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The importance of nuclear magnetic resonance (NMR) spectroscopy for the food industry, including the coffee industry, has led to several studies using this analytical tool to assess coffee in different scenarios. In this context, this chapter presents an overview of 1H NMR-based metabolomics analysis used in coffee quality control. Several articles using 1H NMR associated with multivariate data analysis have been published to investigate aspects related to quality, authenticity, sensory quality, production processes, and origin of coffee. In addition, the present review reports the main chemical shifts of the constituents present in the extracts of green and roasted coffee beans. The results presented confirm the potential and versatility of 1H NMR combined with chemometric tools for coffee analysis, playing an important role in distinguishing and identifying the origins, quality, and purity of coffee. Exploratory data analysis, classification, and calibration are the most used chemometric methods in metabolomics applied in the coffee field. The results show the potential of NMR and multivariate data analysis for coffee authentication.

To tackle the urgency of smarter crop management, the complex nature of agricultural ecosystems needs to be better understood, employing and combining different techniques and technologies. In this study, untargeted metabolomics and agro-meteorological survey were coupled to study the variation of Sorghum bicolor (L.) Moench metabolome during crop development, in response to environmental and anthropic factors. Twelve crop fields in the Emilia-Romagna region, Italy, were monitored and sampled at different stages, seedling (Ss), advanced vegetative (Sv), and ripening (Sr), and subjected to 1H NMR-based metabolomics. The analytical method developed resulted to be successful to quickly analyze different sorghum organs. Dhurrin, a cyanogenic glucoside, resulted to be a biomarker of crop quality and development, and several insights into its turnover and functions were obtained. In particular, p-glucosyloxy-2-hydroxyphenylacetic acid was identified, for the first time, as the main metabolite accumulated in sorghum at Sr, after gradual dhurrin neutralization. During plant life, fertilization and biotic and abiotic stress reflected peculiar metabolomic profiles. Water supply and soil features (i.e., clay content) were correlated to metabolomic variations, affecting dhurrin (and related metabolites), amino acids, organic acids, and carbohydrate content. Increase in chlorogenic acid was registered in consequence of predator attacks. Moreover, grain from three fields presented traces of dhurrin and the lowest antioxidant potential, which resulted in poor grain quality. Metabolomics turned out to be a promising tool in view of smart agriculture for monitoring plant growth status and applying appropriate agricultural practices since the early stage of crop development.

Due to the high prevalence of obesity and type 2 diabetes, adipogenesis dysfunction and metabolic disorders are common features in the elderly population. Thus, the identification of novel compounds with anti-adipogenic and lipolytic effects is highly desirable to reduce diabetes complications. Plants represent an important source of bioactive compounds. To date, the antidiabetic potential of several traditional plants has been reported, among which

The Aeolian Islands (Italy) are a volcanic archipelago in the Tyrrhenian Sea comprising seven main islands, among which are two active volcanoes. The peculiar geological features and the wide variety of environments and soils have an important impact on native plants, and in particular, the Aeolian populations of

Due to the high prevalence of obesity and type 2 diabetes, adipogenesis dysfunction and metabolic disorders are common features in the elderly population. Thus, the identification of novel compounds with anti-adipogenic and lipolytic effects is highly desirable to reduce diabetes complications. Plants represent an important source of bioactive compounds. To date, the antidiabetic potential of several traditional plants has been reported, among which Ficus carica L. is one of the most promising. Considering that plant metabolome changes in response to a number of factors including seasonality, the aim of this study was to evaluate whether Ficus carica leaves extracts collected in autumn (FCa) and spring (FCs) differently modulate lipid metabolism and adipogenesis in 3T3-L1 adipocytes. The 1H-NMR profile of the extracts showed that FCs have a higher content of caffeic acid derivatives, glucose, and sucrose than FCa. In contrast, FCa showed a higher concentration of malic acid and furanocoumarins, identified as psoralen and bergapten. In vitro testing showed that only FCa treatments were able to significantly decrease the lipid content (Ctrl vs. FCa 25 μg/mL, 50 μg/mL and 80 μg/mL; p < 0.05, p < 0.01 and p < 0.001, respectively). Furthermore, FCa treatments were able to downregulate the transcriptional pathway of adipogenesis and insulin sensitivity in 3T3-L1 adipocytes. In more detail, FCa 80 μg/mL significantly decreased the gene expression of PPARγ (p < 0.05), C/EBPα (p < 0.05), Leptin (p < 0.0001), adiponectin (p < 0.05) and GLUT4 (p < 0.01). In conclusion, this study further supports an in-depth investigation of F. carica leaves extracts as a promising source of active compounds useful for targeting obesity and diabetes.

