Thesis submitted in fulfilment of the requirements for the degree
Doctor of Technology: Biomedical Technology
in the
Faculty of Health and Wellness Sciences
at the
Cape Peninsula University of Technology
2014 / According to the World Health Organization, cancer is the leading cause of death in the
developed world, while it is the second leading cause of death in the developing world. In
particular, liver cancer is the fifth most commonly diagnosed cancer in men, however, it is
the second most frequent cause of death, responsible for an estimated 700,000 deaths
annually. General limited access to health services, including treatment and the overall
management of cancer in developing countries often contribute to the increased mortality
rates when compared to developed countries. For centuries, medicinal plants have been
used to prevent, and to a certain extent, treat cancer as a readily available and affordable
alternative. In many instances, the curative or preventative claims still remain anecdotal.
However, increasing evidence suggest that polyphenolic components of plants possess
antioxidant activities, which are credited with curative/beneficial properties of medicinal
plants. The curative properties could either be related to the primary compounds present in
the plant itself, or the bio-activation products of plant components affecting hepatic drug
metabolising and antioxidant enzymes systems related to carcinogen metabolism and
maintaining oxidative homeostasis, respectively. Similarly, chronic consumption of medicinal
plants could also result in hepatotoxicity, either caused by the primary plant components or
bio-activation products. Due to these observations it is paramount to understand the
mechanisms involved in the metabolism of plant components to critically assess beneficial
versus potential harmful properties associated with chronic consumption.
The focus of the current study was aimed at elucidating the bio-activity of four multipurpose
indigenous plants to Southern Africa, i.e. Adansonia digitata, Agathosma betulina,
Siphonochilus aethiopicus and Myrothamnus flabellifolius. Traditionally, A. digitata has been
used as an immunostimulant, anti-inflammatory and analgesic agent, while also as an
antipyretic agent in the treatment of diarrhoea and dysentery. Similarly, traditional medicinal
uses of A. betulina include treatment cholera, haematuria, calculus, kidney diseases, as well
as infections of the bladder, urethra, and prostate among others. S. aethiopicus was
traditionally employed to treat infections associated with pains and fevers, whereas
M. flabellifolius served as treatment of conditions ranging from respiratory ailments,
backache, kidney problems, haemorrhoids, chest pain, and asthma.
In the first part of this study, the polyphenolic contents and antioxidant capacities of the four
plants were characterised. The emphasis was placed on using different solvents, namely
water, ethanol and acetone for the extraction of the plant material and different
methodologies to assess the antioxidant contents and -capacities of the various extracts as
both these factors can influence the outcome. When considering the antioxidant contents,
total polyphenols, flavanols, and flavonols of the different solvent extracts prepared from the
four plants were determined, whereas three different assays were used for the antioxidant
capacities, i.e. oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant
capacity (TEAC) and ferric-reducing antioxidant power (FRAP) assays. The A. digitata
acetone extract had the highest (7.121 mg gallic acid equivalent (GAE)/milligram (mg)
soluble solids), whereas the water extract of the same plant had the lowest total phenolic
content (0.008 mg GAE/mg soluble solids). In general, the acetone extracts demonstrated
the highest total polyphenol, flavanol, and flavonol contents, followed by the ethanol extracts,
with the water extracts having the lowest contents. M. flabellifolius was the only distinct
deviation from this rule, where the water extract demonstrated the highest total polyphenol
content. Considering antioxidant capacities, the acetone extracts provided the highest
antioxidant capacities for all plants when assessed using the TEAC (8.56-32.68 milimole
(mmole) trolox equivalent (TE)/mg soluble solids) and FRAP (5.69-37.39 mmole ascorbic
acid equivalent/mg soluble solids) antioxidant assays, with the exception of M. flabellifolius
where the water extract demonstrated the highest activity (22.73 mmole ascorbic acid
equivalent/mg soluble solids). Antioxidant capacity determinations with TEAC and FRAP
assays followed similar patterns, which were different from capacities determined by the
ORAC (0.46-533.54 mmoleTE/mg of soluble solids) assay. Corroborating the antioxidant
content findings, the acetone extracts also demonstrated the highest antioxidant capacities
(140.41-533.54 mmoleTE/mg of soluble solids), followed by ethanol (94.62-151.29
mmoleTE/mg of soluble solids) and water (0.46-134.02 mmoleTE/mg of soluble solids). Only
M. flabellifolius (TEAC and FRAP) and S. aethiopicus (FRAP) deviated from this trend.
Correlations between the polyphenolic contents and antioxidant capacities indicated that
acetone and ethanol were more effective in extracting polyphenolic compounds than water,
while also providing extracts with superior antioxidant activities. Furthermore, ORAC assay
was the antioxidant capacity determining assay of choice for the aqueous plant extracts,
whereas the TEAC and FRAP assays were more suitable when determining the antioxidant
capacities of the acetone and ethanol plant extracts. These results confirm the notion that no
single assay can comprehensively determine the antioxidant activities of plant extracts and
that a battery of assays should be used, as the various antioxidant capacity determination
techniques use different substrates with different targets for measurement.
