Characterising the Protective Bioactivity of Macroalgal Polyphenols: A study on neuroprotection, epithelial barrier function, ACE-2 expression and activity

Phlorotannins are novel polyphenolic compounds mostly found in brown seaweed and are comprised of variable polymeric chains of phloroglucinol residues (1,3,5-trihydroxybenzene). Biochemical and cell-based assays and some preclinical and clinical studies have highlighted a potential multi-faceted bioactivity of phlorotannins. This thesis aimed at characterizing neuroprotective and epithelial protective bioactivity via cell and enzyme-based assays along with transmission electron microscopy (TEM) and molecular docking of various phlorotannins isolated and identified from the South Australian seaweed Ecklonia radiata using a range of analytical techniques. Firstly, Ecklonia radiata from South Australian coastal regions was collected and its ethanolic extract and solvent-soluble fractions evaluated for neuroprotective activity against the toxic amyloid β protein (Aβ1-42) in neuronal PC-12 cells. The ethyl acetate fraction (EA) comprising 62% phlorotannins demonstrated the most efficacious neuroprotective activity, inhibiting neurotoxicity at all Aβ1-42 concentrations and was associated with Aβ anti-aggregatory properties. Various phlorotannins were tentatively identified via ion mobility mass spectrometry and HPLC quadrupole-time-of flight mass spectroscopy. Two compounds were isolated, purified and identified via high-performance centrifugal partition chromatography (HPCPC) combined with size exclusion chromatography. Nuclear magnetic resonance (NMR) and mass spectroscopy enabled us to identify the major phlorotannins as eckol and dibenzodioxin-fucodiphloroethol (DFD). DFD significantly reduced cell viability in response to Aβ1-42 and significantly reduced Aβ1–42 aggregation, while molecular docking studies revealed DFD binding to key Aβ1-42 residues associated with fibrillisation propensity. DFD also showed moderate cholinesterase inhibitory activity and was also able to significantly scavenge reactive oxygen species (ROS) in PC-12 cells. In addition, eckol, dieckol, phlorofucofuroeckol-A (PFFA) and 974-A previously identified in the E. radiata extract significantly scavenged ROS. However, only PFFA and 974-A protected neuronal PC-12 cells against oxidative stress-evoked neurotoxicity, increasing cell viability significantly in response to both cytosolic (H2O2) and lipid peroxidation-evoked (t-BHP) cell stress and Aβ exposure. Next, the effect of eckol, dieckol, PFFA and 974-A on cytokine-evoked trans-epithelial electrical resistance (TEER) reductions in Caco-2 intestinal epithelial monolayers as a measure of paracellular permeability and epithelial barrier integrity was investigated. Significant improvements in epithelial barrier function occurred at 48 h with eckol, dieckol, and PFFA but not 974-A, indicating a broader capacity of the phlorotannins to support intestinal epithelial barrier function against detrimental proinflammatory cytokines. The effects of these phlorotannins on modulating angiotensin converting enzyme 2 (ACE-2) expression and activity in human epithelial cells (A549 and Caco-2) were also investigated for potential COVID-19 prophylaxis and treatment. Eckol and dieckol had negligible influence on either basal or cytokine-induced ACE-2 expression in Caco-2 cell line, however both significantly reduced cytokine-induced expression of ACE-2 isoform A in A549 cells. PFFA and 974-A significantly reduced ACE-2 expression and downregulated both major ACE-2 isoforms under basal and cytokine-induced conditions, except for isoform B in PFFA-treated A549 cells. Altered expression profiles were accompanied by substantial inhibition of cytokine-augmented ACE-2 enzymatic activity in PFFA and 974-A- treated cells. This thesis demonstrated the widespread biological activity of phlorotannins applied towards neurodegenerative, intestinal and potentially infectious diseases. These findings may inform their further development as nutraceuticals or pharmaceuticals for human health.Thesis (Ph.D.) -- University of Adelaide, School of Biomedicine, 202

Etiology of Acute Diarrheal Disease and Antimicrobial Susceptibility Pattern in Children Younger Than 5 Years Old in Nepal

