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DETERMINATION OF LEVELS OF SOME VITAMINS IN Amaranthus hypochondriacus AND Amaranthus cruenthus LEAVES AND GRAINS FROM SELECTED AREAS OF KENYA By CHERUIYOT NICHOLAS, B. Ed (Sc.) REG. NO. I56/CE/15650/05 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY JUNE 2011 ii DECLARATION I hereby declare that this is my original work and has not been presented for the award of a degree in any other university. Cheruiyot K. Nicholas Reg. No. I56/CE/15650/05 Kenyatta University Signature Date This thesis has been submitted for examination with our approval as university supervisors. Dr. Ruth N. Wanjau Department of Chemistry Kenyatta University Signature Date Dr. Nicholas K. Gikonyo Department of Pharmacy and Complementary/ Alternative Medicine Kenyatta University Signature Date Prof. Hudson Nyambaka Department of Chemistry Kenyatta University Signature Date iii DEDICATION This thesis is dedicated to my late grand father, Mr. Kipsoi Tomno, for his immense academic advice, support and for inspiring me during my childhood education. iv ACKNOWLEDGEMENT This report is an outcome of concerted efforts of many individuals and institutions. It is therefore imperative to acknowledge some of the key contributors. First and foremost may I sincerely thank my supervisors, Dr. Ruth Wanjau, Dr. Nicholas Gikonyo and Prof. Hudson Nyambaka for their guidance, assistance and encouragement during my research. Their positive comments and personal involvement in the preparation of this thesis is immensely appreciated. Much thanks go to the technical staff of the Chemistry department of Kenyatta University for their great assistance. In this regard, I thank Mr. Maina, Mr. Waswa, Mr. Mwangi, Mr. Kang’ethe, Mr. Osoro, Mr. Odhiambo, Mr. Makibia and Mr. Salim for their valued assistance. May I also thank the entire staff of the department of Pharmacy and Complementary/Alternative Medicine and I single out the technicians Mr. Karemi and Mrs. Wangila for their support. The assistance of Mr. Karanja of the department of Food Science and Technology of Jomo Kenyatta University is highly appreciated. I thank Kenyatta University for giving me the opportunity to pursue my studies. I also acknowledge the department of Food Science and Technology of Jomo Kenyatta University for according me the opportunity to use their HPLC equipment for analysis. My appreciation also goes to the Kenya Agricultural Productivity Project (KAPP) grant number (KAPP 06/ PRC-SECBCCI-06-FP2006023) for sponsoring the research work. May I also appreciate my colleagues Mathew Tonui, Stanley Tumbo, Stanley Ngeno, Henry Mwangi, Peter Echesa and Evans Choi just to mention afew for their moral support and company during my studies. Special thanks go to my family members and relatives for their prayers, encouragement and support during the entire study program. Finally, I thank God for granting me sound health, strength and determination to undertake this program successfully. v TABLE OF CONTENTS Contents TITLE PAGE ............................................................ Error! Bookmark not defined. DECLARATION....................................................................................................... ii DEDICATION ......................................................................................................... iii ACKNOWLEDGEMENT ........................................................................................ iv TABLE OF CONTENTS........................................................................................... v LIST OF TABLES ................................................................................................. viii LIST OF FIGURES .................................................................................................. xi ABBREVIATIONS AND ACRONYMS ................................................................. xii ABSTRACT ........................................................................................................... xiii CHAPTER ONE........................................................................................................ 1 1.0 INTRODUCTION ........................................................................................... 1 1.1 Background information .................................................................................. 1 1.2 Problem statement and justification .................................................................. 5 1.3 Hypotheses ...................................................................................................... 6 1.4 Objectives ........................................................................................................ 7 1.4.1 General objective ....................................................................................... 7 1.4.2 Specific objectives ..................................................................................... 7 1.5 Scope and limitations of the study .................................................................... 8 1.6 Significance of the study and anticipated output ............................................... 8 CHAPTER TWO ....................................................................................................... 9 2.0 LITERATURE REVIEW ................................................................................. 9 2.1 Amaranth ......................................................................................................... 9 2.1.1 Grain amaranth ........................................................................................ 10 2.2 Vitamins ........................................................................................................ 13 2.2.1 Beta-carotene (pro-vitamin A) ................................................................. 14 2.2.3 Vitamin B1 (Thiamin)............................................................................... 19 2.2.4 Vitamin B2 (riboflavin) ............................................................................ 21 2.2.5 Vitamin B6 (Pyridoxine) .......................................................................... 23 vi 2.2.6 Vitamin C (ascorbic acid) ........................................................................ 25 2.3 Methods of analysis of vitamins ..................................................................... 27 2.3.1 Principles of HPLC method ..................................................................... 28 2.3.2 Principles of spectrophotometric method.................................................. 30 2.3.3 Principles of titrimetric method ................................................................ 31 CHAPTER THREE ................................................................................................. 33 3.0 MATERIALS AND METHODS ................................................................... 33 3.1 Study areas .................................................................................................... 33 3.2 Study design .................................................................................................. 34 3.3 Planting and management practices of grain amaranth ................................... 35 3.4 Sampling and sample pre-treatment................................................................ 36 3.5 Instrumentation .............................................................................................. 37 3.6 Chemicals ...................................................................................................... 38 3.7 Cleaning of glassware and vials ..................................................................... 39 3.8 Preparation of solutions.................................................................................. 39 3.8.1 Stock solutions of β-carotene and α-tocopherol ........................................ 39 3.8.2 Stock solutions of B vitamins ................................................................... 39 3.8.3 Extraction and standard solutions for AA analysis ................................... 39 3.9 Extraction and analysis of β-carotene and vitamins ........................................ 40 3.9.1 Simultaneous extraction and analysis of β-carotene and α-tocopherol ...... 40 3.9.2 Simultaneous extraction and analysis of thiamin, riboflavin and pyridoxine ......................................................................................................................... 42 3.9.3 Extraction and analysis of AA.................................................................. 44 3.10 Data analysis ................................................................................................ 44 CHAPTER FOUR ................................................................................................... 45 4.0 RESULTS AND DISCUSSION ..................................................................... 45 4.1 Introduction ................................................................................................... 45 4.2 Methods validation......................................................................................... 45 4.2.1 Correlation between spectrophotometric and HPLC methods of β-carotene and α-tocopherol analysis ................................................................................. 45 4.2.2 Linearity and limits of detection ............................................................... 46 4.2.3 Recovery analysis .................................................................................... 47 4.3 Levels of β-carotene/vitamins in A. hypochondriacus and A. cruentus leaves at different ages of growth grown in different areas during wet and dry seasons ...... 47 4.3.1 Beta-carotene levels of A. hypochondriacus and A. cruentus leaves at different ages of growth .................................................................................... 48 4.3.2 Alpha-tocopherol levels of A. hypochondriacus and A. cruentus leaves at different ages of growth .................................................................................... 54 4.3.3 Thiamin levels of A. hypochondriacus and A. cruentus leaves at different ages of growth .................................................................................................. 60 vii 4.3.4 Riboflavin levels of A. hypochondriacus and A. cruentus leaves at different ages of growth .................................................................................................. 65 4.3.5 Pyridoxine levels of A. hypochondriacus and A. cruentus leaves at different ages of growth .................................................................................................. 69 4.3.6 Ascorbic acid levels of A. hypochondriacus and A. cruentus leaves at different ages of growth .................................................................................... 73 4.4 Comparisons of beta-carotene and vitamin levels in A. hypochondriacus and A. cruentus leaves during the wet and dry seasons between different areas ............... 78 4.4.1 Beta-carotene contents in A. hypochondriacus and A. cruentus leaves from different regions ............................................................................................... 79 4.4.2 Alpha-tocopherol contents in A. hypochondriacus and A. cruentus leaves from different areas .......................................................................................... 81 4.4.3 Thiamin contents in A. hypochondriacus and A. cruentus leaves from different regions ............................................................................................... 84 4.4.4 Riboflavin contents in A. hypochondriacus and A. cruentus leaves from different regions ............................................................................................... 85 4.4.5 Pyridoxine contents in A. hypochondriacus and A. cruentus leaves from different regions ............................................................................................... 88 4.4.6 Ascorbic acid contents in A. hypochondriacus and A. cruentus leaves from different areas ................................................................................................... 90 4.5 Levels of beta-carotene and vitamins in amaranth grains from different areas 92 4.5.1 Beta-carotene contents in A. hypochondriacus and A. cruentus grains from different areas ................................................................................................... 92 4.5.2 Alpha-tocopherol contents in A. hypochondriacus and A. cruentus grains from different areas .......................................................................................... 94 4.5.3 Thiamin contents in A. hypochondriacus and A. cruentus grains from different areas ................................................................................................... 96 4.5.4 Riboflavin contents in A. hypochondriacus and A. cruentus grains from different areas ................................................................................................... 97 4.5.5 Pyridoxine contents in A. hypochondriacus and A. cruentus grains from different areas ................................................................................................... 98 4.5.6 Ascorbic acid contents in A. hypochondriacus and A. cruentus grains from different areas ................................................................................................. 100 CHAPTER 5.......................................................................................................... 102 5.0 CONCLUSIONS AND RECCOMENDATIONS ......................................... 102 5.1 Conclusions ................................................................................................. 102 5.2 Recommendations ........................................................................................ 102 5.2.1 Recommendations from this study ......................................................... 102 5.5.2 Recommendations for further study ....................................................... 103 REFERENCES ...................................................................................................... 104 APPENDICES ....................................................................................................... 114 viii LIST OF TABLES Table 2. 1: Beta-carotene content of some vegetables .............................................. 16 Table 2. 2: Alpha-tocopherol content (mg/100 g raw weight) of some foods ............ 18 Table 2. 3: Thiamin composition of some foods in mg/100 g sample raw weight ..... 20 Table 2. 4: Riboflavin content of some foods in mg/100g raw sample weight .......... 22 Table 2. 5: Pyridoxine content of some foods in mg/100g raw sample weight ......... 24 Table 2. 6: Ascorbic acid content of some foods (mg/100g raw sample weight) ....... 26 Table 3.1: Regression lines equations for the thiamin, riboflavin and pyridoxine.....................................................................................................................43 Table 4.1: Comparison of amounts of β-carotene and α-tocopherol in amaranth using spectrophotometric and HPLC methods (n = 24).........................................................46 Table 4. 2: Linearity and limits of detection of the analytical methods ..................... 46 Table 4. 3: Recovery of vitamins/pro-vitamin in amaranth leaves and grains ........... 47 Table 4. 4: Beta-carotene content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons .............................. 48 Table 4. 5: Comparison of mean β-carotene levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing .............. 51 Table 4. 6: Alpha-tocopherol content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons ..................... 55 Table 4. 7: Comparison of mean α-tocopherol levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing .............. 58 Table 4. 8: Thiamin content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons .............................. 61 Table 4. 9: Comparison of mean thiamin levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing .................. 63 ix Table 4. 10: Comparison of mean thiamin contents of A. hypochondriacus and A. cruentus leaves between wet and dry seasons ................................................... 64 Table 4. 11: Riboflavin content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons .............................. 65 Table 4. 12: Comparison of riboflavin mean levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing .............. 67 Table 4. 13: Comparison of mean riboflavin contents of A. hypochondriacus and A. cruentus leaves between wet and dry seasons ................................................... 68 Table 4. 14: Pyridoxine content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons .............................. 69 Table 4. 15: Comparison of pyridoxine levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing .................. 71 Table 4. 16: Comparison of mean pyridoxine contents of A. hypochondriacus and A. cruentus leaves between wet and dry seasons ................................................... 72 Table 4. 17: Ascorbic acid content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons .............................. 73 Table 4. 18: Comparison of AA levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing ................................ 76 Table 4. 19: Beta-carotene content of A. hypochondriacus and A. cruentus leaves from different areas during wet and dry seasons ....................................................... 80 Table 4. 20: Alpha-tocopherol content of A. hypochondriacus and A. cruentus leaves from different areas during the wet and dry seasons ......................................... 82 Table 4. 21: Thiamin content of A. hypochondriacus and A. cruentus leaves from different areas in wet and dry seasons .............................................................. 84 x Table 4. 22: Riboflavin content of A. hypochondriacus and A. cruentus leaves from different areas in wet and dry seasons .............................................................. 86 Table 4. 23: Pyridoxine content of A. hypochondriacus and A. cruentus leaves from different areas in wet and dry seasons .............................................................. 88 Table 4. 24: Ascorbic acid content of A. hypochondriacus and A. cruentus leaves from different regions in wet and dry seasons ........................................................... 90 Table 4. 25: Beta-carotene content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) ............................................ 93 Table 4. 26: Alpha-tocopherol content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) ................................... 94 Table 4. 27: Thiamin content content (mg/100 g DW) of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) ........... 96 Table 4. 28: Riboflavin mean content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) ................................... 97 Table 4. 29: Pyridoxine mean content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) ................................... 99 Table 4. 30: Ascorbic acid content content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) ....................... 100 xi LIST OF FIGURES Figure 2. 1: Amaranth plants: (a) A. cruentus (b) A. hybridus ................................... 10 Figure 2. 2: Amaranth grains ................................................................................... 12 Figure 2. 3: Molecular structure for retinol .............................................................. 15 Figure 2. 4: Molecular structure for beta-carotene .................................................... 15 Figure 2. 5: Molecular structure for alpha tocopherol ............................................... 17 Figure 2. 6: Molecular structure for thiamine ........................................................... 19 Figure 2. 7: Molecular structure for riboflavin ......................................................... 21 Figure 2. 8: Molecular structure for pyridoxine ........................................................ 23 Figure 2. 9: Molecular structure for ascorbic acid .................................................... 25 Figure 2. 10: Block diagram of the components of an HPLC.................................... 29 Figure 2. 11: Block diagram of a single beam spectrophotometer ............................. 30 Figure 3. 1: Grain amaranth plants grown at Bureti: (a) A. cruentus at 25 days (b) A. hypochondriacus at 75 days and the researcher...........................................................35 Figure 3. 2 The HPLC equipment used in the study ................................................. 38 xii ABBREVIATIONS AND ACRONYMS Abbreviation AA AI AIDS AOAC AVC BP CBS DAA DAS DCPIP DCM DRI DW EAR FAD FAO FMN FSV GA HIV HPLC IUPAC KDHS KU MOH MP MPA PLP PLWHA RAE RDA RP RPC SP TPP UI UN UNICEF UV VAD VBP VD WHO WSV Meaning Ascorbic Acid Adequate Intake Acquired Immune Deficiency Syndrome Association of Official Analytical Chemists Association of Vitamin Chemists Bonded Phase Central Bureau of Statistics Dehydroscorbic Acid Days Afer Sowing Dichlorophenolindophenol Dichloromethane Dietary Reference Intakes Dry Weight Estimated Average Requirement Adenine Dinucleotide Food Agricultural Organisation Flavin Mononucleotide Fat Soluble Vitamin Grain Amaranth Human Immuno-defiency Virus High-Performance Liquid-Chromatography International Union of Pure and Applied Chemistry Kenya Demographic and Health Survey Kenyatta University Ministry of Health Mobile Phase Metaphosphoric Acid Pyridoxal Phosphate People Living With HIV and AIDS Retinol Activity Equivalent Recommended Dietary Allowance Reverse Phase Reverse- phase Liquid Chromatography Stationary Phase Thiamin Pyrophosphate Tolerable Upper Intake Level United Nations United Nations Children’s Fund Ultra-violet Vitamin A deficiency Vitamin-binding Protein Vitamin Deficiency World Health Organisation Water Soluble Vitamin xiii ABSTRACT Vitamin deficiencies are health hazards in some parts of Kenya. These are attributed to inadequate access to vitamin-rich foods occasioned by decline in agricultural production due to unfavourable climatic conditions, low purchasing power due to poverty, over reliance on staple foods and dependence on highly refined foods, which are poor in vitamins. Higher intake of vitamin-rich vegetables and grains can alleviate this problem. Grain amaranth (GA), which is relatively a new crop in Kenya, is a dual crop whose seeds are used as grains and leaves as a vegetable. It has been found to have high nutritional and medicinal values, which could combat life-threatening diseases such as HIV/AIDS, diabetes, cancer, hypertension, liver diseases and haemorrhage. However, it is still an underutilized crop. As a leafy vegetable, it can be harvested at different maturity stages of growth, but data on the changes in leaf nutritional value with plant age are scanty. The objective of this study was to determine the levels of β-carotene, thiamin, riboflavin, pyridoxine, ascorbic acid (AA) and α-tocopherol in Amaranthus hypochondriacus (L-) and Amaranthus cruentus (L-) leaves at different ages of growth and their grains grown in different regions of Kenya in wet and dry seasons using high-performance liquid chromatography, spectrophotometric and titrimetric methods. The study areas were Kenyatta University, Bureti, Mt. Elgon, Kisii, Bondo, Meru and Embu districts. The mean vitamin levels (mg/100 g DW) in amaranth leaves were: β-carotene (35.985-49.797), α-tocopherol (29.494-34.779), thiamin (0.573-0.714), riboflavin (2.311-2.755), pyridoxine (2.581-2.921) and AA (440.077-619.210). The mean vitamin contents of leaves were significantly different between the species (P<0.05) except thiamin and riboflavin. Results showed that the amounts of β-carotene and α-tocopherol increased significantly (P<0.05) with the plants’ ages while those of thiamin, riboflavin, pyridoxine and AA decreased significantly (P<0.05). Between the study areas, no clear trend in the amounts of vitamins in the leaves was observed. Significant differences were noted in the leaf vitamin contents between the wet and dry seasons (P<0.05) except thiamin and riboflavin. The amounts of vitamins (mg/100 g DW) in amaranth grain ranged between 4.614-4.633 for β-carotene, 1.055-1.068 for αtocopherol, 0.152-0.166 for thiamin, 0.273-0.285 for riboflavin, 0.223-0.229 for pyridoxine and 4.037-4.303 for AA. The levels of vitamins in amaranth grain were generally not different significantly between the species apart from AA (P<0.05) in some areas. The amounts of vitamins in grain between the seasons were not significantly different except thiamin in some areas (P<0.05). It can be concluded that the plant age, geographical location as well as the season influence the levels of vitamins in amaranth leaves. However, they do not have any pronounced effects on the levels of vitamins in amaranth grains. The results will be used to sensitise the public on the importance of amaranth leaves and grains as a reliable source of vitamins. The results will also be availed to relevant authorities, who may use it for policy formulation. xiv 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background information Malnutrition in Kenya and other developing countries as a result of micronutrient deficiency is widespread (FAO, 2005; Muyonga et al., 2008). It is one of the major causes of poor health and poor child development among low-income populations (Hotz, 2007). Undernutrition accounts for more than 55 