Proteasome inhibition represents an important anticancer strategy. Here, we studied the mechanisms at the basis of the pro-apoptotic activity of the standardized decoction of Hemidesmus indicus, a plant evoking a complex anticancer activity, and explored its inhibition of proteasome activity in human leukemia cells. Additionally, we preliminary tested the cytotoxicity of some H. indicus’s phytochemicals on leukemia cells and their intestinal absorption on a human intestinal epithelium model consisting of a monolayer of differentiated Caco2 cells. We observed a potent antileukemic effect for H. indicus, imputable to the modulation of different critical targets at protein and mRNA levels and the reduction of the 26S proteasome expression. We found that some phytomarkers of H. indicus decoction passed through the enterocyte monolayer. Overall, our study supports the pharmacological potential of H. indicus, which can represent an interesting botanical drug in the oncological area.

Plant cell culture is a biotechnology cultivation method that permit to cultivate plants in a short period of time and to obtain extracts with a high degree of standardization and high safety profile. The aim of our study was to evaluate the anti-inflammatory and neuroprotective activity of a standardized Melissa officinalis L. phytocomplex extract (MD) obtained with an in vitro plant cell culture. The MD has been chemically characterized and the content of total polyphenols was 5.17 ± 0.1 % w/w, with a content of rosmarinic acid (RA), its main constituent, of 4.02 ± 0.1 % w/w. MD was tested in an in vitro model of neuroinflammation, in which microglia cells (BV2) were stimulated with Lipopolysaccharides (LPS; 250 ng/mL) for 24 h and its pharmacological activity was compared with that of RA. MD (10 µg/mL) and RA (0.4 μg/mL) reduced pro-inflammatory factors (NF-kB, HDAC, IL-1ß) in LPS-stimulated BV2 cells and counteracted the toxic effect produced by activated microglia medium on neuronal cells. This work shows the efficacy of MD on reducing microglia-mediated neuroinflammation and promoting neuroprotection, highlighting the innovative use of in vitro plant cell cultures to obtain contaminant-free extracts endowed with marked activity and improved quali-quantitative ratio in the constituents' content.

Background: Muscle atrophy, i.e., the loss of skeletal muscle mass and function, is an unresolved problem associated with aging (sarcopenia) and several pathological conditions. The imbalance between myofibrillary protein breakdown (especially the adult isoforms of myosin heavy chain, MyHC) and synthesis, and the reduction of muscle regenerative potential are main causes of muscle atrophy. Methods: Starting from one-hundred dried hydroalcoholic extracts of medical plants, we identified those able to contrast the reduction of C2C12 myotube diameter in well-characterized in vitro models mimicking muscle atrophy associated to inflammatory states, glucocorticoid treatment or nutrient deprivation. Based on their ability to rescue type II MyHC (MyHC-II) expression in atrophying conditions, six extracts with different phytochemical profiles were selected, mixed in groups of three, and tested on atrophic myotubes. The molecular mechanism underpinning the effects of the most efficacious formulation, and its efficacy on myotubes obtained from muscle biopsies of young and sarcopenic subjects were also investigated. Results: We identified WST (Withania somnifera, Silybum marianum, Trigonella foenum-graecum) formulation as extremely efficacious in protecting C2C12 myotubes against MyHC-II degradation by stimulating Akt (protein kinase B)-dependent protein synthesis and p38 MAPK (p38 mitogen-activated protein kinase)/myogenin-dependent myoblast differentiation. WST sustains trophism in C2C12 and young myotubes, and rescues the size, developmental MyHC expression and myoblast fusion in sarcopenic myotubes. Conclusion: WST strongly counteracts muscle atrophy associated to different conditions in vitro. The future validation in vivo of our results might lead to the use of WST as a food supplement to sustain muscle mass in diffuse atrophying conditions, and to reverse the age-related functional decline of human muscles, thus improving people quality of life and reducing social and health-care costs.

