BIOACTIVITY OF FAMINE FOOD PLANTS FROM THE

FAMILY: AMARANTHACEAE

Alveera Singh

Submitted in fulfillment for the Degree of Master of Technology (Biotechnology) in the

Department of Biotechnology and Food Technology, Durban University of Technology, D urban,

South Africa

Supervisor: Prof. B. Odhav

Co-Supervisor: Dr. L. Reddy

Submitted for Examination: February 2009

 REFERENCE DECLARATION IN RESPECT OF A
MASTER’S DISSERTATION
I, Alveera Singh (Student Number: 20201901), Prof. Bharti Odhav and Dr. L. Reddy do hereby
declare that in respect of the following dissertation:
BIOACTIVITY OF FAMINE FOOD PLANTS FROM THE FAMILY:
AMARANTHACEAE

1. As far as we can ascertain:

(a) No other similar dissertation exists;
(b) The only similar dissertation (s) that exists is/are referenced in my
dissertation as follows:

________________________________________________________________________
________________________________________________________________________

2. All references as detailed in the dissertation are complete in terms of all personal
communications engaged in and public works consulted.

A. Singh March 2009

Student

B. Odhav March 2009

Supervisor

L. Reddy March 2009

Co-Supervisor
 ACKNOWLEDGEMENTS

I would like to extend my sincere appreciation and heartfelt gratitude to:

My supervisor, Professor Bharti Odhav, Department of Biotechnology and Food Technology,

Durban University of Technology, for her remarkable insight, constructive criticism, expert
guidance and support, time and energy, patience, invaluable assistance during the course of my
degree, and for her motivation;

Professor H. Baijnath, School of Botany and Zoology, University of Kwa-Zulu Natal, for his
assistance, time and effort, and for obtaining plant material during the course of this project;

Dr L. Reddy, Department of Biotechnology and Food Technology, Durban University of

Technology, for her contribution towards this project;

The National Research Foundation (South Africa) for funding this project;

Miss Z. Harrichandpersadh and Mr. K. Devchand for their assistance with the nutritional
analysis;

Miss Gayaram, Sykes and lab assistants at the Medical Research Institute (MRC) for their
assistance for the mosquito analysis;

Alex Stuart and Council for Scientific and Industrial Research (Durban) for their assistance with
mineral analysis;

My parents (Veenash and Andhira) for their love, encouragement, support, tolerance and
guidance throughout this period in my life;

My brother, sister and uncle (Aveen, Arisha and Bejai) for their, time, effort and motivation;

Miss V. Hurinathan, Dr. U.S. Akula, Dr Kundasamy, Mr. J.J. Mellem, Mr. V, Mohanlall and
Mrs. L. Govender for their assistance, guidance, friendship and tolerance;
The Barmanandh family for their encouragement and guidance;

Miss F. Peer, Mrs. P. Naicker, Mr. P. Reddy, Mrs. K. Mellem for their friendship;

To GOD for listening to my prayers and guiding me along the correct path;

My dogs Roxy, Bonzai, Bobby, the late Baloo and my cat Ginger for their playfulness and taking
my mind of the stresses of the day.
 CONTENTS

Page

REFERENCE DECLARATION IN RESPECT OF A MASTER’S DISSERTATION i

ACKNOWLEDGEMENTS ii
CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES x
LIST OF APPENDICES xii
LIST OF ABBREVIATIONS xiii
ABSTRACT xiv

CHAPTER ONE: STUDY RATIONALE AND OBJECTIVES 1

CHAPTER TWO: LITERATURE REVIEW 3

2.1 Wild plants and Famine Foods 3

2.1.1 Introduction 3
2.1.2 Categories of Wild Food Plants 5
2.1.3 Usefulness of wild foods 6
2.1.3.1 Agricultural Importance 7
2.1.3.2 Medicinal importance 8

2.2 Amaranthaceae Family 10

2.2.1 Achyranthes aspera 10
2.2.2 Alternanthera sessilis 12
2.2.3 Guilleminea densa 14

2.3 Nutritional and Biological Properties 15

2.3.1 Nutritional evaluation 15
2.3.1.1 Macronutrients 16
2.3.1.2 Micronutrients 17
 2.3.2 Biological Screening 17
2.3.2.1 Antimicrobial activity 17
2.3.2.2 Antimalarial activity 18
2.3.2.3 Anti-inflammatory activity 19
2.3.2.4 Antioxidant activity 20
2.4 Safety Analaysis 21
2.4.1 Toxicity 21
2.4.2 Mutagenicity 22
2.4.3 Cytotoxicity 22

2.5 Micropropagation of Wild Food Plants 23

CHAPTER THREE: METHODOLOGY 26

3.1 Nutritional Analysis 26

3.1.1 Sample Collection and Preparation 26
3.1.2 Determination of Moisture Content 26
3.1.3 Determination of As h Content 27
3.1.4 Determination of Protein Content 27
3.1.5 Determination of Fat Content 28
3.1.6 Carbohydrate Analysis 29
3.1.7 Dietary Fibre Analysis 30
3.1.8 Energy Determination 31
3.1.9 Mineral Analysis 31
 3.1.10 Vitamin Analysis 33
3.1.10.1 Vitamin A 33
3.1.10.2 Vitamin B 1 34
3.1.10.3 Vitamin B 2 35
3.1.10.4 Vitamin B 3 36
3.1.10.5 Vitamin C 37

3.2 Biological Screening 38

3.2.1 Sample Collection and Preparation 38
3.2.2 Preparation of Extracts 38
3.2.3 Determination of Antioxidant Activity 39
3.2.4 Anti-inflammatory properties 40
3.2.5 Antimicrobial Activity 40
3.2.6 Anti Mosquito Activity 42

3.3 Safety Evalution of Plants 46

3.3.1 Cytotoxicity 46
3.3.2 Toxicity 49
3.3.3 Ames Mutagenicity Test 50

3.4 Micropropagation of A.aspera, A.sessilis and G.densa 52

Chapter Four: Results and Discussion 54

4.1 Nutritional Profile 54

4.2 Biological Screening 60

4.2.1 Antioxidant activity of plant extracts 60
4.2.2 Anti-Inflammatory Prope rties 62
4.2.3 Antimicrobial Activity 63
4.2.4 Anti-Mosquito Prope rties 67
4.3 Safety Analysis 72
4.3.1 Cytotoxicity 72
4.3.2 Toxicity to Brine Shrimp 74
4.3.3 Mutagencity 75

4.4 Micropropagation 76
4.4.1 Surface sterilization of Plants 76
4.4.2 Callus induction 77
4.4.3 Shoot Regeneration using callus 78
4.4.4 Rooting of regenerated shoots 79
4.4.5 Hardening of plantlets 79

CHAPTER FIVE: CONCLUSION 83

REFERENCES 85
 LIST OF FIGURES

Fig. 1. Achyranthes aspera 10

Fig. 2. Alternanthera sessilis 12

Fig. 3. Guilleminea densa 14

Fig. 4. Repellency (a) Unfed A. arabiensis females introduced into 44

cup. (b) Rodent anaesthetized using sodium pentobarbital.
(c) Plant extract/Positive control (DEET) applied to
rodent’s abdomens. (d) Unfed A. arabiensis females held in
contact with treated ventral surface of rodent

Fig.5. Insecticidal Assay (a) Potter’s Towe r. (b) Plant extract/ 46

Positive control (K-orithine) s rayed on ceramic non porous
tiles.(c) A. arabiensis (30) females introduced into
bioassay cone.(d) Observed for knockdown after 30 & 60
min of exposure.(e) Transferred to holding cage containing
nutrient solution ove rnight to check for mortality

Fig. 6. Free radical scavenging capacity of methanolic extracts of 61

A. aspera, A. sessilis, G. densa and Rutin

Fig. 7. Repellency activity from the aqueous extracts of A. aspera, 68

A. sessilis and G. densa on A. arabiensis mosquito

Fig. 8. Repellency activity from the methanolic extracts of A. 78

aspera, A. sessilis and G. densa on A. arabiensis mosquito

Fig. 9. Cytotoxic effect of aqueous and methanolic extracts of A. 73

aspera on the K562 cell line
Fig. 10. Cytotoxic effect of aqueous and methanolic extracts of A. 73
sessilis on the K562 cell line

Fig. 11. Cytotoxic effect of aqueous and methanolic extracts of G. 74

densa on the K562 cell line

Fig. 12. A. aspera (a) callus derived from leaf explants (b) plate 80
showing callus derived from leaf explants

Fig. 13. G.densa (a) callus derived from leaf explants (b) plate 80
showing callus derived from leaf explants

Fig. 14. Micropropagation of A. sessilis (a) callus derived from leaf 81

explants (b) plate showing callus derived from leaf explants
(c) shoot formation from callus (d)shoot regeneration in
tissue culture bottle (e) multiple shoot development in a
bottle (f) multiple shoot development on a petri dish

Fig. 15. Root development in A. sessilis (a) Rooting in half MS 82

medium supple mented with 1 mg/L IBA (b) A. sessilis plant
in a sunbag vessel (c) hardened A. sessilis plant (d)
Acclimatized plant
 LIST OF TABLES

Table 1. The proximate values of famine foods A. aspera, A. sessilis 55

and G. densa and cultivated species A. spinosus, A.
hybridus, A. dubius and A. hypochondriacus

Table 2. Comparison of mineral levels of the famine food plants (A. 58

aspera, A. sessilis and G. densa) to the leafy vegetables (A.
spinosus, A. hybridus and A. dubius)

Table 3. Vitamin levels of A. aspera, A. sessilis and G. densa 60

Table 4. Anti-inflammatory properties of aqueous and methanolic 64

extracts of A.aspera, A.sessilis and G. Densa using 5-
lipooxygenases

Table 5. Antibacterial activity of A. aspera, A. sessilis and G. densa 65

Table 6. Minimum Inhibitory Concentration of A. aspera, A. sessilis 65

and G. densa

Table 7. Antifungal activity of A. aspera, A. sessilis and G. densa 66

Table 8. Antifungal minimum inhibitory concentration of A. aspera, 67

A. sessilis and G. densa

Table 9. Larvicidal trials of A. aspera, A. sessilis and G. densa at 69

plant concentration of 1000 µg/ml on A. arabiensis
mosquito

Table 10. % Knockdown and Mortality of A. arabienis with aqueous 71

and methanolic extracts of A. aspera, A. sessilis and G.
densa
Table 11. Mutant frequency of the number of revertants in S. 75
typhimurium strain TA 98 exposed to plant extracts

Table 12. Mutant frequency of the number of revertants in S. 76

typhimurium strain TA 100 exposed to plant extracts

Table 13. Percentage of contamination using different surface 77

sterilizing agents at different exposure times

Table 14. Effect of growth regulators on callus induction from leaves 78

of A. aspera, A. sessilis and G. densa (After 2 weeks of
culture)

Table 15. Effect of growth regulators on shoot differentiation from 79

stem explants of A. sessilis
 LIST OF APPENDICES

Appendix A 104
 LIST OF ABBREVIATIONS

2,4-D : 2,4-Dichlorophenoxyacetic acid

2iP : 2-isopentyl adenine
A : larvae that reached the adult stage
ABA : Abscisic acid
AOAC : Association of Official Analytical Chemists
BAP : Benzylaminopurine
BSL : Brine Shrimp lethality
COX : Cyclooxygenase
DBT : Durban Unive rsity of Technology Culture Collection
GA3 : Giberrilic Acid
IAA : indole-3-acetic acid
IBA : Indole butyric acid
IC50 : concentration of inhibitor necessary for 50% inhibition
ICP : inductively coupled plas ma
K562 : human eythroleukemia cell line
L : Larvae
LOX : Lipoxygenase
MIC : minimum inhibitory concentration
MRC : Medical Research Council
MS : Murashige and Skoog
NAA : 1- Naphthaleneacetic acid
NaN3 : Sodium azide
NRC : National Research Council
P : larvae that reached the pupal stage
PGR’s : Plant growth regulators
RDA : Recommended Daily Allowances
WHO : World Health Organization
 ABSTRACT

Information regarding the nutritional value of wild food plants in Africa and current information
varies from source to source. Prior to commercialization of wild foods the nutritional,
ethnobotanical, medical, chemical, anthropological and toxicity requires investigation. Plants
from the Amaranthaceae family were chosen because the family is characterized by several
species which are used by indigenous communities as a source of nutrition in different plants of
the world. The focus of this study was to investigate the nutritional and bio logical activities of
three plants from the Amaranthaceae family viz. Achyranthes aspera, Alternanthera sessilis and
Guilleminea densa that are considered famine plants.

This study aimed to determine the nutritional value (proximate, minerals and vitamins),
biological activity, toxicity and potential of a tissue culture system for three species from the
family Amaranthaceae. Nutritional analysis comprised of determining moisture, ash, protein, fat,
carbohydrate, dietary fibre and energy. Mineral analysis of calcium, copper, iron, magnesium,
manganese, phosphorus, sodium and zinc was performed by microwave digestion and then
analyzed by ICP Spectrophotometry. Vitamin A, Vitamin B1 , Vitamin B2 , Vitamin B3 and
Vitamin C were also analyzed. For biological and safety analyses aqueous and methanolic
extracts were prepared. Anti-oxidative and anti- inflammatory properties of the extracts were
tested; antimicrobial activity was tested by evaluating the bactericidal, fungal effect and
minimum inhibitory concentration on selected bacteria and fungi using the agar disk diffusion
method. Anti mosquito potential was determined by setting up repellency, larvacidal assay and
insecticidal assay. The safety and toxicity analysis was carried out by measuring cytotoxicity,
toxicity and mutagenicity. The potential of an in vitro tissue culture system of A. aspera, A.
sessilis and G. densa was determined using micropropagation.

A. aspera indicated significant amounts moisture, ash, dietary fibre, protein, vitamin B1 , vitamin
B2 , magnesium and manganese. Plant extracts of A. aspera had antibacterial activity against the
Gram negative bacteria Esherichia coli, Pseudomas aeroginosa and Salmonella typhi; Gram
positive bacteria Staphylococcus epidermis and Staphylococcus aureus. The methanolic extract
had antifungal activity against Sacchromyces cerevisiae and exhibited significant free radical
scavenging activity as well as 85% repellency against Anopheles arabiensis. The aqueous extract
stimulated the growth of the K562 (Chronic Myclogenous Leukaemia) cell line and the plant
extracts showed no mutagenicity or toxicity. A. sessilis indicated significant levels of ash, dietary
fibre, protein, energy, vitamin A, vitamin B1 , vitamin B2 , vitamin B3 , iron, magnesium and
manganese present. Plant extracts of A. sessilis had antibacterial activity against Gram negative
bacteria P. aeroginosa and Gram positive bacteria S. epidermis. The plant also showed antifungal
activity against the yeasts S. cerevisiae and Candida albicans. The methanolic plant extract
showed excellent antioxidant activity. The aqueous plant extract stimulated the growth of the
K562 cell line and the plant extracts possessed no mutagenicity or toxicity. This plant grew well
in a tissue culture system where it was propagated from callus to a fully grown plant able to
survive in environmental conditions. G. densa has ash and dietary fibre, vitamin B2 , vitamin B3
and iron. The plant extracts had antibacterial activity against Gram negative bacteria E. coli, P.
aeroginosa and Klebsiella. oxytoca; Gram positive bacteria Baccilus stereathermophilus and S.
aureus. The plant also has antifungal activity against C. albicans and significant repellency
activity against A. arabiensis where it showed 100% repellency. This plant was not found to be
mutagenic or toxic.

The results obtained from this study show promising potential for the plants to be exploited as
famine food plants. The nutritional value, biological activity and ability to micropropagate A.
aspera, A. sessilis and G. densa indicates a good potential for purposes of harnessing
biotechnological products.
CHAPTER ONE: STUDY RATIONALE AND OBJECTIVES

During times of natural disasters, populations suffering from severe food shortages become
heavily reliant on wild food plants for survival (Leborgne et al., 2002). This gives rise to the
concept of famine plants (Chopak, 2000). Rodale and McGrath (1991) stated that famine plants
have been eaten and utilized for centuries and they can be reintroduced as standard crops to
stabilize the land and mitigate the cycles of famine. Improved strains of native "famine plants,"
edible plants native to Africa that thrive through adverse weather cycles of drought and excess
rainfall, should be reintroduced and cultivated in place of foreign cash crops as these crops are
more nutritious than foods introduced from abroad, are more highly adapted to the climatic
adversity of Africa, are more resistant to local pests and disease, and require relatively little
management (Rodale and McGrath, 1991). Additionally, imported cash crops that have been
introduced into African agricultural systems are also detrimental to the sustainability of African
farming.

Publications (Maundu et al., 1999, Freedman, 2006) showed that wild plants are essential
components of many Africans' diets, especially in periods of seasonal food shortage and
ethnobotanic studies deal with their medicinal properties. The nutritional, antibacterial,
antifungal, anthelmintic, anti-amoebic, antischistosomal, antimalarial, anti- inflammatory and
antioxidant activity, as well as psychotropic and neurotropic activity using appropriate in vitro
tests forms basis for validating the usefulness of famine plants. Final commercialization potential
is then often provided through the isolation of active compounds. It is also important to
investigate how safe, these plants are for consumption, since some are potentially toxic and can
produce toxic constituents during stressful environmental conditions.

In Africa, one of the plants frequently consumed as leafy vegetable belongs to the family
Amaranthaceae. As these are not the usually commercially cultivated vegetables data pertaining
to amaranths is limited to Amaranthus dubius, A. spinosus and A. hybridus. Other members in
this family serve as examples of famine plants that may be superior to crop plants introduced
such as maize. Although, their seeds are smaller than maize, they yield higher nutritional
properties. The average protein content of amaranth seeds is 16% whereas maize yields an
average of only 12%. Additionally, amaranth is considered better nutritionally balanced; having
lysine content three times greater than maize, and its leaves and young stems can be eaten as
greens which have high mineral and vitamin content.

Plants from the Amaranthaceae family are used in indigenous system of medicine for their
antiarthritic, antifertility, laxative, ecbolic, abortifacient, anthelmintic, aphrodisiac, antiviral,
antispasmodic, antihypertensive, anticoagulant, diuretic and antitumour activities. They are used
to treat cough, renal dropsy, fistula, scrofula, skin rash, nasal infection, chronic malaria,
impotence, fever, asthma, amennorrhoea, piles, abdominal cramps and snake bites. Furthermore,
some of the members from this family have important phytochemicals such as rutin which is a
strong antioxidant compound and saponins.