percent of all childhood mortalities (UNICEF, 2008). A Kenyan government study of 2005/2006 (CBS, 2006) on intergrated household budget showed high prevalence of stunting (33 %), wasting (6.1 %) and underweight (20.2 %) among children below five years of age. Assessment of health and nutritional status of children aged five years and below in Siaya district of Western Kenya indicated the prevalence of underweight, stunting and wasting as 30, 47 and 7 per cent respectively (Bloss et al., 2004). Besides children, the other most vulnerable individuals are pregnant and lactating mothers, very old people living alone, refugees and displaced people (FAO, 2005; UNICEF 2008). Malnutrition is caused by food insecurity, which occurs due to unavailability of food, insufficient purchasing power, inappropriate distribution, or inadequate use of food at the household level (Caulfield et al., 2004). Poverty is the major cause of food insecurity in Kenya and consequently high prevalence of micronutrient deficiencies due to inability of the poor in society to access adequate amounts of nutrient-rich foods to meet dietary requirements (Caulfield et al., 2004). It is estimated that 56 % of the Kenyan population is living below the national poverty line and that 47 % does not have secure access to food that can adequately meet their dietary requirements (FAO, 2005). Kenya is plagued by acute food insecurity due to unfavourable climatic 2 conditions such droughts and floods especially in North and North- Eastern (FAO, 2005; UNICEF, 2008). About 80 % of the land area in Kenya is arid and semi-arid while areas with a good agricultural potential represent only about 18 % but support 80 % of the population (FAO, 2005). Problems of access to food due to inadequate market and transport infrastructure have also contributed to food insecurity in Kenya. Poor marketing and trade infrastructures have adversely affected producers’ access to both inputs and markets for selling their products (FAO, 2005). The progression of the Human Immuno-deficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) epidemic is also a major cause of poverty and food insecurity (FAO, 2005; UNICEF, 2008). The Kenya Demographic and Health Survey (KDHS, 2004) estimated that the average prevalence of HIV infection in Kenya is 7 % with 1.2 to 1.5 million people in between the ages of 15 to 49 years infected. The pandemic compromises the immune system resulting in increased susceptibility to severe illness, loss of income and incapacity to work (MOH, 2006; NASCOP, 2006). It also causes a steep rise in the number of orphans due to increased number of adult deaths (UNICEF, 2008). Prevalence of other diseases such as malaria, diabetes, hypertension and cancer also contributes to food insecurity due to loss of income and jobs (KDHS, 2004). Vitamin deficiency (VD) is a health hazard in many parts of Kenya (Jansen et al., 1987). The VD diseases include xerophthalmia, night-blindness, scurvy, pellagra, beriberi and rickets (Jansen et al., 1987). This is attributed to inadequate access to vitamin-rich foods mainly as a result of low purchasing power due to poverty and unfavourable climatic conditions such as prolonged drought. Over-reliance on staple 3 foods like maize, potatoes, sweet potatoes and cassava has also contributed to VD since they cannot adequately supply all the required vitamins (Allen, 2003; FAO, 2005). In urban areas, the dependence on highly refined foods such as rice and readymade maize flour has increased VDs since refining process leads to loss of vitamins (Allen, 2003). A study done in Kenya showed that an average of 94 children who visited Koru Mission Hospital in Nyando district per month in 2001 had nightblindness, bitot’s spots and keratomalacia due to vitamin A deficiency (VAD) (Manuche, 2003). A nutritional survey of Wajir district, North Eastern province revealed high incidences of scurvy and beriberi (Rioba et al., 2000). A significantly high prevalence of xerophthalmia among Kenyan children aged 4-7 years was reported in Mathare slum in Nairobi and Kitui district (Munene, 2003). Data from a study conducted in Nandi district showed that 78 % of breastfeeding mothers had a low level of retinol in breast milk indicating VAD (<1.05 μmol/ L) (Ettyang et al., 2003). The implementation of food supplementation and food fortification programmes in Kenya to avert the VDs amongst the vulnerable groups is costly and insufficient (FAO, 2005). More long-term strategies such as dietary diversification and nutritional education are therefore needed (FAO, 2005). There is therefore a need to identify vitamin-rich foods that can be produced cheaply to meet the vitamin requirements for the vulnerable groups. Fresh vegetables and fruits play a significant role in human nutrition, especially as sources of vitamins (A, E, B6, C, thiamin, riboflavin, niacin and folate), minerals, and dietary fiber (Wargovich, 2000; Kader, 2002). Vegetables and fruits in the daily diet 4 have been associated with reduced risk of some forms of cancer, heart disease, stroke, and other chronic diseases (Prior and Cao, 2000; Southon, 2000; Wargovich, 2000). The levels of nutrients in fruits and vegetables are influenced by factors such as genotype, environmental growing conditions, growth stage and post harvest handling (Gad et al., 1982; Goldman et al., 1999). Among these factors, cultivar and stage of maturity of the plant play a crucial role in the nutritional quality of fruits and vegetables (Kader, 2002; Rodriquez-Amaya and Kimura, 2004; Azevedo and Rodriguez-Amaya, 2005; Bergquist et al., 2007). Azevedo and Rodriguez-Amaya (2005), for instance, reported that carotenoid content of kale leaves increased with with the growth age. The environmental factors that affect the nutrients in plants include altitude, soil quality, soil pH and salinity, production practices, insect injury and plant diseases (Goldman et al., 1999; Kader, 2002). Nitrogen nutrition also has a great influence on the contents of most nutrients in leafy vegetables (Kader, 2002) Grain amaranth (GA) is a crop which has received considerable attention in some parts of the country owing to its high nutritional and nutraceutical values and its ability to withstand diverse environmental conditions (Chenchong, 1997). It is one of the few plants whose leaves are eaten as a vegetable while the seeds are used in the same way as cereals (Morales et al., 1988). The crop has the potential to contribute to addressing the nutritional needs of vulnerable people because of its high content of protein, essential fatty acids and micronutrients (Muyonga, et al., 2008). Studies have reported that amaranth species are rich in vitamins A, B6, C, riboflavin, thiamine and folates and essential minerals (Macrae et al., 1993; Dodok et al., 2006; Platkin, 2008). 5 The dietary composition of amaranth can stimulate the body defense mechanism to retard the progression of HIV/AIDS virus (Ng’ang’a et al., 2008). It has also been found to be having medicinal values, which can reduce or combat diabetes, hypertension, liver disease and haemorrhage (Kim et al., 2006; Martirosyan et al., 2007). Commercial production of GA can serve as a useful tool for poverty alleviation since the revenue generated can contribute significantly to enhancement of household food security (Ng’ang’a et al., 2008). 1.2 Problem statement and justification Grain amaranth is a relatively new crop in Kenya and was registered by the ministry of agriculture as a crop in Kenya in 1991 (Berkelaar and Alemu, 2006). It is a dual crop whose leaves are used as a vegetable and the seeds as a grain (Yarger, 2008). Grain amaranth has unique nutritional and promising economic value due to the variety of uses and benefits to producers, processors and consumers (Muyonga et al., 2008). However, it is still an underexploited plant in Kenya. This could be ascribed to low opinion many people have on the local varieties of amaranth. They are usually considered a poor peoples’ resource, and the plants are often rejected by many. Present interests on GA plant have developed because it offers leaves and grains of high nutritional and nutraceutical values. Many studies on levels of vitamins have been done on vegetable amaranth in Kenya (Nyalili, 1995; Nyambaka, 1988; Onyango, 2008) but little has been reported on GA. The effects of preharvest factors on the nutritional qualities of many fruits and vegetables have been documented by several researchers (Nagy, 1980; Shewfelt, 1990; Weston and Barth, 1997; Kader, 2002; Madakadze and Kwaramba, 2004; 6 Makus et al., 2006). For instance, climatic factors especially temperature and light intensity have been shown to have strong influence on the nutritional values of fruits and vegetables. Consequently, the location and season in which the plants are grown can influence the levels of ascorbic acid (AA), carotene, riboflavin, thiamin and flavanoids (Kader, 2002). Little however, has been reported on the variation of vitamins in the GA leaves and grains grown under different ecological conditions in Kenya. The maturity states of the plants have been shown to have nutritional effects of many vegetables and fruits (Kader, 2002; Rodriqez-Amaya and Kimura, 2004). But very little information is available on the effect of maturity states on the levels of vitamins in the GA leaves. The present study determined the levels of β-carotene, thiamin, riboflavin, pyridoxine, AA and α-tocopherol in Amaranthus cruentus (L-) and Amaranthus hypochondriacus (L-) leaves at 25, 50, 75 days after sowing, and their grains from selected areas of Kenya in wet and dry seasons. 1.3 Hypotheses i. There is no significant difference in the levels of beta-carotene, alphatocopherol, thiamin, riboflavin, pyridoxine and ascorbic acid in A. hypochondriacus and A. cruentus leaves at different ages of growth. ii. The levels of beta-carotene, alpha-tocopherol, thiamin, riboflavin, pyridoxine and ascorbic acid between A. hypochondriacus and A. cruentus leaves and grains do not differ significantly. 7 iii. The levels of beta-carotene, alpha-tocopherol, thiamin, riboflavin, pyridoxine and ascorbic acid in amaranth leaves do not differ significantly between the wet and dry seasons. iv. There is no significant difference in the levels of beta-carotene, alphatocopherol, thiamin, riboflavin, pyridoxine and ascorbic acid in A. hypochondriacus and A. cruentus grains between different areas of Kenya. v. There is no significant variation in the levels of vitamins in amaranth grains between wet and dry seasons. 1.4 Objectives 1.4.1 General objective The general objective of this study was to determine the levels of beta-carotene and some vitamins in A. cruentus and A. hypochondriacus leaves and grains obtained from selected areas of Kenya during wet and dry seasons. 1.4.2 Specific objectives i. To determine the levels of beta-carotene, alpha-tocopherol, thiamin, riboflavin, pyridoxine and ascorbic acid in A. hypochondriacus and A. cruentus leaves harvested from Kenyatta University, Bureti, Mt. Elgon and Kisii at 25, 50 and 75 days of growth in wet and dry seasons.. ii. To determine the levels of beta-carotene, alpha-tocopherol, thiamin, riboflavin, pyridoxine and ascorbic acid in A. hypochondriacus and A. cruentus grains obtained from Kenyatta University, Bureti, Mt. Elgon, Kisii, Bondo, Meru and Embu during wet and dry seasons. 8 1.5 Scope and limitations of the study The study only evaluated six vitamins in the leaves and grains of only two species of grain amaranth. Analyses of vitamins in the leaves were only done at three different ages after sowing in only four study areas namely Kenyatta University, Bureti, Mt. Elgon and Kisii. However, analyses of vitamins in the grains were done in the seven study areas which were Kenyatta University, Bureti, Mt. Elgon, Kisii, Bondo, Meru and Embu.This was due to high cost of analysis and time constraints. The production of the plants at Kenyatta University was done by sowing the seeds in pots in a secured place. This was prompted by the fact that on several occasions the amaranth plants in the Kenyatta University plots were all eaten up by birds as soon as they germinated. However, the soil used for planting in the pots was scooped from the plots where they had initially been planted. 1.6 Significance of the study and anticipated output The results of this study will be published to provide useful information on the importance of amaranth leaves and grains as high sources of vitamins and to encourage the public to grow it in home gardens. The results from this study will also add to the food composition data available to Kenyans. 9 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Amaranth Amaranth is a genus of annual herbs belonging to the family Amaranthaceae (Stephens, 2009). It consists of more than sixty species, most of them wild, and thousands of varieties (Svirskis, 2003; Price and Berkelaar, 2006; Yarger, 2008). Originating in Central and South America, amaranth has been cultivated for more than 8,000 years dating back to Mayan, Incan and Aztec civilisations (Svirskis, 2003; Yarger, 2008). Four types of amaranth are presently recognized: grain, vegetable ornamental and weed (Kauffman and Weber, 1988; Price and Berkelaar, 2006). Many fall into more than one category (Yarger, 2008). Grain amaranth (GA) is represented by A. hypochondriacus (prince’s feather), A. cruentus (purple amaranth) (Figure 2.1a) and A. caudatus (love-lies-bleeding) (Yarger, 2008). Vegetable amaranths grow very well in the hot and humid regions of Africa, Southeast Asia, China and India and are represented by A. hybridus (Figure 2.1b), A. tricolor (tambala), A. viridus, A. dubius, A. cruentus, A. edulis and A. blitum (Muyonga et al., 2008; Yarger, 2008). Although it is a grain type, A. caudatus is often grown more as an ornamental (Ofitserov, 2001; Yarger, 2008). Weedy species are represented by A. retroflexus (redroot pigweed), A. albus (tumbleweed) and A. spinosus (spiny amaranth) (Yarger, 2008). 10 (a) (b) . Figure 2. 1: Amaranth plants: (a) A. cruentus (b) A. hybridus There is no distinct separation between all the types of amaranth since the leaves of their young plants can be eaten as both human and animal food (Muyonga et al., 2008; Yarger, 2008). They are very good sources of vitamins (A, B6, C, E, riboflavin and folate), protein and dietary minerals including calcium, iron, magnesium, phosphorus, potassium, zinc, copper and manganese (Ofitserov, 2001). For instance, the level of β-carotene of 33.3 mg/100 g sample dry weight (DW) reported in GA leaves by Macrae et al. (1993) is comparable to that of A. tricolor (weedy amaranth) leaves which ranged from 35.3-53.1 mg/100 g DW (Yadav and Sengal, 1995). A study in Kenya reported the ascorbic acid (AA) content of vegetable amaranth leaves as 627 mg/100 g DW (Onyango et al., 2008). This is similar to the value of 624.1629.0 mg/100 g reported in weedy amaranth, A. tricolor (Yadav and Sengal, 1995). 2.1.1 Grain amaranth Grain amaranth was a staple food of the Aztecs in Mexico (Price and Berkelaar, 2006). It was incorporated into their religious ceremonies that included eating 11 amaranth mixed with human blood (Price and Berkelaar, 2006; Lara et al., 2007). It was used until 1516 when the Spaniard conquistadors prohibited the growing of amaranth (O’Brien and Price, 1983). In the past 30 years, amaranth has been rediscovered and it has been shown to hold many amazing nutritional qualities (O’Brien and Price, 1983). The two species of GA most commonly grown are A. cruentus and A. hypochondriacus (Figure 2.1) (O’Brien and Price, 1983; Berkelaar and Alemu, 2006). The GA has large, colourful seed heads and can produce over 1000 pounds of grain per acre (Berkelaar and Alemu, 2006). The crop is drought-tolerant, provided there is sufficient moisture during the early growing period (Berkelaar and Price, 2006). Amaranth is not considered as a cereal grain since it does not belong to the grass family (Yarger, 2008). However, because of its flavor and cooking similarities to grains, it is called a pseudocereal (Yarger, 2008). Grain amaranth leaves are highly nutritious. They are especially rich in vitamin C, βcarotene, folic acid, iron, calcium and proteins (Yarger, 2008). Amaranth grain (Figure 2.2) contains exceptionally high protein for plant sources (Berkelaar and Price, 2006; Yarger, 2008). It is also very high in lysine, an essential amino acid that is low in other grains. Compared with many grains, amaranth grain is high in calcium, phosphorus, iron, potassium, zinc, vitamin E, and vitamin B-complex (Ofitserov, 2001; Martirosyan, 2001). The levels of riboflavin, AA, thiamin, and β-carotene in GA grains have been reported as 0.223, 4.47, 0.136 and 4.6 mg per 100 g DW respectively (Macrae et al., 1993). Svirskis (2003) reported the vitamin contents in 12 GA as AA (4.2), thiamine (0.08), riboflavin (0.208) and vitamin E (1.03) mg/100 g DW. Figure 2. 2: Amaranth grains Grain amaranth leaves are used as vegetables for human consumption. They may be eaten raw or cooked like spinach and other greens (Muyonga et al., 2008; Yarger, 2008). Cooking and discarding the water removes potentially harmful oxalates and nitrates (Yarger, 2008). The seeds are eaten as a cereal grain. They are ground into good quality flour used to cook porridge and to make tortillas, pinole or toasted meal, confectionery, pastries, biscuits, atoles, small savoury pancakes, desserts and bread (Muyonga et al., 2008; Yarger, 2008). Alternatively, seeds can be popped like popcorn. Popped amaranth can be used in confections bound with sorghum, molasses or honey (Muyonga et al., 2008). The seeds can also be germinated into nutritious sprouts (Colmenares De Ruiz and Bressani, 1990). Amaranth can also be used as animal feed. Amaranth foliage is used in stockbreeding as a green fodder, silage component and for obtaining protein-vitamin flour and concentrates (Ofitserov, 2001). Research has shown that the use of cooked or 13 autoclaved amaranth grain as chicken feed gives good production results (Yarger, 2008). Amaranth is a major source for obtaining medicinal substances and bioactive food additives (Ofitserov, 2001). Studies have shown that amaranth seed or oil may benefit those with hypertension and cardiovascular disease; regular consumption reduces blood pressure and cholesterol levels, while improving antioxidant status and some immune parameters (Kim et al., 2006; Martirosyan et al., 2007). 2.2 Vitamins Vitamins are a group of complex organic compounds present in small amounts in food that the body requires for its normal metabolism yet cannot be synthesized by the body (Macrae et al., 1993). This definition, however, has some limitations in that certain substances that are considered to be vitamins can be synthesized within the body of some species. For instance, most animal species have the ability to synthesize AA; individuals exposed to modest amounts of sunlight can produce cholecalciferol (vitamin D); and some animal species have the capacity to synthesise choline (Macrae et al., 1993). Members of the same vitamin family are called vitamers for example tocol and tocotrienol are vitamers E while pro-vitamins are precursors of the actual vitamins for example carotenoids are pro-vitamins A (Combs, 1992). Vitamins have been classified into two groups based on their solubility in fat solvents or in water. Fat-soluble vitamins (FSV) include vitamins A, E, D, and K while watersoluble vitamins (WSV) include thiamin, niacin, biotin, folate, riboflavin, vitamin B 6, 14 pantothenic acid, vitamin C and vitamin B12 (Ihekoronye and Ngoddy, 1985; Combs, 1992). Vitamins are required for the maintenance of optimal health, growth and reproduction. Deficiency symptoms (avitaminosis) will occur if a single vitamin is omitted from the diet of a species that requires it (Combs, 1992). Avitaminosis is any disease caused by chronic or long-term VD or by a defect in metabolic conversion, such as tryptophan to niacin (Combs, 1992). Vitamin poisoning, hypervitaminosis or vitamin overdose refers to a condition of high storage levels of vitamins, which can lead to toxic symptoms. With a few exceptions, hypervitaminosis usually occurs more with FSVs, which are stored in the liver and fatty tissues of the body since they build up and remain for a longer time in the body than WSVs (Sizer et al., 2008; Said and Mohamed, 2006; Fukuwatari and Shibata, 2008; Maqbool and Stallings, 2008). The required intakes of vitamins are given in terms of Dietary Reference Intakes (DRIs) which include Recommended Dietary Allowance (RDA), Adequate Intake (AI), Tolerable Upper Intake Level (UI) and Estimated Average Requirement (EAR) (Institute of Medicine, 2000a). The DRIs include levels that may reduce the risk of cardiovascular disease, osteoporosis, certain cancers, and other diseases that are dietrelated (Barr, 2006). 2.2.1 Beta-carotene (pro-vitamin A) Vitamin A is the generic descriptor for compounds with the qualitative activity of retinol, that is, retinoids and pro-vitamin A carotenoids (Combs, 1992). All forms of 15 vitamin A have a beta-ionone ring to which an isoprenoid chain is attached. This structure is essential for vitamin activity (Rodriqez-Amaya, 1997). Retinoids are isoprenoid compounds found in animal products and includes retinol (Figure 2.3), retinyl palmitate, retinal, 3-dehydroretinol, and retinoic acid (Armstrong and Hearst, 1996). The rich sources are liver, milk, butter, fortified margarine, eggs, meat, fish and fish liver oils (Rodriqez-Amaya, 1997). Pro-vitamin A carotenoids are isoprenoid pigments found in plants and some fungi and bacteria with unsubstituted β-ring having 11-carbon polyene chain. These include β-carotene (Figure 2.4), αcarotene, γ-carotene, β-cryptoxanthin, and α-cryptoxanthin of fungus and some bacteria (Armstrong and Hearst, 1996). On a worldwide basis, the highest percentage of dietary vitamin A comes from β-carotene (Simpson 1983; Rodriqez-Amaya, 1997). OH Figure 2. 3: Molecular structure for retinol Figure 2. 4: Molecular structure for beta-carotene The rich sources of β-carotene include carrots, yellow and orange fruits (such as mangoes, papayas, pumpkins, musk melon, peaches), yams, sweet potatoes, crude palm oil and green vegetables like spinach, lettuce, cabbages, broccoli, kale, sweet potato leaves, and sweet gourd leaves (Pamplona-Roger, 2005). The levels of β- 16 carotene in some vegetables are given in Table 2.1. The DRI for β-carotene is between 2.52 mg and 10.8 mg per day (Institute of Medicine, 2000b). Table 2. 1: Beta-carotene content of some vegetables Vegetable Level (mg/100 g) Reference Amaranth leaves 7.54 Weinberger and Msuya , 2004 Night shade 5.02 Weinberger and Msuya, 2004 Spider flower plant 10.05 Weinberger and Msuya, 2004 Bitter lettuce 6.80 Weinberger and Msuya, 2004 Cassava leaves 5.43 Weinberger and Msuya, 2004 Source: Weinberger and Msuya, 2004 Beta-carotene has been linked with enhancement of the immune system and decreased risk of degenerative diseases such as cancer, cardiovascular disease, age-related macular degeneration, and cataract formation (Di Mascio et al., 1989; Courtsoudis, 2001). Epidemiological studies have reported that β-carotene is associated with reduced cardiovascular disease (Street et al., 1994). Inverse relationship between presence of various cancers and carotenoid levels has been reported (Paiva and Russell, 1999). Another study showed major reduction of malaria morbidity with combined vitamin A and zinc supplementation in young children (Zeba et al., 2008). Primary vitamin A deficiency (VAD) occurs among individuals who do not adequately consume yellow and green vegetables, fruits and liver. Early weaning can also increase the risk of VAD (Roncone, 2006). Secondary VAD is associated with chronic malabsorption of lipids, impaired bile production and release, low fat diets, and chronic exposure to oxidants, such as cigarette smoke (Roncone, 2006). The VAD leads to stunted growth, xerophtalmia or thickening of cornea and conjuctiva of the eyes and night blindness (Underwood and Barbara, 2004; Roncone, 2006). 17 Persistent deficiency can cause destruction of cornea (keratomalacia) and total blindness (Roncone, 2006). Excessive dietary intake of β-carotene can lead to carotenodermia, which causes orange-yellow discoloration of the skin (Takita et al., 2006). 2.2.2 Vitamin E (α-tocopherol) Vitamin E is the generic descriptor for all tocol and trienol derivatives exhibiting qualitatively the biological activity of α-tocopherol (Figure 2.5). Vitamers E are hydrophobic and, thus are insoluble in aqueous environments like plasma, interstitial fluids and cytosol (Combs, 1992). Since it is synthesised only by plants, it is found primarily in plant products. Vegetable oils, salad dressings, and margarines are the richest dietary sources of vitamin E. Other good sources are nuts, green leafy vegetables and fortified cereals (Pamplona-Roger, 2005; Hillan, 2006). Table 2.2 gives the α-tocopherol contents of some foods. CH3 HO H H3C CH3 H CH3 O CH3 Figure 2. 5: Molecular structure for alpha tocopherol CH3 CH3 18 Table 2. 