Oregano is one of the most used culinary herb and it is often adult­erated with cheaper plants. In this study, GC–MS was used for identification and quan­tification of metabolites from 104 samples of oregano (Origanum vul­gare and O. onites) adulterated with olive (Olea europaea), venetian sumac (Cotinus coggy­gria) and myrtle (Myrtus communis) leaves, at five different concentration levels. The metabolomics profiles obtained after the two-step derivatization, involving methoxyamination and silanization, were subjected to multivariate data analysis to reveal markers of adulteration and to build the reg­ression models on the basis of the oregano-to-adulterants mixing ratio. Ortho­gonal partial least squares enabled detection of oregano adulterations with olive, Venetian sumac and myrtle leaves. Sorbitol levels distinguished oregano samples adulterated with olive leaves, while shikimic and quinic acids were recognized as discrimination factor for adulteration of oregano with venetian sumac. Fructose and quinic acid levels correlated with oregano adulteration with myrtle. Orthogonal partial least squares discriminant analysis enabled dis­crimination of O. vulgare and O. onites samples, where catechollactate was found to be discriminating metabolite. Оригано је једна од најчешће коришћених кулинарских биљака и често се криво- твори јефтинијим биљкама. У овој студији, гаснa хроматографијa–масенa спектро- метријa коришћена је за идентификацију и квантификацију метаболита из 104 узорка оригана (Origanum vulgare и O. onites) кривотвореног маслином (Olea europea), венеци- јанским сумаком (Cotinus coggygria) и миртом (Myrtus communis), у пет различитих концентрација. Метаболомички профили добијени након двостепене дериватизације, која укључује метоксиаминовање и силанизацију, подвргнути су мултиваријантној ана- лизи података како би се открили маркери кривотворења и направили регресиони модели на основу односа мешања оригана и биљака за кривотворење. Ортогонална дели- мична анализа најмањих квадрата је омогућила детекцију кривотворења оригана лишћем маслине, венецијанског сумака и мирте. Садржај сорбитола разликовао је узорке оригана кривотворених лишћем маслине, док су шикиминска и кининска кисе- лина препознате као фактор разликовања за кривотворење оригана венецијанским сумаком. Садржај фруктозе и кининске киселине у корелацији су са кривотворењем оригана миртом. Ортогонална делимична анализа најмањих квадрата – дискриминантна анализа је омогућила разликовање узорака O. vulgare и O. onites, при чему је одређено да је катехоллактат метаболит који разликује ове две биљне врсте. Part of the theme issue honoring Professor Emeritus Slobodan Milosavljevićs 80th birthday.

Background: Muscle atrophy, i.e., the loss of skeletal muscle mass and function, is an unresolved problem associated with aging (sarcopenia) and several pathological conditions. The imbalance between myofibrillary protein breakdown (especially the adult isoforms of myosin heavy chain, MyHC) and synthesis, and the reduction of muscle regenerative potential are main causes of muscle atrophy. Methods: Starting from one-hundred dried hydroalcoholic extracts of medical plants, we identified those able to contrast the reduction of C2C12 myotube diameter in well-characterized in vitro models mimicking muscle atrophy associated to inflammatory states, glucocorticoid treatment or nutrient deprivation. Based on their ability to rescue type II MyHC (MyHC-II) expression in atrophying conditions, six extracts with different phytochemical profiles were selected, mixed in groups of three, and tested on atrophic myotubes. The molecular mechanism underpinning the effects of the most efficacious formulation, and its efficacy on myotubes obtained from muscle biopsies of young and sarcopenic subjects were also investigated. Results: We identified WST (Withania somnifera, Silybum marianum, Trigonella foenum-graecum) formulation as extremely efficacious in protecting C2C12 myotubes against MyHC-II degradation by stimulating Akt (protein kinase B)-dependent protein synthesis and p38 MAPK (p38 mitogen-activated protein kinase)/myogenin-dependent myoblast differentiation. WST sustains trophism in C2C12 and young myotubes, and rescues the size, developmental MyHC expression and myoblast fusion in sarcopenic myotubes. Conclusion: WST strongly counteracts muscle atrophy associated to different conditions in vitro. The future validation in vivo of our results might lead to the use of WST as a food supplement to sustain muscle mass in diffuse atrophying conditions, and to reverse the age-related functional decline of human muscles, thus improving people quality of life and reducing social and health-care costs.

Castanea sativa cultivation has been present in Mediterranean regions since ancient times. In order to promote a circular economy, it is of great importance to valorize chestnut groves’ by-products. In this study, leaves and spiny burs from twenty-four Castanea trees were analyzed by 1H NMR metabolomics to provide an overview of their phytochemical profile. The Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) performed on these data allowed us to distinguish ‘Marrone’ from ‘Castagna’, since the latter were generally more enriched with secondary metabolites, in particular, flavonoids (astragalin, isorhamnetin glucoside, and myricitrin) were dominant. Knowing that microglia are involved in mediating the oxidative and inflammatory response of the central nervous system, the potential anti-inflammatory effects of extracts derived from leaves and spiny burs were evaluated in a neuroinflammatory cell model: BV-2 microglia cells. The tested extracts showed cytoprotective activity (at 0.1 and 0.5 mg/mL) after inflammation induction by 5 µg/mL lipopolysaccharide (LPS). In addition, the transcriptional levels of IL-1β, TNF-α, and NF-kB expression induced by LPS were significantly decreased by cell incubation with spiny burs and leaves extracts. Taken together, the obtained results are promising and represent an important step to encourage recycling and valorization of chestnut byproducts, usually considered “waste”.