A review of literature showed that many of the medicinal properties are attributed to the common
amaranths i.e. A. dubius, A. spinosus and A. hybridus and through the literature and other sources,
in this study we selected Achyranthes aspera, Alternanthera sessilis and Guilleminea densa
because there is little information available, thus our aim was to evaluate the nutritional and
biological activities of Achyranthes aspera, Alternanthera sessilis and Guilleminea densa and
develop a protocol for cultivating them in a tissue culture. To achieve this we embarked on: (i)
determining the nutritional value i.e. moisture, ash, protein, fat, carbohydrate, dietary fibre,
energy, vitamins and mineral content and compared it to other species from the Amaranthaceae
family; (ii) evaluating the anti microbial activity, anti-oxidant activity, anti- inflammatory activity
and activity against the malaria vector. Anopheles arabiensis; (iii) determining toxicity and
mutagenicity in vitro; and (iv) establishing a protocol for the micropropagation of the plants.
CHAPTER TWO: LITERATURE REVIEW

2.1 Wild plants and Famine Foods

2.1.1 Introduction

Useful wild plants are found all over the world. Some people call wild plants "weeds" although
many have important uses. The term 'wild- food' often implies that there is an absence of human
influence and management, however according to Bell (1995) people have indirectly shaped
many of the plants and some have been largely domesticated in home gardens and in the fields
together with farmers' cultivated food and cash crops. The term 'wild-food' is used to describe all
plant resources outside of agricultural areas that are harvested or collected for the purpose of
human consumption in forests, savannah and other bush land areas. Wild- foods are incorporated
into the normal livelihood strategies of many rural people (Bell, 1995) and is usually considered
as an additional diet to farmers' daily food consumption pattern, generally based on their crop
harvest, domestic livestock products and food purchased from local markets. Not all wild plants
are safe for food or medicine. Plants classified as typical 'famine-food' plants are normally not
consumed due to their limited seasonal availability, local taboos, offensive nature of the plants
such as abundance of thorns and tiny spines, certain unpleasant characteristics and side-effects
such as bad taste, complicated and prolonged preparation, and association with stomach
complaints, constipation, diarrhea and even intoxication. Certain 'wild- foods' are enjoyed and
therefore collected and consumed every time when ripe and these are important 'famine- foods'
during periods of food shortage (Guinard and Lemessa, 2000). Typical 'famine- food' consists of a
variety of plants which contain leafy and tender parts of stalks, pseudo-stems, fruits, berries,
seeds, husks and roots, i.e. tubers and corms.

Many of the wild plants also contribute to herbal medicines which form an important part of the
culture and traditions of African people. Today, most of the populations in urban South Africa, as
well as smaller rural communities, are reliant on herbal medicines for their health care needs.
Apart from their cultural significance, herbal medicines are generally more accessible and
affordable (Mander et al., 1996). As a consequence, there is an increasing trend, worldwide, to
integrate traditional medicine with primary health care.
In the Hausa-speaking regions of the Republic of Niger the majority of people rely on millet as
the staple foodstuff of their diet (Kim et al., 1997). However, during times when crop yields are
low or nonexistent the population must seek other available foods. These foods, referred to as
„„famine foods,‟‟ are consumed during periods of food shortage because they are edible and
available.

In some regions of the Sahel, farmers‟ crop yields are often insufficient to last until the following
harvest season. In many villages in the Republic of Niger, for example, granaries often become
exhausted months before the next planting season begins (Glew et al., 2004). This results in a
„„hungry season‟‟ during which people are forced by circumstances to purchase grain in local
markets at an elevated price. Consequently, during times when staple cereals are scarce or costly,
the inhabitants of rural Niger are compelled to increase their reliance on wild edible plants for
food. In the Sahel region of West Africa wild plant foods are widely consumed to supplement
cultivated cereals such as millet and sorghum (Glew et al., 2004).

In Tanzania, wild vegetables accounted for over 80% of all the leafy vegetables used. Wild
vegetables served as major side dishes or condiments to the meals. Wild vegetables played
central dietary roles where 49% of vegetables consumed were from wild sources and they
supplied significant sources of micronutrients. In Swaziland, wild foods were used throughout the
year. Burkina Faso documented that 20% of all food plants consumed were of the wild species
(Grivetti and Ogle, 2000). A study conducted in Zimbabwe revealed that some poor households
rely on wild fruits as an alternative to cultivated food for a quarter of all dry season's meals
(Balemie and Kebebew, 2006).

Since wild plant foods play an essential role in the survival and health of human populations, it
would be beneficial to understand how these plants contribute to human nutrition and to
recognize their potential for sustaining populations during future food shortages.

2.1.2 Categories of Wild Food Plants

Each registered wild- food plant species has been categorized and listed in one of four proposed
plant categories, depending on their availability and usage (Guinard and Lemessa, 2000);

typical 'famine- food' plants

 'wild- food' plants with 'famine- food' components
wild- food' plants attracting additional consumer categories during food shortage periods
on- farm food crops with 'famine- food' components

Typical 'famine-food' plants

A typical famine- food plant has leaves, stalks, inflorescence, roots (tubers, corms and rhizomes)
or barks (mainly of Acacia sp) that are edible. Many of the root-type famine- food plants are
drought tolerant and can stay in the soil intact for a long time. Most of the leafy-type famine-food
plants are locally referred to and classified as „weeds‟, sprouting and flourishing after rains. They
generally mature within a short period of time. There are two main periods of maximum
consumption of the leaves and tender parts of such “famine- food” plants. The first period is while
farmers are waiting for the upcoming crop harvest and the second is when they run out of food
stocks from the previous harvest. People try to add “famine- food” to local staple foods or to mix
it with other foodstuff to mask the often offensive nature of the food and to reduce any
characteristic and unpleasant side effects.

„Wild-food' plants with 'famine-food' components

Within this category multi-purpose wild-food plants are represented. Fruits plus one or more
additional food products such as leaves and tender parts of stalks or root parts can be used at
different times of the year and at different stages of food shortage.

„Wild-food' plants attracting additional consumer categories during food shortage periods
For most species classified in this category people‟s consumption behavio ur is the same, that is,
only the fruits or the berries are eaten or considered edible. Children consume the fruits in normal
times, but when food is short, adults, as an additional consumer category, will collect and
consume fruits from wild trees and bushes.

On-farm food crops with 'famine-food' components

On-farm crops with famine- food components are few and are likely to be perennial plants. The
famine- food components are normally not consumed because it may imply the total destruction
of the plant. Examples of on- farm crops bearing famine- food components are banana (Musa
paradisica), false banana (Ensete ventricosum), grass pea (Lathyrus sativus), cotton (Gossypium
spp.), cabbage tree (Moringa oleifera), sorghum, beans and tomato plants.

2.1.3 Usefulness of wild foods

Wild food plants represent inexpensive, locally available and versatile food sources capable of
improving nutrition and health quality. Furthermore, they often represent food sources with low
labor requirements, to the extreme that children, aged people and some sick people can
participate in their harvesting, provided adequate indigenous knowledge on their loca lization and
identification has been adequately transferred. Thus, wild food plants can also represent an easy
income source, especially if specific policies and programs were deployed to expand market
niches and support the integration of poor rural house holds in their sustainable harvesting,
processing, and commercialization (FAO, 2003). The wide indigenous knowledge on the
preservation and processing of wild food plants further enhances their ro les in household food
security and nutrition.

Wild foods have both agricultural and medicinal importance.

2.1.3.1 Agricultural Importance

Agricultural development and cultivation in developing countries are primarily based on

subsistence crops and edible wild plant species, and only secondarily, on the cultivation or
utilization of a wide diversity of food crops (Martin, 1984). Dietary utilization of non
domesticated plants has received little attention and a dramatic narrowing of the food base in
many traditional societies has occurred. For example, of the thousands of edib le wild and
domesticated plants documented globally (Tanaka, 1976), as few as 150 enter the commercial
market. Out of these 150 species only 15 constitute main sources of human food energy (Wilkes,
1977). The narrowing of domesticated species used as crops creates a vulnerable position in
which the crops could be destroyed by drought, diseases and pests (Turton, 1977). The
diversification of wild crops can ensure dietary balance and the intake of essential micronutrients.
Wild food plants become a critical food security resource in times when main crops cannot be
harvested especially in dry areas. Wild food plants represent untapped resources with potential to
improve nutrition in arid and semi-arid lands. They play an analogous role to that of vegetable
crops in humid and sub- humid areas (FAO, 2003).

For many years the importance of wild plants in subsistence agriculture in the developing world
as a food supplement and as a means of survival during times of drought and famine has been
overlooked. Generally, the consumption of such so-called 'wild-food' has been under-estimated
(Bell, 1995). Wild food plants represent a versatile agro biodiversity resource, providing different
benefits and opportunities depending on agro-ecological conditions, food security dynamics,
nutritional needs, cultural dimensions, and other circumstances. The most relevant and distinctive
value of wild food plants are that they are:
A supplementary food source, adding nutritional quality and food variety to local diets,
excellent supply of micronutrients (vitamins and minerals), critical food source during
seasonal food shortage periods, which are recurrent among the rural poor dwellings in
arid lands (FAO, 2003).
Inexpensive and easy food source, often requiring low labour inputs.
Vital ingredients in food habits and culinary practices in some areas of Sub-Saharan
Africa, especially for the so-called relishes that accompany main meals.
Emergency food source, for instance in cases of food shortage, during drought, crop
failure, and civil conflict.
Source of income, as some wild food plants may access specific market niches (FAO,
2003).

2.1.3.2 Medicinal importance

Infectious diseases caused by bacteria, fungi, viruses and parasites are a major threat to public
health, despite the tremendous progress in human medicine. Their impact is particularly large in
developing countries due to the relative unavailability of medicines and the emergence of
widespread drug resistance (Okeke et al., 2005). Besides small molecules from medicinal
chemistry, natural products are still major sources of innovative therapeutic agents for various
conditions, including infectious diseases (Clardy and Walsh, 2004). Only a minute portion of the
available diversity among fungi, marine fauna and flora, bacteria and plants has yet been
explored and ample opportunities lie ahead. Current research on natural molecules and products
primarily focuses on plants since they can be sourced more easily and can be selected on the basis
of their ethno- medicinal use (Verpoorte et al., 2005).

Renewed interest in traditional pharmacopoeias has meant that researchers are concerned not
only with determining the scientific rationale for the plant‟s usage, but also with the discovery of
novel compounds of pharmaceutical value. Instead of relying on trial and error, as in random
screening procedures, traditional knowledge helps scientists to target plants that may be
medicinally useful (Cox and Balick, 1994). Already an estimated 122 drugs from 94 plant species
have been discovered through ethnobotanical leads (Fabricant and Farnsworth, 2001).

Wild food plants have a potential in the mitigation of AIDS impact, especially among the rural
poor. In particular, they are relevant in three main concerns by households affected by AIDS;
they improve food access, improve nutrition and health quality, and alleviate labor constrains
related to food production (FAO, 2003). Wild food plants represent a relevant food security and
nutrition option for households suffering labor shortages, such as typically those affected by
HIV/AIDS. In fact, the recognized role of wild food plants in emergency situations is well
extensible to the context of AIDS, which in fact constitutes an emergency scenario (FAO, 2003).
Preserved wild food plants can help AIDS affected households and families to cope with labor
shortages confront sudden food scarcity, or build food reserves for times of acute production
constrains.

2.2 Amaranthaceae Family

The amaranth (or pigweed) family is a large group of dicotyledonous flowering plants known as
the Amaranthaceae. Amaranthaceae is divided into two subfamilies: the Amaranthoideae and the
Gomphrenoideae, based on certain morphological characteristics of their flowers. It is a relatively
large family, having about 65 genera and 900 species. Most of the 900 species of Amaranthaceae
are native to tropical and subtropical regions of Africa, Central America, and South America.
The number of Amaranthaceae species declines as one approaches the northern and southern
temperate zones.

The species in this family are mostly annual or perennial herbs, although a few species are shrubs
or small trees. Many species of Amaranthaceae are considered weeds, since they invade disturbed
areas, such as agricultural fields and roadsides. A. aspera, A. sessilis and G. densa fall under the
category typical 'Famine-Food' plants.

2.2.1 Achyranthes aspera

Description

The flowers are hermaphrodite (have both male and female organs) (Fern, 1996). They are
perennial or annual plants. The stems are 0.4-2 m, pilose or puberulent. They have elliptic, ovate,
or broadly ovate leaf blades. They inflorescences to 30 cm; bracts membranous; bracteoles long-
aristate, spinose; wings attached at sides and base. The flowers occur as tepals 4 or 5, length 3-7
mm; pseudostaminodes with margins fimbriate at apex, often with dorsal scale. The utricles are
cylindric, 2-4 mm, apex truncate or depressed. Below is an illustration of the A. aspera plant
(Fig.1).

Nomenclature
Common Name: Prickly Chaff- flower
Hindi Name: Chirchita, Latjira, Onga
Chemical Constituent: Achyranthine
Useful Parts: All parts of the plants are used
Fig. 1. Achyranthes aspera

Habitat

The plant prefers light sandy, medium loamy, heavy clay soils, can grow in semi-shade (light
woodland) or no shade and requires moist soil (Fern, 1996). It grows as wasteland herb
everywhere. It has been used as folk medicine. It is in flower from July to September, and the
seeds ripen in October.

Nutritional properties and Biological properties

The seeds are rich in protein.

According to Ayurveda, it is bitter, pungent, heating, laxative, stomachic, carminative and useful
in treatment of vomiting, bronchitis, heart disease, piles, itching abdominal pains, ascites,
dyspepsia, dysentery, blood diseases etc. The juice of the plant is used in the treatment of boils,
diarrhea, dysentery, hemorrhoids, rheumatic pains, itches and skin eruptions. The leaf is emetic
and a decoction is used in the treatment of diarrhea and dysentery. A paste of the leaves is applied
in the treatment of rabies, nervous disorders, hysteria, insect and snake bites (Manandhar, 2002).
The ash from the burnt plant, often mixed with mustard oil and a pinch of salt, is used as a tooth
powder for cleaning teeth. The dried twigs are used as toothbrushes.

2.2.2 Alternanthera sessilis

Description

A. sessilis is an annual or perennial herb, grows up to 1 m tall. It sometimes become woody at the
base, prostrate or erect to floating or scrambling. The leaves are opposite and entire. It has
inflorescences heads or short spikes, axillary, sessile or pedunculate, solitary or fasciculate,
bracteate: bracts persistent. The flowers are bisexual, solitary in axils of bracts; 2 bracteoles,
usually shorter than perianth, persistent or not, perianth falling with fruit. There are 4 or 5 tepals,
free or concrescent at the base, glabrous of furnished with smooth or denticulate hairs. There are
2-5 stamens, some occasionally anantherous, hypogynous; filaments monadelphous at the base
into a cup or tube, alternating with large and laciniate to small pseudostaminodes or these rarely
obsolete; anthers thecous. The ovaries are suborbiculare or obovate in outline, usually
compressed; ovule solitary, pendulous, basal; style short or absent; stigma capitellate. The
capsule is thin walled or sometimes corky, in dehiscent. The seeds are lenticular (Glen, 2000). A.
sessilis is unique in the genus in that its tepals are generally shorter than the utricle (Fig. 2). The
utricles of other Alternanthera species sit well below the tops of the tepals (Scher, 2004). Below is
an illustration of the A. sessilis plant (Fig.2).

Nomenclature

Family: Amaranthaceae

Sub Family: Gomphrenoideae,

Common Name: sessile joyweed

Disseminule: Fruit
Fig.2. Alternanthera sessilis

Habitat

It prefers wet conditions, but occurs in both wetlands and uplands and can grow on a variety
of soil types. The plant is propagated by seeds, which are wind and water-dispersed, and by
rooting at stem nodes. Widespread throughout the tropics and subtropics: tropical Africa,
southern Asia to Japan, Southeast Asia, Australia, and the Pacific Islands (Scher, 2004). It is
a pantropical; found in damp shady areas, swamps, pond margins, shallow ditches,
roadsides, low- lying waste places, damp pastures, cultivated areas (Scher, 2004).

Nutritional properties and Biological properties

It is a weed of rice throughout tropical lands, and of other cereal crops, sugarcane, and bananas.
The young tips are eaten as a vegetable. It is a very famous leafy vege table in tribal and rural
areas. The leaf is very rich in iron (17 mg/100 mg), Vitamin A (192 mg/100 mg) and dietary fiber
(12 g/100 g). The plant contains about 5% protein and soups made with the leaf are given to
anaemic patients in rural areas. It contains abundant carotene, therefore it is used for curing night
blindness. The plant has a virtue of galactogogue and enhances the secretion of milk in new
mothers (Naples, 2005). Chemical constituents are Sitosterol, stigmasterol, campesterol, cc-
spinasterol, oleanolic acid rhamnoside, 24- methylene cycloartenol, cycloeucalenol, lupeol, 5-cc-
stigmasta-7-enol and palmitate.

An infusion of the entire plant is used in Indonesia as a remedy against intestinal cramps,
diarrhoea and dysentery (intestinal disorder), and externally as a cooling agent to treat fever. In
Malaysia, it is used internally against intestinal inflammation and fever, and externally to treat
wounds. In Taiwan, this plant is used in local medicine, often in mixtures with other medicinal
plants, to treat hepatitis, congestion, bronchitis and asthma, ailments of the lung, to stop bleeding
and as a hair tonic. It is used in India against dysentery (intestinal disorder), as a cholagogue
(increases bile flow in liver), abortifacient (causes abortion) and febrifuge (reduces fever) and to
treat snake bites, inflamed wounds and boils (Naples, 2005).

2.2.3 Guilleminea densa

Description

It is a woolly, pros trate mat-formi ng perenni al herb. The leaves are narrowly elliptic to broadly ovate;
glabrous on the upper surface and with long matted white hairs on the lower surface. Inflates to 6
mm, dense, ovoid, and whitish (Fig 3) (Hyde and Wursten, 2002).

Nomenclature

Family: Amaranthaceae

Synonyms: Brayulinea densa (Willd.) Small

Fig.3. Guilleminea densa

Habitat

It is found on roadsides, in grassland and open woodland, usually in well- trodden disturbed
places (Hyde and Wursten, 2002). It is naturalized in Australia and South Africa. It is native of
the warmer parts of North and South America, from the southern USA to northern Argentina, and
now a widespread tropical weed (Hyde and Wursten, 2002).

Nutritional properties and Biological properties

There is no information available of nutritional or biological properties.

2.3 Nutritional and Biological Properties

2.3.1 Nutritional Require ments

The basic nutritional needs for humans are to supply energy and raw materials for all the various
activities and processes that occur in the body. In addition to the need for water, humans require
five types of nutrients from their food supply: three of which are required in large amo unts and
are called macronutrients, consisting of carbohydrates, protein and fats. The other two types of
nutrients, vitamins and minerals, are required in small amounts are known as micronutrients.
Although several reports have been published on the nutritional value of famine food plants, the
database of their chemical and nutrient composition is far from exhaustive (Cook et al., 2000).

