A Metabolomic Approach to Investigate the Effect of Phytonutrients on Proteostasis and Metabolic Pathways in Drosophila melanogaster.

The use of Drosophila melanogaster as a biological platform to study the effect of diet and food bioactives on the metabolome remains a highly unexplored subject. Aiming to establish alternative solutions for the investigation of nutritional interventions with bioactive natural products by employing LC-MS-based metabolomics approaches, we assessed the effect of a phytonutrient-rich extract from the endemic Mediterranean plant Cichorium spinosum (stamnagkàthi) on a Drosophila population. The extract's modulating effect on the proteostasis network and metabolism of young D. melanogaster flies was evaluated. Furthermore, an untargeted metabolomics approach, employing a C18 UPLC-ESI-Orbitrap-HRMS/MS platform, permitted the detection of several biomarkers in the metabolic profile of Drosophila's tissues; while targeted amino acid quantification in Drosophila tissue was simultaneously performed by employing aTRAQ labeling and an ion-pairing UPLC-ESI-SWATH-HRMS/MS platform. The detected metabolites belong to different chemical classes, and statistical analysis with chemometrics tools was utilized to reveal patterns and trends, as well as to uncover potential class-distinguishing features and possible biomarkers. Our findings suggest that Drosophila can serve as a valuable in vivo model for investigating the role of bioactive phytoconstituents, like those found in C. spinosum's decoction, on diverse metabolic processes. Additionally, the fruit fly represents a highly effective platform to investigate the molecular mechanisms underlying sex differences in diverse aspects of nutrition and physiology in higher metazoans.

A GC-MS and LC-HRMS perspective on the chemotaxonomic investigation of the natural hybrid Origanum × lirium and its parents, O. vulgare subsp. hirtum and O. scabrum.

INTRODUCTION: The genus Origanum L. (Lamiaceae) is widespread in the Mediterranean region. However, approximately 75% of the species are only encountered in the eastern part. Out of these, a total of nine species (11 taxa) and three natural hybrids occur in Greece. Nevertheless, so far, there is no consensus regarding their precise botanical classification in the literature. In fact, the taxon Origanum × lirium has been proposed both as a separate species as well as natural hybrid between Origanum vulgare subsp. hirtum and Origanum scabrum. OBJECTIVES: In this scope, the aim of the current study is to shed light on the matter through the investigation of the chemical composition of both the essential oils and the polar extracts of the mentioned taxa, collected from different geographical regions of Greece. RESULTS: As it was demonstrated by both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) data, and highlighted by our comparative analysis, it can be stipulated that Origanum × lirium shares its chemotype to a large extent with its parent species concerning both volatile and polar constituents. Additionally, geographical origin conditions stood out as a key factor influencing their chemical composition. CONCLUSION: Altogether, the present work provides useful information on the chemical composition of the taxa under investigation, while our findings support the opinion that Origanum × lirium should be considered not as a separate species, but rather as a hybrid on the way to becoming a species.

Development, Validation and Application of a UHPLC-MS Method for the Quantification of Chios Mastic Gum Triterpenoids in Human Plasma.

Chios mastic gum is the resinous secretion obtained from the barks of the shrub Pistacia lentiscus var. Chia, which is endemic to the Greek island of Chios. Since antiquity, Chios mastic gum has found several uses as a phytotherapeutic remedy, primarily for the treatment of gastrointestinal disorders while recently, Chios mastic gum was also recognized by EMA as an herbal medicinal product with specific indications. Chios mastic gum's biological properties are attributed to triterpenes which comprise the major chemical group (approx. 70%) and notably isomasticadienonic acid and masticadienonic acid. However, due to their structural characteristics, the isolation thereof in high yield and purity is challenging and since they are not commercially available, pharmacological studies aiming to assess their biological properties are limited. In the present work, mastic's phytochemical investigation by UPLC-HRMS is followed by the isolation and characterization of isomasticadienonic acid and masticadienonic acid to be used as analytical standards for their accurate and reliable quantification in human plasma. A UHPLC-tQ-MS method that was developed and validated (in terms of specificity, linearity, limit of quantification, accuracy and precision), for the direct quantification of the targeted compounds in the low ng/mL range of concentration, was subsequently implemented on plasma samples of healthy volunteers thus demonstrating its fitness for purpose. The results presented herein might provide insight to the understanding of this traditional natural product consumed notably for its anti-inflammatory, antioxidant and lipid lowering properties. Moreover, this method might serve as a starting point for any study aiming to monitor bioactive triterpenes in biological fluids.

