Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

Yes / During the years 2003¿2005, a comparative ethnobotanical field survey was conducted on remedies used in traditional animal healthcare in eight Mediterranean areas. The study sites were selected within the EU-funded RUBIA project, and were as follows: the upper Kelmend Province of Albania; the Capannori area in Eastern Tuscany and the Bagnocavallo area of Romagna, Italy; Cercle de Ouezanne, Morocco; Sierra de Aracena y Picos de Aroche Natural Park in the province of Huelva, Spain; the St. Catherine area of the Sinai Peninsula, Egypt; Eastern and Western Crete, Greece; the Paphos and Larnaca areas of Cyprus; and the Mitidja area of Algeria. One hundred and thirty-six veterinary preparations and 110 plant taxa were recorded in the survey, with Asteraceae and Lamiaceae being the most quoted botanical families. For certain plant species the survey uncovered veterinary phytotherapeutical indications that were very uncommon, and to our knowledge never recorded before. These include Anabasis articulata (Chenopodiaceae), Cardopatium corymbosum (Asteraceae), Lilium martagon (Liliaceae), Dorycnium rectum (Fabaceae), Oenanthe pimpinelloides (Apiaceae), Origanum floribundum (Lamiaceae), Tuberaria lignosa (Cistaceae), and Dittrichia graveolens (Asteraceae). These phytotherapeutical indications are briefly discussed in this report, taking into account modern phytopharmacology and phytochemistry. The percentage of overall botanical veterinary taxa recorded in all the study areas was extremely low (8%), however when all taxa belonging to the same botanical genus are considered, this portion increases to 17%. Nevertheless, very few plant uses were found to be part of a presumed "Mediterranean" cultural heritage in veterinary practices, which raises critical questions about the concept of Mediterraneanism in ethnobotany and suggests that further discussion is required. Nearly the half of the recorded veterinary plant uses for mammals uncovered in this survey have also been recorded in the same areas in human folk medicine, suggesting a strong link between human and veterinary medical practices, and perhaps also suggesting the adaptive origins of a few medical practices. Since most of the recorded data concern remedies for treating cattle, sheep, goats, and camels, it would be interesting to test a few of the recorded phytotherapeuticals in the future, to see if they are indeed able to improve animal healthcare in breeding environments, or to raise the quality of dairy and meat products in the absence of classical, industrial, veterinary pharmaceuticals.

No / This article reports on an ethnopharmaceutical field study carried out among Turkish migrants in Cologne, western Germany, which recorded 79 botanical taxa and 115 plant-based preparations, encompassing 167 folk phytotherapeutical uses, as well as a few other biological (animal and mineral derived) remedies. One-fourth of the recorded remedies were represented by food¿medicines. Half of the ingredients quoted came originally from Turkey; only a few plants were gathered from the wild or cultivated in the Cologne area. This article discusses the Turkish migrants¿ frequent use of aerial parts of Sideritis species, the fruits of Pistacia terebinthus and the seeds of Peganum harmala from the perspective of modern phytopharmacology, phytotherapy and toxicology. It considers cultural adaptation related to the use of folk pharmaceuticals by analysing the ingredients of the Turkish folk pharmacopoeia and comparing them with those of the Turkish medical ethnobotany and of the phytotherapy of the host country (Germany). Only one third of the remedies mentioned by Turkish migrants living in Cologne are also known in German evidence-based phytotherapy, and less than 10% of the recorded phytotherapeutic uses are common to both pharmacopoeias. This article concludes by suggesting hypotheses on future changes affecting the knowledge and use of medicinal plants within the Turkish community of Cologne.

