Made available in DSpace on 2016-07-18T17:51:15Z (GMT). No. of bitstreams: 1 Dissertaca Paulo.pdf: 254511 bytes, checksum: c2a84e6eed7079ddc23e53dc978a94f9 (MD5) Previous issue date: 2008-05-28 / The purpose of this experiment was to analyze five irrigation water depth effect on the basil production. The basil is a plant very used as spice to food preparation. The water depths had been based in the class A pan evaporation (CPE): 0% CPE; 50% CPE; 75% CPE; 100% CPE, and 150% CPE. The analyzed variables were dry weight for leaves and flowers; content of essential oil and dry mass and length of root zone. It was calculated the essential oil yield and net return. The experiment was carried out in President Prudente, SP, Brazil, from March to October 2007. The third harvest was possible only under irrigation. The daily excessive water depth was detrimental to the plant development. The 100% CPE water depth showed maximum dry mass productivity (4,248.67 kg ha-1), maximum essential oil yield (58.26 kg ha-1) with the net annual profit of R$ 2,543.82 ha-1. / O objetivo deste experimento foi analisar o efeito de cinco lâminas de irrigação na produção do manjericão, planta muito usada como tempero na preparação de alimentos. As lâminas foram baseadas na evaporação do tanque classe A (ECA) sendo: 0% ECA; 50% ECA; 75% ECA; 100% ECA e 150% ECA. As variáveis analisadas foram a matéria seca das folhas e inflorescências, teor de óleo essencial e massa seca e comprimento de raízes. Foram calculadas a produtividade de óleo essencial e a receita líquida. O experimento foi instalado em Presidente Prudente-SP no período de março a outubro de 2007. A terceira colheita só foi viável utilizando irrigação. A irrigação excessiva diária prejudicou o desenvolvimento da planta. A lâmina de irrigação equivalente a 100% ECA apresentou a máxima produtividade de massa seca (4.248,67 kg ha-1), a máxima produtividade de óleo essencial (58,26 kg ha-1) com a receita líquida anual de R$ 2.543,82 ha-1.

O alto índice de obesidade populacional favorece o aumento das complicações associadas ao excesso de peso corporal, afetando a qualidade de vida do indivíduo. Vários alimentos têm sido investigados com o objetivo de auxiliar no controle do peso, e, dentre esses alimentos, o chá verde, derivado das folhas da planta Camellia sinensis, parece ser eficiente tendo um efeito termogênico, além de promover maior oxidação da gordura corporal. O consumo desse chá possivelmente pode alterar a composição corporal e a taxa metabólica de repouso (TMR), mas até o momento poucos estudos nessa área têm sido realizados e nenhum deles avaliou mulheres com sobrepeso e obesidade. O objetivo deste estudo duplo cego, controlado com placebo, foi avaliar os efeitos do consumo de chá verde e da prática ou não de exercício físico resistido sobre a TMR e a composição corporal em mulheres com Índice de Massa Corporal entre 25 a 35Kg/m2 divididas em quatro grupos (grupo 1 chá verde; grupo 2 placebo; grupo 3 chá verde mais exercícios resistidos; grupo 4 placebo mais exercícios resistidos) durante um período de dois meses. As voluntárias dos grupos 3 e 4 realizaram testes de 1 repetição máxima, sendo estes no supino reto, puxador-costas e leg-press 45º para a realização de um programa de exercícios físicos resistidos. Todas as voluntárias dos quatro grupos foram submetidas às análises bioquímicas e às avaliações da TMR por meio de calorimetria indireta e da composição corporal pela bioimpedância elétrica. Os resultados mostraram que o grupo 1 perdeu uma quantidade de peso relevante para o período de estudo, e apresentaram diminuição da TMR com acompanhamento da perda de peso, diminuíram a gordura corporal, com manutenção da massa magra. As voluntárias do grupo 2, utilizando placebo, não perderam peso, ganharam massa gorda e apresentaram diminuição da massa magra e da TMR. Quando os grupos com exercícios físicos resistidos foram comparados, observou-se que as voluntarias do grupo 3 apresentaram resultados significativamente melhores que as do grupo 4, ou seja, não perderem peso, porém tiveram sua composição corporal modificada, com perda de gordura e ganho de massa muscular; apresentaram aumento da força muscular e redução dos níveis de triglicérides. Os resultados deste estudo sugerem que o consumo de chá verde pode ser um aliado alimentar efetivo para a perda de peso e diminuição da gordura corporal, sem que haja perda da massa muscular. Seu consumo aliado à prática de exercício físico resistido auxilia na redução do triglicérides, ganho de força muscular, ganho de massa magra e na redução da massa gorda. / The high rate of obesity contributes to the greater complications associated with excess