problem in developing countries with pregnant women and infants at risk (WHO 2015). Common micronutrient deficiencies which are of greatest public health significance are iron deficiency, causing variation in degrees of impairment in cognitive performance, lowered immunity to infections, lowered work capacity and reduced psychomotor skills and pregnancy complications e.g. babies with low weight(Stang et a l. 2005). In the semi-arid, Sahara and parts of the dry Savanna areas of Africa, The deficiency of fruits and vegetables in diet is a major cause of and carotene deficiency that causes blindness and death especially in young children (WHO 2015). The correct choice of diet helps in reducing the risk of chronic diseases such as diabetes, cancers and heart diseases which are pre disposing factors of death and disabilities in most developing countries. Wild fruits and other natural foods can reduce major risk factors of chronic diseases such as obesity high blood pressure and high blood cholesterol (Bakru, 2006).

2.6 ESSENTIAL ELEMENTS, TRACE ELEMENTS AND PUBLIC HEALTH

Essential elements are chemical nutrients needed by the body in large or tiny amounts and are vital for growth and development for example Mn, K, Ca, and Na (Ismael et al . 2011). They are usually associated with chemical reactions in the body hence controlling and maintaining body functions (WHO 1996) Through these chemical reactions, the body grows, cells are replaced, energy is produced for essential life processes, reproductive functions are also developed and used, muscles are made to contract and nerve impulses are transmitted. (Adope 2009). Essential elements activate enzymes and vitamins (Soeta et al . 2010) hence lack of these elements present serious health challenges which include clinical and pathological disorders in humans, plants and animals. They are important co-factors of hormones that control chemical reactions in human body (Ismael et al , 2011).

Trace elements are known for their important biological functions in plants and human metabolic reactions (Rabia et al . 2012). In medicinal plants trace elements make up active compounds or

8 take part in reactions which lead to the formation of these compounds. Trace elements play a vital role in the formation of bioactive chemical constituents in medical plants and are therefore responsible for both their medicinal and toxic properties. However trace elements can become toxic to human health if they exceed 100mg/kg (Raymond et al . 2011). This is due to disturbances of nature by environmental pollution (fugitive sources e. g. industrial air emission, agricultural chemicals e. t. c). Most rural and urban environment may accumulate one or more heavy metals which are enough to cause risks to human health, plants and animals as they are transferred from the source of pollution to random environments where higher potentials of direct exposures occur (Raymond et al . 2011:D’Amore et al 2005). There is a strong link between mineral nutrition in plants and humans and the uptake of contaminants in these organisms. The uptake of toxic elements by wild fruits by soil can greatly affect the concept of food security and food safety issues as the fruits may end up causing risks to human health (Okarie et al . 2011)

9

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 DESCRIPTION OF THE STUDY SITE

Bindura is the provincial town of Mashonaland Central and is located in the Mazowe Valley which is approximately 88km north east of Harare. The town lies in geographical region (2b) which is characterised with a rainfall range of 750-1000mm whilst mean annual temperature varies from 19-30 oC thus giving large temperature range of 11 oC (Bindura hydrological map. 2015).

The population for Bindura urban is 44 033 with approximately 11 172 households (ZIMSTATS. 2012). The main sources of potable water for the town are Mazowe and Mwenje dam. Bindura is a rapidly developing small mining and agricultural town. Both urban and peri-urban people practice market gardening to supply a ready market in town with produce. The market opens up to farmers from various surrounding areas as far as Guruve, Muzarabani and Dande. The farmers also come with different exotic and indigenous fruits which are found in their respective areas seasonally. The variety of indigenous fruits includes Ziziphus mauritiana and Uapaca kirkiana. The sale of indigenous fruits has helped local populations fight hunger and poverty as it contributes to livelihoods.

10

STUDY AREA MAP

Fig 3.1 showing the study area

11

Figure 3.2 Mashonaland central districts where indigenous fruits come from.

3.2 SAMPLING AND SAMPLE PREPARATION

Indigenous fruit samples were purposively bought from an open local market in Bindura Town (S17º.31'49" and 31º33'98" E). Sampling was done early in the morning from famers before retailers in order to get the exact places where the fruits came from. Nine samples of each indigenous fruits (U. kirkiana, and Z. mauritiana) were randomly bought from six famers of different places. Famers would bring 80 to 200kgs of indigenous fruits using commuter omnibuses especially from far places such as Muzarabani Mavhuradonha and Dande. Scorch carts were also used to transport fruits especially from places like Dengu, Jingo and Masembura which are close to the town (Personal observation). From each site three people were selected and a sample of $1/ 2 kg was bought. This was done for both fruit species Sample collection was done once on the 10 th of January 2017 between six and eight o’clock in the morning. The fruits and the places where

12 they came from were recorded. Samples were separately packed in clean polythene bags and transported to Bindura University Chemistry laboratory for treatment and analysis.

