impact of various boiling intervals on the antimicrobial efficacy and phytochemical profile of ...

238 Pages·2014·3.61 MB·English

by  Erasmus, Lourens

IMPACT OF VARIOUS BOILING INTERVALS ON THE ANTIMICROBIAL
EFFICACY AND PHYTOCHEMICAL PROFILE OF SELECTED CRUDE AQUEOUS
PLANT EXTRACTS, USED BY BAPEDI TRADITIONAL HEALERS IN THE
TREATMENT OF SEXUALLY TRANSMITTED INFECTIONS

by

Lourens Johannes Christoffel Erasmus

A thesis submitted in fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY
in

BOTANY
in the

FACULTY OF SCIENCE AND AGRICULTURE
School of Molecular and Life Sciences
at the
UNIVERSITY OF LIMPOPO

SUPERVISOR: Prof M.J. Potgieter
CO-SUPERVISOR: Dr. T.E. Tsikhalange
2014
D
ECLARATION

The experimental work described in this thesis was conducted at the Department of
Plant Science, University of Pretoria, under the supervision of Doctor T.E.
Tsikhalange (Antimicrobial aspects), the Biotechnology Unit, School of Molecular
and Life Sciences at the University of Limpopo, under the supervision of Professor I.
Ncube (High Performance Liquid Chromatography Analysis), and the preliminary
phytochemical assessment of Aloe marlothii in the Department of Microbiology,
Biochemistry and Biotechnology, School of Molecular and Life Sciences at the
University of Limpopo, under the supervision of Professor P. Masoko. All research
activities were conducted from January to September 2013. This thesis, submitted
for the degree of Doctor of Philosophy in Botany, in the Faculty of Science and
Agriculture of the University of Limpopo, Mankweng, represents original research
conducted by the author; exceptions are where the work of others is duly
acknowledged in the text. Although the majority of these studies have been
submitted (2013) for publication in peer reviewed journals they have not otherwise
been submitted in any form for any other degree or diploma at any other University.

……………………………...
LOURENS J.C. ERASMUS
22/08/2014

We certify that the above statement is correct:

……..……………………….
PROFESSOR MARTHIENUS JOHANNES POTGIETER
Supervisor
Polokwane

…………...........……………
DOCTOR THILIVHALI EMMANUEL TSHIKALANGE
Co-supervisor
Pretoria
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D
EDICATION

DEDICATION

To my sons Luan, Janco and Ricardo, my joy and inspiration.

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A
CKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

My supervisor, Professor M.J. Potgieter, to whom I’m indebted for the patience and
wisdom with which he guided my discovery and appreciation of the value and
contribution of human physiology within the wonderful field of phytomedicine.

The completion of this thesis was a tremendous logistical challenge, and I would like
to thank the following people who ensured a relatively stable research environment:

Doctor T.E. Tshikalange, my co-supervisor (UP), and Professors I. Ncube and P.
Masosko (UL) who taught me so much about the various analytical procedures
employed in phytomedicine. Thank you for availing your time, expertise and
laboratory facilities.

Colleagues in the Faculty of Science and Agriculture; especially our Executive Dean
Professor H.J. Siweya, a gentleman and leader, who made so many things possible,
including the study leave that made this dream a reality. Professor P.A.S. Olivier for
his encouragement and support. My Head of Department, Doctor M. van Staden who
kept me informed of departemental developments in my absence.

I’m especially grateful to Professor W.J. Luus-Powell, for her dedication in taking
care of the postgraduate students under our supervision, during my study leave.

To the many colleagues who showed a keen interest in my progress. You have been
a constant source of support and laughter.

This research was supported by funds provided by the Departments of Physiology
and Environmental Health and Biodiversity (UL), and the Department of Plant
Science (UP).

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A
CKNOWLEDGEMENTS

I wish to express my gratitude to my wife Elana and my three amazing sons Luan,
Janco and Ricardo for being my pillar of strength and for their love, inspiration and
wisdom.

I thank the Almighty God for being with me and blessing me abundantly with friends
who carried me in prayer during my studies.

