ASSESSMENT OF ZINC AS A MICRONUTRIENT AND HONEY FOR THEIR ROLES IN MANAGEMENT OF VARIOUS DISEASES

Abstract

Micronutrients are essential for metabolism and tissue function. Zinc is an essential micronutrient for the development and growth of all living species, as has been clearly documented over the last several decades. Micronutrient insufficiency is a leading cause of a wide range of health issues in people. The primary cause of micronutrient insufficiency is poor absorption and bioavailability. Analysis of zinc bioavailability from digested food has emerged as a critical stage in the battle against micronutrient deficits. Zinc deficiency is associated to an increased risk of cancer and an imbalance in other cellular functions. Cancer is a set of conditions characterized by uncontrolled proliferation of cells or tissues in any part of the body, which can result in death. Cancer is presently one of the most potentially serious public health challenge, with a substantially higher global fatality rate. Cancer development involves multiple factors and genes. Colorectal cancer is the third most frequent malignant disease worldwide, and researchers are looking towards more cytotoxic chemotherapies and biological agents to combat it. The gastrointestinal tract is the major portion of the body's digestive system. The gastrointestinal mucosa functions as a selective barrier to regulate the movement and absorption of micronutrients, salts, and water from the luminal portion of the small and large intestines into the body's circulatory system. A leaking gut barrier is a major contributor to a wide range of inflammatory gastrointestinal disorders. The current thesis, titled "Assessment of zinc as a micronutrient and honey for their roles in management of various diseases" explores zinc in terms of developing a sensitive and robust cell-based fluorescence assay for evaluating its bioavailability from diverse dietary sources. In addition, the current study also investigated the synergistic effect of zinc with natural phytochemicals in the prevention of colon cancer. The other section of the thesis explores about the use of Indian mustard honey as a nutraceutical in the prevention of inflammation induced intestinal barrier dysfunction. In the beginning, Chapter 1 briefly introduces the role of zinc as a micronutrient in health and various methods, such as measurement of micronutrient bioavailability, to combat its deficiency. This chapter also brief about the chemopreventive effect of zinc and plant derived phytochemicals in the battle against colon cancer, with a special emphasis on i ii

Abstract combinatorial therapeutic approach. The following section of this chapter introduce about the role of the intestinal barrier and tight junction (TJ) proteins in health and disease, as well as some potential preventative approaches such as the use of honey as a nutraceutical to prevent intestinal barrier dysfucntion. Finally, the specific objectives to be addressed in this thesis are described in this chapter. Followed by this, Chapter 2 presents detailed literature review on bioavailability and factors associated with it, various strategies for assessment of nutrient bioavailability, importance of micronutrient in health, importance of zinc in health, intestinal zinc absorption and various in-vitro models to study zinc bioavailability, zinc sensing receptor GPR39 in intestinal health and function, zinc signalling in colon cancer, Combination therapy to fight against colorectal cancer, phytochemicals in prevention of colon cancer, intestinal barrier in health and diseases, importance of tight junction proteins in intestinal barrier integrity, honey in ancient and modern medical science, role of honey as a nutraceutical in prevention of gastrointestinal disorders. All the materials and experimental methods used in this study for achieving the proposed objectives through in-vitro and in-vivo assays are elaborated precisely in Chapter 3. Deficiency of micronutrients like zinc and iron has been linked to the manifestation of hidden hunger. Therefore, it is imperative that development of some rapid screening method for bioavailable zinc in various crops and food commodities would be an essential addition in battle against zinc deficiency related hidden hunger. One such method could be the usage of fluorescence based zinc ion sensing probe which would be robust and convenient for estimating bioavailable zinc. To start with the current work, Chapter 4 presents development of a novel fluorescence based approach for the analysis of zinc bioavailability in Caco-2 cells as an in vitro cellular model. To address this issue, NBD-TPEA, a highly sensitive zinc ion sensing probe, have been used towards the development of assay. The use of this probe showed dose dependent sensitivity towards increasing concentrations of zinc ion uptake by Caco-2 cells. It also showed specificity for zinc ion uptake as compared to other metal ions in-vitro. These observations correlated extremely well with zinc uptake analysis by cell imaging and conventional analytical technique like, ICP-MS. The developed assay was then tested in mushroom and some selected biofortified derivatives of wheat for determining the levels of

