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    Modulation of enteric neural activity and its influence on brain function and behaviour : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatu, New Zealand
    (Massey University, 2024) Parkar, Nabil Jamil
    The gut-brain axis (GBA) facilitates bidirectional communication between the enteric nervous system (ENS) of the gastrointestinal (GI) tract and the central nervous system (CNS). The location of the ENS along the GI tract enables it to serve as a relay station along the GBA. A key regulator of the GBA is the diverse population of microbial communities inhabiting the GI tract, known as the gut microbiota. Due to its proximity to the ENS, gut microbes significantly influence ENS functions, such as gut motility, and also impact brain function and behavior. A diverse and healthy gut microbiota is crucial for normal GI physiology and mental health. Understanding the physiological host factors that influence and control the gut microbiota is essential for grasping its variability in health and states of dysbiosis. Movement of luminal content along the GI tract, primarily driven by rhythmic contractions of GI smooth muscles, affects gut microbiota growth and population dynamics. This research involved a series of ex vivo and behavioral experiments in rodents to better understand ENS control of gut motility and its impact on anxiety-related behavior. Initially, the effect of a specific pharmacological agent on colonic motility patterns was evaluated using ex vivo techniques. Observations from this study provided fundamental insights into ENS function and its regulation of colonic motility, laying the foundation for further research on how altered colonic motility via ENS manipulation affects gut microbiota composition and anxiety-related behavior. The second study investigated whether pharmacological modulation of the ENS, resulting in reduced colonic motility, affected the gut microbiota. Results revealed significant changes in gut microbiota composition, including decreased abundance of certain bacterial species and alterations in community structure. The final study aimed to understand the relationship between ENS manipulation, brain function, and behavior by inducing changes in gut motility. Anxiety-related behavior was assessed in rats using open field and elevated plus maze tests, focusing on those exposed to a pharmacological agent that slowed colonic motility via specific ENS receptors. To determine if behavior changes involved specific neural pathways, brain gene expression in key regions was studied. Additionally, the potential relationship between gut microbiota and brain function was explored, assessing if ENS modulation and behavioral effects correlated with changes in gene expression and microbiota profiles in the large intestine. Findings indicated that ENS modulation altered anxiety-related behavior in a sex-specific manner, with female rats showing increased anxiety and corresponding changes in brain and proximal colon gene expression compared to males. This study highlighted sexually dimorphic gut-brain communication and suggested multiple genes/pathways may influence anxiety-related behavior in females. This comprehensive exploration through three interrelated studies has provided new insights into the regional specificity of ENS receptors in regulating colonic motility, the impact of slowed gut transit on microbiota composition, and the physiological consequences of ENS modulation on brain function and anxiety, with associated sex differences. A combined analysis of these findings discusses their implications for understanding the ENS as a key player in regulating the gut-brain axis.
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    Host-microbiota interactions underlying functional gastrointestinal disorders and the impact of gold kiwifruit consumption : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Albany, Manawatū, Palmerston North, New Zealand
    (Massey University, 2023-05-18) Carco, Caterina
    The microbial ecosystem exists in a mutualistic relationship with its host, contributing to a healthy gastrointestinal tract. Growing evidence supports the role of microbial-immune interactions in functional gastrointestinal disorders (FGIDs), including irritable bowel syndrome (IBS). However, these mechanisms are poorly understood. It has been hypothesised that taxonomic and gene abundance in the faecal microbiota and gene expression of peripheral blood mononuclear cells (PBMCs) could discriminate FGID subtypes (functional constipation (FC), IBS constipation, functional diarrhoea (FD), IBS diarrhoea) from each other and healthy subjects (controls). A second hypothesis was that consuming two gold kiwifruit daily for four weeks had a different effect on the microbial composition and gene abundance than psyllium in constipation predominant FGID subjects or controls. A systems biology approach was used to address these hypotheses. Different microbial compositional and gene abundance profiles were associated with constipation and/or diarrhoea, particularly facultative anaerobes and obligate fermenters, and genes related to tyrosine metabolism, secretion systems and micronutrient utilisation. Differentially PBMC expressed immunoglobulin variable domain genes were shared among FGIDs, except for IBS constipation. Increased expression levels of interferon-induced genes and those linked to the complement system and platelet functions characterised the immune signature of functional constipation. Increased expression levels of immunoglobulin variable domain associated with immunoglobulin E/G receptor-mediated pathways characterised the immune signature of IBS diarrhoea and FD. Further analyses showed that computationally selected microbial, immune gene and symptomatic variables were associated with constipation or diarrhoea predominant FGIDs, and that symptoms remain the best way to discriminate among FGIDs or controls than PBMC genes or microbial taxa except for FC, which was best discriminated from other FGIDs or controls by selected PBMC genes. Eggerthella and Bacteroides were the only genera that differed between subjects consuming gold kiwifruit or psyllium or between each intervention compared to pre-intervention levels, regardless of the digestive health status of the subjects. This PhD thesis presents novel insights into the host-microbiota interactions underlying FGIDs and the microbiota responses to daily consumption of two gold kiwifruit over four weeks in constipation predominant FGID subjects. The knowledge generated can be used for future research on food-based treatments supporting gastrointestinal health and comfort.