In this thesis I synthesized different aspects related to diversity, distribution, uses and conservation of medicinal plants in Nepal and also have attempted to recommend guidelines for sustainability of two highly used alpine plant species. The over-harvesting or human induced activities are not the only problem for biodiversity but recently invasion of alien species has also emerged as serious problem in Nepal. I thus also attempted to analyze the effect of invasive species on community composition in the last paper. The first two papers deal with diversity, distribution, uses and harvesting. Paper I showed that medicinal plants in Nepal have unimodal relationship with elevation and the maximum total species richness is at 1000 m. Paper II which deals with the uses of medicinal plants in the Humla region, west Nepal showed that there are 161 medicinal plant species belonging to 61 families and 106 genera used for treating 72 human and 7 veterinary ailments. Medicinal plants in Humla were mostly collected in wild. This induces a serious threat to diversity of the medicinal plants and it is therefore necessary to develop proper management guidelines for their harvesting in wild and/or their domestication. Rheum australe, an endemic plant to west Himalayan region, is widely used plant in traditional...

Chan, Wing Hin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-143). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of Content --- p.viii / List of Figures --- p.xiv / List of Tables --- p.xvi / Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Food and herb authentication --- p.1 / Chapter 1.1.1 --- Background and definition --- p.1 / Chapter 1.1.2 --- Importance of species identification in food and herb authentication --- p.2 / Chapter 1.1.2.1 --- Primary health care --- p.2 / Chapter 1.1.2.2 --- Food and herb safety --- p.3 / Chapter 1.1.2.3 --- Conservation --- p.4 / Chapter 1.1.3 --- Methods for species identification in food and herb authentication --- p.4 / Chapter 1.1.3.1 --- Morphological identification --- p.5 / Chapter 1.1.3.2 --- Chemical analysis --- p.6 / Chapter 1.1.3.3 --- Molecular analysis --- p.9 / Chapter 1.1.4 --- Legislation --- p.11 / Chapter 1.1.4.1 --- Labeling ´ب --- p.11 / Chapter 1.1.4.2 --- Chinese medicine : --- p.12 / Chapter 1.1.4.3 --- Conservation --- p.12 / Chapter 1.2 --- Dongchongxiacao --- p.13 / Chapter 1.2.1 --- Background information of Dongchongxiacao --- p.13 / Chapter 1.2.2 --- Classification of fungal part of Dongchongxiacao --- p.14 / Chapter 1.2.3 --- Dongchongxiacao as a Traditional Chinese Medicine. --- p.15 / Chapter 1.2.4 --- The Dongchongxiacao market --- p.16 / Chapter 1.2.5 --- Adulteration and contamination of Dongchongxiacao --- p.18 / Chapter 1.2.6 --- Authentication of Dongchongxiacao --- p.19 / Chapter 1.2.6.1 --- Morphological identification --- p.19 / Chapter 1.2.6.2 --- Chemical analysis --- p.20 / Chapter 1.2.6.3 --- Molecular analysis --- p.22 / Chapter 1.2.6.3.1 --- "FINS analysis with genomic ITS, nrLSU, EF-lα and rpbl regions for fungal analyses" --- p.22 / Chapter 1.2.6.3.2 --- FINS analysis with mitochondrial CytB and COI regions for caterpillar analyses --- p.24 / Chapter 1.3 --- Abalone --- p.26 / Chapter 1.3.1 --- Background information of abalone --- p.26 / Chapter 1.3.2 --- Abalone as food --- p.27 / Chapter 1.3.3 --- The abalone market --- p.28 / Chapter 1.3.4 --- Adulteration of abalone --- p.31 / Chapter 1.3.5 --- Authentication of abalone --- p.32 / Chapter 1.3.5.1 --- Morphological identification --- p.32 / Chapter 1.3.5.2 --- Chemical analysis --- p.32 / Chapter 1.3.5.3 --- Molecular analysis --- p.33 / Chapter 1.3.5.3.1 --- FINS analysis with mitochondrial COI and 16S rDNA --- p.33 / Chapter 1.3.5.3.2 --- Haliotis-specific detection --- p.34 / Chapter 1.4 --- Aim and Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.36 / Chapter 2.1 --- Materials used in this sutdy --- p.36 / Chapter 2.1.1 --- Dongchongxiacao and Cordyceps samples --- p.36 / Chapter 2.1.2 --- Downloaded sequences from NCBI database included in Dongchongxiacao study. --- p.45 / Chapter 2.1.3 --- Abalone and gastropod samples --- p.48 / Chapter 2.1.4 --- Downloaded