body weight, affecting the individual\'s life quality. Several considered functional foods have been investigated in order to help weight control, and among these foods, green tea, derived from the leaves of the Camellia sinensis plant, appear to be an ally with a thermogenic effect and increases the oxidation of fats. Consumption of green tea could possibly alter body composition and metabolic rate (BMR), but so far few studies have been conducted in this area and none of them to evaluate women with overweight and obesity. The aim of this double-blind, placebo-controlled trial was to evaluated the effects of green tea consumption and the practice or not of resistive exercise on BMR and body composition in women with body mass index between 25 35Kg/m2 divided into four groups (group 1 green tea; group 2 placebo; group 3 green tea plus resistance training; group 4 placebo plus resistance exercises) during a period of two months. The subjects of groups 3 and 4 trials of 1-repetition maximum, which are the bench press, puller-back and leg press 45 ° to carry out a program of resistive exercise. All the volunteers of the four groups were evaluated at biochemical analysis and at evaluations of BMR by indirect calorimetry and body composition by bioelectrical impedance analysis. The results showed that group 1 lost a significant amount of weight for the period of study, and showed a decrease in TMR with accompanying weight loss, decreased body fat while maintaining lean body mass. The subjects in group 2, using placebo, have not lost weight, gained fat mass and showed a decrease in lean body mass and BMR. When groups with resistance exercise were compared, it was observed that the subjects in group 3 showed significantly better results than those of Group 4, in other words, not losing weight but your body composition had changed, with fat loss and gain muscle mass had increased muscle strength and reducing triglyceride levels. The results of this study suggest that consumption of green tea can be an effective ally food for weight loss and decreased body fat without losing muscle mass. Consumption combined with the practice of resistive exercise helps in reducing triglycerides, gain muscle strength gain lean mass and reducing fat mass.

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2019 / The emergence of drug resistance to the first line drugs complicates the treatment of tuberculosis (TB), especially in parts of sub-Saharan Africa where accessibility to quality health care is limited. The search for alternative medication has been the centre of research for years due to challenges posed by infectious organisms including drug resistance, lengthy treatment periods and lack of quality health care in developing countries. Stomatostemma monteiroae is used in traditional medicine to treat TB and related symptoms. The aim of this study was to isolate and characterise compounds with antimycobacterial activity from Stomatostemma monteiroae. The plant materials were collected from Ga-Madiga village in Limpopo province of South Africa. Different plant parts namely: leaves, twigs, roots, tuber and tuber-peels were separated, washed, dried and milled to a fine powder. Several solvents (n-hexane, dichloromethane, acetone and methanol) were used to extract the plant material using various extraction methods such as maceration, defatting, and extract enrichment procedure and phytochemical analysis was done using standard chemical tests and thin layer chromatography. The qualitative antioxidant activity was determined by the thin layer chromatography (TLC) based 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity and quantitative antioxidant activity was determined using colorimetric DPPH free radical scavenging and ferric reducing power assay. Antimycobacterial activity of the extracts was assessed using bioautography and micro dilution method tested on Mycobacterium smegmatis (ATCC 1441), Mycobacterium tuberculosis (ATCC 25177) and M. tuberculosis H37Rv (ATCC 27294). The cytotoxic effects of the extracts were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. The compounds with antimycobacterial activity were isolated using bioassay-guided fractionation and purified using preparative thin layer chromatography and thereafter identified using NMR spectroscopy to elucidate the structure. Various phytochemical constituents were detected in different plant parts, with the leaves and twigs possessing more of the phytoconstituents analysed. The TLC profile of S. monteiroae indicated that more compounds are non-polar to intermediate in polarity. The antioxidant activity analysis on TLC plates indicated that all the plant parts have