2: Alpha-tocopherol content (mg/100 g raw weight) of some foods Vegetable Content Reference Amaranth leaves 0.50 Akubugwo et al., 2007 Amaranth grain 1.03 Svirskis, 2003 Asparagus 1.07 Pamplona-Roger, 2005 Broccoli 1.44 Pamplona-Roger, 2005 Cabbage(white) 0.21 Pamplona-Roger, 2005 Wheat 1.44 Pamplona-Roger, 2005 Buckwheat 1.03 Pamplona-Roger, 2005 Millet 0.18 Pamplona-Roger, 2005 Vitamin E is useful in protecting deleterious reactions of highly oxidizing species, formation and functioning of red blood cell, protection of fatty acids, protection against cancer and arteriosclerosis, minimising cataract development, prevention of disorders like haemolytic anaemia of pre-maturity (Combs, 1992; Pamplona-Roger, 2005). Some studies have revealed that vitamin E is capable of delaying tumor formation or retarding carcinogenesis (Shklar, 1982; Schwartz et al., 1985). The clinical deficiency of vitamin E leads to haemolytic anaemia in infants. Vitamin E is one of the least toxic of the vitamins but isolated reports of negative effects in humans consuming up to 1000 IU vitamin E per day include fatigue, nausea, double vision, muscular weakness, mild creatinuria and gastro-intestinal distress (Combs, 1992). The RDI of vitamin E ranges from 5 mg/day for children below four years of age to 16 mg/day for lactating mothers (Institute of Medicine, 2000b). 19 2.2.3 Vitamin B1 (Thiamin) Thiamin (Figure 2.6), also called aneurin, is a WSV whose phosphate derivatives are involved in many cellular processes (Combs, 1992). The best-characterized form is thiamine pyrophosphate (TPP). Others include thiamine monophosphate (ThMP), thiamine triphosphate (ThTP), adenosine thiamine triphosphate (AThTP) and adenosine thiamine diphosphate (AThDP). Chemically, it consists of substituted pyrimidine and thiazole rings linked by a methylene bridge (Institute of Medicine, 1998). NH2 N N S H3C N H3C OH Figure 2. 6: Molecular structure for thiamine Thiamin is found in a variety of foods, although in small amounts. Sunflower seeds, wheat germ, whole grains, brewer’s yeast and lean pork are the best sources (Pamplona-Roger, 2005). Vegetables contain generally less than 0.1 mg/100 g of thiamin (Tee et al., 1997). Table 2.3 gives the levels of thiamine in various foods. 20 Table 2. 3: Thiamin composition of some foods in mg/100 g sample raw weight Food Thiamin content Reference Amaranth leaves 0.080 Yarger, 2008 Amaranth grain 0.100 Muyonga et al., 2008 Spinach 0.078 Pamplona-Roger, 2005 Asparagus 0.140 Pamplona-Roger, 2005 Broccoli 0.065 Pamplona-Roger, 2005 Cabbage 0.050 Pamplona-Roger, 2005 Cauliflower 0.057 Pamplona-Roger, 2005 Sunflower seeds 2.290 Pamplona-Roger, 2005 Peanut 0.640 Pamplona-Roger, 2005 Corn 0.200 Pamplona-Roger, 2005 Soybean 0.874 Pamplona-Roger, 2005 Wheat 0.394 Pamplona-Roger, 2005 Thiamine functions as the coenzyme TPP in the metabolism of carbohydrates and branched-chain amino acids (Pamplona-Roger, 2005). Thiamin is also important in aiding the correct functioning of the heart and nervous system (Pamplona-Roger, 2005). Thiamine deficiency results in the disease called beriberi (Ihekoronye and Ngoddy, 1985; McCormick and Greene, 1994). Beriberi occurs in human-milk-fed infants whose nursing mothers are deficient, adults with high carbohydrate intakes and with intakes of anti-thiamine factors, such as the bacterial thiaminases that are in certain ingested raw fish (McCormick and Greene, 1994). In industrialized nations, the neurologic manifestations of Wernicke-Korsakoff syndrome are frequently associated with chronic alcoholism (McCormick and Greene, 1994). Some cases of thiamine deficiency have been observed with patients who are hypermetabolic, are on parenteral nutrition, are undergoing chronic renal dialysis, or have undergone a 21 gastrectomy. Hypervitaminosis B1 may cause headache, convulsions, weakness, paralysis, cardiac arrhythmia and allergic reactions (Combs, 1992). The recommended intakes of thiamin are 0.2-0.9 mg/day for children and 1.2 mg/day for adults (FAO/WHO, 2002). 2.2.4 Vitamin B2 (riboflavin) Riboflavin (Figure 2.7) is the trivial designation of the compound 7,8-dimethyl-10(1’-D-ribityl)isoalloxazine (Combs, 1992). The metabolically active forms are flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (Combs, 1992). Riboflavin is a yellow tricyclic molecule that is usually phosphorylated to FMN and subsequently to FAD in biological systems (Combs, 1992). Riboflavin is widely distributed in foods mainly in the form of FMN and FAD (Combs, 1992). Table 2.4 gives the riboflavin contents of some foods. The rich sources include yeast extract, liver and kidney, wheat bran, eggs, meat, milk, and cheese (Ball and George, 2006; Elaine, 2006). O H3C N NH H3C N N O OH OH HO OH Figure 2. 7: Molecular structure for riboflavin 22 Table 2. 4: Riboflavin content of some foods in mg/100g raw sample weight Food Riboflavin level Reference Amaranth leaves 0.160 Yarger, 2008 Amaranth grains 0.223 Macrae et al., 1993 Spinach 0.189 Pamplona-Roger, 2005 Carrot 0.059 Pamplona-Roger, 2005 Cabbage 0.040 Pamplona-Roger, 2005 Peas 0.132 Pamplona-Roger, 2005 Peanut 0.135 Pamplona-Roger, 2005 Corn 0.060 Pamplona-Roger, 2005 Soybean 0.870 Pamplona-Roger, 2005 Rice 0.048 Pamplona-Roger, 2005 Wheat 0.096 Pamplona-Roger, 2005 Buckwheat 0.425 Pamplona-Roger, 2005 Fresh egg 0.508 Pamplona-Roger, 2005 Bran 0.577 Pamplona-Roger, 2005 Riboflavin is important in carbohydrates, proteins and fat metabolism and in releasing energy into the cells (Macrae et al., 1993; Pamplona-Roger, 2005). It is also necessary for the formation of pigments in the retina involved with vision (Pamplona-Roger, 2005). The recommended intake of riboflavin is between 0.3 and 1.2 mg/day (FAO/WHO, 2002). Riboflavin deficiency (Ariboflavinosis) is relatively common when dietary intake is insufficient (McCormick, 1994). A deficiency of riboflavin can be primary due to poor vitamin sources in one's daily diet or secondary, as a result of conditions that affect absorption in the intestine, or an increase in the excretion of the vitamin from the body (McCormick, 1994). Ariboflavinosis leads to fatigue, weakness, apathy, 23 vision disorders, seborrheic dermatitis, angular cheilitis, dry and scaling skin and irondeficiciency anaemia (Pamplona-Roger, 2005). Riboflavin deficiency is classically associated with the oral-ocular-genital syndrome (McCormick, 1994). The effects of long-term sub-clinical riboflavin deficiency in children result in reduced growth. Subclinical riboflavin deficiency has also been observed in women taking oral contraceptives, in the elderly, in people with eating disorders, and in disease states such as HIV, inflammatory bowel disease, diabetes and chronic heart disease (Powers, 2003). Since toxicity of riboflavin is very low, problems of hypervitaminosis are not expected. This is because excess riboflavin is excreted in the urine (Zempleni et al., 1996; Gropper et al., 2009). 2.2.5 Vitamin B6 (Pyridoxine) Vitamin B6 is the generic descriptor for all 2-methyl-3,5-dihydroxymethylpyridine derivative exhibiting the biological activity of pyridoxine (Figure 2.8) (Combs, 1992). The biologically active analogues of pyridoxine are pyridoxal and pyridoxamine. The metabolically active form of vitamin B6 is pyridoxal phosphate (PLP) (Combs, 1992). H3C N OH H3C OH Figure 2. 8: Molecular structure for pyridoxine 24 Good sources of vitamin B6 are meat, fish, wheat, whole grain products, green vegetables and nuts (Pamplona-Roger, 2005; McCormick, 2006). The levels in some foods are presented in Table 2.5. Table 2. 5: Pyridoxine content of some foods in mg/100g raw sample weight Food Pyridoxine content Reference Amaranth leaves 2.330 Akubugwo et al., 2007 Amaranth grains 0.223 Svirskis, 2003 Spinach 0.195 Pamplona-Roger, 2005 Cabbage 0.096 Pamplona-Roger, 2005 Wheat 0.272 Pamplona-Roger, 2005 Soybean 0.377 Pamplona-Roger, 2005 Potato 0.260 Pamplona-Roger, 2005 Peas 0.169 Pamplona-Roger, 2005 Asparagus 0.131 Pamplona-Roger, 2005 Peanut 0.348 Pamplona-Roger, 2005 Corn 0.055 Pamplona-Roger, 2005 Quinoa 0.223 Pamplona-Roger, 2005 Millet 0.384 Pamplona-Roger, 2005 The primary role of vitamin B6 is to act as a coenzyme in the metabolism of proteins, carbohydrates and fats (Pamplona-Roger, 2005). It is also involved in energy production within the cells of the nervous system and the synthesis of hemoglobin (Pamplona-Roger, 2005). A deficiency of vitamin B6 alone is relatively uncommon and often occurs in association with other vitamins of the B-complex. The elderly, alcoholics, renal patients undergoing dialysis have an increased risk of vitamin B6 deficiency (Bowman 25 and Russel, 2006). Deficiency symptoms include fatigue, nervousness, anaemia and skin disorders (Pamplona-Roger, 2005). Vitamin B6 deficiency also results in an impairment of the immune system (McCormick and Greene, 1994). Toxicity of vitamin B6 is relatively low, although high acute doses have been shown to cause sensory neuropathy (Combs, 1992). 2.2.6 Vitamin C (ascorbic acid) Vitamin C is the generic descriptor for all compounds exhibiting qualitatively the biological activity of L-ascorbic acid (Figure 2.9) (Davey et al., 2000; Deutsch, 2000). The term L-ascorbic acid is the trivial designator for the compound 2,3didehydro-L-threo-hexano-1,4-lactone (Combs, 1992). The AA is reversibly oxidised to form L-dehydroascorbic acid (DAA), which also exhibits biological activity (Davey et al., 2000; Deutsch, 2000). HO HO O O H HO OH Figure 2. 9: Molecular structure for ascorbic acid Vitamin C is widely distributed in both plants and animals, occurring as both AA and DAA. The best plant sources are fresh fruits (particularly citrus fruits, tomatoes, and green peppers), baked potatoes and leafy vegetables (Davey et al., 2000; Deutsch, 2000). Animal sources include organ meats such as liver and kidney, and milk (Macrae et al., 1993; Pamplona-Roger, 2005). Table 2.6 summarises the AA contents of some common foods. 26 Table 2. 6: Ascorbic acid content of some foods (mg/100g raw sample weight) Vegetable Ascorbic acid level Reference Amaranth leaves 80.0 Yarger, 2008 Amaranth grain 4.2 Svirskis, 2003 Cabbage 32.2 Pamplona- Roger, 2005 Cauliflower 46.4 Pamplona- Roger, 2005 Onion 6.4 Pamplona- Roger, 2005 Broccoli 93.2 Pamplona- Roger, 2005 Soybean 6.0 Pamplona- Roger, 2005 Alfafa seeds, sprouted 8.2 Pamplona- Roger, 2005 Chick pea 4.0 Pamplona- Roger, 2005 Vitamin C is important in protecting against cancers, improving the body immune system, disease prevention, maintaining of bone and teeth, strengthening of blood vessels, wound healing, increasing the bioavailability of iron in foods, neutralising the actions of toxic substances and minimising cold symptoms (Pamplona-Roger, 2005). Epidemiological studies indicate that diets with high vitamin C content have been associated with lower cancer risk, especially for cancers of the oral cavity, oesophagus, stomach, colon, and lung (Byers and Mouchawar, 1998; Schorah, 1998). Clinical studies showed that high-dose of vitamin C can improve symptoms and prolong life in patients with terminal cancer (Padayatty et al., 2006). Acute vitamin C deficiency results in scurvy (Combs, 1992). It also causes defects in collagen formation leading to impaired wound healing and edema, and weakening of collagenous structures in bone, cartilage, teeth and connective tissues (Combs, 1992). Mild deficiency results in several non-specific pre-scorbutic signs including lassitude, fatigue, anorexia, muscular weakness and increased susceptibility to infection 27 (Pamplona-Roger, 2005). Megadoses of vitamin C can cause renal stones, enhanced destruction of vitamin B12 in the gut and enhanced enteric absorption of non-heme iron leading to Fe-over-load (Davies et al., 1991; Combs, 1992). The recommended intake (RDA) of vitamin C is 20 mg for children and 30 mg for adults per day (Combs, 1992). 2.3 Methods of analysis of vitamins Some methods used in the analysis of vitamins have been tested by interlaboratory trials and accepted as official by the Association of Official Analytical Chemists (AOAC), the Association of Vitamin Chemists (AVC) and other official organizations such as International Standards Organisation (ISO) and Food and Agricultural Organisation (FAO)/World Health Organisation (WHO) (Macrae et al., 1993). These include bioassays, microbiological methods, enzymatic methods, immunoassays, protein-binding assays, radioassays, chemical methods (titrimetric, fluorometric and spectrophotometric) and chromatographic methods (open column, thin-layer chromatography, gas chromatography and high-performance liquid-chromatography) (Davies et al., 1991; Ponder et al., 2004; Casella et al., 2006; Luo et al., 2006; Batt et al., 2008 ; France and Labor, 2008). Although all the assays are useful, the choice is made according to the specific reasons for the analyses (Macrae et al., 1993). The high-performance liquid-chromatography (HPLC) technique was used in the analysis of thiamin, riboflavin and pyridoxine. This was because it has high specificity, high resolution of vitamers, high sensitivity, excellent precision and accuracy (Krystulovic and Brown, 1982). In addition, it operates at ambient temperature, does not require derivatization prior to analysis and simultaneous 28 determination of vitamins can be done in a single step (Krstulovic and Brown, 1982; Qian and Sheng, 1998). The method is, however, expensive, requires skilled personnel and standardization of clean-up procedures is necessary for each food matrix (Wilson and Walker, 1994). Analysis of β-carotene and α-tocopherol was done using spectrophometric technique because of the availability of the spectrophotometer and due to its high degree of precision, sensitivity, accuracy, short analysis time and low cost as it requires less reagents (Rutkowski and Grzegorczyk, 2007; Abd El-Baky et al., 2008). The method has been applied and compared well with the HPLC method in the determination of vitamins A, D, E and carotenoids (Blanco et al., 2004; Murthy et al., 2005). Ascorbic acid was analysed using titrimetric method. This method was chosen since it is simple, inexpensive and reasonably accurate when many replicates are performed. Furthermore, it is rapid and thus the samples could be analysed immediately after extraction minimising the losses of AA due to oxidation. This method has been reported in the analysis of AA in some vegetables (Hui, 1992; Reiss, 1993; Mnkeni et al., 2007; Onyango et al., 2008). 2.3.1 Principles of HPLC method This method relies on the separation of solutes in the mobile phase (MP) flowing through a stationary phase (SP) due to differential retention by the SP (Lough and Wainer, 1996). This depends on the solute-solute, solute-MP, solute-SP and SP-MP interactions. The solute-SP interactions may be hydrogen bonding, van der Waal’s forces, electrostatic forces or hydrophobic forces (Lough and Wainer, 1996). The 29 variety of SPs used results in a variety of separation modes: Adsorption chromatography, partition chromatography, ion-exchanged chromatography and size exclusion-chromatography (Wilson and Walker, 1994). Partion chromatography mode was used in the analysis based on reverse phase chromatography (RPC). The RPC uses a non-polar SP and a relatively polar MP. The most commonly used SP is octadecylsilyl silica (ODS, C18). The MP is usually water or aqueous buffers, methanol, acetonitrile, tetrahydrofuran or mixtures of them. Chromatographic separation of analytes occurs due to hydrophobic interactions between the analytes and the SP (Horvath, 1980; Wilson and Walker, 1994). Polar analytes elute first and non-polar analytes last. This is the most widely used form of chromatography because of its flexibility and high resolution. The main components of HPLC instrument (Figure 2.10) are a high-pressure pump, a column, injector system and a detector. Other components are solvent reservoir, inline filters, pressure gauges, recorders; integrators may be required (Lough and Wainer, 1996). Figure 2. 10: Block diagram of the components of an HPLC 30 2.3.2 Principles of spectrophotometric method Spectrophotometry involves the use of a spectrophotometer, which is a device for measuring light intensity as a function of the wavelength of light (Kirk and Sawyer, 1991). There are two major classes of devices: single beam and double beam. A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted (Kirk and sawyer, 1991). A spectrophotometer contains a light source, a means of isolating a particular wavelength band of the light source, a sample holder and a device to measure light intensity (Figure 2.11). Figure 2. 11: Block diagram of a single beam spectrophotometer The spectrophotometer quantitatively compares the fraction of light that passes through a reference solution and a test solution. Then the intensity of the transmitted light is measured with a photodiode or other light sensor, and the transmittance value for this wavelength is then compared with the transmission through a reference sample. In some instruments, the signal is presented as absorbance on the digital display for quantitative work (Kirk and Sawyer, 1991). Within small concentration ranges, the Beer-Lambert’s law (Eq. 1) holds and the absorbance between samples vary with concentration linearly. 31 A bc (Eq. 1) Where A is the absorbance, is the molar absorptivity, b is the path length and c is the concentration of the sample. Absorbance measurement is used to determine the concentration by the using standard calibration curve (Blanco et al., 2004; Saushkina and Karpenko, 2005; Rutkowski and Grzegorczyk, 2007) 2.3.3 Principles of titrimetric method Titrimetric analysis is based on the complete reaction between the analyte and a reagent, the titrant as per equation 2. aA tT products (Eq. 2) Where A and T represent the analyte and titrant, respectively, and a and t are the stoichiometric coefficients. Titrations are often classified by the nature of this titration reaction: acid-base, redox, precipitation and complexation reactions are the most common reaction types. For volumetric titrations, the amount, nA, of analyte in the sample can be calculated using equation 3. a nA CTVT (Eq. 3) t Where CT is the concentration of the titrant, and VT is the volume of titrant needed to reach the endpoint. The titrant solution must be standardized either by preparing it using a primary standard or, more commonly, titrating it against a solution prepared with a primary standard. Redox titrations can be used to analyse a wide variety of analytes. Ascorbic acid may be assayed by titration with iodine or titration with a redox indicator, 2,6dichlorophenolindophenol (DCPIP) in acid solution. The latter method was used in 32 the analysis because it is reasonably accurate, rapid and convenient (AOAC, 1990; Reiss, 1993). Titrimetric assay of AA is based on the reducing capacity of L-ascorbic acid. The method relies on chemical reduction of the dye 2,6-dichlorophenol into a colorless reduced form by ascorbic acid (Onyango et al., 2008). The solution will remain colorless as more DCPIP is added until the entire AA has reacted when the solution will turn pink at end-point. The DCPIP solution must first be standardized against a known amount of AA. This can be accomplished by titrating the dye with a solution containing 1.0 ml of standard AA solution (Reiss, 1993). The sample must be rapidly prepared and analyzed since AA is oxidized very rapidly and may be destroyed by heat and oxygen exposure. However, AA stability can be increased by acidifying extracts or by using metal chelators (AOAC, 1990). 33 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study areas The study was done in seven regions of Kenya namely Kenyatta University Main Campus, Bureti, Mt. Elgon, Kisii, Bondo, Meru and Embu. This was to determine the effects of different areas on the levels of vitamins in amaranth. Further, the regions were chosen based on the availability of plots for sowing the samples, availabilty of willing personnel who could undertake the prescribed production and management practices, accessibility and convenience of sample collection for analyses. Kenyatta University is located in Nairobi, the capital city of Kenya, which has an altitude of 1,600-1,850 m above sea level. It has a temperate tropical climate with mean annual rainfall of 850-1,050 mm and a mean daily temperature of 12-26 °C. The soils of the Nairobi area are mainly the andosols. Bureti district is situated in Rift Valley province and has an altitude of 1,800-3,000 m above the sea level. Its climate is highland sub-tropical with high rainfall ranging from 1,700-2,020 mm annually. It experiences a temperature between 16 oC-20 oC. The main type of soil found in this area is nitosol. Mt. Elgon district is located in Western province and has an altitude of 1,200-2,000 m above the sea level. It has a tropical climate with a mean annual rainfall of 1,500-2,100 mm and temperature of 14 oC-24 oC. Kisii district is in Nyanza province and lies at an altitude of 1,190-2,130 m. It has a highland equatorial climate. The district experiences a mean annual rainfall of 1,500-2000 mm and temperature ranges of 16.4 °C-28.4 °C. Humic nitisol is the main type of soil found in this area. Bondo district is situated in Nyanza province and has altitude ranging between 1,140m and 1,400m above the sea level. The district has a modified equatorial climate 34 with mean annual rainfall ranging between 800-1,600 mm and mean temperature of 22.5 ºC. The soil types found range between black-cotton, sandy loams and laterites. Meru district is located in Eastern Province. It has an altitude of 600-5,200 m above the sea level. The district recieves rainfall ranging from 500-2,200 mm and temperatures of 14 oC-27 oC. The soils of Meru district are mainly nitisols and andosols. Embu district, located in Eastern province, has an altitude of between 1,200 and 2,000 m above the sea level. It has a mean annual rainfall of 1,000-2,000 mm and average temperature of between 15 oC and 21 oC. The soils in the region vary form volcanic humic andosols to rhodic and humic nitisols (CBS, 2002). 3.2 Study design The experimental design was factorial based on a Randomized Complete Block (RCB) with three replications. The factors were the species, plant ages, areas of study and seasons of planting. Certified seeds of A. cruentus and A. hypochondriacus were sown in April 2008 for the wet season and in December the same year for the dry season. The seeds were sown in plastic pots at Kenyatta University Main Campus on 2nd April 2008 and 1st December for the wet season and dry seasons respectively. This was used as a control experiment. In the other areas, sowing was done in seedbeds measuring 4 m by 3 m between 5th and 30th of April 2008 for the wet season and between 5th and 30th December the same year for the dry season at an interval of 5 days between each areas. The difference in the sowing dates in all the study areas was to facilitate the harvesting and subsequent analysis of the vitamins in the leaves at exactly the same plants’ ages. The leaves were harvested for the analysis of vitamins at 25, 50 and 75 days after sowing (DAS) from 4 out of the 7 areas of study (Kenyatta University, Bureti, Mt. Elgon and Kisii). This was to determine the effect of plant age 35 on the vitamin content of amaranth leaves. The leaves in the upper parts of the plants were harvested at 25 (Figure 3.1a), 50 and 75 (Figure 3.1b) DAS for analysis of vitamins. The grains were harvested from all the study areas after 90 days of plants’ growth, when a few seeds had started dropping off, for analysis of vitamins. (a) (b) Figure 3. 1: Grain amaranth plants grown at Bureti: (a) A. cruentus at 25 days (b) A. hypochondriacus at 75 days and the researcher 3.3 Planting and management practices of grain amaranth The certified seeds of A. hypochondriacus variety NUK 117 golden coloured and A. cruentus variety 320 white coloured were obtained from Ngong Amaranth Company in Nairobi. These were sown at Kenyatta University in 8 plastic pots for each and in seed-beds in the other regions. Before sowing was done at Kenyatta University, the soil was thoroughly mixed with organic manure in the ratio 1:2 (manure: soil) using a shovel. The soil-manure mixture was then placed in all the pots and the pots labelled for each species. The mixture was watered thoroughly before planting was done. The seeds were planted at 36 the spacing of 15 cm between plants and about 1.3 cm deep. They were then covered lightly with soil. The plants were thinned after two weeks, so that four plants remained per pot. Weeding was done carefully when the plants were young using a piece of stick. The seeds of the two species were planted in the other regions in tagged seedbeds measuring 4 m by 3 m. The seedbeds were prepared by cultivating the farms three times. Large pieces of soil or hard crusts in the soil were broken so that the final seedbed consisted of tiny fine soil particles. The soil was mixed with organic manure and watered before sowing was done. The seeds were sown in rows, which were 76 cm from each other at a depth of about 1.3 cm and covered lightly with soil. After two weeks, the plants were thinned so that they were 10-15 cm apart. The plants were weeded when they were still young by hand. The grains were harvested after three months of sowing when a few seeds had started dropping off. The top parts of the plants containing the awns were cut off. The awns were threshed to release the grains. The grains were separated from the chaff by winnowing. 