Diets of various types can be devised to meet recognized nutritional needs based on
Recommended Daily Allowances (RDA). The National Research Council defines the levels of
intake of essential nutrients that, on the basis of scientific knowledge, are judged by the Food and
Nutrition Board to be adequate to meet the known nutrient needs of practically all healthy
persons (NRC, 1989). In principle the RDAs are based on various kinds of evidence but in
practice, there is only limited data on which estimates of nutrient requirements can be used.
According to the Food and Nutrition Board, RDAs should be provided from a se lection of foods
that are acceptable and palatable to ensure consumption. In a few cases where deficiency is
commonly observed, food fortification and individual supplementation are appropriate. Attention
and public interest have also been focused on the possible effects of nutrients, often at high
intakes, on conditions other than those associated with specific deficiencies. At higher levels of
intake, both the toxicity and the pharmacological action of specific nutrients must be considered
(NRC, 1989).

2.3.1.1 Macronutrients

Human energy requirements (1200-3200 calories per day) vary with age, sex, and activity level
of the individual. A calorie is the measure of energy (amount of energy needed to raise the
temperature of one gram of water by one degree Celsius). Although all the macronutrients can be
used as a source of energy, normally only carbohydrates and fats do so, while proteins provide
the raw materials or building blocks, required for the synthesis of essential metabolites, growth
and tissue maintenance (Arntzen and Ritter, 1984). Previous studies on leafy vegetables (Odhav
et al., 2007) showed that other species from the Amaranthaceae family namely A. dubius, A.
hybridus and A. spinosus had significant moisture and carbohydrate values. A comparison was
later performed between the famine food plants and the leafy vegetables from the Amaranthaceae
family in this study.

2.3.1.2 Micronutrients
Micronutrients are essential for proper nutrition but are required in much smaller amounts. There
are two categories of micronutrients, the organic compounds known as vitamins and the
inorganic compounds known as minerals (Arntzen and Ritter, 1984). Odhav et al. (2007) reported
significant calcium values for A. spinosus and A. hybridus; significant sodium and manganese
values for A. dubius and A. hybridus. A. spinosus, A. dubius and A. hybridus possessed substantial
quantities of magnesium.

2.3.2 Biological Screening

2.3.2.1 Antimicrobial activity

Many countries ravaged by war and poverty do not have the sanitation and hygiene levels
comparable to those of First World countries which exposes the people to a wider array of
microbial pathogens. These communities have to depend on local and indigenous plants as these
are often the only available means of treating such infections (Taylor et al., 2001).

Many plants have been screened to rationalize their use in traditional medicines (Rabe and van
Staden, 1997; Lin et al., 1999; Luyt et al., 1999; Shale et al., 1999; Kelmanson et al., 2000;
Tetyana et al., 2002). Antibacterial compounds were isolated from Warburgia salutaris which is
traditionally used as a topical application for sores and inflammation (Rabe and van Staden.
2000). Reid and coworkers in 2001 showed vernodalin was extracted from the leaf extract of
Vernonia colorata (Willd.) Drake (Asteraceae). Rabe et al. (2002) isolated sesquiterpene
lactones. Pillay et al. (2001) showed that bark extracts of Erythrina lysistemon Hutch. (Fabaceae)
had good antibacterial activity and was attributed to the compound wighteone. In studies
conducted by Grace et al. (2002) on the fruits of Kigelia africana (Lam.) Benth. (Bignoniaceae)
they found activity against Bacillus subtilis which was due to the presence of the antibacterial
compound palmitic acid.

Plants may also offer protection against some opportunistic fungal infections such as Candida
albicans where Western medicines are unavailable in HIV patients (Fan-Harvard et al., 1991).
Motsei et al. (2003) reported that the aqueous extracts of Tulbaghia violacea Harv., Allium
sativum L. (both Alliaceae), Polygala myrtifolia L. (Polygalaceae) and Glycyrrhiza glabra L.
(Fabaceae) exhibited anti-candidial activity.

2.3.2.2 Antimalarial activity

Malaria is the world‟s leading killer among the infectious diseases. It is estimated that more than
two million people die from the disease each year (Prozesky et al., 2001).

One of the approaches for control of mosquito-borne diseases is the interruption of disease
transmission by killing or preventing mosquitoes from biting humans. Herbal products with
proven potential as an insecticide or repellent can play an important role in the interruption of the
transmission of mosquito-borne diseases at the individual as well as at the community level.
Some herbal products such as nicotine obtained from tobacco leaves, Nicotiana tabacum,
anabasine and lupinine, the alkaloids extracted from Russian weed Anabasis aphylla (Campbell
et al., 1993), rotenone from Derris elliptica and pyrethrums from Chrysanthemum cinererifolium
flowers have been used as natural insecticides even before the discovery of synthetic organic
insecticides (Jacobson and Crosby, 1971). The discovery, development and use of synthetic
organic chemicals with persistent residual action not only overshadowed the use of herbal
products against mosquitoes but also became the major weapon for mosquito control. Since the
discovery of DDT (Dichloro-Diphenyl-Trichloroethane), mosquito control approach has been
almost completely based on synthetic organic insecticides. But the extensive use of synthetic
organic insecticides during the last five decades has resulted in environmental hazards and also in
the development of physiological resistance in major vector species. This has necessitated the
need for search and development of environmentally safe, biodegradable, low cost, indigenous
methods for vector control, which can be used with minimum care by individuals and
communities in specific situations.

Prozesky et al., (2001) showed plants to have antiplasmodial activity against a chloroquine-
resistant strain of Plasmodium falciparum. Ajuga remota Benth. (Lamiaceae) and Caesalpinia
volkensii Harms (Fabaceae) are two medicinal plants commonly used by traditional healers in
Kenya to treat malaria (Kuria et al., 2001). Petroleum ether extracts of Morinda lucida Benth.
(Morindaceae) inhibited the maturation of Plasmodium falciparum in vitro (Awe and Makinde,
1998). A recent report by Fennel et al. (2004) revealed that 134 South African plant species were
tested for in vitro antiplasmodial activity against a Plasmodium falciparum strain D10 using the
parasite lactate dehydrogenase assay. The species selected were based on their traditional use. Of
the 134 species 49% showed promising antiplasmodial activity (IC 50 ≤ 10 µg/ml). A further 23
species were found to be highly active with IC 50 values of ≤5 µg/ml and warrant further
investigation as possible plant-based antimalarial drugs. At present many of the African
medicinal plants tested for antiplasmodial activity have shown potential to be developed as new
antimalarial drugs.

2.3.2.3 Anti-inflammatory activity

To evaluate the efficacy of a plant in reducing pain and inflammation, extracts can be tested for
prostaglandin synthesis, these are involved in the complex process of inflammation and are
responsible for the sensation of pain inhibitory activity in an in vitro assay (White and Glassman,
1974). Plants used in the treatment of pain and inflammation have been screened for COX-1 and
COX-2 inhibition in an attempt to rationalize their traditional usage (Jäger et al., 1995, McGaw et
al., 1997, Lindsey et al., 1999, Shale et al., 1999). To classify a plant as active, the minimum
inhibition by aqueous extracts tested at a final concentration of 250 µg per test solution must be
59% and for ethanolic extracts, 70%, when tested at a final concentration of 250 µg per test
solution.

Attention has recently focused on developing safer anti- inflammatory drugs following new
evidence for the metabolism of arachidonic acid. Arachidonic acid metabolism through
cyclooxygenase (COX) and lipoxygenase (LOX) pathways generates various biologically active
intermediates that play an important role in inflammation, thrombosis and tumor progression. In
addition to arachidonic acid‟s conversion to prostaglandins by the COX enzymes, it may also be
converted to leukotrienes by the action of 5-lipoxygenase. Drugs that are able to block both COX
and 5-LO metabolic pathways (dual inhibitors) equally well, seem to be the best option in terms
of NSAIDs. (Fiorucci et al., 2001)

2.3.2.4 Antioxidant activity

Antioxidants are substances that may protect cells from the damage ca used by unstable molecules
known as free radicals. Antioxidants interact with and stabilize free radicals and may prevent
some of the damage caused by free radicals. These plant-derived antioxidants have been shown to
function as singlet and triplet oxygen quenchers, peroxide decomposers, enzyme inhibitors and
synergists (Choi et al., 2002). There are increasing suggestions by considerable evidence that the
free radicals induce oxidative damage to biomolecules (lipids, proteins and nucleic acids), the
damage which eventually causes atherosclerosis, ageing, cancer, diabetes mellitus, inflammation,
AIDS and several degenerative diseases in humans (Choi et al., 2002). Examples of antioxidants
include β-carotene, lycopene, vitamins A, C, E, and other substances. Plants contain a wide
variety of free radical scavenging molecules, such as flavonoids, anthocyanins, carotenoids,
dietary glutathionine, vitamins and endogenous metabolites and such natural products are rich in
antioxidant activities (Choi et al., 2002).

In addition to the antioxidant vitamins, edible wild plants are rich in phenols and other
compounds that increase their antioxidant capacity. According to Simopolous (2004), it is
important to systematically analyze the total antioxidant capacity of wild plants and promote their
commercialization in both developed and developing countries. Studies by Lindsey et al. (2002)
reveal that the plants that are eaten as foods in Southern Africa were shown to provide important
health benefits in the form of antioxidant activity. (Lindsey et al., 2002).

Extracts with inhibition values greater than 70%, and thus with high antioxidant activity, were
found in the potherbs Amaranthus sp. (Amaranthaceae), Sisymbrium thellungii O.E. Schulz
(Brassicaceae) and Urtica dioica L. (Urticaceae). Traditionally, these plants are used in the
preparation of „imfino‟ which forms an important part of the diet. High activity (greater than
70%) was also shown by the tuberous Colocasia esculanta Schott (Araceae) and the teas Galium
aparine L. (Rubiaceae) and Aspalathus linearis (Burm.f.) R. Dahlgren (Fabaceae) (rooibos)
(Fennell et al., 2004).
2. 4 Safety Analaysis

Significant risks are associated with the inappropriate use of medicines in all healthcare systems.
In South African traditional healthcare, these risks are of greater consequence due to the form in
which plant medicines are sold. Medicinal plants, most commonly bark bulbs and roots (Mander
et al., 1996) are dried and sold as semi and processed products, ranging from plant organs to
crude powders. Products are seldom labeled but bulk stock may be identified by signage
indicating a local vernacular name, and packaging is rudimentary. Botanical identification of
medicinal plant products using vernacular nomenclature is notably difficult. Furthermore,
taxonomically reliable characters are usually lost through desiccation during drying; rendering
medicinal plant products extremely difficult to identify using morphological characters (Fennell
et al., 2004). Frequently used plants in traditional medicine are assumed safe, due to their long-
term use (Elgorashi et al., 2002) and are considered to have no side effects because they are
„natural‟ (Popat et al., 2001). However, scientific research has shown that many plants used as
food or in traditional medicine are potentially toxic, mutagenic and carcinogenic (Schimmer et
al., 1988, Higashimoto et al., 1993, Kassie et al., 1996, De Sã Ferrira and Ferrão Vargas, 1999).

2. 4.1 Toxicity

The eggs of the brine shrimp, Artemia salina, have been used in the Brine Shrimp lethality (BSL)
assay, which is a simple bench top bioassay used to test toxicity of compounds. A. salina, with
same purine metabolism as that of mammalian cells and the DNA-dependent RNA polymerases
of A. salina are also similar to the mammalian type (McLaughlin, 1991, Solis et al., 1993).

2.4.2 Mutagenicity

The Ames test also called the Salmonella/microsome assay (Maron and Ames, 1983) is used as it
is widely used in the determination of possible gene mutations caused by extracts. A positive
response in any single bacterial strain either with or without metabolic activation is sufficient to
designate a substance as a mutagen (Zeiger, 2001). The assay acts by detecting back mutations in
the His operon to (→His+) when growing Salmonella typhimurium bacteria in a His-poor
medium. Elgorashi et al. (2003) reported that Crinum macowanii, Chaetacme aristata Planch.
(Celastraceae), Plumbago auriculata Lam. (Plumbaginaceae), Catharanthus roseus (L.) G.Don.
(Apocynaceae) and Ziziphus mucronata Willd. (Rhamnaceae) had mutagenic effects in the
Salmonella/microsome assay

2.4.3 Cytotoxicity

Scientific strategies for the in vitro evaluation of natural products with biological activity have
changed in the past few years. Interest in a large number of traditional natural products has
increased (Cordell, 1995, Kurokawa et al., 1993, Vlietinck et al., 1995, Taylor et al., 1996). MTT
(Tetrazolium blue) colorimetric assays are used to evaluate the reduction of viability of cell
cultures in presence and absence of the extracts (Betancur-Galvis et al., 1999). It has been
suggested that aqueous and ethanolic extracts from plants used in allopathic medicine are
potential sources of antiviral and antitumor agents (Chung et al., 1995, Vlietinck et al., 1995).
These techniques which considered quick and inexpensive for the evaluation of antitumor
(Carmichael et al., 1987, Rubinstein et al., 1990) and antiviral activity (Weislow et al., 1989) of a
large number of natural product extracts. They can be used to guide the isolation and purification
of their biologically active principles (Cordell, 1995).

2.5 Micropropagation of Wild Food Plants

For many years the importance of wild plants in subsistence agriculture in the developing world
as a food supplement and as a means of survival during times of drought and famine has been
overlooked. Native plants are disappearing from their habitat in an alarming rate. In addition to
their contribution to the integrity of the environment, plants are invaluable sources of useful
genes for genetic improvement of crop plants. Conservation of the components of biological
diversity is vital in the arid zone, where renewable natural resources are scarce. While the
contribution of native wild plants to the preservation of the environment has been well
recognized, development of methods for tissue culturing of many native wild plants is lagging
behind. Lack of well defined economic or commercial interests may be the cause for little interest
in these plants. Cell and tissue culture of native plants is envisaged as a mean for germplasm
conservation to ensure the survival of endangered plant species, rapid mass propagation for large
scale revegetation with perennial plants and for genetic manipulation studies. Among the
requirements for an applicable large-scale propagation system for native plants are cost
effectiveness, reproducibility and simplicity.

Tissue culture is basically defined as in vitro growth of plantlets from any part of the plants in
suitable nutritive culture medium. It is also known as `micropropagation' in scientific technology
(Ghatnekar and Kavian, 2000). The application of tissue culture for large-scale plant production
meant for commercial purposes is well demonstrated in the case of several crops and horticulture
species. Many varieties have grown remarkably well in vitro, allowing various kinds of
experimental manipulations. In many instances, the objectives of the investigations have been
more than fulfilled and many solutions have been found to problems of plant growth,
differentiation, morphogenesis and in the application of different growth substances (Ghatnekar
and Kavian, 2000 ).

There are currently no published literatures available on micropropagation of A. aspera, A.

sessilis and G. densa. Literature related to tissue culture on Amaranthus spp. belonging to the
family Amaranthaceae is available.

Flores et al. (1982) developed in vitro culture system for some species and varieties of
Amaranthus. Leaf disks and hypocotyls segments from 2-3 week old A. hypochondriacus, A.
cruentus and A. tricolor seedlings were cultured in B5 (Gamborg) and MS media supplemented
with 2,4-D, NAA, BA and zeatin in various combinations. 2,4-D induced callus and abnormal
roots in the first two species. Flores and Teutonico (1986) reported that in vitro technology may
provide very useful methods for the genetic improvement of grain Amaranthus species. In
particular the micropropagation and regeneration of plants from primary explants offers the
possibility to multiply superior genotypes as biotic and abiotic stress resistant plants, selected
plants for increased production of proteins or specific amino acids or male sterile plants.

Bagga et al. (1987) found that hypocotyl segments of A. paniculatus callused on B5 medium
supplemented with Kn (0.5 ppm) and NAA (0.1 ppm) and even without transfer, shoots were
formed in such cultures. About 20% of the cultures produced multiple shoots. In medium with 1
ppm each of Kn and NAA direct shoots were formed at one end of the hypocotyl segment and
callusing was initiated at the other end. The plants obtained in either medium formed roots and
could be transferred to soil for further growth.
Callus induction, callus growth and organogenetic processes were investigated in hypocotyl and
stem cultures of four species of Amaranthus each of which comprised several varieties by
Bennici et al. (1992). The combinations of NAA plus BAP or 2,4-D plus Kinetin were very
effective in causing callus formation. As far organogenesis-based on a few varieties - only A.
caudatus and A. hypochondriacus responded well forming shoots from callus when cultured in
presence of IAA plus Kinetin and/or IAA plus BAP. Root regeneration was also observed in
several varieties (Bennici et al., 1992).

Bennici et al. (1997) studied in vitro behaviour of A. cruentus, A. hybridus and A.

hypochondriacus and found that callus formation occurred from explants from almost all the
lines tested. The results obtained demonstrate that in Amaranthus the genotype, growth regulator
dose and combination, the type and the physiological stage of explants are factors of great
importance for in vitro plant regeneration.

In general, the genus Amaranthus shows the great potential with regard to dedifferentiation and
morphogenetic processes and the possibility to micropropagate selected genotypes via direct or
indirect regeneration or via. somatic embryogenesis. The data obtained from different
experiments provide unequivocal evidence that plant regeneration from primary explants varies
with the genotype. Optimum conditions for shoot induction are high cytokinin/auxin ratio. Strong
cytokinin such as BAP or 2-iP seem to be effective agents for shoot regeneration. The
endogenous auxin / cytokinin balance and age dependant competence of explants tissues may
play an important role in regeneration (Bennici and Schiff, 1997).
CHAPTER THREE: METHODOLOGY

3.1 Nutritional Analysis

All analyses were conducted in duplicate and the reagents used were that of analytical grade.
Results are based on fresh weight per 100 g of sample.

3.1.1 Sample Collection and Preparation

A. aspera, A. sessilis and G. densa were collected from the greater Durban area, Kwa-Zulu Natal
in 2006. The plants were collected and identified by Prof. Baijnath using taxonomic keys.

Upon receipt, the leaves were separated and washed several times with distilled water until no
foreign material remained and air dried for 24 h. Thereafter, they were dried in an oven
(Memmert, South Africa) at 25°C for 7 days. The dried leaves were powdered using an industrial
grinder (Retsch Gmbh, West Germany) and stored in Schott bottles until use.

3.1.2 Determination of Moisture Content

Moisture analysis was carried out using the drying oven method (AOAC, 1990). Porcelain
crucibles were weighed and their masses recorded. In this study 4 g of each fresh plant sample
was weighed into pre-weighed crucibles and dried in the drying oven (Memmert, South Africa) at
105°C for 3 h. These were cooled in desiccators for 1 h and reweighed.

