University of Macau / Institute of Chinese Medical Sciences

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Chronic inflammation is suggested to contribute to cancer development and therefore a potential target for chemoprevention. In the skin, keratinocytes and macrophages play an integral part in acute and chronic inflammation, with interleukin 1-α (IL-1α) and tumor necrosis factor α (TNF-α) as key cytokines governing this process. Green tea (Camellia sinensis) and the South African herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.) displayed antiinflammatory effects in mouse and human skin. To further investigate the antiinflammatory properties of green tea and the herbal teas, rooibos and honeybush (C. subternata and C. maculata) herbal teas, suitable cell culture models were developed and validated utilising human keratinocytes (HaCaT) and monocyte (THP- 1) derived macrophages. Aqueous extracts of the green tea and unfermented herbal teas were prepared and their chemical composition determined by high performance liquid chromatography (HPLC) and the antioxidant activity characterised utilising different antioxidant assays. Green tea and rooibos exhibited similar antioxidant activities while C. maculata displayed the lowest overall antioxidant activity of all the extracts, despite possessing the highest mangiferin level, the major polyphenol in honeybush. The modulation of cytokine release was studied in (i) an UVB-induced pre-exposure HaCaT model monitoring the accumulation of IL-1α and (ii) a LPS stimulated THP-1 macrophage model monitoring the TNF-α release, utilising both a pre-exposure and co-exposure extract regimens. In the pre-exposure HaCaT inflammatory model the UVB-induced IL-1α was decreased by the green tea extract while a far weaker response was obtained with the rooibos extract. Both the honeybush extracts displayed a significant effect in the reduction of IL-1α with C. subternata exhibiting a slight increased protection at a lower extract concentration. In the pre-exposure THP-1 derived macrophage model, green tea and the herbal tea extracts inhibited TNF-α release in a dose dependent manner in the absence of an overt loss in cell viability and apoptosis at lower extract concentrations, suggesting a typical anti-inflammatory effect. In the co-exposure model, the different extracts also exhibited an anti-inflammatory effect at the lowest concentrations in the absence of apoptosis while at higher extract concentrations the effect was masked by a decrease in cell viability and increased apoptosis. C. maculata exhibit differential effects when considering the inhibition of cytokine production and, depending on the cell model, either exhibited a weaker or stronger effect when compared to C. subternata and rooibos. Phenolic diversity of the different teas is likely to explain the differential effects in the antioxidant assays and cell culture models with respect to the regulation of the production of the inflammatory markers. Proposed mechanism for the anti-inflammatory effects include the modulation of oxidative stress via various pathways and the subsequent down regulation of nuclear factor kappa β (NFκB) and activated protein-1 (AP-1) which are key regulators of cytokine production governing the inflammatory response. / AFRIKAANSE OPSOMMING: Kroniese inflammasie van die vel kan bydra tot die ontwikkeling van kanker en is dus ’n potensiële area om te teiken in die voorkoming van velkanker. Keratinosiete en makrofage speel ’n integrale rol in akute en chroniese inflammasie van die vel en TNF-α en IL-1α is die belangrikste sitokiene wat hierdie proses inisieer. Dit is bekend dat ekstrakte van groen tee (Camellia sinensis) en die Suid-Afrikaanse kruietees, rooibos (Aspalathus linearis) en heuningbos (Cyclopia spp.) ‘n anti-inflammatoriese effek op die vel van muise en mense het. Om die anti-inflammatoriese aktiwitieit van groen tee, rooibos en 2 heuningbos kruietees (C. subternata en C. maculata) verder te ondersoek en te definieer is geskikte selkultuurmodelle ontwikkel en gevalideer deur gebruik te maak van menslike keratinosiete (HaCaT) en monosiet (THP-1) afgeleide makrofage. Water ekstrakte van groen tee en ongefermenteerde kruietees is voorberei en die chemiese samestelling deur hoë druk vloeistof chromatografie (HDLC) bepaal. ‘n Verskeidenheid van antioksidant bepalingstoetse is gebruik om die antioksidant aktiwiteit van die ekstrakte te meet. Groen tee en