At the laboratory, fruit samples were washed with distilled water to remove soil and dust particles (Akan et al , 2013). About (500g) of each fruit was used to prepare a laboratory sample. The edible parts of the fruits were separated from the seeds using a knife and oven dried at 80 °C for 72hrs, cooled and crushed in a mortar and sieved through a (1mm) sieve ( Shehata, et al . 2010). A split sample of 0.5g of the powder was put into an acid washed porcelain crucible and ashed in a muffle furnace at 500 °C for four hours (Zhang et al, 1998). After cooling, 6MHCI (10ml) was added and the crucible was heated over a steam bath for 15 minutes whilst covered. Concentrated HNO 3 (1ml) was added and evaporated to dryness by continuously heating for an hour in order to dehydrate silica and completely digest compounds. (Shaker et al , 2005). Finally 6MHCI (5ml) and deionised water (5:10 v/v) were added and the mixture was heated over a steam bath to complete dissolution. The mixture was cooled and filtered through a (Whatman no 1) into a 25ml volumetric flask which was then made to the mark with deionised water (Khan et al . 2011).

3.3 SAMPLE ANALYSIS

Fruit digests were analysed for selected essential and trace elements (Cd, Cu, Cr, Zn, Fe, Mn, Mg, Cd, Ca and K) using F AAS (Model ICE 3000 Series) following procedures as described by (Zhang et a l.1998) Analyses of samples was done in triplicate. Operating conditions of the instrument were adjusted (wavelengths, slit width, lamp, burner, height and flow rate) for the analysis of the selected essential and trace elements following the manufacturers instruction. Elemental stock solutions (1000 mg/l) were prepared by dissolving weighed amounts of appropriate salts of each respective element in deionised water (Khan et al. 2014). Calibration standards were prepared by serial dilutions of the stock solutions using deionised water (Srikanth 2013). Procedural blank solutions were made as described for the elements but without any analyte (Sharma et al. 2006). Elements concentrations in the experimental solutions were determined by extrapolation based on an established calibration curves (Khan et al. 2014).

3.4 DATA ANALYSIS

The mean values of essential and trace elements of the two indigenous ( Z. mauritiana and U. kirkiana) were compared against the means of two exotic fruits which are commonly eaten ( Citrus

13 sinensis and Malus domestica ). (Mahapatra et al, 2012). Mean values per 100g of essential and trace elements (Ca, Fe, Mg, K, Na, Zn, Cu, Fe, Cd and Mn) of two exotic fruits were taken from USDA National Nutrient Database for standard Reference (2015).

3.5 QUALITY ASSURANCE AND QUALITY CONTROL

Standard methods of analysis were used in sample collection and analytical procedures (WHO/APHA. 2006). Calibration with standards, analysis of replicates and analysis of blanks were done. All sample analysis where carried out in triplicates and results were expressed as means of triplicate samples (Zhuang and Gao.2014). All glassware was soaked in 105 HHO3 for 12 hours before use (Somnath et al. 2012)

3.6 STATISTICAL ANALYSIS

A one sample t-test was used to determine test if there was any significant difference between the means of the essential and trace elements found in wild fruits and those of exotic fruits. Data was presented in the form of table. One-way ANOVA was used to determine whether there were any significant differences in measured mean element concentrations from the different places of origin of the fruits. Post hoc was used to separate treatment means at < 0.005. All statistical analysis were analysed using SPSS version 20 0f 2007.

14

CHAPTER FOUR

4.0DATA PRESENTATION AND ANALYSIS

4.1 VARIATION OF ESSENTIAL AND TRACE ELEMENTS IN WILD FRUITS

Table 4.1 Concentrations of essential and trace elements in fruits of U. kirkiana and Z. mauritiana bought at an open market in Bindura. Values are expressed as Mean±SD mg/kg DW of triplicate measurements .