The percentage moisture content was determined according to the formula:

Percentage moisture = initial mass (g) – mass after 3 h (g) 100

mass of sample (g) 1

3.1.3 Determination of As h Content

Ash content was analysed as per method outlined in the AOAC (1990). Dried porcelain crucibles
were weighed and their masses recorded. In this study 3 g of each plant sample was weighed into
the crucibles. An aliquot of 7 ml glycerol: methanol (1:1 v/v) was added to the crucibles. The
crucibles were ignited and burnt until all organic material volatilised. The samples in the
crucibles were ashed by placing them in a muffle furnace (Labcon, Laboratory Consumables and
Chemical Supplies) at 600°C for 6 h. The crucibles were placed in desiccators and allowed to
cool. The resulting crucibles were weighed and the percentage ash content was determined as
follows:

percentage ash = mass of ash (g) _ 100

mass of sample (g)

3.1.4 Determination of Protein Content

The Kjeldahl method (AOAC, 1990) was used for protein analysis using a Buchi 430 Digestor
(Switzerland). This method is based on the assumption that a mixture of pure proteins will
contain 16% nitrogen. The nitrogen concentration is determined by converting the nitrogen
present in the sample to ammonium sulphate by digesting it in concentrated sulphuric acid
(H2 SO4 ). The digested sample is then made alkaline with 32% sodium hydroxide (NaOH) (m/v).
The ammonia is distilled into excess 2% boric acid solution and is determined by titration with
standardized 0.1N H2 SO4 . Protein content was obtained by multiplying the percentage
determined nitrogen by the appropriate factor, which was 6.25.

In this study 0.5 g of sample, 4 g of catalyst mixture (192 g anhydrous sodium sulphate, 7 g
copper sulphate crystals and 0.71 g selenium powder were crushed using a mortar and pestle) and
20 ml of concentrated H2 SO4 were weighed into clean dry digestion tubes (Buchi, Switzerland).
The digestion tubes were connected to a NaOH trap for absorbing the noxious fumes and a
vacuum was used to draw the fumes into the NaOH trap. The tubes containing the sample
mixture were heated and the heating power was maintained such that the samples were always
boiling. Digestion occurred for 45 min and was completed when the solution turned light green.
The digested samples were distilled by inserting the digestion tubes into the preheated Buchi 321
Distillation Unit (Switzerland) and were diluted with distilled water (1:3 v/v) followed by the
addition of 70 ml of a 32% NaOH (w/v) solution. An aliquot of 60 ml of 4% boric acid and 6
drops of methyl red indicator was added to Erlenmeyer flasks that were placed in the distillation
unit. Samples were distilled for 3 min. The distillate was titrated with 0.1N H 2 SO 4 and the end
point was reached when the light blue solution turned colourless to grey. The percentage nitrogen
content was calculated as follows:

% N= (sample titre in ml – blank in ml) 1.4 x normality of acid 100

mass of sample x 10

The percentage protein content was calculated as follows:

% Protein = 6.25 %N

3.1.5 Determination of Fat Content

The Soxhlet method was used to determine total fat (AOAC, 1990). This method is based on acid
hydrolysis that liberates the bound fat followed by solvent extraction of the fat.

Acid hydrolysis was achieved by adding 3 g of sample, 5 g of Celite 545 (Capital Lab Suppliers,
New Germany) and 100 ml of 4N hydrochloric acid (HCl) to clean, dry digestion flasks (Buchi,
Switzerland) and swirled to mix contents. 10 g of sea sand (Merck, Germany) and 5 g of celite
545 were added into filter crucibles (Buchi hydrolysis unit B-425 Switzerland). The raise/lower
device was placed in the top boil position and the filter crucibles were placed in the preheated
digestion block. Sample aspiration tubes as well as the water jet pump for the vacuum extraction
system was connected and samples were filtered into filter crucibles. After filtration was
complete, the heater was switched off. Empty digestion tubes were rinsed out with aliquots of
warm distilled water (50 C) into filter crucibles until no sample remained. Filter crucibles were
dried in a drying oven at 80 C overnight.

The fat was extracted using a Buchi 810 Soxhlet system (Switzerland). The system was switched
on, water tap opened and glass steam generator tap closed. Pre-dried beakers together with 2
boiling chips in each were weighed and their masses recorded. Beakers were filled with
petroleum ether and inserted into the system. Filter crucibles containing samples were sealed with
cotton wool and inserted into the extraction chamber. Beakers were consta ntly topped up with
petroleum ether (40-60 C) using a plastic hypodermic sponge during the 6 hour extraction period.
At the end of the extraction period, the side lever was opened which allowed the solvent to drain
out of the beakers. Heating continued until all the solvent evaporated from the beakers. These
were placed in a drying oven overnight at 105°C, cooled in desiccators and weighed. Results
were analyzed according to the formula:

Percentage fat = mass of flask after drying - initial mass of flask 100
mass of sample

3.1.6 Carbohydrate Analysis

Carbohydrate analysis was performed using the Phenol Sulphuric Acid Carbohydrate Assay
Method (Dubois, 1956)

Plant samples were dissolved in distilled water and filtered using a C18 Sep-Pak Plus cartridge
(Waters Corp. Wilford, Mass. USA), previously conditioned with 10 ml dis tilled water (MillQ.
R.G. Millipore, USA) and 45 µl nylon filter (Scheicher & Schuell, Germany).

100 µl of plant sample or glucose standards were added to 50 µl of 80 % phenol followed by 2 ml

of concentrated sulphuric acid. Dilutions were made from the standard solution in the range of 10
µg/ml to 100 µg/ml to construct a standard curve. These solutions were kept at room temperature
for 10 min and the resultant absorbance was measured at 490 nm using a spectrophotometer
(Ultraspec 2). The concentration of the sugars was determined from the standard curve obtained.

3.1.7 Dietary Fibre Analysis

Procedure was carried out according to Scheizer and Wuersch (1979) with some modifications.

The moisture of the samples was determined as described in 3.1.2. In this study 1 g sample was
weighed into a 250 ml beaker and the fat was extracted with 3 × 25 ml portions of hot petroleum
ether. The defatted sample was extracted with 3 × 25 ml portions of 80% ethanol. 50 ml distilled
water was added to sample and autoclaved for 1 h at 121°C to gelatinize the starch. The sample
was cooled and the pH was adjusted to 1.5 using dilute HC l. 50 mg Pepsin and 2 ml of
chloroform was added and this mixture was incubated for 20 h at 37 °C. After 16 h the pH was
readjusted to 1.5 and a further 25 mg Pepsin was added. After pepsin digestion the pH was
changed to 6.0 with dilute NaOH solution. 25 ml phosphate buffer (pH 6) containing 100 mg
pancreatin and 50 mg glucoamylase was added to the mixture. This mixture was incubated for 18
h at 37°C. A few drops of thymol solution prevented bacterial growth during incubation.

The digested sample was centrifuged at 3000 rpm for 30 min and the supernatant was transferred
to a 600 ml beaker. The residue was transferred to a glass sintered crucible with distilled water,
acetone and diethyl ether. This was Fraction A. Four volumes of ethanol were added to the
beaker containing the supernatant (Fraction B) to precipitate the soluble fibres. The supernatant
was drained off and the precipitate was transferred to a glass sintered crucible and filtered
(Fraction C). Fraction C was washed with 80% ethanol, acetone and diethyl ether. The crucibles
containing fraction A and C were dried for 16 h in an oven at 105°C. The crucibles were weighed
(W1) and placed in a furnace at 45°C for 2 h. These were cooled in desiccators and reweighed
(W2). The difference between the weights W1 and W2 of fractions A and C represent the
insoluble and soluble fibres respectively. The sum of the two represents the total dietary fibre. A
reagent blank was used during the procedure.

The calculations were as follows:

% Insoluble Fibre = (W1 – W2) x 10

mass sample

% Soluble Fibre = ((W1 – W2) – Blank) x 100

mass sample

Calculation for sample with < 2% fat:

Total dietary fibre

% Dietary fibre = sample mass X 100

Calculation of % dietary fibre on dried, defatted samples expressed on:

% Dietary Fibre = (Total dietary fibre x 100) X (100 - % moisture) X (100 - % fat)

mass of sample 100 100

3.1.8 Energy Determination

The Atwater system was used to determine the energy values. This system uses factors to
estimate available energy from the protein, fat, carbohydrate, and alcohol components of food
items. Energy was calculated using the general Atwater‟s factors of 4 kilocalorie (kcal) per g
protein, 9 kcal per g fat and 4 kcal per g carbohydrate. These conversion factors were multiplied
by 4.186 in order to obtain energy values in kilojoules (kJ) (WHO, 1985):

Energy (kJ) = (4 kcal/g g protein 4.186) + (4 kcal/g g carbohydrate

4.186) + (9 kcal/g g fat 4.186)

3.1.9 Mineral Analysis

The mineral metallic elements calcium, copper, iron, magnesium, manganese, zinc, sodium and
phosphorus were determined on dried samples that were digested in a microwave digester using
the method of Milestone Microwave Lab Systems (MMLS, 1999). The concentrations of the
minerals were determined with an Inductively Coupled Plasma (ICP) spectrometer (Perkin-
Elmer). Sample solutions were quantified against standard solutions of known concentrations that
were analyzed concurrently according to Perkin Elmer (Perkin, 1996). All assays were carried out
in duplicate. Mean values and standard deviations are based on these results.

Sample Digestion

Duplicate aliquots (0.5 g) from each of the dried plant specimens were weighed into teflon
vessels to which 5 ml concentrated nitric acid and 2 ml hydrogen peroxide were added. Each
vessel was closed with its teflon cover and adapter and tightened with a spring disc. Vessels were
positioned on the rotor and were secured by placing a circular safety band around them. The rotor
was placed onto its base and each vessel was tightened using a torque wrench. The microwave
oven and the fume extractor were switched on and the rotor was transferred to the microwave
oven. The appropriate program from the instrument user manual was selected and the following
parameters were entered:

Step 1 Step 2 Step 3 Step 4 Step 5

Power(W) 250 0 250 400 600

Time (Min ) 1 2 5 5 5

Once the operation was complete, the oven was switched off a nd the rotor was taken out of the
oven. The vessels were allowed to cool and the contents were transferred into 50 ml volumetric
flasks and made up to 50 ml using double deionised water (MMLS, 1999).

Mineral Analysis

Mineral analysis by Inductively Coupled Plasma Spectrophotometry was carried out according to
protocols obtained from Perkin (1996) at ALEX STEWART TES BRETBY (KZN) (Pty) Ltd.

Stock solutions of the mineral elements were prepared from standard solutions of 1 mg/ml. The
ICP spectrometer was ignited and the ICP 400 software program (Perkin, 1996) was loaded. The
extractor fan, argon gas and the spectrometer were switched on. The peristaltic pump was turned
on and deionised water was aspirated for 1 min. The torch was ignited and the nebulizer argon
flow was set by performing the bullet test. This was achieved by aspirating a 1000 mg/ml
solution of sodium. The plasma was examined through the viewing window of the torch
compartment door and a yellow-orange bullet, extending from the base of the discharge to a point
about 2-3 mm above the top of the RF coil, was visible in the central channel of the discharge. A
satisfactory bullet height was achieved by adjusting the nebulizer argon flow incrementally using
the nebulizer adjustment knob. After setting the nebulizer argon flow, the system was allowed to
stabilize for 1 hour before running samples.

A method was developed by entering the element parameters into the element mode and the
samples, standards and blanks were aspirated and read. The calibrated wavelengths as well as the
element parameters were stored in the element mode whilst the element parameters and element
file names were entered and stored in the method mode. The method file name was accessed and
the standards, blank and samples were aspirated and read. A quality control standard of known
concentration of each element analyzed was determined after every five samples in order to
verify accuracy of the procedure. The concentrations of minerals were calculated using the
concentration from the ICP analysis reports using the formula:

Concentration (mg/100 g) = concentration (ppm) volume (ml) X 100

mass of sample (g)

3.1.10 Vitamin Analysis

3.1.10.1 Vitamin A

Vitamin A analysis was carried out using the Carr Price Method (Hoffman, 1969).

In this study 5 g of the plant sample and 5 g of the retinol standard were weighed individually
into a 100 ml flask containing 63 ml of the alkali alcohol solution (7.5 g potassium hydroxide in
63 ml ethanol). The solution was heated for 25 mins under nitrogen. Then the contents of the
flask were transferred into a separating funnel and allowed to cool. 100 ml of petroleum ether
was added to the funnel. Following which the air was displaced using nitrogen. Shook the funnel
vigorously and the cap was removed to allow the layers to separate. The contents were
centrifuged for 5 min at 3000 rpm to separate the layers. Pipetted out 20 ml of the petroleum
ether layer and filtered through a dried funnel containing anhydrous sodium sulphate. The filtrate
was evaporated using a rotary evaporator (Buchi RE Rotoevaporator including a Buchi 461 water
bath) and 5 ml of chloroform was added to the flask. From this 1 ml of the sample and standard
was taken and added to 2 ml of antimony trichloride solution. The absorbance was measured at
620 nm. Each test was carried out in duplicate.

The amount of Vitamin A present in the samples was calculated based on the following formula :

Vitamin A (I.U.) = E A x C x 5x 100 x100

EB x 20 x sample weight

EA = average extinction of the sample preparation

EB =average extinction of the standard preparation

C =I.U. of vitamin A contained in 1 ml of the vitamin A standard solution

3.1.10.2 Vitamin B 1

Vitamin B1 analysis was carried out using a fluorescence method described as the Thiochrome
Method (Hoffman, 1969).

2 ml of each plant sample and standard was taken separately into a 100 ml flask containing 50 ml
distilled water and 20 ml of 10% HCl. This solution was heated for 10 min in a water bath at
60°C. The solution was cooled and then volume was made up to 100 ml using distilled water.
The solution was centrifuged at 5000 rpm for 10 min. After centrifugation, 5 ml of the
supernatant was added to 100 ml of distilled water and then 5 ml of Solution A (30 g of
potassium hydroxide in 100 ml distilled water) and Solution B (300 mg of potassium ferricyanide
in 6 ml distilled water) was also added. Then, after 90 s, 10 ml of isobutanol was added and the
samples were centrifuged for 5 min at 5000 rpm to separate the layers. 5 ml of the upper layer
was pipetted out into a test tube and 2 ml of alcohol was added and mixed. The fluorescence of
the sample and standard were measured with a fluorimeter using a primary filter (maximum
transmission at 360-365 mol) and a secondary filter (maximum transmission at 460-480 mol).

Vitamin B1 was calculated according to the following formula:

Vitamin B1 (µg/ml) = FA x 0.8

FB

FA = average fluorescence of the sample preparations

FB = average fluorescence of the standard preparations

0.8 = µg/ml of thiamine hydrochloride contained in 1 ml of the thiamine standard solution

 3.1.10.3 Vitamin B 2

Vitamin B2 analysis was carried out using the Fluorimetric method (Hoffman, 1969)

In this study 0.8 mg of plant sample and standard solution was added to a 100 ml volumetric
flask containing 80 ml of solvent mixture (pyridine: glacial acetic acid : distilled water, 10:1:40
v/v) The solution was heated on a water bath at 60°C for 10 min. The flask was cooled and
diluted to 100 ml with the solvent mixture. Thereafter, 20 ml of the solution was transferred to a
centrifuge tube and 1 g of purified kieselguhr was added. This was centrifuged at 5000 rpm for
10 min. 5 ml of the supernatant solution was diluted with 100 ml of the solvent mixture (pyridine:
glacial acetic acid: distilled water, 10:1:40 v/v). The fluorescence of the sample and the riboflavin
standard solution was measured with a fluorimeter, using a primary filter (maximum transmission
at 400-420 mu.) and a secondary filter (maximum transmission at 550-700 mu).

Vitamin B2 was calculated according to the following formulae:

Vitamin B2 (µg/ml) = FA x 0.4

FB

FA = average fluorescence of the sample solution

FB = average fluorescence of the riboflavine standard solution

0.4= µg of riboflavine contained in 1 ml of the riboflavine standard solution

3.1.10.4 Vitamin B3

Vitamin B3 analysis was carried out using a method described by (Horowitz, 2000).

Pipetted out 0, 5, 10, 15 and 20 ml of standard solution of Niacin in a series of flasks and 5 g of
the sample was weighed in a separate flask. 1.5 g of calcium hydroxide and 60 ml of distilled
water was added to each of the flasks and autoclaved at 121°C for 15 min. The contents were
made up to 100 ml and centrifuged at 5000 rpm for 10 min followed by placing it in an ice bath
for 15 min. From this, 20 ml of the supernatant was transferred into separate centrifuge tubes
containing 8 g ammonium sulphate (NH4 )2 S04 and 2 ml phosphate buffer solution. The tubes
were placed in a water bath at 55°C for 15 min and then centrifuged at 5000 rpm for 15 min and
filtered through Whatman No. 1 filter paper. To the supernatant 10 ml distilled water was added
and placed in an ice bath for 30 min. 1 ml sulphanilic acid was added and placed into the ice bath
for 15 min. For the reagent blank, 10 ml cyanogen bromide and 1 ml sulphanilic acid was added
and placed in an ice bath for 15 min. The absorbance was measured at 470 nm.

Vitamin B3 was calculated as follows:

mg Vitamin B3 / 100g sample = Concentration

10 × weight of sample

3.1.10.5 Vitamin C

Vitamin C analysis was carried out using a method described by Hoffman (1969).

Pipetted out 2 ml of Vitamin C solution (Ascorbic Acid) and 5ml of 1% oxalic acid was added to
a conical flask. This solution was titrated against the indicator (0.050 g Phenol- indo- 2:6 -
dichloro - phenol was dissolved in 200 ml of hot water containing 0.042 g Sodium bicarbonate)
until a light pink end point was obtained. This is A, the average of the duplicate standard titres.

Pipetted out 7 ml of 1% Oxalic acid and 16ml distilled water was added into a conical flask. This
solution was titrated against the indicator until a light pink end point was obtained. This is B, the
average of the duplicate blank titres.

For the sample, 10 g of plant sample was dissolved in 1% Oxalic acid. Then it was diluted in 100
ml distilled water and filtered. Pipetted out 10 ml of the filtered sample and titrated against the
indicator until a light pink end point. This is C, the average of the duplicate sample titres.

Vitamin C was calculated as follows:

Weight of Standard (W) = Weight of Vitamin C (g)

100 ml of Standard Vitamin C Solution

mg Vitamin C/100g = 100 × W × 2 × 100 × C

(A - B) × Weight of sample

3.2 Biological Screening

3.2.1 Sample Collection and Preparation

The plants were collected and stored as described in Section 3.1.1.

3.2.2 Preparation of Aqueous and Methanolic Extracts

Preparation of the Aqueous and Methanolic Extracts

Aqueous extraction of the dried plant material was carried out according to the procedure
outlined by (Jeremy and Whiteman, 2003) with minor modifications. 20 g of the dried plant
material was stirred for 24 h in 200 ml of distilled water. The slurry was filtered using Whatman
No. 1 filter paper and the supernatant was collected. This was then concentrated by placing the
supernatant in a biofreezer (Snijders Scientific, Holland) at -70°C and then freeze dried (Virtis
Benchtop Freeze Dryer). The freeze dried material was used as a stock and working solutions
were prepared for appropriate applications.