rooibos het soortgelyke antioksidant aktiwiteite getoon, terwyl C. maculata die swakste algehele aktiwiteit getooon het, ten spyte van die teenwoordigheid van hoёr vlakke van mangiferin, die belangrikste polifenoliese verbinding in heuningbos. Modulasie van sitokiene is verder bestudeer in (i) ’n UVB-geïnduseerde vooraf-blootstelling HaCaT model, waartydens akkumulering van IL-1α gemonitor is en (ii) ‘n lipopolisakkaried (LPS)-gestimuleerde THP-1 makrofaag model, waar die vrystelling van TNF-α gemonitor is. Vir die THP-1 model is beide die voor en gelyktydige blootstelling benaderings vir die ekstrakte met LPS gebruik. In die keratinosiet model, waar die selle aan ekstrakte blootgestel is voor UVB bestraing, is IL-1α beduidend verlaag deur die groen tee ekstrak, terwyl ’n swakker reaksie gesien is met rooibos. Beide heuningbos ekstrakte het ’n beduidende invloed in die vermindering van IL-1α getoon, waar C. subternata ’n effense verhoogde beskerming teen selsterfte by ‘n laer ekstrakkonsentrasie toon. Blootstelling van die makrofage aan al vier ekstrakte voor LPS stimulasie (vooraf-blootstelling), het gelei tot inhibisie van TNF-α vrystelling op ’n dosis afhanklike wyse en die afwesigheid van apoptose en selsterftes by lae ekstrak konsentrasievlakke. Hierdie waarnemings dui op ’n tipiese antiinflammatoriese effek. In die gelyktydige-blootstelling model verlaag al die ekstrakte TNF-α vrystelling teen die laagste ekstrak konsentrasievlakke, in die afwesigheid van apoptose en met geen effek op seldood nie. Hoёr ekstrak konsentrasievlakke het sitotoksisiteit en verhoogde apoptose getoon, dus was die anti-inflammatoriese effek gemaskeer. C. maculata toon ‘n variërende effek met betrekking tot antioksidant aktiwiteit en die bekamping van sitokien produksie, afhangend van die model wat bestudeer is. Die verskeidenheid fenoliese verbindings teenwoordig in die verskillende tee ekstrakte is waarskynlik die rede vir die effekte wat waargeneem is tydens antioksidant toetsing en selkultuurmodelle. Die anti-inflammatoriese meganismes wat deur hierdie studie voorgestel word sluit die modulasie van oksidatiewe stres via verskeie metaboliese paaie in. Modulasie van oksidatiewe stres lei tot af-regulering van kernfaktor-kappaB (NF-κB) en aktiveerderproteïen- 1(AP-1), wat sleutel reguleerders van sitokien produksie tydens inflammatoriese respons is.

Abstract Introduction: Current evidence suggests that the lungs of infants with the debilitating disorder, bronchopulmonary dysplasia (BPD), react to the challenges of extra-uterine adaptation with inappropriately aggressive inflammation. The reasons for this are not entirely clear and this study hypothesizes that a deficiency of interleukin (IL)-10, a potent anti-inflammatory mediator, leads to the functional and architectural changes characteristic of BPD. Aim: To characterize the behaviour of IL-10 and neutrophil apoptosis in the tracheal fluids (TF) of infants at risk of developing BPD. Method: TF from intubated infants of varying gestations at the Royal Hospital for Women, Randwick was spun and ILs 8, 10 and 16 were measured in the supernatant. The residual pellets of white cells were used to determine differential white cell counts and neutrophil apoptosis. Results: None of the 20 TF specimens from the extremely premature infants with BPD (n=11) had detectable IL-10, compared to 14/20(70%) of the specimens from preterm infants without BPD (n=20) and to 5/19 (26%) of the specimens from term infants (n=19). BPD infants also had a significantly lower number of apoptotic neutrophils during the 1st week of life. Premature infants with TF IL-10 &gt5pg/ml did not develop BPD. Levels of IL-8, a neutrophil chemotaxin, and white cell counts, while not differing significantly between the groups, increased considerably towards the end of the first week of life in the BPD group. IL-16, a chemotaxin for inflammatory CD4+ cells, was also detected in more BPD than non-BPD specimens (BPD: 16/46 (35%) v 1/30 (0.3%) non-BPD preterm and 2/7 (28%) term TF specimens). Conclusions: Extremely premature infants prone to BPD have decreased pulmonary anti-inflammatory activity as demonstrated by decreased IL-10 and apoptotic neutrophils in tracheal fluids. The lack of a counter-regulatory response to the inflammatory processes that are an inevitable consequence of extra-uterine adaptation may therefore place the extremely premature newborn infant at a considerable risk of developing BPD.

