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dc.contributor.authorPanwar, Richa-
dc.guidePruthi, Vikas-
dc.guideDutt, Dharam-
dc.description.abstractUse of naturally occurring phytochemicals have attracted a great deal of interest as therapeutic agents against a wide range of ailments. Hydroxycinnamic acid derivatives are a major category of phenolic acids which provide significant benefits as natural antioxidants. Ferulic acid (FA; C10H10O4) is the most abundant hydroxycinnamic acid bound to lignin and polysaccharides via ester/ether linkages in plant cell walls. FA is present in various cereals, fruits, crop residues (wheat bran, corn bran, sugarcane bagasse) and possess a potent antioxidant capacity due to its structural characteristics. It exhibits an array of therapeutic properties including anti-inflammatory, anticancer, anti-diabetic, antimicrobial along with prevention of cardiovascular disorders. Most of these activities could be directly attributed to the free radical scavenging potential of FA. Although having valuable therapeutic characteristics, this important plant derived nutraceutic display major drawback in the form of low bioavailability and clinical efficacy. FA exhibits a small plasma retention time both in rats and humans and is quickly eliminated from the body, mostly via urinary excretion. Polymeric encapsulation of FA would enhance its plasma retention time, bioavailability and stability against degradation in gastrointestinal tract. Chitosan (CS) is a non-toxic biocompatible polymer, used extensively for encapsulation of bioactive molecules. CS acts as a shell material to envelop these compounds bearing multiple negative charges via cationic crosslinking to generate stable, biodegradable nanosized particles through ionic cross linking when comes in contact with specific polyanions such as sodium-tripolyphosphate. These nanoparticles are extensively used to deliver various compounds including drugs, proteins, nutrients and phenolics into the biological systems. Present investigation was carried out to synthesize ferulic acid encapsulated chitosan nanoparticles (FANPs) having enhanced plasma retention time along with investigation of their effects in biological disorders such as cancers, diabetes, inflammations. Chapter 2 deals with synthesis and characterization of FANPs at optimized CS conc. 1 mg/ml, CS:TPP mass ratio 4 and FA conc. 0.0528 and 0.1056 mg/ml. Particles diameter/morphology of resulting nanoparticles were measure and visualized through zeta sizer and field emission- scanning electron microscopy (FE-SEM) analysis. Approximately 50% encapsulation efficiencies were achieved for optimized nanoformulations that exhibited enhanced solubility, higher thermal degradation range along with stability of FA antioxidant potential during encapsulation. ii Biochemical characterization of FANPs through the Fourier transform infrared spectra (FT-IR) provided an insight about its secondary interactions with chitosan nanoparticles (CSNPs). Proton nuclear magnetic resonance (1H NMR) spectroscopy revealed the functional groups shifts in the chemical structures of different formulations that have taken place during encapsulation process while the X-ray diffractometry (XRD) established the successful encapsulation of FA. Subsequently, in Chapter 3, pharmacokinetic and urinary excretion profile analyses were carried out for FA in free form (non-encapsulated) and FANPs using healthy Wistar albino rats. FA in its free form was found to eliminate quickly from the circulation owing to its rapid and extensive metabolism and is mostly excreted through urine. The encapsulated FA displayed extended plasma retention time and maximum plasma conc. was recorded at 60 min which implied enhancement of Tmax to four times higher compare to free FA. The elimination of compound from rat’s body also displayed a similar pattern where the peak urinary excretion of FA from nanoformulations was measured at 4 h contrary to 2 h in case of free FA. In the same context, rats with carrageenan-induced paw edema showed a better recovery when treated with FANPs establishing them as a highly promising and cost-effective nutraceutical with commendable safety profile. The ability of FA to regulate cell growth and proliferation, scavenge free radicals, stimulate cytoprotective enzymes and inhibit cytotoxic systems in both in-vitro and in-vivo experimental models account for the