sequences from NCBI database included in abalone study --- p.54 / Chapter 2.2 --- Reagents and equipments : --- p.56 / Chapter 2.2.1 --- Chemical test on the presence of potassium alum in Dongchongxiacao --- p.56 / Chapter 2.2.2 --- Sample preparation and DNA extraction --- p.57 / Chapter 2.2.3 --- Polymerase Chain Reaction --- p.57 / Chapter 2.2.4 --- Agarose gel electrophoresis and Gene Clean --- p.57 / Chapter 2.2.5 --- Cloning --- p.58 / Chapter 2.2.6 --- Cycle sequencing --- p.58 / Chapter 2.3 --- Experimental procedures --- p.58 / Chapter 2.3.1 --- Morphological observation of Dongchongxiacao and abalone --- p.59 / Chapter 2.3.2 --- Chemical test of potassium in Dongchongxiacao --- p.59 / Chapter 2.3.3 --- Sample preparation and DNA extraction --- p.60 / Chapter 2.3.4 --- Polymerase Chain Reaction --- p.61 / Chapter 2.3.5 --- Agarose gel electrophoresis and Gene Clean --- p.64 / Chapter 2.3.6 --- Cloning --- p.65 / Chapter 2.3.7 --- Cycle sequencing --- p.67 / Chapter 2.3.8 --- Sequence analyses --- p.67 / Chapter 2.3.9 --- Haliotis-specific primer design and PCR test --- p.68 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Dongchongxiacao --- p.71 / Chapter 3.1.1 --- Morphological observations --- p.71 / Chapter 3.1.2 --- Chemical test of potassium alum --- p.77 / Chapter 3.1.3 --- Sequence analyses --- p.79 / Chapter 3.1.4 --- The dendrograms --- p.81 / Chapter 3.2 --- Abalone --- p.91 / Chapter 3.2.1 --- Morphological observations --- p.91 / Chapter 3.2.2 --- Sequence analyses --- p.92 / Chapter 3.2.3 --- The dendrograms --- p.94 / Chapter 3.2.4 --- Haliotis-specific PCR --- p.96 / Chapter Chapter 4 --- Discussion --- p.98 / Chapter 4.1 --- Dongchongxiacao --- p.98 / Chapter 4.1.1 --- Species identification of Dongchongxiacao and related Cordyceps species --- p.98 / Chapter 4.1.1.1 --- Ophiocordyceps sinensis --- p.98 / Chapter 4.1.1.2 --- Cordyceps gunnii --- p.100 / Chapter 4.1.1.3 --- Metacordyceps taii --- p.102 / Chapter 4.1.1.4 --- Cordyceps militaris --- p.103 / Chapter 4.1.2 --- Adulteration of Dongchongxiacao and labeling --- p.104 / Chapter 4.1.3 --- Hosts of Dongchongxiacao fungi and relationship between them --- p.107 / Chapter 4.2 --- Abalone --- p.109 / Chapter 4.2.1 --- Species identification of abalones and other gastropod species by FINS analysis --- p.109 / Chapter 4.2.1.1 --- Haliotis species --- p.109 / Chapter 4.2.1.1.1 --- Haliotis diversicolor --- p.110 / Chapter 4.2.1.1.2 --- Haliotis discus --- p.110 / Chapter 4.2.1.1.3 --- Haliotis asinina --- p.111 / Chapter 4.2.1.1.4 --- Haliotis rufescens --- p.111 / Chapter 4.2.1.1.5 --- Haliotis midae --- p.111 / Chapter 4.2.1.1.6 --- Haliotis madaka --- p.112 / Chapter 4.2.1.1.7 --- Haliotis rubra --- p.113 / Chapter 4.2.1.1.8 --- Haliotis iris --- p.113 / Chapter 4.2.1.1.9 --- Haliotis corrugata --- p.114 / Chapter 4.2.1.2 --- Concholepas concholepas --- p.114 / Chapter 4.2.1.3 --- Hemifusus species --- p.115 / Chapter 4.2.1.4 --- """Dried abalone slice"" samples (D1 to D3) and canned top-shell (E5)" --- p.115 / Chapter 4.2.2 --- Haliotis-speciflc PCR --- p.115 / Chapter 4.2.3 --- Adulteration of abalone and labeling --- p.116 / Chapter 4.3 --- Significance and limitation of molecular approaches in authentication of food and herbs --- p.117 / Chapter 4.3.1 --- FINS analysis --- p.117 / Chapter 4.3.1.1 --- High interspecific variability but low intraspecific variations --- p.118 / Chapter 4.3.1.2 --- Amplification with universal primers --- p.118 / Chapter 4.3.1.3 --- Insufficient DNA sequence available in database --- p.119 / Chapter 4.3.1.4 --- Contamination by foreign DNA and amplification of undesirable DNA in sample mixture --- p.120 / Chapter 4.3.1.5 --- Amplification of degraded DNA --- p.121 / Chapter 4.3.1.6 --- Suggested regions for authentication of Dongchongxiacao and abalone based on FINS analysis results --- p.121 / Chapter 4.3.2 --- PCR with specific primers for targeted amplicons --- p.122 / Chapter 4.3.3 --- Other limitations of molecular approaches in authentication of food and herbs --- p.123 / Chapter 4.4 --- Further investigation --- p.124 / Chapter 4.5 --- Conclusion --- p.124 / References : --- p.126 / Chapter Appendix 