Methanolic extracts of the dried plant material were carried as above but the sample was
extracted in 80% methanol. The supernatant was concentrated using a Buchi RE Rotoevaporator
including a Buchi 461 water bath set at a temperature of 50°C. The concentrate was placed in a
biofreezer and freeze dried using a Virtis Benchtop Freeze Dryer. Aliquots were prepared from
the dried crude extract and dissolved in methanol, acetone or dimethyl sulfoxide (DMSO)
depending on the experimental protocol.

The yield per 100 g of plant material was calculated using the following equation:
Amount of dry extract per 100 g = Amount of Final Product X 100

Amount of Sample Added

3.2.3 Determination of Antioxidant Activity

The anti-oxidative properties of the crude extracts were tested using the DPPH (1.1-diphenyl-2-
picrylhydrazyl radical) photometric assay (Choi et al., 2002).

Preparation of plant material

The freeze dried aqueous and methanolic plant material (1000 g/ml) were diluted to final
concentrations of 500 g/ml, 250 g/ml, 100 g/ml, 50 g/ml, 10 g/ml and 1 g/ml in ethanol.

Preparation of Standard

Rutin (Sigma) found in the buckwheat plant Fagopyrum esculentum, was used as a comparative
standard.

DPPH Photometric Assay

1 ml of 0.3 mM DPPH in ethanol, was added to 2.5 ml of plant sample solution of different
concentrations and were allowed to react at room temperature for 30 min. 1.0 ml ethanol plus
plant extract solution (2.5 ml) was used as a blank, while DPPH solution and 2.5 ml ethanol was
used as a negative control. The positive control was DPPH solution (1 ml) plus 2.5 ml 1mM
Rutin. Each test was carried out in triplicate and results are expressed as the mean and standard
deviation of the mean. The absorbance values were measured in a Varian Cary 1E UV- visible
spectrophotometer at 518 nm and the average absorbance values was converted into the
percentage antioxidant activity, using the following equation:
Scavenging capacity %=100- (Abs of sample-Abs of blank) × 100

Abs of negative control

 3.2.4 Anti-inflammatory properties

Determination of the 5- lipoxygenase activity.

Lipoxygenase is known to catalyse the oxidation of unsaturated fatty acids containing 1-4 diene
structures. The conversion of linoleic acid to 13-hydroperoxy linoleic acid was followed
spectrophotometrically by the appearance of a conjugate diene at 234 nm on a UV/Visible
spectrophotometer (Varion Cary 1E UV- Visible spectrophotometer). Nordihydroguaiaretic acid
(NDGA) and Rutin known inhibitors of soybean lipoxygenase, were used as controls. The
reaction was initiated by the addition of aliquots (50 μl) of a soybean lipoxygenase solution
(prepared daily in potassium phosphate buffer 1M pH 9.0) in a sufficient concentration to give an
easily measurable initial rate of reaction to 2.0 ml of sodium linoleate (100 μM) in phosphate
buffer. The enzymatic reactions were performed in absence or in presence of inhibitor and their
kinetics were compared. The inhibitors were dissolved in DMSO in such a manner that an aliquot
of each (30 μl) yielded a final concentration of maximum 100 ppm in each assay. The initial
reaction rate was determined from the slope of the straight line portion of the curve and the
percentage inhibition of the enzyme activity was calculated by comparing with the control (using
30 μl of phosphate buffer (pH 9.0) instead of 30 μl of the inhibitor solution). Each inhibitor
concentration was tested in triplicate and the results averaged; the concentration that gave 50%
inhibition (IC 50 ) was calculated from the outline of the inhibition percentages as a function of the
inhibitor concentration (Njenga and Viljoen, 2006). Aqueous extracts (IC 50 ≥100 μg/ml) were not
taken in this study. All the analysis were carried out in triplicate and the results were expressed
the mean ±SD. Regression analysis was used to calculate IC 50 , defined as the concentration of
inhibitor necessary for 50% inhibition of the enzyme reaction.

3.2.5 Antimicrobial Activity

The antimicrobial activity of methanolic and aqueous plant extracts were carried out on selected
bacteria and fungi by evaluating the bactericidal and anti fungal effect and the minimum
inhibitory concentration on selected bacteria and fungi in a petri dish using the agar disk
diffusion method (Vlotman, 2003).

Determination of Antibacterial Activity

The ten bacteria used as test organisms were as follows: Bacillus cereus (DBT*_F), B.
stearothermophilus (DBT*_Q), Escherichia coli (DBT*_L), Klebsiella oxytoca (DBT*_AM),
Micrococcus sp. (DBT*_AR), Pseudomonas aeruginosa (DBT*_D), Proteus mirabilis
(DBT*_O), Salmonella typhimurium (DBT*_AF), Staphylococcus aureus (DBT*_E) and S.
epidermis (DBT*_ Q). * DBT is a reference for the Durban University of Technolgy Culture
Collection which is based at the Department of Biotechnology and Food Technology.

Stock cultures were prepared from the Culture Collection and stored in micro bank vials us ing
50% glycerol. When required the cultures were plated out on Tryptone Soya Agar (Biolab) plates
and were subsequently grown in Tryptone Soya Broth (Biolab) for 24 h at 37°C. The absorbance
of bacterial cells was adjusted to MacFarland Standard of 0.5 which corresponded to 108
CFU/ml.

Molten (45°C) sterile tryptone agar (10 ml) in a flask was inoculated with a 0.1 ml of 108 cfu/ml
of each of the respective bacterial strains. This was poured over the base plates containing 10 ml
tryptone agar in sterile 9 cm Petri dishes. 50 l of plant extracts at different concentrations (1000
µg/ml, 500 µg/ml, 250 µg/ml, 100 µg/ml, 10 µg/ml, 1 µg/ml) were pipetted on 5 mm sterile filter
paper disks (Whatman No 1); and air dried in a biological safety cabinet laminar. Sample
containing discs were placed on the surface of the bacterial plates inoculated and incubated at
37°C for 24 h except for B. stearothermophilus, which was incubated at 50°C. Control disks with
ethanol (5 l) served as the negative control, whilst Cipro floxacin (5 mg per disk) was the
positive control. All tests were carried out in triplicate. The minimum inhibitory concentration
(MIC) was taken as the lowest concentration of plant extract that inhibited growth after
incubation.

Determination of Antifungal Activity

The two yeasts and seven fungi used as test organisms were: Candida albicans (DBT*_AB) and
Saccharomyces cerevisiae (DBT*_R), Aspergillus flavus (DBT*_AR), Cladosporium sp
(DBT*_AS), Fusarium verticilloides (DBT*_AT), Geotrichum sp (DBT*_AA), Penicillium sp
(DBT*_AC), Rhizopus sp (DBT*_Y) and Trichoderma sp (DBT*_AU).
The yeast cultures were recovered by growing from stock cultures in Sabourand Dextrose Broth
for 24 h at 37°C. The moulds were grown on Sabourand Dextrose Agar at 28°C for 4 to 7 days
until sporulation. The spores were collected in 10 ml sterile distilled water, counted in a counting
chamber (Neubauer) and the concentration adjusted to 10 6 spores/ml. Sterile distilled water
containing the fungal spores (106 spores/ml) were poured over the Sabourand Dextrose Agar
(SDA) base plates (Biolab). 50 µl of each extract (1000 μg/ml, 500 μg/ml, 250 μg/ml, 100
μg/ml, 10 μg/ml, 1 μg/ml) was transferred onto each of three sterile 9 mm discs (Whatman No.
1). 50 µl of ethanol served as the negative control, and 5 μg/ml Amphotericin B (Fluka,
Biochemika), was used as a positive control. Each plant extract and control was tested in
triplicate. The plant extracts and ethanol impregnated discs were dried in sterile Petri dishes and
incubated at 30°C. Antifungal activities were recorded as the width (mm). The minimum
inhibitory concentration (MIC) was taken as the lowest concentration that inhibited growth.

3.2.6 Anti Mosquito Activity

Mosquito repellency, larvicidal and insecticidal activity against Anopheles arabiensis were
carried out at the Medical Research Council, South Africa under the guidance of Ms R. Gayaram.

Determination of Mosquito Repellency

Repellent activity was assessed by topical application of 1000 µg/ml of plant extract to the skin
and subsequent exposure of the treated areas of skin to unfed female mosquitoes. The rodent
Mastomys coucha was the test animal used for the screening of extracts for repellency activity.

The repellency analysis followed the South African Medical Research Council standard protocols
and is depicted by figures. 4a-4d below. Ethical approval for the use of Mastomys in these trials
was obtained from the MRC‟s Ethics Committee for Research on Animals.

Animal preparation

For each plant extract four adult Mastomys were weighed individually and injected
intraperitoneally with sodium pentobarbital (Fig. 4b). The anesthetized rodents were shaved on
the ventral surface and 1000 µg/ml of plant extract was applied to each of two rodent‟s abdomens
(Fig. 4c). The third animal served as a negative control and the fourth animal was a positive
control (N, N-Diethyl- meta-Toluamide (DEET)).

Repellency assay

Paper cups (500ml) were modified by replacing the base of the cup with mosquito netting held in
place with a rubber band and covering the mouth of the cup with transparent plastic film. Thirty
unfed 4-day old A. arabiensis females (Fig. 4a) were placed in the cup and held in contact with
the treated ventral surface of each rodent. Mosquito activity was observed through the transparent
plastic film. After a period of two mins the numbers of mosquitoes probing were recorded (Fig.
4d). The rodent was then returned to the animal facility and allowed to recover from anesthetic.
Each rodent was monitored for 7 days for adverse reactions to the applied plant extracts.

Repellency of the extracts was calculated using the formula:

% mosquito repelled = number repelled × 100

number introduced
 A B

D C

Fig. 4: Repellency (a) Unfed A. arabiensis females introduced into cup. (b) Rodent
anaesthetized using sodium pentobarbital. (c) Plant extract/Positive control (DEET)
applied to rodent’s abdomens. (d) Unfed A. arabiensis females held in contact with
treated ventral surface of rodent

Larvicidal assay

The larvicidal bioassay followed the WHO standard protocols (WHO, 1981b) with slight
modifications. 1 ml of the extract solution was added to polypropylene containers (10 cm x10
cm) containing 0.25 liters of distilled water. Thirty 3 rd instar larvae of A. arabiensis were placed
in the container. A negative control was set up in which solvent was o nly added instead of
extract. A positive control was set up using Mostop, an organo-phosphate (used by the malaria
control program as a larvicide). Each container was monitored for larval mortality over a 7 day
period.

Insecticidal Assay

The adulticidal effect was assayed following a slightly modified version of the WHO standard
method (WHO, 1981a) depicted by figures 5a-5e below.

1 ml extract solution or 1 ml of the positive control (K-Orithrine) or 1 ml negative control

(acetone or distilled water) was sprayed onto a clean dry non-porous ceramic tile using the pre-
calibrated Potter‟s Tower. The negative controls in this experiment were acetone and distilled
water. The Potter‟s Tower (Fig. 5a) was cleaned with acetone between each different extract
application. The sprayed tiles (Fig. 5b) were air dried and assayed within 24 h of spraying. A
standard bioassay cone was fixed in place over the sprayed tile and thirty blood- fed A. arabiensis
females 3-5 days old were introduced into the cone (Fig. 5c). The mosquitoes were then observed
for knockdowns after 30 and 60 min of exposure (Fig. 5d). The test species were thereafter
removed from the bioassay cone and transferred to a holding cage containing a nutrient solution
(Fig. 5e). After 24 h the number of dead mosquitoes was recorded and percentage mortality
calculated.
 A B C

E D

Fig.5: Insecticidal Assay (a) Potter’s Tower. (b) Plant extract/ Positive control (K-orithine)
sprayed on ce ramic non porous tiles. (c) A. arabiensis (30) females introduced into
bioassay cone. (d) Observed for knockdown after 30 & 60 min of exposure. (e)
Transferred to holding cage containing nutrient solution overnight to check for
mortality

3.3 Safety Evalution of Plants

3.3.1 Cytotoxicity

Cell proliferation inhibition was assessed by the MTT assay (Hanelt et al., 1994).

Cell Line
Human chronic myelogenous leukaemia (K562) was used in this study. The K562 cell line was
purchased from Highveld Biological, Modderfontein, South Africa. The cells were received in an
active state and immediately incubated at 37ºC in a humidified incubator (Snjiders Hepa, United
Scientific group, Cape Town South Africa) with 5% CO 2 . When the cells were 80% confluent,
they were sub-cultured, and stock cultures were stored at -70ºC until required.

Cell maintenance

Cell maintenance was performed according by protocols obtained from Freshney (1987).

All cell culture procedures were carried out in a laminar flow cabinet containing a UV light,
(Scientific Engineering INC). The unit was swabbed /sterilized with 70% ethanol (Merck, South
Africa) before each use.

The cells were grown aseptically in 75 cm2 tissue culture flasks (T 75) (Greiner, Germany) using
filter sterilized (0.22 mm) 10% Complete Culture Medium (CCM) which comprised of
Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum and
supplemented with antibiotics (penicillin: 10 000 U/ml, streptomycin sulphate 10 000 U/ml) and
1 mM sodium pyruvate). Cells were incubated in a humidified incubator under 5% CO2 at 37°C
and passaged weekly. All the above media and chemicals were obtained from Highveld
Biological, South Africa. The cultures were incubated at 37°C in a humidified incubator (Snjiders
Hepa, United Scientific group, Cape Town South Africa) with a 5% CO 2 atmosphere. The culture
flasks were examined for colour changes and turbidity of the media on a daily basis. This
determined the frequency of media changes. The culture was examined under an inverted
microscope (Nikon) for cell growth

The cells were harvested when the culture was 80% confluent and divided into two separate
flasks, then more medium added to each culture flask and incubated at 37°C humidified incubator
(Snjiders Hepa, United Scientific group, Cape Town South Africa) with a 5% CO 2 atmosphere.

The cells were enumerated using a haemocytometer. The cell suspension was mixed with equal
aliquot of 0.2% Trypan Blue [Biowhittaker, Wakersville (USA)] (v/v 1:1). This mixture was
drawn across the grid by capillary action. The volume of cell suspension that occupied one
primary square is 0.1mm3 (1.0mm2 ´ 0.1mm/ 1.0 ´ 104 mL). Only the viable (translucent) cells
that lay within or that touched, the left or top boundary was counted. The number of viable cells
per ml in the original sample was calculated as follows:

Cells/ml = Average number of cells per primary square × 104 dilution factor

Storage of cells

Storage of cells was performed according by protocols obtained from Freshney (1987).

The cells were pelleted and washed twice with pre-warmed Phosphate Buffered Saline, pH 7.2
(PBS). Resuspended in 0.5 ml FCS and cooled on ice. A 20% dimethylsulphoxide (DMSO) in
DMEM (V/V 1:4) was prepared as the cryoprotecting agent and also placed on ice. Equal
aliquots (0.5ml) of the cell suspension and the cryoprotective agent were added to a cryotube
(Corning , South Africa ). The tubes were transferred to the thermos flask and kept overnight at -
20°C. The cells were subsequently transferred to a –70°C bio- freezer and stored until required.

Regeneration of cells

Regeneration of cells was performed according to protocols obtained from Freshney (1987).

Cells were removed from the –70°C biofreezer, swabbed with 70% ethanol and rapidly thawed.
The cells were then transferred to 20 ml of pre-warmed 10% CCM in 75 cm2 tissue culture flasks
and incubated at 37°C humidified incubator with a 5% CO 2 atmosphere.

MTT assay

The MTT cytotoxity assay was conducted according to Mosmann, (1983) with minor
modifications. The assay was carried out in 96 well, flat bottomed microtitre plates (Cellstar,
Greiner, Germany). 200 µl of ±1.2 x103 of cells was added into each well, 20 µl of the plant
(1000 µg/ ml, 100 µg/ ml 10 µg/ ml) extracts were added to the respective wells. In the control
wells 20 µl DMSO and 20 µl media only respectively were added. The plate was incubated in a
37°C humidified incubator with a 5% CO 2 atmosphere for 20 h. Then 20 μl of MTT reagent
(Sigma, St Louis, USA) was added, the plates were then incubated for a further 4 hours at 37ºC in
a humidified incubator with 5% CO 2 atmosphere. Subsequently 100 µl of 100% DMSO was
added to each well and the plate was incubated for an additional 1 h. The absorbance was read at
578 nm on an ELISA plate reader (Digital Analogue Systems, Italy ).

The percentage viability was determined using the formula below:

% Cell Viability = Absorbance of treated cells x 100

Absorbance of untreated cells

3.3.2 Toxicity

The brine shrimp lethality assay was used with minor modifications (Meyer et al., 1982).

Sample Preparation

10, 100, and 1000 µg/ml of plant extract were dissolved in DMSO and 50 µl was impregnated on
filter paper disks. The disks were allowed to dry in an open sterile Petri dish in a biological safety
cabinet with a vertical laminar flow for 1 h. Control disks were prepared using only DMSO.
Three replicates of each dose and the control were tested.

Hatching the shrimp

25 mg of Class C Artemia salina eggs (Natures Petland, Durban, South Africa) was added to
artificial salt water (23 g NaCl, 11g MgCl2 ·6H2 O, 4 g Na2 SO4 , 1.3 g CaCl2 ·2H2 O, 0.7 g KCl in
1000 ml distilled water) and kept at room temperature. The pH was adjusted to 9.0 using Na2 CO3
to avoid risk of death to the Artemia larvae by decrease of pH during incubation. This was
incubated in a hatching chamber at room temperature. After 24 h, 15 ml of yeast solution was
added to the chamber for every litre of salt water in order to feed the larvae, 48 h after the eggs
were incubated, the larvae were extracted by picking up the moving larvae and visibly counted.

Bioassay
Every vial with 100 µl of plant sample at different concentration (10, 100, 1000 µl) contained 10
larvae of brine shrimp, including the control group, and was filled to 5 ml total volume with
artificial salt water. A drop of yeast suspension (3 mg in 5 ml sea water) was added to each vial.
The vials were then incubated at 27°C for 24 h. After 24 h, dead larvae were counted and
percentage death determined.