Coffee quality is determined by several factors and, in the chemometric domain, the multi-block data analysis methods are valuable to study multiple information describing the same samples. In this industrial study, the Common Dimension (ComDim) multi-block method was applied to evaluate metabolite fingerprints, near-infrared spectra, sensory properties, and quality parameters of coffee blends of different cup and roasting profiles and to search relationships between these multiple data blocks. Data fusion-based Principal Component Analysis was not effective in exploiting multiple data blocks like ComDim. However, when a multi-block was applied to explore the data sets, it was possible to demonstrate relationships between the methods and techniques investigated and the importance of each block or criterion involved in the industrial quality control of coffee. Coffee blends were distinguished based on their qualities and metabolite composition. Blends with high cup quality and lower roasting degrees were generally differentiated from those with opposite characteristics.

In search for natural products of cosmetic interest, a hundred plant extracts were in vitro tested against elastase and tyrosinase. The inhibitors of these enzymes find application as skin whitening, anti-ageing, anti-wrinkle agents as well as in the treatment of dermatological disorders. Among the tested samples, seventeen extracts resulted strongly active. In particular, eleven out of them were capable to inhibit both enzymes, five showed a strong activity only against tyrosinase and one only against elastase. The IC50 values of the selected samples ranged from 7 to 100 μg/mL and from 20 to 100 μg/mL against elastase and tyrosinase, respectively. Leaves extract of Pistacia lentiscus emerged as the most potent elastase inhibitor and, together with Cytinus hypocistis (aerial parts) and Limonium morisianum (aerial parts), it showed also the lowest IC50 of tyrosinase inhibition. The tested plants were collected in India, Africa and Mediterranean area. Interestingly, among the most active ones, two are endemic and exclusive of Sardinia Island (Italy), namely: Limonium morisianum and Hypericum scruglii, moreover, the latter resulted the only plant which hydroalcoholic extract was capable to inhibit elastase selectively. Moreover, a positive correlation was established among the potency of enzymatic inhibitions and the total phenolic and flavonoid content of the samples. The presence of these aromatic compounds in the most active plants confers them a potential additional value as skin protectors from oxidative damage.

This paper describes the use of 1H NMR profiling and chemometrics in order to facilitate the selection of medicinal plants as potential sources of collagenase inhibitors. A total of 49 plants with reported ethnobotanical uses, such as the healing of wounds and burns, treatment of skin-related diseases, rheumatism, arthritis, and bone diseases, were initially chosen as potential candidates. The in vitro collagenase inhibitory activity of hydroalcoholic extracts of these plants was tested. Moreover, their phytochemical profiles were analyzed by 1H NMR and combined with the inhibitory activity data by an orthogonal partial least squares model. The results showed a correlation between the bioactivity and the concentration of phenolics, including flavonoids, phenylpropanoids, and tannins, in the extracts. Considering the eventual false-positive effect on the bioactivity given by tannins, a tannin removal procedure was performed on the most active extracts. After this procedure, Alchemilla vulgaris was the most persistently active, proving to owe its activity to compounds other than tannins. Thus, this plant was selected as the most promising and further investigated through bioassay-guided fractionation, which resulted in the isolation of a flavonoid, quercetin-3-O-β-glucuronide, as confirmed by NMR and HRMS spectra. This compound showed not only a higher activity than other flavonoids with the same aglycone moiety, but was also higher than doxycycline (positive control), the only Federal Drug Administration-approved collagenase inhibitor. The approach employed in this study, namely the integration of metabolomics and bioactivity-guided fractionation, showed great potential as a tool for plant selection and identification of bioactive compounds in natural product research.