Abstract their bioavailable zinc using Caco-2 cells. The data as obtained with these food samples in our developed bioassay correlated well with the other sophisticated analytical techniques thus validating our cell based assay. Hence, the developed assay could serve as a simple but sensitive tool for determining bioavailable zinc in various food samples. Rice is the most important staple crop because it nourishes approximately half of the world's population. In Chapter 5, study was carried out to screen a set of eight rice varieties for phytate concentration and total grain Zn and Fe content along with their bioavailability in differentiated Caco-2 cells Using atomic absorption spectrophotometry (AAS), the total grain Zn and Fe content, as well as their bioaccessibility, were determined. The cell-based fluorescence assays and Caco-2 cell ferritin synthesis were used to determine Zn and Fe bioavailability. The transcriptional expression of micronutrient transporters has been analysed in Caco-2 cells in response to the uptake of Zn and Fe from in-vitro digested rice samples. The findings of this study revealed a comparative analysis of polished rice of eight different varieties, three of which were shown to have low phytate content and high grain Zn content, as well as significantly greater Zn bioavailability in differentiated Caco-2 cells. The outcome of this study provides an easy and convenient approach for the screening of food items or cereals based on micronutrient bioavailability. Chapter 6 depicts the synergistic anti-cancer effect of zinc and berberine on HT-29 adenocarcinoma cancer cells. Anti-proliferative activity of berberine and zinc were determined by cell viability assay and colony forming assay. Combination index (CI) and Drug reduction index (DRI) values of zinc and berberine co-treatment were estimated by CompuSyn software. Flow cytometry was used to analyse cell cycle distribution and Annexin V/PI assay. Expression of apoptosis and zinc signalling markers were analysed by RT-qPCR and immunoblotting. Berberine decreased the viability of colon cancer cells in a dose-dependent manner but zinc had no significant influence on it. However, zinc and berberine co-treatment resulted in a synergistic anti-cancer action which was demonstrated by G2/M phase arrest of cell growth at a lower dose of berberine. Annexin V assay demonstrated that the synergistic impact of zinc and berberine boosted the number of apoptotic cells. Gene expression analysis at both transcriptional and translational levels showed the upregulation of apoptotic (Caspase-3 and caspase-8) and a zinc-sensing receptor (GPR39) gene with concomitant downregulation of anti apoptotic genes like proliferating cell nuclear antigen (PCNA) and clusterin. Overall, the iii Abstract findings of this study showed that the combination of zinc and berberine has synergistic anticancer efficacy and thus could be used as a potential therapeutic option for colon cancer. A leaky gut barrier is an important factor leading to a variety of inflammatory gastrointestinal disorders. The nutritional value of honey and variety of health benefits have long been recognized. In Chapter 7, study was undertaken to see if Indian mustard honey may prevent lipopolysaccharide (LPS)-induced intestinal barrier dysfunction in an in-vitro and in- vivo model system. LPS was used to induce intestinal barrier damage in trans-well model of Caco-2 cells and in Swiss albino mice. Gas chromatography-mass spectrometry (GC-MS) and Liquid chromatography-mass spectrometry (LC-MS) were used to analyse sugar and phenolic compounds of honey. Caco-2 cell monolayer integrity was evaluated by transepithelial electrical resistance (TEER) and paracellular permeability assays. Histopathology of intestinal tissue was analysed by haematoxylin and eosin dual staining. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Protein expression was analysed by immunofluorescence and western blot techniques. In-vitro data showed that dose-dependent effect of honey prevented LPS induced intestinal barrier dysfunction as measured by TEER and paracellular flux of FITC-dextran dye. In-vivo data showed that prophylactic effect of orally administered honey prevented loss of intestinal barrier integrity and villus structure. Cellular localization and expression of tight junction proteins were upregulated along with down regulation of pro-inflammatory cytokine genes in response to administration of honey along with LPS. The findings of this study suggest a propitious role of honey in the maintenance of TJ protein integrity, thereby preventing LPS-induced intestinal barrier disintegration. Finally, Chapter 8 summarizes the major findings of this thesis and highlights the possible future scope of in the respective areas. The scientific conclusion addressed in this thesis may be useful to future scholars working in this field. Last but not the least, Chapter 9 enlists the whole bibliography consulted over the course of the current work.