3.4 Sampling and sample pre-treatment The leaf samples of A. hypochondriacus and were A. cruentus were obtained from the areas of study at 25, 50 and 75 DAS. Sampling at Kenyatta University was done by picking atleast 4 leaves from each of the potted plants of each species and combining them to form a batch of about 250 g. In the other areas of study, atleast 4 leaves of each species were picked from different plants chosen at random in all the rows and 37 combined to obtain a batch of about 500 g per species. All the samples were transported in cool iceboxes to the laboratory of the Department of Chemistry, Kenyatta University for extractions and preparation for the analysis within five hours. The grains of each species after harvest from each of the 7 study areas were mixed thoroughly and atleast 200 g placed into the polythene bags and transported in cool ice-box to the laboratory for extractions and preparation for analysis. The leaves in each batch were prepared for the analysis by trimming with a sharp knife to remove the inedible parts. They were then mixed well and three sub-samples removed from the edible portions. The samples were then homogenised by crushing using a pestle and mortar. The grains were homogenised by grinding with a warring blender and the ground samples refregerated at -4 ○C before extractions were done. 3.5 Instrumentation The HPLC system (Figure 3.2) was a Shimadzu (Kyoto, Japan) consisting of a column oven (model CTO-10 AVP), a UV-visible diode-array detector (Waters 2996), a degasser (Model, DGU-14A), an LC pump (model, LC-10 ADVP) and RP18 column (VP-ODS, 150 mm× 4.6 mm× 5 µm). A 20-μL syringe (HamiltonBonaduz, Schweiz) was used for sample injection. Empower programme software was used for processing the chromatographic peaks. 38 Figure 3. 2 The HPLC equipment used in the study The UV-visible spectrophotometer was a CECIL CE 2041 2000 series model. A quartz cuvette was used for holding the prepared samples. 3.6 Chemicals The HPLC grade reagents including n-hexane, acetone, acetonitrile, methanol dichloromethane and 1-hexanesulphonic acid sodium salt were obtained from Merck, Germany. Vitamin standards (L-ascorbic acid, pyridoxine, thiamine, riboflavin, ( )α-tocopherol and β-carotene), ethanol, metaphosphoric acid, orthophosphoric acid, acetic acid, potassium di-hydrogen phosphate, 2,6-dichclorophenolindophenol, hydrochloric acid, sodium chloride, anhydrous sodium sulphate, potassium hydroxide pellets, sodium acetate and takadiastase were of analytical grade from Sigma (SigmaAldrich, Germany). Distilled deionised water was used in all the experiments. 39 3.7 Cleaning of glassware and vials The glass apparatus and vials were cleaned with chromic acid and subsequently by a detergent. They were then rinsed with distilled or de-ionised water and dried in an oven at 105 oC. 3.8 Preparation of solutions 3.8.1 Stock solutions of β-carotene and α-tocopherol The stock solutions of β-carotene and α-tocopherol were prepared by dissolving 0.100 g of each of the crystalline standard reagents in n-hexane and made up to 100 mL. A working solution was prepared by diluting portions of the stock solution to appropriate concentrations. A series of standard solutions were prepared from the working solution by diluting with n-hexane and treating each according to the procedure described in section 3.9.1. 3.8.2 Stock solutions of B vitamins The stock solutions containing 100 mg/L of thiamin, riboflavin and pyridoxine were prepared by weighing 0.010 g of each of the standard reagent into a 100 mL volumetric flask. About 50 mL of 0.1 M HCl was added and shaken to dissolve the reagent and then diluted to the mark. These stock solutions were kept under refrigerating conditions. A series of standard solutions were prepared each day of analysis by diluting the stock solution with 0.1 M HCl appropriately. They were filtered using 0.45 µm pore size filters before injecting into the HPLC. 3.8.3 Extraction and standard solutions for AA analysis The extraction solution for AA analysis consisting of 3 % metaphosphoric acid (MPA) and 8 % acetic acid was prepared by dissolving 15.000 g of MPA in 40 mL 40 acetic acid and 200 mL water, and diluted to 500 mL with water. It was then filtered using ashless whatman No. 42 filter paper and kept for use in the extraction of AA. The standard AA was prepared by dissolving 0.050 g of AA in 45 mL of the extraction solution and made up to 50 mL. This was prepared immediately before use. The indophenol standard solution was made by dissolving 0.050 g of DCPIP in 50 mL water containing 0.042 g of sodium bicarbonate and diluted to 200 mL with water then filtered using ashless whatman No. 42 filter paper. It was then standardized by titrating against 2 mL of standard AA solution added to 5 mL of the extraction solution. 3.9 Extraction and analysis of β-carotene and vitamins 3.9.1 Simultaneous extraction and analysis of β-carotene and α-tocopherol A 5.000 g of the pre-treated vegetable or 2.500 g of ground grain sample was weighed into into a 100 mL conical flask covered with aluminium foil. An exactly 50 mL extraction mixture consisting of acetone and hexane in the ratio 3:2 v/v containing 1 mL of 0.1 % butylated hydroxytoluene (BHT) was added and the flask corked. The mixture was shaken at a moderate speed with a mechanical shaker for 10 minutes. The mixture was centrifuged and transferred to a separating funnel in a dim room. It was re-extracted two more times. The combined extract was saponified by adding 25 mL 0.5 M ethanolic KOH, shaken vigorously and allowed to settle for 30 minutes. It was washed with 100 mL of 10 % NaCl followed by four 100 mL portions of distilled water to remove acetone while discarding the aqueous layer continuosly. The extract was dried by filtering over anhydrous sodium sulphate. It was then evaporated using rotary evaporator at 45 oC while protecting it from light, with the final traces of solvent being removed in a stream of nitrogen. The residue was immediately 41 dissolved in absolute ethanol and made upto 50 mL volumetric flask mark for leaves samples but to 5 mL for grain samples (Brubacher et al., 1985; Kirk and Sawyer, 1991; Macrae et al., 1993; Blanco et al., 2004). Analysis of β-carotene was done by determining the absorbance of extracted samples and the standards using a UV-visible spectrophotomer at 451.5 nm against a blank treated using the same procedure. Determination of α-tocopherol was done after development of a red colour by oxidation to red tocoquinone using nitric acid (Kirk and Sawyer, 1991). The aliquots of the solutions of the samples and standards were transferred to a 20 mL volumetric flask. Exactly 5 mL absolute ethanol, followed by 1 mL concentrated nitric was added. The flask was then placed on a water bath at 90 οC for 3 minutes from the time the alcohol started to boil. It was then cooled rapidly under running water and the volume was adjusted with absolute ethanol. Absorbance was measured at 298 nm against a blank containing 5 mL absolute ethanol and 1 mL concentrated nitric acid treated in a similar manner (Brubacher et al., 1985; Fidanza, 1991). The standard solutions were used to obtain the calibration graphs. The regression lines for β-carotene and α-tocopherol in this expereriment are given by equations 4 and 5 respectively. Absorbance 0.0758x 0.0019 Absorbance 0.0298x (Eq. 4) (Eq. 5) Where x is the concentration in ppm. Recovery analysis was done by adding 0.005 g of β-carotene and α-tocopherol standards each to the pre-treated sample and treating it according to the procedures and analysis described above. This was done in triplicates. Reliability of this method 42 was also ascertained by analysing 24 samples using the HPLC method and relating them using the t-test and Pearson correlation coefficient. The HPLC analysis of β-carotene and α-tocopherol was done by injecting 20 µL of samples and standards into the system and detections done at 450 nm and 298 nm for β-carotene and α-tocopherol respectively. The MP consisted of methanol: dichloromethane: water in the ratio 79: 18: 3. Analysis was done isocratically at a flow rate of 1.0 mL/min. Vitamins were identified by comparing their retention times with those of standards while quantification was done using the calibration curves of peak areas versus the concentrations of standards in ppm. The retention times were 33 minutes for β-carotene and 8 minutes for α-tocopherol. The regression lines for βcarotene and α-tocopherol in this analysis are given by equations 6 and 7 respectively. Peak area = 39202 x 2892.5 (Eq. 6) Peak area = 84413x 701.8 (Eq. 7) Where x represents the concentration in ppm. 3.9.2 Simultaneous extraction and analysis of thiamin, riboflavin and pyridoxine Exactly 5.000 g of fresh leaves or 2.500 g of ground grains were extracted by hydrolyzing with 20 mL of 0.26 M HCl over boiling water for 45-60 minutes. The mixture was cooled over tap water and the pH adjusted to 4.5 with 2.5 M sodium acetate solution using a pH meter. A 0.1 g of takadiastase was added and then incubated for 2 ½ hours at 45 oC, cooled to room temperature and filtered (Nyambaka, 1988; Kirk and Sawyer, 1991; Jedlicka and Klimes, 2004). Before the HPLC analysis, all the samples were filtered through 0.45 µm pore size filters. 43 The MP was prepared by mixing 0.01 M phosphate buffer plus 0.005 M 1hexanesulphonic acid (PH 7.0), methanol and acetonitrile in the ratio 80: 12: 8. The mixture was then filtered through a millipore filter and degassed under a vacuum for 15 minutes. The column was operated at room temperature. Analysis was carried out isocratically at a flow rate of 1mL/min (Anyakora et al., 2008). A 20 µL aliquot of samples and standards was injected into the HPLC system and monitored with a photodiode-array detector at 230 nm for thiamin, 266 nm for riboflavin and 290 nm for pyridoxine. Identification of compounds was achieved by comparing their retention times with those of standards. The concentrations of vitamins were determined using the equations of regression lines obtained by plotting the peak areas against the concentration of standards in ppm (Nyambaka, 1988; Jedlicka and Klimes, 2004). The equations of peak area versus the concentration of standards for thiamin, riboflavin and pyridoxine are given in Table 3.1. Table 3. 1: Regression lines equations for the thiamin, riboflavin and pyridoxine Vitamin Equation of line of regression R2 Thiamin y = 325,226x-872.94 Riboflavin y = 240,637x-4803.5 0.9995 R2 0.9996 Pyridoxine y = 2,000,006x 0.9977 Validation of the method was done by performing recovery analysis in triplicates using the standard addition method. This was carried out by by adding 0.005 g, 0.003 g and 0.005 g of thiamin, riboflavin and pyridoxine standards respectively to the pretreated sample and treating it according to the above procedures. 44 3.9.3 Extraction and analysis of AA The sample was prepared by macerating 10.000 g of the sample with 50 mL water. An equal volume of extraction solution was added, mixed and then filtered rapidly (Basu and Schorah, 1982; Kirk and Sawyer, 1991; Reiss, 1993; Macrae et al., 1993). A 10 mL aliquot of the sample was titrated with standard DCPIP until a rose pink colour persisted for about 15 seconds (Kirk and Sawyer, 1991; Hui, 1992; Reiss, 1993; Macrae et al., 1993). Recovery analysis using the standard addition technique was done in triplicates by adding 0.050 g of the AA standard to the pre-treated sample before the extraction and analysis was done. 3.10 Data analysis The data was analysed using the SPSS computer package. One-way analysis of variance (ANOVA) was used to compare the levels of vitamins in the amaranth leaves and grains between the areas of study and between different ages of the plants for the leaves. The amounts of vitamins between the species and between the seasons were determined using independent t-tests. 45 CHAPTER FOUR 4.0 RESULTS AND DISCUSSION 4.1 Introduction Vitamins and pro-vitamin A carotenoid (β-carotene) were analysed in A. hypochondriacus and A. cruentus leaves and grains from selected regions during the wet and dry seasons. Leaves were analysed for β-carotene and vitamins at three ages of plant growth (25, 50 and 75 DAS). Beta-carotene and α-tocopherol were analysed using spectrophotometric and HPLC methods. The HPLC method was used to determine the levels of thiamin, riboflavin and pyridoxine. Vitamin C was analysed using titrimetric method. All the vitamin levels are reported in mg/100 g of sample dry weight (DW). The results of the analyses are discussed in the following sections. 4.2 Methods validation 4.2.1 Correlation between spectrophotometric and HPLC methods of β-carotene and α-tocopherol analysis Spectrophotometric method was used in the analysis of FSVs due to the availability of a spectrophotometer, low cost as it requires less reagents, immediate analysis due to susceptibility of FSVs to oxidation, short analysis time and thus many samples could be analysed within a very short period. This was validated using the HPLC technique and the two methods related using the t-test and Pearson correlation coefficient (Table 4.1). This was done to ascertain the reliability of spectrophotometric method in the analysis of FSVs. 46 Table 4.1: Comparison of amounts of β-carotene and α-tocopherol in amaranth using spectrophotometric and HPLC methods (n = 24) Vitamin/ method Mean ± SE pro-vitamin β-Carotene α-Tocopherol Spectrophotometric 33.988±0.673 HPLC 35.326±0.619 Spectrophotometric 31.287±0.248 HPLC 31.456±0.237 t- P- Pearson calc. value correlation 1.463 0.150 0.995 0.491 0.626 0.992 T-critical (df = 46) = 2.015 at P = 0.05 The results (Table 4.1) point out that the two analytical techniques had high correlation coefficients when used in the analysis of β-carotene and α-tocopherol in the same samples (R = 0.995 for β-carotene and R = 0.992 for α-tocopherol). Therefore, spectrophotometric method can reliably be used for routine analysis of βcarotene and α-tocopherol in food samples. 4.2.2 Linearity and limits of detection Linearity and limits of detection (LoDs) of the analytical procedures are given in Table 4.2. Table 4. 2: Linearity and limits of detection of the analytical methods Vitamin/pro-vitamin Concentration range (ppm) LoD (μg/mL) β-Carotene 1.00-10.00 0.17 α-Tocopherol 1.00-10.00 0.09 Thiamin 0.05-1.00 0.03 Riboflavin 0.10-1.20 0.05 Pyridoxine 0.10-1.20 0.06 47 The analytical methods (Table 4.2) were linear over the concentration ranges given. The LoDs ranged from 0.03 to 0.17 mg/ mL. These values compare well with what have been reported (Jedlicka and Klimes, 2004; Zhao, 2004). 4.2.3 Recovery analysis Recovery experiments were performed using the standard addition method and the results presented in Table 4.3. Table 4. 3: Recovery of vitamins/pro-vitamin in amaranth leaves and grains Vitamin/pro-vitamin Amount of standard X ±sd (mg) X ±sd % added (mg) recovered (n = 3) recovery β-Carotene 5.00 4.75 ± 0.05 94.94±0.11 α-Tocopherol 5.00 4.57 ± 0.05 91.47±0.10 Thiamin 5.00 4.83 ± 0.04 96.67±0.08 Riboflavin 3.00 3.15 ± 0.04 105.08±0.12 Pyridoxine 5.00 5.08 ± 0.03 101.68±0.05 AA 50.00 44.40 ± 0.12 88.74±0.02 The recoveries were between 88.74 % and 105.08 %. The results showed the recovery of the proposed methods were satisfactory. The recoveries obtained in this study are similar to what has been reported by other workers (Rutkowski and Grzegorczyk, 2007; Amidzic et al., 2005). 4.3 Levels of β-carotene/vitamins in A. hypochondriacus and A. cruentus leaves at different ages of growth grown in different areas during wet and dry seasons Vegetables are normally harvested and consumed at different ages of plant growth because there is a preffered age when the flavour and palatability of the leaves are favourable for human consumption (Modi, 2007). However, for most vegetables the age at harvest decisively affects their nutritional values (Rodriquez-Amaya and 48 Kimura, 2004). To determine this effect, β-carotene and vitamins in A. hypochondriacus and A. cruentus leaves harvested from the areas of study during the wet and dry seasons were analysed at 25, 50 and 75 days after sowing (DAS). The results of each vitamin are discussed in the following sub-sections. 4.3.1 Beta-carotene levels of A. hypochondriacus and A. cruentus leaves at different ages of growth The mean levels of β-carotene in A. hypochondriacus and A. cruentus leaves obtained from the study areas during the wet and dry seasons at 25, 50 and 75 DAS are presented in Table 4.4. Table 4. 4: Beta-carotene content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons Study areas Species Season AH Wet Mean content ± sd (mg/100 g DW) at 25 DAS (n = 3) 50 DAS (n = 3) 75 DAS (n = 3) 36.663±0.340a 38.250±0.434b 42.868±0.666c Dry 38.288±0.297a 40.592±0.074b 45.831±0.132c Wet 35.985±0.062a 36.863±0.107b 41.554±0.523c Dry 38.030±0.117a 40.747±0.543b 45.192±0.146c Wet 36.965±0.056a 39.212±0.093b 43.616±0.884c Dry 40.363±0.148a 42.243±0.363b 48.341±0.126c Wet 36.347±0.572a 37.327±0.305b 43.715±0.231c Dry 38.591±0.152a 41.013±0.183b 45.828±0.261c Wet 36.476±0.067a 37.495±0.109b 44.028±0.087c Dry 39.335±0.172a 41.541±0.324b 49.797±0.414c Wet 36.349±0.073a 37.375±0.099b 42.045±0.235c Dry 39.800±0.560a 42.205±0.466b 46.725±0.292c Wet 37.610±0.042a 38.423±0.971a 42.310±0.073b Dry 39.943±0.254a 42.211±0.396b 47.841±0.174c Wet 37.096±0.131a 38.030±0.491b 42.434±0.504c Dry 38.179±0.210a 41.391±0.140b 47.071±0.182c Kenyatta AC University AH Bureti AC AH Mt. Elgon AC AH Kisii AC AH and AC stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same row do not differ significantly (P>0.05) 49 Table 4.4 shows that the mean amounts of β-carotene in the leaves of both species harvested from all the areas of study during both wet and dry seasons increased with plant age. These amounts were significantly different between all the plant ages (P<0.05) except between 25 and 50 DAS in A. hypochondriacus leaves picked from Kisii during the wet season. Thus, it can be deduced that the amounts of β-carotene in GA leaves regardless of the species and the season in which they are planted are affected by the age of the plant. This trend is in agreement with what was reported in lettuce, endive leaves, squash leaves and pumpkin leaves (Arima and RodriguezAmaya, 1988; Rodriguez-Amaya and Kimura, 2004). Similarly, Bergquist et al. (2007) reported a significant increase in the carotenoid content of ‘baby’ spinach between the time of sprouting and 3 ½ weeks. Maturation in vegetables has been shown generally to be accompanied by enhanced carotenogenesis hence the higher levels of β-carotene at advanced ages of growth when the leaves have become yellow (Rodriguez-Amaya and Kimura, 2004). The mean level (mg/100 g DW) of β-carotene present in A. hypochondriacus leaves ranged from 36.347-49.797 while that of A. cruentus leaves ranged from 35.98547.071. The amounts of β-carotene in the leaves of the two species regardless of the age of the plants are higher than the value of 33.3 mg/100 g DW reported in some amaranth species (Macrae et al., 1993). The mean amounts at all ages of the plants in this study were within the range of 35.3- 53.1 mg/100 g sample DW reported in A. tricolor (Yadav and Sengal, 1995). Raju et al. (2007) reported the amounts of βcarotene in A. viridis as 58.95 mg/100 g DW which is higher than what this study found. This difference may be ascribed to factors such as species difference, climatic conditons, age of the plant at harvest and farming practices (Kader, 2002; Rodriguez- 50 Amaya and Kimura, 2004). Comparatively, the leaves of both species are higher in βcarotene contents than most of the leafy vegetables such as nightshade, chili pepper leaves and cassava leaves (Weinberger and Msuya, 2004). However, the level is significantly lower (P<0.05) than 63.5 mg/100 g DW reported in spinach (Simopoulos, 2004). The DRI of vitamin A for an average adult is 500-625 μg retinol activity equivalent (RAE) or 6-7.5 mg of β-carotene (1 RAE = 12 μg of β-carotene) (Institute of Medicine, 2001). Therefore, consumption of upto 50 g on DW amaranth leaves of each species at any plant’s age per day is sufficient to meet vitamin A requirements of all the individuals. This can help alleviate the problems associated with high-blood pressure, cancer, blindness and age-related muscular degeneration (O’Brien and Price, 1983; Ali et al., 2009; Ofori et al., 2009; Ogunlesi et al., 2010). The mean amounts of β-carotene between A. hypochondriacus and A. cruentus leaves obtained from the study areas at different plant ages during wet and dry seasons were compared and the results presented in Table 4.5. 51 Table 4. 5: Comparison of mean β-carotene levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing Study areas DAS Kenyatta University Bureti Mt. Elgon Kisii Wet season (n = 3) Dry season (n = 3) t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 3.398 0.678 0.027* 1.399 0.258 0.235 50 5.373 1.388 0.006* 0.489 0.155 0.650 75 2.689 1.314 0.055 5.617 0.639 0.005* 25 1.862 0.618 0.136 14.466 1.772 <0.001* 50 10.229 1.885 0.001* 5.244 1.230 0.006* 75 0.188 0.099 0.860 15.045 2.512 <0.001* 25 2.234 0.128 0.089 1.373 0.465 0.242 50 1.410 0.120 0.231 2.025 0.664 0.113 75 13.699 1.983 0.050* 10.505 3.072 <0.001* 25 6.485 0.513 0.003* 9.278 1.764 0.001* 50 0.625 0.393 0.566 3.382 0.820 0.028* 75 0.422 0.124 0.695 5.299 0.770 0.006* P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) The leaves of A. hypochondriacus harvested from all the study areas at all the growth ages during both seasons generally had higher β-carotene levels than A. cruentus leaves. However, higher levels in A. cruentus leaves were noted in the samples picked from Kenyatta University (KU) at 50 DAS during the dry season, Bureti at 75 DAS during the wet season, Mt. Elgon at 25 and 50 DAS during the dry season, and Kisii at 75 DAS during the wet season. The results (Table 4.5) indicate that the levels of βcarotene in the leaves harvested from KU at 25 and 50 DAS, Bureti at 50 DAS, Mt. Elgon at 75 DAS and Kisii at 25 DAS during the wet season between the species were significantly different (P<0.05). Similarly, during the dry season the levels of βcarotene in the samples obtained from KU at 75 DAS, Bureti at all DAS, Mt. Elgon at 75 DAS and Kisii at all DAS differed significantly between the two species (P<0.05). 52 These differences could be attributed to the variation in the genotypic compositions between the two species (Kader, 2002; Rodriguez-Amaya and Kimura, 2004; Ofori et al., 2009). A study on β-carotene content of sweet potato cultivars, for example, showed a wide variation of 10 to 26,600 μg/100 g of the sample (Rodriguez-Amaya and Kimura, 2004). The results of comparison of the levels of β-carotene in the leaves obtained from study areas at 25, 50 and 75 DAS between the wet and dry seasons are given by Beta-carotene level in mg/100 g Figures 4.1 and 4.2 for A. hypochondriacus and A. cruentus respectively. 50.000 40.000 30.000 S e ason s W et Dry 20.000 DAS 10.000 KU Bureti Mt Elgo n 25 50 75 Kisii Study a reas Figure 4. 1: Graph of mean β-carotene contents in A. hypochondriacus leaves from different areas at 25, 50 and 75 DAS during wet and dry seasons 53 Beta-carotene level in mg/100 g 40.000 30.000 S e ason s W et Dry 20.000 DAS 10.000 KU Bureti Mt Elgon S tudy are as 25 50 75 Kisii Figure 4. 2: Graph of mean β-carotene contents in Acruentus leaves from different areas at 25, 50 and 75 DAS during wet and dry seasons The mean levels of β-carotene in the leaves of both species (Figures 4.1 and 4.2) picked at the same age of growth from all the study areas were significantly higher in the dry season than in the wet season (P<0.05). The higher levels observed in the dry season could be due to higher temperatures and light intensities experience throughout the country especially in January and February 2009 (ROK, 2009). High temperature and higher light intensity enhances biosynthesis of β-carotene in vegetables (Rodriquez-Amaya and Kimura, 2004). The wet season of 2008 was characterised by cool conditions over most parts of the country including the areas of study especially during the months of June and July with some areas like Nairobi recording as low as 13 oC in some days (ROK, 2009). Most of the areas in this season also had persistent cloud cover leading to low sunlight intensity. However, during the dry season, most parts of the country experienced generally sunny and dry weather conditions and slightly higher than average daytime temperatures during January to February 2009 (ROK, 2009). Occasional light to moderate rainfall, however, occurred over some 54 parts of the country including the areas covered in this study except Nairobi area in which Kenyatta University falls (ROK, 2009). A study done on kales, for example, pointed out that with increased exposure to sunlight and high temperatures, there was a corresponding increase in carotenoid accumulation in the leaves (Azevedo et al., 2005). However, if exposure to sunlight and temperature is too high, photodegradation may lead to the reduction to the carotenoid concentration (RodriquezAmaya and Kimura, 2004). 4.3.2 Alpha-tocopherol levels of A. hypochondriacus and A. cruentus leaves at different ages of growth The levels of α-tocopherol in the leaves of A. hypochondriacus and A. cruentus harvested at different ages of growth during the wet and dry seasons are presented in Table 4.6. 55 Table 4. 