Development of innovative methods to preserve a new line of infant food supplements free from synthetic additives

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

Annurca Apple Polyphenols Ignite Keratin Production in Hair Follicles by Inhibiting the Pentose Phosphate Pathway and Amino Acid Oxidation

Patterned hair loss (PHL) affects around 50% of the adult population worldwide. The negative impact that this condition exerts on people’s life quality has boosted the appearance of over-the-counter products endowed with hair-promoting activity. Nutraceuticals enriched in polyphenols have been recently shown to promote hair growth and counteract PHL. Malus pumila Miller cv. Annurca is an apple native to Southern Italy presenting one of the highest contents of Procyanidin B2. We have recently shown that oral consumption of Annurca polyphenolic extracts (AAE) stimulates hair growth, hair number, hair weight and keratin content in healthy human subjects. Despite its activity, the analysis of the molecular mechanism behind its hair promoting effect is still partially unclear. In this work we performed an unprecedented metabolite analysis of hair follicles (HFs) in mice topically treated with AAE. The metabolomic profile, based on a high-resolution mass spectrometry approach, revealed that AAE re-programs murine HF metabolism. AAE acts by inhibiting several NADPH dependent reactions. Glutaminolysis, pentose phosphate pathway, glutathione, citrulline and nucleotide synthesis are all halted in vivo by the treatment of HFs with AAE. On the contrary, mitochondrial respiration, β-oxidation and keratin production are stimulated by the treatment with AAE. The metabolic shift induced by AAE spares amino acids from being oxidized, ultimately keeping them available for keratin biosynthesis

In vivo bioavailability and in vitro toxicological evaluation of the new butyric acid releaser N-(1-carbamoyl-2-phenyl-ethyl) butyramide

A large body of evidence suggests that supplementation of butyric acid exerts beneficial intestinal and extra-intestinal effects. Unfortunately, unpleasant sensorial properties and unfavourable physico-chemical properties strongly limit its use in food supplements and foods for medicinal purposes. N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) is a new butyric acid releaser in solid form with neutral sensorial properties. The aim of this investigation is to provide preliminary information on its pharmacokinetic and toxicological properties through the study of a) in vivo bioavailability of FBA administered by oral gavage to male and female Swiss CD1 mice in comparison with sodium butyrate, b) the influence of digestion on FBA stability through an in vitro simulated oro-gastro-duodenal digestion process, and c) in vitro toxicological profile by means of the Ames Test and Micronucleus Test. The results reveal that FBA is a good butyric acid releaser, being able to increase butyrate serum concentration in a dose and time dependent manner in both male and female mice with a pharmacokinetic profile similar to that obtained from sodium butyrate as such. These data are confirmed by investigating the influence of digestion on FBA, which undergoes extensive hydrolysis following oro-gastro-duodenal digestion, especially in duodenal conditions, with a residual concentration of less than 10% of the initial FBA concentration. Finally, in the Ames and Micronucleus Tests, FBA does not show any in vitro genotoxicity as it is non mutagenic in the Ames Test and results to be unable to induce chromosome breaks in the Micronucleus Test. In conclusion, FBA is a new butyric acid releaser that can overcome the disadvantages of butyric acid while maintaining the same pharmacokinetic properties and safety profile, as shown by the results of the preliminary in vitro toxicological studies performed in this investigation

A boost in mitochondrial activity underpins the cholesterol-lowering effect of annurca apple polyphenols on hepatic cells