Ptilostemon casabonae (L.) Greuter is a Mediterranean endemism traditionally used for its health-giving properties. Little is known about this species, therefore this study provides additional information about the phytochemical and biomolecular patterns of this plant, to have a combined fingerprint as a taxonomic tool. Several P. casabonae specimens were therefore collected from three different sites, two from Sardinia (Italy) and one from Corsica and the hydroalcoholic extracts of their aerial parts were investigated through HPLC-PDA-MS/MS analysis to study the phenolic composition. Quercetin, luteolin, kaempferol, apigenin and diosmetin O-glycosides, and caffeoylquinic acid derivatives were found as main components. Samples from the three sites showed similar phenolic profiles, although statistical analyses highlighted some quantitative differences for several compounds. The biomolecular analysis included amplification and sequencing of ITS, 5S-rRNA-NTS and psbA regions. No difference was found in the nucleotides among the P. casabonae samples from different geographical origins; however, a comparison with other Ptilostemon species sequences from Genbank, revealed an interspecific variability of ITS and psbA regions. The combination of the results of the phytochemical and biomolecular studies provide information on P. casabonae useful to depict this little-known plant, which can also be applied for future investigations and to obtain a fingerprint of it. Moreover, the stability of the phenolic profile within the species affords to identify a set of specialised metabolites useful for its chemotaxonomic characterization. At the same time, the stability of the biomolecular profile of P. casabonae, and the identification of sequences specific for this species, enables to identify useful biomolecular markers to distinguish it unequivocally.

This chapter presents the correlation between coffee compounds identified by 1H Nuclear Magnetic Resonance (1H NMR) spectroscopy with psychological attention tests in order to verify which compounds are related to the focus and/or diffuse attention. Psychometric tests applied by a clinical psychologist, before and after coffee intake, were the focus attention AC-vector and the diffuse attention TADIM, and the focus attention TACOM-B and the diffuse attention TEDIF, respectively. Different tests to measure the attention before and after coffee intake were used to avoid learning effects. After AC-vector and TADIM tests, each volunteer consumed a total of 40 mL of coffee with different cup qualities (four different coffee blends – 10 mL per beverage) and indicated the order of preference in relation to the smell. This approach was used to create a greater metabolic variation between the samples tested, allowing to build a robust chemometric model. For each preferred coffee, a 1H NMR spectrum was obtained and a chemometric data treatment based on Partial Least Squares (PLS) regression and Variable Importance in Projection scores (VIP scores) was used to correlate the spectra with the psychological test results and to verify which metabolites of the coffee beverage could be related to the focus or diffuse attention. In general, our results showed that coffee intake attenuated diffuse attention and improved focus attention in most volunteers. The major metabolites that contributed to both diffuse and focus attention were caffeine, trigonelline, chlorogenic acids, acetate, lipids, lactate, γ-quinide, and polysaccharides. Among metabolites exclusively important to focus attention, formate, choline, myo-inositol, citrate, and malate were the most important. Therefore, the 1H NMR profile, in combination with chemometric tools, is interesting to assess the correlation between coffee compounds and human attention.

Sardinia (Italy), with its wide range of habitats and high degree of endemism, is an important area for plant-based drug discovery studies. In this work, the antitumor activity of 35 samples from Sardinian plants was evaluated on human osteosarcoma cells U2OS. The results showed that five plants were strongly antiproliferative: Arbutus unedo (AuL), Cynara cardunculus (CyaA), Centaurea calcitrapa (CcA), Smilax aspera (SaA), and Tanacetum audibertii (TaA), the latter endemic to Sardinia and Corsica. Thus, their ability to induce cell cycle arrest and apoptosis was tested. All extracts determined cell cycle block in G2/M phase. Nevertheless, the p53 expression levels were increased only by TaA. The effector caspases were activated mainly by CycA, TaA, and CcA, while AuL and SaA did not induce apoptosis. The antiproliferative effects were also tested on human umbilical vein endothelial cells (HUVEC). Except for AuL, all the extracts were able to reduce significantly cell population, suggesting a potential antiangiogenic activity. The phytochemical composition was first explored by 1H NMR profiling, followed by further purifications to confirm the structure of the most abundant metabolites, such as phenolic compounds and sesquiterpene lactones, which might play a role in the measured bioactivity.

Berberine (BBR) is a natural active principle with potential antitumor activity. The compound targets multiple cell signaling pathways, including proliferation, differentiation, and epithelial–mesenchymal transition. The aim of this study was to elucidate the mechanisms behind the anticancer activity of BBR by comparing the effects of purified BBR with those of the extract of Tinospora cordifolia, a medicinal plant that produces this metabolite. The expression levels of a panel of 44 selected genes in human colon adenocarcinoma (HCA-7) cell line were quantified by real-time polymerase chain reaction (PCR). BBR treatment resulted in a time- and dose-dependent down regulation of 33 genes differently involved in cell cycle, differentiation, and epithelial–mesenchymal transition. The trend was confirmed across the two types of treatment, the two time points, and the different absolute dosage of BBR. These findings suggest that the presence of BBR in T. cordifolia extract significantly contributes to its antiproliferative activity.