6: Alpha-tocopherol content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons Study areas Species Season Mean content ± sd (mg/100 g DW) at 25 DAS (n = 3) 50 DAS (n = 3) 75 DAS (n = 3) AH Kenyatta AC University AH Bureti AC AH Mt. Elgon AC AH Kisii AC Wet 34.075±1.596a 33.539±0.127a 34.431±1.193a Dry 32.342±0.321a 33.348±0.078a 33.129±0.911a Wet 30.846±0.524a 32.497±0.579ab 34.181±2.410b Dry 32.176±0.131a 32.372±0.304a 34.141±0.080b Wet 31.635±0.299a 33.415±0.491b 33.493±0.147b Dry 32.537±0.148a 33.388±0.055b 33.583±0.075b Wet 31.728±0.639a 32.183±0.147a 34.779±0.090b Dry 32.032±0.026a 32.285±0.054b 33.913±0.072c Wet 29.847±0.132a 32.468±0.336b 33.637±0.248c Dry 32.041±0.064a 32.638±0.104b 33.053±0.069c Wet 29.801±0.035a 31.082±0.243ab 32.395±1.382b Dry 32.178±0.052a 32.834±0.027b 33.392±0.090c Wet 30.270±0.245a 30.403±0.399a 33.503±0.398b Dry 32.456±0.051a 32.950±0.030b 33.783±0.018c Wet 29.494±0.412a 30.684±0.839b 31.004±0.435b Dry 32.116±0.092a 32.965±0.012a 32.238±1.458a AH and AC stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same row do not differ significantly (P>0.05) The results (Table 4.6) indicate that α-tocopherol contents in the leaves of both species increased with the growth of the plants during the two seasons considered except that of A. cruentus from KU in the wet season, which recorded the highest mean level at 50 DAS. The amounts of α-tocopherol in A. hypochondriacus leaves harvested from Bureti during both wet and dry seasons at 25 DAS differed significantly from those picked at 50 and 75 DAS (P<0.05). The leaves of the same species obtained from Mt. Elgon during both seasons had significantly different levels 56 between all the DAS (P<0.05). The samples of A. hypochondriacus from Kisii analysed during the wet season at 75 DAS recorded α-tocopherol levels which were significantly different from those analysed at 25 and 50 DAS (P<0.05) while those analysed during the dry season had significantly different levels between all the DAS (P<0.05). The amounts of α-tocopherol in A. cruentus leaves obtained from KU during the wet season were different significantly between 25 and 75 DAS (P<0.05). The leaves of the same species harvested at 75 days from the same area during the dry season had mean level which was significantly different from those recorded at 25 and 50 DAS (P<0.05). The A. cruentus leaves sampled from Bureti during the wet season at 75 DAS differed significantly (P<0.05) in the mean α-tocopherol content from those sampled at 25 and 50 DAS while those picked during the dry season were significantly different between all the DAS (P<0.05). The A. cruentus leaves picked from Mt. Elgon at 25 and 75 DAS during the wet season as well as those picked at all DAS during the dry season, had significantly different levels of α-tocopherol between them (P<0.05). The samples of A. cruentus obtained from Kisii during the wet season at 25 DAS contained mean α-tocopherol, which was significantly different from those noted at 50 and 75 DAS. The trend observed indicates that the level of α-tocopherol in amaranth leaves is influenced by the age of the plants. Increased in the levels of antioxidants with plant age have been reported (Modi, 2007). Similarly, Kunert and Ederer (1985) reported a significant increase in the levels of α-tocopherol in beech leaves (Kunert and Ederer, 1985). The mean levels of α-tocopherol in this study ranged from 29.847-34.431 and 29.49434.779 mg/100 g DW for A. hypochondriacus and A. cruentus leaves respectively. The level of α-tocopherol reported in vegetable amaranth in an earlier study was 57 6950-7040 μg/100 g raw sample weight (Nyambaka, 1988). The percentage moisture content of vegetable amaranth was reported as 83.48 % (Akubugwo et al., 2007). Based on this value, when the amount is given in DW basis, it was ranging from 42.070-42.615 mg/100 g. These were significantly higher (P<0.05) than the values contained by A. hypochondriacus and A. cruentus leaves in this study. This could be due to the factors such as species variation, the age of the plants at harvest, climatic conditions and agricultural practices (Rodriquez-Amaya and Kimura, 2004; Modi, 2007). The α-tocopherol mean contents in the leaves of both species are higher than that found in most vegetables like cabbage, asparagus and broccoli (Pamplona-Roger, 2005). However, it is significantly lower (P<0.05) than the level reported in spinach (36.0 mg/100 g DW) (Simopoulos, 2004). The DRI of vitamin E ranges from 5 mg for children below four years of age to 12 mg for average adults and 16 mg for lactating mothers per day (Institute of Medicine, 2000b). Consumption of 50-100 g of amaranth leaves daily can suffice to meet vitamin E requirements for all healthy individuals. This will be beneficial in protection against cancer, cardiovascular diseases, cataract development and haemolytic anaemia in infants (Simopoulos, 2004; Pamplona-Roger, 2005). The levels of α-tocopherol in the leaves harvested at 25, 50 and 75 DAS during the wet and dry seasons between A. hypochondriacus and A. cruentus were compared using an independent t-test and the results given in Table 4.7. 58 Table 4. 7: Comparison of mean α-tocopherol levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing Study areas DAS Kenyatta University Bureti Mt. Elgon Kisii Wet season (n = 3) Dry season (n = 3) t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 3.330 3.230 0.029* 0.829 0.166 0.454 50 3.044 1.042 0.038* 5.392 0.976 0.006* 75 0.161 0.250 0.880 1.917 1.012 0.128 25 0.228 0.093 0.831 5.846 0.506 0.004* 50 4.162 1.232 0.014* 24.828 1.103 <0.001* 75 12.919 1.287 <0.001* 5.510 0.330 0.005* 25 0.577 0.046 0.595 2.870 0.137 0.046* 50 5.794 1.386 0.004* 3.167 0.196 0.034* 75 1.533 1.242 0.200 5.175 0.339 0.007* 25 2.805 0.776 0.049* 5.579 0.339 0.005* 50 0.524 0.281 0.628 0.791 0.015 0.473 75 7.341 2.499 0.002* 1.835 1.545 0.140 P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) The results (Table 4.7) show that the levels of α-tocopherol in the leaves picked from KU at 25 DAS and 50 DAS during the wet season and 50 DAS during the dry season were significantly different between A. hypochondriacus and A. cruentus (P<0.05). The leaves harvested from Bureti at 50 and 75 DAS during the wet season and at all DAS during the dry season also had significantly different α-tocopherol contents between the species (P<0.05). Those obtained from Mt. Elgon contained significantly different amounts between the two species at 50 DAS during the wet season and at all DAS during the dry season (P<0.05). In addition, the samples obtained from Kisii at 25 and 75 DAS during the wet season as well as at 25 DAS during the dry season had significantly different levels of α-tocopherol between the species (P<0.05). The 59 differences in the concentrations of α-tocopherol between the species could be attributed to cultivar or varietal difference (Kader, 2002; Ofori et al., 2009). Figures 4.3 and 4.4 compares the levels of α-tocopherol in the leaves of A. hypochondriacus and A. cruentus respectively picked from the study areas at different ages of growth between the wet and dry seasons. Alpha-Tocopherol level in mg/100 g 30.000 20.000 S e ason s W et Dry 10.000 DAS 0.000 KU Bureti Mt Elgon 25 50 75 Kisii Study areas Figure 4. 3: Graph of mean α-tocopherol contents in A. hypochondriacus leaves from different areas at 25, 50 and 75 DAS during wet and dry seasons Alpha-Tocopherol level in mg/100 g 40.000 30.000 Seasons W et Dry 20.000 DAS 10.000 KU Bureti Mt Elgon S tudy are as 25 50 75 Kisii Figure 4. 4: Graph of mean α-tocopherol contents in A. cruentus leaves from different areas at 25, 50 and 75 DAS during wet and dry seasons 60 From the results (Figure 4.3), it was noted that between the wet season and dry seasons, a significant difference in the α-tocopherol contents of A. hypochondriacus leaves harvested from Bureti at 25 DAS existed (P<0.05). Similarly, the samples of the same species obtained from Mt. Elgon at 25 and 75 DAS as well as Kisii at 25 and 75 DAS had mean α-tocopherol amounts which were different significantly between the two seasons (P<0.05). The amounts in A. cruentus (Figure 4.4) leaves obtained from Kenyatta University at 25 DAS, Bureti at 75 DAS, Mt. Elgon at 25 and 75 DAS, and Kisii at 25 and 75 DAS were also significantly different between the seasons (P<0.05). These differences might have resulted from other factors such as postharvest handling as opposed to climatic conditions brought about by the change of seasons. 4.3.3 Thiamin levels of A. hypochondriacus and A. cruentus leaves at different ages of growth Thiamin contents of A. hypochondriacus and A. cruentus leaves harvested from the sampling areas at different ages of the plants during the wet and the dry seasons are summarised in Table 4.8. 61 Table 4. 8: Thiamin content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons Study Species Season Mean content ± sd (mg/100 g DW) at 25 DAS (n = 3) 50 DAS (n = 3) 75 DAS (n = 3) areas AH Kenyatta AC University AH Bureti AC AH Mt. Elgon AC AH Kisii AC Wet 0.680±0.008c 0.661±0.008b 0.594±0.009a Dry 0.678±0.003c 0.662±0.008b 0.625±0.008a Wet 0.691±0.011c 0.668±0.012b 0.598±0.005a Dry 0.675±0.005c 0.654±0.003b 0.618±0.008a Wet 0.714±0.011c 0.686±0.002b 0.593±0.006a Dry 0.695±0.007c 0.678±0.003b 0.607±0.003a Wet 0.702±0.005c 0.685±0.003b 0.590±0.006a Dry 0.686±0.010c 0.669±0.003b 0.604±0.005a Wet 0.675±0.013b 0.662±0.013b 0.588±0.005a Dry 0.686±0.010c 0.665±0.011b 0.596±0.002a Wet 0.660±0.007c 0.642±0.004b 0.591±0.006a Dry 0.664±0.003c 0.646±0.003b 0.609±0.008a Wet 0.689±0.006c 0.652±0.007b 0.573±0.002a Dry 0.688±0.005c 0.668±0.003b 0.610±0.003a Wet 0.678±0.004c 0.668±0.004b 0.598±0.005a Dry 0.681±0.002c 0.673±0.002b 0.612±0.004a AH and AC stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same row do not differ significantly (P>0.05) From the results (Table 4.8), it was noted that the mean thiamin levels in the leaves of the two species picked from all the sampling areas declined with the age of the plants. The differences in their mean contents between the ages of the plants were significant (P<0.05) except in A. hypochondriacus harvested from Mt. Elgon at 25 and 50 DAS. These differences may be attributed to factors such as insect injuries and physical injuries during harvesting (Kader, 2002). 62 The mean amounts (mg/100 g DW) of thiamin in this study ranged from 0.573-0.714 in A. hypochondriacus leaves and 0.590-0.702 in A. cruentus leaves. The amount of thiamin reported earlier in GA leaves was 0.68 g/100 g DW (Macrae et al., 1993). This value is comparable to what this study reported at 25 and 50 DAS. The DRI of thiamin range from 0.4 mg for children aged between 1-3 years old to 0.9 mg for adults per day while that for pregnant and lactating mothers is 1.2 mg per day (Institute of Medicine, 1998). According to Yarger (2008), GA leaves may be eaten raw in salads or cooked like spinach. From the results of this study, consumption of upto 200 g of raw amaranth leaves regardless of the species can be able to meet the vitamin requirements of all individuals. Cooking of the leaves and then discarding the water removes potentially harmful oxalates and nitrates (Yarger, 2008). However, cooking of vegetables results in loss of about 25 % thiamin (Pamplona-Roger, 2005). Thus, about 300 g of cooked taken daily is sufficient to meet the vitamin requirements of all the individuals. Comparison of the levels of thiamin in the leaves picked from the study areas at 25, 50 and 75 DAS between A. hypochondriacus and A. cruentus species was done and the results presented in Table 4.9. 63 Table 4. 9: Comparison of mean thiamin levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing Study areas DAS Kenyatta University Bureti Mt. Elgon Kisii Wet season (n = 3) Dry season (n = 3) t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 1.487 0.012 0.211 0.896 0.003 0.421 50 0.773 0.007 0.483 1.582 0.008 0.189 75 0.600 0.003 0.581 1.100 0.007 0.333 25 1.665 0.012 0.171 1.283 0.009 0.269 50 0.406 0.001 0.706 3.738 0.009 0.020* 75 0.726 0.004 0.508 0.768 0.002 0.485 25 1.797 0.015 0.147 3.648 0.022 0.022* 50 2.537 0.020 0.064 2.916 0.019 0.043* 75 0.731 0.003 0.505 2.654 0.013 0.057 25 2.685 0.011 0.055 1.913 0.006 0.128 50 3.592 0.015 0.023* 2.615 0.005 0.059 75 7.410 0.024 0.002* 0.735 0.002 0.503 P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) The results presented in Table 4.9 indicate that the mean amounts of thiamin in the leaves obtained from Kisii at 25 and 75 DAS during the wet season were significantly different between A. hypochondriacus and A. cruentus (P<0.05). In dry season, the leaves sampled from Bureti at 50 DAS and Mt. Elgon at 25 and 50 DAS differed significantly betweent the two species (P<0.05). These differences may be attributable to post-harvest handling since these are the only differences that were noted (Kader 2000). Table 4.10 compares the thiamin contents in the leaves of each species harvested from the sampling areas at three growth ages between the wet and the dry seasons. 64 Table 4. 10: Comparison of mean thiamin contents of A. hypochondriacus and A. cruentus leaves between wet and dry seasons A. hypochondriacus Regions DAS Kenyatta A. cruentus t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 0.259 0.001 0.809 2.353 0.016 0.078 University 50 75 0.138 0.001 0.897 1.831 0.014 0.141 4.726 0.031 0.009* 3.958 0.021 0.017* 25 2.490 0.019 0.068 2.519 0.016 0.065 50 3.852 0.008 0.018* 6.209 0.016 0.003* 75 3.530 0.013 0.024* 3.262 0.015 0.031* 25 1.199 0.011 0.297 0.887 0.004 0.425 50 0.311 0.003 0.771 1.411 0.004 0.231 75 2.449 0.008 0.071 3.022 0.017 0.039* 25 0.199 0.001 0.852 1.386 0.003 0.238 50 3.863 0.016 0.018* 2.235 0.005 0.089 75 16.675 0.037 <0.001* 4.086 0.015 0.015* Bureti Mt. Elgon Kisii P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) The t-test results (Table 4.10), indicate that A. hypochondriacus leaves obtained from all the study areas had thiamin amounts which were not significantly different between the wet and dry seasons except those obtained from KU at 75 DAS, Bureti at 50 and 75 DAS, and Kisii at 50 and 75 DAS (P<0.05). Similarly, A. cruentus leaves had insignificantly different thiamin levels between the two seasons except the samples harvested from KU at 75 DAS, Bureti at 50 and 75 DAS, Mt. Elgon at 75 DAS and Kisii at 75 DAS (P<0.05). This points out that the amounts of thiamin in amaranth leaves are not significantly influenced by the change of climatic conditions. The observed differences could have arisen due to post-harvest handling of the samples (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 65 4.3.4 Riboflavin levels of A. hypochondriacus and A. cruentus leaves at different ages of growth The trends in the amounts of riboflavin in A. hypochondriacus and A. cruentus leaves harvested from the areas of study at different ages of growth during the wet and dry seasons are presented in Table 4.11. Table 4. 11: Riboflavin content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons Study Species Season Mean content ± sd (mg/100 g DW) at 25 DAS (n = 3) 50 DAS (n = 3) 75 DAS (n = 3) areas AH Kenyatta AC University AH Bureti AC AH Mt. Elgon AC AH Kisii AC Wet 2.715±0.041c 2.500±0.007b 2.336±0.009a Dry 2.594±0.010c 2.524±0.011b 2.472±0.006a Wet 2.732±0.043c 2.638±0.039b 2.536±0.046a Dry 2.564±0.028b 2.550±0.033b 2.486±0.005a Wet 2.722±0.040c 2.623±0.016b 2.572±0.002a Dry 2.605±0.008c 2.563±0.023b 2.486±0.005a Wet 2.755±0.022c 2.649±0.004b 2.425±0.020a Dry 2.625±0.008c 2.598±0.014b 2.481±0.002a Wet 2.527±0.014c 2.355±0.011b 2.311±0.014a Dry 2.527±0.008b 2.505±0.012b 2.431±0.050a Wet 2.478±0.053c 2.352±0.029b 2.277±0.006a Dry 2.510±0.003c 2.496±0.003b 2.391±0.004a Wet 2.631±0.009b 2.588±0.006b 2.477±0.041a Dry 2.549±0.007b 2.532±0.005b 2.385±0.032a Wet 2.578±0.008b 2.544±0.027b 2.389±0.015a Dry 2.519±0.008b 2.508±0.008b 2.388±0.005a AH and AC stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same row do not differ significantly (P>0.05) Table 4.11 shows that the levels of riboflavin in the leaves of both species sampled in all the areas of study decreased with the plants’ age. The differences in the riboflavin 66 mean amounts were significant between all DAS (P<0.05) with the exceptions of A. hypochondriacus leaves harvested from Mt. Elgon at 25 and 50 DAS during the dry season and leaves of both species picked from Kisii at 25 and 50 DAS during both wet and dry seasons. The differences may be attributed factors such as mechanical and insect injuries as the plants grew (Kader, 2002). The mean amount of riboflavin in this study ranged from 2.311-2.722 mg in A. hypochondriacus leaves and 2.277-2.755 mg in A. cruentus leaves per sample DWs. These values are comparable to the mean amount of 2.24 mg/100 g reported in vegetable amaranth by Macrae et al. (1993). However, Akubugwo et al. (2007) reported a significantly higher (P<0.05) mean riboflavin content of 4.24 mg/100 g DW in A. hybridus leaves. These differences could be due to factors such as the species variation, climatic conditions, plants’ age at harvest and agricultural practices (Rodriquez-Amaya and Kimura, 2004; Raju et al., 2007). Domestic cooking of vegetables results in 10-20 % loss of riboflavin (Pamplona-Roger, 2005). Therefore, a daily intake of 30-50 g of cooked amaranth leaves of both species harvested between the time of sprouting and 50 days and 50-100 g at the plant’s advanced ages is sufficient to meet riboflavin requirements of 0.4-1.0 mg for all the healthy individuals (Institute of Medicine, 1998). The results of comparison of the levels of riboflavin in the leaves harvested at different DAS from the sampling areas during the wet and the dry seasons between the species are given in Table 4.12. 67 Table 4. 12: Comparison of riboflavin mean levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing Study DAS University Bureti Mt. Elgon Kisii Dry season (n = 3) t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 0.507 0.017 0.639 1.729 0.029 0.159 50 6.056 0.138 0.004* 1.310 0.026 0.260 75 7.451 0.201 0.002* 3.273 0.014 0.031* 25 1.230 0.033 0.286 3.197 0.020 0.033* 50 2.638 0.025 0.058 2.271 0.035 0.086 75 12.983 0.147 <0.001* 1.800 0.005 0.146 25 1.541 0.049 0.198 3.326 0.017 0.029* 50 0.188 0.003 0.860 1.301 0.009 0.263 75 3.946 0.034 0.017* 1.354 0.039 0.247 25 8.121 0.053 0.001* 5.325 0.031 0.006* 50 2.713 0.044 0.053 4.323 0.024 0.012* 75 3.480 0.088 0.025* 0.143 0.003 0.893 areas Kenyatta Wet season (n = 3) P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) From the results presented in Table 4.12, significant differences between the species in the samples analysed during the wet season were observed in the leaves picked from KU at 50 and 75 DAS, Bureti at 75 DAS, Mt. Elgon at 75 DAS and Kisii at 25 and 75 DAS (P<0.05). The leaves obtained during the dry season from KU at 25 DAS, Bureti at 25 DAS, Mt. Elgon at 25 DAS, and Kisii at 25 and 50 DAS had riboflavin contents which differed between the species significantly (P<0.05). These differences noted could not have been due to cultivar difference since the two species had insignificantly different riboflavin amounts in most of sampling times. Thus, other factors such as mechanical damages of the leaves during post-harvesting 68 handling may have significantly caused the variation of riboflavin between the species. Comparisons of the levels of riboflavin in A. hypochondriacus and A. cruentus leaves obtained from the study areas at different ages of growth between wet and dry seasons are presented in Table 4.13 Table 4. 13: Comparison of mean riboflavin contents of A. hypochondriacus and A. cruentus leaves between wet and dry seasons Study areas Kenyatta A. hypochondriacus DAS 25 50 University 75 25 50 Bureti 75 25 50 Mt. Elgon 75 A. cruentus t-cal. Mean diff. P-value t-cal. Mean diff. P-value 4.927 3.229 21.350 4.939 3.719 0.121 0.024 0.136 0.117 0.060 0.008* 0.032* <0.001* 0.008* 0.021* 5.734 2.974 1.884 9.617 6.279 0.168 0.088 0.050 0.130 0.051 0.005* 0.041* 0.133 0.001* 0.003* 28.915 0.000 15.651 4.010 0.086 0.000 0.150 0.120 <0.001* 1.000 <0.001* 0.016* 4.964 1.051 8.672 28.099 0.056 0.032 0.144 0.115 0.008* 0.353 0.001* <0.001* 25 13.235 0.082 <0.001* 9.664 0.059 0.001* 50 12.684 0.056 <0.001* 2.194 0.036 0.093 Kisii 75 3.049 0.092 0.038* 0.151 0.001 0.887 P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) The results (Table 4.13) indicate that levels of riboflavin in A. hypochondriacus leaves harvested in wet and dry seasons from all the areas of study were significantly different (P<0.05) except Mt. Elgon. The samples of A. cruentus leaves also showed significant differences in the amounts of riboflavin between wet and dry seasons (P<0.05) apart from KU at 75 DAS, Mt. Elgon at 25 DAS and Kisii at 50 and 75 69 DAS. Factors such as climatic conditions, mechanical damage of the leaves as well as post-harvest handling might have contributed to these differences (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 4.3.5 Pyridoxine levels of A. hypochondriacus and A. cruentus leaves at different ages of growth The amounts of pyridoxine in A. hypochondriacus and A. cruentus leaves harvested from the study areas at plants’ different days of growth after sowing are given in Table 4.14. Table 4. 14: Pyridoxine content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons Study areas Kenyatta University Bureti Species Mean content ± sd (mg/100 g DW) at 25 DAS (n = 3) 50 DAS (n = 3) 75 DAS (n = 3) AH AC AH AC Mt. Elgon Season AH AC Wet Dry Wet 2.866±0.031c 2.753±0.011c 2.920±0.008c 2.822±0.008b 2.734±0.006b 2.867±0.005b 2.624±0.006a 2.695±0.007a 2.789±0.008a Dry Wet Dry Wet 2.807±0.007b 2.921±0.008c 2.838±0.006c 2.840±0.046b 2.740±0.002a 2.857±0.036b 2.810±0.011b 2.855±0.011ab 2.732±0.003a 2.801±0.004a 2.761±0.007a 2.784±0.030a Dry Wet Dry Wet Dry 2.831±0.002c 2.799±0.005c 2.656±0.009b 2.860±0.011c 2.723±0.008b 2.747±0.009b 2.828±0.007b 2.720±0.024ab 2.782±0.005b 2.718±0.003b 2.723±0.015a 2.734±0.011a 2.581±0.061a 2.763±0.009a 2.669±0.009a Wet 2.824±0.014a 2.799±0.005a 2.798±0.068a Dry 2.698±0.013c 2.739±0.002b 2.664±0.006a AC Wet 2.872±0.015c 2.749±0.003b 2.727±0.007a Dry 2.687±0.004b 2.653±0.008a 2.645±0.005a AH and AC stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same row do not differ significantly (P>0.05) Kisii AH 70 Table 4.14 shows that the levels of pyridoxine in the leaves of both species harvested from all the areas during both seasons decreased with the plants’ age except A. cruentus leaves from Bureti during the wet season, A. hypochondriacus leaves from Mt. Elgon during both seasons and A. hypochondriacus leaves from Kisii during the dry season. These had higher levels at 50 DAS than at 25 DAS. The study revealed that the amounts of pyridoxine in A. hypochondriacus leaves sampled from KU and Bureti during both seasons were significantly different between all the DAS (P<0.05). In addition, A. hypochondriacus leaves harvested from Mt. Elgon during the wet season and Kisii during the dry season contained mean pyridoxine contents that were significantly different between all the DAS (P<0.05). It was also noted that A. cruentus leaves harvested from KU during both wet and dry seasons had pyridoxine mean amounts which were significantly different between all the DAS (P<0.05). Similarly, the leaves picked from Bureti during the dry season, Mt. Elgon during the wet season and Kisii during the wet season had pyridoxine levels which differed significantly between all the DAS (P<0.05). These differences may be attributed to the mechanical damages during the plants’ growth (kader, 2002). From this study, the mean pyridoxine contents (mg/100 g DW) ranged from 2.5812.921 in A. hypochondriacus leaves and 2.645-2.920 in A. cruentus leaves. These are higher than the mean value of 2.33 mg/100 g DW reported in A. hybridus (Akubugwo et al., 2007). This difference may be ascribed to factors including genotypic variation between the two species, climatic conditions, agricultural practices and post-harvest handling of the samples (Kader, 2002; Raju et al., 2007). The DRIs of vitamin B6 for healthy individuals range from 0.4 mg for children aged 1-3 years old to 1.4 mg for male adults per day (Institute of Medicine, 1998). Studies have shown that cooking of 71 vegetables result in 25-50 % degradation of pyridoxine content (Pamplona-Roger, 2005). Thus taking a meal containing 100 g of cooked amaranth leaves based on DW daily is enough to provide vitamin B6 for all the individuals. The results obtained when the levels of pyridoxine present in the leaves at 25, 50 and 75 DAS during the wet and dry seasons between A. hypochondriacus and A. cruentus were compared are presented in Table 4.15. Table 4. 15: Comparison of pyridoxine levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing Study areas Wet season (n = 3) 25 50 University 75 25 2.989 8.649 27.923 2.975 Mean diff. 0.054 0.045 0.165 0.080 50 75 25 50 0.097 0.992 -8.805 9.784 0.002 0.017 0.061 0.047 DAS Kenyatta Bureti Mt. Elgon t-cal. Dry season (n = 3) P-value t-cal. P-value 7.169 1.866 8.245 1.943 Mean diff. 0.054 0.006 0.037 0.007 0.040* 0.001* <0.001* 0.041* 0.927 0.377 0.001* 0.001* 8.044 3.945 9.778 0.143 0.063 0.037 0.068 0.002 0.001* 0.017* 0.001* 0.894 0.002* 0.136 0.001* 0.124 75 3.438 0.029 0.026* 2.469 0.088 0.069 25 3.950 0.048 0.017* 1.452 0.012 0.220 50 14.148 0.050 <0.001* 17.976 0.086 <0.001* Kisii 75 1.814 0.072 0.144 4.427 0.019 0.011* P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) From the results (Table 4.15), it was noted that the levels of pyridoxine in the leaves at all the ages of growth during wet season were significantly different between the species (P<0.05) except those which were picked from Bureti at 50 and 75 DAS, and Kisii at 75 DAS after sowing. Similarly, the samples analysed during the dry season had significantly different pyridoxine amounts between the species (P<0.05) except 72 those obtained from KU at 50 DAS, Bureti at 25 DAS, Mt. Elgon at 50 and 75 DAS, and Kisii at 25 DAS. Factors like species variation and post-harvest handling could have caused these differences (Kader, 2002; Raju et al., 2007). Comparisons of the levels of pyridoxine in the leaves of each species harvested from the areas of study at three ages of growth after sowing between the two seasons considered in the study are presented in Table 4.16. Table 4. 