Reduction in cholesterol blood levels represents one of the therapeutic goals to achieve in order to reduce the occurrence of cardiovascular diseases. Commonly, this goal is attempted by promoting healthy lifestyle behaviors and low-fat diets. Recently, several nutraceuticals have been shown to possess cholesterol-lowering properties and are becoming common over the counter products. Among others, apple polyphenols efficiently lower total cholesterol levels in humans and impact overall lipid metabolism. Malus Pumila Miller cv Annurca is an apple native to Southern Italy presenting one of the highest content of procyanidin B2, a dimeric procyanidin. Tested in clinical trials, the oral consumption of an Annurca polyphenolic extract (AAE) exerted a cholesterol-lowering effect similar to the statins Atorvastatin and Simvastatin. Despite AAE activity, the analysis of the molecular mechanism behind its cholesterol-lowering effect is unclear. Using isotope labeling and high-resolution mass spectrometry approaches we here performed a metabolic profiling of in vitro cultured human hepatocytes treated with AAE to reveal its mechanism of action. The results show that AAE acts differently than statins. The extract reprograms hepatic cell metabolism and promotes mitochondrial respiration, lipolysis and fatty acid β-oxidation. Citrate and acetyl-CoA, both necessary for the production of cholesterol, are diverted to the Krebs Cycle by AAE, that, ultimately, lowers cholesterogenesis and fatty acid synthesis

Annurca apple polyphenols protect murine hair follicles from taxane induced dystrophy and hijacks polyunsaturated fatty acid metabolism toward β-oxidation

Chemotherapy-induced alopecia (CIA) is a common side effect of conventional chemotherapy and represents a major problem in clinical oncology. Even months after the end of chemotherapy, many cancer patients complain of hair loss, a condition that is psychologically difficult to manage. CIA disturbs social and sexual interactions and causes anxiety and depression. Synthetic drugs protecting from CIA and endowed with hair growth stimulatory properties are prescribed with caution by oncologists. Hormones, growth factors, morphogens could unwontedly protect tumour cells or induce cancer cell proliferation and are thus considered incompatible with many chemotherapy regimens. Nutraceuticals, on the contrary, have been shown to be safe and effective treatment options for hair loss. We here show that polyphenols from Malus Pumila Miller cv Annurca are endowed with hair growth promoting activity and can be considered a safe alternative to avoid CIA. In vitro, Annurca Apple Polyphenolic Extract (AAE) protects murine Hair Follicles (HF) from taxanes induced dystrophy. Moreover, in virtue of its mechanism of action, AAE is herein proven to be compatible with chemotherapy regimens. AAE forces HFs to produce ATP using mitochondrial β-oxidation, reducing Pentose Phosphate Pathway (PPP) rate and nucleotides production. As consequence, DNA replication and mitosis are not stimulated, while a pool of free amino acids usually involved in catabolic reactions are spared for keratin production. Moreover, measuring the effect exerted on Poly Unsaturated Fatty Acid (PUFA) metabolism, we prove that AAE promotes hair-growth by increasing the intracellular levels of Prostaglandins F2α (PGF2α) and by hijacking PUFA catabolites toward β-oxidation

Salvigenin, a Trimethoxylated Flavone from Achillea Wilhelmsii C. Koch, Exerts Combined Lipid-Lowering and Mitochondrial Stimulatory Effects

Phytochemical analysis of the Iranian plant Achillea wilhelmsii led to the isolation of 17 pure secondary metabolites belonging to the classes of sesquiterpenoids and phenolics. Two of these compounds, named wilhemsin (7) and wilhelmsolide (9), are new sesquiterpenoids, and the first shows undescribed structural features. Their structures were elucidated through extensive spectroscopic analysis, mainly based on 1D and 2D NMR, and chemical derivatization. Starting from plant traditional use and previous reports on the activity of the plant extracts, all the pure compounds were evaluated on endpoints related to the treatment of metabolic syndrome. The sesquiterpene hanphyllin (8) showed a selective cholesterol-lowering activity (−12.7% at 30 µM), santoflavone (13) stimulated glucose uptake via the GLUT transporter (+16.2% at 30 µM), while the trimethoxylated flavone salvigenin (14) showed a dual activity in decreasing lipid levels (−22.5% palmitic acid biosynthesis at 30 µM) and stimulating mitochondrial functionality (+15.4% at 30 µM). This study further confirms that, in addition to the antioxidants vitexin, isovitexin, and isoschaftoside, A. wilhelmsii extracts contain molecules that can act at different levels on the metabolic syndrome symptoms