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    Drivers of obesity : associations of physical activity, sedentary behaviour and diet on metabolic health and the gut microbiota : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Tāmaki Makaurau, Aotearoa New Zealand
    (Massey University, 2023) Slater, Joanne
    Background: Regular physical activity (PA) and limited time spent sedentary are important for almost all aspects of health, including prevention and treatment of obesity. Aim: To describe the PA and sedentary behaviour (SB) of healthy, lean and obese, Pacific and NZE women, aged 18-45 years; and to explore the associations of PA and SB with diet, BF%, biomarkers of metabolic health, and gut microbiota composition. Methods: Pacific (n = 142) or NZE (n = 162) women aged 18–45 years with a self- reported body mass index of either 18.5–25.0 kg/m2 or ≥30.0 kg/m2 were recruited. Whole body dual-energy X-ray absorptiometry was used to subsequently stratify participants as either low (<35%) or high (≥35%) BF%. Eight-day accelerometery assessed PA and SB levels. Meeting the PA guidelines was defined as accumulation of ≥ 30 minutes of moderate or greater intensity activity on ≥ 5 days per week OR 150 minutes of moderate to vigorous PA (MVPA) per week. Dietary intake was assessed using a 5-day food record. Fasting blood was analysed for biomarkers of metabolic health, and whole body dual-energy X-ray absorptiometry was used to estimate body composition. Bulk DNA was extracted from faecal samples and the metagenomic sequences associated with the microbiota were analysed using MetaPhlAN and QIIME2 software. Adjusted multivariate regression models were conducted to explore the associations between PA, SB and diet, body composition and biomarkers of metabolic health, and between PA, SB and gut microbiota composition. Results: Less than half Pacific women were meeting the PA guidelines (high-BF%; 39% and low-BF%; 47%) versus 81% of low-BF% and 65% of high-BF% NZE women. Low-BF% Pacific women were more sedentary than all other women (p<0.05): Pacific low- 10.4 and high-BF% 9.93 and NZE low- 9.69 and high-BF% 9.96 hours/day. Every additional 10-minutes spent in MVPA was associated with 0.9% lower total and trunk fat and 0.7% lower gynoid fat in all women (p<0.05). Among Pacific women; every 100 cpm increase in total PA was associated with 6% lower fasting plasma insulin. Every 10-minute increase in MVPA was associated with 8% lower fasting plasma insulin in both ethnic groups (p<0.05). Among NZE women, every one-hour increase in sedentary time was associated with 0.8% higher gynoid fat (p<0.05), and longer weighted median sedentary bout length was associated with higher BF% (gynoid fat 0.3%, total body 0.4%, trunk 0.4%, android 0.4% and visceral fat 0.4% (p<0.05)) and 14% higher C-reactive protein (CRP) (p<0.05). No associations between SB and body composition or metabolic markers were found among Pacific women. There was no significant difference in average total energy intake between Pacific and NZE women or BF% groups. No women were consuming more than the carbohydrate AMDR (>65% total energy). Pacific women’s mean daily starch intake was significantly higher than NZE women (g/day, and % total energy intake). Only the NZE low-BF% groups mean fibre intake was above the recommended daily intake of ≥25g/day. All the women that were in the lowest quartile of fibre intake, and particularly the NZE women, had a lower odds of meeting the PA guidelines (OR 0.72 (p=0.008) and OR 0.66 (p=0.021) respectively) compared to women in the top three quartiles of fibre intake. All the women that were in the lowest quartile of polyunsaturated fat intake, especially Pacific women had a lower odds of meeting the PA guidelines compared women in the top