ANTIBACTERIAL, ANTIOXIDANT AND PHYTOCHEMICAL INVESTIGATION OF

ACACIA ARENARIA, ALOE ESCULENTA, AND PECHUEL-LOESCHEA LEUBNITZIAE.

A THESIS SUBMITTED IN FULFILMENT

OF THE REGUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE (BIOCHEMISTRY)

OF

THE UNIVERSITY OF NAMIBIA

BY

DANIEL NDONGO

200732838

August 2017

Main Supervisor: Dr. Petrina T Kapewangolo

Co-supervisors: Prof. Martha Kandawa-Schulz and Ms. Celine Mukakalisa

ABSTRACT

Natural products present in medicinal plants are an important source of therapeutic agents and

many research groups are currently screening different biological activities of plants. This study

aimed to investigate the antibacterial, antioxidant properties and phytochemical analysis of Acacia

arenaria (Fabaceae), Aloe esculenta (Alliaceae) and Pechuel-Loeschea leubnitziae (Asteraceae)

that are used in traditional settings. The antimicrobial activity of the methanol-dichloromethane

(DCM) (1:1) extracts of these medicinal plants were tested against Shigella sonnei (ATCC 25931),

Serratia marcescens (ATCC 8100), Enterococcus faecalis (ATCC 7080) and Alcaligenes faecalis

(ATCC 8750). The minimum inhibitory concentration (MIC) of the extracts against afore

mentioned microbes were determined. The antioxidant activity of the extracts was determined

using α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging and reducing power assays. The

phytochemical analysis of A. arenaria, A. esculenta and P. leubnitziae was done using qualitative

and quantitative phytochemical analysis. Extracts demonstrated anti-microbial activity against S.

sonnei, A. faecalis, E. faecalis and S. marcescens. The best activity was recorded with A. arenaria

leaves extract, with MIC values ranging from 0.1 to 5.0 mg/mL against all tested bacteria. All

extracts exhibited antioxidant activity for both assay techniques. The antioxidant activity

correlated with the quantitative phytochemical presence of phenolic content ranging from 0.5 ±

0.1 to 5.7 ± 0.1 mg of GAE/g extract obtained in the extracts as well as condensed tannins ranging

from 0.1 ± 0.0 to 72.6 ± 0.0 mg of TAC/g extract. Qualitative phytochemical analysis showed the

presence of steroids, terpenoids, saponins, flavonoids, anthraquinones, coumarins, alkaloids,

phenols and tannins. The results obtained in the present study indicated that the leaves, stem and

roots of A. esculenta, and A. arenaria, P. leubnitziae are potential sources of natural antioxidants

ii
and antimicrobials. Further studies could look into the characterization of the bioactive compounds

present in these plants.

iii
Table of Contents

Abstract...............................................................................................................................ii

Table of Contents…………………………………………………………………………iv

List of figures......................................................................................................................viii

List of tables ......................................................................................................................x

Acknowledgements ............................................................................................................xi

Declaration..........................................................................................................................xii

Dedication...........................................................................................................................xiii

List of abbreviations............................................................................................................xiv

INTRODUCTION…………………………………………………………………………1

1.1. Orientation of the study.................................................................................................1

1.2. Statement of the problem..............................................................................................2

1.3. Objectives of the study..................................................................................................3

1.4. Research hypothesis…..................................................................................................3

1.5. Significance of the study...............................................................................................4

LITERATURE REVIEW…………………………………………....................................5

2.1. Plant description, geographical location and scientific studies....................................5

2.2. Traditional uses of A. arenaria, A. esculenta and P. leubnitziae.................................9

2.3. Natural Products as source of drugs …………………………………………………10

2.4. Phytochemicals……………………………………………………………………….12

2.4.1. Phenolics …………………………………………………………………..13

iv
 2.4.2. Flavonoids ………………………………………………………………....14

2.4.3. Alkaloids ………………………………………………………………......15

2.4.4. Terpenoids………………………………………………………………….16

2.4.5. Saponins ……………………………………………………………….......16

2.4.6. Tannins …………………………………………………………………….16

2.4.7. Anthraquinones ……………………………………………………………17

2.4.8. Coumarins………………………………………………………………….17

2.4.9. Steroids…………………………………………………………………….18

2.5. Oxidative stress...........................................................................................................19

2.5.1. Free radicals.................................................................................................19

2.5.2 The role of antioxidants in diseases prevention............................................22

2.6. α, α-Diphenyl-1-PicrylHydrazyl (DPPH) Radical .....................................................24

2.7. Reducing Power Assay……………….......................................................................26

2.8. Quantitative determination of phytochemicals ..........................................................26

2.8.1. Total phenolics content...............................................................................27

2.8.2. Total condensed tannins..............................................................................27

2.9. Phytochemicals as antibacterial agents......................................................................28

2.9.1. Shigella sonnei............................................................................................29

2.9.2 Alcaligenes faecalis ……………………………………………………... 30

2.9.3. Enterococcus faecalis…………………………………………………….30

2.9.4. Serratia marcescens……………………………………………………...31

v
METHODS.....................................................................................................................33

3.1. Research design ......................................................................................................33

3.2. Plant specimen and collection.................................................................................33

3.3. Phytochemical screening.........................................................................................34

3.4. Extraction of dried powder samples........................................................................36

3.5. Quantitative phytochemicals analysis ………..…………………………………..36

3.5.1. Total Phenolic Content.............................................................................36

3.5.2. Total Condensed Tannins ........................................................................37

3.6. Antioxidant Activity...............................................................................................37

3.6.1. DPPH Radical Scavenging Activity Assay..............................................37

3.6.2. Reducing Power Assay ………………………………………………...38

3.7. Antibacterial Activity …………………………………………………………....38

3.7.1. Disc Diffusion Assay…………………………………………………...38

3.7.2. Minimum inhibitory concentration (MIC) ……………………..……...39

3.8. Data analysis .........................................................................................................39

RESULTS .....................................................................................................................40

4.1. Phytochemical screening ……...............................................................................40

4.2. Quantitative phytochemical analysis………………………………………….......41

4.2.1. Total Phenolic and total condensed tannins content…………………………….41

4.3. Antibacterial Activity results ……………………………………………………...43

4.3.1. Disc diffusion result……………………………………………………………...43

vi
4.3.2. MIC Data………...…………………………….………………………………...45

4.4. Antioxidant Activity results......................................................................................46

4.4.1 DPPH Assay results………………………………………………………46

4.4.2. Reducing Power Assay ……....………………………………………….51

DISCUSSION..................................................................................................................54

CONCLUSION………………………………………………………………………....58

RECOMMENDATIONS ................................................................................................59

REFERENCE LIST .........................................................................................................60

Appendix A: L-Ascorbic Acid Calibration Curve ……………………………………...79

Appendix B: Gallic Acid Calibration Curve………………..…………………………...80

Appendix C: Tannic Acid Calibration Curve……………………………………………81

Appendix D: Ethical clearance certificate……………………………………………….82

Appendix E: Research collecting/permit………………………………………………...83

Appendix F: Research permission letter…………………………………………………84

vii
List of figures

Figure 1: Acacia arenaria (whole plant)………………………………………………….6

Figure 2: Aloe esculenta (whole plant)……………………………………………………7

Figure 3: Pechuel-Loeschea leubnitziae (whole plant)……………………………………8

Figure 4: Example of major classes of flavonoids isolated from plants ………………..15

Figure 5: Core chemical structures of coumarins. A=simple coumarins, B= pyrano coumarins,

C= furanocoumarins...........................................................................................................18

Figure 6: Structure of a typical steroid…………………………………………………...19

Figure 7: DPPH• free radical conversion to DPPH by anti-oxidant compound

…………………………………………………………………………………………….25

Figure 8: Schematic research design flow diagram ………………………………………33

Figure 9: DPPH scavenging activity of A. arenaria’s roots, leaves and stem extracts as compare
to Ascorbic Acid …………………………………………………………………………..47

Figure 10: DPPH scavenging activity of A. esculenta’s roots, leaves and stem extracts as
compare to Ascorbic Acid …………………………………………………………………48

Figure 11: DPPH scavenging activity of P. leubnitziae roots, leaves and stem extracts as
compared to Ascorbic Acid ………………………………………………………………..49

Figure 12: Reducing power activity of A. arenaria roots, leaves and stem extracts
………………………………………………………………………………………………51

Figure 13: Reducing power activity of A. esculenta roots, leaves and stem extracts
………………………………………………………………………………………………52

Figure 14: Reducing power activity of P. leibnitziae roots, leaves and stem extracts
……………………………………………………………………………….……………...53

Figure 15: L- Ascorbic Acid Calibration Curve ……………………………………………79

viii
Figure 16: Gallic Acid Calibration Curve ………………………………………………….80

Figure 17: Tannic Acid Calibration Curve …………………………………………………81

ix
List of tables

Table 1: Phytochemical screening results A. esculenta, A. arenaria, and P. leubnitziae’s roots,

stems and leaves …………………………………………………………..…………….....41

Table 2: Total phenolic content and total condensed tannins of leaves, roots and stem extract

from A. arenaria, A. esculenta and P. leubnitziae …………………..……………………..42

Table 3: Antimicrobial screening of plants against A. faecalis (ATCC 8750), E. faecalis (ATCC

7080), S. sonnei (ATCC 25931) and S. marcescens (ATCC 8100) using disc diffusion

method.………………………………………….………………………………………….44

Table 4: MIC values (mg/mL) as determined by the microdilution method ……...……….45

Table 5: DPPH free radicals A. arenaria, A. esculenta and P. leubnitziae leaves, roots and stem

extracts ……………………………………..………………………………………………50

x
ACKNOWLEDGEMENT

I would like to express my sincere gratitude and appreciation to my Lord and Savior, Jesus Christ, for

the love, mercy, power, wisdom and strength He gave me throughout my life. It is by His grace and

encouragement that I was able to complete this research. My God put my feet on solid ground and

lifted me up. He gave me new hope for tomorrow and put a new song in my heart. How I love you

Lord. I thank you for your faithfulness, loving, kindness and patience. Praise the Lord! A deep

appreciation is extended to Dr. Petrina Kapewangolo, my supervisor, for her support, encouragement,

patience and kind assistance throughout this project and my stay at UNAM. I thank Dr. Martha

Kandawa-Schulz and Ms. Celine Mukakalisa, my co-supervisors, for their direction, guidance and

patience as my research advisors. I express special gratitude to Ministry of Agriculture Water and

Forestry and Department of Chemistry and Biochemistry of the Faculty of Science, UNAM for the

facilities to conduct my research. I gratefully acknowledge my parents, Mr. A. Kondo and Mrs.

Johanna T. France-Edhiya, my brothers and my sisters, and Mr. N. Edhiya and my grandfather Mr. A.

Edhiya and their families, for their encouragement, prayers, magnificent support, love and patience

throughout my years of education. I am so blessed to have you as family. I am sincerely grateful for

the help and support I received from my family in this research. A deep appreciation is extended to my

fellow Christian sisters and brothers in Namibia for their prayers, friendship, support and encouraging

words. In addition a special thanks to Mr. Japhet Edhiya, Ms. A. Kamukuto, Ms Sarlote Edhiya, Ms

Wilka Edhiya, Ms. Maria Anguku, Ms. Frieda Kankoshi and Mr. Silvanus Kamukuto for their support,

patience, advice, prayers, understanding and encouragement throughout this degree program. To God

is the glory. Great things He has done!

xi
DECLARATION

I, Daniel Ndongo, declare hereby that this is a true reflection of my own research, and that this
work, or part thereof has not been submitted for a degree in any other institution of higher
education.

No part of this thesis may be reproduced, stored in any retrieval system, or transmitted in any form,
or by any mean (e.g. electronic, mechanical, photocopying, recording or otherwise) without the
prior permission of the author or The University of Namibia in that behalf.

I, Daniel Ndongo, grant The University of Namibia the right to reproduce this thesis in whole or
part, in any manner or format, which The University of Namibia may deem fit, for any person or
institution requiring it for study and research; providing that The University of Namibia shall
waive this right if the whole thesis has been or is being published in a manner satisfactory to the
University.