Fruit tree species Z. mauritiana U. kirkiana Element A B C 1 2 3 Cd* ND ND ND ND ND ND

Cr* ND ND ND ND ND ND Cu* 0.89±0.21 a 0.80±0.08 b 1.13±0.04 c 354±61.02 a 0.86±0.24 b 0.94±0.23 c Fe* 43.5±11.4 a 38.1±10.9 b 24±5.56 c 26.5±2.02 a 25.5±2.75 b 14.6±1.23 c Zn* 6.28±0.62 a 5.38±2.46 b 7.04±1.38 c 7.89±0.87 a 6.65±5.34 b 5.13±1.23 c Mg 15.3±3.81 a 17.9±1.68 b 19.03±1.62 c 19.7±0.08 a 16.1±3.73 b 12.8±10.1 c

Ca 118.±16 a 131±19.2 b 94.23±37.85 c 137±7.95 a 112±14.7 b 122±16.9 c Na 29.7±16.02 a 30.40±8.74 b 41.5±41.4 c 52.330±2.645 a 28.8±10.2 b 34.4±13.4 c Mn 4.75±5.56 a 1.59±0.48 b 644±0.49c 1.96±0.58 a 1.64±0.67 b 2.13±0.57 c K 87.9±2.57 a 91.9±1.39 b 89.3±6.02 c 88±0.42 a 87.6±1.98 b 86.2±2.24 c * - trace element

ND- Not detected

Different superscripts (a, b and c) along a row for a specific plant species and element are significantly different for the three places of origin (p <0.05)

15

Places: A-Mavhuradonha B-Dande C-Muzarabani Places 1 -Masembura 2– Jingo 3 - Dengu

Table 4.1 shows the concentrations of selected essential and trace elements in fruits of Z. mauritiana and U. kirkiana collected at an open market in Bindura. Results show that trace elements Cr and Cd were not detected in both indigenous fruits under study. The mean concentration of Cu (354±61.02mg/kg) was very high in Place (1) of U. kirkiana as compared to the other two places whereas in Z. mauritiana the highest Cu concentration was (1.13±0.04mg/kg). Fe was also detected in both fruits with concentration significantly different from site to site (p<0.05). The mean concentrations of Fe are showing that Z. mauritiana has the highest concentrations as compared to U. kirkiana . Mean concentrations of Zn were detected in all the fruit species and were varying from site to site.

Results in the above table also show that all essential elements tested in the indigenous fruits were detected. Ca has the highest concentration levels in both fruits with (p<0.05) across all sites. The range of Ca mean concentrations of U. kirkiana (112.51±141.72 mg/kg to137.05±7.95mg/kg) is higher than Z. mauritiana (94.23±37.85mg/kg to130.91±19.15 mg/kg). Highest K concentration levels are found in Z. mauritiana (l91.89±1.39mg/kg), In U. kirkiana, the highest concentration levels is found in Masembura area (88±0.42mg/kg). Very high levels of Mn are detected in Z. mauritiana fruits which are found in the Mavhuradonha area whereas in other areas the concentrations are very low. Mg is detected in all the fruit species with concentrations significantly different from site to site (p<0.05). Concentrations of essential elements are higher than those of trace elements in indigenous fruit species

16

TABLE 4.2 Concentrations of essential and trace elements in Z. mauritiana and U. kirkiana collected from an open market place in Bindura compared against concentrations in exotic fruits (apple and orange). Values are in mg/100g DW.

. Trace element Essential elements Fruit Cd Cr Cu Zn Fe Mg Ca Na Mn K Z. ND ND 0.09 a 0.62 a 3.52 a 1.74 a 11.57 a 3.39 a 214.74 a 8.9 a mauritiana U. kirkiana ND ND 0.10 b 0.66 b 2.22 b 1.6 b 13.01 b 3,85 b 0.19 b 8.7 b

C. sinensis ND ND 0.045 c 0.07 c 0.10 c 10 c 40 c 0c 0.025 c 181 c

M.domestic ND ND 0.021 d 0.05 d 0.12 d 5d 7d 1d 0.037 d 108 d a.

4.2 COMPARISON OF ELEMENTAL CONCENTRATIONS IN WILD AND EXOTIC

FRUITS

Table 4.2 shows comparison of elemental concentrations in wild and exotic fruits

Results show that the detected levels of Mn in Z. mauritiana and U. kirkiana are significantly higher than the levels found in M. domestica (apple) fruit. Ca concentration of M domestica fruits (7mg/100g) is significantly lower than the concentrations found in the two indigenous fruits Z. mauritiana (11.57mg/100g) and Uapaca kirkiana (13mg/100g).Levels of Cu has a significant variation among all the fruits with U. kirkiana having the highest levels of (0.10mg/100g ), M. domestica (0.021mg/100g), Z. mauritiana (0.094mg/100g). M. domestica has the highest level of Mg (5mg/100g) as compared to indigenous fruits Ziziphus mauritiana (1.742mg/100g) and Uapaca kirkiana (1.62mg/100g). High levels of K are also detected in the exotic fruit, (10g/100g) as compared to Z. mauritiana (8.97mg/100g) and Uapaca kirkiana (8.79mg/100g). Z. mauritiana has the highest level of Fe as compared to U. kirkiana (2.22 mg/100g) and M. domestica fruit (0.12mg/100g).Indigenous fruits Ziziphus mauritiana and Uapaca kirkiana have shown high levels