3.3.3 Ames Mutagenicity Test

The Salmonella mutagenicity assay was conducted according to the method described by Maron
and Ames, 1983 with minor modifications. The tester strains TA 98 and TA 100 were obtained
on disc cultures from MOLTOXT M. The disc cultures described were prepared from master
cultures obtained from Dr. B.N. Ames (Berkeley, California, USA)

From the frozen disc culture of the S. typhimurium TA 98 and TA 100 tester strain, broth cultures
were made. Using a flamed bacteriological needle, one of the culture disks was aseptically
removed and inoculated into a sterile 250 ml Erlenmeyer flask containing 25 ml of nutrient broth
(Oxoid) and 78 µl of 8 mg/ml Ampicillin (to maintain the stability of the plasmid). The flask was
incubated on a shaker (150 rpm) at 37ºC for 16 h to obtain an optical density at 660 nm of
between 1.2 and 1.4.

In a sterile test tube, 100 µl of culture was added to 2 ml of 0.05 mM histidine/0.05 mM biotin
top agar (Appendix A), vortexed and plated onto a minimal glucose agar plate. The plate was
incubated at 37ºC for 48 h. Well separated colonies were used from this plate for initial broth
cultures.

Broth cultures of S. typhimurium were made by inoculating nutrient broth with master plate
colonies. These cultures were incubated on a shaker (150 rpm) at 37 ºC for 24 h. The plant
extracts were dissolved in DMSO to obtain concentrations of 100 µg/ml, 1000 µg/ml and 10 000
µg/ml. Sodium azide (NaN 3 ) is a highly mutagenic compound and was used as a positive control.
NaN 3 was dissolved in DMSO to obtain concentrations of 5 µg/ml, 10 µg/ml and 20 µg/ml.
Sterile distilled water was used as a negative control.
Three plates were prepared for each concentration of test compound. In a sterile test tube, 100 µl
of bacterial culture, 100 µl of test compound and 2.9 ml of soft agar (Appendix A) held at 45 ºC,
were added. This was briefly mixed with a vortex mixer and poured onto glucose minimal agar
plates (Appendix A). Once the agar overlay solidified, the plates were inverted and incubated at
37ºC for 48 h, after which the number of revertant colonies (i.e. histidine dependant) colonies
were counted and the mutant frequency determined. The mutant frequency was expressed as the
quotient of the number of revertant colonies over the number of colonies in the negative control.

This can be expressed in the formula:

Mutant Frequency = Revertant number of colonies

Negative control

3.4 Microprogation of A.aspera, A.sessilis and G.densa

Plant Material

A. aspera, A. sessilis and G. densa were used in this study. The plant material was collected and
the leaves were harvested and washed thoroughly in tap water to remove the impurities and
subsequently washed three times in sterile distilled water.

Surface sterilization of explants

The leaves were disinfected either for 5 min in 0.1% Mercury Chloride and for 20 min in 40%
Sodium Hypochlorite or for 5 min in 0.1% Mercury Chloride and for 15 min in 30% Sodium
Hypochlorite or for 20 min in 30% Sodium Hypochlorite only. It was then thoroughly rinsed
three times with sterile distilled water under a laminar flow hood.

Callus induction

Preparation of media for callus induction

The MS medium (Murashige and Skoog, 1962) purchased from Sigma was used in this study.
For callus initiation the basal MS media was manipulated with auxin (2, 4-D) and cytokinin
(BAP) in different concentrations. The different combinations from 0.5 mg/L to 1 mg/L were
used. Fresh stock solutions were made twice a month. The pH of the media was always verified
to be 5.8. Cefotaxamine 25 mg/L and Fungizone 25 mg/L were the antibiotics used to prevent
microbial contamination.

After sterilization the leaves were cut into square pieces using sterile scalpel blades. Five leaf
squares were placed on each Petri plate containing the basal salt MS medium (42.2 g/L MS) and
plant growth regulators. All inoculations were performed under the laminar flow to maintain
aseptic conditions. All cultures were maintained at 25 °C in a 16 h photoperiod (16 h light/16 h
dark) in a growth chamber.

Shoot Regeneration using Callus

Callus obtained from MS medium supplemented with 1 mg/L 2, 4-D and 1 mg/ L BAP were
transferred on shoot regeneration medium having different concentrations of hormones. This was
kept in a growth chamber (Polychem Supplies, South Africa) with a 16 h photoperiod and
observed for shoot formation. All cultures were transferred every four weeks unless
contamination was noted. After shoot formation had occurred the individual shoots were
transferred into tissue culture grade bottles and the shoots were allowed to further multiply.

Root induction

Regenerated shoots are separated from the cultures individually and used for root induction. The
media used for root induction was half strength MS (21.1 g/L) supplemented with different
concentrations of NAA and IBA respectively to determine the best root induction medium.

Hardening of regenerated plantlets

When the plantlets attained adequate growth by producing 4-6 leaves with sufficient root system,
they were removed from the culture medium and washed in running tap water carefully to
remove the media adhering to roots. They were subsequently transplanted in bottles containing
sterilized soil. The plants were covered with polyethylene bags and kept in the culture room.
After 15 days, the polyethylene bags were removed and well established plants were transferred
to greenhouse.
CHAPTER FOUR: RESULTS AND DISCUSSION

4.1 Nutritional profile

Proximate composition

The carbohydrate, protein, moisture, energy, fat, dietary fibre and ash levels of the famine food
plants and those of other edible species from Amaranthaceae family are shown in Table 1. A.
sessilis had the highest energy value (636.11±8.56 g/100 g), followed closely by A. aspera
(513.99±9.88 g/100 g) and G. densa (341.37±2.63 g/100 g).

The moisture levels in A. aspera, A. sessilis and G. densa were 83.24±2.5 g/100g, 65.5±5.0
g/100g and 54±1.4 g/100g respectively. The high moisture content of A. aspera is keeping with
its succulent nature and the low moisture content in G. densa is due its woody nature.

The carbohydrate values for A. aspera. A. sessilis and G. densa were 4.29±0.03 g/100 g,
4.01±0.25 g/100 g and 3.07±0.03 g/100 g respectively. The highest protein levels were found in
A. sessilis (27.70±0.12 g/100 g) when compared to A. aspera (21.87±0.10g/100g) and G. densa
(12.95±0.08g/100g). The fat content of A. aspera was marginally lower than A. sessilis followed
by G. densa (2.06±0.16 g/100 g, 2.8±0.06 g/100 g and 1.95±0.05 g/100 g respectively). G. densa
(48.84±1.43) contained the highest amount of dietary fibre in comparison to the other famine
food plants A. aspera and A. sessilis. Odhav et al. (2007) reported low dietary fibre contents for
A. spinosus, A. hybridus and A. dubius. The famine food plants analyzed in this study contained
higher dietary fibre contents than the leafy vegetables from the Amaranthaceae family.

A large proportion of our daily energy and nutritional needs is obtained from plants. The
proportion of energy from plant foods varies from about 96% in poor countries to as low as 50%
in urban societies. During famine conditions or food shortages, non commercialized wild plants
may be the only source of energy. The nutrients in the plants that provide energy are
carbohydrates, fats and proteins. Vitamins and minerals are present in small amounts and are
important in maintaining specific functions in the body. The energy value depends on the water
content and a combination of carbohydrates, fats and proteins. Adults require about 1 500 to 3
000 kilocalories per day. Carbohydrates, fats and proteins and provide about 400 kcal per 100 g
carbohydrates, 900 kcal per 100 g fat and 400 kcal per 100 g protein. The amount of water
 (moisture) in the food has an important influence on the calorific value. Nuts and grains have a
low water content (5 to 10%) and therefore high energy value, while starch tubers and fruits have
high water content (80 to 90%) and thus lower energy value.

These results correlate well with the recently published results of Freedman (2006) for A. sessilis
where the values for moisture, fat and protein are similar. For A. aspera and G.densa we did not
find any published data, these results in Table 1 below are the first. A comparison of the energy
values with cultivated species in the Amaranthaceae family viz. A. spinosus, A. hybridus, A.
dubius and A. hypochondriacus indicate that the famine species A. aspera and A. sessilis in this
study have twice as much energy and also higher values than Maundu et al. (1999) on indigenous
leafy vegetables as well as those reported by Odhav et al. (2007). Furthermore, in A. sessilis the
protein quantity is sufficient to make up 50% of the RDA.

Table 1. The proximate values of famine foods A. aspera, A. sessilis and G. densa and
cultivated species A. spinosus, A. hybridus, A. dubius and A. hypochondriacus
Plants Moisture Ash Dietary CHO Fat Protein Energy
(g) (g) Fi bre (g) (g) (g) (g) (kJ)

A. aspera 83.24±2.5 13.50±1.03 40.85 4.29±0.03 2.06±0.16 21.87±0.10 513.99±9.88

A. sessilis 65.5±4.9 15.6±1.81 38.44±0.47 4.01±0.25 2.8±0.06 27.70±0.12 636.11±8.56
G. densa 54±1.4g 12.51±1.05 48.84±1.43 3.07±0.03 1.95±0.05 12.95±0.08 341.37±2.63
COMPARATIVE STUDY
A. spinosusa 91.36±0.25 2.76±0.34 2.48±0.08 1.16±0.16 0.60±0.01 4.12±0.06 111.02±1.12
A. hybridusa 82.55±0.07 4.91±0.11 2.81±0.03 6.09±0.31 0.53±0.08 5.92±0.04 221.07±1.29
A. dubiusa 84.59±0.38 3.42±0.03 2.87±0.04 7.86±0.55 0.24±0.06 3.89±0.08 205.79±5.68
A. sessilisb 70.40 20.54 11.47 47.99 3.65
A. hypochondriacusc 8.81 15.8 365
a b c
Data are mean ± SD( n=2), (Odhav et al, 2007), (Freedman, 2006), (van Wyk, 2005)

Mineral content

A. aspera and G. densa had similar levels of calcium present (142.97±10 mg/100 g and
140.84±2.5 mg/100 g respectively) and A. sessilis had a lower value (83.97±0.5 mg/100 g) (Table
3). The RDA of calcium for adults is 800 mg per day (NRC, 1989). Thus 100g/day of the famine
plants in this study do not meet the RDA. These values reported in this study are low. Calcium is
an important mineral involved in the building of rigid structures to support the body (Aletor et
al., 2002). Reports of calcium levels in other famine plants Ximenia americana, A. viridus, and
the leaves of the baobab tree (Adansonia digitata) contained higher quantities of calcium (Glew
et al., 2005).

A. aspera and A. sessilis had similar amounts of copper present with values of 0.22 mg/100 g and
0.20 mg/100 g respectively (Table 3). G. densa had a slightly lower amount of copper present
than the other two famine food plants with a concentration of 0.17 mg/100 g (Table 3).
According to the National Research Council, the daily requirement for copper is 2 mg per day
(NRC, 1989). A. aspera and A. sessilis meet about 10 % of the RDA for adults. In comparison to
the leafy vegetables (A. hybridus, A. dubius and A. spinosus) in the Amaranthaceae family
reported by Odhav et al. (2007), the famine food plants in this study had low copper
concentrations. Plants which have high copper concentrations could be useful in preventing a
deficiency of copper which normally results in anemia and bone problems (Arntzen and Ritter,
1984).

A. sessilis and G. densa had iron concentrations of 8.57±3 mg/100 g and 8.71±0.5 mg/100 g
respectively and A. aspera had 4.70±1.6 mg/100 g (Table 3). The RDA for iron is 10 mg per day
adults (NRC, 1989). A. sessilis and G. densa satisfied more than 85% and 87% respectively of the
iron requirement for the RDA for adults. Iron is necessary for the optimal immune function
(Glew et al., 2005). There is a relatively high prevalence of iron deficiency, anaemia among all
age groups in the rural populations (Glew et al., 2005). Odhav et al. (2007) reported very high
levels of iron in the leafy vegetables in the Amaranthaceae family.

In G. densa we found low levels of magnesium (45.27±2.9 mg/100 g), however in A. aspera and
A. sessilis the magnesium concentration were 73.03±2.6 mg/100 g and 53.23±0.2 mg/100 g
respectively (Table 3). The daily consumption of magnesium should amount to 120 mg (NRC,
1989). The famine food plants A. aspera satisfies 60% and A. sessilis satisfies 53% of the RDA
for magnesium. Magnesium is needed for healthy bones and teeth, proper nervous system
functioning, and energy metabolism.

In G. densa we found very low levels of manganese (0.31 mg/100 g) and A. aspera and A. sessilis
had amounts of 5.00±0.2 mg/100 g and 4.47±0.1 mg/100 g respectively (Table 3). The RDA for
manganese is 7 mg (NRC, 1989). A. aspera and A. sessilis meet more than 50% of the daily
 recommended and have a greater manganese concentration than the food plant A. spinosus (Table
3). Manganese is needed for enzyme structure.

A. sessilis had the highest amount (53.58±2.4 mg/100 g) of phosphorus present, followed by G.
densa (35.08±1.0 mg/100 g) and thereafter A. aspera (26.41±0.6 mg/100 g) (Table 3). The RDA
for phosphorus is 800 mg for adults. According to Odhav et al. (2007) the leafy vegetables
analyzed from the Amaranthaceae family satisfied more than half the RDA for phosphorus,
however the plants analyzed in this study had very low phosphorous concentrations (Table 3).
Phosphorus is needed for healthy bones and teeth, energy metabolism, and acid-base balance in
the body.

A. aspera (114.72±6.7 mg/100 g) had a very high concentration of sodium in comparison to G.

densa which only had 12.76±0.7 mg/100 g (Table 3).The RDA for sodium is 300 mg. No
deficiency of sodium is found in the human diet, however excessive intake of sodium can result
in high blood pressure (Jaworska and Kmiecik, 1999).

A. sessilis had the highest concentration of zinc (2.65±1.9 mg/100 g) compared to the other two
famine food plants which had values of 1.85±0.1 mg/100g and 1.01 mg/100g for A. aspera and
G. densa respectively (Table 3). The RDA for zinc is 10 mg per day for adults (NRC, 1989).
Thus, the famine plants from this study do not meet the RDA. Poor zinc status is widespread,
especially amongst populations that consume cereal based diets (Brown and Wuehler, 2000).

Table 2. Comparison of mineral levels of the famine food plants (A. aspera, A. sessilis
and G. densa) to the leafy vegetables (A. spinosus, A. hybridus and A. dubius)
Calcium Copper Iron Magnesium Manganese Phos phorus Sodium Zinc
Plants mg/ ml

A. aspera 142.97±10 0.22 4.70±1.6 73.03±2.6 5.00±0.2 26.41±0.6 114.72±6.7 1.85±0.1

A. sessilis 83.97±0.5 0.20 8.57±3 53.23±0.2 4.47±0.1 53.58±2.4 72.94±2.1 2.65±1.9
G. densa 140.84±2.5 0.17 8.71±0.5 45.27±2.9 0.31 35.08±1.0 12.76±0.7 1.01
COMPARATIVE STUDIES
A. spinosusa 3930.58±15.3 3.36±0.3 31.92±2.4 1165.64±2. 3.02 628.60±7.2 392.92±3 15.47±0.8
A. hybridusa 2363.26±0.4 2.38±0.3 21.2±0.5 1316.88±2 24.38±0.9 603.94±1 427.1±0.1 17.93±0.2
A. dubiusa 1686.2±0.4 2.76±0.1 25.11±0.3 805.63±2.1 81.92±0.9 487.40±0.2 346.99±1 56.12±1.4

Data are mean± standard deviation (n=2) a (Odhav et al., 2007)

 Vitamin composition

The famine food plant A. sessilis had the highest amount of Vitamin A (982.32±33.07 mg/ml)
followed by G. densa (929.46±11.60 mg/ml) and A. aspera (806.55±27.34 mg/ml). According to
the Food and Nutrition Board, the recommended daily allowance is 800 micrograms for adult
females and 1000 micrograms for adult males (NRC, 1989). Vitamin A has widespread
physiological functions in the body. Apart from its effects on vision, the role of vitamin A in
maintaining the structural and functional integrity of mucosal epithelial cells is important.
Vitamin A controls cellular proliferation and differentiation, and thus has significant effects on
the immune system (Bhaskaram, 2002). Deficiency of Vitamin A causes night blindness,
xerophthalmia and keratinisation of skin.

A. aspera had the highest content of Vitamin B1 compared to the other famine food plants tested,
almost twice the amount of A. sessilis and G. densa had 11.08±0.12 mg (Table 2). The RDA for
Vitamin B1 is 1.1-1.5 mg. Vitamin B1 (thiamine) is a coenzyme in pyruvate biosynthesis and for
2-oxo-glutarate dehydrogenases, and transketolase and it also regulates Cl−channel in nerve
conduction. Deficiencies in Vitamin B1 result in peripheral nerve damage or central nervous
system lesions (Bender, 2003).

The Vitamin B2 concentrations in this study ranged from 9.7±0.4 mg in A. sessilis to 41.75±0.35
mg in G. densa (Table 2). This meets more than the recommended daily allowance for Vitamin
B2 . The recommended daily allowance is 1.2-1.7 mg (Bender, 2003). Vitamin B2 (riboflavin)
which acts as a coenzyme in the oxidation and reduction reaction is also a prosthetic group of
flavoproteins. Deficiencies of riboflavin result in lesions of the corner of the mouth, lips, and
tongue and sebhorreic dermatitis.

A. sessilis and G. densa had similar Vitamin B3 levels with values ranging between 8.96±0.10 mg
and 9.05±0.08 mg respectively (Table 2). A. aspera‟s Vitamin B3 level was only 5.98±0.16 mg.
A. sessilis and G .densa meet only 60 % of the RDA. According to the Food and Nutrition Board,
the recommended daily allowance is 15-19 mg (NRC, 1989). Vitamin B3 acts as a coenzyme in
oxidation and reduction reactions, it is a functional part of NAD and NADP and plays a role in
intracellular calcium regulation and cell signalling. Deficiencies lead to pellagra-photosensitive
dermatitis and depressive psychosis (Bender, 2003).
 Vitamin C levels range from 16.27 mg in G. densa to 43.57 mg in A. aspera (Table 2). According
to the Food and Nutrition Board, the RDA for Vitamin C is 60 mg. The famine food plants
studied do not meet the daily allowance standard set for Vitamin C by the Food and Nutrition
Board (NRC, 1989). Vitamin C (ascorbic acid) functions as a coenzyme in hydroxylation of
proline and lysine in collagen synthesis; as an anti-oxidant and enhances absorption of iron.
Deficiencies result in scurvy, loss of dental cement and subcutaneous haemorrhage (Bender,
2003).

The vitamins of relevance to plant foods are the fat soluble vitamins (A, D, E and K) and the
water soluble vitamins (B1 , B2 , B6 and B12 , biotin, C, folic acid, niacin and pantothenic acid).
The recommended daily allowance is the quantity which is required to prevent disease.
Micronutrient deficiencies and infectious diseases often coexist and exhibit complex interactions
leading to the vicious cycle of malnutrition and infections among underprivileged populations of
the developing countries, particularly in preschool children. Several micronutrients such as
vitamin A, β-carotene, folic acid, vitamin B12 , vitamin C, riboflavin, iron, zinc, and selenium,
have immunomodulating functions and thus influence the susceptibility of a host to infectious
diseases and the course and outcome of such diseases (Bhaskaram, 2002).