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A
BSTRACT

ABSTRACT

Ethnobotanical studies are currently a major contributor to the identification of plant
species used in the treatment of a large assortment of diseases, including but not
limited to sexually transmitted infections. Some of these studies are comprehensive
enough that detail regarding the traditional health care practice is captured;
especially information related to the combination of different plant species or plant
parts in the preparation of remedies, as well as preparation procedures employed.
However, relevant information is often lost because a number of studies, due to poor
planning and execution do not cover these aspects.
It is true that large variations exist regarding these preparation procedures.
Such an example includes the preference of different boiling intervals when
preparing an extract. Scientific sources highlight the fact that these variations are
often regulated by cultural norms, but can also include healer specific preferences. In
the process of scientifically validating the use of such extracts, innovative
approaches are required. The current study is an example of such an effort, where it
was noted that the Bapedi traditional healers in the Limpopo Province of South
Africa, used boiling periods ranging from 5 to 20 minutes when they prepare
aqueous extracts from the plant species (Aloe marlothii, Catharanthus roseus,
Hypoxis hemerocallidea, Tribulus terrestris and Ziziphus mucronata) selected for this
study.
Fresh plant material was collected and added to a pot of boiling tap water. At 5
minute intervals 500 ml was removed to a maximum of 20 minutes, starting with time
zero, without topping-up the volume in the pot. The aim was to collect the highest
possible yield, and to subsequently subject the freeze-dried particulates to
antimicrobial assays (micro-dilution), using a 50 mg/ml stock solution. Pathogens
included in this study were: Candida albicans, Neisseria gonorrhea, Proteus vulgaris
and Staphylococcus aureus. A small volume (2 ml) of each crude aqueous extract
was also assessed via High Performance Liqiud Chromatography, to determine
fluctuations in alkaloid, phenolic and terpenoid levels.
Aloe marlothii leaf and root extracts exhibited a wide range (MIC 0.09‒12.5
mg/ml) of inhibitory activities against the above mentioned pathogens. Results
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A
BSTRACT

indicated that this plant species is not only underutilized from a research perspective,
its therapeutic value is completely underestimated. The fact that its combined root
and leave aqueous extract had an minimum inhibitory concentration (MIC) <0.09
mg/ml at time zero should focus attention on further phytopharmaceutical analyses,
to establish its potential value in future drug development.
Ziziphus mucronata, in combination with either H. hemerocallidea or T.
terrestris, exhibited very poor antimicrobial activity. The fact that Z. mucronata
featured in previous research as a common denominator in a number of multi-plant
extracts, created the impression that this species might play a role in the possible
antimicrobial activity of these crude extracts. This theory could not be confirmed in
our study; indicating that the inclusion of this species might be related to a non-
pathogenic function.
Catharanthus roseus, a species exclusively used to treat gonorrhea, showed
potential in the inhibition of N. gonorrhea, with all MICs recorded at 0.78 mg/ml. This
level of activity does not fall within the excellent or even clinically relevant range, yet
its continued traditional use supports the notion that its therapeutic impact might
involve far more than only antibacterial capabilities.
High performance liquid chromatography results established the presence of
time-dependent extraction relationships for alkaloids, phenolics and terpenoids.
Combining this data with the antimicrobial results highlighted the following.
Terpenoids present in A. marlothii and Z. mucronata / T. terrestris extracts were
implicated in its antigonococcal efficacy. This was different from C. roseus, where
alkaloids were identified as the possible source of antigonococcal activity. Proteus
vulgaris showed a particular sensitivity for alkaloids and terpenoids present in A.
marlothii extracts. Candida albicans displayed a diverse reaction when exposed to
the various crude extracts. Alkaloids present in A. marlothii root and leaf extracts,
and terpenoids in the A. marlothii leave / root and Z. mucronata / H. hemerocallidea
combinations showed potential as anti-fungal compounds.
This study found that some plant species require an extended boiling period to
exhibit antimicrobial activity; however, a few of the species showed this potential with
limited boiling. Current findings partially support the use of some of these species,
such as C. roseus and A. marlothii subsp. marlothii, in the treatment of STIs. The
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A
BSTRACT

fact that very poor antimicrobial activity was detected for a number of these extracts,
and that these plants species remain highly used in traditional medicine, opens up
the possibility that they might contribute to the alleviation of symptoms via a non-
pathogen dependent mechanism.