17 of Zn 0.62mg/100g and 0.66mg/100g respectively which is higher than the concentration in M. domestica fruit (0.05mg/100g). Cd was not detected in the two indigenous fruits and its levels in M domestica fruits could not be found in the database

The results presented in the above table show that C. sinensis (orange) fruits have high levels of Ca (40mg/100g) as compared to the two indigenous fruits Z. mauritiana (11.571mg/100g) and Uapaca kirkiana (13.003mg/100g). A high level of K (181mg) is also observed in C. sinensis which is higher than the concentration in indigenous fruits Z. mauritiana (8.968mg/100g) and Uapaca kirkiana (8.790mg/100g). Indigenous fruits show levels of Na which is not detected in orange fruits . Z. mauritiana (3.40mg/100g) and U. kirkiana (3.85mg/100g). This shows that there is a significant difference in levels of Na between indigenous fruits and C. sinensis fruits. C sinensis fruits show high levels of Mg (10mg) as compared to indigenous fruits with (1.742mg/100g for Z. mauritiana and (1.620mg/100g) for U. kirkiana. Z. mauritiana fruits have the highest level of Mn (214.742mg/100g) and C sinensis having the lowest concentration of (0.025mg/100g). There is a significant difference in Fe levels of indigenous fruits and C. sinensis fruits. Z. mauritiana has the highest level of Fe (3.519mg/100g), C. sinensis fruits having the lowest level of (0.10mg/10

18

CHAPTER FIVE

5.0 DISCUSSION OF RESULTS

5.1 VARIATION OF ESSENTIAL AND TRACE ELEMENTS IN WILD FRUITS The results of trace and essential elements in Ziziphus mauritiana and Uapaca kirkiana fruits are shown in table 4.1.The results show that there is variation between essential elements and trace elements of each fruit species according to their places of origin. Many studies have shown similar findings that have shown the distribution of essential and trace elements in wild fruits consumed by people in India (Mahapatra et al.2012, Nazurudeen et al 2010), Saudi Arabia (Hegazy et al . 2013), Ethiopia (Teketay et al . 2010).

The analyzed fruits have low concentrations of trace elements as compared to essential elements.Trace elements are required by the body in very small amounts (Ismael et al . 2011).Also trace elements tend to bio-accumulate in the body with some of them causing carcinogenic effects to the body (Sobukola et al . 2010) Some of these trace elements aids to absorb some of the elements needed in the body for example a relatively amount of Ni is required to aid in the absorption of Fe in the body. However, high concentrations of Ni concentrations can interfere with zinc, magnesium and calcium utilization and metabolism (Ismael et al , 2011).

Concentrations of Cd and Cr were not detected both the wild fruits under study. This may be because there are no toxic substances in the areas that they grow (Sobukola et al . 2010) excessive content of elements like Cd in food is associated with etiology of a number of diseases especially with cardiovascular diseases especially with kidney, cardiovascular, nervous as well as bone diseases (WHO. 1992). Copper is high in Uapaca kirkiana fruits of Masembura area as compared to the other two places. This might be due to difference in soil composition or soil disturbance due to anthropogenic factors as soil acidity and seasons also affect mineral uptake by plants (Soetan et al, 2010). Other Cu concentrations from the other two sites were lower than the concentrations reported by (Nyanga et al . 2010) This might as well be explained by the difference in soil type and the genetic make- up of the fruit trees in absorbing elements from the soil (Nyangito et al , 2010).

19

Fe is also high in both indigenous fruits which were analyzed with Z. mauritiana ranging U. kirkiana . The results were in agreement with the ones obtained by (Mahapatra et al , 2012) which showed that most wild /indigenous fruits have high iron concentrations as compared to most exotic or cultivated fruits. These results also agree with the results obtained by Nazurudeen, 2010, were he finds some of the indigenous fruits which he tested having high iron concentrations. In a case study carried out by Ebenezer ,(2012) comparing mineral content of fruits, Ziziphus mauritiana had a high level of iron concentration of 1g/100g whilst other fruits ranged from 0,1 to 0.7g (Ebenezer et al , 2012). The results show that naturally Z. mauritiana fruits are rich in Fe.