Signature...............................Date...............................2017

Daniel Ndongo

xii
DEDICATION

I dedicate this work to my family and friends for always believing in me and encouraging me even

in instances when I felt like giving up. To my brothers and sisters, where there is a will there is a

way. Thus, you can go as far as your imagination goes. Do not give up on your dreams.

xiii
ABRIVIATION

AIDS: Acquired Immune Deficiency Syndrome

ATP: Adenosine Triphosphate

CAT: Catalase

DCM: Dichloromethane

DNA: Deoxyribonucleic Acid

DPPH●: α, α-Diphenyl-β-picrylhydrazyl radical

GAC: Gallic Acid Equivalent

IC50: 50% Inhibition Concentration

MeOH: Methanol

MET: Ministry of Environment and Tourism

MIC: Mimimum Inhibitory Concentration

NBRI: National Botanical Research Institute

TAC: Tannic Acid Equivalent

TCT: Total Condensed Tannins

TPC: Total Phenolic Content

RNS: Reactive Nitrogen Species

ROS: Reactive Oxygen Species

xiv
RPC: Research and Publication Committee

SOD: Superoxide Dismutase

WHO: World Health Organization

xv
INTRODUCTION

1.1. Orientation of the study

Medicinal plants are reservoirs of curative elements used by a large population of Africans in the

treatment of various infections (Okigbo, Anuagasi, & Amadi, 2009; Akhter, Hossain, Haque,

Shahriar, & Bhuryan, 2012). A medicinal plant is any plant with healing capacity. For centuries,

man has used plants to treat common communicable diseases (Alabri, Musalami, Hossian, Weli,

& Al-Riyami, 2013). Furthermore, an herbal product from medicinal plants is produced through a

number of processes such as extraction, fractionation, purification, and concentration of plant

materials (Alabri et al., 2013). According to recent studies conducted by the World Health

Organisation (WHO), about 80% of the world’s population relies on traditional medicine (Moudgil

& Khalil, 2016). There is over 350,659 medicinal plants globally of which only 2% have been

explored and tested for their bioactivity (Ghosh, Ramakrishna, & Ramakrishna, 2014).

Medicinal plants reportedly contain biologically active secondary metabolites that include

saponins, tannins, essential oils, flavonoids and alkaloids (Okigbo et al., 2009). In many cases,

these metabolites serve as plant defence mechanisms against predation by microorganisms, insects

and herbivores (T. S. Geetha & Geetha, 2014). Some of these plant-derived compounds have

demonstrated good antiviral, antibacterial, antioxidant activities just to mention few (Suneetha,

Gopinath, Divya, Amaashankar, & Narasimha, 2013). A number of in vitro studies on medicinal

plants have revealed antiviral as well as antibacterial potential (Patel, Bessong, & Liu, 2011;

Fobofou et al., 2015; Zohra, Meriem, Samira, & Alsayadi, 2012). In addition, many medicinal

plants have also shown preservative effects due to the presence of antioxidants (Akbarirad, Gohari,

Kazemeini, & Mousavi, 2016).

1
Namibia reportedly has over 4,334 plant taxa some of which are utilized as herbal medicines

(Chinsembu, Hedimbi, & Mukaru, 2011). In the present study antibacterial, antioxidant activities

and phytochemical profiles of A. arenaria, A. esculenta and P. leubnitziae were investigated.

Acacia arenaria and A. esculenta have never been investigated for antibacterial, antioxidant

properties neither their phytochemical screening was conducted. On the other hand, phytochemical

screening and antibacterial study of P. leubnitziae were previously reported (Hedimbi, Kaputjaza,

Hans, Mumbengegwi, & Bӧck, 2012a; Hedimbi, Ndjoze-Sirika, & Han, 2012b). However, there

were no studies on the antioxidant activity done on P. leubnitziae. Acacia arenaria, A. esculenta

and P. leubnitziae were selected based on known traditional uses reported in literature such as

cold, cough and intestinal related conditions (Curtis & Mannheimer, 2005; Mannheimer & Curtis,

2009; Steyn, 2007; Von Koenen, 2001).

1.2. Statement of the problem

Many people in African, Asian communities greatly rely on medicinal plants to alleviate symptoms

of various diseases. In Namibia, many residents in rural areas rely on medicinal plants to treat

several diseases (Chinsembu, Hijarunguru, & Mbangu, 2015). In most cases, the exact mechanism

of action of the extracts is unknown; hence, it is therefore crucially important to determine whether

actual pharmacological effects support the traditional claims. A. arenaria, A. esculenta and P.

leubnitziae are traditionally used as antibacterial agents in both humans and animals, Von Koenen

(2001) but minimal scientific knowledge is available to support the traditional claims. Studying

these plants therefore assisted in approving or disapproving the anecdotal use, and contributed to

the on-going search for new antibacterial agents because of the continuous emergence of drug

resistant bacterial strains.

2
1.3. Objectives of the study

The specific objectives of this study were to:

a) Investigate phytochemicals present in the crude extracts of A. arenaria, A. esculenta and

P. leubnitziae using qualitative and quantitative methods.

b) Investigate the antibacterial property of A. arenaria and A. esculenta extracts against

Alcaligenes faecalis, Shigella sonnei, Serratia marcescens and Enterococcus faecalis

c) Determine the antioxidant activity of A. arenaria, A. esculenta, and P. leubnitziae using

DPPH antiradical scavenging and reducing power assays

1.4. Research hypothesis

a) H0: The crude extracts of A. arenaria, A. esculenta and P. leubnitziae do not have

phytochemicals.

Ha: The crude extracts of A. arenaria, A. esculenta and P. leubnitziae have phytochemicals.

b) H0: Crude extracts of A. arenaria, A. esculenta and P. lebnitziae do not exhibit antibacterial

against A. faecalis, S. sonnei, S marcescens and E. faecalis.

Ha: Crude extracts of A. arenaria, A. esculenta, and P. leubnitziae exhibit antibacterial

property against A. faecalis, S. sonnei, S. marcescens and E. faecalis.

c) H0: Crude extracts of A. arenaria, A. esculenta and P. leubnitziae do not exhibit antioxidant

property.

Ha: Crude extracts of A. arenaria, A. esculenta and P. leubnitziae exhibit antioxidant

property.

3
1.5. Significance of the study

The findings of this study were expected to provide in vitro evidence to justify the traditional use

of A. arenaria, A. esculenta and P. leubnitziae. The antibacterial and antioxidant results from the

present study would support for further investigation to identify active compounds through

bioassay guided fractionation from the bioactive extracts of A. arenaria, A. esculenta and P.

leubnitziae. Synergistic interactions between compounds in crude extracts have been reported to

take place and that is why it was important to first test crude extracts before purification of

bioactive constituents. The study findings will also create more awareness on the Namibian A.

arenaria, A. esculenta, and P. leubnitziae and add to the scientific value of these species.

4
LITERATURE REVIEW

2.1. Plants description, geographical location and scientific studies done

2.1.1. Acacia arenaria

Acacia arenaria, commonly known as Sand-thorn in English and Omulyamenye in Oshiwambo,

belongs to the sub-family Mimosoidae under the family Fabaceae (Curtis & Mannheimer, 2005;

Mannheimer & Curtis, 2009; Steyn, 2007). According to Steyn (2007), about 1350 Fabaceae

species have been described of which 954 are found in Australia, 130 in Africa and the rest

elsewhere (Abdel-Farid, Sheded, & Mohamed 2014). However, regardless of the large number of

Acacia species, minimal research has been done on the phytochemistry of these plants (Abdel-

Farid et al., 2014). Acacia arenaria, a small tree with a short stem, has zigzag branches with thin

thorns (Curtis & Mannheimer, 2005; Mannheimer & Curtis, 2009; Steyn, 2007; Von Koenen,

2001). The plant have bipinnate leaves, whitish flowers that are borne in heads, and sickle-shaped

pod fruit somewhat constricted between seeds (Von Koenen, 2001). Acacia arenaria is commonly

distributed across northern Namibia (Curtis & Mannheimer, 2005; Mannheimer & Curtis, 2009).

Chemical composition of Acacia seeds has been reported in Botswana, since Acacia trees are of

significant to livestock nutrition in that country (Aganga, Tsopito, Yeboah, Mokgoko, & Manne,

1997). Acacia arenaria seeds reportedly have dry matter (92.55%), dry matter digestibly (53.13%),

crude protein (19.93%), ash (4.05%) and extractable fat (5.00%) in g/100 g dry matter (Aganga et

al., 1997). The mineral composition of the seeds has been reported: calcium (0.37), phosphorus

(0.48), potassium (1.39), sodium (0.05) and magnesium (0.20) in g/100g dry matter. The

microminerals determined were copper (21), iron (77), manganese (9) and zinc (54) in mg/kg dry

5
matter (Aganga et al., 1997). Figure 1 shows A. arenaria picture that was taken during sample

collection.

Figure 1: Acacia arenaria (whole plant)

2.1.2. Aloe esculenta

Aloe esculenta, which belongs to the family Alliaceae, is commonly known as endombo in

Oshiwambo. There are 400 species of this plant of which 31 are found in Namibia (Retief, 2013;

Sahu et al., 2013). Aloe esculenta is a succulent herb of 80-100 cm in height, which matures in 4-

6 years and survives for about 50 years under favourable conditions (Sahu et al., 2013). This plant

belongs to the great masses of plants found on the sandy flat soils (Klopper, Matos, Figueiredo, &

Smith, 2009). Von Koenen (2001) described A. esculenta as a perennial succulent plant with a

short stem inside a rosette of leaves. The candelabra-like inflorescence of this plant reportedly

bears orange-yellowish flowers, and the capsule contains a number of seeds (Von Koenen, 2001).

In Namibia, A. esculenta can only be found in the northern part of the country (Klopper et al,

2009).

6
There exist few reports in literature on experimental studies conducted on A. esculenta. Aloe

esculenta extract has demonstrated antiplasmodium activity (Amoo, Aremu, & Staden, 2014). The

most studied Aloe species is Aloe vera, and vast literature is available on this plant. The chemical

composition of A. vera revealed that the active components possibly present in this plant are

anthraquinones, chromones, polysaccharides, enzymes, vitamin C, vitamin E, lecithin,

glycoprotein and amino acids (Ghayempoura, Montazera, & Rad, 2016; Sahu et al., 2013). In

addition, the medicinal properties of A. vera include antioxidant, antitumor, anti-ulcer, anti-

neoplastic, antiviral and antiiflammatory activities (Ghayempoura et al., 2016; Soltanizadeh &

Ghiasi-Esfahani, 2014). There are about 75 active components in A. vera that makes it attractive

to cosmetic and medicinal industries (Soltanizadeh & Ghiasi-Esfahani, 2014). Figure 2 below

shows the picture of A. esculenta that was taken during sample collection.

Figure 2: Aloe esculenta (whole plant)

7
2.1.3. Pechuel-Loeschea leubnitziae

Pechuel-Loeschea leubnitziae belongs to the family Asteraceae (Curtis & Mannheimer, 2009;

Hedimbi et al., 2012b). This shrub is commonly known as Bitter bush in English and Oshizimba

in Oshiwambo (Hedimbi et al., 2012a). It is a small shrub with silver-grey felt-like pubescence on

stems and leaves, with leaves tapering petiole-like towards the base, lanceolate, entire and up to

about 3 cm long (Von Koenen, 2001). The plant is found throughout Namibia except in the far

north-eastern part (Mannheimer & Curtis, 2009).

Phytochemical analysis of P. leubnitziae’s roots, leaves and stem extracts reportedly contain

saponins, anthraquinones, flavonoids, and polyphenols (Hedimbi et al., 2012a). Moreover, stem,

leaves, and roots extracts of this plant were reported to possess antimicrobial activities against a

number of microbes such as Neisseria meningitidis, Shigella flexneri, Proteus vulgaris,

Pseudomonasa aeruginosa, Enterobacter aerogenes, Staphylococcus aureus, Escherichia coli,

and Candida albicans (Hedimbi et al., 2012a; Hedimbi et al., 2012b). Figure 3 below shows the

P. leubnitziae picture that was taken during sample collection.

Figure 3: Pechuel-Loeschea leubnitziae (whole plant)

8
2.2. Traditional uses of A. arenaria, A. esculenta and P. leubnitziae

The use of traditional medicine has been widely witnessed in most developing countries as a first

prescription for the maintenance of good health (Cheikhyoussef, Mapaure, & Shapi, 2011). The

plants investigated in this study are used by rural people in the treatment of various diseases in

both human and animals (Von Koenen, 2001).