Table 3. Vitamin levels of A. aspera, A. sessilis and G. densa

Plants Vitami n A Vitami nB 1 Vitami n B 2 Vitami n B 3 Vitami n C

mg/100 g of dry weight

A. aspera 806.55±27.34 20.02±0.12 22.2±0.57 5.98±0.16 43.57

A. sessilis 982.32±33.07 10.98±0.24 9.7±0.42 9.05±0.08 27.19

G. densa 929.46±11.60 11.08±0.12 41.75±0.35 8.96±0.10 16.27

Data are mean± standard deviation (n = 2)

4.2 Biological Screening

4.2.1 Antioxidant activity of plant extracts

Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen
species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite.
An imbalance between antioxidants and reactive oxygen species results in oxidative stress,
leading to cellular damage (Cheng et al., 2003). Oxidative stress has been linked to cancer, aging,
atherosclerosis, ischemic injury, inflammation and neurodegenerative diseases (Parkinson's and
Alzheimer's). Flavonoids may help provide protection against these diseases by contributing,
along with antioxidant vitamins and enzymes, to the total antioxidant defense.

In this study, we used the DPPH assay as it determines the activities of both hydrophilic and
lipophilic chemicals. Rutin, the positive control used reduced 92.43 ±0.22 % whilst the famine
food plants A. aspera, A. sessilis and G. densa reduced 83.86±2.63%, 86.51±0.37 % and 54±55
respectively (Fig. 6). The minimum concentration at which these two plants exhibited free radical
scavenging activity was 100 µg/ml. The aqueous extracts of the three plants showed no
antioxidant activity. Akula and Odhav (2008) reported high antioxidant activities from plants in
the Amaranthaceae family. A. hybridus was reported to have 90.5±0.24%, A. spinosus had
88.2±0.22% and A. dubius had 78.4±0.22% (Akula and Odhav, 2008).

The recognized dietary antioxidants are vitamin C, vitamin E, selenium, and carotenoids. In our
study A. sessilis and A. aspera had vitamin C present. This may explain the high level of anti-
oxidative properties displayed. Furthermore, there is a connection between plant stress levels and
the production of secondary metabolites, including many polyphenols and antioxidants. There is
substantial agreement among plant pathologists, physiologists, and entomologists that relatively
higher levels of antioxidant secondary plant metabolites are produced by plants in response to
biotic and abiotic stress. Famine plants grow in stress conditions and hence it is plausible that
they have better anti-oxidative capacity than commercially cultivated (amaranth species).
 100 A.aspera
A.sessilis
90
G.densa
80

% Scavenging Capacity
Rutin
70
60
50
40
30
20
10
0
1 10 100 250 500 1000
plant concentration (ug/ml)

Data are mean ± standard deviation, n=3

Fig. 6. Free radical scavenging capacity of methanolic extracts of A. aspera, A. sessilis, G.
densa and Rutin

4.2.2 Anti-Inflammatory Prope rties

The in vitro inhibition of soybean lipoxygenase constitutes a good model for the screening of
plants with anti- inflammatory potential (Abad et al., 1995). In our study, we used NDGA as a
standard for the comparison of anti- inflammatory potential of the three famine plants. The IC50
value of NDGA was 2.48 µg/ml and the famine food plants A. aspera, A. sessilis and G. densa
were 246 µg/ml, 341.1 µg/ml and 582.1 µg/ml respectively (Table 4). These results indicate that
the famine food plants analyzed had low anti- inflammatory activity. Reduced IC50 values suggest
better inhibitory action on 5 LOX. A negative result in the lipoxygenase assay does not
necessarily mean that the plant is without anti- inflammatory activity. The active compounds
could work at other sites in the complex process of inflammation (Jäger et al., 1995).

Previous work in our laboratory on anti- inflammatory activity with methanolic extracts among
leafy vegetables from commonly consumed leafy Amaranthaceae species showed that A. dubius
had IC50 value of 69.4 µg/ml and A. spinosus had 57.3 µg/ml (Akula and Odhav, 2008). These
results suggest that the plants of Amarantheceae family do not have chemicals that can induce
anti- inflammatory activity in this specific site. It has been reported by Gokhale et al. (2002) that
the ethanolic extract of A. aspera possessed anti- inflammatory and anti-arthritic activity and he
also reported that A. aspera is used in the indigenous system of medicine for the treatment of
inflammatory conditions; however, there is no reported literature on detailed investigation for
rationality behind their use in inflammation.

Table 4: Anti-inflammatory properties of aqueous and methanolic extracts of A.aspera,

A.sessilis and G. Densa using 5- lipooxygenases
5-LOX IC50 (µg/ml )

Plant Aqueous Methanolic Positi ve Control

A . aspera 0 246 nd

A. sessilis 0 343.1 nd

G. densa 0 582.1 nd

NDGA nd nd 2.48

(n=2), nd: not determined

4.2.3 Antimicrobial Activity

Under many famine and food shortage circumstances, a wide array of microbial pathogens, and a
low immunity increases susceptibility to bacterial and fungal infections. Wild plants are often the
only available means of treating such infections (Taylor et al., 2001). Fennel et al., (2004)
suggested that the use of the plants in treating fungal infections could be even more widely used
as an alternative to the expensive and often unobtainable Western drugs.

The aqueous extract of A. aspera showed zones of inhibition against the Gram negative bacteria
E. coli, P. aeruginosa and S. typhi (Table 5) with MIC values of 250 µg/ml, 100 µg/ml and 100
µg/ml respectively (Table 6) and against two Gram positive bacteria S. epidermis and S. aureus
(Table 6) with MIC values of 1000 µg/ml and 1000 µg/ml respectively (Table 6). In the studies
of Perumal Samy et al. (1998) the aqueous leaf extract of A. aspera did not show any activity
against E. coli, K. oxytoca and P. aeruginosa at lower doses whereas, it exhibited activity against
P. bulgaricus at higher doses of 4000 and 5000 ppm. Similar results related to Perumal Samy et
al. (1998) were reported by Belachew Desta (1993). The extracts of A. aspera analyzed in our
study showed activity against E. coli, P. aeruginosa, K. oxytoca and S. typhi which is
contradictory to Perumal Samy et al. (1998) and Belachew Desta (1993). Lamikarna (1999)
reported that Gram negative bacteria have an impervious cell envelope which makes them
resistant to many antibacterial agents. Although the A. aspera plant extracts did not inhibit fungal
growth, the methanolic extract showed inhibition against the yeast S. cerevisiae (Table 7) at a
minimum inhibitory concentration of 500 µg/ml (Table 8).

The aqueous extracts of A. sessilis showed activity against P. aeruginosa and S. epidermis (Table
5) and the methanolic extracts showed activity against P. aeroginosa and S. aureus. The aqueous
extract of A. sessilis also showed the best inhibition against C. albicans
(Table 7) at a minimum inhibitory concentration of 500 µg/ml (Table 8) in comparison to the rest
of the plant extracts when determining the zone of inhibition. The aqueous and methanolic extract
of A. sessilis showed antifungal activity against S. cerevisiae (Table 7) at a minimum inhibitory
concentration of 500 µg/ml and 250 µg/ml respectively (Table 8).

G. densa extracts showed activity against the Gram negative E. coli, P. aeroginosa and K.
oxytoca (Table 5) as well as the Gram positive B. sterathermophilus and S. aureus (Table 5). The
methanolic and aqueous extracts of G. densa displayed antifungal activity against C. albicans
(Table 7) at minimum inhibitory concentrations of 500 µg/ml and 250 µg/ml respectively (Table
8).
 Table 5. Antibacterial activity of A. aspera, A. sessilis and G. densa
Zone of Inhi bition (mm)
Plant Extracts (1mg/ ml) Ec Pa Ko Pm St Se Bs Bc M Sa

A .aspera (aqueous) 15 11.7±1.5 na na 11.3±1.2 11.7±1.5 na na na 10

A. aspera (methanolic) 13±2 12.7±0.6 12±1 na 11±1.7 Na na na na 11±1.7

A. sessilis (aqueous) na 8.7±0.6 na na na 11±3.46 na na na na

A. sessilis (methanolic) na 13 na na na na na na na 12

G. densa (aqueous) 12±2 11.3±1.5 11.3±0.6 na na na 12.3±2.5 na na na

G. densa (methanolic) 10 7.3±1.5 na na na na na na na 13
Ethanol 8.7±0.6 7±1 10.7±0.6 5 4 10 10 15 15 7
Ci profloxacin 25 30.7±1.2 35 ±1 37±1 32±1 34±1 42.3±2.5 30±1 25±1 30±1

Data are mean± S D (n=3); na: no activity Ec- Escherichia coli, Pa- Pseudomonas aeroginosus,
Ko- Klebsiella oxytoca, Pm- Proteus mirabilis, St- Salmonella typhi, Se-Staphylococcus
epidermis, Bs- Bacillus stereathermophilus, Bc- Bacillus cereus, M-Micrococcus, Sa-
Staphylococcus aureus

Table 6. Minimum Inhibitory Concentration of A. aspera, A. sessilis and G. densa

Mi ni mum Inhi bi tory Concentration
(µg/ ml)
Plant Extracts Ec Pa Ko Pm St Se Bs Bc M Sa

A. aspera (aqueous) 250 100 na na 100 1000 na na na 1000

A. aspera (methanolic) 250 100 1000 na 10 Na na na na 500

A. sessilis (aqueous) na 500 na na na 1000 na na na na

A. sessilis (methanolic) na 250 na na na Na na na na 1000

G. densa (aqueous) 250 250 1000 na na Na 500 na na na

G. densa (methanolic) 1000 1000 na na na Na na na na 500

Data are mean± S D (n=3); na: no activity Ec- Escherichia coli, Pa- Pseudomonas aeroginosus,
Ko- Klebsiella oxytoca, Pm- Proteus mirabilis, St- Salmonella typhi, Se-Staphylococcus
epidermis, Bs- Bacillus stereathermophilus, Bc- Bacillus cereus, M-Micrococcus, Sa-
Staphylococcus aureus
Table 7. Antifungal activity of A. aspera, A. sessilis and G. densa
Zone of inhi bi tion (mm)

Plant Extracts (1 mg/ml) Sc F P Ca R T C G Af

A. aspera (aqueous) na na na na na na na na na

A. aspera (methanolic) 18.33±2.8 na na na na na na na na

A. sessilis (aqueous) 15 na na na na na na na na

A. sessilis (methanolic) 17.33±2.5 na na 15 na na na na na

G. densa (aqueous) na na na 15 na na na na na

G. densa (methanolic) na na na 16±1 na na na na na

Amphotericin B 25 25 25 26 20 20 20 20 20

Ethanol 10 10 10 10 0 0 0 0 0

Data are mean±SD (n=3); na: no activity; F- Fusarium, P- Penicillium, Ca- Candida albicans, R-
Rhizopus, T- Trichoderma, C- Cladosporium, G- Geotrichum, Af- Aspergillus flavus
Table 8: Antifungal minimum inhibitory concentration of A. aspera, A. sessilis
and G. densa
Mi ni mum Inhi bi tory Concentration (µg/ ml)

Plant Extract Sc F P Ca R T C G Af

A. aspera (aqueous) na na na na na na Na na na

A. aspera (methanolic) 500 na na na na na Na na Na

A. sesslis (aqueous) 500 na na na na na Na na Na

A. sessilis (methanolic) 250 na na 500 na na Na na na

G. densa (aqueous) Na na na 250 na na Na na na

G. densa (methanolic) Na na na 500 na na Na na na

Data are mean±SD (n=3); na: no activity; F- Fusarium, P- Penicillium, Ca- Candida albicans, R-
Rhizopus, T- Trichoderma, C- Cladosporium, G- Geotrichum, Af- Aspergillus flavus

4.2.4 Anti-Mosquito Prope rties

Repellent Activity

The repellency against A. arabiensis with the aqueous and methanolic extracts of the three famine
plants is depicted in Fig. 7 and Fig. 8. The methanolic extract of G. densa and A. aspera has
repellency effects against A. arabiensis and these can be used for personal protection against the
mosquitoes by individuals. Further evaluation of G. densa and A. aspera is required before this
could be used as an alternative mosquito repellent. When compared to the positive control which
was DEET a well known synthetic organic insecticide, G. densa displayed 100% repellency and
A. aspera displayed 85% repellency.
 120

100

% repellency
80

60

40

20

0
A. aspera A. sessilis G. densa DEET
aqueous plant extracts and positive control

Data are mean± SD (n=2)

Fig. 7. Repellency activity from the aqueous extracts of A. aspera, A. sessilis and G.
densa on A. arabiensis mosquito

120

100

80
% repellency

60

40

20

0
A. aspera A. sessilis G. densa DEET
methanolic plant extracts and positive control

Data are mean±SD: (n=2)

Fig. 8. Repellency activity from the methanolic extracts of A. aspera, A. sessilis and G.
densa on A. arabiensis mosquito
Larvicidal Activity

For larvacidal trials none of the plant extracts have exhibited any significant e ffect on the
immature stages of the Anopheline mosquito during the seven day exposure period, compared to
 the positive control which in this case was Mostop, a commercial organophosphate.
Transformation of larvae to pupae and subsequently to adult stage occurred as normal (Table 9).
However recent studies by Bagavan et al. (2008) reported that the ethyl acetate extract of A.
aspera showed larvicidal activity against the early fourth- instar larvae of Aedes aegypti L and
Culex quinquefasciatus Say.

Table 9. Larvicidal trials of A. aspera, A. sessilis and G. densa at plant concentration of

1000 µg/ml on A. arabiensis mosquito
Larvici dal acti vity/ Survi val
Plant Extracts Day0 Day1 Day2 Day3 Day4 Day5 Day6 Day7

A. aspera (Aqueous) L L L L P P A A
A. aspera (Methanolic) L L L L P P A A
A. sessilis (Aqueous) L L L L P P A A
A. sessilis (Methanolic) L L L L P P A A
G. densa (Aqueous) L L L L P P A A
G. densa (Methanolic) L L L L P P A A
Acetone L L L L P P A A
Distilled Water L L L L P P A A
Mostop (Organophos phate) 100 L L L P P A A

L- Larvae, P- Larvae that reached the pupal stage, A- Larvae that reached the adult stage

Insecticidal Activity

K orthrine, a commercial insecticide exhibited between 98.36±2.35 and 100% knockdown during
the 60 min exposure time and 100% mortality (Table 10). The aqueous extract of A. aspera
exhibited between 26 and 38.5% (Table 10) knockdown activity against the Anopheline
mosquitoes during the initial 60 min of exposure. Mortality measured after 24 h has shown a
decrease in activity as only 21.67±7.07% (Table 10) of the test species had been affected by the
extract. The methanolic extract of A. aspera exhibited between 30 and 32% (Table 10)
knockdown activity against the Anopheline mosquitoes during the initial 60 min of
exposure. Mortality measured after 24 h showed a decrease in activity as only 16.67% (Table 10)
of the test species was affected by the extract. Results showed that the aqueous extract of A.
sessilis exhibited between 30 and 54% (Table 10) knockdown activity against the Anopheline
mosquitoes during the initial 60 min of exposure. Mortality measured after 24 h showed a
 decrease in activity as only 21.67±7.07% (Table 10) of the test species was affected by the
extract. The methanolic extract of A. sessilis exhibited between 15 and 37% (Table 10)
knockdown activity against the Anopheline mosquitoes during the initial 60 min of
exposure. Mortality measured after 24 h showed a decrease in activity as only 5±2.36% (Table
10) of the test species was affected by the extract. The aqueous extract of G. densa has
exhibited between 23 and 32% (Table 10) knockdown activity against the Anopheline
mosquitoes during the initial 60 min of exposure. Mortality measured after 24 h showed a
decrease in activity as only 11.67±7.07% (Table 10) of the test species was affected by the
extract. The methanolic extract of G. densa exhibited 18% (Table 10) knockdown activity against
the Anopheline mosquitoes during the initial 60 min of exposure. Mortality measured after 24 h
displayed that only 18% (Table 10) of the test species had been affected by the extract. None of
the above plants can be used to kill the Anopheline vector as the level of knockdowns by the
extracts are lower than the recommended standards set by the World Health Organization
(WHO, 1981a).

Table 10: % Knockdown and Mortality of A. arabienis with aqueous and methanolic
extracts of A. aspera, A. sessilis and G. densa
Knock down Mortality
Plant Extracts 30 mins 60 mins 24 hrs

A. aspera (Aqueous) 26.67±9.43 38.36±16.50 21.67±7.07

A. aspera (Methanolic) 31.67±2.35 30±4.71 16.67

A. sessilis (Aqueous) 30±14.14 53.33±28.28 21.67±7.07

A. sessilis (Methanolic) 15±2.35 36.67±9.43 5±2.36

G. densa (Aqueous) 23.33±14.14 31.67±7.07 11.67±7.07

G. densa Methanolic) 18.36±2.35 18.36±2.35 18.36±11.79

Acetone 8.36±2.35 5±2.36 10±4.71

Distilled Water 1.67±2.35 10 11.67±2.35

K- Orithrine 98.36±2.35 100 100

Data are mean± S D (n=2)

Mosquitoes are the major vectors for the transmission of malaria, dengue fever, yellow fever,
filariasis and several diseases (James, 1992 ). Effective use of controlling agents has disrupted
natural biological control systems and led to the outbreaks of insect species showing resistance.
According to Yang et al. (2002) it has provoked undesirable effects including toxicity to non-
target organisms and fostered environmental and health concerns. These problems have
highlighted the need for the development of alternate insecticides which will be effective,
environmentally safe, biodegradable, safe and cheap. According to Wink (1993) extracts of plants
may be alternate source of controlling agents since they constitute a rich source of bioactive
compounds that are biodegradable into nontoxic products and potentially suitable for use in
integrated pest management programs. The initial repellency activity of G. densa and A. aspera
was as effective as DEET (positive control), a synthetic organic insecticide, with the methanolic
plant extracts showing 100% and 85% respectively. A. aspera is used as an antimalarial and
antisplasmodic agent (Goyal et al., 2007). For larvacidal trials none of the plant extracts exhibited
any effect on the immature stages of the Anopheline mosquito during the seven day exposure
period. The results for the insecticidal screening reflected in this study has not been very
encouraging since the criteria set for determining if an extract is a potential insecticide according
to the World Health Organization (WHO, 1981a) standards is 95% knockdown and greater than
80% mortality after 24 h.