The aim of this study was to determine the in vitro antimicrobial activity of crude extracts of three plants from Ayurveda tradition (Tinospora cordifolia, Alstonia scholaris, Crataeva nurvala) against reference microbial strains and clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenemase-producing Klebsiella pneumoniae. IC50 values were obtained by micro-dilution methods meeting the requirements of the NCCLS standard. The cytotoxicity of the extracts was also investigated on a mammalian cell line. Extracts displayed a variable degree of antimicrobial activity and did not interfere with mammalian cell proliferation. T. cordifolia and A. scholaris exhibited a higher inhibitory activity against clinical isolates of MRSA and carbapenemase-producing K. pneumoniae compared with reference strains, while C. nurvala exhibited a different behaviour. An antifungal activity towards Candida albicans was observed for A. scholaris extract. Results indicate that constituents from T. cordifolia and A. scholaris may be a potential source of new therapeutic strategies for infectious diseases.

Sambucus nigra L. (Caprifoliaceae) is wide spread in temperate and sub-tropical zones. The consumption of its berries has been associated with health benefits especially for its high content of natural antioxidants such as polyphenols, in particular anthocyanins. In this work we investigated the polyphenolic composition and the in vitro antioxidant activities (ABTS, DPPH, BCB and FRAP-ferrozine (FRAP-FZ) assays) of S. nigra berries, collected in four different Sicilian areas (Italy). Elderberries are considered one of the fruits with highest anthocyanins content, the amount of phenolic compounds, other than anthocyanins, is approximately 1.5 times greater than the latter. The LC–MS analyses have revealed an opposite trend in the polyphenols pattern in Sicilian populations. Moreover, a statistical correlation was found between cyanidin-3- sambubioside-5-glucoside and antioxidant activity evaluated by FRAP-FZ and ABTS assays. In conclusion, Sicilian S. nigra berries are appealing for its antioxidant potential and for its particularly high content of anthocyanins.

Vitis vinifera L. (Vitaceae family) is a popular woody perennial plant commonly used in the winemaking. Unfortunately, viticulture generates huge amounts of residues (i.e. leaves, twigs, seeds, skins), which can cause environmental issues when discarded in open areas. It is therefore extremely important to evaluate the potential reuse of these matrices, to reduce the amount of waste at landfill sites [1],[2]. Among the main grapevine by-products, no data on the green pruning residues (GPRs) produced by the annual spring pruning have been reported. Thus, the aim of this work was the analysis and valorization of GPRs from 16 red and white V. vinifera cultivars from Piedmont (Italy), by comparing the results with those obtained from the leaves of the same cultivars. An experimental design optimization was performed and the obtained hydroalcoholic extracts where then quali- and quantitatively analyzed by HPLC-PDA-MS/MS, with similar polyphenolic profiles for both GPRs and leaves. Quercetin 3-O-glucuronide, caftaric acid and quercetin 3-O-glucoside were the main components of the investigated matrices, although in variable proportions. Considering the prevalent presence of polyphenols in the analyzed extracts their antioxidant activity was evaluated with colorimetric in-vitro assays, off-line combined with HPLC-PDA analysis. In this way the contribution of each compound to the antioxidant activity, in terms of radical scavenging abilities, was determined. These findings could suggest GPRs as a potential source of natural compounds, and therefore promote their use in the food field, as food supplements, which could increase their economic value together with a positive effect on the environment.

In this paper, thirty-six extracts from Sardinian plants were evaluated in vitro for their antimicrobial activity towards a panel of reference strains, Staphylococcus aureus, S. epidermidis, Klebsiella pneumoniae and Escherichia coli, and for their cytotoxicity on mammalian cells. The biological data, together with total phenolic and flavonoid content of the extracts, were treated by PCA (Principal Components Analysis), which highlighted the positive correlation among total phenolic content and increasing antibacterial activities, and a possible involvement of flavonoids in mitigate the cytotoxicity. Thirteen extracts displayed relevant IC50 values (half maximal inhibitory concentration) on S. aureus (IC50 from 1.4 to 153.6 μg/mL), ten out of them were active also against S. epidermidis (IC50 from 3.9 to 150 μg/mL), seven against K. pneumoniae (IC50 from 28.5 to 97.5 μg/mL), and two against E. coli (IC50 74.9 and 156.3 μg/mL). In particular, three extracts obtained from Pistacia terebinthus ssp. terebinthus, Cytinus hypocistis and Limonium morisianum emerged as promising antibacterial candidates. They exhibited remarkable inhibitory activity towards bacterial strains from clinical specimens and presenting different antibiotic-resistance profiles.