Diarrhea is a common cause of morbidity and mortality among children younger than 5 years in developing countries. Children from 3 to 60 months of age were recruited from two hospitals in Nepal— Bharatpur Hospital, Bharatpur, and Kanti Children’s Hospital, Kathmandu—in 2006 to 2009. Stool specimens collected from 1,200 children with acute diarrhea (cases) and 1,200 children without diarrhea (control subjects) were examined for a broad range of enteropathogens by standard microbiology, including microscopy, enzyme immunoassay for viral pathogens (adenovirus, astrovirus, and rotavirus) and protozoa (Giardia, Cryptosporidium, and Entamoeba histolytica), as well as by using reverse transcription real-time polymerase for norovirus. Antimicrobial susceptibility testing was performed using the disk diffusion method. Overall, rotavirus (22% versus 2%), norovirus (13% versus 7%), adenovirus (3% versus 0%), Shigella (6% versus 1%), enterotoxigenic Escherichia coli (8% versus 4%), Vibrio (7% versus 0%), and Aeromonas (9% versus 3%) were identified significantly more frequently in cases than control subjects. Campylobacter, Plesiomonas, Salmonella, and diarrheagenic E. coli (enteropathogenic, enteroinvasive, enteroaggregative) were identified in similar proportions in diarrheal and non-diarrheal stools. Campylobacter was resistant to second-generation quinolone drugs (ciprofloxacin and norfloxacin), whereas Vibrio and Shigella were resistant to nalidixic acid and trimethoprim/sulfamethoxazole. This study documents the important role of rotavirus and norovirus in acute diarrhea in children younger than 5 years, followed by the bacteria Shigella, enterotoxigenic E. coli, Vibrio cholera, and Aeromonas. Data on the prevalence and epidemiology of enteropathogens identify potential pathogens for public health interventions, whereas pathogen antibiotic resistance pattern data may provide guidance on choice of therapy in clinical settings.publishedVersio

Seasonally Separated Logistic Models to Assess the Impact of Climate Variables on Occurrence of Rainfall over the Bagmati River Basin of Nepal

Aims: This paper aims to develop prediction models for forecasting rainfall occurrence over the Bagmati river basin of Nepal based upon climate related predictor variables. Study Design: Time series design with statistical downscaling of large scale daily climate data and observed rainfall data. Place and Duration of Study: Study was conducted at Central Department of Statistics, Tribhuvan University, Kirtipur, Nepal, between 2013 and 2015. Methodology: A day is considered as a wet day if area weighted daily rainfall (AWDR) is more than 1 mm. Extreme rainfall is determined by the 98thpercentile of AWDR. Binary logistic regression models are built with 13 possible principal components (PCs) of 7 climate related predictor variables using daily data for 1981-2000 period. Thereafter, built models are validated for 2001-2008 period. Results: Nine separate seasonal logistic models are fitted with Hosmer-Lemeshow tests having at least 0.207 p-values. The first PC of Air surface temperature has the greatest influence with odds ratio (OR) of 4.757 in predicting a wet day during post-monsoon across four models. It is followed by the first PC of Relative humidity with OR (4.112) in winter, first PC of Relative humidity with OR (3.443) in pre-monsoon and second PC of Relative humidity with OR (3.601) in monsoon. Similarly, second PC of Relative humidity has the highest contribution with OR (7.395) in predicting extreme rainfall in post-monsoon across all five models. It is followed by the first PC of Air surface temperature with OR (7.194) in monsoon, first PC of Relative humidity in winter with OR (6.820) and pre-monsoon with OR (5.076), and second PC of Relative humidity with OR (3.186) for the non-seasonal model. Conclusion: The developed logistic regression models are applicable in forecasting rainfall occurrence seasonally in the Bagmati river basin of Nepal

Nonparametric Distribution Fitting of Age at First Marriage of Nepalese Women through Scatter Plot Smoother

Age at first marriage is one of the important aspects for studying fertility behavior of women since it is believed that the number of children ever born is inversely related to age at first marriage. An attempt has been made to fit the Nepal Demographic and Health Survey (NDHS)-2006 data for age at first marriage of Nepalese women. The lack of fitting of some parametric distributions like lognormal and weibull was detected when applied to population census data of Nepal-2001 and the NDHS data-2006. Even though there is possibility of fitting more complex theoretical distributions, this highlighted the need to explore for alternative methods. In the process, it was identified that nonparametric smoothing is perhaps one of the viable solutions to this problem apart from fitting more sophisticated parametric distributions. Consequently, two nonparametric smoothers were examined for their suitability for distributional fitting, namely locally weighted regression smoother (LOESS) and cubic smoothing splines. Finally, cubic smoothing splines was chosen and fitted to NDHS data for Nepal and also separately for different ecological belts of Nepal since it fitted better than LOESS. Key words: cubic smoothing splines, locally weighted regression smoother, Nepal demographic and health survey, nonparametric smoothing, theoretical probability distribution