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ABLE OF ONTENTS

DEDICATION ............................................................................................................. ii
ABSTRACT ............................................................................................................... v
GENERAL INTRODUCTION ..................................................................................... 1

CHAPTER 1 ............................................................................................................... 2

1.1 GENERAL LITERATURE REVIEW................................................................... 2
1.1.1 Traditional healers ........................................................................................... 2
1.1.2 Phytomedicine ................................................................................................. 3
1.2 SEXUALLY TRANSMITTED INFECTIONS ...................................................... 3
1.2.1 General introduction ........................................................................................ 3
1.2.2 Neisseria gonorrhea ....................................................................................... 4
1.2.2.1 General description ................................................................................ 4
1.2.2.2 Epidemiology .......................................................................................... 5
1.2.2.3 Traditional medicine ............................................................................... 6
1.2.2.3.1 Diagnosis........................................................................................ 6
1.2.2.3.2 Treatment ....................................................................................... 6
1.2.3 Proteus vulgaris ............................................................................................... 7
1.2.3.1 General description ................................................................................ 7
1.2.3.2 Epidemiology .......................................................................................... 8
1.2.3.3 Traditional medicine ............................................................................... 9
1.2.3.3.1 Diagnosis........................................................................................ 9
1.2.3.3.2 Treatment ....................................................................................... 9
1.2.4 Candida albicans ............................................................................................. 9
1.2.4.1 General description ................................................................................ 9
1.2.4.2 Epidemiology ........................................................................................ 10
1.2.4.3 Traditional medicine ............................................................................. 11
1.2.4.3.1 Diagnosis...................................................................................... 11
1.2.4.3.2 Treatment ..................................................................................... 11
1.3 MEDICINAL PLANTS USED IN THIS STUDY ................................................ 11

CHAPTER 2: ALOE MARLOTHII ............................................................................ 13
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ABLE OF ONTENTS

2.1 BOTANICAL DESCRIPTION .......................................................................... 13
2.2 HABITAT AND DISTRIBUTION ...................................................................... 13
2.3 USES ............................................................................................................... 14
2.3.1 Plant parts ..................................................................................................... 14
2.3.2 Therapeutic and prophylactic application of Aloe marlothii ............................ 14
2.4 PHYTOCHEMICAL CONSTITUENTS ............................................................. 17

CHAPTER 3: CATHARANTHUS ROSEUS ............................................................. 13

3.1 BOTANICAL DESCRIPTION .......................................................................... 13
3.2 HABITAT AND DISTRIBUTION ...................................................................... 13
3.3 USES ............................................................................................................... 13
3.3.1 Plant parts ..................................................................................................... 13
3.3.2 Therapeutic and prophylactic application of Catharanthus roseus ................ 14
3.4 PHYTOCHEMICAL CONSTITUENTS ............................................................. 17

CHAPTER 4: HYPOXIS HEMEROCALLIDEA ........................................................ 28

4.1 BOTANICAL DESCRIPTION .......................................................................... 28
4.2 HABITAT AND DISTRIBUTION ...................................................................... 29
4.3 USES ............................................................................................................... 29
4.3.1 Plant parts ..................................................................................................... 29
4.3.2 Therapeutic and prophylactic application of Hypoxis hemerocallidea ............ 29
4.4 PHYTOCHEMICAL CONSTITUENTS ............................................................. 32

CHAPTER 5: TRIBULUS TERRESTRIS ................................................................. 36

5.1 BOTANICAL DESCRIPTION .......................................................................... 36
5.2 HABITAT AND DISTRIBUTION ...................................................................... 36
5.3 USES ............................................................................................................... 36
5.3.1 Plant parts ..................................................................................................... 36
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