Introduction The argan tree (Argania spinosa) is an endemic species from south-western Morocco. Argan-based preparations have been widely used in Moroccan traditional medicine for their biological properties, as well as for several cosmetic purposes. Whereas kernel, pulp of fruit and trunk have been extensively studied for their nutritional and pharmacological effects, relatively little is known about argan tree leaves. Objective The main purpose of the present study is to investigate and characterise the bioactive phenolic fractions in both crude and aqueous extracts derived from argan tree leaves. Methodology A qualitative profile of the antioxidant phenolic compounds in argan leaves was obtained by means of structural hypothesis based on UV spectra and mass spectrometric fragmentation patterns. Moreover, selected phenolics were quantified in argan leaves by using a fully validated method based on liquid chromatography coupled to diode array detection and tandem mass spectrometry (LC-DAD-MS/MS). All the extracts were purified by a fast and reliable microextraction by packed sorbent (MEPS) procedure, before analysing them by LC-MS/MS. Results Based on retention times, mass spectrometric fragmentation and UV spectra, 13 phenolic compounds were identified or tentatively elucidated from crude and aqueous extracts derived from Argania spinosa leaves, while seven compounds were quantified in both extracts. Conclusion The obtained results could represent a first step towards a complete characterisation of the argan plant, its bioactive profiling and the valorisation of its by-products as a source of potentially beneficial bioactive molecules. Copyright © 2015 John Wiley & Sons, Ltd.

The phytochemical profile, antioxidant activity and α-glucosidase inhibitory activity of Hypericum scruglii Bacchetta, Brullo et Salmeri and Hypericum hircinum L. were analyzed and compared to those of the best-known Hypericum perforatum L. Samples were prepared using three different extraction methods (maceration in 70% ethanol, extraction in MeOH/phosphate buffer and decoction), the influence of which on the metabolic profile and bioactivities are discussed. H. scruglii and H. hircinum extracted in 70% ethanol strongly inhibited α-glucosidase (IC50 7.25 and 14.05 μg/ml, respectively), which is a valuable enzymatic target for treating metabolic disorders, while H. perforatum was found to be less powerful than the other two species in all the performed biological tests. The phytochemical profile was analyzed by NMR, HPLC-DAD and HPLC-FLD, revealing remarkable differences among the species. In particular, H. scruglii, which is a species endemic to Sardinia Island (Italy), was particularly enriched in two phloroglucinol-derivatives identified by means of 2D NMR and LC/MS/MS experiments as 3-geranyl-1-(2′-methylbutanoyl)-phloroglucinol and 3-geranyl-1-(2′-methylpropanoyl)-phloroglucinol.

Despite the availability of several anti-retrovirals, there is still an urgent need for developing novel therapeutic strategies and finding new drugs against underexplored HIV-1 targets. Among them, there are the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) function and the cellular α-glucosidase, involved in the control mechanisms of N-linked glycoproteins formation in the endoplasmic reticulum. It is known that many natural compounds, such as pentacyclic triterpenes, are a promising class of HIV-1 inhibitors. Hence, here we tested the pentacyclic triterpene Lupeol, showing that it inhibits the HIV-1 RT-associated RNase H function. We then performed combination studies of Lupeol and the active site RNase H inhibitor RDS1759, and blind docking calculations, demonstrating that Lupeol binds to an HIV-1 RT allosteric pocket. On the bases of these results and searching for potential multitarget active drug supplement, we also investigated the anti-HIV-1 activity of Hemidesmus indicus, an Ayurveda medicinal plant containing Lupeol. Results supported the potential of this plant as a valuable multitarget active drug source. In fact, by virtue of its numerous active metabolites, H. indicus was able to inhibit not only the RT-associated RNase H function, but also the HIV-1 RT-associated RNA-dependent DNA polymerase activity and the cellular α-glucosidase.