1 --- Sequence alignment of 16S rDNA gene sequences of abalone for Haliotis-specific primer design --- p.144 / Chapter Appendix 2 --- Accession numbers of sequences of Dongchongxiacao and Cordyceps samples in this study --- p.149 / Chapter Appendix 3 --- Search results of CytB sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.150 / Chapter Appendix 4 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.151 / Chapter Appendix 5 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.152 / Chapter Appendix 6 --- Sequence alignment of ITS sequences of Cordyceps samples and related sequences --- p.153 / Chapter Appendix 7 --- Sequence alignment of nrLSU sequences of Cordyceps samples and related sequences --- p.161 / Chapter Appendix 8 --- Sequence alignment of EF-lα sequences of Cordyceps samples and related sequences --- p.168 / Chapter Appendix 9 --- Sequence alignment of rpbl sequences of Cordyceps samples and related sequences --- p.173 / Chapter Appendix 10 --- "Sequence alignment of combined dataset of three regions (nrLSU, EF-lα and rpbl) of Cordyceps samples and related sequences" --- p.179 / Chapter Appendix 11 --- Sequences alignment of CytB sequences of caterpillar host of Cordyceps samples and related sequences --- p.188 / Chapter Appendix 12 --- Sequence alignment of COI sequences of caterpillar host of Cordyceps samples and related sequences --- p.191 / Chapter Appendix 13 --- Sequence alignment of COI sequences of Cordyceps samples D12-2 and D14 and related sequences --- p.195 / Chapter Appendix 14 --- Sequence distance matrix of ITS sequences of Cordyceps samples and related samples based on K2P algorithm --- p.196 / Chapter Appendix 15 --- Sequence distance matrix of nrLSU sequences of Cordyceps samples and related samples based on K2P algorithm --- p.203 / Chapter Appendix 16 --- Sequence distance matrix of EF-lα sequences of Cordyceps samples and related samples based on K2P algorithm --- p.208 / Chapter Appendix 17 --- Sequence distance matrix of rpbl sequences of Cordyceps samples and related samples based on K2P algorithm --- p.213 / Chapter Appendix 18 --- "Sequence distance matrix of combined dataset of three regions (nrLSU, EF-lα and rpbl) sequences of Cordyceps samples and related samples based on K2P algorithm" --- p.217 / Chapter Appendix 19 --- Sequence distance matrix of CytB sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.219 / Chapter Appendix 20 --- Sequence distance matrix of COI sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.223 / Chapter Appendix 21 --- Sequence alignment of chloroplast trnH-psbA sequences of Cordyceps sample D12-2 and related sequences --- p.226 / Chapter Appendix 22 --- Accession numbers of sequences of abalone and gastropod samples in this study --- p.227 / Chapter Appendix 23 --- Search results of 16S rDNA sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.228 / Chapter Appendix 24 --- Search results of COI sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.229 / Chapter Appendix 25 --- Search results of COI sequences of the abalone and gastropod samples based on BOLD-IDS --- p.230 / Chapter Appendix 26 --- Sequence alignment of 16S sequences of abalone samples and related sequences --- p.231 / Chapter Appendix 27 --- Sequence alignment of COI sequences of abalone samples and related sequences --- p.234 / Chapter Appendix 28 --- Sequence alignment of COI sequences of abalone product sample D2 and related sequences --- p.238 / Chapter Appendix 29 --- Sequence distance matrix of 16S sequences of abalone samples and related samples based on K2P algorithm --- p.239 / Chapter Appendix 30 --- Sequence distance matrix of COI sequences of abalone samples and related samples based on K2P algorithm --- p.243

Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2017 / Cancer is a broad group of various diseases characterised by unregulated cell proliferation which leads to the formation of tumours (Vickers, 2004). Some tumours remain confined to their site of origin while some gain the ability to spread to other parts of the body, a process known as metastasis (Weiss, 1990). The burden of cancer continues to rise, due to inefficient prevention strategies and serious side effects, as well as the cost of cancer regimens (Sondhi et al., 2010). Medicinal plants represent a reservoir of bioactive compounds that can be useful in the management of cancer with less or no side effects (Wong et al., 2012). The aim of this study was to investigate the anti-cancer effects of M. balsamina leaf extract in breast MCF-7 cancer cells. In this study, M. balsamina leaves powder was extracted using acetone. The biological effect of the extract was assessed on the viability of MCF-7 cells using the MTT assay. The extract’s ability to induce apoptosis was assessed using the Hoechst/propidium iodide dual staining method. Its anti-metastatic potential was investigated by determining its effect on MCF-7 cell migration, attachment and invasion using wound healing, adhesion, invasion assay, respectively. The human apoptosis antibody and human angiogenesis antibody array kits were used to determine the effect of the extract on the expression levels of proteins involved in apoptosis and metastasis, respectively. Treatment of MCF-7 cells with different concentrations of the extract showed a significant decrease in cell viability after 48 h incubation at 10 - 20 µg/ml. The decrease in cell viability was associated with the induction of apoptosis as seen by nuclear condensation and loss of membrane permeability in cells treated with the extract. Inhibition of migration, adhesion and invasiveness of the MCF-7 cells was seen in the treated cells. The extract also modulated proteins implicated in cell apoptosis, adhesion, migration and invasion such as Bcl-2 family of proteins, IGFBP, uPA, MMPs. In conclusion, based on the results, the extract show pro-apoptotic and anti-metastasis potential. Thus M. balsamina can be considered as a potential source of compounds with anti-cancer activity

Thesis (Ph. D. (Microbiology)) --University of Limpopo, 2019 / Tuberculosis (TB) is one of the top ten diseases that causes morbidity and mortality worldwide. Although TB is curable, the main problem currently with TB is development resistance to the current chemotherapy. Medicinal plants, as a source of drugs, have been found to cause less or no resistance. Medicinal plants are studied and considered for their efficacy and safety because they possess bioactive compounds with various biological activities. The aim of the study was to isolate and characterise bioactive compounds from selected seven plant species [A. dimidiata (LNBG 1969/46), A. afra (LNBG 2010/27), Z. capense (LNBG 1969/100), C. herorense (LNBG 1977/71), L. javanica (LNBG 1969/460), E. camaldulensis and C. lemon (UNIN 12330)] with activity against Mycobacterium smegmatis, Multi- drug resistant tuberculosis starain and H37Rv Mycobacterium tuberculosis strain. It was also imperative to determine whether crude extracts, sub- fractions of the extracts and the isolated bioactive compounds are cytotoxic or not. Leaves of the seven selected plants were collected from South African National Botanical Institute (SANBI) at Nelspruit, Mpumalanga Province, South Africa. The leaves were dried and milled to fine powder. The leaves of each plant were extracted using solvents of varying polarity (i.e. hexane, dichloromethane, acetone and methanol). Phytochemical screening was done using Thin Layer Chromatography (TLC) developed in three mobile phases varying in polarity and then sprayed with vanillin sulphuric acid in methanol heated at 110oC for optimal colour development. Qualitative antioxidant activity was determined by using 1,2- diphenylpicryl hydrazyl (DPPH) assay on TLC plates. Antimycobacterial activity for all the plant extracts was done using bioautography assay in qualitative analysis of the active compounds and for quantitative analysis, the microplate dilution assay was used. The plants which showed better activity (C. lemon, C. hereroense and A. dimidiata) with the microplate dilution assay and bioautography were further subjected to solvent- solvent fractionation as the first step towards isolation of bioactive compounds. Synergistic, additive, indifferent and antagonistic effects of the crude extracts combinations of the three selected plants was