Levels of trace and essential elements were significantly varying from site to site of the fruit origin (p<0.05) like in a case of Cu in U. kirkiana . Results have shown higher levels of Cu in one site and the other sites which have very low concentrations. This shows differences in soil composition as soil acidity and seasons also affect mineral uptake by plants (Soetan et al, 2010). Other Cu concentrations for Z. mauritiana were different from concentrations reported by (Nyanga et al 2013 and Mahapatra et al , 2012). This is due to the difference in soil type, the differences in climatic conditions of the fruit origin and the genetic make-up of the fruit trees in absorbing elements from the soil (Feyssa and Nyangito. 2010)

The results show that the fruits have high concentrations of essential elements than trace elements. Essential elements are found in large quantities in fruits needed by the body in large quantities for biological and physiochemical functions (Ismail et al , 2011) Na concentrations in Z. mauritiana . Were lower and different from the ones obtained by (Nyanga et al , 2010) where he finds that potassium was higher than the other essential elements under his study. The difference in the availability of concentrations may be due to the difference in weather and climate, availability of elements in the soil and the ability of the fruit trees to absorb the elements from the soil. The concentration of calcium were the highest among the essential elements under with U. kirkiana concentrations having higher concentrations as compared to Z. mauritiana . High concentrations of calcium in the two fruits shows that the soil in which the places where the samples were rich in calcium and also that the fruit trees are good in. According to (Ismail et al , 2011), essential elements such as Ca and Mg are required fairly on large as they maintain body electrolytes and tissue homeostasis, they are also essential for bone structure development and necessary for carbohydrate and protein metabolism. Calcium, magnesium and Potassium are essential for

20 repairing worn out cells, building of red blood cells and maintaining body mechanisms (WHO, 1996). Their absence in diet might result in weak stunted growth and poor bone development (Effiong and Udo, 2010).

5.2 COMPARISON OF ELEMENTAL CONCENTRATIONS IN WILD AND EXOTIC FRUITS The comparison between two indigenous fruits and two exotic fruits shows that indigenous fruits have more nutritional benefits compared to exotic fruits. Ziziphus mauritiana and Uapaca kirkiana . The results show that there is significant difference on the mean concentrations of Ziziphus mauritiana , Uapaca kirkiana and those of apple fruit with Ca, Na, Fe, Cu, Mn, Zn of indigenous fruits higher than concentrations found in apples. Only two elements (K and Mg) in the M. domestica (Apple) fruit had high concentrations as compared with the indigenous fruits.

Mean concentration of Citrus sinensis fruit were also compared with the mean concentrations of two indigenous fruits listed above. The comparison reveals that most of the essential and trace elements levels in orange fruits are lower than those found in the indigenous fruits except for Ca, Na and Mg. This shows that indigenous fruits can be highly nutritious than exotic fruits having more nutrition and food security benefits as supported by literature. Mahapatra et al also agree that most of the wild or indigenous fruits qualify as high nutrient and mineral content comparable to most cultivated counterparts (Mahapatra et al, 2010). In his study it is clear that wild or indigenous fruits such as Ziziphus encolpia and Mimusops elengi have nutrient contents which are higher than mangoes, apples and guavas. The observation by Mahapatra seems to agree with observations made by Nazurudeen, (2010). In the research, the results also show that wild/ indigenous fruits under study Garcinia gummi –gutta , ,Debressa longifolia and Tamilnada uliginosa are with high fiber and mineral content compared to mango , apple and paw-paw fruits.

21

CHAPTER SIX

RECOMMENDATIONS AND CONCLUSIONS

6.1 CONCLUSIONS The results have highlighted that indigenous fruits under study have a low risk of toxic elements. Most of the trace elements which were analyzed were less than the essential elements which means that the fruits have a high probability of food safety and are safe to consume. It was found that indigenous fruits can have higher nutritional benefits in terms of essential and trace elements than some domesticated popular fruits such as M. domestica and C . sinensis. This makes the fruits Ziziphus mauritiana and Uapaca kirkiana potential contributors towards a balanced diet for the residents of Bindura especially children, pregnant women and adolescence that are at high risk of malnutrition deficiency. Consumption of Ziziphus mauritiana and Uapaca kirkiana needs to be promoted as these fruits contribute significantly to the diet and income of people inside and around Bindura where the fruits can be found and also where people cannot afford expensive food supplements

6.2 RECOMMENDATIONS Based on the findings from the current study, it can be recommended that

People need to be sensitised on the nutritive value of indigenous fruits and their role in human health. Since most of the people in Zimbabwe cannot afford food supplements and balanced diets. Hence encourage people to forage for wild plant products

Promotion on the preservation of wild plant species which are presently under threat by human activities needs to be done. This can be achieved by promoting domestication of indigenous fruit trees in areas where they are growing

Future researches should look on other indigenous fruits and vegetables consumed by the residents of Bindura needs to be carried out in order to investigate factors that affect bioavailability of essential and trace elements.

