Download determination of levels of some vitamins in grain amaranth leaves

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
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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)