Bioassay-guided isolation of BACE1 inhibitors from Crataegus pinnatifida

Background: Crataegus pinnatifida, functional food has been used in traditional Chinese medicine due to its significant pharmacological effects on various metabolic disorders, neurodegenerative diseases, as well as anti-cancer and anti-viral properties. Despite numerous studies reporting its anti-Alzheimer's disease (AD) activity, there is currently no research on the potential of its extract or constituents to inhibit β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Methods: The study aimed to evaluate the inhibitory effects of the methanolic extract of C. pinnatifida and its solvent-soluble fractions on cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Following a bioassay-guided isolation, we identified several compounds, including two triterpenoids [corosolic acid (1), euscaphic acid (2)], β-sitosterol glucoside (3), three flavonoids [astragalin (4), hyperoside (5), isovitexin (6)], and four phenolic acid derivatives [gentisic acid (7), protocatechuic acid (8), p-hydroxybenzoic acid (9), p-coumaric acid (10)] from EtOAc fraction. Additionally, two triterpenoids (3-epicorosolic acid (11) and ursolic acid (12)) and β-sitosterol (13) were isolated from the CH2Cl2 fraction, while isoorientin (14) and hymenoside X (15) were isolated from n-BuOH fraction. Notably, compounds 2, 7, 10, and 14 were reported for the first time from the fruit of C. pinnatifida. Results: We observed that 3-epicorosolic acid (11) exhibited promising inhibitory activity against BACE1 with IC50 values of 40.12 ± 1.72 µM. Hyperoside (5) showed moderate activity with IC50 values of 98.50 ± 4.20 µM, while corosolic acid (1) and astragalin (4) displayed mild BACE1 inhibitory activity, with IC50 values of 179.55 ± 1.57 and 192.30 ± 5.14 µM, respectively. We also investigated the structure activity relationship of ursane-type triterpenoids based on in vitro and in silico results, which revealed that the hydroxyl groups position of the triterpenes have a significant impact on their potency. Our findings suggest that α‑hydroxyl group at the C-2 and C-3 positions and the absence of α‑hydroxyl group at the C-19 position are essential for BACE1 inhibitory activity. Conclusions: This study highlights the significance of the configuration and position of hydroxyl groups and their impact on BACE1 inhibitory activity. The findings of this study could be leveraged to develop novel therapeutic approaches for Alzheimer's disease

Structure Related Inhibition of Enzyme Systems in Cholinesterases and BACE1 In Vitro by Naturally Occurring Naphthopyrone and Its Glycosides Isolated from Cassia obtusifolia

Cassia obtusifolia Linn. have been used to improve vision, inflammatory diseases, and as hepatoprotective agents and to promote urination from ancient times. In the present study, we investigated the influence of glycosylation of components of C. obtusifolia and structure-activity relationships (SARs) with respect to the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are related to Alzheimer’s disease (AD). All six C. obtusifolia-derived compounds, rubrofusarin (1), rubrofusarin 6-O-β-d-glucopyranoside (2), rubrofusarin 6-O-β-d-gentiobioside (3), nor-rubrofusarin 6-O-β-d-glucoside (4), isorubrofusarin 10-O-β-d-gentiobioside (5), and rubrofusarin 6-O-β-d-triglucoside (6) showed promising inhibitory activity against AChE/BACE1. Compounds 3 and 4 showed most significant inhibition against AChE and BACE1, respectively. The SARs results emphasized the importance of gentiobiosyl moiety in the rubrofusarin for AChE inhibition, whereas the presence of hydroxyl group at C-8 and the glucosyl moiety at the C-6 position in the nor-rubrofusarin appeared to largely determine BACE1 inhibition. Kinetics and docking studies showed the lowest binding energy and highest affinity for mixed-type inhibitors, 3 and 4. Hydrophobic bonds interactions and the number of hydrogen bonds determined the strength of the protein-inhibitor interaction. These results suggest that C. obtusifolia and its constituents have therapeutic potential, and that the SARs of its active components are further explored with a view towards developing a treatment for AD

Structure Related Inhibition of Enzyme Systems in Cholinesterases and BACE1 In Vitro by Naturally Occurring Naphthopyrone and Its Glycosides Isolated from Cassia obtusifolia

Cassia obtusifolia Linn. have been used to improve vision, inflammatory diseases, and as hepatoprotective agents and to promote urination from ancient times. In the present study, we investigated the influence of glycosylation of components of C. obtusifolia and structure-activity relationships (SARs) with respect to the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are related to Alzheimer’s disease (AD). All six C. obtusifolia-derived compounds, rubrofusarin (1), rubrofusarin 6-O-β-d-glucopyranoside (2), rubrofusarin 6-O-β-d-gentiobioside (3), nor-rubrofusarin 6-O-β-d-glucoside (4), isorubrofusarin 10-O-β-d-gentiobioside (5), and rubrofusarin 6-O-β-d-triglucoside (6) showed promising inhibitory activity against AChE/BACE1. Compounds 3 and 4 showed most significant inhibition against AChE and BACE1, respectively. The SARs results emphasized the importance of gentiobiosyl moiety in the rubrofusarin for AChE inhibition, whereas the presence of hydroxyl group at C-8 and the glucosyl moiety at the C-6 position in the nor-rubrofusarin appeared to largely determine BACE1 inhibition. Kinetics and docking studies showed the lowest binding energy and highest affinity for mixed-type inhibitors, 3 and 4. Hydrophobic bonds interactions and the number of hydrogen bonds determined the strength of the protein-inhibitor interaction. These results suggest that C. obtusifolia and its constituents have therapeutic potential, and that the SARs of its active components are further explored with a view towards developing a treatment for AD