22

REFERENCES Adepoju OT, Oyewole EO (2008. Nutrient composition and acceptability of fortified jam from Spondias mombin (Hog plum, (Iyeye)) fruit pulp.Niger. J Nutr Sci 29(2): 1 80-1 89.

Akan , J.C. Kolo, B G, Yikala B.S. (2013). Determination of some heavy metals in vegetable samples from Biu local Government area, Borno State , North Eastern Nigeria, International Journal of Environmental Monitoring and Analysis 1(2); 40-46

Al Jassir, M.S., Shaker, A., Khaliq, M.A., 2005. Deposition of heavy metals on green leafy vegetables sold on roadsides of Riyadh city,Saudi Arabia. Bulletin of Environment Contamination and Toxicology 75, 1020–1027.

American Journal of Clinical Nutrition (2006). Whole grain intake is inversely associated with the metabolic syndrome and mortality in older adults; 83: 248-257.

Bakhru, H. (2006). Healing through natural fruits, Jaico press, Mumbai; pp. 34-39

Bharucha, Z. and Pretty, J. (2010). The roles and values ofwild foods in agricultural systems. Philosophical Transactions of the Royal Society London B: BiologicalSciences 365: 2913-2926

Brown M.E and CC and Funk, 2008.Food security under Climate Change. Science 319(5868);580- 581.http//dx.doi.org/10.1126/science. 1154102.

Chu, Y.F.; Sun, J.; Wu, X.Z.; Liu, R.H. 2002Antioxidant and Antiproliferative activities of common vegetables J. Agric. Food Chem. 50, 6910–6916.

Edible Fruits of Deciduous Forests of India: an Explorative Study towards Non Conventional BioNutrition. Advance Journal of Food Science and

Effiong, G.S. and I.F. Udo, 2010. Nutritive value of indigenous wild fruits in southeastern Nigeria.Electronic J. Environ. Agric. Food Chem., 9(7):1168-1176.

FAO (1997). Brahui Women’s indigenous knowledge of medicinalplants. Inter regional project for participatory upland conservation anddevelopment. GCP/INT/542/ITN, Balochistan, .

23

FAO (Food and Agriculture Org) 2011b. The state of food insecurity in the world: how does International pricing volatility affect domestic economies and food security? FAO Rome,Italy [online] URL http//www.fao.org/docrep/014/12330e/i2336e00.htm.

FAO (Food and Agriculture Org) 2011c.The state of food insecurity in the world: how does International pricing volatility affect domestic economies and food security? FAO Rome, Italy [online] URL http//www.fao.org/docrep/014/12330e/i2336e00.htm.

FAO (Food and Agriculture Org) 2012.The state of food insecurity in the world: economic growth necessary but not sufficient to accelerate reduction of hunger and malnutrition? FAO Rome Italy [online] URL http//www.fao.org/publications/sofi/2012.en

FAO 2011. (Food and Agriculture Org). Forests for improved Nutrition and food security. FAO, Rome, Italy [online]URL; http//www.fao.org.docrep//014/i2011e00pd.

FAO, 2014 [Food and Agriculture Organization]. State of Food Insecurity

Feyssa, D. H., J. T. Njoka, Z. Asfaw, and M. M. Nyangito. 2011. Seasonal availability and consumption of wild edible plants in semiarid Ethiopia: implications to food security and climate change adaptation. Journal of Horticulture and Forestry 3 (5):138-149

Food and Agriculture Organization– http://www.fao.org/3/a-i4030e.pdf

Godfray, H. C. J., J. R. Beddington, I. R. Crute, L. Haddad, D. Lawrence, J. F. Muir, J. Pretty, S. Robinson, S. M. Thomas, and Ecology and Society 19 (4): 34 C. Toulmin. 2010. Food security: the challenge of feeding 9 billion people. Science 327(5967):812-818.

Institute of Medicine. 2001. Dietary reference intakes for vitamin A, vitamin K, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc.

Washington D.C: National Academy Press 2001; 290-442 .