2.2.1. Acacia arenaria

The Aawambo people, one of the big tribes in Namibia, have used A. arenaria roots decoction for

coughs, and colds (Von Koenen, 2001). Goats, and cattle consume leaves and shoots of this plant

(Curtis & Mannheimer, 2009). Furthermore, A. arenaria has also been used by Aawambo people

for the treatment of intestinal conditions in cattle (Von Koenen, 2001).

2.2.2. Pechuel-Loeschea leubnitziae

The Herero people of Namibia reportedly use P. leubnitziae to delay menstruation by

administering steam baths with leaf decoctions of the plant, which are used for thorough washing

of the abdomen (Von Koenen, 2001). Pechuel-Loeschea leubnitziae shrub is an effective mosquito

repellent. Ovahimba are indigenous people living in the Kunene region of Namibia, and also in

the neighbouring country Angola use P. leubnitziae finely grinded roots for cosmetic purposes

(Curtis & Mannheimer, 2005). The Nama tribe of Namibia stuff P. leubnitziae into their shoes to

alleviate sweaty feet (Von Koenen, 2001). Boiled roots and leaves are used for enemas in cases

of intestinal conditions, or as steam bath with colds (Von Koenen, 2001). In addition, Aawambo

people in Namibia, heat the plant materials, and use it as facial compress for cold to treat infection

(Von Koenen, 2001).

9
2.2.3. Aloe esculenta

The Aawambo people in Namibia, use A. esculenta for various traditional applications. Its leaf sap

can be applied to cuts and burns (Von Koenen, 2001). Aloe esculenta are also used to manage skin

rashes and decoction is taken orally to treat coughs (Chinsembu, Negumbo, Likando, & Mbangu,

2014; Chinsembu et al., 2015). Dried roots are pulverized and mixed with water to make a paste

that is massaged onto painful and swollen body parts (Von Koenen, 2001). It is also used in

facilitating weaning of babies/toddlers by applying very bitter leaf sap to the mother’s nipples

(Retief, 2013). In addition, A. esculenta is used in the treatment of red water disease and other

ailments in cattle and sheep. The plant is also added to drinking water to improve the general health

of animals and by reducing parasite load, as well as blood purification by making it sour for

invading pathogens to treat acne in animals (Retief, 2013).

2.3. Natural products as sources of drugs

Secondary metabolites are defined as low-molecular weight compound that do not play a role in

growth and development but rather play a major in defensive system of the plant (T. S. Geetha &

Geetha, 2014). However, so far only roughly 20% of higher plants are investigated. A natural

product is defined as a chemical component or substance produced by a living organism that is

found in nature (Khalil, Diab, & Moudgil, 2016). Natural products may have pharmacological or

biological activities that can be of therapeutic benefit in treating diseases (Khalil et al., 2016).

Natural products are seldom utilized as precursors or starting materials for new drugs discovery

due to their efficacy and safety (Khalil, et al., 2016).

Plants have been the source of crude drugs/extracts as well as precursors of synthesized

compounds that have made large influences to human health and well-being. Their role has

10
significantly increased in the upgrading of new drugs (T. S. Geetha & Geetha, 2014): (1) they may

turn out to be the base for the development of a medicine, a natural blueprint for the development

of new drugs, or; (2) a phytomedicine to be utilized for the treatment of disease. There are several

examples of plant derivative drugs such as vinblastine, vincristine, vinca alkaloids, comptothecin

and so forth (Gurung, Bhattacharjee, & Ali, 2016).

Many drugs derived from plants on the market were discovered based on their traditional uses

(Phani, Raj, Rupesh, & Shashi, 2010). For thousands of years, natural products have played a

critical role in health care and prevention of diseases (Ilonga, 2012). The earliest known written

document is a 4000-year-old Sumerian clay tablet that records remedies for various illnesses

(Moudgil & Khalil, 2016). It is estimated that approximately 80 % of the entire world population

rely on traditional medicine for their primary health care (Alabri et al., 2013; T. S. Geetha &

Geetha, 2014). Nowadays people all over the world are trying to keep away from poor eating

habits, pollution and synthetic drugs, addictions (tobacco and alcohol) and so forth because all

these lead to chronic oxidative stress (Glόd, Wantusiak, Piszcz, Lewczuk, & Zarzycki, 2014; Phani

et al., 2010).

Roughly about 5-18% drugs given to patients in the world today are derived from natural sources,

whereby 90% of which are either directly or indirectly from plant sources (Moudgil & Khalil,

2016). Moreover, 47% of anticancer drugs on the market are derived from natural products or are

mimics of natural product (Moudgil & Khalil, 2016). These anticancer drugs include

podophyllotoxin, etoposide, teniposide, vincristine, vinblastine and paclitaxel (Gurung et al., 2016;

Popoola et al., 2016).

11
An interest in natural antioxidant and antibacterial agents has risen recently due to their positive

impact on the human body (Glόd et al., 2014, Kaur & Mondal, 2014). Plant based products are

reportedly rich in bioactive compounds such as vitamins, terpenoids, phenolic acids, lignins,

stilbenes, tannins, flavanoids, quinones, coumarins, alkaloids, amines, betalains and other

metabolites which are good antioxidants (Annapoorani & Manimegalai, 2013; Jayanthi & Lalitha,

2011; Ugochukwu, Uche, & Ifeanyi, 2013). Antioxidants scavenge a variety of free radicals and it

can be extremely important in inhibiting oxidative mechanisms that lead to degenerative diseases

(Glod et al., 2014).

The increasing interest in natural products derived therapeutic agent is becoming skeptical for

various reasons. Reasons for doubting includes lack of proper documentations about source and

formulation used, standardization of the composition, batch-to-batch consistency, documented

safety of herbal products, and information about the mechanisms of action of these products

(Moudgil & Khalil, 2016).

2.4. Phytochemicals

The three major groups of phytochemicals are nitrogen-containing substance, terpenes and

phenolics. Roughly over fourteen thousand nitrogen containing secondary metabolites have been

described so far. The main compounds of this group are: alkaloids, amines, non-protein amino

acids, cyanogenic glycoside and glucosinolate (Nafuka, 2014).

More than 4000 phytochemicals have been cataloged and are classified by their function, structural

and chemical characteristics (Scalbert et al., 2011). However, only about 150 phytochemicals have

been studied in detail (M. Saxena, Saxena, Nema, Singh, & Gupta, 2013). Phytochemicals are

12
divided into various classes and each class’s description is based on the structure and function

(Irhhaiya et al., 2014).

Medicinal plants contain bioactive compounds that can be used in the treatment of various animals

and human diseases (Heneman & Zidenberg-Cherr, 2008). Phytochemicals are divided into two

categories which are primary and secondary metabolites (Ilonga, 2012). Primary metabolites are

such as chlorophyll, proteins sugar and amino acids on the other hand Secondary metabolites

contain terpenoids, phenolics, alkaloids and so forth (Wadood et al., 2013). According to

literature, phytochemicals are responsible for medicinal activity of plants (Savithramma, Rao, &

Suhrulatha, 2011). Phytochemicals are plant constituents responsible for protecting it from

diseases and damage and contribute to the plant’s color, aroma and flavour (Saxena et al., 2013).

Studies carried have revealed that phytochemicals have an important role in preventing chronic

diseases such as cancer, diabetes, coronary heart diseases (Saxena et al., 2013; Saidulu,

Venkateshwar, & Gangadhar, 2014). Phytochemicals act by neutralizing free radicals, inhibiting

enzymes that activate carcinogens and activate enzymes that detoxify carcinogen (Saidulu et al.,

2014).

The classes of phytochemicals investigated in the present study are described in the following

subsections.

2.4.1 Phenolics

Phenolics are the largest group of phytochemicals and the most widely distributed in the plant

kingdom (Irchhaiya et al., 2014; Kabera, Semana, Mussa, & He, 2014). The three most essential

groups of dietary phenolics are polyphenols, phenolics acids and flavonoids (Irhhaiya et al., 2014).

Among the different classes of phenolic compounds, phenol is considered the smallest class

13
(Kabera et al., 2014; Saxena et al., 2013). Phenolic reportedly to exhibit antibacterial and

antioxidant activities (Pratibha, Sushma, & Gupta, 2012).

Phenolic acids is a diverse group that is divided into two groups namely hydroxybenzoic and

hydroxycinnamic acids (Irhhaiya et al., 2014; Kabera et al., 2014). Phenolic polymers, termed

tannins, are also phenolic compounds with higher molecular weight and are divided into two

groups; hydrolysable and condensed tannins (Saxena et al., 2013). Phenolic compounds are

gaining more attention with researchers due to their medicinal properties against oxidative stress.

Oxidative stress is the cause of various degenerative diseases such as coronary heart diseases,

hypertension, cancer and diabetes

2.4.2. Flavonoids

Flavonoids are the largest group of plant phenolics and most studied as compared to other

phenolics due to their pharmacological and biological activity (Saxena et al., 2013). Flavonoids

are phenolic compounds that are ubiquitous within the plant kingdom (Nafuka, 2014). About 3000

flavonoid compounds have been described (Percival, 1998). They function as protective agents

against environmental stress in plants (Percival, 1998). Flavonoids are also regarded as the most

abundant dietary polyphenolic and medicinal phytochemical (Nafuka, 2014). They have gained

more interest due to their biological and pharmacological activities (Saxena et al., 2013). This

group of compounds reportedly has multiple biological properties including: antibacterial,

cytotoxicity, antioxidant, antiinflammatory, antitumor, anti-allergenic, anti-carcinogenic, anti-

aging and antiviral activities (Saxena et al., 2013).

14
Flavonoids are categorized according to chemical structures, into flavones, flavonols, flavanals,

inflavanones, isoflavones, isoflavones and anthocyanins (Iikasha, 2016; Nafuka, 2014). Figure 1

show the major classes of flavonoids (Iikasha, 2016).

Figure 4: Example of major classes of flavonoids isolated from plants (Adapted from Iikasha,

2016).

2.4.3. Alkaloids

Alkaloids are basic compounds containing one or more heterocyclic nitrogen atoms, derived from

amino acids and are pharmacologically active (Saxena et al., 2013). Alkaloids are mostly

synthesized by plants but some can also be found in animal tissues e.g. amphibian’s skin serves as

a source of alkaloids (Nafuka, 2014). Alkaloids are associated with a wide range of

pharmacological activities such as anesthetics, central nervous system stimulants, antibacterial,

anticancer and so forth (Saxena et al., 2013).

15
2.4.4. Terpenoids

Terpenoids are defined as a group of natural products derived from five-carbon isoprene units

(Irchhaiya et al., 2014; Saxena et al., 2013). Carotenoids, steroids and gibberellic acid are just

some of its members (Kabera et al., 2014). In addition, they are classified as mono-, di-, tri-, and

sesquiterpenoids depending on the number of carbon atoms they have. Moreover, terpenoids

reportedly have medicinal properties such as anti-carcinogenic, antimalarial, antihypertensive,

insecticidal, antifungal, antiviral, anti-ulcer and antibacterial (Kabera et al., 2014; Saxena et al.,

2013).

2.4.5. Saponins

Saponins are a group of phytochemicals widely distributed in the plant kingdom (Saxena et al.,

2013; Sidana, Singh & Sharma, 2016). Saponins are defined as glycosides of steroids or terpenes

and include the group of cardiac glycosides as well as steroidal alkaloids Elekofehinti, 2015;

Mikołajczyk-Bator, Błaszczyk, Czy˙zniejewski, & Kachlicki, 2016). Saponins reportedly have

antibacterial, antifungal, antidiabetic, adjuvant, immunostimulant, hypocholesterolemic,

antitumor and antiinflammatory activities (Ajayi, Ajibade, & Oderinde, 2011, Elekofehinti, 2015;

Ramachandran, Kamaraj, Subramani, & Jeyakumar 2014; Sidana et al., 2016).