Ethnopharmacological relevance Four Indian plants, traditionally used in Ayurvedic medicine: Asparagus racemosus Willd., Emblica officinalis Gaertn., Hemidesmus indicus R. Br., and Rubia cordifolia L. were selected on the basis of their ethnobotanical use and of scientific evidence that suggests a potential efficacy in the treatment of bone-loss diseases. The antiresorptive properties of the four plants have been investigated. The aim was to provide adequate evidence for the exploitation of natural compounds as alternative therapeutics for the treatment of diseases caused by increased osteoclast activity. Materials and methods Decoctions were prepared from dried plant material according to the traditional procedure and standardization by HPLC was performed using marker compounds for each species. Total polyphenols, flavonoids and radical scavenging activity of the decoctions were also determined. The bioactivity of the plant decoctions was evaluated in subsequent phases. (1) A cytotoxicity screening was performed on the mouse monocytic RAW 264.7 cell line to define the concentrations that could be utilized in the following step. (2) The antiresorptive properties of plant decoctions were compared with that of a “gold standard” drug (alendronate) by measuring osteoclastogenesis inhibition and osteoclast apoptosis. (3) The toxic effect on bone forming cells was excluded by evaluating the impact on the proliferation of osteogenic precursors (mesenchymal stem cells, MSC). Results All the decoctions inhibited osteoclastogenesis similarly to alendronate at the highest doses, but Hemidesmus indicus and Rubia cordifolia were also effective at lower concentrations. Apoptosis increased significantly when cells were exposed to the highest concentration of Emblica officinalis, Hemidesmus indicus, and Rubia cordifolia. All concentrations of Emblica officinalis tested inhibited the proliferation of osteogenic precursors, while only the highest doses of Asparagus racemosus and Rubia cordifolia were toxic. On the contrary, Hemidesmus indicus did not affect osteogenic precursor growth at any concentration tested. Conclusion Among the medicinal plants included in the study, Hemidesmus indicus showed the greatest antiosteoclastic activity without toxic effect on osteogenic precursors. Therefore, Hemidesmus indicus exhibits the properties of an antiresorptive drug and represents the ideal candidate for further clinical investigations.

Argania spinosa (L.) Skeels is an endemic Moroccan species belonging to Sapotaceae family. In this work, lipophilic and aqueous extracts were obtained from leaves and subjected to a chemical profiling by MS and LC-MS/MS. Pentacyclic terpenoids were identified and quantified in the lipophilic fraction, while phenolic compounds (mainly belonging to flavonols and flavan-3-ols) were identified in the aqueous fraction. The antibacterial activities of fractions were evaluated in vitro against both reference Gram-positive and -negative bacterial strains and clinical isolates of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA); in addition, the compounds quantified as main components in each extract were assayed against reference strains. A relevant antibacterial activity was observed against reference MSSA and MRSA strains of S. aureus: for the lipophilic fraction, MIC50 values obtained were 177.8 and 170.6 μg/mL for the former and the latter, respectively, while for the aqueous fraction were 215.5 and 233.3 μg/mL. These inhibitory activities could be mainly ascribed to ursolic and oleanolic acids, among pentacyclic terpenoids, and to quercetin concerning phenolic compounds. A remarkable antibacterial activity was also observed against clinical isolates, thus argan leaves can be considered of interest in the chemotherapy of human infections.

Teucrium chamaedrys, one of the most common and investigated species of the genus Teucrium, has been used for centuries in traditional medicine for many purposes. Its phytochemical components comprise, among others, phenylethanoid glycosides (PGs) and neo-clerodane diterpenoids. Several reports have demonstrated a wide range of beneficial biological and pharmacological activities of the phenylethanoid components, while the diterpenes were shown to be strongly hepatotoxic. In this work, in vitro cultures were established from leaf explants of T. chamaedrys. Both solid (callus) and liquid (cell suspension) cultures maintained the capacity to produce PGs, with teucrioside (TS) representing the most abundant one. Cell suspensions had a lower TS content than that found in leaf extracts, but higher than that of calli. An NMR-based metabolomics approach was used to compare the product profile of intact plants vs. cell suspension cultures, and results showed that neo-clerodane diterpenes, present in the intact plant, were not detected in cell cultures. Several elicitors were supplied to cell cultures with the aim of increasing TS production, and elicitation was tested at different growth phases and by exposing cells for different periods. Methyl jasmonate and fungal mycelia from Trichoderma viridae and Fusarium moniliforme were able to significantly increase TS production if supplied at the early-exponential growth phase for 24 h. Based on the proposed link between proline and the phenylpropanoid pathways, proline accumulation in cell cultures was followed throughout a 14-day culture period, showing that it strictly reflected that of TS production. Moreover, exogenously supplied proline, and its analogue hydroxyproline, turned out to be very effective in increasing teucrioside production. © 2012 Elsevier Ltd. All rights reserved.