J.C Akan. B.G Kolo, B.S Yikala, V.O Ogugbuaja. 2014. Determination of some heavy metals in vegetable samples. International Journal of Environmental monitoring and analysis (12 and 13)

Khan A, Mahmoud IS, Khan RU, Khan SU (2013). Study of wild medicinal xerophytes of district Bannu, KPK. J. Med. Plants Stud.1(4):79-86.

24

Khan B, Abdukadir A, Qureshi R, Mustafa G (2011 ). Medicinal uses of plants by the inhabitants of Khunjerab National Park, Gilgit, Pakistan.Pak. J. Bot. 43(5):2301 -2310

Li JW, Shan L, Liu YF, Fan LP, Ai LZ. Screening of a functional polysaccharide from Zizyphus jujuba cv.jinsixiaozao and its property. Int J Biol Micromole

Li, F.; Li, S.; Li, H.B.; Deng, G.F.; Ling, W.H.; Xu, X.R. 2013 Antiproliferative activities of tea and herbal infusions. Food Funct., 4, 530–538. [

Liu, R.H. Li, F.; Li, S.; Li, H.B.; Deng, G.F.; 2003, Health benefits of fruits and vegetables are from additive and synergistic combinations of phytochemicals. Am. J. Clin. Nutr. ,78, 517S–520S.

Mahapatra, A.K., Satarupa, M. , Basak, U.C., Panda, P.C. (2012). Nutrient analysis of some selected edible fruits of deciduous forests of India :an explorative study towards nonconventional bio-nutrient.4(1): 15-21

Mahapatram, A.K., Mishra, S., Basak, U.C. and Panda, P.C.(2012). Nutrient Analysis of Some Selected Wild

Moseley.W.G 2012. Famine myths five understandings related to the 2011 hunger crisis in the horn of Africa Dollars and sense March

Muller L.A.M Almedon.2008 ‘what is famine/?’.Distinguishing between traditional vegetables and special foods for times of hunger/ scarcity (Boumba Nigeria) Human ecology

Muller, C, W Cramer W. L Hare and Lotze Compen 2011.Climate Change risks for Africa Agriculture. Proceedings for the National Academy of Sciences.

Nazarudeen, A., 2010. Nutritional composition of some lesser-known fruits used by ethnic communities andlocal folks of Kerela. Ind. J. Traditional Knowl. 9(2):398-402.

Neudeck, L., L. Avelino, P. Bareetseng, B.N. Ngwenya, D. Teketay & M.R. Motsholapheko. 2012. The contribution of edible wild plants to food security, dietary diversity and income of households in Shorobe Village, northern Botswana. Ethnobotanical Research and Applications 10:449–462 .

Rabia et al, 2012 Journal of applied pharmaceutical Science 02903),

25

Rathore, M., 2009. Nutrient content of important fruit trees from arid zone of Rajasthan. J. Horti

Forestry,1(7):

Rhan K.Y, Khan M.A et al. 2011 Element content analysis of genus ficus using Atomic Absorption Spectroscope .African Journal of pharmacy and pharmacology.

Rsaissi .N, E.L Kamili (1), B. Bencharki (1), L. Hillali(1) & M. Bouhache & M. Bouhache.2013. Antimicrobial activity of fruits extracts of the wild " Ziziphus Lotus (L.) International Journal of Scientific & EngineeringResearch,Volume4, Issue9,15212229-5518.

Sobukola, O.P, Adeniran, M.O. Odedairo, A.A. and Kajihausa, O.E. 2010. Heavy metal levels of some fruits and vegetables from selected markets in Lagos, Nigeria. African Journal of Food Science 4: 389-393

Soetan et al, 2010.The importance of minerals elements for human domestic animals and plants. African journal of food science Volume 4

Soetan KO (2008). Pharmacological and other Beneficial effects of Antinutritional Factors in Plants- A Review. Afr. J. Biotechnol Vol.7(25): 471 3-4721 .

Somnath B, Badal K. D and Ajay K.S (2012) Determination of mineral content and heavy metal content of some traditionally important aquatic plants of Tripura, India using atomic absorption spectroscopy. Journal of Agricultural Technology 8(4): 1467-1476

Teketay, D., F. Senbeta, M. Maclachlam, M. Bekele & P. Barklund. 2010. Edible Wild Plants in Ethiopia. Addis Ababa University Press, Addis Ababa.

UNSCN (2010) 6th Report on the world nutrition situation: Progress in nutrition, United Nations System – Standing Committee on Nutrition, Geneva, Switzerland, http://www.unscn.org/files/Publications/RWNS6/report/SCN_report.pdf, accessed July

WHO, 1996. Trace Elements in Human Nutrition and Health. World Health Organization, Geneva.