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M. Tech. / Plantar fasciitis is the most common cause of heel pain for which professional care is sought (Singh, 2008). Plantar fasciitis is a common injury, and one which is known to be stubborn to many forms of treatment. The aim of the study was to determine the most effective treatment for individuals suffering with plantar fasciitis comparing three protocols, i.e. mobilisation and manipulation of the foot and ankle with cross friction massage of the plantar fascia versus stretching of the gastroc-soleus complex with cross friction massage of the plantar fascia or a combination of the aforementioned treatments. Participants in the study were recruited from information pamphlets, the University of Johannesburg Chiropractic and Podiatry Day Clinics and running clubs. Only those participants that conformed to the selection criteria were allowed to participate in the study. A total of forty five participants were included. These participants were randomly placed into one of three groups of fifteen participants in each group. Group one received manipulation and mobilisation therapy to the foot and ankle with cross friction massage of the plantar fascia. Group two received cross friction massage of the plantar fascia and stretching of the gastroc-soleus muscle. Group three received a combination of the aforementioned therapies. Subjective data was collected using the Short Form McGill Pain Questionnaire (SF-MPQ) and the Foot Function Index (FFI). Objective data was collected using the algometer to test differences in pain at the plantar fascia as well as ankle range of motion in dorsiflexion and plantarflexion which was measured using a goniometer. The results of this study indicate that cross friction massage of the plantar fascia and stretching of the gastroc-soleus complex would appear to have shown the greatest overall improvement in terms of reducing the pain and disability and ankle dorsiflexion range of motion. Manipulation of the foot and ankle, cross friction massage of the plantar fascia and stretching of the gastroc-soleus complex proved to have shown the greatest overall improvement in plantarflexion only. Based on the results of the study, cross friction massage and gastroc-soleus stretching is the most effective treatment protocol for plantar fasciitis.

D. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences)|, Vaal University of Technology. / Medicinal plants provide valuable alternative sources of drugs and drug discovery because many have been used in traditional practices for centuries to manage or treat various forms of ailments. The aim of this study was to evaluate the biological activities of nine medicinal plants used by Zulus in Mabandla village, KwaZulu-Natal province, South Africa to treat inflammation and to isolate selected active compounds against studied pathogens from Alepidea amatymbica and Pomaria sandersonii. The plants were selected on the basis of an ethnobotanical survey based on questionnaire response and verbal interviews that were conducted in Mabandla village with the local traditional healers and herbalists. The isolation of compounds from Alepidea amatymbica and Pomaria sandersonii was based on the bioassay based study which was carried out in this study. Bioassay guided study involving in vitro anti-inflammatory measurement using soya bean derived 15 Lipoxygenase, free radical scavenging capacity against the ABTS●+ radical cation and DPPH● radicals; antimicrobial and bioautography assays against Staphylococcus aureus, ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Escherichia coli, ATCC25922, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus were carried out using the plants extracts, fractions and pure compounds. Isolation of compounds displaying biological activity was carried out by using open column chromatography and preparative thin layer chromatography (PTLC). The compounds were characterised by use of Nuclear Magnetic resonance, (NMR) and Mass Spectrometry (MS). The DPPH sprayed TLC showed that all the nine plants contained antioxidants. Most of which were contained in polar fractions of acetone and methanol. Results of the assays displayed a range of biological activities comparable to the positive controls used for each assay. DPPH● scavenging displayed EC50 values ranging between 1.008 and 467 Kg/ml. The highest activity was observed with the methanol fraction of Berkheya setifera with an EC50 value of 1.008 Kg/ml followed by the crude extract of Gunnera perpensa with EC50 value of 1.069 Kg/ml. Carissa bispinosa hexane fraction had the lowest activity of 467.7 Kg/ml. The Pomaria