Nutraceutical formulation for immune system modulation: Active constituents, in vitro antibacterial and immunomodulatory activity, and metabolomics analysis.

There is a large demand for nutraceuticals in the market and studies related to their action are needed. In this paper, the antimicrobial activity and the immunomodulatory effect of a nutraceutical formulation containing 14.39% of ascorbic acid, 7.17% of coenzyme Q10, 1.33% of Echinacea polyphenols, 0.99% of pine flavan-3-ols, 0.69% of resveratrol and 0.023% of Echinacea alkylamides were studied using in vitro assays and cell-based metabolomics. Chromatographic analysis allowed us to study the nutraceutical composition. The antibacterial activity was evaluated on S. aureus, K. pneumoniae, P. aeruginosa, E. coli, H. influenzae, S. pyogenes, S. pneumoniae and M. catarrhalis. The immunomodulatory activity was assessed on human macrophages and dendritic cells. The production of IL-1ß, IL-12p70, IL-10 and IL-8 was evaluated on culture medium by ELISA and the activation/maturation of dendritic cells with cytofluorimetric analysis. Treated and untreated macrophages and dendritic cell lysates were analysed by liquid chromatography coupled with high-resolution mass spectrometry, and results were compared using multivariate data analysis to identify biological markers related to the treatment with the food supplement. The food supplement decreased K. pneumoniae, P. aeruginosa, E. coli, Methicillin-resistant Staphylococcus aureus (MRSA) and M. catharralis growth, reduced the inflammatory response in macrophages exposed to lipopolysaccharide (LPS) and modulated the activation and maturation of the dendritic cells. Oxidized phospholipids were identified as the main biological markers of treated cell lysates, compared with controls.

Linking traditional medicine to modern phytotherapy: Chemical characterization and assessment of antioxidant and anticholinesterase effects in vitro of a natural Persian remedy for dementia.

Several natural remedies are used in the Traditional Persian Medicine (TPM) to prevent dementia, but their efficacy is debated. In this work, an improved "Safoof-e-Nesyan" formulation described in the "Qarabadin-e-Azam" pharmacopoeia was developed, and its chemical composition and antioxidant and anti-cholinesterase properties were assessed. The formulation contains a mixture (FM) of Cinnamomum cassia (CC), C. verum (CV), Pistacia lentiscus (PL), Rheum palmatum (RP), Syzygium aromaticum (SA), and Zingiber officinalis (ZO) powdered plants. Its total phenolic content is 110.45 mg GAE/g, while the total flavonoid content is 6.28 mg RE/g. 66 secondary metabolites (mainly tannins, flavonoids, anthraquinones, and gingerols) were identified by UPLC-QToF-MS analysis. FM exerts antioxidant effects by scavenging radicals, and by reducing and chelating metals such as Mb, Cu and Fe. The anticholinesterase activity of one gram of the FM equals that of 3.60 mg of the reference drug galantamine, on both acetyl- and butyryl-cholinesterase. Correlations between specific compounds and bioactivities were highlighted by multivariate analysis of data: lyoniresinol 9'-glucoside strongly correlates with antiradical activities on DPPH and ABTS and reducing activity on Cu, and with anti-AChE effects. Most of the identified flavonoids and the ellagic acid derivatives positively correlate with the reducing activity on Fe and Mb, and with anti-BChE effects. Finally, a tablet formulation of the FM was developed, and its physical properties were preliminarily assessed. Overall, our results indicate that the FM may be a useful natural remedy for dementia, although further safety and efficacy assessments in vivo are required.

"Smart Extraction Chain" with Green Solvents: Extraction of Bioactive Compounds from Picea abies Bark Waste for Pharmaceutical, Nutraceutical and Cosmetic Uses.