2.4.6. Tannins

Tannins are a heterogeneous group of high molecular weight polyphenolic compounds with the

capacity to form reversible and irreversible complexes with proteins and other macromolecules

(Salminen & Karonen, 2011; Saxena et al., 2013). The pharmacological activities of tannins

include antioxidant, anticancer and antibacterial (Ramachandran et al., 2014).

16
2.4.7. Anthraquinones

Anthraquinones are secondary metabolites nitrogen atom and about 750 types are reported in

higher plants (Irchhaiya, et al., 2014). Anthraquinones are characteristics of the plant families:

Fabaceae, Asphodelaceae, Liliaceae, Hypericaceae, Polygonaceae, Rhamnaceae, Rubiaceae,

Scrophulariaceae, and Zanthorrhoeaceae (Wink, 2015). Most anthraquinones possess a phenolic

OH group that makes them possible to interact with proteins and metals thereby forming hydrogen

and ionic bonds respectively in the same way polyphenols do thereby modulate their conformation

which explain their broad biological activities (Wink, 2015). The protective benefits of

anthraquinones include antifungal, antibacterial, laxative, hepatoprotective, antioxidant,

antiinflammatory, anticancer, antidiabetic and antiviral activities (Duvala, Pecher, Poujol, &

Lesellier, 2016).

2.4.8. Coumarins

Coumarins are plant derived compounds which have attracted more interest due to their diversity

and pharmacological properties (Skalicka-Woźniak, Orhan, Cordell, Nabavi, & Budzyńska, 2015).

Coumarins can be identified by the presence of a benzopyrone ring as the shown in figure 2A.

Coumarins are further categorized into four sub groups namely: simple coumarins,

furanocoumarins, pyranocoumarins and pyrone-substituted coumarins (Nafuka, 2014). Currently

only about 2% of coumarins search in plants can be obtained as they are only common in certain

genera of the following families Apiaceae, Fabaceae, Poaceae and Rubiaceae (Wink, 2015).

Coumarins and its derivatives possess antiinflammatory, anti-edemic, anti-tumor, antiviral, anti-

coagulant, antioxidant effect, enzyme inhibition and antibacterial properties (M. R. Patel & Patel,

17
2011). Caumarins are aromatic and can be incorporated in cosmetics and beverages (Wink, 2015).

The diagrams below show the common core structures of coumarins:

A B

Figure 5: Core chemical structures of coumarins. A= simple coumarins, B = pyrano coumarins, C

= furanocoumarins (Adapted from Nafuka, 2014).

2.4.9. Steroids

Steroids is a subgroup belonging to the class terpenoids and have been found to possess a broad

spectrum of biological activities (Du preez, 2012). Du preez (2012), further stated that due to their

chemical structure they show pharmaceutical activities including some antimalarial activity in

vitro. Steroids specifically exert multiple biological effects due to their antioxidant and free radical

scavenging abilities (Ilonga, 2012). The mechanism of action of these bioactive compounds as

antimalarial agents is that they exerted through the prevention stage of the developmental stages

18
of plasmodium parasites from rings to trophozoites (Du preez, 2012). Figure 6 below shows the

general structure of steroids.

Figure 6: Structure of a typical steroid (Adapted from Du preez, 2012).

2.5. Oxidative Stress

Oxidative stress may be defined as the imbalance between the production of free radicals and the

ability to neutralize their harmful effects by antioxidants (Lephart, 2016). Oxidative stress has been

found to lead to several diseases such as cancer, heart diseases, brain dysfunction, age related

degenerative condition, declination of the immune system, coronary arteriosclerosis,

carcinogenesis, gastric ulcer and DNA damage (Kambli, Patil, Chithrashree, & Keshava, 2014).

Natural antioxidants such as phenolics, flavonoids and tannins can delay or inhibit the oxidation

of biomolecules by regulation of oxidative chain reactions (Mothana, Abdo, Hasson, Althawab,

Alaghbari, & Lindequist, 2010).

2.5.1. Free radicals

Free radicals are atoms or molecules with an unpaired electron, they are highly reactive molecular

species with an unpaired electron (Buonocore, Perrone, & Tataranno, 2010; Sivanandham, 2011).

19
Free radicals react with and modify protein, nucleic acids and fatty acids in plasma membrane and

lipoprotein (Buonocore et al., 2010; Kammeyer & Luiten, 2015). Free radicals’ unpaired electron,

causes them to seek out and capture electrons from other substances in order to neutralize

themselves (Sen, Chakraborty, Sridhar, Reddy, & De, 2010).

Free radicals are well studied and documented for playing a major role in the body as both

deleterious and beneficial species (Sen et al., 2010). In their low or moderate concentrations, free

radicals partake in normal physiological functions but in their excess and/or decrease of

antioxidants level, it can lead to oxidative stress (Sen et al., 2010). Numerous synthetic drugs taken

for the treatment of some diseases are reported to generate free radicals, as a result causing other

diseases such as cancer, cardiovascular disease, cataracts, immune system decline, and brain

dysfunction (Sen et al., 2010; Sivanandham, 2011). Plants are found to be rich in antioxidants that

can work in scavenging free radicals thereby terminating oxidative damage of the body

(Kapewangolo, Tawha, Nawinda, Knott, & Hans, 2016; Sen et al., 2010). Phytochemicals

reportedly possess fewer side effects therefore, more funds are needed to be spend on these types

researches (Sen et al., 2010).

There are different sources of free radicals and oxygen is one of them (Sarma, Mallick, & Ghosh,

2010). Oxygen is an essential element needed by aerobic organisms for the process of respiration

to occur (Lephart, 2016). When oxygen is supplied from the atmosphere through the lungs at a

high concentration it becomes toxic. Oxygen in its ground state is unreactive; but its partial

reduction gives rises to active oxygen species (AOS) such as singlet oxygen, super oxide radical

anion and hydrogen peroxide (Thomas, S. D. Kamat, & Kamat, 2014). Other sources of free

radicals include (Mandal, S. Yadav, Yadav, & Nema, 2009; Sarma et al., 2010; Sen et al., 2010;

Sivanandham, 2011):

20
  Metal-catalyzed reactions

 UV radiations, X-rays, gamma rays and microwave radiations.

 Neutrophils stimulated by exposure to microbes

 In mitochondria-catalyzed electron transport reaction, oxygen free radicals produced as by

product.

 Oxygen free radicals in the atmosphere considered as pollutants.

 Inflammation initiates neutrophils and macrophages to produce ROS and RNS

 ROS generated by the metabolism of arachidonic acid, platelets, macrophages and smooth

muscle cells.

 ROS formed from several sources like mitochondrial cytochrome oxidase, xanthine

oxidases, and neutrophils and by lipid peroxidation

 Industrial effluents excess chemicals, alcoholic intake, certain drugs, asbestos, certain

pesticides and herbicides, some metal ions, fungal toxins and xenobiotics.

 Interaction with chemicals, automobile exhausts fumes, smoking of cigarettes, cigars,

beedie.

 Burning of organic matter during cooking, forest fires, volcanic activities.

Examples of radicals are such as radicals such as superoxide (O2•−), hydroxyl (OH•), peroxyl

(RO2•), hydroperoxyl (HO2•), alkoxyl (RO•), peroxyl (ROO•), nitric oxide (NO•), nitrogen dioxide

(NO2•) and lipid peroxyl (LOO•); and non-radicals like hydrogen peroxide (H2O2), hypochlorous

acid (HOCl), ozone (O3), singlet oxygen (1Δg), peroxynitrate (ONOO−), nitrous acid (HNO2),

dinitrogen trioxide (N2O3), lipid peroxide (LOOH)11 (Sen et al., 2010). Non radicals are also

termed as oxidants and capable to lead free radical reactions in living organisms easily. Radicals

derived from oxygen are characterized as the most important class of radical species generated in

21
living systems (Sen et al., 2010). Free radicals can be formed from both exogenous and endogenous

substances. They are continuously forming in cell and environment.

2.5.2. The role of antioxidants in disease prevention

Natural products represent a rich source of biologically active compounds. Specifically, plants

kingdom offers a wide range of natural antioxidants (Addai, 2016; Garcia et al., 2012).

Antioxidants prevent cell damage by neutralizing or scavenging the free radicals interactively and

synergistically to form harmless by products (Jiménez-Zamora, Delgado-Andrade, & Rufián-

Henares, 2015). In the body, an antioxidant may be water-soluble, lipid-soluble, and insoluble or

bound to the cell wall (Marinova & Batchvarov, 2011). Plants are found to be rich sources of free

radical scavenging molecules (antioxidants) namely terpenoids, phenolic acids, vitamins (A, C &

E), stilbenes, lignins, tannins, alkaloids, quinones, coumarins, flavonoids, amines, betalains and

other metabolites which possess antioxidant activity (Jayanthi & Lalitha, 2011).

There are two types of antioxidants the synthetic and the natural one. However, most researchers

are diverting from synthetic antioxidants due to their toxicity (Elaasser, Abdel-Aziz, & El-Kassas,

2011). Natural antioxidants play a major role in health maintenance as well as prevention of

chronic and degenerative diseases namely atherosclerosis, cardiac, cerebral ischema,

carcinogenesis, neurodegenerative disorders, diabetic pregnancy, rheumatic disorder, DNA

damage and ageing (Akbarirad et al., 2016). A human body can naturally produce antioxidants but

they can be are insufficient to suppress the oxidation stress. Plants reportedly contain enough

antioxidants that man can use to cover antioxidant deficiency. Therefore, eating of fruits and

vegetables and the use of natural supplement is highly recommended.

22
Within a human body there are three main defence systems namely free radicals detoxifying

enzymes, metal binding protein and antioxidants (Lephart, 2016). The free radicals detoxifying

enzymes are: catalase which is involved in the conversion of hydrogen peroxide to harmless water

and molecular oxygen. Superoxide dismutase (SOD) is an enzyme involved in the conversion of

superoxide (●O2) to either molecular oxygen or hydrogen peroxide (Lephart, 2016). Glutathione

peroxide is another class of enzymes involved in converting hydrogen peroxide into water and

oxygen (Krishnamurthy & Wadhwani, 2012). Metal binding protein such as ferritin, albumin,

lactoferrin, and ceruloplasmin that seize free iron and copper that are capable of catalysing

oxidative stress reaction (Lephart, 2016).

Dietary antioxidants, namely vitamin C, vitamin E, and beta-carotene are the most widely studied

antioxidants (Mandal et al., 2009). Vitamin C is considered the most significant hydrophilic

antioxidants in the extracellular fluid. It has the potential of neutralizing free radicals in the

aqueous phase prior lipid peroxidation is initiated (Percival, 1998). Vitamin E a major hydrophobic

antioxidant embedded in the cell membrane where it functions to protect the membrane fatty acids

from lipid peroxidation (Percival, 1998). Vitamin C has also been reported to possess the potential

of regenerating vitamin E. Moreover, beta-carotene other carotenoids are also believed to offer

antioxidant protection to the lipid-rich tissue. It is reported that beta-carotenes work synergistically

with vitamin E (Henríquez et al., 2010).

According to Phani et al., (2010) there are four most commonly used antioxidants namely

butylated hydroxyamisole (BHA), butylated hydroxytoluene (BHT), propylgallate (PG) and tert-

butylhydroxytoluene (TBHQ). Nevertheless, these antioxidants are suspected of being responsible

for liver damage and carcinogenesis in laboratory animals (Phani et al., 2010). Therefore, the

23
development and utilization of more effective and less toxic antioxidants of natural origin

particularly from plants are encouraged (Elaasser et al., 2011; Phani et al., 2010).

Antioxidants neutralize free radicals through two different mechanisms. Both mechanisms lead to

similar reduction but differ in their kinetics and propensity (Marxen et al., 2007). The two

mechanisms are single electron transfer whereby the antioxidant transfer an electron to the radical

and hydrogen atom transfer whereby the antioxidant quench the free radicals by donating a

hydrogen atom (Marxen et al., 2007; Senguttuvan, Paulsamy, & Karthika, 2014).