three quartiles (0.76, p=0.027 and OR 0.67, p=0.030 respectively). Among NZE women, every one SD increase in total PA (197 cpm/day) was associated with 36.3% higher relative abundance of Erysipelotrichaceae (p=0.031) and 37.9% lower relative abundance of Verrucomicrobiaceae (p=0.029). Every one SD increase in SB (1.45 hours/day) was associated with a 28% lower relative abundance of Erysipelotrichaceae (p=0.030). Every one SD increase in NZE women’s total PA was associated with 23.1% higher Firmicutes:Bacteroidetes ratio (p=0.031), whereas among Pacific women, every 1 SD increase in MVPA was associated with 22.8% lower (p=0.034) Firmicutes:Bacteroidetes ratio. Conclusion: Increased time spent in PA of all intensities and breaking-up prolonged SB was associated with healthier body composition and lower metabolic disease risk in Pacific and NZE women. Compared to NZE, the impact of increased total PA on fasting insulin may be greater in Pacific women and inflammation may be a pathway through which SB impacts cardiovascular risk, especially for NZE women. Although higher total PA and lower SB was associated with some aspects of the gut microbiota composition, more needs to be known about the mechanisms driving associations between PA SB and the gut microbiota to enable these findings to be interpreted.
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    The links between human breath methane, dietary fibre digestion, and the gut microbiota : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutrition Science at Massey University, Manawatu, New Zealand
    (Massey University, 2022) Payling, Laura Marie
    The concentration of methane that is exhaled in human breath has been associated with the composition and fibre-fermenting function of the human colonic microbiota. The current research aimed to investigate whether composition and fibre fermentation function of the colonic microbiota differ in individuals who are low breath methane emitters (LE) or high breath methane emitters (HE). Healthy adult individuals (18) were recruited and breath tested. Unexpectedly, they showed positive correlations between breath hydrogen and methane. Then, the highest and lowest breath methane emitters provided faecal samples used for shotgun metagenomic sequencing and as faecal inocula for in vitro colonic fermentations with dietary fibres (β-glucan and lignocellulose). Individuals who were LE reported higher dietary vitamin E, fibre, and fat intakes and a Bacteroides-driven microbiota composition compared to HE individuals who reported a greater starch intake and a Prevotella-driven microbiota composition. The faecal microbiota from individuals who were HE had a greater abundance of taxa from the Methanobrevibacter genus and more methane gas production during in vitro colonic fibre fermentation compared to the microbiota of individuals who were LE; however, the results were variable within the HE group. There was a greater rate and extent of dietary fibre fermentation in LE compared to HE individuals during in vitro colonic fermentation, which aligned with the greater fibre intakes of LE individuals. Furthermore, the faecal microbiota of LE individuals showed increased beneficial organic acid production and a greater abundance of functional pathways related to amino acid metabolism compared to HE during in vitro colonic fermentation. These results did not align with published research on human breath methane and the gut microbiota. However, there was consensus with emerging hypotheses suggesting that there are important pathways involved in hydrogen sulphide production and hydrogen utilisation that are largely unexplored. Further investigations in these areas could help redefine our understanding of fibre fermentation by the human colonic microbiota.