Phytochemical Composition of the Decoctions of Greek Edible Greens (Chórta) and Evaluation of Antioxidant and Cytotoxic Properties.

Wild or semi-wild edible greens (chórta) are an integral part of the traditional Greek Mediterranean diet due to their nutritional value, containing various phytonutrients beneficial to human health. Water-based decoctions of chórta are widely consumed in Greek alternative medicine as health promoting agents. This study examined the chemical profile of the decoctions of eight edible plants, Cichorium intybus, C. endivia, C. spinosum, Crepis sancta, Sonchus asper, Carthamus lanatus, Centaurea raphanina, and Amaranthus blitum, by UPLC-ESI-HRMS and HRMS/MS analysis, to determine possibly bioactive constituents. The profiles of the plants from the Asteraceae family are dominated by the presence of phenolic acids and flavonoid derivatives, whereas the A. blitum decoction is rich in triterpene saponins. Interestingly, the Centaurea raphanina decoction was found to be extremely rich in flavanones, particularly in the aglycone pinocembrin. Further phytochemical investigation and fractionation of this extract resulted in the isolation and identification of five compounds: phlorin (1), syringin (2), pinocembrin (3), pinocembroside (4), and pinocembrin-7-O-neohesperidoside (5). The extracts were also tested for their antioxidant and differential cytotoxic activity against tumor cells. C. raphanina was found to be differentially toxic against metastatic tumor cells. In conclusion, we found that Greek edible greens are a rich source of bioactive secondary metabolites and their consumption could contribute to the maintenance of overall health.

Plasma Amino Acids in NAFLD Patients with Obesity Are Associated with Steatosis and Fibrosis: Results from the MAST4HEALTH Study.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have been linked to changes in amino acid (AA) levels. The objective of the current study was to examine the relationship between MRI parameters that reflect inflammation and fibrosis and plasma AA concentrations in NAFLD patients. Plasma AA levels of 97 NAFLD patients from the MAST4HEALTH study were quantified with liquid chromatography. Medical, anthropometric and lifestyle characteristics were collected and biochemical parameters, as well as inflammatory and oxidative stress biomarkers, were measured. In total, subjects with a higher MRI-proton density fat fraction (MRI-PDFF) exhibited higher plasma AA levels compared to subjects with lower PDFF. The concentrations of BCAAs (p-Value: 0.03), AAAs (p-Value: 0.039), L-valine (p-Value: 0.029), L-tyrosine (p-Value: 0.039) and L-isoleucine (p-Value: 0.032) were found to be significantly higher in the higher PDFF group compared to lower group. Plasma AA levels varied according to MRI-PDFF. Significant associations were also demonstrated between AAs and MRI-PDFF and MRI-cT1, showing the potential utility of circulating AAs as diagnostic markers of NAFLD.

Effect of Mastiha supplementation on NAFLD: The MAST4HEALTH Randomised, Controlled Trial.

SCOPE: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease with poor therapeutic strategies. Mastiha possesses antioxidant/anti-inflammatory and lipid-lowering properties. The authors investigate the effectiveness of Mastiha as a nonpharmacological intervention in NAFLD. METHODS AND RESULTS: Ninety-eight patients with NAFLD in three countries (Greece, Italy, Serbia) are randomly allocated to either Mastiha or Placebo for 6 months, as part of a multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trial. The authors assess NAFLD severity via magnetic resonance imaging (MRI) scanning and LiverMultiScan technique and evaluate the effectiveness of Mastiha through medical, anthropometric, biochemical, metabolomic, and microbiota assessment. Mastiha is not superior to Placebo on changes in iron-corrected T1 (cT1) and Liver Inflammation Fibrosis score (LIF) in entire patient population; however, after BMI stratification (BMI ≤ 35 kg m-2 and BMI > 35 kg m-2 ), severely obese patients show an improvement in cT1 and LIF in Mastiha versus Placebo. Mastiha increases dissimilarity of gut microbiota, as shown by the Bray-Curtis index, downregulates Flavonifractor, a known inflammatory taxon and decreases Lysophosphatidylcholines-(LysoPC) 18:1, Lysophosphatidylethanolamines-(LysoPE) 18:1, and cholic acid compared to Placebo. CONCLUSION: Mastiha supplementation improves microbiota dysbiosis and lipid metabolite levels in patients with NAFLD, although it reduces parameters of liver inflammation/fibrosis only in severely obese patients.

