Iannuzzo, Fortuna (2023) Grape seed extract as a source of proanthocyanidins with potential effects on human health. [Tesi di dottorato]

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Abstract

Proanthocyanidins (PACs) are a group of bioactive molecules found in a variety of plants and foods that have numerous beneficial effects on human health. Their bioavailability and metabolism depend on their molecular size. Although studies in the literature are quite contradictory, it is generally reported that monomers and dimers of PACs are more bioavailable than those with a higher degree of polymerization (DP). High molecular weight PACs are converted by the colon microflora into lower molecular weight and absorbable metabolites with health-promoting properties. In this regard, if polymeric PACs could be depolymerized into oligomeric ones, their bioaccessibility and bioavailability could also significantly increase. Several depolymerization methods of PACs are described in the literature, but none of them is applicable in the agri-food industry due to the difficulty of execution, high costs and the impossibility to produce food-grade nutraceutical products. One of the most typical sources of PACs is represented by grapes, especially its seeds. Grape skins and seeds are waste products of wine production, nevertheless, they represent a still rich source of bioactive molecules, especially antioxidants. This aspect is part of the green-economy concept and involves the use of agri-food waste as an alternative raw material for the extraction of bioactive compounds to be included in nutraceuticals and dietary supplements. In light of these considerations, this Ph.D. thesis aimed to develop a food-grade, as well as, rapid and economical method for the large-scale production of PACs with high bioavailability and bioaccessibility from a grape seed extract (GSE ) (Vitis Vinifera L.). Therefore, GSE was subjected to alkaline treatment to depolymerize PAC polymers into low molecular weight monomers and oligomers. The different chemical composition of GSE and its version processed under alkaline conditions (ATGSE) was evaluated using a molecular networking (MN) approach based on results obtained from HPLC-ESI HRMS/MS characterization analysis. The network analysis mainly noted the PAC cluster with about 142 PAC compounds identified. In particular, the obtained results showed a higher content of monomeric and dimeric PACs in ATGSE compared to GSE, with 58% and 49% monomers and 31% and 24% dimers, respectively. Conversely, trimeric (9%), polymeric (4%), and galloylated PACs (14%) were more abundant in GSE than in ATGSE (6%, 1%, and 4%, respectively). Moreover, in vitro antioxidant and anti-inflammatory activities were investigated, showing the high beneficial potential of both extracts. In addition, to evaluate the different intestinal bioactivity and bioaccessibility, GSE and ATGSE were subjected to a human gastrointestinal digestion-colonic fermentation process in vitro, carried out at the Department of Nutrition and Bromatology, University of Granada (Spain). Fermentation was carried out using fecal material from five different donors (healthy adults and children, obese, celiac, and cow's milk protein allergic children). For this purpose, the antioxidant profile and short-chain fatty acid (SCFA) production were determined. After in vitro gastrointestinal digestion, the bioaccessible polyphenol fraction and antioxidant capacity were higher in the ATGSE sample than in the GSE, while this trend was reversed after in vitro fermentation. Nevertheless, ATGSE fermentation showed a higher modulatory effect on the composition and functionality of the gut microbiota of healthy and pathological subjects, which was reflected in a greater increase in SCFA concentration. Literature studies show that structural variations of PACs seem to play an important role in determining their effectiveness. However, it is not possible to make broad generalizations about the structure-bioactivity correlation of PACs, as it is highly dependent on the biological systems in which these molecules act. Therefore, the second part of my Ph.D. project aimed to investigate the potential biological effects of the two extracts with different PAC compositions on human health disorders. In particular, the interest was directed toward diseases of the urogenital system. One of the most important diseases of the urogenital system in men is benign prostatic hyperplasia (BPH), which leads to non-physiological enlargement of the prostate, especially in old age. Therefore, a first evaluation of the different bioactivity of the two extracts was performed through a study on an animal model of BPH induced by subcutaneous administration of testosterone. At the end of the four weeks of treatment, the weight of the prostates removed microsurgically from the mice was evaluated and hematological and biochemical analyses of blood and tissues were performed. The data showed that oral administration of ATGSE (250 mg/kg) was more effective in reducing prostate weight, PSA levels, and expression of major pro-inflammatory cyto-chemokines in the prostate homogenate. On the other hand, the most common pathology of the urogenital system in women is cystitis, which is caused mainly by the attack of uropathogenic Escherichia coli (UPEC) in the bladder epithelium. Therefore, the different bioactivity of the two extracts was tested in an in vitro model to evaluate their ability to inhibit the growth and invasion of UPEC (ATCC 700928) in human bladder cells (HT-1376). ATGSE at a dose of 25 mg/mL proved to be more effective than GSE in inhibiting bacterial invasion, showing colony forming units (CFU) of 105 and 107, respectively. Considering these promising biological effects of ATGSE, the Ph.D. project included a research period at the Department of Galenic Technique of the company S.I.I.T. S.R.L.-INNOVATIVE HEALTHCARE PRODUCTS in Milan, where the most appropriate technological process was developed to obtain a nutraceutical formulation based on ATGSE, through the best possible selection of excipients, ingredients, and strategies for releasing the active ingredient. This technological process allowed to significant increase the density of ATGSE and turn it into a compressible powder. Considering the nature of this PAC-rich food matrix and its sensitivity to gastric pH, the gastro-resistant tablet proved to be the most appropriate choice to increase the bioaccessibility of the active ingredient in the small intestine. In conclusion, the results obtained suggest that ATGSE could represent an innovative nutraceutical formulation rich in bioavailable and bioaccessible PACs with potential beneficial properties on human health. However, our results highlight the need for further in vitro and in vivo, before efficacy can be tested in humans.

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