low antioxidant activity, this was also confirmed by xxii quantitative tests. The leaves of S. monteiroae had antimycobacterial activity when analysed using bioautography, while other plant parts had no active bands. The minimum inhibitory concentration values were much higher than the positive control rifampicin and the roots (0.31 mg/mL) followed by the leaves (0.83 mg/mL) had lower inhibitory concentrations when tested against M. smegmatis. The MIC values of extracts against TB causing strains varied greatly, the leaves and the roots had even higher MIC value. Toxicity analysis indicated that all plant parts were non-toxic towards Vero cells (LC50 > 0.02 mg/mL). Bioassay-guided fractionation enabled isolation of one antimycobacterial pure compound from the leaves extracts. The isolated compound was identified using NMR and was found to be a sitosterol derivative 8,9-dehydro-4-methyl-24-vinylobtusifoliol. This compound had a noteworthy activity against M. smegmatis. The present study validates the use of S. monteiroae in the treatment of TB related symptoms traditionally. Further studies are required to analyse the cytotoxic effects of the isolated compound and also testing the antimycobacterial activity of the isolated compound on TB causing pathogens. / National Research Foundation (NRF)

Thesis (DPhil (Biomedical Science))--Cape Peninsula University of Technology, 2019. / Diabetes mellitus (DM) is a group of metabolic disorders characterised by persistent high blood glucose levels together with abnormal metabolism of macromolecules. If the hyperglycemia is not controlled, adverse metabolic changes could occur leading to the progressive development of severe complications. Formation of reactive oxygen/nitrogen species and inflammatory responses are principal mechanisms that have been implicated in the development of hyperglycemia-induced tissue damage. The commercially available drugs utilised in the treatment of diabetes have been linked to detrimental side effects hence the need to discover alternative medicines especially from medicinal plants. Catharanthus roseus is both a medicinal and ornamental plant that is traditionally used to treat various diseases. It has been reported to possess antidiabetic, anticancer, antimicrobial and antioxidant properties. The plant has been shown to possess more than 100 monotepernoid indole alkaloids which were linked to the plants’ antihyperglycemic and antioxidant effects. Therefore, this study was carried out to investigate the effect of vindoline; a bioactive compound derived from C. roseus against type 2 diabetes–induced complications. The study also investigated the effects of Catharanthus roseus extracts in RIN-5F cell line. The study was carried out in two parts: viz in vitro and the in vivo assessments. The in vitro study initially investigated the polyphenolic content and antioxidant activities of vindoline and the 3 extracts (methanolic, aqueous and the dichloromethane) of C.roseus. The assays used to evaluate the antioxidant capacity of the extracts include oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibitory assay. Among the evaluated extracts, the methanolic extract demonstrated both high total polyphenolic content and antioxidant capacity. The HLPC analysis of the extracts was performed and showed highest concentrations of vindoline in the dichloromethane extract and the aqueous extract exhibited the least. The antioxidant activities of vindoline were determined and compared to a known antioxidant, ascorbic acid. Vindoline revealed stronger ORAC activity than ascorbic acid however the ferric reducing antioxidant power did not show any significant differences (p < 0.05). Insulin secretion studies were performed in a β-cell insulinoma cell line- RIN-5F exposed to different concentrations of glucose (high, low and in the absence of glucose). The studies were carried out to compare the β-cell stimulatory effect of vindoline to the extracts. After performing cytotoxic experiments, concentrations that resulted in about 80% cell viability were used to determine the insulin secretory effects. In cells that exposed to glucotoxicity (50 mM glucose), vindoline showed the highest β-cell stimulatory effect (p < 0.05) when compared to the untreated controls and to the cells that were treated with the methanolic extract. In cells that were exposed to a low glucose concentration, vindoline additionally showed significant β-cell stimulatory effect at p < 0.05 when compared to the aqueous and the methanolic extracts. Thereafter, the intracellular reactive oxygen species assay (ROSA) was performed in glucotoxicity-induced cells after treatment with vindoline and the respective