2.6. α, α-Diphenyl-1-picrylhydrazyl radical scavenging activity

α, α- diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging offers the first approach for

determining the antioxidant capacity of a compound, extract or biological substances (Kedare &

Singh, 2011; MacDonald-Wicks, Wood, & Garg, 2006; Moon & Shibamoto, 2009). DPPH

molecule is classified as a stable free radical due to that there is delocalization of the spare electron

over the entire molecule, so that the molecule does not dimerize like most other free radicals

(Kedare & Singh, 2011). The delocalization gives rise to the deep violet colour, with an absorption

in ethanol solution at around 520 nm. The following equation Z● + AH = ZH + A● represents the

primary equation of free radical neutralization. In the equation Z● represent DPPH radical and AH

represent the donor molecule leading to ZH reduced form and A● is free radical produced in the

first (Kedare & Singh et al., 2011).

DPPH is a commercial standard free radical often used in measuring the antioxidant activity of

various chemical components including plant extracts like in case of this study. To determine the

antioxidant potential of a chemical component different experimental approaches are always used.

24
In this study two methods were used to screen for antioxidant activity; DPPH radical scavenging

assay and reducing power method.

DPPH radical scavenging assay is said to be simple, accurate, inexpensive, rapid, sensitive and a

convenient method independent of samples’ polarity, for screening many samples for radical

scavenging activity (Kedare & Singh, 2011; Marinova & Batchvarov, 2011; Marxen et al., 2007).

The fore mentioned advantages made the DPPH method suitable for testing medicinal plants as

natural sources of scavengers of free radicals thereby discovering lead candidates for commercial

purposes. Methanol and ethanol are the most used solvents for determination of radical scavenging

activity by DPPH (Marinova & Batchvarov, 2011).

The DPPH assay was developed by Blois in 1958 with the aim to determine antioxidant capacity

using α, α-diphenyl-β-picrylhydrazyl (DPPH; C18H12N5O6, M= 394.33) (Kedare & Singh, 2011).

The principle behind DPPH is that the odd electron of nitrogen atom in DPPH is reduced by

receiving a hydrogen atom from antioxidants. (Kedare & Singh, 2011; MacDonald-Wicks et al.,

2006; Moon & Shibamoto, 2009). Figure 7 shows the mechanism of how 2, 2-diphenyl-1-

picrylhydrazyl radical (DPPH●) was neutralized to DPPH by an antioxidant.

Figure 7. DPPH• free radical conversion to DPPH by anti-oxidant compound (Adapted from

Moon & Shibamoto, 2009).

25
2.7. Reducing Power Assay

The principle behind reducing power assay lies in the substances such as antioxidant with

reduction potential to react with potassium ferricyanide (3+) forming potassium ferrocyanide that

may serve as important indicators of antioxidant activity (Senguttuvan et al., 2014). In this assay,

there is a change of colors from yellow to blue and green depending on the reducing power of the

extract or specimen (Jayanthi & Lalitha, 2011). Furthermore, in the presence of reductones

(antioxidants), free radicals are being scavenged by accepting hydrogen atom (Jayanthi & Lalitha,

2011). In case of the plant extracts, the reductones in the extract cause the reduction of Fe3+/ Ferric

cyanide complex to ferrous form that is Fe2+, which can be measured spectrophotometrically at

700 nm (Senguttuvan et al., 2014). The rationale behind reducing power assay is that there is a

direct relationship between reducing power and samples/ extracts concentration such that reducing

power increases with an increase in extracts concentration. This is a significant indicator of its

potential antioxidant activity (Senguttuvan et al., 2014).

2.8. Quantitative determination of phytochemicals

The discovery of new drugs from natural products is normally based on phytochemical and

pharmacological approaches (Santhi & Sengottuvel, 2016). These chemicals are such as phenolics,

terpenoids, alkaloids, flavonoids, tannins, anthraquinones etc. (Kaur & Mondal, 2014). In addition

bioactive chemicals of plant have various activities such as antibacterial, antioxidant,

antiinflammatory, antifungal, antiviral etc, (Obiang et al., 2016). Quantitative phytochemicals

screening is the first step toward extraction, purification and identification the bioactive

compounds for useful aspects (T. S. Geetha & Geetha, 2014).

26
2.8.1. Total Phenolic content

Phenolic compounds are found to be good antioxidants (Azlim et al., 2010). Therefore, it is

important to determine the content of phenolic compounds present in an extract or sample. The

high the content of phenolic compounds the greater antioxidant activity a plant has. The total

phenolic content (TPC) assay using the Folin-Ciocalteu (FC) reagent is one of the most regularly

utilized methods to quantify the phenolic content of a plant extract (Henríquez et al., 2010). Upon

reaction with reducing agents, a blue-coloured complex is formed between the molybdenum and

tungsten present in FC, which can then be measured through spectrophotometry (Tan & Lim,

2015). The high sensitivity, reproducibility and convenience of this single electron transfer-based

assay has made it prevalent in routine screening of natural products (Tan & Lim, 2015).

2.8.2. Total condensed tannins

The present study only focused on condensed tannins (proanthocyanidins) as they are proven to

be one of the groups of compounds with promising health-promoting properties (Zarin, Wan, Isha,

& Armania, 2016). Furthermore, condensed tannins reportedly assist in wound healing, reducing

pain for pancreatitis, reduce insulin resistance in diabetics, assist in protection from drug toxicity

and also can help lower the levels of low- density lipoprotein or in other word bad cholesterol

(Zarin et al., 2016). Vinillin (in 4% methanol) assay is a reported method used often to determined

total condensed tannins (Rebaya et al., 2014).

Condensed tannins are thought to have strong antioxidant properties making them potential

phytoconstituents in preventing diseases, health promoters and anti-ageing agents (Tamilselvi,

Krishnomoorthy, Phamotharan, Arumagam, & Sagadevan, 2012). In addition to antioxidant

properties, condensed tannins reportedly possess antiinflammatory, anti-asthmatic, anticancer,

27
antiviral, anti-carcinogenic, anti-allergy, antibacterial, antihypertension and cardiovascular system

protective properties (Zarin et al., 2016).

2.9. Phytochemicals as antibacterial agents

Bacterial infections are of particular concern globally due to current bacterial strains that are

becoming resistant to antibiotics (Tchinda, Voukeng, Beng, & Kuete, 2016). Epidemic due to

bacteria strains resistance is still an issue even in developed nations with understanding in

microbiology and their control (Dabur et al., 2007). Plants have been a good source of antibacterial

agents and still continue to be highly effective instruments in fighting bacterial infections (Tchinda

et al., 2016). Phytochemical derivatives have shown pronounced promise in the relief of intractable

infectious diseases including opportunistic Acquired Immune Deficiency Syndrome (AIDS)

infections (Rebaya et al., 2014).

Antibacterial drugs are substances that interfere with the growth and spreading of microbes in the

host (Saga & Yamaguchi, 2009). Antibacterial action can be bacteriostatic such that it can inhibit

the growth of the microbes or bactericidal meaning that it can kill the microbe (Taukoorah, Lall,

& Mahomoodally, 2016). Historically the first antibacterial was a synthetic drug discovered by

Ehrlich in 1910 for the remedy of syphilis “Salvarsan” (Saga & Yamaguchi, 2009). In 1935 another

synthetic drug “Sulfonamide” was discovered by Domagk and other researchers (Saga &

Yamaguchi, 2009). Since both of these drugs were synthetic their major limitations included safety

and efficacy (Saga & Yamaguchi, 2009).

Antibacterial drugs derived from medicinal plants are such atropine, quinine, morphine and

tubocurarine (Abdallah, 2011). Investment in natural product antibacterial drugs studies have been

neglected and significantly decreased over the past two decades (Brown, Lister, & May-Dracka,

28
2013). According to records, only seven new natural chemical entities have been approved for

therapy for the treatment of bacterial infections (Brown et al., 2013). The significant decline in

investment in natural products antibacterials as well as approval of antibacterial agents could be

due to certain factors which includes change of drug regulatory procedures, increased drugs safety

standard and failure of modern drugs discovery techniques i.e., high throughput screening against

certain bacterial genome and so forth (Brown et al., 2013).

Microorganisms investigated in the study

2.9.1. Shigella sonnei

According to Massenet, Vohod, Hamadicko, and Caugant (2011), Shigella species are found to be

the major cause of diarrhoea worldwide. Shigella sonnei is a gram negative pathogenic bacterium

that can cause an infection called shigellosis, also known as bacillary dysentery, an infection of

the digestive system (Anand, Pande, & Gore, 2013). Shigella species are non-spore forming, rod

shaped bacteria and are a member of Enterobacteriaceae family (Gaurav, Singh, Gill, Kumar, &

Kumar, 2013). These species are more prevalent in human than in any other living organisms and

they are often found in small children. However, this species cannot survive long enough outside

the host (Baydack & Ens, 2011).

Shigella species are found in the faeces of infected person and often spread through the faecal-oral

route by direct contact with an infected person, eating contaminated food by food handlers, eating

contaminated food by flies carrying infection, drinking water (Baydack & Ens, 2011). Outbreaks

are also reported to occur in areas with severe crowding and/or poor hygiene (Gaurav, Singh, Gill,

Kumar, & Kumar, 2013). S. sonnei is 1 serotype it causes mildest form of foodborne disease

(Massenet et al., 2011).

29
2.9.2 Alcaligenes faecalis

The taxonomy of the genus Alcaligenes is closely linked with the taxonomy of the genus

Achromocbater, and several Alcaligenes species have now been reclassified as Achromobacter

species (Win et al., 2006). Alcaligenes faecalis, a species of gram-negative, rod shaped bacterium

commonly found in the environment is the most frequently isolated member of the Alicaligenaceae

family in the clinical laboratory (Mordi, Yusuf, Onemu, Igeleke, & Odjadjare, 2013). It is reported

that members of this family produce strong alkaline reaction in all carbohydrates media. Most of

these strains form characteristic colonies with a thin spreading irregular edge (Win et al., 2006).

Alcaligenes faecalis is reported to cause meningitis in new born, bacteremia in cancer patients, has

been associated with pancreatic abscess and corneal ulcer (Mordi et al., 2013).

2.9.3. Enterococcus faecalis

Members of the genus Enterococcus, collectively called enterococci, are recognized by the as

indicator organisms for bacteriological water quality in fresh and saline waters (Harwood et al.,

2004). Their presence, specifically at an increased level, is an indication of faecal pollution from

animal or humans (Tyne, Martin, & Gilmore, 2013). These organisms are normally found in the

gastrointestinal, biliary tracts, in lower number of the vigina and male urethra (Win et al., 2006).

Enterococci are increasingly becoming significant agents of many human diseases mainly because

of their resistance to antibacterial agents (Tyne et al., 2013). Enterococci are found to be the second

most prevalent cause of nosocomial urinary tract and wound infections (Sood, Malhotra, Das, &

Kapil, 2008; Win et al., 2006). Even though Enterococcus faecalis was once regarded as

nonpathogenic, this opportunistic gram-positive coccus now ranks among the most worrisome

hospital pathogens (Ogihara, Saito, Sawabe, Hagihara, & Tohda, 2016). It has an instinct resistance

30
to many antibiotics and a remarkable potential for developing resistance to others (Jain, Mulay, &

Mullany, 2016). These species are resistant to pencicillins, cephalosporins, and aminoglycosides

and have developed resistance to vancomycin (Win et al., 2006).

2.9.4. Serratia marcescens

The Serratia species are special among the Enterobacteriaceae family (Yang, Cheng, Hu, Zhu, &

Li, 2012). Furthermore, these species are resistant to colistin and cephalothin (Win et al., 2006).

Ten species of Serratia genus were recognised whereby 7 of which have been recovered from

human clinical specimens (Win et al., 2006).