sandersonii DCM extract had the highest ABTS●+ radical scavenging activity by Pomaria sandersonii DCM extract, (1.273 Kg/ml) for the ethyl acetate, (5.973 Kg/ml) while the hexane fraction from Eucomis autumnalis had the lowest activity (929.4 Kg/ml). The activity of Pomaria sandersonii extracts and fractions demonstrated that the plant contains antioxidants that react with both DPPH and ABTS radicals although higher activities were shown by ABTS as displayed by the lower EC50 values. All the crude fractions and extracts had high to moderate antibacterial activities (20-625 Kg/ml) and anti-fungal activities (20-2500 Kg /ml). Pomaria sandersonii crude and fractions had the highest antimicrobial activity compared to other plants. Some MIC values for P. sandersonii dichloromethane and ethyl acetate fractions (80 Kg/ml in each case) compared well with gentamycin (4 Kg/ml) since they showed same values against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudonomus aeruginosa. The dichloromethane, acetone and methanol fractions were also active (20 Kg/ml) against both Candida albicans and Aspergillus fumigatus. Inhibition of pathogen growth demonstrated by the polar fractions of the studied plants suggested that some of the active compounds would be soluble in water. A total of seven compounds were isolated from Alepidea amatymbica and Pomaria sandersonii. We propose three were new compounds after considering literature search involving closely related research to this investigation. These were two diterpenes from Alepidea amatymbica, namely, 14-acetoxo-12-oxokaur-16-en-19-oic acid labelled as 0657 and 16-hydroxy-kaur-6-en-19-oic acid given the label 06-2 in this study. The third suspected new compound is the chalcone dimer, which is referred to as EM86 in this study from Pomaria sandersonii. EM80-2 was obtained as a mixture of the cis and trans of 2’, 4, 4,’-trihydroxychalcone or 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one, from Pomaria sandersonii. The three diterpenes, 14-acetoxokaur-16-en-19-oic acid (0652), 13-hydroxy-16-kauren-19-oic acid (06B) and 14-oxokaur-16-en-19-oic acid (06431) were isolated from Alepidea amatymbica for the first time. Isolated compounds were further tested as individual compounds and results showed that 16-hydroxy-kaur-6-en-19-oic acid (06-2) had weak activity against tested bacteria and fungi with the MIC: Staphylococcus aureus (320 Kg/ml) and Candida albicans, (320 Kg/ml). On the other hand 13-hydroxy-kaur-16-en-19-oic acid (06B) was more active against, Staphylococcus aureus (160 Kg/ml) and Aspergillus fumigatus (40 Kg/ml). The yellow compound that was isolated from Pomaria sandersonii, 1-(2, 4-ihydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one was antimicrobial with the following MICs: Candida albicans: 80 Kg/ml; Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus: 160 Kg/ml and Aspergillus fumigatus: 625 Kg/ml. There were two mixtures referred to as EM 49 and EM 77 from Pomaria sandersonii which were difficult to purify but had anti-microbial inhibitory activities worth reporting. EM49 had MIC against Candida albicans of: 160μg/ml; Pseudomonas aeruginosa: 320 Kg/ml, Escherichia coli: 80μg/ml, Enterococcus faecalis 80μg/ml, and Staphylococcus aureus: 80μg/ml and Aspergillus fumigatus: 320μg/ml. EM 77 had MIC against Escherichia coli: 80 Kg/ml and Cryptococcus neoformans: 80μg/ml. Further work on their purification need to be done since in this research we are just reporting on their high MIC activities. The medicinal plants used to treat inflammation under different disease conditions in the Zulu community of Mabandla village, Kwa-Zulu Natal, South Africa have some relevant biological activities. The various antimicrobial, antioxidant and anti-inflammatory activities support the validity of their healing capacities that the traditional healers of the community claim to possess. Although there is evidence of good antimicrobial, antioxidant and anti-inflammatory activities by the crude extracts, the high levels of sucrose in P. prunelloides and glucose in G. perpensa should be borne in mind when using their decoctions in traditional medicine particularly by diabetic patients. In vitro results for the antioxidant, antinflammtory and antimicrobial activities carried out in this investigation illustrate that the plants can be a source of treatment and management for inflammation related conditions. These therefore justify their use in Zulu traditional medicine. However, in vivo assays should be carried out in order to completely validate claims by the traditional healers that they treat inflammation related conditions. / Vaal University of Technology