extracts. The results were compared to the untreated control: vindoline, methanolic and the dichloromethane extracts indicated significant reduction in ROS generation (p < 0.05). Further measurement of the release of TNF-α, a pro-inflammatory cytokine in the cells following treatment, the results were not significant among the groups at p < 0.05. The carbohydrate enzymes inhibitory activity of vindoline and extracts of C.roseus (50, 25, 12.5 and 6.125 mg/ml) were measured. The alpha glucosidase inhibitory activities of the extracts at 50 mg/ml resulted in < 30% enzyme inhibition with no significant differences among the groups at p < 0.05. At lower concentrations, the dichloromethane extract exhibited significantly lower inhibitory activities when compared to the methanolic and the aqueous extract (p < 0.05). The alpha amylase inhibitory activity of the methanolic extract was significantly increased at all concentrations; recording the highest enzyme inhibition of approximately 40% (p < 0.5). However, the dichloromethane extract did not show any enzyme inhibitory activity. The enzyme inhibitory activity of vindoline was compared to acarbose-a known standard drug, for both enzymes; vindoline did not show appreciable enzyme inhibition when compared to acarbose (p < 0.05). In vivo studies were performed in a type 2 diabetes (T2DM) rat model in which T2DM was induced in 6 weeks old male Wistar rats by having them drink 10% fructose solution ad libitum for 14 days followed by a single intraperitoneal injection of streptozotocin (STZ 40 mg/kg) in freshly prepared 0.1 M citrate buffer (pH 4.5). Animals were randomly divided into six groups (n=8) and received daily treatments for 6 weeks with the vehicle, vindoline (20 mg/kg) or glibenclamide (5 mg/kg) via oral gavage. The effects of the treatments on blood glucose, insulin, body weight, organ weight, serum biochemical parameters, oxidative status, inflammatory markers and tissue histology were assessed in diabetic and non-diabetic rats. Administration of vindoline significantly (p < 0.05) reduced the fasting blood glucose in diabetic rats by 15% and significantly increased serum insulin levels when compared to the diabetic controls. Vindoline and glibenclamide significantly (p < 0.05) reduced the levels of circulating hepatic enzymes in T2DM; the results were significant when compared to the diabetic controls. Treatment with vindoline significantly improved the hepatic antioxidant status as indicated by increased ORAC, superoxide dismutase and catalase activities, indicative of the protective effect of vindoline in diabetes-induced hepatic injury. Assessment of the levels of pro-inflammatory cytokines in the hepatic tissue indicated remarkable reduction of TNF-ɑ by (-41%) and IL-6 (-28%) in diabetic rats treated with vindoline when compared to the diabetic controls (p < 0.05). The serum lipid profile showed marked increases in the levels of serum lipids (triglycerides, low density lipoproteins, total cholesterol and very low density lipoproteins) in diabetic controls when compared to all treatment groups (p < 0.05). Therefore, vindoline and glibenclamide showed possible protective effects against diabetes-induced cardiovascular disease. Kidney function assessment revealed increased levels of urea and creatinine in the diabetic control group. Vindoline and glibenclamide significantly reduced the urea and creatinine levels in diabetic rats. Vindoline additionally improved the FRAP in diabetic hearts. The SOD activity and ORAC were increased while lipid peroxidation was reduced in the kidneys of diabetic rats treated with vindoline when compared to the diabetic control (p < 0.05). Histopathological assessment in diabetic rats showed severe damage of the liver, kidney and pancreas. Treatment of diabetic rats with vindoline restored the structure of these organs which was indicated by minimum structural changes. The expression of pro-apoptotic marker caspase 9 in response to glucose stress was significantly higher in the diabetic control group when compared to all the treatment groups. Treatment with vindoline showed remarkable reduction of caspase 9 expression in the diabetic rats. In conclusion, persistent high blood glucose levels resulted in free radical induced tissue damage in the type 2 diabetes rat model. Vindoline demonstrated protective effects against diabetes induced hepatic, cardiac, pancreatic and nephritic injuries. In addition, vindoline improved insulin secretion in both in vitro and in vivo setups hence the findings suggest that vindoline could be an important agent that can be considered in the treatment and management of diabetes and diabetic complications.