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    An investigation into the interaction of the microbiome-gut-brain axis with stress : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Bear, Tracey
    This thesis aimed to investigate whether changes in the gut microbiota and associated biomarkers were associated with stress-induced anxiety-like and depressive-like behaviour. Two studies used the unpredictable chronic mild stress (UCMS) over 4 or 6 weeks (vs no UCMS, control) in Sprague-Dawley rats. Depressive-like behaviour was measured in female rats using the sucrose preference test, and the Porsolt swim test. Anxiety-like behaviour was measured with the light-dark box test. Faecal corticosterone, caecal microbiota (composition and organic acids), serum gut permeability (lipopolysaccharide-binding protein, LBP) and plasma inflammation (12 cytokines) markers were measured. Atypical behaviours were observed in female rats following UCMS and no depressive-like behaviours were observed. The circulating concentration of cytokines, but not plasma LBP or caecal organic acids, was higher in UCMS-exposed female rats. Relative abundance of taxa from the Clostridiales order and Desulfovibrionaceae family correlated with anxiety-like behaviours and plasma cytokine concentrations, regardless of UCMS. Studies of these atypical behaviours in female rats confirmed expected patterns of sucrose intake in the sucrose preference test and no decreased depressive-like behaviours in the Porsolt swim test with antidepressant citalopram and imipramine drugs. A further study also showed differences in baseline behaviour in male versus female rats, leading the second UCMS study to be in male rats. Increased faecal corticosterone and anxiety-like behaviours were observed in male UCMS-exposed and control rats at week 4 of UCMS compared to baseline. Plasma cytokine concentrations were higher in the UCMS group but higher faecal corticosterone concentrations and anxiety behaviours in control rats suggest that they were more stressed than treated rats. Caecal neurotransmitter concentrations did not differ between treatments nor correlate with serum neurotransmitter, cytokines or LBP concentrations or behaviour. The findings showed an association between the gut microbiota and anxiety-like behaviours, which was not stress dependent. No measured biomarkers explained the observed anxiety-like behaviours. Caecal digesta neurotransmitter profiles were dissimilar to serum profiles indicating it may not be an important influence on serum levels. Despite the atypical behavioural results following the interventions, the results still provided useful and unique information which contributes to the body of Microbiome Gut Brain Axis research.
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    The gut‐bone axis in coeliac disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Schraders, Katie Elizabeth Coad Pedley
    Coeliac Disease (CD) is a lifelong autoimmune disease, highly prevalent in New Zealand, triggered by ingestion of gluten in genetically susceptible individuals. It leads to villous atrophy and nutrient malabsorption which often compromises bone mineral density (BMD). Although a gluten‐free diet (GFD) usually improves symptoms, BMD is often not fully resolved. Persistent low BMD may be due to ongoing gut inflammation and malabsorption, or the uncoupling of bone formation and resorption. Previous research indicates that individuals with CD and low BMD may have altered bone signalling pathways, particularly in the expression of receptor activator of NF‐κβ ligand (RANKL) and osteoprotegerin. RANKL is implicated in both the initiation of the immune response and persistent low BMD because it has a role in bone resorption, via the differentiation of osteoclasts, and a possible role in translocating gluten across the gut, via the expression of microfold cells. The objective of this PhD was to examine underlying mechanisms underpinning low BMD in individuals with CD using a small intestinal organoid model which allows for investigation of otherwise inaccessible gut cells and signalling pathways implicated in the gut‐bone axis. A further study, Close to the Bone, investigated BMD in premenopausal women with CD compared to healthy controls. A third online A Gut Feeling study investigated dietary advice that individuals consuming a GFD in New Zealand receive, focusing on bone health. Although the organoid research was interrupted due to the SARS‐CoV‐2 pandemic, the model was established using murine tissue and future research opportunities were identified. The results of the Close to the Bone study demonstrated no differences in BMD between the coeliac group and healthy controls but identified further research into bone density in people with CD in New Zealand was warranted. This study raised concerns about iodine intake and status in people with CD. The A Gut Feeling study found inconsistencies in advice given to individuals diagnosed with CD. The research identified that the organoid model offers potential for future study of the gut‐bone axis and that individuals with CD are at risk of nutritional deficiencies but often are not advised well or referred for a bonescan.