determined. The combinations where A. dimdiata was also part of the combinations frequently showed synergistic effect. On the other hand, with the combinations of C. hereroense and A. dimdidata (CH-AD) there was no antagonistic effect observed. The combinations of crude extracts of C. lemon and A. dimidiata all showed synergistic effect, except for only three combinations. Based on the synergistic effect observed and the bioactivity on the bioautography and microplate dilution assay of the sub- fractions, A. dimidiata was chosen for further analysis for antimycobacterial activity using the MDR- TB strain and M. tuberculosis H37Rv strain. The sub- fractions of A. dimidiata with the most activity were hexane and butanol. Hexane and butanol fractions both showed good MIC activity against the TB isolated M. tuberculosis field strain and H37Rv strain of 0.47 and 031 mg/ml, respectively. Butanol fraction was further taken for isolation using open colum chromatography doing bioassay guided isolation. The isolated compounds, together with the crude were tested for their biological activity using MTT assay to determine their cytotoxicity and antimycobacterial activity assay to confirm their activity against M. smegmatis and M. tuberculosis. Cytotoxicity assay showed that the crude extracts of A. dimidiate were toxic against the Vero kidney cells and the subfractions (i.e. butanol and hexane) became moderate to non-toxic and one compound (oleanolic acid) from the butanol sub-fraction was non- cytotoxic. This indicates that the isolation of the crude extracts tends to become non- toxic to the cells. The study suggests the use of pure compounds to fight against TB as compared to crude extracts since they are both bioactive and non- cytotoxic. Crude extracts combinations were effective in killing Mycobacterium as compared to single crude extracts. The present study recommends the use of A. dimidiata plant leaves crude extracts combinations as they mostly exhibit synergistic effect. Furthermore, Mycobacterium and also contain non- cytotoxic antimycobacterial compound (oleanolic acid). The study serves as a scientific proof for the use of this plant in traditional medicine for TB treatment.

Diabetes mellitus (DM) has become a global threat in developing and developed countries, where diabetic patients are more prone to cardiovascular complications, a condition called diabetic cardiomyopathy. Studies have shown a direct link between hyperglycaemia and an increase in the production of reactive oxygen species in cardiac cells leading to diabetic cardiomyopathy. This study tests oleanolic acid, a bioactive compound from the plant Syzigium aromaticum as an antioxidant which could have a potential role in management of DM. Aims i) To extract Oleanolic acid (OA) from Syzigium aromaticim, ii) Investigate the antioxidant effects of plant derived OA in an in-vitro model of hyperglycaemia induced oxidative stress. Methods The flower buds of the Syzigium aromaticim [(Linnaeus) Merrill & Perry] (Myrtaceae) plant (commonly called cloves) were used to isolate OA. The ethyl acetate solubles from the cloves were subjected to chromatographic fractionation to yield OA powder. Spectroscopic analysis was done using 1D and 2D 1H and 13C NMR techniques for the identification of the structure of the compound. This compound was then used in vitro to test for its antioxidative properties. H9C2 cardiac myoblasts were employed which were treated with normoglycaemic (5.5 mM) and hyperglycaemic (33 mM) glucose conditions. The cells were then treated with oleanolic acid to test for its antioxidant properties. We looked at a dose-dependent (0, 20, 50 μM) and time-dependent effects of OA treatment (6 and 24 hrs) following 48 hours glucose exposure. ROS levels were measured using H2DCF-DA fluorescence staining using microscopy and flow cytometry techniques for analysis. xviii Results Recrystallisation of the powder with ethanol and inspection of the 1 and 2- dimensional 1H- and 13C-NMR spectra of the compound with comparison to literature data confirmed OA molecular structure and IUPAC numbering similar to that of literature characterized and confirmed the structure of