Badolati, Nadia (2022) Development of innovative methods to preserve a new line of infant food supplements free from synthetic additives. [Tesi di dottorato]

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In last two decades, research studies on nutraceuticals, functional foods and natural products have acquired increasing scientific importance. Unfortunately, this is not only for the interest in this scientific field, but also because the discoveries regarding the healthy effect of these products have the power to further boost enormous profit of the nutraceutical market. Natural products, such as plants and fruit extracts, provide unlimited opportunities for the development of pharmaceuticals and nutraceuticals. This is due to the amazing chemical diversity and the plethora of physiological activity of their chemical components. The areas of application for nutraceuticals, botanicals, and herbal products are thus becoming more and more varied: from maintenance of well-being to cosmetic applications, from reestablishment of homeostasis to prevention of cardiovascular and dysmetabolic pathologies, from neuro- to chondroprotection, from strengthening the immune system to preserving regular intestinal functions, form weight loss to improvement of muscle performance. However, being “natural” does not necessarily mean “being safe”. Despite the considerable interest received by nutraceuticals, the proof of their biological activity and of their therapeutic effects is mostly postulated based on the traditional usage but almost never confirmed by scientifically sound approaches. Nowadays, the European Commission (EC) has proposed harmonized approaches, made of in vitro and in vivo experimental pipelines, necessary to demonstrate and/or confirm the effectiveness of almost every pharmaceutical preparation before the latters can be sold on the market. The approval procedure is very complex, as it requires “companies' requests” to be collected by the responsible national institutions of the various member countries and sent to EC. Final decisions are based on the "opinions" of an external Authority, the European Food Safety Authority (EFSA), which periodically organizes meetings of experts’ panels, judging data and results presented and expressing a favorable or unfavorable opinion. Some nutraceuticals complete preclinical and clinical pharmaco-toxicology phases, and meta-analysis of their clinical trials are sometimes available. However, in many cases, specific data on the individual nutraceutical, which can characterize it as an effective aid in medical practice, are absent. In this scenario, it is therefore necessary to perform preclinical researches on nutraceuticals in order to confirm their activity and their safety in a scientifically sound, valid and reproducible way. This thesis presents and discusses the development and the application of biological platforms aimed to: 1) validate the health-promoting potential of phytocomplexes and nutraceuticals; 2) investigate their mechanism of action; 3) confirm their biotoxicological safety. The results are presented in five chapters. CHAPTER 1, describes the regulatory framework that is relevant for the marketing of nutraceuticals and functional foods in the European Union (EU), and the regulatory authorities who manage the state-of-art worldwide. It also defines how this legislation was developed as well as some practical consequences for manufacturers, marketers and consumers. In this chapter it is explained the research methodologies to be applied and the scientific endpoints to aim during preclinical and clinical phases of the development on nutraceuticals, based on a step-by-step approach. CHAPTER 2, describes platforms, pipelines, procedures and results obtained by performing the risk assessment of three nutraceuticals and a novel food. Implementing the approaches suggested by OECD guidelines, I present data on the genotoxic and cancerogenic potential of these products confirming their safety. In particular, the aim of this investigation is to provide preliminary information of their pharmacokinetic and toxicological properties through the study of their in vitro toxicological profile and in vivo bioavailability. In particular, I herein perform and optimize the most commonly used methods to investigate gene point mutations and chromosome aberrations: bacterial reverse mutation assay in Salmonella typhimurium and Escherichia coli (OECD 471) and in vitro micronucleus assay (OECD 487). CHAPTER 3, describes experimental platform aimed at the identification of plant extracts endowed with antimicrobial potential and discusses their use in a new line of supplements. I confirm that phytochemicals’ broad-spectrum antibacterial activity represents a possible source of effective, cheap and safe antimicrobial agents. Specifically, I perform and optimize extraction procedure on five plants commonly used in herbal products for early childhood: Melissa officinalis; Matricaria chamomilla; Mentha spicata; Melissa officinalis and Mentha sylvestris. To evaluate antioxidant activity of the herbal extracts a DPPH assay is performed. In order to identify their antibacterial activity, the sensitivity to these compounds of the Gram-positive microorganism Staphylococcus Aureus (strain ATCC 25923) and the Gram-negative Escherichia Coli (strain ATCC 25922) is evaluated, through the determination of the MIC and MBC. CHAPTER 4 presents biological platforms aimed at the identification of the molecular mechanism underpinning nutraceuticals’ health claims. The use of botanicals is often validated simply by tradition of usage, but there is a lack of modern specific assays to confirm their pharmacological activity. Herein, I present several in vitro and in vivo experimental approaches useful to identify pro-metabolic properties of phytocomplexes. To reach the purpose, (1) I make use of isotope labeling and high-resolution mass spectrometry approaches, Deuterium labeling gas chromatography-mass spectrometry (GC/MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR), as potential validated platform to highlight molecular mechanism of nutraceuticals exerting cholesterol and lipid lowering properties. Metabolomic approaches are extremely useful tools for probing any change in metabolism accompanying drug treatments and provide invaluable insights in the mechanism of action of complex mixtures and phytocomplexes. (2) To evaluate Mitochondrial boosting activity of a nutraceutical formulation, I assess a platform on both in vitro cultured hepatic cells and in vivo on mice fed a High Fat Diet and treated with the nutraceutical by using the mitochondrial selective probe MitoTracker CMXRos. (3) To demonstrate the insulin-like properties of a natural products, I perform a pipeline assessed the ability of nutraceuticals to modulate glucose uptake via glucose transporter (GLUT), by measuring the uptake of 2-NBDG, a fluorescent analogue of deoxy-glucose covalently linked to the fluorescent molecule nitro blue tetrazolium (NBT). In CHAPTER 5, I describe my work on nutraceuticals as supplements during anti-cancer treatments to support chemotherapy therapies. The aim of this thesis chapter is to present three studies in which I set up new approaches to test nutraceuticals and investigate their mechanism of action. In these studies, I perform a metabolite analysis using Direct Infusion Fourier Transform-ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS) to investigate the molecular mechanism underpinning their nutraceutical activity.

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