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    Robotic capsule for sampling gut microbiota : design, development and evaluation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Rehan, Muhammad
    In this research, a pill-sized robotic capsule was developed that can collect gut microbiota both from the gut lumen (capsule surroundings) and intestinal wall (mucosa layer). Initially, the peristaltic forces exerted on the robotic capsule inside the gut were quantified so the working environment of the capsule could be understood. Secondly, a unique sampling mechanism was developed that could gently scrape the content from the gut lining and could provide a full length assessment of microbiota after capsule retrieval. Thirdly, the design of shape memory alloy (SMA) spring actuator was realised that could apply sufficient force to overcome peristaltic and frictional forces for sample collection at the target-site. Furthermore, an actuation system was devised by tackling the high-drain current requirement of SMAs. Fourthly, a sealing mechanism was developed to secure the collected sample from cross contamination and to assure successful encapsulation. Fifthly, the robotic capsule was rigorously tested in various in vitro simulators replicating the gut environment and a dedicated gut simulator that mimicked the in-vivo environment to ensure successful and safe travel of the capsule along the gastrointestinal tract. Finally, an in vitro experimental setup that kept an intestine alive for 6 hours was used to optimise the sample collection process. The robotic capsule collected sufficient quantities of sample (more than 100 µL) for microbiota analysis from living intestines of three animal species (pig, sheep and cow) during the trials. The study of gut microbiota is gaining increasing attention due to its direct impact on human health. Gut microbiota can provide comprehensive information about the health of a host, and it can help in the early diagnosis of diseases like cancer, diabetes, obesity, etc. The robotic capsule prototype, developed in this work, has a potential to become a vital apparatus for clinicians and scientists to sample human and animal gut in the future.
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    Complementary feeding to nourish the infant gut microbiome : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) McKeen, Starin
    The transition from milk-feeding to consuming a range of solid foods during the first 1000 days of life constitutes the most dramatic dietary shift in the human lifespan. This period coincides with the development of host-microbial processes. This PhD dissertation aimed to identify relationships between complementary feeding and characteristics of the developing microbiome of the infant using faecal samples as a proxy of the large intestine. The composition and gene abundances of the microbiome and the metabolites of dietary, host, and/or microbial origins were detected and analysed. The Nourish to Flourish pilot clinical study was conducted to inform subsequent study designs and develop sampling and processing protocols for research in weaning infants. The faecal samples were analysed to characterise the microbiome and metabolome at 4, 9, and 12 months of age. These data revealed greater shifts during the introduction of solid foods between 4 and 9 months of age than during the continued diversification of solid foods in the diet between 9 and 12 months of age. The changes occurring in the microbiome and metabolome in faecal samples were then interpreted in the context of milk-feeding behaviours and associated dietary diversification. The associations between the faecal microbiome and metabolome and dietary nutrient composition estimates were identified and evaluated. The patterns of association that emerged were varied and showed low prediction values but supported published evidence about relationships between specific nutrients, microbial taxa, and predicted functional pathways based on microbial gene abundance. Correlation networks between dietary food groups and relative abundance of microbes, KEGG pathway abundance, and metabolites revealed patterns that implicate the food matrix by protein-rich foods compared to the relatively weak effect of starchy foods on the luminal environment. This research demonstrates the window of opportunity for the greatest influence of solid foods on the infant gastrointestinal environment is prior to 9 months of age. However, the impact of continued milk feeding behaviours is likely to be relatively greater than solid foods later in the complementary feeding window. This research provides a basis for selecting food products that can benefit the infant's development by modulating the microbiome and metabolome in the gastrointestinal tract.