Secondary metabolites from the sawmill waste Picea abies bark were extracted using an innovative two-step extraction that includes a first step with supercritical CO2 (SCO2) and a second step using green solvents, namely ethanol, water, and water ethanol mixture. Maceration (M), ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE) techniques were applied in the second step. A total of nineteen extract were obtained and yield were compared. Bark extracts were characterized by LC-DAD-MSn and classes of compounds were quantified as abietane derivatives, piceasides, flavonoids, and phenolics to compare different extractions. Obtained extracts were studied by in vitro assay to evaluate potential pharmaceutical, nutraceutical and cosmetic uses assessing the antioxidant activity as well as the inhibitory activity on target enzymes. Results show that the "smart extraction chain" is advantageous in term of yield of extraction and phytoconstituent concentration. SCO2 extract, presenting a unique composition with a large amount of abietane derivatives, exerted the best activity for amylase inhibition compared to the other extracts.

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 species­are 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.

Characterization of PACs profile and bioactivity of a novel nutraceutical combining cranberry extracts with different PAC-A oligomers, D-mannose and ascorbic acid: An in vivo/ex vivo evaluation of dual mechanism of action on intestinal barrier and urinary epithelium.

In this paper, an A-type procyanidin (PAC)-rich cranberry extract (CB-B) was obtained mixing different extracts and was formulated with D-mannose and ascorbic acid to obtain a novel nutraceutical (URO-F) aimed at preventing non-complicated bacterial urinary tract infections (UTIs). To assess the bioactivity of CB-B and URO-F, urine samples collected from six healthy volunteers undergoing a 2-days oral consumption of 0.41 g/day of CB-B or 10 g/day of URO-F (corresponding to 72 mg/day of PACs) were tested against uropathogenic E. coli (UPEC) incubated on urinary bladder epithelial cells (T24). Urinary markers of CB-B and URO-F consumption were assessed in the same urine output by UPLC-QTOF-based untargeted metabolomics approach. CB-B and URO-F were evaluated for their ability to promote the intestinal barrier function by restoring the trans-epithelial electrical resistance (TEER) and to inhibit the production of inflammatory cytokines in intestinal epithelial Caco2 cells. CB-B was characterized by a high PAC-A content (70% of total PACs) and a broad distribution of different PACs polymers (dimers-hexamers). Urine from subjects consuming CB-B and URO-F showed a significant effect in reducing the adhesion of UPEC to urothelium in vitro, supporting their efficacy as anti-adhesive agents after oral intake. CB-B inhibited the release of cytokine IL-8, and both products were effective in restoring the TEER. Overall, our results show that the beneficial effects of CB-B and URO-F on UTIs are not only due to the antiadhesive activity of cranberry on UPEC in the urothelium, but also to a multi-target activity involving anti-inflammatory and permeability-enhancing effects on intestinal epithelium.

Analysis of Monacolins and Berberine in Food Supplements for Lipid Control: An Overview of Products Sold on the Italian Market.

The use of dietary supplements for the prevention and management of diseases associated with excess of lipids is spreading in Western countries. Supplements containing red yeast rice (RYR) and extracts from Berberis species, characterized, respectively, by the active compounds monacolin K (MK) and berberine (BBR), are sold in pharmacies as over the counter medicines (OTC) and in regular markets without the need of medical prescription and medical surveillance. However, MK is chemically identical to lovastatin, a drug commonly used to treat hypercholesterolemia, and is characterized by the same mechanism of action, pharmacokinetic profile and toxicity. On the other hand, although BBR-containing supplements are considered to be well-tolerated and safe, they frequently show poor standardization of active ingredients, and this could lead to lack of effects. In this work, with the aim to give an overview on the potency of RYR- and BBR-containing supplements available on the Italian market, we analyzed a pool of supplements bought from both local pharmacies and markets. Results confirm the data already published by other authors, showing scarce standardization of bioactives and discrepancy between the doses of bioactives reported by the manufacturers and the amounts resulting from analysis of the same products. Overall, our data represent a further proof that a strict legislation regulating the production and marketing of dietary supplements and a close monitoring of these products by food and drug regulatory organs is mandatory.

Rosa chinensis in vitro cell cultures: a phytocomplex rich of medium molecular weight polysaccharides with hydrating properties.

