Plants: From Farm to Food and Biomedical Applications

Historically, mankind has used plants and their derivatives as food and medicine for thousands of years [...

Metabolomics in natural products research: application to in vivo bioactivity studies involving nutraceuticals

The analysis of urinary metabolite changes can provide information on the effects of food supplements or health-promoting products on healthy subjects or animal models. Specimen collection is non-invasive, long-term experiments can be easily conducted and urinary biomarkers of oxidative stress can also be measured, offering the opportunity to study the effects of a nutritional intervention and evaluate the redox status of the considered organism. In this thesis, three natural extracts were selected, namely Polygonum cuspidatum Sieb. et Zucc. (rich in resveratrol), Vaccinium macrocarpon Aiton (cranberry, rich in type A procyanidins) and green Coffea canephora Pierre ex Froehn. beans (GCBE, rich in chlorogenic acids), on the basis of their distribution on the market and on the basis of the information regarding their in vivo activity actually available in literature. In the first part of the work, the effects of P. cuspidatum dry extract were studied in healthy adult rats during a 49-days supplementation, using a combined 1H NMR and UPLC-HRMS metabolomics approach. Because of the reported antioxidant activity of resveratrol, the urinary amounts of two oxidative stress biomarkers were measured by targeted HPLC–MS/MS analyses and, due to the supposed “anti-aging” effects of resveratrol, multivariate models were designed in order to compare the aging effects between control and treated animals. Specific biomarkers were then selected and identified, and their amounts in urine were monitored throughout the experimental period. UPLC-MS metabolomics approaches were used to evaluate the mode of action of cranberry against uropathogenic Escherichia coli in two independent experiments, using an animal model and enrolling healthy adult volunteers, respectively. The experimental design was similar for the two trials, and the aim was to observe if the results obtained from the first animal experiment were reproducible in humans, being cranberry supplements claimed for treatment of UTIs in human consumers. In the first experiment, healthy Sprague−Dawley rats were orally supplemented with a standardized cranberry extract for 35 days, to mimic a prolonged consumption of cranberry by healthy subjects. 24-h urinary outputs were collected weekly during the experiment, and samples were subjected to UPLC−MS analysis using an untargeted approach. In a second experiment on the same animal model, a single dose of cranberry was administered to animals and the changes of urinary composition at 2, 4, 8, and 24 h after extract administration were monitored. Anti-adhesive properties of all the urine samples were studied. Furthermore, the markers related to cranberry intake were discovered using a multivariate data analysis approach. Finally, a specific chromatographic method was developed for the measurement of unmodified PAC-A in urine. In the experiment involving human volunteers, these consumed an oral sachet containing 360 mg of dry cranberry extract and 100 mg of quercetin. Urine samples were collected at 2, 4, 6, 8 ad 24 hours after product administration and the anti-adhesive properties of urine samples were tested using an in vitro assay on E. coli. In order to correlate possible observed bioactivity with modification of urinary composition, LC-MS-based targeted and untargeted metabolomics approaches were used. Finally, a clinical trial on a small number of healthy adult volunteers was performed to study the effects of a prolonged (30 days) supplementation with 400 mg of green coffee bean extract. The 24-h urinary samples were collected weekly, and analyzed by LC-MS. Multivariate data analysis approaches were applied and also targeted analysis were performed to measure urinary oxidative stress biomarkers, namely allantoin and 8-hydroxydeoxyguanosine (8-OHdG), in order to assess the potential antioxidant activity of GCBE in vivo

Secondary metabolites of alchemilla persica growing in Iran (East Azarbaijan)

Phytochemical investigations of Alchemilla persica Rothm. growing in Iran were performed taking into account both the volatile and polar constituents. The hydrodistilled essential oil was analysed by GC-MS that revealed the presence of diterpenoids (19.6 %) and sesquiterpenes (17.1%) as the major constituents, while tannins and flavonol glycosides were identified as the most abundant constituents of the methanol extract by HPLC-MS. A. persica can be a valuable source of ellagitannins and polyphenols

Development of an LC-DAD-MS-Based Method for the Analysis of Hydroxyanthracene Derivatives in Food Supplements and Plant Materials

Products based on plants containing hydroxyanthracene derivatives (HADs)-such as Rheum, Cassia, and Aloe species-are widely used in food supplements or nutraceuticals due to their laxative effects. A more restricted control of HAD contents in food supplements has been implemented by EU Regulation 2021/468, in order to increase the safety of these preparations. Due to their toxicity, aloin A, aloin B, aloe emodin, emodin, and the synthetic derivative danthron have been listed as prohibited substances in food supplements, being tolerated in amounts < 1 mg kg(-1) in marketed products. In this work, we report the development of a sensitive and fast LC-DAD-MS-based procedure for the determination of these five compounds in food supplements and plant materials or extracts. The entire procedure includes a simple sample preparation step, where target analytes are concentrated by means of solvent extraction and evaporative concentration (solid samples), or by lyophilisation (liquid samples). The average LOQ of 0.10 mg/L, LOD of 0.03 mg/L, accuracy, and precision with CVs below 12.72 were obtained for the studied analytes. This method is suitable for assessing the compliance of commercial products and raw materials with EU Regulation 2021/468. Furthermore, the proposed method can represent a starting point for the development of a unique and standardised analytical approach for the determination of other HADs under the attention of EU authorities

Total phytochemical analysis of Thymus munbyanus subsp. coloratus from Algeria by HS-SPME-GC-MS, NMR and HPLC-MSn studies