4.3 Safety Analysis

4.3.1 Cytotoxicity

The cytotoxic effect of aqueous and methanolic extracts of A. aspera on the K562 cell line is
depicted in Fig. 9. The aqueous extract of A. aspera stimulated the growth of the K562 cell line.
As the concentration of the extract increased, there was a slight decrease in cell viability. The
methanolic extract of A. aspera showed some differentiation where at concentrations of 10 and
1000 µg/ml there was stimulation of cells but at a concentration of 100 µg/ml there was a
decrease in cell viability. The same trend was observed for A. sessilis. The cytotoxic effect of
aqueous and methanolic extracts of A. sessilis on the K562 cell line is illustrated in Fig. 10. The
cytotoxic effect of aqueous and methanolic extracts of G. densa on the K562 cell line is shown in
Fig. 11. The aqueous extract of G. densa stimulated the growth of the cell at low concentration
but was toxic to the cells at a high concentration of 1000 µg/ml. The methanolic extracts
stimulated the growth of cells at low concentrations and as the concentration increased there was
a decrease in the cell viability.

This particular cell line was chosen because K562 cells express the membrane complement
regulatory proteins CD59, CD55 and CD46. Simpson and coworkers in 1997 reported that CD46
and CD55 inhibit the deposition of C3 fragments on the cell surface and thereby limit
complement-dependent cellular cytotoxicity. CD59 prevents the formation of membrane attack
complexes and the subsequent osmotic lysis of the target cell (Simpson et al., 1997). Peer et al.
(2005) reported that A. spinosus and A. hybridus from the Amaranthaceae family were cytotoxic
to the HepG2 cell line.

A.aspera (aqueous)
180
160 A.aspera(methanolic)

140
% Cell Viability

120
100
80
60
40
20
0
10 100 1000
Plant Concentration (ug/ml)

Data are mean± Standard Deviation (n=3)

Fig.9. Cytotoxic effect of aqueous and methanolic extracts of A. aspera on the K562
cell line
 250

200
% Cell Viability
150 A. sessilis (Aqueous)
A. sessilis (Methanolic)
100

50

0
10 100 1000
Plant Concentration (ug/ml)

Data are mean± Standard Deviation (n=3)

Fig.10. Cytotoxic effect of aqueous and methanolic extracts of A. sessilis on the K562
cell line

200
180
G. densa (Aqueous)
160
G. densa (Methanolic)
% Cell Viability

140
120
100
80
60
40
20
0
10 100 1000
Plant Concentration (ug/ml)

Data are mean± Standard Deviation (n=3)

Fig.11. Cytotoxic effect of aqueous and methanolic extracts of G. densa on the K562 cell
line
 4.3.2 Toxicity to Brine Shrimp

None of the plants showed any level of toxicity with all the plants showing 100% of shrimp
survival whereas the positive control which was an organophosphate showed 100 % mortality. A
study performed by Peer et al., (2005) showed that other plants from the Amaranthaceae family
such as A.dubius, A spinosus and A. hybridus has some toxic effects. Gayathri et al. (2006)
reported that histopathological testing revealed degenerative and necrotic changes in the liver and
kidney in Swiss mice, caused by oral administration of water extract of A. sessilis in high doses.
He suggested that this could be due to the effects of cytotoxic substances in A. sessilis, however,
in our study no toxic activity was noted.

4.3.3 Mutagencity

The mutant frequency is expressed as the quotient of the number of revertant colonies over the
number of colonies in the negative control. Table 11 shows the mutant frequency of the plant
extract tested on the S. typhimurium TA 98 strain. Table 12 shows the mutant frequency of the
plant extract tested on the S. typhimurium TA 100 strain. The greater the number of revertant
colonies, the greater the mutant frequency. According to Maron and Ames (1983) a mutagenic
potential is assumed if the mutant frequency is greater than 2; a possible mutagenic potential is
assumed if the mutant frequency ranges between 1.7 and 1.9; and no mutagenic potential is
assumed if the mutant frequency is lower than 1.6. None of the plant extracts up to concentrations
of 1000 µg/ml showed any mutagenic potential. Sodium azide was the chosen mutagen used in
this experiment and it showed a mutagenic potential; as the concentration increased so did the
number of revertant colonies. Thus the extracts of A. aspera, A. sessilis and G. densa do not have
compounds that may a potential to be a carcinogenic agent. Peer et al. (2005) reported that none
of the leafy vegetables (A. dubius, A. spinosus and A. hybridus) had any mutagenic activity.
Table 11: Mutant fre quency of the number of revertants in S. typhimurium strain TA 98
exposed to plant extracts
Mutant frequency of revertants at different concentrati ons
5µg/ml 10µg/ml 20µg/ml 100µg/ml 1000µg/ml
Plant Extract

A. aspera(aqueous) nd 0.5 nd 0.7 1.1

A. aspera(methanolic) nd 0 nd 0 0

A. sessilis(aqueous) nd 0.8 nd 1 1.4

A. sessilis(methanolic) nd 0 nd 0 0

G. densa(aqueous) nd 0.3 nd 0.4 0.8

G. densa(methanolic) nd 0 nd 0 0

Sodium Azi de (positi ve control) 1.6 2.9 5.2 nd nd

nd: not determined

Table 12: Mutant fre quency of the number of revertants in S. typhimurium strain TA 100
exposed to plant extracts
Mutant frequency of revertants at different concentrati ons
Plant Extract 5µg/ml 10µg/ml 20µg/ml 100µg/ml 1000µg/ml

A. aspera(aqueous) nd 0.6 nd 0.7 0.9

A. aspera(methanolic) nd 0 nd 0 0

A. sessilis(aqueous) nd 0.6 nd 0.9 1

A. sessilis(methanolic) nd 0 nd 0 0

G. densa(aqueous) nd 0.5 nd 0.7 0.9

G. densa(methanolic) nd 0 nd 0 0

Sodium Azi de (positi ve control) 1.7 2 2,5 nd nd

nd: not determined

4.4 Micropropagation

4.4.1 Surface sterilization of explants

To identify the ideal surface sterilization conditions of the explants, different combinations of
chemical sterilants viz., sodium hypochlorite and mercury chloride were used. Results are
presented in Table 13 given below. Plants were exposed to sterilizing agents for varying
durations. Maximum contamination (86±1.41%) was observed when explants are treated with
30% sodium hypochlorite (v/v) for 20 min, followed by combination of 0.1% mercury chloride
(m/v) for 5 mins and 30% sodium hypochlorite (v/v) for 15 min. 0.1% mercury chloride for 5
min and 40% sodium hypochlorite for 20 min showed no contamination and were used to
sterilize the leave explants for further use.
Table 13. Percentage of contamination using different surface sterilizing agents at different
exposure times

Surface sterilizing agent Duration of Exposure % Contamination

30 % (v/v) Sodiu m hypochlorite 20 minutes 86±1.41

0.1 % (m/v) Mercury chloride 5 minutes

+ +
52.5±3.53
30 % (v/v) Sodiu m hypochlorite 15 minutes

0.1 % (m/v) Mercury chloride 5 minutes

+ +
5.5±2.12
40 % (v/v) Sodiu m hypochlorite 20 minutes
Data are mean±SD (n=2)

4.4.2 Callus induction

To determine the best callus induction response, various concentration and combinations of
growth regulators were used. The leaves of the plants were used as explants. Callus initiation was
observed on the surface or cut ends of the explants after 14 days of inoculation. The effect of
different plant growth regulators and their concentration on callus induction is summarized in
Table 14. The best callus induction response for leaf explants of A. aspera (Fig.12a-b) A. sessilis
(Fig.14a-b) and G. densa (Fig.13a-b) were observed on MS medium supplemented with 1 mg/L–
2, 4-D and 1 mg/ L BAP. No callus induction was noted on 1 mg/L 2, 4-D or 1 mg/L BAP only,
the explants just withered away. When equal concentrations of auxins and cytokinins were
supplemented in the media high frequencies of callus ind uction were noted. When a high
concentration of a strong auxin viz., 2,4-D was used in combination with a lower concentration of
a cytokinin viz., BAP a low frequency of callus induction was noted. Similar reports of 2,4-D
plus kinetin or NAA plus BAP induced callus development was observed in amaranthus by
Flores et al. (1982) and Bennici et al. (1992). When a high concentration of cytokinin was used
with a low concentration of auxin a low frequency of callus induction was noted. Hence the calli
obtained from MS medium supplemented with 1 mg/L 2, 4-D and 1 mg/ L BAP were used for
further analyses. The calli were subcultured onto fresh medium every two weeks.
Table 14: Effect of growth regulators on callus induction from leaves of A. aspera, A. sessilis
and G. densa (After 2 weeks of culture)
MS mediu m plus % of leaves forming callus
A.aspera A.sessilis G.densa
1 mg/ L 2, 4-D + 1 mg/ L BAP 98.5±2.12 98±2.82 97.5±2.12
1 mg/ L 2, 4-D 0 0
1 mg/ L BA P 0 0
0.5 mg/ L 2, 4-D + 1 mg/ L BAP 37±2.82 49.5±6.36 27±2.82
0.5 mg/ L 2, 4-D + 0.5 mg/ L BAP 74±1.41 77.5±3.53 79.5±6.36
1 mg/ L 2, 4-D + 0.5 mg/ L BAP 54±5.65 53.5±2.12 40.5±0.70

Data are mean±SD (n=2)

4.4.3 Shoot regeneration from callus

Callus obtained from A. sessilis in MS medium supplemented with 1 mg/L 2, 4-D and 1 mg/ L
BAP were transferred onto shooting media which consisted of half strength MS medium, 1 mg/L
IAA and 1 mg/ L BAP. After 4 weeks, structures were observed with their basal ends embedded
on the callus. These structures turned into green colored shoot buds (Fig. 14c). These structures
were transferred into tissue culture bottles consisting of different combinations of growth
hormones (Fig. 14d).

After 21 days the individual shoots were cut off and transfer red into tissue culture bottles
containing MS medium supplemented with 1 mg/L IAA and 1 mg/L BAP (Fig. 14e- f). This
combination of growth regulators showed maximum shoot multiplication of 10 shoots per culture
(Table 16). Bennici et al. (1997) found that high cytokinin:auxin ratio favours shoot regeneration
in Amaranthus. The lowest number shoot regeneration was observed on half strength MS
medium supplemented with 1 mg/L IAA only. No shoot multiplication was observed on half
strength MS medium supplemented with 1 mg/ L BAP. Shoot multiplication was noted 14 days
after the cultures were inoculated into the medium. A. aspera and G. densa did not successfully
form shoots from callus.
Table 15: Effect of growth regulators on shoot differentiation from ste m explants of A.
sessilis
MS medium Shoot mul ti plicati on of A.sessilis

1 mg/ L IAA + 1 mg/ L BAP 10 shoots per culture

1 mg/ L IAA 2 shoots per culture

1 mg/ L BA P 0

4.4.4 Rooting of regenerated shoots

For rooting, the shoots developed from the callus of A. sessilis were cultured on rooting medium
consisting of half strength MS medium containing different concentrations of either NAA or
IBA. Optimal rooting was observed on half strength MS medium supplemented with 1 mg/L IBA
(Fig. 15a). Some rooting response was also observed on MS medium supplemented with 1mg/L
NAA. Bagga et al. (1987) found that hypocotyls segments of A. paniculatus formed roots on B5
medium supplemented with NAA and Bennici et al. (1992) reported that Amaranthus responded
well forming roots in IAA plus kinetin and/or IAA plus BAP.

4.4.5 Hardening of plantlets

The regenerated plantlets from the explant with healthy root and shoot system were transferred
after washing with distilled water to remove the media traces from the roots. They were
transferred into bottles containing sterilized soil. They were covered with sunbag vessels (Fig.
15b) to maintain a high humidity environment. These were maintained for one week. Thereafter
the sunbags were removed and the plants were transferred into bottles containing normal soil and
they were grown under full sunlight (Fig. 15d). To summarize, in all the three plants, callus
formation was observed in most of the growth regulator combinations but the differences in
callus growth were observed depending on the growth hormone combinations used. The best
medium for callus induction was found to be MS medium supplemented with 1mg/L 2,4-D and 1
mg/L BAP. Shoot regeneration from the callus of A. sessilis was found in MS medium
supplemented with 1mg/L IAA and 1 mg/L BAP. Optimal rooting was observed in MS medium
with 1mg/L IBA. The results obtained from our study demonstrate that A. sessilis has great
potential with regard to dedifferentiation and morphogenetic processes and the possib ility to
micropropagate these plants.
Fig.12. A.aspera (a) callus derived from leaf explants (b) plate showing callus derived from
leaf explants.

Fig.13. G.densa (a) callus derived from leaf explants (b) plate showing callus derived from leaf
explants.
 A
B

C D

E F

Fig.14. Micropropagation of A. sessilis (a) callus derived from leaf explants (b) plate
showing callus derived from leaf explants (c) shoot formation from callus (d)shoot
regeneration in tissue culture bottle (e) multiple shoot development in a bottle (f)
multiple shoot development on a petri dish
 A B

C D

Fig.15 . Root development in A. sessilis (a) Rooting in half MS medium supple mented with
1 mg/L IBA (b) A. sessilis plant in a sunbag vessel (c) hardened A. sessilis plant (d)
Acclimatized plant
CHAPTER FIVE: CONCLUSION

The focus of this study was to investigate the nutritional, bio logical and safety of three
plants from the Amaranthaceae family viz. A. aspera, A. sessilis and G. densa that are
considered as famine plants.

The nutritional profile of A. aspera indicated that moisture, ash, carbohydrates, protein,
fat, fibre and energy meet with the RDA levels. The plant extracts had high levels of
Vitamin B1 , Vitamin B2 and Vitamin C. The nutritional composition indicates that this
neglected plant can be a valuable source of nutrients under famine conditions and the
high levels of some vitamins and minerals can be used to prevent diseases. This plant can
be cultivated on a large scale due to the fact that the extracts have high antioxidant
activity. This activity can be used to prevent diseases of unknown aetiology such as
cancer, ageing, atherosclerosis, ischemic injury and neurodegenerative diseases. The
extracts can also provide an alternative source for current infectious diseases caused by
multi drug resistant bacteria and can act as an agent for malarial control.

A. sessils has high level of ash, carbohydrates, protein, dietary, fat, fibre and energy
needed to nourish individuals suffering from malnutrition. This plant can help eradicate
malnutrition in areas rife where people are starving. The plant had high levels of Vitamin
A and Vitamin B3 and also high levels of magnesium, manganese and iron present,
therefore, this plant can aid in proper nervous system functioning, energy metabolism,
enzyme structuring and optimal immune functioning especially in rural populations
where anemia is prevalent. The plant extracts also show high anti-oxidative activity. As
far as the antibacterial activity results are concerned, it can be used to prevent diseases
caused by P. aeruginosa and S. aureus and due to its fungicidal effect on C. albicans, this
plant can be used against Candidiasis. A further advantage of cultivating this plant on a
large scale like the other members from the family is that it is conducive for
micropropagion by callus culture to a fully grown plantlet able to survive environmental
conditions.
G. densa had sufficient amounts of ash, fibre and fat. This plant has excessive amounts of
Vitamin B1 and Vitamin B2 therefore they could prevent deficiencies that cause
peripheral nerve damage or central nerve system lesions, lesions of the corner of the
mouth, lip and tongue and seborrheic dermatitis. Due to the sufficient amounts of
Vitamin A and Vitamin B3 this plant can fight illnesses such as night blindness,
xerophthalmia, keratinisation of skin, pellagra- photosensitive dermatitis and depressive
psychosis. The high iron levels found can be used to treat/prevent diarrhea. The plant has
antibacterial effects against a range of bacteria so it can prevent diarrhea, vomiting and
bronchitis and repellent properties against the mosquito whish could be used for personal
protection especially by rural communities where chemical protection is very expensive.

A. aspera, A. sessilis and G. densa are also considered safe as they showed no toxicity or
mutagenicity.

In conclusion the three plants from the Amaranthaceae family viz. A. aspera, A. sessilis
and G. densa that are considered as famine plants should be cultivated on a large scale
due to their nutritional and biological value.

(Gokhale et al., 2002, Pillay et al., 2001, Perumal Samy et al., 1998, Cheng et al., 2003,
Wink 1993, MMLS, 1999, Yang et al., 2002, Akula and Odhav, 2008)
((Flores and Teutonico, 1986, Edmonds and Chweya, 1997, Rabe and van Staden, 2000,
Rabe et al., 2002, Reid et al., 2001, Motsei et al., 2003, Grace et al., 2002, Elgorashi et
al., 2003, Simopuolos, 2004, Bennici and Schiff, 1997, Bennici et al., 1997, Bennici et
al., 1992, Bagga et al., 1987, Flores et al., 1982, Odhav et al., 2007)
(WHO, 1985, Murashige and Skoog, 1962, Bagavan et al., 2008, Peer et al., 2005,
Gayathri et al., 2006, Belachew Desta, 1993, Bhaskaram, 2002, Lamikanra, 1999)
(Freshney, 1987, Hanelt et al., 1994, Scheizer and Wuersch, 1979, Abad et al., 1995,
Njenga and Viljoen, 2006, Brown and Wuehler, 2000)
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APPENDIX A

1. 0.5mM histidine/0.5mM biotin solution for the top agar in mutagenicity test

Ingredient per 250 ml

D-Biotin 30.5 mg
L-histidine 26.2 mg
Dd H20 250 ml

Dissolved biotin in hot water first, filter sterilized and stored at 4°C in glass bottle.

2. Vogel –Bonner medium E (50 x strength stock) for minimal agar base
Ingredient per 1000 ml
Warm ddH20 670 ml
MgSO4.7H2O 10 g
Citric acid monohydrate 100 g
K2HPO4 500 g
NaHNH4PO4.4H2O 175 g

Added salts in the order indicated to warm water in a 2 liter flask placed on a magnetic
stirring hot plate. Allowed each salt to dissolve completely before adding the next.
Adjusted the volume to 1 litre. Distributed into two 1 liter glass bottles. Autoclaved for
20 min at 121°C.

3. 40 % Glucose (autoclave sterile)

Ingredient per 1000 ml

Glucose 400 g
ddH2O 1000 ml
4. Minimal Glucose agar plates for mutagencity test

Ingredient per 1000 ml

Agar 15 g
Ddh20 930 ml
VB medium E stock
(autoclaved sterile) 20 ml
40 % glucose
(autoclaved sterile) 50 ml

Added agar to dd h20 in a 2 liter flask. Autoclaved at 121°C for 20 min using slow
exhaust. When the solution cooled slightly, added sterile VB medium E stock and sterile
40% glucose.

5. Top Agar for mutagenicity test (soft agar)

Ingredient per 1000ml

Agar 6g
NaCl 5g
Dd H2O to 1000 ml

Microwaved to dissolve the agar. Mixed thoroughly and made 100 ml aliquots to 250 ml
glass bottles with screw caps. Autoclaved at 121°C for 20 min with loosed caps.

* added 1/10 volume (10 ml) of the 0.5 mM histidine/0.5 mM biotin solution to the
molten top agar.