16: Comparison of mean pyridoxine contents of A. hypochondriacus and A. cruentus leaves between wet and dry seasons Study A. hypochondriacus A. cruentus t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 50 University 75 25 6.054 15.913 13.134 14.306 0.113 0.088 0.071 0.083 0.004* <0.001* <0.001* <0.001* 18.521 42.139 11.325 0.330 0.113 0.127 0.057 0.009 <0.001* <0.001* <0.001* 0.758 50 75 25 50 75 2.204 8.451 23.907 7.489 4.283 0.047 0.040 0.143 0.108 0.153 0.092 0.001* <0.001* 0.002* 0.013* 13.200 3.157 17.617 18.623 12.561 0.108 0.060 0.136 0.063 0.094 <0.001* 0.034* <0.001* <0.001* <0.001* areas DAS Kenyatta Bureti Mt. Elgon 25 11.209 0.126 <0.001* 20.265 0.185 <0.001* 50 19.080 0.060 <0.001* 19.187 0.096 <0.001* Kisii 75 3.417 0.135 0.027* 16.334 0.082 <0.001* P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) The results (Table 4.16) point out that the levels of pyridoxine in the leaves of A. hypochondriacus and A. cruentus plants at different ages harvested during the wet season were significantly higher than those harvested during the dry season (P<0.05) 73 except A. hypochondriacus leaves at 50 DAS and A. cruentus leaves at 25 DAS obtained from Bureti. 4.3.6 Ascorbic acid levels of A. hypochondriacus and A. cruentus leaves at different ages of growth Analysis of AA in A. hypochondriacus and A. cruentus leaves harvested from the areas of study at three varied ages of plants’ growth after sowing in the two seasons were done and the results presented in Table 4.17. Table 4. 17: Ascorbic acid content of A. hypochondriacus and A. cruentus leaves from different areas at different ages during wet and dry seasons Study areas Kenyatta University Species Season Mean content ± sd (mg/100 g DW) at AH AC AH Bureti AC AH Mt. Elgon AC AH 25 DAS (n = 3) 50 DAS (n = 3) 75 DAS (n = 3) Wet Dry Wet Dry 619.210±0.486b 618.055±2.844c 528.670±1.567b 581.970±4.489c 609.767±8.168b b 607.787±4.370 b 4 526.660±5.587b 556.867±4.595b 465.213±2.918a 495.220±2.907a 448.383±8.576a 485.383±3.833a Wet Dry Wet Dry Wet 607.430±0.703c 610.630±1.630c 538.353±0.595c 598.130±0.883c 536.577±7.264c 596.373±3.660b 597.863±2.435b 529.850±4.799b 566.730±1.365b 524.033±6.624b 443.900±7.365a 583.917±2.742a 440.810±2.425a 494.417±3.119a 447.710±0.295a Dry Wet Dry Wet 598.150±2.763c 497.237±0.595c 587.250±0.615c 572.380±2.725b 567.373±0.935b 489.733±4.974b 566.433±1.927b 589.707±1.750c 487.733±0.338a 440.077±0.117a 483.820±5.285a 450.123±1.030a Dry 595.873±2.917c 582.707±2.115b 490.457±0.653a Kisii AC Wet 501.440±5.446b 519.347±2.073c 441.830±0.221a Dry 586.777±1.265c 569.403±2.027b 491.933±0.068a AH and AC stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same row do not differ significantly (P>0.05) The results (Table 4.17) revealed a decline in the levels of AA in the leaves of both species harvested from all the study areas with ages of growth apart from A. 74 hypochondriacus leaves and A. cruentus leaves picked from Kisii at 50 DAS during the dry season which were higher than those picked at 25 DAS. The difference in the levels of AA in the leaves of both species obtained from Bureti, Mt. Elgon and Kisii were significant between all the DAS (P<0.05). The samples of both species harvested from KU at 75 DAS also had levels which differed from those harvested at 25 and 50 DAS significantly (P<0.05). Studies have reported that the leaf AA content in vegetables is strongly influenced by both the maturity at harvest and soil nutrition (Lee and kader, 2000; Bergquist et al., 2007). Bergquist et al. (2007) found out that the content of vitamin C in ‘baby’ spinach decreased significantly between 2 ½ weeks and 3 ½ weeks after planting. Decrease in the AA contents of amaranth leaves with age could be as a result of accumulation of nitrate (Lee and Kader, 2000). Increase in nitrate increases the plant foliage, which may reduce the light intensity on the leaves, thereby reducing accumulation of AA in the shaded plant parts (Lee and Kader, 2000). The mean AA content (mg/100 g DW) of A. hypochondriacus leaves was 443.900619.210 while that of A. cruentus leaves was 440.077-598.130. The levels of AA at 25 and 50 DAS from all the study areas except Mt. Elgon at 50 DAS are within the values of 496.5-630.9 mg/100 g DW reported in some amaranth accessions (Mnkeni et al., 2007). Lower mean amounts of AA outside this range were reported in the leaves of both species at 75 DAS except that of A. hypochondriacus leaves harvested from Bureti during the wet season. Macrae et al. (1993) reported the mean value of AA in grain amaranth leaves as 570.7 mg/100 g DW. This is comparable to the values noted in A. cruentus leaves obtained from all the study areas at 25 and 50 DAS during the dry season and A. hypochondriacus leaves at 25 and 50 DAS obtained from Mt. 75 Elgon during the dry season as well as Kisii during both seasons. But the amounts present in A. hypochondriacus leaves sampled from KU and Bureti at 25 and 50 DAS during both seasons were higher. A study done on vegetable amaranth leaves in Kenya reported higher AA content (629 mg/100 g DW) than both species considered in this study. Similarly, Yadav and Sengal (1995) reported the amounts of AA ranging from 624.1-629.0 mg/100 g DW in A. tricolor. These differences could be attributed to factors such as cultivar difference, climatic conditons, stage of plant development and agricultural practices (Kader, 2002; Modi, 2007). The AA requirements per day for healthy individuals in terms of DRIs range from 13 mg for young children aged 1-3 years old to 60 mg for adult females while for pregnant and lactating mothers the values range from 66-100 mg per day (Institute of Medicine, 2000b). Cooking of vegetables as per the common household practice as been reported to result in a loss of 15-55 % AA due to leaching into the cooking water (Pamplona-Roger, 2005). To meet vitamin C requirements of children and adults, a daily intake of upto 50 g of cooked amaranth is reccomended. However, for pregnant and lactating mothers, consumption of about 100 g is sufficient for their vitamin C requirements. The levels of AA in the leaves harvested from the areas considered in this study at different ages of the plants during the wet and dry seasons were compared using an independent t-test and the results shown in Table 4.18. 76 Table 4. 18: Comparison of AA levels between A. hypochondriacus and A. cruentus leaves from the study areas at different days after sowing Study areas DAS Kenyatta University Bureti Mt. Elgon Wet season (n = 3) Dry season (n = 3) t-cal. Mean diff. P-value t-cal. Mean diff. P-value 25 95.590 90.540 <0.001* 11.761 36.085 <0.001* 50 75 25 50 75 14.546 3.218 129.915 19.090 0.690 83.107 16.830 69.077 66.523 3.090 <0.001* 0.032* <0.001* <0.001* 0.528 13.908 3.542 11.678 19.321 37.330 50.920 9.837 12.500 31.133 89.500 <0.001* 0.024* <0.001* <0.001* <0.001* 25 50 75 25 9.349 7.171 41.636 20.178 39.340 34.300 7.633 70.940 0.001* 0.002* <0.001* <0.001* 6.669 0.760 1.280 4.955 10.900 0.940 3.913 9.097 0.003* 0.490 0.270 0.008* 50 44.923 70.360 <0.001* 7.866 13.303 0.001* 75 13.636 8.293 <0.001* 3.898 1.477 0.058 P-values marked * are significantly different (P<0.05); t-crit. = 2.776 (df = 4) Kisii From the results (Table 4.18), the AA contents of A. hypochondriacus leaves harvested during the wet season from all the areas of study at varying ages of plants after sowing were significantly higher than those of A. cruentus leaves (P<0.05) except those noted in Bureti at 75 DAS. Similarly, significantly higher amounts of AA in the dry season were recorded in A. hypochondriacus leaves compared to A. cruentus leaves in all the study areas (P<0.05) except Mt. Elgon at 50 and 75 DAS, and Kisii at 75 DAS. These differences could be due to cultivar or varietal differences (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 77 The results of comparison of the AA amount in the leaves of each species harvested from the areas of study at varied plant ages after sowing between the wet and the dry seasons are given shown in Figures 4.5 and 4.6. Ascorbic acid level in mg/100 g 600.000 400.000 S e ason s W et Dry 200.000 DAS 25 50 75 0.000 KU Bureti Mt Elgo n S tudy are as Kisii Figure 4. 5: Graph of mean AA contents in A. hypochondriacus leaves from different areas at 25, 50 and 75 DAS during wet and dry seasons 600.000 Ascorbic acid level in mg/100 g 400.000 S e ason s W et Dry 200.000 DAS 0.000 KU Bureti Mt Elgo n S tudy are as 25 50 75 Kisii Figure 4. 6: Graph of mean AA contents in A. hypochondriacus leaves from different areas at 25, 50 and 75 DAS during wet and dry seasons 78 Figures 4.5 and 4.6, shows that the levels of AA in the leaves of both species were higher during the dry than the wet season at all plants’ ages except in A. hypochondriacus leaves sampled from KU at 25 and 50 DAS as well as Kisii at 50 DAS. The differences in the levels of AA in the leaves of both species obtained from all the areas of study were significant between the wet and dry seasons (P<0.05) except A. hypochondriacus leaves from KU at 25 and 50 DAS and also Bureti at 50 DAS. This could be due to varied climatic conditions brought by seasonal changes. Studies have shown that the higher the temperature the higher the AA content of plant tissues (Kader, 2002). This is because temperature influences the uptake and metabolism of mineral nutrients by plants since transpiration increases with higher temperatures. Furthermore, rainfall affects the water supply to the plant, which may influence the composition of the leaves and its susceptibility to the mechanical damage during harvesting (Kader, 2002). Temperature and light also play an important role in antioxidant metabolism. Thus, increase in temperature results in increase in levels of AA. This explains the high levels of AA in the dry season. During the wet season, most parts of the country especially the Western, Northeastern and some parts of Central Kenya including Nairobi experienced heavy rainfall. The dry season was characterised by sunny and dry weather conditions from January to February 2009. However, light rains were experienced in December 2008 (ROK, 2009). 4.4 Comparisons of beta-carotene and vitamin levels in A. hypochondriacus and A. cruentus leaves during the wet and dry seasons between different areas Environmental factors such as climatic conditions, soil quality, soil pH and salinity, production practices, insect injury and plant diseases have a marked influence on on the nutritional quality of vegetables (Goldman et al., 1999; Kader, 2002; Khader and 79 Rama, 2003). Climatic factors especially temperature and light intensity, affect the vitamin level of the leafy vegetables. Thus, the location in which they are planted can determine the amounts of vitamins in the same species of vegetable (Kader, 2002). Soil nutrition particularly nitrogen has a great influence on the yield and the contents of most nutrients in leafy vegetables. Comparison of the levels of β-carotene and vitamins in A. hypochondriacus and A. cruentus leaves at different ages of their growth sown in four different areas was therefore undertaken to determine these effects. The results are highlighted in the following sub-sections. 4.4.1 Beta-carotene contents in A. hypochondriacus and A. cruentus leaves from different regions The levels of β-carotene in A. hypochondriacus and A. cruentus leaves from the sampling areas at different ages of growth obtained during the wet and dry seasons are presented in Table 4.19. 80 Table 4. 19: Beta-carotene content of A. hypochondriacus and A. cruentus leaves from different areas during wet and dry seasons Species Season Study Mean content ± sd (mg/100 g DW) at Wet areas KU Bureti 25 DAS 36.663±0.340ab 36.965±0.056b 50 DAS 38.250±0.434ab 39.212±0.093b 75 DAS 42.868±0.666ab 43.616±0.884bc Dry Mt. Elgon Kisii KU Bureti Mt. Elgon 36.476±0.067a 37.610±0.042c 38.288±0.297a 40.363±0.148c 39.335±0.172b 37.495±0.109a 38.423±0.971ab 40.592±0.074a 42.243±0.363c 41.541±0.324b 44.028±0.087c 42.310±0.073a 45.831±0.132a 48.341±0.126c 49.797±0.414d Kisii KU Bureti Mt. Elgon Kisii 37.610±0.042c 35.985±0.062a 36.347±0.572a 36.349±0.073a 37.096±0.131b 38.423±0.971ab 36.863±0.107a 37.327±0.305a 37.375±0.099a 38.030±0.491b 42.310±0.073a 41.554±0.523a 43.715±0.231c 42.045±0.235c 42.434±0.504c KU Bureti Mt. Elgon Kisii 38.030±0.117a 38.591±0.152a 39.800±0.560b 38.179±0.210a 40.747±0.543a 41.013±0.183a 42.205±0.466b 41.391±0.140a 45.192±0.146a 45.828±0.261b 46.725±0.292c 47.071±0.182c AH Wet AC Dry AH and AC and stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same column for each species in the same season do not differ significantly (P>0.05) The results (Table 4.19) show that during the wet season, the mean amount of βcarotene in A. hypochondriacus leaves at 25 DAS from Kisii was significantly the highest (P<0.05). The levels in the leaves between Bureti, Mt. Elgon and Kisii were also significantly different (P<0.05). At 75 DAS, A. hypochondriacus leaves picked from Mt. Elgon had significantly higher β-carotene level compared to those obtained from KU and Kisii (P<0.05). In addition, the samples harvested from Kisii compared with those harvested from Bureti and Mt. Elgon had significantly lower β-carotene content (P<0.05). In the dry season, the amounts of β-carotene present in A. hypochondriacus leaves harvested at 25 and 50 DAS from Bureti and Kisii were 81 significantly different from those found in the samples obtained from KU and Mt. Elgon (P<0.05). The leaves of A. hypochondriacus species harvested at 75 DAS had significantly varied concentrations of β-carotene between all the areas of study (P<0.05). The leaves of A. cruentus species analysed in the wet season from Kisii at 25 and 50 DAS had significantly higher β-carotene content compared to other areas (P<0.05). The leaves harvested from KU at 75 DAS had significantly the least amount of β-carotene (P<0.05). With regard to the dry season, it was noted that A. cruentus leaves obtained at 25 and 50 DAS from Mt. Elgon had significantly higher β-carotene content than those picked from the other areas of study (P<0.05). The A. cruentus leaves harvested at 75 DAS from Mt. Elgon and Kisii had β-carotene amounts that were insignificantly different between them but differed significantly from those obtained from KU and Bureti (P<0.05). The observed differences may be attributed to factors like pre-harvest climatic conditions, soil conditions (soil type, soil nutrients, pH and salinity) and insect injuries (Goldman et al., 1999). Climatic conditions, particularly temperature and light intensity, have an effect on β-carotene content of vegetables (Rodriquez-Amaya and Kimura, 2004). High temperature and higher light intensity favours carotenogenesis in vegetables (Rodriquez-Amaya and Kimura, 2004). Higher levels of nitrogen in the soil results in higher amounts of β-carotene in vegetables (Weston and Barth, 1997; Onyango, 2010). 4.4.2 Alpha-tocopherol contents in A. hypochondriacus and A. cruentus leaves from different areas The results of analysis of α-tocopherol in A. hypochondriacus and A. cruentus leaves harvested from sampling areas at different ages of plant after sowing during the wet and dry seasons are tabulated in Table 4.20. 82 Table 4. 20: Alpha-tocopherol content of A. hypochondriacus and A. cruentus leaves from different areas during the wet and dry seasons Species Season Wet AH Dry Wet AC Dry Study Mean content ± sd (mg/100 g DW) at areas 25 DAS 50 DAS 75 DAS KU 34.075±1.596c 33.539±0.127c 34.431±1.193a Bureti 31.635±0.299b 33.415±0.491c 33.493±0.147a Mt. Elgon 29.847±0.132a 32.468±0.336b 33.637±0.248a Kisii 30.270±0.245ab 30.403±0.399a 33.503±0.398a KU 32.342±0.321ab 33.348±0.078c 33.129±0.911a Bureti 32.537±0.148b 33.388±0.055c 33.583±0.075a Mt. Elgon 32.041±0.064a 32.638±0.104a 33.053±0.069a Kisii 32.456±0.051b 32.950±0.030b 33.783±0.018a KU 30.846±0.524b 32.497±0.579b 34.181±2.410b Bureti 31.728±0.639c 32.183±0.147b 34.779±0.090b Mt. Elgon 29.801±0.035a 31.082±0.243a 32.395±1.382ab Kisii 29.494±0.412a 30.684±0.839a 31.004±0.435a KU 32.176±0.131a 32.372±0.304a 34.141±0.080b Bureti 32.032±0.026a 32.285±0.054a 33.913±0.072b Mt. Elgon 32.178±0.052a 32.834±0.027b 33.392±0.090ab Kisii 32.116±0.092a 32.965±0.012b 32.238±1.458a AH and AC and stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same column for each species in the same season do not differ significantly (P>0.05) The results (Table 4.20) show that during the wet season A. hypochondriacus leaves harvested at 25 DAS from KU had significantly the highest α-tocopherol mean level (P<0.05). Furthermore, the levels between the leaves picked at the same age from Bureti and Mt. Elgon were significantly different (P<0.05). The A. hypochondriacus leaves obtained at 50 DAS between Bureti and Mt. Elgon had mean α-tocopherol values which differed significantly (P<0.05). Considering the dry season, it was noted 83 that A. hypochondriacus leaves obtained at 25 DAS were significantly different in the amounts of α-tocopherol between Bureti and Mt. Elgon (P<0.05). The leaves of the same species picked at 50 DAS between KU and Bureti were not different significantly in their mean levels of α-tocopherol but differed when compared with those obtained from Mt. Elgon and Kisii significantly (P<0.05). As for A. cruentus leaves analysed during the wet season, the leaves harvested at 25 DAS from Mt. Elgon and Kisii contained mean α-tocopherol contents that were insignificantly different between them but they differed significantly from those obtained from KU and Bureti (P<0.05). The leaves harvested at 50 DAS from KU and Bureti versus Mt. Elgon and Kisii were different significantly in their mean αtocopherol levels (P<0.05). At 75 DAS, A. cruentus leaves harvested from Kisii had significantly different mean α-tocopherol concentration compared with those from KU and Bureti (P<0.05). With respect to the dry season, it was noted that A. cruentus leaves at 50 DAS obtained from KU and Bureti versus those obtained from Mt. Elgon and Kisii had levels which were significantly different (P<0.05). In addition, the mean amount of α-tocopherol in the leaves harvested at 75 DAS from Kisii was significantly different from the mean amounts of those picked from KU and Bureti (P<0.05). The trend in the levels of α-tocopherol between the areas of study indicates that the amounts of α-tocopherol in amaranth leaves are influenced by pre-harvest factors like climatic conditions, soil nutrients and pH as well as pos-harvest handling of samples (Goldman et al., 1999; Kader, 2002; Rutkowski and Grzegorczyk, 2007). 84 4.4.3 Thiamin contents in A. hypochondriacus and A. cruentus leaves from different regions Thiamin concentrations in A. hypochondriacus and A. cruentus leaves harvested at 25, 50 and 75 DAS during the wet and dry seasons from the study areas were compared are presented in Table 4.21. Table 4. 21: Thiamin content of A. hypochondriacus and A. cruentus leaves from different areas in wet and dry seasons Species AH AC Season Study Mean content ± sd (mg/100 g DW) at areas 50 DAS 0.661±0.008a 0.686±0.002b 0.662±0.013a 0.652±0.007a 75 DAS 0.594±0.009b 0.593±0.006b 0.588±0.005b 0.573±0.002a Wet KU Bureti Mt. Elgon Kisii 25 DAS 0.680±0.008a 0.714±0.011b 0.675±0.013a 0.689±0.006a Dry Wet KU Bureti Mt. Elgon Kisii KU 0.678±0.003a 0.695±0.007b 0.686±0.010ab 0.688±0.005ab 0.691±0.011c 0.662±0.008a 0.678±0.003b 0.665±0.011ab 0.668±0.003ab 0.668±0.012b 0.625±0.008c 0.607±0.003b 0.596±0.002a 0.610±0.003b 0.598±0.005a Dry Bureti Mt. Elgon Kisii KU 0.702±0.005c 0.660±0.007a 0.678±0.004b 0.675±0.005b 0.685±0.003c 0.642±0.004a 0.668±0.004b 0.654±0.003b 0.590±0.006a 0.591±0.006a 0.598±0.005a 0.618±0.008b Bureti 0.686±0.010b 0.669±0.003c 0.604±0.005a Mt. Elgon 0.664±0.003a 0.646±0.003a 0.609±0.008ab Kisii 0.681±0.002b 0.673±0.002c 0.612±0.004ab AH and AC and stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same column for each species in the same season do not differ significantly (P>0.05) The results (Table 4.21) point out that the levels of thiamin in A. hypochondriacus leaves obtained during the wet season from Bureti at 25 and 50 DAS afer sowing contained significantly higher amounts of thiamin than other areas (P<0.05). In addition, the mean thiamin concentration in the leaves obtained from Kisii at 75 DAS 85 was significantly lower than those from the other areas (P<0.05). With respect to the dry season, significant differences in the levels of thiamin in A. hypochondriacus leaves between KU and Bureti at 25 and 50 DAS were noted. The mean thiamin values of leaves obtained at 75 days between Bureti and Kisii after sowing were insignificantly different but were different significantly when compared with those harvested from KU and Mt. Elgon (P<0.05). Amanthus cruentus leaves harvested during the wet season at 25 DAS from KU and Bureti contained mean thiamin amounts that were not significantly different between them but they differed with those picked from Mt. Elgon and Kisii significantly (P<0.05). The levels of thiamin present in the leaves of A. cruentus at 50 DAS sampled during the same season from KU and Kisii were insignificantly different between them but differed significantly with those recorded in the leaves obtained from Bureti and Mt. Elgon (P<0.05). Considering the dry season, A. cruentus leaves harvested at 25 DAS from Mt. Elgon had significantly lower level of thiamin than the other areas of study (P<0.05). The leaves analysed at 50 DAS depicted significantly different mean thiamin values between KU and Mt. Elgon (P<0.05). The differences observed in the levels of thiamin between the areas of study could have resulted from factors such as soil conditions (soil type, pH and soil nutrients), climatic conditions especially rainfall and post-harvest handling of samples (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 4.4.4 Riboflavin contents in A. hypochondriacus and A. cruentus leaves from different regions The results of comparisons of the levels of riboflavin in A. hypochondriacus and A. cruentus leaves harvested at different plants’ ages from the study areas during the wet and dry seasons areas given in Table 4.22. 86 Table 4. 22: Riboflavin content of A. hypochondriacus and A. cruentus leaves from different areas in wet and dry seasons Species Season Wet AH Dry Wet AC Dry Study Mean content ± sd (mg/100 g DW) at areas KU Bureti 25 DAS 2.715±0.041c 2.722±0.040c 50 DAS 2.500±0.007b 2.623±0.016d 75 DAS 2.336±0.009a 2.572±0.002c Mt. Elgon Kisii KU Bureti 2.527±0.014a 2.631±0.009b 2.594±0.010c 2.605±0.008c 2.355±0.011a 2.588±0.006c 2.524±0.011a 2.563±0.023b 2.311±0.014a 2.477±0.041b 2.472±0.006b 2.486±0.005b Mt. Elgon Kisii KU Bureti Mt. Elgon 2.527±0.008a 2.549±0.007b 2.732±0.043c 2.755±0.022c 2.478±0.053a 2.505±0.012a 2.532±0.005a 2.638±0.039c 2.649±0.004c 2.352±0.029a 2.431±0.050ab 2.385±0.032a 2.536±0.046c 2.425±0.020b 2.277±0.006a Kisii KU Bureti Mt. Elgon 2.578±0.008b 2.564±0.028b 2.625±0.008c 2.510±0.003a 2.544±0.027b 2.550±0.033b 2.598±0.014c 2.496±0.003a 2.389±0.015b 2.486±0.005b 2.481±0.002b 2.391±0.004a Kisii 2.519±0.008a 2.508±0.008a 2.388±0.005a AH and AC and stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same column for each species in the same season do not differ significantly (P>0.05) The amounts of riboflavin (Table 4.22) in A. hypochondriacus leaves sampled during the wet season from KU and Bureti areas at 25 DAS were not significantly different between them but differed significantly from those in the leaves harvested from Mt. Elgon and Kisii (P<0.05). Significant differences in the levels of riboflavin in A. hypochondriacus leaves between all the areas of study were noted at 50 DAS during the wet season (P<0.05). The leaves of the same species picked at 75 DAS showed insignificant differences in the mean riboflavin content between KU and Mt. Elgon. There were significant differences in the amounts contained in the leaves between 87 these two areas versus Bureti and Kisii (P<0.05). With respect to the dry season, the same trend in the concentrations of riboflavin in leaves at 25 DAS as in the wet season was observed. At 50 DAS, the leaves picked from Bureti had riboflavin content which was significantly different from those recorded in other areas of study (P<0.05). The leaves of the same species harvested from Kisii at 75 DAS had riboflavin concentration which was significantly lower than the other study areas (P<0.05). Considering A. cruentus, the results of analysis during the wet season revealed that riboflavin contents in the leaves picked at 25 and 50 DAS between KU and Bureti were not significantly different but were different significantly compared to those recorded in Mt. Elgon and Kisii (P<0.05). The leaves sampled at 75 DAS from Bureti and Kisii had the mean amounts of riboflavin which were not significantly different between them but were different significantly from those obtained from KU and Mt. Elgon (P<0.05). During the dry season, the leaves harvested at 25 and 50 DAS from Bureti had riboflavin concentrations which were significantly higher than the other areas of study (P<0.05). The samples analysed at 75 DAS obtained from KU and Bureti versus those obtained from Mt. Elgon and Kisii contained riboflavin amounts which were significantly different between them (P<0.05). The variations in the leaf riboflavin contents of both species depicted between some areas at the same plants’ ages may be ascribed to factors like climatic conditions, soil pH, soil nutrients and post-harvest handling (Goldman et al., 1999; Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 88 4.4.5 Pyridoxine contents in A. hypochondriacus and A. cruentus leaves from different regions Table 4.23 gives the results of comparisons of mean pyridoxine levels in A. hypochondriacus and A. cruentus leaves harvested from the study areas at different plants’ ages during the wet and the dry seasons. Table 4. 