oleanolic acid. In cell specific data high glucose treatments on H9C2 cells showed increased ROS production (22 ± 6 % and 20 ± 7 % n= 3 p< 0.01) for 6 and 24 hrs treatments, respectively, compared to their normoglycaemic control groups. The 6 h OA treated group showed a decrease in ROS production with 26.6 ± 17.4 % for the 20 μM while for 50 μM there was a 37.7 ± 14.3% decrease. A ROS reduction trend was observed in the normoglycaemic group, but this was significant at 24 hrs with 46.8 ± 45.3% and 57.3 ± 9 % for both 20 and 50 μM treatments, respectively. The 24 hrs OA treated group showed a dose-dependent decrease in ROS with 50 μM more pronounced (80.7% ± 4.5 %). The 20 μM OA treatments also showed a 15.7 ± 19 % decrease in ROS. Discussion In the present study, we have evaluated the antioxidant effects of OA in vitro following extraction of the compound from Syzigium aromaticim. The oxidative stress induced by hyperglycaemia was attenuated by oleanolic acid and this also translated into decreased ROS suggesting its use as an antioxidant in alleviating cardiovascular complications associated with diabetes mellitus.

Schumanniophyton magnificum and Glypheae brevis are important medicinal plants growing wild in the West African rain forest. They are used in folkloric medicine in the treatment of epilepsy and convulsion as well as for some other diseases. The purpose of this work was to investigate the aspect of folkloric use in order to support folkloric claims and document the findings. The extracts were prepared from ground plant material by a continuous extraction method. Five hundred grams of ground plant material were continuously de-fatted with 2 L petroleum ether (60°- 80°) in a Soxhlet apparatus for about 5 h. The resulting marc was dried and the chemical constituents extracted hot in a Soxhlet apparatus for about 8 to 10 h with 2 L aqueous ethanol (70%). The efficacy of the extraction method was confirmed using standard bioassays and phytochemical analyses. The anti-convulsant activity of the crude extracts was evaluated in vivo against chemically induced convulsions using three different animal models, namely the strychnine, the picrotoxin and the pentylenetetrazole tests. The acute and delayed toxicity test results showed that in all the animal models investigated very high doses, about four times higher than the protective doses of the extracts, were required to kill 50% of the population of animal used. Phytochemical assays of the extracts indicated the presence of alkaloids only in S. magnificum root extract and glycosides in extracts from both species. The glycosides were positive to Baljet, Xanthydrol and Keller-Kiliani tests for cardiac glycosides. S. magnificum and G. brevis chemical constituents were initially isolated with a sequential fractionation method starting with a highly non-polar solvent and gradually increasing to a more polar solvent. The fractions were pooled on the basis of TLC similarity profiles when viewed under the UV light at 254 and 366 nm and were found to have two and four major UV absorbing fractions for S. magnificum and G. brevis respectively. Radio-receptor binding tests were used to assess the anti-convulsant activities of the hydro-alcoholic crude extracts, the organic and aqueous fractions of the crude extracts, partially purified components and pure components in in vitro tests against some standard GABA[A] receptor antagonists, muscimol and isoguvacine respectively. The anti-convulsant activities resided in the aqueous fractions of the hydro-alcoholic crude extracts of both plants. The purely organic fractions of G. brevis demonstrated no activity while all the fractions of the aqueous component demonstrated some degree of activity. The anti-convulsant activity of S. magnificum was found only in one fraction-Fraction 1. This Fraction was further investigated and one of the components appear to be responsible for the activity. The structure of the active constituent was 5,7dihdroxy-2 methylbenzopyran-4-one, a noreugenin. A second bioactive compound, schumanniofoside, was identified from Fraction M[5.2] from S. magnificum. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.