Annurca apple polyphenols ignite keratin production in hair follicles by inhibiting the pentose phosphate pathway and amino acid oxidation

Patterned hair loss (PHL) affects around 50% of the adult population worldwide. The negative impact that this condition exerts on people’s life quality has boosted the appearance of over-the-counter products endowed with hair-promoting activity. Nutraceuticals enriched in polyphenols have been recently shown to promote hair growth and counteract PHL. Malus pumila Miller cv. Annurca is an apple native to Southern Italy presenting one of the highest contents of Procyanidin B2. We have recently shown that oral consumption of Annurca polyphenolic extracts (AAE) stimulates hair growth, hair number, hair weight and keratin content in healthy human subjects. Despite its activity, the analysis of the molecular mechanism behind its hair promoting effect is still partially unclear. In this work we performed an unprecedented metabolite analysis of hair follicles (HFs) in mice topically treated with AAE. The metabolomic profile, based on a high-resolution mass spectrometry approach, revealed that AAE re-programs murine HF metabolism. AAE acts by inhibiting several NADPH dependent reactions. Glutaminolysis, pentose phosphate pathway, glutathione, citrulline and nucleotide synthesis are all halted in vivo by the treatment of HFs with AAE. On the contrary, mitochondrial respiration, β-oxidation and keratin production are stimulated by the treatment with AAE. The metabolic shift induced by AAE spares amino acids from being oxidized, ultimately keeping them available for keratin biosynthesis

The Hepatoprotective Effect of Taurisolo, a Nutraceutical Enriched in Resveratrol and Polyphenols, Involves Activation of Mitochondrial Metabolism in Mice Liver

Liver diseases affect millions of people worldwide. In most of the cases, severe hepatic dysfunction and liver cancer stem from mild and common clinical signs including hepatic steatosis, insulin resistance, liver inflammation, and oxidative stress, all together referred to as Nonalcoholic Fatty Liver Disease (NAFLD). Nutraceuticals endowed with antioxidant activity have been shown to reduce NAFLD risk factors and exert hepatoprotective effects. Here, we test the protective effect exerted on liver by the antioxidant Taurisolo, a nutraceutical formulation produced by grape pomace and enriched in Resveratrol and Polyphenols. We analyze the effect of Taurisolo on liver cells by profiling the metabolome of in vitro cultured hepatic HuH7 cells and of C57BL-6J mice fed a High Fat Diet and treated with the nutraceutical. Both in vitro and in vivo, we provide evidence that Taurisolo reduces risk factor markers associated with NAFLD. Taurisolo stimulates glucose uptake and reduces hepatic cholesterol and serum triglycerides. Furthermore, we give new insights into the mechanism of action of Taurisolo. The nutraceutical increases mitochondrial activity and promotes respiration and ATP production, fostering catabolic reactions like fatty acid β-oxidation and amino acid catabolism. On the contrary, Taurisolo reduces anabolic reactions like biosynthesis of cholesterol, bile acids, and plasma membrane lipids

A Negative Allosteric Modulator of WNT Receptor Frizzled 4 Switches into an Allosteric Agonist

The WNT pathway interconnects a network of signaling events involved in a huge plethora of cellular processes, from organogenesis to tissue homeostasis. Despite its importance, the exiguity of organic drugs directly targeting the members of the Frizzled family of WNT receptors has hampered progress across the whole spectrum of biological fields in which the signaling is involved. We here present FzM1.8, a small molecule acting as an allosteric agonist of Frizzled receptor FZD4. FzM1.8 derives from FzM1, a negative allosteric modulator of the receptor. Replacement of FzM1 thiophene with a carboxylic moiety induces a molecular switch in the lead and transforms the molecule into an activator of WNT signaling. We here show that, in the absence of any WNT ligand, FzM1.8 binds to FZD4, promotes recruitment of heterotrimeric G proteins, and biases WNT signaling toward a noncanonical route that involves PI3K. Finally, in colon cancer cells, we prove that the FZD4/PI3K axis elicited by FzM1.8 preserves stemness and promotes proliferation of undifferentiated cells