23: Pyridoxine content of A. hypochondriacus and A. cruentus leaves from different areas in wet and dry seasons Species AH AC Season Study Mean content ± sd (mg/100 g DW) at areas 50 DAS 2.822±0.008ab 2.857±0.036b 2.828±0.007ab 2.799±0.005a 75 DAS 2.624±0.006a 2.801±0.004b 2.734±0.011b 2.798±0.068b Wet KU Bureti Mt. Elgon Kisii 25 DAS 2.866±0.031b 2.921±0.008c 2.799±0.005a 2.824±0.014a Dry Wet KU Bureti Mt. Elgon Kisii KU 2.715±0.041c 2.722±0.040c 2.527±0.014a 2.631±0.009b 2.920±0.008b 2.500±0.007b 2.623±0.016d 2.355±0.011a 2.588±0.006c 2.867±0.005c 2.336±0.009a 2.572±0.002c 2.311±0.014a 2.477±0.041b 2.789±0.008b Dry Bureti Mt. Elgon Kisii KU 2.840±0.046a 2.860±0.011a 2.872±0.015a 2.807±0.007c 2.855±0.011c 2.782±0.005b 2.749±0.003a 2.740±0.002c 2.784±0.030b 2.763±0.009b 2.727±0.007a 2.732±0.003c Bureti 2.831±0.002d 2.747±0.009c 2.723±0.015c Mt. Elgon 2.723±0.008b 2.718±0.003b 2.669±0.009b Kisii 2.687±0.004a 2.653±0.008a 2.645±0.005a AH and AC and stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same column for each species in the same season do not differ significantly (P>0.05) According to the results (Table 4.23) of the wet season in A. hypochondriacus, the mean pyridoxine amounts in the leaves obtained at 25 DAS between Mt. Elgon and Kisii were not significantly different but differed significantly from the amounts noted at KU and Bureti (P<0.05). The leaves obtained at 50 DAS were only significantly 89 different in the pyridoxine content between Bureti and Kisii (P<0.05). Comparison of the levels of pyridoxine at 75 DAS between the areas indicates that A. hypochondriacus leaves harvested from KU had significantly the least mean level (P<0.05). As for the dry season, the mean pyridoxine concentrations of A. hypochondriacus leaves harvested at 25 DAS between KU and Bureti were not significantly different. However, when these levels were compared with those contained in the samples from Mt. Elgon and Kisii, significant differences were noted (P<0.05). The A. hypochondriacus leaves analysed at 50 DAS had significantly different concentrations of pyridoxine between all the areas of study (P<0.05). The leaves at 75 DAS harvested from KU and Mt. Elgon had mean pyridoxine amounts which were not significantly different between them but were significantly different from those found in the leaves picked from Bureti and Kisii (P<0.05). Amaranthus cruentus leaves obtained at 25 DAS from KU during the wet season depicted the highest amount of pyridoxine which was different significantly compared to those noted in the other areas (P<0.05). The leaves picked at 50 DAS from KU and Bureti displayed the levels which did not differ significantly between them but differed significantly from those contained in the samples obtained from Mt. Elgon and Kisii (P<0.05). The leaves harvested at DAS from Kisii had the least mean amount of pyridoxine which was significantly different from those noted in the other study areas (P<0.05). As regard to the dry season, A. cruentus leaves picked at 25 DAS showed significant differences between all the study areas in the mean pyridoxine contents (P<0.05). The leaves harvested at 50 and 75 DAS from KU and Bureti had levels which did not differ significantly between them but differed significantly from those contained in the samples from Mt. Elgon and Kisii (P<0.05). 90 The differences which were noted in the levels of pyridoxine in the two amaranth species between some areas may be attributed to climatic conditions, soil conditions and post-harvest handling (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 4.4.6 Ascorbic acid contents in A. hypochondriacus and A. cruentus leaves from different areas The mean amounts of AA in A. hypochondriacus and A. cruentus leaves sampled and analysed during the wet and dry seasons at different days of plants’ growth after sowing are shown in Table 4.24. Table 4. 24: Ascorbic acid content of A. hypochondriacus and A. cruentus leaves from different regions in wet and dry seasons Species Season Study areas Wet AH Dry Wet AC Dry Mean content ± sd (mg/100 g DW) at KU Bureti 25 DAS 50 DAS d 619.210±0.486 609.767±8.168c 607.430±0.703c 596.373±3.660b 75 DAS 465.213±2.918b 443.900±7.365a Mt. Elgon Kisii KU Bureti Mt. Elgon 536.577±7.264a 572.380±2.725b 618.055±2.844c 610.630±1.630b 598.150±2.763a 524.033±6.624a 589.707±1.750b 607.787±4.370d 597.863±2.435c 567.373±0.935a 447.710±0.295a 450.123±1.030a 495.220±2.907b 583.917±2.742c 487.733±0.338a Kisii KU Bureti Mt. Elgon Kisii 595.873±2.917a 528.670±1.567c 538.353±0.595b 497.237±0.595a 501.440±5.446a 582.707±2.115b 526.660±5.587bc 529.850±4.799c 489.733±4.974a 519.347±2.073b 490.457±0.653a 448.383±8.576a 440.810±2.425a 440.077±0.117a 441.830±0.221a KU Bureti Mt. Elgon Kisii 581.970±4.489a 598.130±0.883c 587.250±0.615b 586.777±1.265b 556.867±4.595a 566.730±1.365b 566.433±1.927b 569.403±2.027b 485.383±3.833ab 494.417±3.119c 483.820±5.285a 491.933±0.068bc AH and AC and stands for A. hypochondriacus and A. cruentus respectively. Mean values with the same superscript in the same column for each species in the same season do not differ significantly (P>0.05) 91 The results (Table 4.24) show that the mean levels of AA in A. hypochondriacus leaves obtained from KU were the highest at all the growth ages in both seasons except at 75 DAS in the dry season. The leaves harvested in the wet season at 25 DAS had levels of AA which were different significantly between the areas of study (P<0.05). The AA contents of leaves analysed at 50 DAS between KU and Bureti displayed no significant differences but was found to differ significantly from those in the samples picked from Mt. Elgon and Kisii (P<0.05). The leaves of A. hypochondriacus plants at 75 DAS sampled from KU had significantly higher AA levels than the other areas of study (P<0.05). Considering the dry season, the AA contents of A. hypochondriacus leaves picked at 25 DAS between Mt. Elgon and Kisii areas were insignificantly different. However, these differed significantly when compared with those contained in the leaves obtained from KU and Bureti (P<0.05). The samples analysed at 50 DAS had AA amounts which were different between all the study areas significantly (P<0.05). The results of analysis in A. cruentus during both the wet and the dry seasons showed that the leaves collected from Bureti at all DAS contained the highest amounts of AA except at 75 DAS during the wet season. The results recorded during the wet season indicate that A. cruentus leaves picked at 25 DAS from Mt. Elgon and Kisii had AA levels which were not different significantly between them but were different significantly from those present in the leaves harvested from Kenyatta University and Bureti (P<0.05). The mean amount of AA present in the leaves picked from Mt. Elgon at 50 DAS was significantly the lowest (P<0.05). In addition, A. cruentus leaves sampled from Kisii at the same age after sowing showed a significant difference in the mean AA content from those in the leaves sampled from Bureti and Mt. Elgon 92 (P<0.05). Considering the dry season, A. cruentus leaves obtained from Kenyatta University at 25 DAS had significantly the least mean AA value (P<0.05). The leaves from Bureti also had mean AA concentration which differed significantly from those contained in the leaves picked from the other areas (P<0.05). On the other hand, the levels of AA in the leaves between Mt. Elgon and Kisii were insignificantly different. The mean amount of AA in the samples harvested from KU at 50 DAS was significantly the lowest (P<0.05). Between the other study areas, no significant differences in the levels of AA were recorded during this time. The plants at 75 DAS in Bureti and Mt. Elgon had significantly different mean values of AA in their leaves (P<0.05). There were no significant differences between KU and Kisii as well as between Bureti and Kisii with respect to AA content of A. cruentus leaves. 4.5 Levels of beta-carotene and vitamins in amaranth grains from different areas Beta-carotene and vitamins in the ground samples of newly harvested A. hypochondriacus and A. cruentus grains obtained from the sampling areas were analysed as per the procedures highlighted in chapter 3. The amounts of β-carotene and each vitamin in the grains harvested during the wet and dry seasons are presented and discussed in the following sub-sections. 4.5.1 Beta-carotene contents in A. hypochondriacus and A. cruentus grains from different areas The β-carotene contents in A. hypochondriacus and A. cruentus grains harvested from the areas of study during the wet and the dry seasons are presented in Table 4.25. 93 Table 4. 25: Beta-carotene content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) Study areas A. hypochondriacus mg/100 g DW) ( X ±sd A. cruentus ( X ±sd mg/100 g DW) Wet season Dry season Wet season Dry season KU 4.627±0.002ab 4.631±0.002b 4.624±0.003b 4.617±0.018a Bureti 4.629±0.005b 4.633±0.002b 4.624±0.003b 4.625±0.002a Mt. Elgon 4.621±0.002a 4.619±0.002a 4.614±0.007a 4.618±0.003a Kisii 4.622±0.004a 4.629±0.007b 4.619±0.002ab 4.625±0.002a Bondo 4.621±0.007a 4.621±0.002a 4.614±0.008a 4.624±0.003a Meru 4.621±0.002a 4.622±0.001a 4.621±0.005ab 4.620±0.003a Embu 4.620±0.001a 4.620±0.003a 4.616±0.005ab 4.619±0.003a Mean values with the same superscript in the same column do not differ significantly (P>0.05) Table 4.25 indicates that the mean amounts of β-carotene in A. hypochondriacus grains analysed during the wet season between Bureti and KU were not significantly different but these differed from those contained in the grains from the other study areas (P<0.05). The levels present in A. hypochondriacus grains analysed during the dry season between KU, Bureti and Kisii versus Mt. Elgon, Bondo, Meru and Embu were significantly different (P<0.05). Amaranthus cruentus grains harvested during the wet season from Mt. Elgon and Bondo had least mean amounts of β-carotene that were significantly different from those noted in the grains sampled from KU and Bureti (P<0.05). Generally, the levels of β-carotene in amaranth grains were not significantly influenced by varied environmental conditions of the study areas. The mean amounts of β-carotene (mg/100 g DW) ranged from 4.619-4.633 and 4.6144.625 in A. hypochondriacus and A. cruentus grains respectively. These are comparable to the value of 4.6 mg/100 g DW reported earlier (Macrae et al., 1993). Thus, consumption of between 150-200 g of amaranth grains per day is sufficient to 94 provide vitamin A requirements of 210-630 μg/day or β-carotene of 2.52-7.56 mg/day (Institute of Medicine, 2000b). The amounts of β-carotene in the grains harvested from all the areas of study between the two species were not significantly different except between those obtained from Bureti during the dry season (P<0.05). The levels of β-carotene in the grains of both species were also not significanty different between the two seasons except A. hypochondriacus and A. cruentus grains obtained from KU and Kisii respectively. These differences could be as result of post-harvest handling of the samples (Kader, 2002, Rodriquez-Amaya and Kimura, 2004). 4.5.2 Alpha-tocopherol contents in A. hypochondriacus and A. cruentus grains from different areas The results obtained after the analysis of α-tocopherol in A. hypochondriacus and A. cruentus grains harvested from the study areas during the two seasons considered are presented in Table 4.26. Table 4. 26: Alpha-tocopherol content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) Study areas A. hypochondriacus mg/100 g DW) ( X ±sd A. cruentus ( X ±sd mg/100 g DW) Wet season Dry season Wet season Dry season KU 1.067±0.002a 1.068±0.003a 1.063±0.003a 1.058±0.002a Bureti 1.066±0.003a 1.067±0.004a 1.064±0.004a 1.062±0.001a Mt. Elgon 1.062±0.003a 1.065±0.003a 1.061±0.005a 1.059±0.005a Kisii 1.066±0.003a 1.067±0.004a 1.061±0.002a 1.060±0.005a Bondo 1.062±0.004a 1.066±0.002a 1.060±0.002a 1.063±0.008a Meru 1.062±0.003a 1.064±0.004a 1.059±0.003a 1.060±0.006a Embu 1.063±0.005a 1.065±0.005a 1.062±0.004a 1.055±0.002a Mean values with the same superscript in the same column do not differ significantly (P>0.05) 95 From Table 4.26, it is apparent that the study areas did not significantly influence the levels of α-tocopherol in the grains of both species. The mean values ranged from 1.062-1.068 mg in A. hypochondriacus grains and 1.055-1.064 mg in A. cruentus grains per 100 g (DWs). These values compare favourable with 1.030 mg/100 g reported in amaranth grain in an earlier study (Svirskis, 2003). The mean amounts in this study are also within the range of 2.97-15.65 mg/kg DW reported in amaranth grains in another study (Lehman et al., 2006). The amounts of α-tocopherol are higher than that found in many cereal grains (Svirskis, 2003; Muyonga et al., 2008). For instance, the levels reported in millet and barley in a study was 0.180 and 0.600 mg per 100 g (DW) respectively (Svirskis, 2003). However, they were lower than the amounts contained in peanut and rye, which were reported as 9.13 and 1.44 mg per 100 g (DWs) respectively (Pamplona-Roger, 2005). Consumption of whole grains has been associated with protection against cancer and cardio-vascular diseases due to high levels of antioxidants such as vitamin E (Pamplona-Roger, 2005). The amounts of α-tocopherol in grains harvested from the study areas between the two species were only significanly different (P<0.05) in the samples obtained from Kenyatta University and Embu signifying that they were not influenced by the species/cultivar variation. The levels of α-tocopherol in the grains of both species were also not affected by the seasons since the only significant differences (P<0.05) between the seasons were noted in A. cruentus grains from Kenyatta University and Embu. The observed differences could have resulted from post-harvest handling of the samples (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 96 4.5.3 Thiamin contents in A. hypochondriacus and A. cruentus grains from different areas Thiamin mean contents of thiamin in the samples of newly harvested grains from all the areas of study during the wet and dry seasons are presented in Table 4.27. Table 4. 27: Thiamin content content (mg/100 g DW) of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) Study areas A. hypochondriacus mg/100 g DW) ( X ±sd A. cruentus ( X ±sd mg/100 g DW) Wet season Dry season Wet season Dry season KU 0.158±0.004a 0.166±0.004a 0.158±0.003b Bureti 0.160±0.004a 0.162±0.002a 0.155±0.001ab 0.160±0.001a Mt. Elgon 0.157±0.002a 0.161±0.004a 0.152±0.003a Kisii 0.157±0.002a 0.163±0.003a 0.154±0.003ab 0.160±0.003a Bondo 0.157±0.002a 0.163±0.003a 0.155±0.001ab 0.160±0.002a Meru 0.156±0.003a 0.162±0.003a 0.152±0.002a 0.159±0.002a Embu 0.156±0.006a 0.161±0.004a 0.152±0.001a 0.155±0.004a 0.160±0.002a 0.157±0.003a Mean values with the same superscript in the same column do not differ significantly (P>0.05) The results (Table 4.27) displayed no significant differences in the levels of thiamin between all the study areas in A. hypochondriacus grains in both seasons as well as in A. cruentus grains in the dry season. In the wet season, the samples analysed from KU only differed with those from Meru and Embu significantly (P<0.05). Furthermore, mean thiamin contents of amaranth grains sampled from all the areas of study were not significantly different between the two species. It was also found out that they were not influenced largely by the seasonal changes. However, A. hypochondriacus grains harvested from Kisii, Bondo and Meru had significantly different thiamin levels between the wet and the dry seasons (P<0.05). As for A. cruentus, the grains obtained from Bureti, Bondo and Meru had mean thiamin amounts which differed 97 significantly between the two seasons (P<0.05). These differences could be due to post-harvest handling of the samples (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). The mean thiamin contents (mg/100 g DW) of A. hypochondriacus and A. cruentus grains were 0.156-0.166 and 0.152-0.160 respectively. Macrae et al. (1993) reported the mean thiamin value of 0.136 mg/100 DW in amaranth grain; this was comparable to what this study recorded. However, Muyonga et al. (2008) reported a significantly lower amount (P<0.05) of 0.10 mg/100 g. This may be attributed to factors like climatic conditions and agricultural management practices (Kader, 2002; Raju et al., 2007). 4.5.4 Riboflavin contents in A. hypochondriacus and A. cruentus grains from different areas Table 4.28 gives the mean levels of riboflavin in A. hypochondriacus and A. cruentus grain samples obtained from sampling areas during the wet and dry seasons. Table 4. 28: Riboflavin mean content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) Study areas A. hypochondriacus mg/100 g DW) ( X ±sd A. cruentus ( X ±sd mg/100 g DW) Wet season Dry season Wet season Dry season KU 0.279±0.004a 0.281±0.003a 0.276±0.003a 0.276±0.003a Bureti 0.275±0.001a 0.281±0.003a 0.275±0.005a 0.275±0.004a Mt. Elgon 0.277±0.002a 0.279±0.004a 0.273±0.003a 0.276±0.002a Kisii 0.282±0.003a 0.281±0.002a 0.277±0.004a 0.276±0.004a Bondo 0.285±0.013a 0.280±0.004a 0.275±0.002a 0.275±0.004a Meru 0.275±0.005a 0.278±0.005a 0.274±0.004a 0.274±0.003a Embu 0.275±0.004a 0.278±0.003a 0.277±0.001a 0.276±0.003a Mean values with the same superscript in the same column do not differ significantly (P>0.05) 98 The results (Table 4.28) indicate that the levels of riboflavin in the grains of both A. hypochondriacus and A. cruentus species were not significantly different between all the study areas during the two seasons. It was also observed that riboflavin amounts were not significantly different between the species in all the study areas. In addition, riboflavin content of the amaranth grain were insignificantly different between the two seasons apart from A. hypochondriacus sampled from Bureti (P<0.05) which might have been due to post-harvest handling of the samples (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). The mean amounts (mg/100 g DW) of riboflavin ranged from 0.275-0.285 in A. hypochondriacus grains and 0.273-0.277 in A. cruentus grains. These values are significantly higher (P<0.05) than 0.223 mg/100 g (DW) reported earlier (Svirskis, 2003). The mean levels of riboflavin in the grains of both species are comparable to that contained in barley (0.285 mg/100 g) and millet (0.290 mg/100 g) but higher than that found in corn, wheat, rice, peanut and wheat (Svirskis, 2003; Pamplona-Roger, 2005). 4.5.5 Pyridoxine contents in A. hypochondriacus and A. cruentus grains from different areas Table 4.29 gives the mean levels of pyridoxine in the samples of A. hypochondriacus and A. cruentus grains obtained from different areas during the wet and dry seasons. 99 Table 4. 29: Pyridoxine mean content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) Study areas A. hypochondriacus mg/100 g DW) ( X ±sd A. cruentus ( X ±sd mg/100 g DW) Wet season Dry season Wet season KU 0.227±0.002a 0.228±0.002b 0.229±0.002ab 0.228±0.003a Bureti 0.228±0.002a 0.227±0.001ab 0.230±0.002b 0.228±0.001a Mt. Elgon 0.224±0.002a 0.225±0.002ab 0.226±0.001a 0.227±0.002a Kisii 0.225±0.002a 0.225±0.002ab 0.226±0.002a 0.226±0.002a Bondo 0.226±0.002a 0.226±0.004ab 0.226±0.003a 0.225±0.002a Meru 0.224±0.003a 0.225±0.002ab 0.226±0.003a 0.227±0.002a Embu 0.223±0.004a 0.223±0.002a 0.225±0.001a 0.226±0.001a Dry season Mean values with the same superscript in the same column do not differ significantly (P>0.05) The results (Table 4.29) showed a significant difference in the mean pyridoxine contents of A. hypochondriacus existed only between KU and Embu areas during the dry season (P<0.05). With respect to A. cruentus, the grains obtained from Bureti during the wet season had pyridoxine mean amount that differed significantly (P<0.05) from those recorded in other areas apart from KU. It was also noted that the levels of pyridoxine in amaranth grain regardless of the area of study were not affected by the species/cultivar difference as well as the seasons. The mean pyridoxine contents (mg/100 g DW) of A. hypochondriacus and A. cruentus grains ranged from 0.223-0.228 and 0.225-0.229 respectively. These values compare favourably with the amount of 0.223 mg/ 100 g reported in an earlier study (Svirskis, 2003). The pyridoxine contents are also comparable to that of quinoa (0.223 mg/100 g) but higher than found in corn (0.055 mg/100 g) (Pamplona-Roger, 2005). 100 4.5.6 Ascorbic acid contents in A. hypochondriacus and A. cruentus grains from different areas The results of comparison of the levels of AA in A. hypochondriacus and A. cruentus grains harvested and analysed during both wet and dry seasons between the areas of study are presented in Table 4.30. Table 4. 30: Ascorbic acid content content of A. hypochondriacus and A. cruentus grains from different areas during wet and dry seasons (n = 3) Study areas A. hypochondriacus mg/100 g DW) ( X ±sd A. cruentus ( X ±sd mg/100 g DW) Wet season Dry season Wet season Dry season KU 4.303±0.050b 4.300±0.050b 4.200±0.020b 4.220±0.061a Bureti 4.277±0.050ab 4.343±0.023b 4.217±0.038b 4.240±0.010a Mt. Elgon 4.233±0.040ab 4.227±0.040a 4.200±0.044b 4.217±0.061a Kisii 4.240±0.036ab 4.307±0.025b 4.203±0.021b 4.227±0.042a Bondo 4.233±0.021ab 4.303±0.012b 4.173±0.015b 4.203±0.012a Meru 4.207±0.015a 4.230±0.040a 4.037±0.051a 4.197±0.012a Embu 4.200±0.090a 4.213±0.006a 4.183±0.065b 4.187±0.012a Mean values with the same superscript in the same column do not differ significantly (P>0.05) According to the results (Table 4.30), A. hypochondriacus grains harvested from KU during the wet season had the mean AA amount which was only significantly different from those present in the samples obtained from meru and Embu (P<0.05). With respect to the dry season, A. hypochondriacus grains sampled from KU, Bureti, Kisii and Bondo had mean amounts of AA which were insignificantly different between them but differed significantly when compared to those contained in the grains sampled from Mt. Elgon, Meru and Embu (P<0.05). Considering A. cruentus, it was observed that the grains harvested from Meru during the wet season had significantly the least mean AA amount (P<0.05). This trend shows that the AA levels 101 of amaranth grains were not affected by the area in which they were planted. Thus, the observed differences might have been caused by post-harvest factors (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). The mean levels (mg/100 g DW) of AA were 4.200-4.303 in A. hypochondriacus grains and 4.037-4.240 in A. cruentus. The amounts reported in the grains of both species compare well with what has been reported in other studies. Macrae et al. (1993) reported the AA mean value of 4.47 mg/100 g while Svirskis (2003) reported the mean value of 4.2 mg/100 g in amaranth grain. When the levels of AA in the grains were comapared between A. hypochondriacus and A. cruentus species, it was observed that those harvested during the wet season from KU, Bondo and Meru were different significantly (P<0.05). Similarly, significant differences in the mean amounts of AA were noted in the grains harvested from Bureti, Kisii, Bondo and Embu during the dry season (P<0.05). Comparison of mean amounts of AA in the grains between the two seasons, showed that they only differed significantly (P<0.05) in A. hypochondriacus and A. cruentus grains sampled from Bondo and Meru respectively. These differences may be attributed to postharvest handling of the samples (Kader, 2002; Rodriquez-Amaya and Kimura, 2004). 102 CHAPTER 5 5.0 CONCLUSIONS AND RECCOMENDATIONS 5.1 Conclusions With respect to the results obtained from this study, the following conclusions can be made:i. Beta-carotene and vitamin contents in the leaves between A. hypochondriacus and A. cruentus were significantly different (P<0.05) except thiamin. ii. The levels of β-carotene and α-tocopherol in amaranth leaves increased significantly (P<0.05) with the plant’s age while those of thiamin, riboflavin, pyridoxine and AA declined significantly (P<0.05). iii. With respect to the study areas, significant differences (P<0.05) in the amounts of β-carotene and vitamins except thiamin in the leaves of both species were noted. iv. Beta-carotene and vitamin amounts in the leaves of both species differed significantly (P<0.05) between the wet and the dry season that of thiamin. v. The grains of A. hypochondriacus had significantly higher mean amount of AA than A. cruentus (P<0.05). 5.2 Recommendations 5.2.1 Recommendations from this study i. Amaranth leaves and grains contain appreciable amounts of β-carotene, which can adequately meet the vitamin requirements of all individuals. ii. Grain amaranth leaves should be consumed at plant’s tender age of between 25 and 50 DAS since they are high in WSVs although low in FSVs as 103 compared to advanced ages. This is because the amaranth plant accumulates potentially harmful nitrates in the leaves as it grows. iii. Efforts to market and promote amaranth should be undertaken based on their vitamin content. This can be done through development of products that are of greater nutrional values and acceptable higher tastes. iv. Because the use of food supplements and food fortification to mitigate vitamin deficiency incidences amongst the vulnerable groups are costly undertakings and beyond the reach of the poor majority, the public should be encouraged to use leafy vegetables like amaranth. 