Parkinson's disease (PD) is the second most common chronic neurodegenerative disease characterized by dopamine decrease in the substantia nigra. Currently, there is no promising cure for PD and this has resulted in extensive research into alternative medicines. The aim of this study was to investigate the effect of methanol and ethyl acetate extracts of Lannea schweinfurthii (Engl. Engl) (Anacardiaceae), Zanthoxylum capense (Thunb. Harv) (Rutaceae), Scadoxus puniceus ((L.) Friis & Nordal) (Amaryllidaceae) and Crinum bulbispermum (Burm. f.) Milne-Redh. & Schweick) (Amaryllidaceae) on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells. The latter which mimics PD symptoms in vitro. Cytotoxicity of the plant extracts was assessed using sulforhodamine B (SRB) assay. Intracellular reactive oxygen species (ROS) were measured fluorometrically with the use of the fluorescent dye 2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA). Intracellular glutathione content was measured fluorometrically after staining with monochlorobimane (MCB). Fluorescent dye 5,5‟ ,6,6‟ -tetrachloro-1,1‟ ,3,3‟ -tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to assess the mitochondrial membrane potential (MMP) status of cells. Apoptosis was assessed by determining caspase-3 activity through detection of 7-amino-4-methylcoumarin (AMC) which is a product of caspace-3 substrate, acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Ac-DEVD-AMC), cleaved by the caspase-3 enzyme. Rotenone was used as an in vitro model to induce PD-like symptoms. Cytotoxicity studies for methanol extract of Zanthoxylum capense revealed the highest IC50 value of 121.3 μg/mL, indicating low toxicity. The ethyl acetate extract of Crinum bulbispermum was observed to have no effect on the normal proliferation of the SH-SY5Y cells and produced an IC50 value >100 μg/mL. The calculated IC50 value obtained from rotenone cytotoxicity studies was 112 iv nM. Zanthoxylum capense and Scadoxus puniceus plant extracts were observed to be neuroprotective against rotenone-induced toxicity. A decrease in intracellular glutathione content as well as MMP was also observed in cells exposed to rotenone alone (50 nM). There was no intracellular ROS generation observed in cells exposed to rotenone alone (50 nM) after 24 h and 72 h. However, apoptotic cell death was observed in cells treated with rotenone (50 nM). Intracellular ROS production was observed to be elevated by methanol and ethyl acetate extracts of C. bulbispermum. Methanol extracts of Z. capense was observed to increase intracellular glutathione content. MMP was increased effectively following treatment with ethyl acetate extract of C. bulbispermum. Moreover, both methanol and ethyl acetate plant extracts were found to decrease caspase-3 activity significantly (p<0.05), in cells exposed to 50 nM rotenone. Z. capense methanol extract reduced caspase-3 activity the most effectively. Treatment with plant extracts was protective and decreased cell death. Furthermore, L. schweinfurthii, Z. capense, S. puniceus and C. bulbispermum, demonstrated strong antioxidant and anti-apoptotic effects against rotenone-toxicity, making them potential agents in developing therapies for treating PD. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Department of Botany / MSc(Botany)

MSc (Botany) / Department of Botany / See the attached abstract below