In-vitro cell cultures of selected Rosa chinensis meristematic cells cultivated with an innovative CROP® (Controlled Release of Optimized Plants) platform, allowed obtaining a stable and standardized phytocomplex rich of medium molecular weight polysaccharides. The polysaccharides profile of the rose extract has been analysed with the size exclusion chromatography (HPLC-ELSD-SEC) both in the in vitro extract and in the dried petals of Rosa chinensis. The polysaccharides content in the extract was ≥20%, higher than in the dried petals. The 65-80% of total polysaccharides have a medium molecular weight (1000 Da), known for their moisturizing and anti-age properties. Reconstructed human epidermis in homeostatic conditions was used to evaluate its moisturizing action and the ability to maintain homeostasis. The Rosa chinensis extract increased the Aquaporin-3 expression and cell membrane localization and demonstrated to regulate hydration either in topical and systemic exposure.

Phytochemical investigations on Artemisia alba Turra growing in the North-East of Italy.

Artemisia alba Turra (Asteraceae) is an Euro-Mediterranean plant used in Veneto (North-East of Italy) as traditional medicine for the treatment of various diseases. A. alba is a taxonomically problematic species, characterised by common polymorphism leading to a quite high variability in secondary metabolites content. Nonetheless, the phytochemical knowledge on its phytoconstituents, especially non-volatile components, is limited. In the present paper, the phytochemical composition of a tincture obtained from the aerial parts of A. alba growing in Veneto is presented. Extensive chromatographic separations led to the isolation of three new sesquiterpene derivatives, whose structures were elucidated by 1D and 2D NMR experiments and mass spectrometry. Furthermore, flavonoid composition and volatile constituents of the tincture of A. alba were preliminary studied by HPLC-MSn and GC-MS, respectively.

Natural Deep Eutectic Solvents (NADES) as a Tool for Bioavailability Improvement: Pharmacokinetics of Rutin Dissolved in Proline/Glycine after Oral Administration in Rats: Possible Application in Nutraceuticals.

There is a need for innovation in plant-derived pharmaceuticals, food supplements and nutraceutical products regarding the use of more eco-sustainable solvents for their extraction. Furthermore, the poor oral bioavailability of several phytochemicals with health promoting effects stimulates the research in the field of pharmaceutical formulations. Natural Deep Eutectic Solvents (NADES) are formed by natural compounds, and can be considered as future solvents being especially useful for the preparation of nutraceuticals and food-grade extracts. In this paper various NADES were prepared using sugars, aminoacids and organic acids. Rutin (quercetin-3-O-α-l-rhamnopyranosyl-(1→6))-ß-d-glucopyranose) was used as a model compound to study NADES. Moreover, the effect of various eutectic mixtures on rutin's water solubility was studied. Proline/glutamic acid (2:1) and proline/choline chloride (1:1) mixtures have a solubility comparable to ethanol. The proline/glutamic acid (2:1) eutectic containing rutin was used in a pharmacokinetic study in Balb/c mice while bioavailability was compared to oral dosing of water suspension. Plasmatic levels of rutin were measured by HPLC-MS/MS showing increased levels and longer period of rutin permanence in plasma of NADES treated animals. This paper reports the possible use of non-toxic NADES for pharmaceutical and nutraceutical preparations.

Nutraceuticals, A New Challenge for Medicinal Chemistry.

"Nutraceuticals" are food-derived products largely used for their presumed healthpromoting or disease-preventing effects. In the recent years, many efforts have been aimed at assessing nutraceutical efficacy and safety, but these factors are difficult to address because of the complex chemical compositions and multiple mode of actions. Thus, the study of nutraceutical ingredients poses several challenges for the medicinal chemistry field, some of which are related to extraction and chemical characterization, some to in vitro and in vivo bioactivity evaluation, and some to the bioavailability and interaction of these natural mixtures with organs and microbiota. Furthermore, because of their nature as medicinal and food products, these nutraceuticals can also be considered as a valuable source of new "lead compounds", creating the opportunity to discover new classes of therapeutic agents. This review provides information on these themes, showing the new challenges that comprehensive medicinal chemistry research is called to answer in the field of nutraceuticals.