Serratia marcescens, a gram-negative bacterium, is the most significant of the genus Serratia and

in many cases is associated with a variety of human infections specifically pneumonia and

septicaemia in patients with reticuloendothelial malignancies who are taking chemotherapeutic

agents (Win et al., 2006). This microorganism was used as a harmless relationship where one

organism benefit living another unaffected to trace environmental contamination, basically due to

their characteristic red pigmentation of some strains was easy to spot in culture media (Win et al.,

2006). Currently, S. marcescens is considered an important pathogen with invasive properties and

they have a tendency to resist many prevalently utilized antibiotics (Koh et al., 2007). S.

marcescens can also be considered a significant nosocomial opportunist as evidenced by a case of

childhood meningitis following the utilization of contaminated benzalkonium chloride disinfectant

solution (Win et al., 2006).

31
METHODS

3.1. Research design

The study investigated antibacterial, antioxidant and screened for phytochemicals profile of A.

esculenta, A. arenaria and P. leubnitziae using qualitative and quantitative methods. Figure 8

below shows the schematic research design flow diagram.

Collectio
Antibacte
n of
Qualitativ rial Assay
plants
Ethical e nd (Disc Antioxida
materials
approval Organic quantitati agar nt Assay
from
& permit Extractio- ve diffussion (DPPH &
Ovambol
(RPC n phytoche method & Reducing
and and
&MET) mical Microdilu power)
identificat
screening tion
ion
method
(NBRI)

Figure 8: Schematic research design flow diagram

3.2. Plant specimen and collection

The fresh leaves, stems and roots of A. arenaria, A. esculenta and P. leubnitziae were collected

from Omutala village in Oshana region, Namibia in November 2014. Research ethical approval

was obtained from the University of Namibia (Appendix D). Collection permit was obtained from

the Ministry of Environment and Tourism (Appendix E) and Research and Publication Committee

of the University of Namibia (RPC) (Appendix F). The plants were identified and authenticated at

the National Botanical Research Institute (NBRI), Windhoek. Voucher specimen numbers: A.

arenaria (300), A. esculenta (305) and P. leubnitziae (310). The fresh leaves, stems, and roots

were air dried at room temperature. The dried plant leaves, stems and roots were powdered using

a blender and stored at room temperature until further analysis.

32
3.3. Phytochemical screening

Phytochemical tests were carried out on the on the powdered specimen using standard procedures

to identify constituents (Ajayi et al., 2011; Alabri et al., 2013; Alaga, Edema, Atayese, & Bankole,

2014; Arya, Thakur, & Kashyap, 2012; Maregesi, Mwakalukwa, Mwangomo, & Nondo, 2013;

Shakeri, Hazeri, Vlizadeh, Ghasemi, & Tavallae, 2012; Suneetha et al., 2013; Upadhyay, Singh,

& Kumar, 2010)

3.3.1. Test for steroids: the powder samples (1 mg) were dissolved in chloroform (10 ml) and

added concentrated sulphuric acid (1 ml) into the test tube by wall sides. The colour of the upper

layer turned red and the sulphuric acid layer showed yellow with green fluorescence. This

indicated the presence of steroids.

3.3.2. Test for tannins: the grounded 0.5 g of each sample was separately boiled with 10 ml

distilled water for five minutes in a water bath and was filtered while hot. 1 ml of cool filtrate was

distilled to 5 ml with distilled water and a few drops (2-3) of 10% ferric chloride were observed

for any formation of precipitates and any colour change. A bluish-black or brownish-green

precipitate indicated the presence of tannins.

3.3.3. Test for saponins: the blended 1 g of each dried stain was separately boiled with 10ml of

distilled water in a bottle bath for 10min. The mixture was filtered while hot and allowed to cool.

The following test was then carried out. Demonstration of frothing: 2.5 ml of filtrate was diluted

to 10ml with distilled water and shaken vigorously for 2minutes (frothing indicated the presence

of saponin in the filtrate).

33
3.3.4. Test for terpenoids: exactly 5 ml of each extract was mixed in 2 ml of chloroform. 3 ml of

concentrated Sulphuric acid (H2SO4) was then added to form a layer. A reddish brown precipitate

colouration at the interface formed indicated the presence of terpenoids.

3.3.5. Test for flavonoids: the powdered 1 g of dried leaves of each specimen was boiled with 10

ml of distilled water for 5 minutes and filtered while hot. Few drops of 20 % sodium hydroxide

solution were added to 1 ml of the cooled filtrate. A change to yellow colour which on addition of

acid changed to colourless solution depicted the presence of flavonoids.

3.3.6. Test for anthraquinones: chloroform (5 ml) was added to 0.5 g of the powdered dry

samples of each specimen. The resulting mixture was shaken for 5 mins after which it was filtered.

The filtrate was then shaken with equal volume of 10 % ammonia solution. The presence of a

bright pink colour in the aqueous layer indicated the presence of free anthraquinones.

3.3.7. Test for alkaloids: one gram of powdered sample of each specimen was separately boiled

with water and 10 ml hydrochloric acid on a water bath and filtered. The pH of the filtrate was

adjusted with ammonia to about 6-7. A very small quantity of Mayer’s reagent was added to about

0.5 ml of the filtrate in two test tubes and observed. The test tubes were observed for coloured

precipitates or turbidity.

3.3.8. Test for coumarins: With each concentrated alcoholic extract of drugs, few drops of alcohol

ferric chloride (FeCl3) solution were added. The formation of deep green colour, which turned

yellow on the addition of concentrated Nitric acid (HNO3), indicated the presence of coumarins.

3.3.9. Test for Phenols: the extracts were dissolved in alcohol and a drop of neutral ferric chloride

was added to these. The intense colour indicated the presence of phenolic compounds.

34
3.4. Extraction of dried powder samples

The extraction of the sample was carried out according to a method previously described by Naz

and Bano (2013), with minor modification. The dry powder samples (20 g) were extracted with

MeOH-DCM (1:1) solvent (200 mL) for 48 h. After extraction, the samples were filtered using

filter paper (Whatman No.1). The extraction solvent was evaporated using a rotary evaporator

(Heidolph Instruments GmbH & Co. KG, country) at 40oC. The filtrate was transferred with little

volume of ethyl acetate into pre-weighed vails for further drying at room temperature.

3.5. Quantitative phytochemical analysis

3.5.1. Total phenolics content

The total phenolic content was estimated by Folin Ciocalteu method as described by Rebaya et al.

(2014) with slight modifications. The extracts (1mL) or the standard solution of Gallic acid

(Sigma-Aldrich, Germany) (100, 200, 300, 400, and 500μg/ml) were mixed with 5 mL of distilled

water, 1 mL of sodium carbonate (20%) and 1mL of Folin Ciocalteu reagent (Merck, Germany).

The mixture was allowed to stand in a water bath for 30 min at 40°C. The absorbance was

measured at 765 nm using microplate reader spectrophotometer. The total phenolic content was

expressed as mg of gallic acid equivalents per g dry matter (mg GAE. g-1DM). The experiment

was run in triplicate and analysis was carried out.

35
3.5.2. Total Condensed Tannins

The tannin content was determined by a method described by Rebaya et al. (2014) with minor

modification, using tannic acid (Sigma-Aldrich, Germany) as a reference compound. A volume of

400 μL of extract was added to 3 mL of a solution of vanillin (4% in methanol) and 1.5 mL of

concentrated hydrochloric acid. After 15 min of incubation the absorbance was read at 500 nm

using micro plate reader. The condensed tannin was expressed as mg TAE/g.

3.6. Antioxidant activity

Due to the complex nature of phytochemicals as well as the antioxidant activity determination

dependence on reaction mechanisms, it is advisable for antioxidant activity to be evaluated using

multiple assays (Zou, Chang, Gu, & Qian, 2011). Therefore, in this study the antioxidant property

of plant extracts was determined using two antioxidant methods; DPPH free radical scavenging

assay and reducing power assay.

3.6.1. DPPH radical scavenging assay

The DPPH radical scavenging activity assay described by Azlim et al. (2010) was adopted with

slight changes. Different concentrations of extracts (7.81, 15.63, 31.25, 62.50, 125, 250, 500

µg/mL) were prepared. DPPH solution (0.012 mg/mL) was prepared by dissolving 1.18mg of

DPPH (Sigma-Aldrich, Germany) in 100 mL ethanol in a foil covered bottle. Distilled water was

added in the fi all the wells. Extracts and controls were added to the first row of wells. Ascorbic

acid (Sigma-Aldrich, Germany) was used as a standard. Double dilute 100 µL from the first row

downwards (discard the final 100 100 µL). DPPH solution (100 µL) was added to all triplicate

wells except the wells of the extract control. The mixture was shaken vigorously and was left

covered with a foil to stand in the dark for 30 min. The absorbance of the resulting solution was

36
measured spectrophotometrically at 517 nm using micro plate reader (manufactured by Molecular

Devices LLC).

3.6.2. Reducing power assay

The reducing power was assayed as described by Deore et al. (2009) with some modifications.

Different concentrations of ethanolic extracts (0.0625, 0.125, 0.25 and 0.5 mg/mL) were mixed

with 1.25 ml of phosphate buffer (50 mM, pH 7.0) and 1.25 mL of 1% potassium ferricyanide.

The mixture was then incubated at 50oC for 20 min. After incubation, 1.25 ml of trichloroacetic

acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally,

1.25 mL of the supernatant was mixed with 1.25 mL of distilled water and 0.25 mL FeCl3 solution

(0.1%, w/v). The absorbance was measured at 700 nm. The assays were carried out in triplicate

and the results were expressed as mean values ± standard deviations.

3.7. Antibacterial Activity

3.7.1. Disk diffusion assay

Antibacterial screening was performed using the disk diffusion method (Zarin et al., 2016).

Antibacterial activity was based on clear zone formed around the disk. Complete inhibition was

indicated by a clear zone, while partial inhibition was indicated by a semi-clear zone. Imipenem

(10 µg) (OXOID LTD, England) was used as the reference antibiotic for antibacterial activity.

Sterile distilled water (dH2O) was used as negative control. The test microorganisms: A. faecalis

(ATCC 8750), E. faecalis (ATCC 7080), S. sonnei (ATCC 25931) and S. marcescens (ATCC

8100) obtained from Department of Biological Sciences of the University of Namibia were seeded

onto nutrient agar medium at 25oC by using sterilized micropipette. The filter paper discs

impregnated with the extracts of different concentrations (6.3, 3.2 and 1.6 mg/mL) were placed on

37
test microorganism-seeded plates. The plates were incubated at 37oC for 24 h. All tests were

performed in triplicate and the antibacterial activity was expressed as the mean of inhibition

diameters (mm) produced.

3.7.2. Minimum inhibitory concentration (MIC).

MIC was determined by microdilution method with nutrient broth and Muller Hinton agar with

few modification (Obiang et al., 2016). Briefly, nutrient broth (100 µL/wells) was distributed into

wells of a microplate. One hundred microliters of extracts were added to the first row of wells and

a two-fold dilution was added into other wells to make up 0.1-5.0 mg/mL concentration of each

extract. Ninety microliters of nutrient broth and 10 µL of inocula were added into wells to prepare

a total volume of 200 µL in each well. The plates were slightly shaken and incubated 37 °C

overnight. Inhibition was assessed by observing the absence of turbidity in the wells. Wells without

extract were used as negative control and Gentamycin (OXOID LTD, England) was used as a

positive control.

3.8. Data analysis

The experimental results were expressed as mean ± standard deviation. All experiments were

replicated three times. The 50% inhibitory concentration (IC50) values were calculated using a

nonlinear regression analysis in the Graphpad prism 6 software (Graphpad Software Inc.,

California, USA).

38
RESULTS

4.1. Phytochemical Screening

The present study revealed that extracts of leaves, roots and stem of A. arenaria, A. esculenta, and

P. leubnitziae contained steroids, terpenoids, saponins, flavonoids, anthraquinones, coumarins,

alkaloids, phenols, coumarins and tannins (Table 1). However, anthraquinones were only detected

in A. esculenta root and stem as presented in Table 1. On the other hand, phytochemical such as

steroids, phenols and tannins were found to be present in all extracts. In addition, tannins and

phenols were detected in appreciable amounts (+++). Most phytochemicals were found to be

present with less amount (+) in the A. arenaria root and stem extracts and were in appreciable

amount (+++) in leaves extract (Table 1). Appreciable was measured according to the change in

colours’ intensity.