The water solubility of drug molecules plays an important role in consideration of formulation development to treat a wide range of diseases. In this project, two kinds of drug delivery systems, cyclodextrins and liposomes, were developed for insoluble drug delivery to treat Alzheimer's disease (AD) and colorectal cancer (CRC), respectively. AD is an irreversible neurodegenerative disorder associated with the accumulation of amyloid-beta (A??) fibrils. Approximately 10% of people aged 65 and above have AD. Crocetin (CRT) is an active compound isolated from the fruits of gardenia (Gardenia jasminoides Ellis) and the stigmas of saffron (Crocus sativus L.). It has been reported to show various neuroprotective activities. However, poor water solubility and bioavailability are the major obstacles in developing pharmaceutical formulations of CRT. To address the issues, CRT liposomal formulations and CRT-cyclodextrin inclusion complexes were developed and evaluated. CRT-cyclodextrin inclusion complexes significantly increased the water solubility of CRT from the range ??g/mL to mg/mL. The CRT-??-cyclodextrin inclusion complex (1:3 molar ratio of CRT/??-cyclodextrin) was chosen for further studies as it showed the highest encapsulation efficiency (94.73 ?? 0.86%). The formulation had no toxicity to neuronal cells nor AD model cells within the experimental concentration range (0.625 to 100 ??M of CRT). It could downregulate the expression of C-terminus fragments and decrease both intracellular and extracellular levels of A??, which are hallmarks of AD. It also showed dose-dependent neuroprotective and antioxidant effects against H2O2-induced cell death. Pharmacokinetics and biodistribution studies showed that this CRT-??-cyclodextrin inclusion complex was suitable for intravenous administration. The formulation significantly increased the bioavailability of CRT and facilitated CRT crossing the blood-brain barrier to enter the brain. Similar to AD, CRC is increasingly prevalent with aging populations. Approximately 60% of CRC patients are aged 70 and above. Halofuginone (HF) is an active pharmaceutical ingredient (API) originated from Chinese quinine (Dichroa febrifuga Lour.) and artesunate (ART) is a semi-synthetic derivative of artemisinin (ATS) extracted from annual wormwood (Artemisia annua L.). Both APIs show anticancer activities by inhibiting the growth of CRC. However, low aqueous stability limits their applications. Liposome formulation with surface functionalization by CPP2 cell-penetrating peptide was developed to deliver HF and ART for targeted CRC therapy. CPP2 is a peptide that can selectively penetrate colon cancer cells. The liposomal drug formulations had uniform particle size (about 100 nm), high encapsulation efficiency (over 80%) and good stability upon 14 days of storage. In cellular uptake study, CPP2-modified liposome showed stronger permeability and selectivity to colon cancer lines without inducing lysosomal degradation. CPP2 surface-modified liposomal drugs demonstrated greater anticancer activities than free form of drugs or conventional liposomal drugs. Combinations of HF and ART formulations notably decreased cancer cell viability as compared to single formulation alone, which indicated that HF and ART formulations exhibited synergistic anticancer effects at specific ratios. To conclude, the drug delivery systems, cyclodextrins and peptide-modified liposomes, which were developed for AD and CRC treatment, successfully improved the aqueous solubility of insoluble APIs extracted from Chinese medicinal plants.

Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Cancer remains one of the leading causes of morbidity and mortality worldwide with an estimated 9.9 million deaths in 2020. Cancer treatment regimens such as chemotherapy and radiotherapy have over the years fallen short due to drug resistance, toxicity, damage to normal healthy cells and tissues surrounding the treatment area. Moreover, they have shown very limited survival benefits for most advanced staged cancers such as colorectal cancer, which in 2020 was responsible for 3 728 deaths with a 6.8% incidence rate. Despite the many efforts in developing alternative chemotherapeutic strategies, cancer of the colon and cancer, in general, remains a burden. For centuries, plants and plant derivatives have been exploited for their nutritional and medicinal properties and now serve as chemical scaffolds or templates for designing and synthesising products with pharmacological importance. Herbal medicines are claimed to enhance therapeutic effects and are often used in combination with chronic medication. However, the concurrent use of herbal medicines and synthetic drugs may affect the pharmacokinetic profile of therapeutic drugs or trigger unexpected and undesirable effects. This study aimed to characterise the leaf extracts (crude water and crude methanol) of Momordica balsamina and investigate their potential anticancer activity on HT-29 colon cancer cells. The study also aimed to asses the effect of the extracts on drug metabolising enzymes (CYP450), specifically those which metabolise 5-Fluorouracil (5-FU) prodrugs or are inhibited by 5-FU since it is one of the first-line treatments for colon cancer. Dried powdered leaves were extracted using water and absolute methanol to obtain crude water and crude methanol extracts, respectively. For characterisation, the extracts were spotted on thin-layer chromatography (TLC) plates and further screened using chemical tests. The ferric ion reducing power assay and Liquid chromatographymass spectrometry were used to determine the antioxidant activity of the extracts and to identify prominent or abundant compounds in each extract, respectively. To assess the cytotoxic effect of the extracts and 5-FU, HT-29 colon cancer cells and C2C12 muscle cells, which were used as a model for normal cells, were exposed to concentrations that ranged from 0 to 2000 µg/ml for the water (H2O) extract, 0 to 300 µg/ml for the methanol (MeOH) extract or 0 to 100 µg/ml of 5-FU for 24 and 72 hours, and subjected to the MTT assay. The effect of the extracts on the efficacy of 5-FU was xxi assessed using the MTT assay by combined treatments of the extract and 5-FU. Genotoxicity of the extracts was assessed on the C2C12 cells using the Muse™ MultiColour DNA Damage kit. The generation of intracellular reactive oxygen species (ROS) was assessed by flow cytometry using the DCFH-DA assay. The JC-1 and acridine orange (AO)/propidium iodide (PI) staining assays were used to assess the effect of the extracts on the mitochondrial potential as well as cell and nuclear morphology, respectively. Apoptosis was quantified by flow cytometry using annexin V/PI and caspase activation assessed using the Caspase-8 and Caspase-9 colourimetric assay kits. The pro-apoptotic mechanism(s) was determined by assessing the expression profiles of selected apoptosis regulatory proteins using the human apoptosis antibody array kit. Cell cycle analysis by flow cytometry was conducted to determine the effect of the extract on the cell division cycle. Moreover, to determine the potential of herb-drug interactions, the Vivid® CYP450 Screening kits and P-gp-GloTM Assay Systems with P-glycoprotein were used to assess the effect on the activity of drug metabolising enzymes and drug transportation, respectively. The results showed that the MeOH extract possessed fewer polar compounds, higher ferric iron-reducing power, and a relative abundance of flavonol glycosides, cucurbitane-type triterpenoid aglycones, and cucurbitane-type glycosides than the H2O extract. The MeOH extract was further selectively cytotoxic to the HT-29 colon cancer cells at 24 hours of treatment and selectively induced genotoxicity in HT-29 cells. The H2O extract, however, was not cytotoxic to the HT-29 cells at all the tested concentrations at 24 and 72 hours of treatment. Analysis of nuclear and cell morphology suggested that the decrease in the percentage viability of MeOH extracttreated cells was associated with apoptotic cell death. Apoptosis was further confirmed by the loss of mitochondrial potential, increase in ROS production, caspase-8 and -9 activities as well as Annexin-V/PI-stained cells. Cell cycle analysis revealed cell cycle arrest at