The second part of this study comprised an in vivo experimental animal model to assess the
potential toxicity, antioxidant status and modulation of the hepatic phase 2 drug metabolising
enzymes following chronic consumption of the various plant extracts in male Fisher rats.
Rats consumed aqueous extracts of the various plants (2% and 5% (w/v)) as the sole source
of drinking fluid for 90 days, and the serum chemical pathology parameters for monitoring
liver and kidney function conducted. These included alkaline phosphatase (ALP), aspartate
transaminase (AST), alanine transaminase (ALT), total iron (Fe), and creatinine (CREA).
Parameters for blood and hepatic redox status included total polyphenols, ORAC, reduced
glutathione (GSH), oxidised glutathione (GSSG), their ratio (GSH:GSSG), conjugated dienes
(CD) and thiobarbituric acid reactive substances (TBARS). Assessment of the phase 2
hepatic xenobiotic metabolising enzymes included glutathione S-transferase (GST) and
activity in the cytosolic fraction and, UDP-glucuronosyltransferase (UDP-GT) activity in
liver microsomes. When considering the liver and kidney function none of the plant extracts
induced any significant toxicity, while 2% A. digitata significantly increased serum Fe. When
considering the redox status, the whole blood and liver samples yielded similar results, with
significant decreases in oxidised glutathione (GSSG) in rats consuming the 2% M.
flabellifolius (82.76 mole/L) and 5% A. digitata (90.42 mole/L) with a resultant significant
increase in the glutathione redox status (GSH:GSSG ratio of 5.69 and 5.64, respectively)
when compared to rats consuming water (4.77). The GSH:GSSG ratio was also significantly
increased by consumption of 2% A. betulina (8.45) and 5% S. aethiopicus (5.99). The
consumption of all plant extracts, except 5% A. betulina and M. flabellifolius, significantly
increased lipid peroxidation in the plasma CDs assay. These results indicated an increased
antioxidant capacity in the liver with/without an associated reduced cellular oxidative stress
status, which could be interpreted as a reduced susceptibility to oxidative damage. When
considering the phase 2 hepatic enzymes, none of the plant extracts caused any significant
changes in GST, GST or UDP-GT activities.
The third part investigated the chemoprotective properties against cancer promotion in the
liver utilising diethylnitrosamine (DEN) as cancer initiator and maize culture material of
Fusarium verticillioides, containing the fumonisin B mycotoxins, as promoters in male
Fischer rats. The rats consumed 2% (w/v) aqueous extracts of A. digitata, A. betulina, and
S. aethiopicus over 28 days after cancer initiation and liver sections subjected to
glutathione-S-transferase placental form positive GSTP+ staining and pre-cancerous liver foci
categorised according to size. In addition, blood and liver analyses were done as described
in the chronic feeding study above. Consumption of the A. digitata and, to a certain extent,
S. aethiopicus extracts, altered the oxidative stress status in the liver as indicated by the
increased lipid peroxidation, as determined by significantly increased liver CDs and the
decreased GSH:GSSG ratio in the blood. This can be related to a subchronic toxicity due to
the high total polyphenol intake as mentioned above. These underlying sub chronic toxic
effects of A. digitata and S. aethiopicus are likely to be responsible for the observed
inhibitory effect on the proliferation of GSTP+ minifoci in the liver. Hepatic phase 2
metabolising enzyme activities were not significantly altered by A. digitata and S. aethiopicus
consumption, while GST activity was significantly increased by A. betulina treatment.
Based on the findings of the current study, aqueous extracts of A. digitata, A. betulina, and
S. aethiopicus may serve as hepatoprotectors with a potential to modulate liver
carcinogenesis, specifically cancer promotion. To our knowledge, no other studies have
attempted to describe the possible chemoprevention mechanisms of these indigenous
medicinal plants. Assessments of phase 1 hepatic enzymes and other antioxidant enzymes
are suggested for future studies to further describe biochemical and molecular mechanisms
associated with consumption of these extracts. Additionally, identifying main compounds
present in the plant extracts could culminate in development of drugs and novel
nutraceuticals. It is also recommended that increasing concentrations of the plant extracts
and/or the ethanol extracts to be used in future studies to better describe dose-responses of
the different plants in liver carcinogenesis.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:cput/oai:localhost:20.500.11838/1524 |
| Date | January 2014 |
| Creators | Hikuam, Willem Christoph |
| Publisher | Cape Peninsula University of Technology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | http://creativecommons.org/licenses/by-nc-sa/3.0/za/ |
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