39
Table 1: Phytochemical screening results A. esculenta, A. arenaria, and P. leubnitziae’s roots,

stems and leaves.

Plant constituent Plants Extracts

Aloe esculenta Acacia arenaria Pechuel-Loeschea leubnitziae

Leaves Root Stem Leaves Root Stem Leaves Root Stem

Steroids + +++ +++ ++ + ++ ++ + ++

Terpenoids ++ ‒ ‒ + ‒ ‒ ‒ + +++

Saponins ‒ + ‒ +++ + ‒ + ++ +

Flavonoids + ++ ‒ +++ ‒ ‒ +++ + ‒

Anthraquinones ‒ +++ ++ ‒ ‒ ‒ ‒ ‒ ‒

Coumarins + + + + + + + + +

Alkaloids + ++ + + + + ‒ + +

Phenols +++ +++ ++ +++ + + +++ ++ +

Tannins +++ +++ ++ +++ + + +++ ++ +

(-) = absent, (+) = present, (++) = moderately present, (+++) = appreciable amount

4.2. Quantitative phytochemical analysis

4.3.1. Total phenolic and total condensed tannins content

The total phenolic content (TPC) was expressed as gallic acid equivalents (Table 2). The presence

of TPC among the nine tested crude extracts was observed. The TPC was in the range of (0.5 ±

0.1 to 5.7 ± 0.1 mg GAE/g extract). Phenolic compounds are ubiquitous secondary metabolites in

plants. They are known to possess antioxidant activity and it is likely that the activity of these

extracts is due to this compounds (Rebaya et al., 2015; Motlhanka & Nthoiwa, 2012). The results

40
obtained in this study showed the greatest total phenolic content in A. arenaria leaves extract

(5.7±0.1 mg of GAE/g extract) followed by A. esculenta root extract (4.8 ± 0.0 mg of GAE/g

extract) and A. esculenta stem was the least with (0.5 ± 0.1 mg of GAE/g extract) as presented in

table 2. The results on the other hand showed that P. leubnitziae stem, A. arenaria leaves and A.

esulenta leaves extracts had high levels of TCT and low levels of TPC. The results also showed A.

esculenta stem extract with the least TCT (0.1 ± 0.0 mg of GAE/g extract) table 2 below show the

results.

Table 2: Total phenolic content and total condensed tannins of leaves, roots and stem extract

from A. arenaria, A. esculenta and P. leubnitziae.

Samples TCT (mg of TAE/g Extract)±SD TPC (mg of GAE/g Extract)±SD

P. leubnitziae
Stem 71.6 ± 0.0 1.6 ± 0.1
Root 1.0 ± 0.3 1.6 ± 0.0
Leaves 2.1 ± 0.1 3.1 ± 0.0
A. arenaria
Stem 0.8 ± 0.2 2.9 ± 0.1
Root 0.1 ± 0.0 2.0 ± 0.1
Leaves 66.5 ± 0.1 5.7 ± 0.1
A. esculenta
Stem 0.1 ± 0.0 0.5 ± 0.1
Root 0.9 ± 0.1 4.8 ± 0.0
Leaves 4.2 ± 0.0 1.4 ± 0.0

TCT: Total condensed tannins; TAE: Tannic acid equivalent; TPC: Total phenolics content;

GAE: Gallic acid equivalent; SD: Standard deviation

41
4.3. Antibacterial Activity

4.3.1. Disc diffusion results

In vitro antibacterial activity of MeOH-DCM (1:1) crude extracts of nine extracts against the four

bacteria was assessed on the presence or absence of inhibition zones. The various crude extracts

from dry leaves, stems and roots of A. arenaria, A. esculenta and P. leubnitziae exhibited

antibacterial potential against one gram positive bacterium (E. faecalis) and three gram negative

bacteria (A. faecalis, S. marcescens and S. sonnei) at three concentrations; 6.3, 3.2 and 1.6 mg/ml.

Not all the tested concentrations inhibited the tested microbes. However, A. arenaria root and A.

esculenta leaves extracts showed the greatest inhibition (4.5 ± 0.7 mm) and (4.5 ± 0.0 mm) against

S. sonnei and A. faecalis respectively at 6.3 mg/ml. Furthermore, A. arenaria stem extract showed

inhibition (4.0 ± 0.0 mm) against S. marcescens and E. faecalis at 3.2 and 1.6 mg/ml respectively.

In a similar manner P. leubnitziae root and A. esculenta leaves extracts showed (4.0 ± 1.4 mm)

and (4.0 ± 0.0 mm) respectively against E. faecalis microbe. Additionally, A. esculenta root extract

inhibited most microbes at different concentrations while A. arenaria leaves extract inhibited less

microbes at different concentrations as compared to other extracts. Table 3 below shows the

(inhibition zone±Standard Deviation) measurements were done in doublet and averaged. As

expected, Imipenem the positive control demonstrated good inhibition at 10 µg/mL concentration.

42
Table 3: Antimicrobial screening of plants against A. faecalis (ATCC 8750), E. faecalis (ATCC 7080), S. sonnei (ATCC 25931) and

S. marcescens (ATCC 8100) using disc diffusion method.

Extract concentrations (mg/mL)

6.3 3.2 1.6
Extract Af Ss Ef Sm Af Ss Ef Sm Af Ss Ef Sm
AcaL - - 3.0 ± 1.4 - 2.5 ± 0.7 - - - - - 3.0 ± 1.4 -
AcaR 3.0 ± 0.0 4.5 ± 0.7 2.0 ± 0.0 - 2.0 ± 0.0 - - 2.0 ± 0.0 2.5 ± 2.1 - 1.5 ± 0.7 -
AcaS 1.0 ± 0.0 3.5 ± 2.1 2.5 ± 0.7 - - - - 4.0 ± 0.0 - - 4.0 ± 0.0 2.5 ± 0.7
PecL 2.5 ± 0.7 - 2.0 ± 0.0 - 2.5 ± 0.7 - - - 2.5 ± 0.7 2.5 ± 0.7 2.0 ± 0.0 -
PecR 3.0 ± 1.4 - 4.0 ± 1.4 1.0 ± 0.0 - - - 2.0 ± 0.0 - - 3.0 ± 0.0 -
PecS - 3.5 ± 3.5 1.3 ± 0.4 3.5 ± 0.7 - - - - 3.5 ± 3.5 - 2.0 ± 0.0 -
AloL 4.5 ± 2.1 - 4.0 ± 0.0 - 3.5 ± 0.7 - - - - 2.5 ± 2.1 2.0 ± 0.0 2.0 ± 0.0
AloR 3.5 ± 0.7 - 2.0 ± 0.0 - 3.5 ± 0.7 - - 2.0 ± 0.0 2.5 ± 0.7 3.0 ± 1.4 3.5 ± 2.1 3.5 ± 0.7
AloS 3.5 ± 0.7 3.0 ± 0.0 - 2.0 ± 0.0 3.0 ± 1.4 - 3.0 ± 0.0 - - - - 3.5 ± 0.7
IMI 14.0 ± 0 24.0 ± 0 - 14.0±0.0
AcaL: Acacia leaves, AcaR: Acacia roots, AcaS: Acacia Stem, PecL: Pechuel leaves, PecR: Pechuel roots, PecS: Pechuel stem, AloL:

Aloe leaves, AloeR: Aloe roots, AloS: Aloe stem, Af: A. faecalis, Ss: S. sonnei, Ef: E. faecalis, Sm: S. marcescens, (-) = no activity

detected, units for values are in mm and IMI: Imipenem antibiotic.

44
4.3.2. MIC data

The result showed in Table 4 revealed the MIC values of MeOH-DCM (1:1) extracts of A.

arenaria, A. esculenta and P. leubnitziae. The best activity was recorded with A. arenaria leaves

extract, with MIC values ranging from 0.1 to 5.0 mg/mL against all tested bacteria. MIC values

below or equal to 5.0 mg/mL were also recorded with P. leubnitziae leaves extract and Gentamycin

antibiotic respectively against E. faecalis, S. marcescens and S. sonnei. The lowest MIC value (0.1

mg/mL) was recorded with the extract from A. arenaria leaves against S. marcescens and S. sonnei

as well as the standard gentamycin against S. sonnei.

Table 4: MIC values (mg/mL) as determined by the microdilution method

Extract A. faecalis E. faecalis S. marcescens S. sonnei

MIC MIC MIC MIC

Acacia leaves 5.0 5.0 0.1 0.1

Acacia stem >5.0 >5.0 >5.0 >5.0

Aloe leaves >5.0 >5.0 >5.0 >5.0

Aloe stem >5.0 >5.0 >5.0 >5.0

Aloe root >5.0 >5.0 >5.0 >5.0

Pechuel leaves >5.0 >5.0 >5.0 5.0

Gentamycin >5.0 5.0 1.3 0.1

45
4.4. Antioxidant Activity

4.4.1. DPPH Assay

The free radical scavenging activity of A. arenaria, A. esculenta and P. leubnitziae crude extracts

was assessed by the DPPH assay. Figure 9, 10 and 11 illustrates a significant decrease in the

concentration of DPPH radical due to the scavenging ability of A. arenaria, A. esculenta and P.

leubnitziae. The results show that P. leubnitziae roots extract had the highest DPPH radical

scavenging activity with an IC50 value of 25.4 µg/mL followed by A. arenaria leaves with an IC50

value of 26.6 µg/mL. A. arenaria roots showed the least IC50 value of 196.8 µg/mL as shown in

(table 5). The IC50 value of the positive control ascorbic acid was 83.7 µg/mL.

46
 140
Aca L Aca R Aca S Ascorbic Acid
120

100
%Inhibition

80

60

40

20

0
Vit 50 500 250 125 61.5 31.3 15.3 7.8
Concentrtion (µg/mL)

Figure 9: DPPH scavenging activity of A. arenaria’s roots, leaves and stem extracts as compare

to Ascorbic Acid. Aca L: A. arenaria leaves, Aca R: A. arenaria root and Aca S: A. arenaria

stem.

47
 120
Aloe L Aloe R Aloe S Ascorbic Acid

100

80
%Inhibition

60

40

20

0
Ascorbic 500 250 125 61.5 31.3 15.3 7.8
Acid
Concentration (µg/mL)

Figure 10: DPPH scavenging activity of A. esculenta’s roots, leaves and stem extracts as

compare to vitamin C. Aloe L: A. esculenta leaves; Aloe R: A. esculenta roots; Aloe S: A.

esculenta stem; STD: standard, Ascorbic Acid.

48
 120
Pechuel L Pechuel R Pechuel S Ascorbic Acid

100

80
%Inhibition

60

40

20

0
Acorbic 500 250 125 61.5 31.3 15.2 7.8
Acid
Concentration (µg/mL)

Figure 11: DPPH scavenging activity of P. leubnitziae roots, leaves and stem extracts as compared

to vitamin C. Pechuel L: P. leubnitziae leaves; Pechuel R: P. leubnitziae root; Pechuel S: P.

leubnitziae stem; STD: standard, Ascorbic Acid.

49
Table 5 is showing the IC50 values of leaves, roots and stem extracts of A. arenaria, A. esculenta

and P. leubnitziae. A comparable scavenging activity was observed between the extracts and the

standard, Ascorbic Acid. The IC50 of extracts ranged between (25.4 ± 0.7 µg/mL) of the root

extract of P. leubnitziae greater than that of the Standard and (196.8 ± 14.6 µg/mL) of the root

extract of A. arenaria lower than that of the standard.

Table 5: DPPH free radicals A. arenaria, A. esculenta and P. leubnitziae leaves, roots and stem

extracts.