In this article, we report a comprehensive characterization of volatile and polar constituents extracted from the aerial parts of Thymus munbyanus subsp. coloratus, a shrub that is used as culinary ingredient and as traditional medicine in Algeria, mainly to treat respiratory and gastrointestinal disorders and endocrine dysfunctions. Headspace solid phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to assess volatile constituents, whereas the phytochemical composition of solid residues obtained from extraction with solvents at diffrent polarity was obtained by an integrated Nuclear Magnetic Resonance (NMR) and liquid chromatography coupled with tandem mass spectrometry (LC-MSn) approach. Fourty-five apolar ccompounds were identified, mainly oxygenated monoterpenes (65.8%), sesquiterpene hydrocarbons and nonoterpene hydrocarbons (18.6 and 14.5%, respectively). On the other hand, LC-MSn and NMR analyses revealed the presence of aglyconic and glycosilated flavonoids, phenylpropanoid derivatives and triterpenoid acids related to oleanolic acid, mainly in the methanol, dichloromethane and hexane extracts. Overall, these data indicate that Thymus munbyanus subsp. coloratus could be a potential source of antioxidants and bioactive compounds, and our results represent a starting point for further research on this plant species

Solid-State Fermentation of Trichoderma spp.: A New Way to Valorize the Agricultural Digestate and Produce Value-Added Bioproducts

In this study, the agricultural digestate from anaerobic biogas production mixed with food wastes was used as a substrate to grow Trichoderma reesei RUT-C30 and Trichoderma atroviride Ta13 in solid-state fermentation (SSF) and produce high-value bioproducts, such as bioactive molecules to be used as ingredients for biostimulants. The Trichoderma spp. reached their maximum growth after 6 and 3 SSF days, respectively. Both Trichoderma species were able to produce cellulase, esterase, and citric and malic acids, while T. atroviride also produced gibberellins and oxylipins as shown by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) profiling. Experimental evaluation of germination parameters highlighted a significant promotion of tomato seed germination and root elongation induced by T. atroviride crude extracts from SSF. This study suggests an innovative sustainable use of the whole digestate mixed with agro-food waste as a valuable substrate in fungal biorefineries. Here, it has been applied to produce plant growth-promoting fungi and bioactive molecules for sustainable agriculture

Exploring the Molecular Pathways Behind the Effects of Nutrients and Dietary Polyphenols on Gut Microbiota and Intestinal Permeability: A Perspective on the Potential of Metabolomics and Future Clinical Applications.

The gut microbiota is involved in the regulation of the intestinal permeability (IP), whose disruption is a frequent condition in older people and is associated with the development of several diseases. The diet can affect the gut microbiota and IP, although the molecular mechanisms involved are unclear. Metabolomics is one of the suitable approaches to study the effects of diet on gut microbiota and IP, although, up to now, the research has focused only on a few dietary components. The aim here was to review the most recent literature concerning the application of metabolomics to the study of the diet-induced alterations of gut microbiota and the effects on IP, with a particular focus on the molecular pathways involved. An additional aim was to give a perspective on the future research involving dietary polyphenols, because despite their potential use in the management of increased IP, few studies have been reported to date

Role of a Polyphenol-Rich Dietary Pattern in the Modulation of Intestinal Permeability in Older Subjects: The MaPLE Study

The inevitable rise of the proportion of people aged >65 years worldwide is paralleled by an increased burden of chronic diseases often associated with low-grade systemic inflammation. Recent findings suggest a link between inflammation and intestinal permeability (IP), a condition characterized by an impairment of intestinal barrier function which enables the translocation of dietary and bacterial factors into the blood activating the host immune system [1,2]. Dietary components can be significant modulators of inflammation and IP, and can also affect the intestinal microbial ecosystem. In the context of a diet-microbiota-IP axis in older subjects, dietary bioactives such as polyphenols may play a significant protective role due to their widely reported antioxidant and immunomodulatory properties and potential to regulate IP [3-6]

Intestinal permeability modulation through a polyphenol-rich dietary pattern in older subjects: MaPLE project outcomes and perspectives

In recent years, research has been focusing on strategies to counteract inflammatory processes and age-related diseases(1). During ageing, a low-grade systemic inflammation is often associated to an altered intestinal permeability (IP) a condition that has been shown to promote inflammation possibly through the translocation of dietary and bacterial factors into the blood stream that activates the immune system(2).In this regard, dietary pattern and environmental factors could play a fundamental role because of their potential ability to modulate inflammation, IP and the gut microbial ecosystem (GME). Moreover, it has been hypothesized that bioactive compounds such as polyphenols may affect IP and GME(3).The MaPLE project (Microbiome mAnipulation through Polyphenols for managing gut Leakiness in the Elderly) aimed to investigate the hypothesis that a polyphenol-rich diet can improve IP condition in a target population with beneficial changes at intestine and systemic level. To this aim, a randomised, controlled, crossover dietary intervention study (8-week polyphenol-rich diet versus 8-week control diet, separated by a wash-out period) was carried out in a group of older subjects (> 60 years) living in a well-controlled setting (i.e. nursing home). Markers related with IP, inflammation, oxidative stress, vascular function and intestinal microbial ecosystem were investigated in serum, urine and/or fecal samples. Moreover, blood bacteria DNAemia, and serum/urine metabolomics has been assessed. Moreover, a consistent nutritional evaluation of the standard menu (provided by the nursing home) and of weighed food diaries was performed, providing also data on actual polyphenol intake during the intervention. The results show there were higher levels of IP in the older subjects, and that the polyphenol-enriched diet changed the levels of serum zonulin, a marker of IP. In addition, an association between zonulin and blood bacterial load was demonstrated. Ongoing in vitro and in vivo experiments are exploring the potential effects of different polyphenols on IP and the mechanisms involved. The MaPLE project will generate new data to improve the understanding on the role of polyphenols in the modulation of intestinal microbiome and its interactions with the host