potential adjuvant role of FA in cancer therapy. Nevertheless, the unfavorable pharmacokinetic and low bioavailability of FA necessitate its encapsulation into CS, in order to increase bioavailability and at the same time maintain or enhance its antiproliferative action. Chapter 4 emphasized on the in-vitro evaluation of anticancer potential of FANPs on ME-180 human cervical carcinomas and PC-3 human prostate cancer cell lines. Initially, the in-vitro release of FA from its nanoformulations was evaluated at different pH, followed by screening of both cell lines with different conc. of FANPs to estimate minimum dose required for their 50% inhibition (IC50). Subsequently, the effect of nanoformulations on cell proliferation were assessed via MTT assay and flowcytometry. Morphological changes occurring within the formulation treated cells were visualized through FE-SEM and fluorescence microscopy. Finally, the cytocompatibility of corresponding formulations were checked on Human Embryonic Kidney (HEK-293) cell lines. Cytotoxicity evaluation of FANPs established them as strong antiproliferative agents against ME-180 human cervical and PC-3 human prostate cancer cell lines without any potential toxicity to normal healthy cells. In subsequent chapter testing of antibiofilm potential of FANPs against Candida albicans biofilms was performed. C. albicans is the major multidrug tolerant species, iii causing life-threatening infections that display striking ability to form drug-resistant biofilms. C. albicans biofilms are reported to be 4000 times more resistant to antifungal drug fluconazole when compared to planktonic or to its free-floating counterparts. Compared to conventional synthetic antifungal drugs that have become unyielding towards the biofilm matrix, there was a less likelihood of the development of resistance by biofilm cells against naturally occurring FA. Hence, it was hypothesized that FANPs could easily penetrate the biofilm cells and matrix owing to their high surface area to volume ratio. These FANPs are believed to disrupt or alter the permeability of fungal cell plasma membrane and could reduce the cell population. Cytocompatible FANPs at conc. 40 μM and 80μM reduced C. albicans biofilm cells viability upto 30 and 21.4% respectively, thus emphasizing its potential as a powerful antifungal agent. Positive zeta potential of FANPs was proposed to be a crucial factor facilitating their binding with negatively charged membrane of fungal cell, thus disrupting its integrity which eventually lead to leakage of intracellular materials and biofilm inhibition. The intriguing synergistic effect of FANPs on prevention of C. albicans biofilm might offer a new hope in the management of biofilm related infections. Chapter 6 confined to the evaluation and comparison of in-vivo anti-diabetic activities of native FA and FANPs in streptozotocin (STZ) induced diabetic Wistar albino rats. Diabetes mellitus is a condition which is characterized by various pathological disorders, all or most of which are integrated with chronic hyperglycemia. The hyperglycemic disorders are very likely to cause overproduction of free radicals, giving rise to an oxidative stress inside the body tissues. FA displayed a strong anti-diabetic behavior by regulating a large number of biochemical or physiological pathways involved in hyperglycemia. It is found to stimulate insulin secretion by pancreatic β-cells while inhibiting lipid peroxidation in diabetic mice resulting in the improvement of hyperglycemia. FANPs with a higher cellular availability and enhanced plasma retention time exhibited a positive effects on diabetes associated symptoms such as lowering of blood glucose levels, enhancement in body weight along with reduction in total cholesterol, low density lipoprotein cholesterol and triglycerides conc. in diabetic rats. FANPs also resisted a sharp decline in insulin level, which allowed further regulation of cholesterol, triglycerides and overall lipid profile in induced diabetes. Positive impact of FANPs in improving the hyperglycemic condition prevalent in diabetic rats might provide new avenues for the treatment of diabetes mellitus and helps to avoid the secondary complications associated with synthetic drugs.en_US
dc.description.sponsorshipBIOTECHNOLOGY IIT ROORKEEen_US
dc.subjectFerulic aciden_US
dc.subjectBiochemical characterizationen_US
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