Sample IC50±SD (µg/mL)

A. arenaria
Leaves 26.6 ± 12.9
Roots 196.8 ± 14.6
Stem 59.0 ± 2.1
A. esculenta
Leaves 97.9 ± 4.3
Roots 68.0 ± 5.9
Stem 139.2 ± 3.2
P. leubnitziae
Leaves 96.0 ± 3.6
Roots 25.4 ± 0.7
Stem 95.5 ± 2.0

50
4.5.2. Reducing power assay

The reducing power was found to be concentration dependent (Fig. 12-14). The reducing power

of A. arenaria, A. esculenta and P. leubnitziae leaves, root and stem extracts except P. leubnitziae

leaves increased with an increase in concentration. At 0.5 mg/mL, the leaves extract of A. arenaria

showed an absorbance of 0.269 (Fig. 12) as the highest followed by P. leubnitziae leaves extract

at 0.062 mg/mL with an absorbance of 0.083 (Fig. 13). Aloe esculenta and P. leubnitziae root

extracts exhibited the lowest reducing ability at 0.125 mg/mL and absorbance of 0.00 (Fig. 13 and

14).

0.3
Roots Leaves Stem

0.25
Absorbance at 700 nm

0.2

0.15

0.1

0.05

0
0.0625 0.125 0.25 0.5
Concentration (mg/mL)

Figure 12: Reducing power activity of A. arenaria roots, leaves and stem extracts. Aca R- A.

arenaria roots; Aca L- A. arenaria leaves and Aca S- A. arenaria stem.

51
 0.07 Aloe S Aloe L Aloe R

0.06

0.05
Absorbance at 700nm

0.04

0.03

0.02

0.01

0
0.0625 0.125 0.25 0.5
-0.01
Concentration (mg/mL)

Figure 13: Reducing power activity of A. esculenta roots, leaves and stem extracts. Aloe S- A.

esculenta stem; Aloe L- A. esculenta leaves; Aloe R- A. esculenta roots.

52
 0.09
Leaves Stem Roots
0.08

0.07
Absorbance at 700 nm

0.06

0.05

0.04

0.03

0.02

0.01

0
0.0625 0.125 0.25 0.5
Concentration (mg/mL)

Figure 14: Reducing power activity of P. leibnitziae roots, leaves and stem extracts. Bitter L- P.

leubnitziae leaves; Bitter R- P. leubnitziae; Bitter S- P. leubnitziae stem.

53
DISCUSSION

Phytochemicals are bioactive, non-nutrient and naturally occurring compounds in plants

(Cheikyoussef, Summers, & Kahaka, 2015). Several phytochemical compounds detected in A.

arenaria, A. esculenta and P. leubnitziae plants extracts (Table 1) are known to have health

benefits, physiological activities and medicinal significance (Iikasha, 2016). Steroids, saponins,

terpenoids, flavonoids, coumarins, phenols and tannins were tested positive in all three plants.

These results are in agreement to the previous studies done on other species by Abdel-Farid et al.

(2014) and Atiku, Oladipo, Forcados, Usman, and Mancha (2016) who found that Acacia nilotica

and Acacia seyal have saponins, phenolics and flavonoids and Acacia seiberiana possess

flavonoids, tannins, steroids, alkaloids and saponins. It is also in agreement with Hedimbi et al.

(2012a) who reported on the presence of saponins, flavonoids and polyphenols in P. leubnitziae.

On the contrary the qualitative phytochemical results of A. esculenta slightly differed with those

of Aloe vera, the extensively research Aloe plant. Most phytochemicals detected in this study in A.

esculenta were not reported in A. vera (Lawrence, Tripathi, & Jeyakumar, 2009). However,

anthraquinones were only found present in A. esculenta roots and stem extracts but absent in A.

arenaria and P. leubnitziae. The results suggested that there are similarities and differences among

species of the same genus. This is in agreement with Cheikyoussef et al. (2015) who indicated that

there are differences and similarities in the chemical profiles of two species as well as variation

between populations from different regions.

In the present study, quantitative phytochemical results clearly indicated (Table 2) that amongst

all test extracts, A. arenaria leaves extract had the highest amount of phenolic compounds and the

lowest phenolic content was obtained in A. esculenta stem extract. The high phenolic content in

the A. arenaria leaves extract correlates with those in other species from the same family such as

54
Acacia nilotica (9.5 mg GAE/g), Acacia seyal (10.2 mg GAE/g) and Acacia laeta (9.9 mg GAE/g)

(Abdel-Farid et al., 2014). The high content of phenolic compounds in Acacia species may be the

reason for increasing the potentialities of theses extracts as antioxidants. Since antioxidant activity

of many plants extracts is attributed to the presence of phenolic compounds (Abdel-Farid et al.,

2014). Condensed tannins are polyphenolic compounds that play an important role in stabilizing

lipid oxidation and are also associated with antioxidative property (Tamilselvi et al., 2012). The

condensed tannin content of the extracts in tannic acid equivalents was obtained. The highest

condensed tannin content was observed in P. leubnitziae stem extract and the lowest was observed

in A. esculenta stem extract. The quantified TPC and TCT correlated to the observed antioxidant

activity in all the tested extracts. Tannins reportedly have a good stringent properties (Pratibha et

al., 2012). In addition, tannins are known to fasten the healing of wounds and inflamed mucous

membrane (Yadav, Chatterji, Gupta, & Watal, 2014). Condensed tannins have been associated

with the antioxidant activity of food and plant extracts (Cheikyoussef et al., 2015).

The in vitro antioxidant activity of extracts under study were determined using the DPPH assay.

Antioxidant activity of all extracts showed that when gradually increasing the samples

concentration there was an increase in the % inhibition. A lower IC50 value is an indicative of good

antioxidant activity (Pratibha et al., 2012). The dose-dependent from previous study showed that

IC50 for A. nilotica and A. seyal leaves extracts were higher than that of A. arenaria leaves extract

(Abdel-Farid et al., 2014). The A. esculenta leaves extract (97.9 µg mL¯1) (table 5) was also found

to possess a lower IC50 than that of the A. vera leaves extract (5, 200 µg mL-1) the most studied

species of Aloe species (Bawankar et al., 2013). In the present study, the experimental data indicated

that though all the tested extracts demonstrated good radical scavenging activity, the highest DPPH

radical scavenging activity was observed in P. leubnitziae, followed by A. arenaria leaves and A.

55
arenaria stem whereas A. arenaria roots’ extract demonstrated moderate DPPH radical

scavenging activity. Acacia arenaria, A. esculenta and P. leubnitziae extracts showed a dose-

dependent reducing power activity.

Screening for in vitro antibacterial activity using imipenem (10 µg) as a positive control clearly

indicated that A. arenaria, A. esculenta and P. leubnitziae leaves, stem and roots extracts had

antibacterial activities against the four test microorganisms namely A. faecalis, E. faecalis, S.

sonnei and S. marcescens. According to Alabri et al. (2014), the antibacterial activity of plant crude

extracts depends on the dose and the type of bacterial strains employed. This might be the reason

why variation in the inhibition zones at different concentration was obtained. The highest zone of

inhibition (4.5 mm) was observed for A. arenaria root extract and A. esculenta (6.3 mg/mL) against

S. sonnei and A. faecalis (table 3). Little ethyl acetate was used to remove concentrated extracts

from the round bottom flask the study inhibition zones are correlating to the values of ethyl acetate

A. vera extract (1.0-9.0 mm) inhibition against gram negative and positive bacteria (Thiruppathi,

Ramasubramanian, Sivakumar, & Thirumalai, 2010). According to Thiruppathi et al. (2010), ethyl

acetate extract was found to give the best antibacterial results against most pathogenic bacteria.

Furthermore, generally the extracts showed greater antibacterial activity against Gram-positive as

compared to Gram-negative bacteria (Lawrence et al., 2009). The reason behind this is that, Gram-

negative bacteria have additional lipopolysaccharide layer (Lawrence et al., 2009). In this study,

results of the extracts at 1.6 mg/mL are almost in agreement to the above reason. A strong

relationship between the total phenolic content and antioxidant activity due to its attachment to the

cell walls, cell membranes and interference with membranes functions like electron transport,

protein synthesis and enzyme activity it made it to be a good antibacterial agents. Thus, phenolic

56
compounds could lead to the destruction of pathogens (Pratibha et al., 2012), which explains the

antibacterial activity observed in the present study.

The findings of this study also showed a difference in MIC of plants extracts against S. sonnei, S.

marcescens, A. faecalis and E. faecalis.It is important to note that MIC value lower or equal to 5

mg/mL was recorded for A. arenaria leaves extract against all tested bacteria. The lowest MIC

value recorded was 0.1 mg/mL with A. arenaria and gentamycin against S. marcescens and

S.sonnei. The MIC values ranged from 0.1-5.0 mg/mL of the acacia leaves were almost closer to

that of A. nilotica in previous study which ranged from 1.6–3.1 mg/mL (Sadiq et al., 2017).

Although traditional uses for selected plants in this study are documented, scientific validations,

antioxidant and antibacterial efficacies of A. arenaria, and A. esculenta do not exist. In addition

antioxidant efficacy of P. leubnitziae also does not exit. Nevertheless, the information provided by

traditional healers and other knowledge holders during ethanobotanical surveys serves as an

important goal toward documentation of the uses, of many medicinal plants. All these validates

that traditional plant knowledge is of important value, not just for local uses, but also to researchers

for the development of novel antioxidant and antibacterial medicines. The findings of this study

served as justification to validate the use of A. arenaria, A. esculenta and P. leubnitziae in

combating infections causing pathogens as well as scavenging free radicals in Namibia. On the

other hand, it verified the presence of phytochemical compounds associated with antioxidant and

antibacterial activities.

57
CONCLUSION

This study contributed to the evaluation of Namibian ethnomedicinal plants for search of new

chemical entities with known antioxidant and antibacterial properties. Alkaloids, flavonoids,

terpenoids, saponins, phenols, tannins and steroids were found to be present in the A. arenaria, A.

esculenta and P. leubnitziae. Furthermore, anthraquinones were only found present in A. esculenta.

In addition, the study demonstrated total phenolics and condensed tannins contents in three

selected plants. All plants showed good antibacterial activity against A. facaelis, E. faecalis, S.

sonnei and S. marcescens and also possessed antioxidant activity. Alkaloids, saponins, phenols,

tannins, steroids, terpenoids, flavonoids, anthraquinones and coumarins were identified as possible

classes of phytochemicals responsible for the antibacterial and antioxidant activity of A. arenaria,

A. esculenta and P. leubnitziae.

The study findings supported the use of these plants to treat bacterial infections in traditional

settings. The further identification of the bioactive components in these plants will serve as a basis

for in-depth pharmacological evaluation and isolation of antibacterial and antioxidant

phytochemicals for possible drugs development. In addition, the results can assist in herbal drug

formulations of the plants studied.

58
RECOMMENDATION

A number of aspects of this research need to be further investigated to confirm the observed

activities. The assays used in this study provided good antioxidant and antibacterial results. In

order to confirm the activities of the promising extracts, more in-depth studies are necessary. These

includes making use of other antioxidant assays such as total antioxidant, total flavonoids,

superoxide etc. The antioxidant potential of the extracts will then be compared through different

methods. A bioassay-guided fractionation and purification of the crude extracts is also

recommended as this will give more information on the specific compounds active in the crude

extracts. Testing several Namibian medicinal plants (as numerous as possible) should be carried

out in order to establish a national database of possible lead plants. Several solvents can be used

for extraction to ensure both polar and non-polar compounds are extracted. Due to escalating

deforestation in our country there is a need that the communities be educated on the importance of

these plants in order to preserve them.

59
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APPENDIX A: L-Ascorbic Acid Calibration Curve

1.6
y = 2.9163x + 0.0388
1.4 R² = 1
1.2
Absorbance nm

0.8

0.6

0.4

0.2

0
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration mg/ml

Figure 15: L- Ascorbic Acid Calibration Curve

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APPENDIX B: Gallic Acid Calibration Curve

Figure 16: Gallic Acid Calibration Curve

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APPENDIX C: Tannic Acid Calibration Curve

0.14
0.12 y = 0.0166x + 0.0314
Absorbance at 500 nm

R² = 0.9952
0.1
0.08
0.06
0.04
0.02
0
0 1 2 3 4 5 6
Concentration (mg/mL)

Figure 17: Tannic Acid Calibration Curve

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APPENDIX D: Ethical clearance certificate

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APPENDIX E: Research/collecting permit.

83
EPPENDIX F: Research permission letter

84