World Health Organization (1996b). Trace elements in human nutrition and health, Geneva; pp.72- 103.

26

Yan. O, Lui.Y and Li Y, 2014. Analysis of Essential and Toxic Elements in Jujube Fruits collected from different locations in China Tropical Journal of Pharmaceutical Research; 13 (4) :607-611

Zhao GR, Xiang ZJ, Ye TX, Yuan YJ, Guo ZX (2006).Antioxidant activities of Salvia miltiorrhiza and Panaxnotoginseng. Food Chem.99:676-774

Zhou, Y.; Li, Y.; Zhou, T.; Zheng, J.; Li, S.; Li, H.B. 2016 Dietary natural products for prevention and treatment of liver cancer. Nutrients, 8, 156.

APPENDICES

APPENDIX 1 Mean nutritional content of Exotic fruits [USDA Release 28, (2015)]

FRUIT Ca Fe Mg K Na Zn Cu Mn Apple 7 0.12 5 108 1 0.05 0.021 0.037 Orange 40 0.10 10 181 0 0.07 0.045 0.025

APPENDIX 2 Preparation of Elemental 100ml/l Stock Solutions.

Element Salt used Formula mass Mass Volume of Final (g/mol) weighed Distilled concentration and Water of stock dissolved solution (g)

27

Manganese Manganese MnCl2.4H20 3.6607g 1 litre 1000ppm chloride Zinc Zinc oxide ZnO 1.2450g 1 litre 1000ppm

Copper Copper CU(NO30)2.3H20 3.7980g 1 litre 1000ppm Nitrate Cadmium Cadmium CdCl2 2.0360g 1 litre 100ppm Chloride Magnesium Magnesium MgCl2.4H20 3.9160g I litre 100ppm Chloride Iron Iron FeCl3.6H20 4.8400g 1 litre 1000ppm Chloride Sodium Sodium NaCl 2.5420g 1 litre 1000ppm Chloride Chromium Chromium Cr(NO3)3.9H20 7.6960g 1 litre 1000ppm nitrate Calcium Calcium CaCo3 2.4973g 1 litre 100ppm Carbonate Potassium Potassium KCl 11.9070g 1 litre 1000ppm chloride

APPENDIX THREE Determination of trace and essential elements in Indigenous fruits

There is drying of salts in the block digester at the temperature of 105degrees celcius. i. Operating conditions: Establish instrumental detection limit, precision, optimum background correction positions, linear dynamic range and interference for each analytical line. Verify that the instrument configuration and operating conditions satisfy the analytical requirements. An atom- to-ion emission intensity ratio can be used to reproduce optimum conditions for multi element analysis precisely. The Pb/Cd intensity ratio may be incorporated into the calibration procedure, including specifications for sensitivity and for precision.

28 ii .Instrumental calibration: Warm up for 30 min. For polychromators, perform an optical alignment using the profile lamp or solution. Check alignment of plasma torch and spectrometer entrance slit. Make Pb/Cd or similar intensity ratio adjustment. Calibrate instrument using calibration standards and blank. Aspirate the standard or blank for a minimum of 15 s after reaching the plasma before beginning signal integration. Rinse with calibration blank or similar solution for at least 60 s between each standard to eliminate any carryover from the previous standards. Use average intensity of multiple integration of standards or samples to reduce random error. Before analysing samples, ensure the instrument check that concentration values obtained should not deviate from the actual values by more than ± 5%. The wavelength for the element are as suggested below. iii. Analysis of samples: Analyse the samples using calibration blank. This permits a check of the sample preparation reagents and procedures for contamination. Analyse samples, alternatively with analyses of calibration blank. Rinse for at least 60s with dilute acid between samples and blanks. After introducing each sample or blank let system equilibrate before starting signal integration. Examine each analysis of the calibration blank to verify that no carryover memory effect has occurred. If carryover is observed, repeat rinsing until proper blank values are obtained. Make appropriate dilutions of the sample to determine concentrations beyond the linear calibration. iv. Spike test: To the known volume of digested sample, add known volume of the standard. Shake well and aspirate through ICP. Check the increase in concentration of metal for added quality of standards.

Instrument quality control: Analyse instrument check standard to confirm proper recalibration. Reanalyse one or more samples analysed just before termination of the analytical run. Results should agree with 15% error. Analyse instrument quality control sample within everyone run. Use this analysis to verify accuracy and validity of the calibration standards.

29

.

30