5.5.2 Recommendations for further study i. Determination of the bioavailability of vitamins from leaves and grains of A. hypochondriacus and A. cruentus should be done. ii. Studies on the effects of specific pre-harvest factors on vitamin contents of amaranth such as the soil type, soil pH and farming practices should be done. iii. The effects of processing on the levels of vitamins in A. hypochondriacus and A. cruentus leaves and grains should be evaluated. iv. Analysis of vitamins in the stem and the roots should be undertaken in order to ascertain the significance of the whole plant in the provision of vitamins. v. Evaluation of other micronutrients as well as macronutrients in amaranth should be done. 104 REFERENCES Abd El-Baky, H., El Baz, F. K. and El-Baroty, G.S. (2008). Evaluation of marine alga ulva lactuca L. as a source of natural preservative ingredient. EJEAFChe.7: 33533367. Akubugwo, I.E., Obasi, N.A., Chinyere, G.C. and Ugbogu, A.E (2007). Nutritional and chemical value of Amaranthus hybridus L. leaves from Afikpo, Nigeria. Afr. J Biotech. 6: 2833-2839. Ali, B., Khandaker, L. and Oba, S. (2009). Comparative study on functional components, antioxidant activity and color parameters of selected colored leafy vegetables as affected by photoperiods. J. Food, Agric. and Env. 7: 392-398. Allen, L.H. (2003). Interventions for Micronutrient Deficiency Control in Developing Countries: Past, Present and Future. J. Nutr. 133. Pp 1-15. Amidzic,R., Boric, J.B., Cudina, O. and Vladimirov, S. (2005). RP-HPLC Determination of vitamins B1, B3, B6, folic acid and B12 in multivitamin tablets. J. Serb. Chem. Soc. 70: 1229-1235. Anyakora, C., Afolami, I., Teddy Ehianeta, T. and Onwumere, F. (2008). HPLC analysis of nicotinamide, pyridoxine, riboflavin and thiamin in some selected food products in Nigeria. Afr. J. Pharm. Pharmacol. 2: 29-36. AOAC. (1990). Official methods of analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, Arlington VA. Pp 1058-1059. Armstrong, G.A. and Hearst, J.E. (1996). Carotenoids 2: Genetics and molecular biology of carotenoid pigment biosynthesis. Faseb J. 10: 228-237. Arroyave, G., Mejia, L.A. and Aguilar, J.R. (1981). The effect of vitamin A fortification of sugar on the serum vitamin A levels of preschool Guatemalan children: a longitudinal evaluation. J. Nutr. 34: 41-49. Azevedo, C.H., Rodriguez-Amaya and Delia B. (2005). Carotenoid composition of kale as influenced by maturity, season and minimal processing. J. Sci. Food Agr. 85: 591-597. Ball, F.M. and George (2006). Riboflavin in Foods, Analysis, Bioavailability, and Stability. Taylor and Francis Group, New York. Pp 168-175. Barr, S.I. (2006). Applications of Dietary Intakes in dietary assessment and planning. Appl. Physiol. Nutr. Metab. 31: 66-73. Basu, T.K. and Schorah, C.J. (1982). Vitamin C and Disease. AVI publishing Co., Westport Connecticut. Pp 38; 48-59. 105 Batt, R.M., McLean, L., Rutgers, H.C. and Hall, E.J. (2008). Validation of a radioassay for the determination of serum folate and cobalamin concentrations in dogs. J. of Small Animal Practice. 32: 221-224. Bergquist, S.Å.M., Gertsson, U. and Olsson, M. (2007). Bioactive compounds and visual quality of baby spinach-Changes during plant growth and storage. Acta Hort. 744: 343- 347. Berkelaar, D. and Alemu, J. (2006). Grain amaranth. Christian Reformed World Relief, Nairobi. Pp 2-3. Blanco, M., Coello, H. and Iturriaga (2004). Simultaneous spectrophotometric determination of fat-soluble vitamins in multivitamin pharmaceutical preparations. J.Food analysis. 351: 315-319. Bloss, E., Wainaina, F. and Bailey, R.C. (2004). Prevalence and predictors of underweight, stunting, and wasting among children aged five and under in Western Kenya. J. tropical pediatr. 50: 260-270. Bowman, B.A. and Russell, R.M. (2006). Present Knowledge in Nutrition. ILSI Press, Washington, DC. Pp 273-279. Brubacher, G., Muller-mullot, W. and Southgate, D.A.T. (1985). Methods for the determination of vitamins in Foods. Elsevier Applied Science Publishers Ltd., London. Pp 34-39; 98-105; 120-127; 130-138. Byers, T. and Mouchawar, J. (1998). Antioxidants and cancer prevention in 1997. Springer, Milan. Pp 29-40. Casella, L., Gullotti, M., Marchesini, A. and Petrarulo, M. (2006). Rapid Enzymatic method for vitamin C Assay in Fruits and Vegetables using peroxidase. J. food sci. 54: 374-375. Caulfield, L.E., de Onis, M., Blossner, M., and Black, R.E. (2004). Undernutrition as underlying cause of child deaths associated with diarrhea, pneumonia, malaria, and measles. Am. J clin nutr. 80: 193-198. CBS (2002). Economic survey. Ministry of planning and National development. Government printers, Nairobi. CBS (2006). Economic survey. Ministry of planning and National development. Government printers, Nairobi. Chenchong, C. (1997). Evaluation of six Amaranth varieties. Kasetsart University, Bangkok. Pp 1-7. Colmenares De Ruiz, A.S. and Bressani, R. (1990). Effect of Germination on the Chemical Composition of Amaranth Grain. Cereal chem. 67: 519-522. 106 Combs, G.F. (1992). The Vitamins: Fundamental aspects in nutrition and health. Academic press ltd, New York. Pp 61-76; 83; 121-147; 179-198; 225-244; 253-265; 271-284; 311-324; 377-388. Courtsoudis, A. (2001). The Relationship between Vitamin A Deficiency and HIV Infection: Review of Scientific Studies. Food and Nutrition Bulletin. 22: 3. Daniel, W.W. (1999). Biostatistics: A Foundation for analysis in the health sciences. John Wiley and Sons, New York. Pp 9-12. Davey, M. W., Van Montagu, M., Inzé, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I. J. J., Strain, J. J., Favell, D., and Fletcher, J. (2000). Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food and Agr. 80: 825-860. Davies, M.B., John, A. and partridge, D.A. (1991). Vitamin C: Its chemistry and biochemistry. Royal society of chemistry, Cambridge, Pp 80-81; 98-101; 116. Deutsch, J. C. (2000). Dehydroascorbic acid. J. Chrom. A. 881: 299-307. Di Mascio, P., Kaiser, S. and Sies, H. (1989). Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274: 532538. Dodok, L., Modhir, A.A., Halasova, G., Pola, I. and Hozova, B. (2006). Importance and utilization of amaranth in food industry. Faculty of Chemical Technology and Research Institute of Foodstuffs Industry and Services, Bratislava. Pp 378-381. Elaine, R.T. (2006). Facts about Riboflavin. Food Science and Human Nutrition Department, Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, Gainesville. Pp 1-2. Ettyang, G.A., Van Marken, W.D., Oloo, A. and Saris, W.M. (2003). Serum retinol, Iron status and body composition of Lactating women in Nandi, Kenya. Ann. Nutr. Metab. 47: 276-283. FAO/WHO. (2002). Thiamin, riboflavin, niacin, vitamin B6, pantothenic acid and biotin.In: Human Vitamin and Mineral Requirements. Report of a Joint FAO/WHO Expert Consultation. FAO, Rome. Pp 27-30. FAO (2005). Nutrition country profile: Republic of Kenya. Rome. Pp 3-4; 9-15; 2127; 31-32. Fidanza, F. (1991). Nutritional status Assessment. Chapman and Hall, London. Pp 210-211. Fukuwatari, T. and Shibata, K. (2008). Urinary water-soluble vitamins and their metabolite contents as nutritional markers for evaluating vitamin intakes in young Japanese women. J. Nutr. Sci. Vitaminol. 54: 223-239. 107 Goldman, I.L., Kader, A.A. and Heintz., C. (1999). Influence of production, handling, and storage on phytonutrient content of foods. J. Nutr. Rev. 57: 46-52. Gropper, S.S., Smith, J.L., and Groff, J.L. (2009). Riboflavin in Advanced Nutrition and Human Metabolism. CENGAG Learning, Wadsworth. Pp 329-333. Hillan, J. (2006). Facts about vitamin E. Department of Family, Youth and Community Sciences, Cooperative, Florida. Pp 1-5. Horvath, C. (1980). High-performance liquid chromatography: advances and perspectives. Academic press, New York. Pp 62-63; 76-77. Hotz, C. (2007). An agricultural strategy to contribute to improved micronutrient adequacy among poor, rural populations. Harvest Plus International Food Policy Research Institute,Washington. Pp 41-43. Hui, Y.H. (1992). Encyclopedia of Food Science and Technology. John Wiley and sons, New York. Pp 301-305; 2736-2737. Ihekoronye, A.I. and Ngoddy, P.O. (1985). Intergrated Food Science and Technology for the Tropics. Macmillan, Hong Kong. Pp 86-89. Institute of Medicine. (1998). Dietary Reference Intakes for thiamin, riboflavin, niacin, vitamin b6, folate, vitamin B12, pantothenic acid, biotin, and choline. National Academies Press, Washington, D.C. Pp 17-23; 66-79; 105-112; 165-179; 186-187. Institute of Medicine. (2000a). Dietary Reference Intakes: applications in dietary assessment. National Academies Press, Washington, D.C. Pp 325- 400. Institute of Medicine. (2000b). Dietary Reference Intakes for vitamin C, vitamin E, selenium and carotenoids. National Academies Press, Washington, D.C. Pp 134; 187; 226; 358- 360; 507. Jansen, A.A., Horelli, H.T. and Quinn, V.J. (1987). Food and Nutrition in Kenya. Department of community, faculty of medicine, college of Health sciences, University of Nairobi, Nairobi. Pp 17; 436-437. Jedlicka, A. and Klimes, J. (2004). Determination of water- and Fat-soluble vitamins in different matrices using High-Performance Liquid Chromatography. Department of pharmaceutical chemistry and drug control, Charles University, Prague. Pp 204-220. Kader, A.A. (2002). Pre- and post-harvest factors affecting fresh produce quality, nutritional value, and implications for Human Health. Department pomology, University of California, Davis. Pp 109-117. Kauffmann, C. S and Weber, L. Ε. (1988). Grain amaranth: Advances in new crops: Proceedings of the First National Symposium of New Crops: Research, Development and Economics. Timber Press, Oregon. Pp 127-139. 108 KDHS (2004). Kenya Demographic and Health Survey 2003. CBS/ MOH/ KEMRI/ NCPD/ ORC MACRO, Nairobi. Pp 2-3. Khader, V. and Rama, S. (2003). Effects of maturity on macro-mineral content of selected leafy vegetables. Asia pacific J Clin Nutr. 12: 45-49. Kim, H.K., Kim, M.J. and Shin, D.H. (2006). Antioxidative and anti-diabetic effects of amaranth in streptozotocin-induced diabetic rats. J. cell biochem. 24: 195-199. Kirk, S.R. and Sawyer, R. (1991). Composition and Analysis of Foods. Longman, London. Pp 243-245. Krystulovic, A.M. and Brown, P.R. (1982). Reversed- phase high-performance liquid chromatography: Theory, practice, and biomedical applications. John Wiley and sons, New York. Pp 243-245; 248-252. Kunert, K. J. and Ederer, M. (1985). Leaf aging and lipid peroxidation: The role of the antioxidants vitamin C and E. Physiologia Plantarum. 65: 85-88. Lara, N., Mejia, A. and Cangas, A. (2007). Popped amaranth grain and its products breakfast cereal and crunchy bars: Popping process, nutritive value and shelf life. Department of nutrition and quality, Ecuador. Pp 1-4. Lehmann, J.L., Putnam, D.H. and Qureshi, A.A. (2006). Vitamin E isomers in grain amaranths (Amaranthus spp). J. Lipids. 29: 177-181. Lough, W.J. and Wainer, I.W. (1996). High performance liquid chromatography: fundamental principles and practice. Blackie Academic and professional, London. Pp 5-6; 16; 44; 61-62; 64; 70-72; 74-76. Luo, X., Chen, B., Ding, L., Tang, F. and Yao, S. (2006). HPLC-ESI-MS analysis of Vitamin B12 in food products and in multivitamins-multimineral tablets. State Key Laboratory of Chemo/Biosensing and Chemometrics and PR China Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Hunan Normal University, Changsha. Pp 185-189. Macrae, R., Robinson, R.K. and Sadler, M.J. (1993). Encyclopedia of Food science , Food technology and nutrition. Academic press, London. Pp 4816-4820; 4822-4826. Madakadze, R. M. and Kwaramba, J. (2004). Effect of Pre-harvest Factors on the Quality of Vegetables produced in the Tropics. Kluwer Academic Publishers, Harare. Pp 1-36. Makus, D., Zibilske, L. and Lester, G. (2006). Effect of light intensity, soil type, Lithium addition on spinach and mustard greens leaf constituents. Subtropical plant sciences. 58: 36-43. Manuche, W.E. (2003). Vitamin A content in the traditionally preserved indigenous vegetables: A case study of Koru location, Nyando district. M.Sc Thesis, Kenyatta University, Nairobi. Pp 55-56. 109 Maqbool, A. and Stallings, V.A. (2008). Update on fat-soluble vitamins in cystic fibrosis. Curr Opin Pulm Med. 14: 574–581. Martirosyan, D.M., Miroshnichenko, L.A., Kulakova, S.N., Pokojeva, A.V. and Zoloedov, V.I. (2007). Amaranth oil application for coronary heart disease and hypertension. J. lipids in health and disease. 6: 1-11. McCormick, D.B. (1994). Riboflavin. Lea & Febiger, Philadelphia. Pp 366-375. McCormick, D.B and Greene, H.L. (1994). Vitamins. WB Saunders, Philadelphia. Pp1275-1316. McCormick, D.B. (2006).Vitamin B6. International Life Sciences Institute, Washington, D.C. Pp 270-273. Mnkeni, A.P., Masika, P. and MMaphaha, M. (2007). Nutritional quality of vegetable and seed from different accessions of Amaranthus in South Africa. Agricultural Rural Development Research Institute, University of Fort Hare. Pp 377-380. Modi, A.T. (2007). Growth temperature and plant age influence on nutritional quality of Amaranthus leaves and seed germination capacity. University of KwaZulu-Natal Press, Pretoria. Pp 369-374. MOH (2006). Kenya National Guidelines on Nutrition and HIV/ AIDS. Pp 1-12. Morales, E., Lembcke, J. and Graham, G.G. (1988). Nutritional value for young children of grain amaranth and maize amaranth mixture: effect of processing. J. Nutr. 118: 75-78. Moriyama, M. and Oba, K. (2004). Sprouts as antioxidant food resources and young peoples’ taste for them. J. Biofactors 21: 241-247. Munene, R.M., Adala, H.S., Masinde, M.S. and Rana, F.S. (2003). Vitamin A deficiency among Kenyan children as detected by conjuctival impression cytology. East Afr med j. 9: 476-479. Murthy, K.N., Vanitha, A., Rajesha, M., Swamy, M., Sowmya, P. R. and Ravishankar A. G. (2005). In vivo antioxidant activity of carotenoids from Dunaliella salina-a green microalga. LifeSci. 76: 1381-1390. Muyonga, J.H., Nabakabya, D., Nakimbudgwe, D.N. and Masinde, D. (2008). Efforts to promote Amaranth production and consumption in Uganda to fight malnutrition. Department of Food Science and Technology, Makerere University, Kampala. Pp 1-9. Nagy, S. (1980). Vitamin C contents of citrus fruit and their products. J. Agr. and Food chem. 28: 8-18. NASCOP. (2006). Kenyan National Guidelines on Nutrition and HIV/ AIDs. MOH, Nairobi. Pp 4-8; 12-14. 110 Ng’ang’a, M., Ohiokpehai, O., Muasya, R., Omani, E. and Sanginga, N. (2008). Effect of intercropping on nutritional value of grain amaranth and soybean among HIV/AIDS affected rural households of western Kenya. Moi University, Eldoret. Pp 237-238; 241. Nyalili, M. (1995). Determination of Ascorbic Acid in kenyan fruits and vegetables by Pulse Anodic stripping Voltametry (DPASV) and High Performance Chromatography (HPLC). Kenyatta University, Nairobi. Pp 90-97. Nyambaka, H.N. (1988). High-performance liquid-chromatography (HPLC) determination of four vitamins in some Kenyan Foods. M.Sc Thesis, Kenyatta university, Nairobi. Pp 92-94. O’Brien, G.K. and Price, M.L. (1983). Amaranth: Grain and Vegetable Types. Echo Technical Note, Florida. Pp 1-14. Ofitserov, N.E. (2001). Amaranth: Perspective raw material for Food-processing and Pharmaceutical Industry. Chemistry and computational simulation, Tatarstan. Pp 1-4. Ofori, G., Oduro, I., Ellis, W.O. and Dapaah, K.H. (2009). Assessment of vitamin A content and sensory attributes of new sweet potato (Ipomoea batatas) genotypes in Ghana. Afr. J. Food Sci. 3: 184-192. Ogunlesi, M., Okiei, W., Azeez, L., Obakachi, V., Osunsanmi, M. and Nkenchor, G. (2010). Vitamin C contents of tropical Vegetables and Foods determined by Voltammetric and Titrimetric methods and their relevance to the Medicinal uses of the plants. Int. J. Electrochem. Sci. 5: 105-115. Onyango, C.M., Shibairo, S.I., Imungi, J.K. and Harbinson, J. (2008). The PhysicoChemical Characteristics and Some Nutritional Values of Vegetable Amaranth Sold in Nairobi-Kenya. Ecology of Food and Nutrition, Nairobi. Pp, 382-398. Onyango, C.M. (2010). Preharvest and Postharvest Factors Affecting Yield and Nutrient Contents of Vegetable Amaranth Horticultural Supply Chains Group of Wageningen University, Wageningen. Pp 45-53. Padayatty, S.J., Riordan, H.D., Hewitt, S.M., Katz, A., Hoffer, L.J. and Mark L. (2006). Intravenously administered vitamin C as cancer therapy: three cases. Med. Assoc. J. 174: 956-957. Paiva, S.A. and Russel, R.M. (1999). Beta-carotene and other carotenoids as antioxidants. J. Am. Coll. Nutr. 18: 426-433. Pamplona-Roger, G.D. (2005). Encyclopedia of Foods and their Healing power. Editorial Safeliz, Madrid. Pp 27-29; 60-69; 92-111; 389-397. Platkin, C.S. (2008). Amaranth: Grains you might have never heard of or tried, but should. Diet Detective, Texas. Pp 1-2. 111 Ponder, E.L., Fried, B., and Sherma, J. (2004). Thin-layer chromatographic analysis of hydrophilic vitamins in standards and from helisoma trivolvis snails. Departments of Chemistry and Biology, Lafayette College, Easton, Pennsylvania. Pp 70-74. Powers, J.H. (2003). Riboflavin and health. Am J. Clin. Nutr. 77: 1352-1360. Price, M. and Berkelaar, D. (2006). Grain Amaranth. ECHO Development Notes, Florida. Pp 1-8. Prior, R.L. and Cao, G. (2000). Antioxidant phytochemicals in fruits and vegetables; diet and health implications. Hortscience. 35: 588-592. Qian, H. and Sheng, M. (1998). Simultaneous determination of fat-soluble vitamins A, D, E and pro-vitamin D2 in animal feeds by one-step extraction and highperformance liquid chromatography analysis. J. chromatography. 825: 127-133. Quebedeaux, B. and Eisa, H.M. (1990). Horticulture and human health. Contributions of fruits and vegetables. Hortscience. 25: 1473-1732. Raju, M., Varakumar, S., Lakshminanarayana, R., Krishnakantha, T.P. and Baskaran, V. (2007). Carotenoid composition and vitamin A activity of medicinally important green leafy vegetables. Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Cheluvamba Mansion and Department of Biochemistry, Sri Venkateswara University, New Delhi. Pp 1-8. Reiss, C. (1993). Measuring the Amount of Ascorbic Acid in Cabbage. Division of Biological Sciences, Cornell University, New York. Pp 85-96. Rioba, J., Sheikh, M. and Stevens, D. (2000). A report of Nutritional survey in North and West Wajir District in North Eastern Kenya. MOH, Nairobi. Pp 14-15. Rodriquez-Amaya, D.B. (1996). Nature and distribution of carotenoids in foods. Elsevier. Science Publishers, Amsterdam. Pp 547-589. Rodriquez-Amaya, D.B. (1997). Carotenoids and food preparation: The retention of provitamin A carotenoids in prepared, processed and stored foods. Departmento deciencias de Alimentos, Campinas. Pp 2; 5-22; 32. Rodriquez-Amaya, D.B. and Kimura, M. (2004). Harvest plus Handbook for Carotenoid Analysis. International Food Policy Research Institute (IFPRI) and International Center for Tropical Agriculture (CIAT), Washington. Pp 3-4. ROK (2009). Review of of the Weather in Kenya. Ministry of Environmental and Mineral Resources Kenya Meteorological Department, Nairobi. Pp 1-3. Roncone, D.P. (2006). Xerophthalmia secondary to alcohol-induced malnutrition. Optometry. 77: 124-133. 112 Rutkowski, M. and Grzegorczyk, K. (2007). Modifications of spectrophotometric methods for antioxidative vitamins determination convenient in analytic practice. Medical University of Lodz. Pp 17-18; 23-24. Said, H.M. and Mohammed, Z.M. (2006). Intestinal absorption of water-soluble vitamins: an update. Curr. Opin. Gastroenterol. 22: 140-146. Saushkina, A. S. and Karpenko, V. A. (2005). Developing Methods for the quantitativeAnalysis of Olazol preparation. Pharm. Chem. J. 39: 54-56. Schorah, C.J. (1998). Vitamin C and gastric cancer prevention. Springer, Milan. Pp 41-49. Schwartz, J., Odukoya, O., Stoufi, E. and Shklar, G. (1985). Alpha tocopherol alters the distribution of langerhans cells in DMBA-treated Hamster cheek pouch epithelium. J. Dent. Res. 64: 117-121. Shewfelt, R. (1990). Sources of variation in the nutrient content of agricultural commodities from the farms to the consumers. J. Food. Qual. 13: 37-54. Shklar, G. (1982). Inhibition of Oral Mucosal Carcinogenesis by Vitamin E. J Natl Cancer Inst. 68: 791-797. Simopoulos, A.P. (2004). Omega-3 Fatty Acids and Antioxidants in Edible Wild Plants. Biol Res. 37: 263-277. Simpson, K.L. (1983). Relative value of carotenoids as precursors of vitamin A. Proc. Nutr. Soc. 42: 7-17. Sizer, Sienkiewicz, F. and Whitney, E. (2008). Nutrition: Concepts and Controversies. Thomson Wadsworth. Pp 221-235. Southon, S. (2000). Increased fruit and vegetable consumption within the EU: potential health benefits. Food Res. Intl. 33: 211-217. Stephens, J.M. (2009). Amaranth- Amaranthus spp. IFAS, University of Florida, Florida. Pp 1-2. Street, D.A., Comstock, G.W., Salkeld, R.M., Schuep, W. and Klag, M.J. (1994). Serum antioxidants and myocardial infection: are low levels of carotenoids and βtocopherol risk factors for myocardial infection? Circulation. 90: 1154-1161. Svirskis, A. (2003). Investigation of amaranth cultivation and utilisation in Luthuania. Siauliai University. Pp 253-262. Takita, Y., Ichimiya, M., Hamamoto, Y. and Muto, M. (2006). A case of carotenemia associated with ingestion of nutrient supplements. J. Dermatol. 33: 132-134. Underwood and Barbara, A. (2004).Vitamin A Deficiency Disorders: International efforts to control a preventable Pox. J. Nutr. 134: 231-236. 113 UNICEF (2008). UNICEF Humanitarian Action: Kenya in 2008. New York. Pp 1-4. Wargovich, M.J. (2000). Anticancer properties of fruits and vegetables. Hortscience. 35: 573-575. Weinberger, K. and Msuya, J. (2004). Indigenous vegetables in Tanzania: significance and prospects. AVRDC publisher, Dar es Salaam. Pp 9-10; 12; 65. Weston, L.A. and Barth, M.M. (1997). Preharvest factors affecting postharvest quality of vegetables. Hortscience. 32: 812-816. Wilson, K. and Walker, J. (1994). Practical Biochemistry; Principles and techniques. Cambridge university press, Great Britain. Pp 480-497. Yadav, S.K., Sengal, S. (1995). Effect of home processing on ascorbic acid and betacarotene content of spinach (Spinacea oleracia) and amaranth (Amaranth tricolor). Plant Foods Hum. Nutr. 47: 125-131. Yarger, L. (2008). Amaranth: Grain and Vegetable Types. Echo Technical Note, Florida. Pp 1-14. Zhao, B., Tham, S., Lu, J., Lai, M.H., Lee, K.H. and Moochhala, S.M. (2004). Simultaneous Determination of vitamins C, E and Beta-Carotene in Human Plasma by HPLC with Photodiode-Array Detection. Defence Medical and Environmental Research Institute, Singapore. Pp 1-5. Zeba, A.N., Sorgho, H. and Rouamba, N. (2008). Major reduction of malaria morbidity with combined vitamin A and zinc supplementation in young children in Burkina Faso: a randomized double blind trial. Nutr J. 7: 7-12. Zempleni, J. and Galloway, J.R. and McCormick, D.B. (1996). Pharmacokinetics of orally and intravenously administered riboflavin in healthy humans. Am J Clin Nutr. 63: 54-66. 114 APPENDICES APPENDIX I: Thiamin calibration curve Peak area Thiamin calibration curve 400000 y = 325226x - 872.94 2 R = 0.9995 300000 200000 100000 0 -100000 0 0.5 1 1.5 Concentration (ppm) APPENDIX II: Riboflavin calibration curve Riboflavin calibration curve 600000 y = 240637x - 4803.5 R2 = 0.9996 Peak area 500000 400000 300000 200000 100000 0 -100000 0 0.5 1 1.5 Concentration (ppm) 2 2.5 115 APPENDIX III: Pyridoxine calibration curve pyridoxine calibration curve 2500000 Peak area 2000000 y = 2E+06x - 37319 R2 = 0.9982 1500000 1000000 500000 0 -500000 0 0.5 1 1.5 Concentration (ppm) APPENDIX IV: Beta-carotene spectrophotometric calibration curve Absorbance Beta-carotene spectrophotometric curve y = 0.0758x + 0.0019 R2 = 0.9998 0.8 0.6 0.4 0.2 0 0 5 10 Concentration (ppm) 15 116 APPENDIX V: Beta-carotene HPLC calibration curve Peak area HPLC calibration curve for beta-carotene y = 39202x + 2892.5 R2 = 0.9976 600000 400000 200000 0 0 5 10 15 Concentration (ppm) APPENDIX V: Alpha-tocopherol spectrophotometric calibration curve Absorbance Alpha-Tocopherol calibration curve 0.4 0.3 0.2 y = 0.0301x - 0.0019 R2 = 0.9999 0.1 0 -0.1 0 5 10 Concentration (ppm) 15 117 APPENDIX VII: Alpha-tocopherol HPLC calibration curve HPLC Alpha-tocopherol calibration curve y = 84413x + 701.8 1000000 Peak area 2 R = 0.998 800000 600000 400000 200000 0 0 5 10 Conce ntration (ppm) 15 118 APPENDIX VIII: Chromatogram of β-carotene standard 119 APPENDIX IX: HPLC profile of β-carotene and α-tocopherol in amaranth grain 1- β- Carotene; 2- α-Tocopherol. Peaks not designated by numbers were unidentifie 120 APPENDIX X: Chromatogram of thiamin standard 121 APPENDIX XI: HPLC profile of B vitamins in amaranth grain sample 1- Thiamin; 2-Riboflavin; 3- Pyridoxine. Peaks not designated by numbers were unidentified 122 APPENDIX XII: HPLC profile of B vitamins in amaranth leaf sample 1- Thiamin; 2-Riboflavin; 3- Pyridoxine. Peaks not designated by numbers were unidentified 123 APPENDIX XIII: DRI of vitamins: Estimated Average Requirements per day for Groups Life stage group children Age Vit A (µg) Vit C (mg) Vit E (mg) Thiamin (mg) Riboflavin (mg) Vit.B6 (mg) 1-3 210 13 5 0.4 0.4 0.4 4-8 275 22 6 0.5 0.5 0.5 males 9-13 445 39 9 0.7 0.8 0.8 14-18 630 63 12 1.0 1.1 1.1 19-30 625 75 12 1.0 1.1 1.1 31-50 625 75 12 1.0 1.1 1.1 51-70 625 75 12 1.0 1.1 1.4 >70 625 75 12 1.0 1.1 1.4 Females 9-13 420 39 9 0.7 0.8 0.8 14-18 485 56 12 0.9 0.9 1.0 19-30 500 60 12 0.9 0.9 1.1 31-50 500 60 12 0.9 0.9 1.1 51-70 500 60 12 0.9 0.9 1.3 >70 500 60 12 0.9 0.9 1.3 pregnancy 14-18 530 66 12 1.2 1.2 1.6 19-30 550 70 12 1.2 1.2 1.6 31-50 550 70 12 1.2 1.2 1.6 Lactation 14-18 885 96 16 1.2 1.3 1.7 19-30 900 100 16 1.2 1.3 1.7 31-50 900 100 16 1.2 1.3 1.7 Sources: Institue of Medicine (1998); Institute of Medicine (2000b); Institute of Medicine (2001)