the S phase in MeOH extract-treated cells. Analysis of protein expression profiles revealed that the extract modulated various proteins that play a role in the promotion or inhibition of apoptosis. Moreover, the MeOH extract was shown to inhibit the activity of CYPs 1A2, 2A6, 2C8, and 2C9, while the H2O extract showed no significant inhibitory effects on the activity of all tested CYPs and 5-FU only significantly inhibited the activity of CYP2C9. However, combinatory treatments with 5-FU and the MeOH extract were shown to have no additive or diminishing effects on the efficacy of 5-FU on the activity of all the tested CYP enzymes. Treatment with 5FU (0.008 – 32 μg/ml) and the H2O extract (0.02 – 200 μg/ml) was shown to stimulate the ATPase activity of P-gp, while the MeOH extract significantly inhibited its activity with concentrations of 0.2, 2, and 20 μg/ml. In conclusion, the MeOH extract selectively induced cancer cell toxicity, genotoxicity as well as S phase cell cycle arrest and apoptosis via the intrinsic and extrinsic pathways. The anticancer activity of the MeOH leaf extract of M. balsamina as well as its antioxidant potential may be attributed to the presence and relative abundance of flavonol glycosides, cucurbitane-type triterpenoid aglycones, and cucurbitane-type glycosides. Although the MeOH extract may potentially reverse the effects of P-gp multidrug resistance by decreasing its activity, its inhibition of the activity of CYPs 1A2, 2A6, 2C8 and, 2C9, which are involved in the metabolism of more than 80% of the drugs in clinical use may suggest that co-administration of the MeOH extract may still result in increased plasma levels of drugs, thereby resulting in toxicity. The H2O extract, although not pro-apoptotic as the MeOH extract may still have the potential to be developed as a nutraceutical as it was shown to exhibit no adverse drug interactions and because this species is known to possess a wide variety of nutritional and medicinal values. / South African Medical Research Council (SAMRC) Research Capacity Development Initiative.

Thesis (M. Sc. (Biochemistry)) -- University of Limpopo, 2021 / Plant-derived products have been used for years in the treatment of various ailments with low or no side effects. Thus, screening of medicinal plants for potential anticancer activity, in vitro, could help identify plant extracts or compounds that can be developed for use as anticancer agents with less or no side effects. The aim of this study was to investigate the probable anticancer effects and induced mechanism of action of Momordica balsamina crude leaf acetone extract in lung A549 cancer cells. The effect of the extract on cell viability, proliferation and cell division cycle were determined using Muse count & viability, Ki67 proliferation and cell cycle assay kits, respectively. The presence of biochemical and morphological features associated with apoptosis were analysed by Muse annexin-V & dead cell assay kit and Acridine orange/Ethidium bromide dual staining. The effect of the extract on the mRNA expression levels of cell cycle regulatory genes was determined using RT PCR. Proteome profiler antibody array was used to determine the effect of the extract on the protein expression levels of apoptosis regulatory genes. The findings revealed that the crude leaf acetone extract of M. balsamina decreased the percentage viability of lung A549 cells with less effect on the percentage viability of normal cells (KMST-6). Furthermore, a significant anti-proliferative effect in extract treated A549 cells was observed. Characteristic nuclear and morphological features of apoptosis such as chromatin and nuclear condensation, externalisation of phosphatidylserine and loss of cell membrane function were observed in A549 cells treated with the extract. Although there was no relative upregulation of Bax and Bad protein expression, a downregulation of the Bcl-xl and Bcl-2 protein expression was observed in extract-treated cells. This led to the release of Cytochrome c and HTRA2/Omi leading to pro-caspase-3 cleavage. Furthermore, presence of HTRA2/Omi in the cytosol inhibited the functions of IAPs such as XIAP and cIAP1/2. Phosphorylation of p53 at different serine residues led to upregulated protein expression levels of p27/Kip1 protein which resulted in the cell division cycle arrest at G0/G1-phase. Reverse transcriptase polymerase chain reaction results showed that the extract modulated mRNA expression levels of p53, p21, cyclin B and cdc2 genes. In summary, M. balsamina extract induced cell division cycle arrest and apoptosis in A549 cells through intrinsic apoptosis pathway via p53-mediated mechanism. / South African Medical Research Council (SAMRC)