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    Understanding host and microbial metabolites in functional gut disorders : a thesis presented in particle fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) James, Crystal Shanalee
    Interactions between diet, host, and the gut microbiome can result in beneficial or detrimental effects on human health. Functional gut disorders (FGDs) are an example of the negative effects of these interactions. However, an understanding of the mechanisms behind FGDs remains unknown. Metabolomics is a powerful tool to understand possible mechanisms. It was hypothesised that key metabolite groups in faecal samples would differ between or within FGD subtypes and healthy controls reflective of mechanistic perturbations. This PhD aimed to characterise the metabolite profile of FGD individuals (functional constipation (FC), IBS-constipation (IBS-C), functional diarrhoea (FD), IBS-diarrhoea (IBS-D), IBS-mixed (IBS-M)) and healthy controls. The concentration of faecal bile acids and plasma amino acids were quantified to ascertain changes in known metabolites associated with FGDs. The faecal metabolome was characterised to potentially identify wider perturbations, and this was then integrated with dietary intake, plasma metabolome abundance and faecal microbiota composition for a systems biology analysis. Constipation (FC + IBS-C) and diarrhoea (FD + IBS-D) were combined to determine differences between healthy controls and disease states. Faecal bile acid concentrations differed between all FGD participants and healthy controls. In the combined analysis of the diarrhoea (FD+IBS-D) and constipation (FC+IBS-C) groups, the diarrhoea group had a higher concentration of bile acids than the constipation group or healthy control group. The concentration of plasma amino acids did not differ between FGD participants and healthy controls. Furthermore, analysis of key amino acid groups showed that only the concentration of branched chain amino acids were different between all subtypes and healthy controls. Characterisation of faecal polar, semi-polar and lipid metabolites showed a differential relative abundance of some polar and semi-polar metabolites (e.g., riboflavin, nicotinic acid) between diarrhoea and healthy control groups, and constipation and healthy control groups. Substantial changes in the abundance of some lipids (e.g., ceramides, triglycerides) were evident between constipation and healthy control groups, and diarrhoea and healthy control groups. Integration of the faecal metabolome with other datasets (faecal microbiome, plasma metabolome, dietary intake) showed the faecal metabolome and microbiome separated healthy controls from constipation or diarrhoea. Additionally, differential positive and negative correlations were observed between faecal lipids (triglycerides and diglycerides) and microbial species (Firmicutes and Bacteroidetes). This PhD thesis presents novel insights into the metabolite signature characterising FGD participants and healthy controls and provides directions for future research.
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    Discovery of novel plant based compounds to address the drug resistance problem in nematode infested ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Gupta, Arka
    In this thesis, the discovery of novel compounds from plant extracts towards addressing the drug resistance issue in nematode infested ruminants is presented. The nematocidal efficacy of the plant extracts was tested against the L3 stage nematode larvae through bioassay-guided fractionation and chromatographic separation. Quebracho crude powder (QCP) is the only commercially available source of polyphenols. The efficacy of a separated fraction of the QCP was found to be higher (P<0.05) than the crude extract against batches of L3 larvae. However, the research with QCP encountered many challenges and attention was shifted towards medicinal plants found in New Zealand. From a screening study, the Māori plant Piper excelsum (Kawakawa) was found to be the most effective. Of its different components, the leaf component was found to have the highest nematocidal efficacy. Kawakawa (KK) leaf samples were collected over three seasons and the nematocidal efficacy of the leaf samples was found to be independent. From the bioassay-guided fractionation study, the Water and MeOH solvent fractions were found to be most effective, and they were subjected to further reverse-phase chromatographic separation. It was found that the separated fractions had better anthelmintic efficacy than the parent crude solvent fractions (P<0.05). An improvised separation technique named ‘Hand Controlled Countercurrent Separation’ (HCCCS) based on the principle of CCS was developed. It was found that the fractions obtained from the HCCCS study had better anthelmintic efficacy than the parent MeOH-Fraction-4 and the anthelmintic ivermectin (IVM) (P<0.05). A series of combination formulations were made with the separated fractions and IVM. It was observed that these formulations had better efficacy than IVM and the individual HCCCS Fractions (P<0.05). The HCCCS Fractions were subjected to LC-MS/MS investigation and 34 compounds were identified. Of which, 8 were reported in the literature from published KK research. However, the anthelmintic properties of these compounds were not previously reported. The cytotoxicity evaluation of a series of fractions did not reveal any toxic effect to mammalian epithelial cells. Therefore, there is further potential towards isolating leaf fractions into pure isolates which may possess higher nematocidal efficacy compared to the fractions presented in this thesis.