Traditional uses, phytochemistry and pharmacology of Chios mastic gum (Pistacia lentiscus var. Chia, Anacardiaceae): A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Chios mastic gum constitutes a unique Greek product, produced exclusively in the southern part of the island of Chios. References about its use from local populations for the treatment of gastrointestinal disorders or as a cosmetic agent can even be encountered in ancient texts of Galen, Theophrastus and Dioscorides. Nowadays, this versatile resin has been rediscovered, not only as a traditional remedy and aromatic agent, but as a potent phytotherapeutic product with various biological properties. AIM OF THE STUDY: The aim of this study is to quote the summation of the ethnopharmacology, phytochemical profile and pharmacological properties of the resin of Pistacia lentiscus var. Chia and thus provide the scientific community with a summary of the research conducted so far. Furthermore, perspectives and uses are being discussed and studied so as to broaden the field of its applications. MATERIALS AND METHODS: A comprehensive review of the literature on Pistacia lentiscus var. Chia was performed using as resources scientific databases such as Scopus, Sciencedirect, Pubmed and Web of science, studies and traditional books provided by the Chios Mastiha Growers Association as well as PhD and Master' s theses. RESULTS: Chios mastic gum has been used as a traditional medicine over the last 2500 years. More than 120 chemical compounds have been identified in the resin and the major components are a natural polymer, acidic and neutral triterpenes and volatile secondary metabolites. Several plant extracts and compounds have been studied for their antibacterial, anti-inflammatory, antioxidant, anti-ulcer, anti-diabetic, cardioprotective and anti-cancer properties in vitro and in vivo. Clinical interventions and trials have also showed the therapeutic potential of Chios mastic gum. In 2015 Pistacia lentiscus L., resin (mastic) was recognized as a herbal medicinal product with traditional use by the European Medicines Agency (EMA) with two therapeutic indications (mild dyspeptic disorders & skin inflammation/healing of minor wounds). Over the last years, Chios mastic gum is widely involved in medicinal products, food supplements and cosmetics and has become object of study, also in the field of Pharmacotechnology. CONCLUSIONS: Chios mastic's beneficial properties have been demonstrated in the treatment of gastrointestinal disorders, wound healing, skin inflammations, plasma lipid and blood sugar reduction and oral care. These properties are attributed to triterpenes and volatile compounds. However, because of the resin's chemical complexity and the lack of commercial standards for its main compounds, there is a notable gap in literature concerning the biological evaluation of CMG's isolated components. Therefore, future research should focus on the development of efficient extraction, isolation and analysis techniques in order to unravel CMG's full pharmacological potential.

Quantification of bioactive lignans in sesame seeds using HPTLC densitometry: Comparative evaluation by HPLC-PDA.

Sesamin and sesamolin constitute the main bioactive secondary metabolites of sesame seeds (Sesamum indicum L., Pedaliaceae). In the present work, a rapid HPTLC-based methodology was developed in compliance with the requirements of the European Pharmacopoeia for the quantification of these two lignans in sesame seeds. A comparative study was simultaneously performed with HPLC-PDA for assessing the sesamin and sesamolin content of diverse samples. Both methods were validated and the results were subsequently subjected to statistical analysis in order to compare their performance as well as to investigate possible correlations. The methods were shown to be adequately correlated in terms of performance, as revealed by Pearson's rank correlation coefficients (>0.99 for sesamin and >0.98 for sesamolin) and Bland-Altman analysis (relative method bias 0.06-0.21, SD of bias 0.05-0.07). HPTLC densitometry could thereby serve as a valid and reliable tool for the rapid determination of the major lignans in sesame seed samples.

Extracts from the Mediterranean Food Plants Carthamus lanatus, Cichorium intybus, and Cichorium spinosum Enhanced GSH Levels and Increased Nrf2 Expression in Human Endothelial Cells.

The Mediterranean diet is considered to prevent several diseases. In the present study, the antioxidant properties of six extracts from Mediterranean plant foods were assessed. The extracts' chemical composition analysis showed that the total polyphenolic content ranged from 56 to 408 GAE mg/g dw of extract. The major polyphenols identified in the extracts were quercetin, luteolin, caftaric acid, caffeoylquinic acid isomers, and cichoric acid. The extracts showed in vitro high scavenging potency against ABTS•+ and O2 •- radicals and reducing power activity. Also, the extracts inhibited peroxyl radical-induced cleavage of DNA plasmids. The three most potent extracts, Cichorium intybus, Carthamus lanatus, and Cichorium spinosum, inhibited OH•-induced mutations in Salmonella typhimurium TA102 cells. Moreover, C. intybus, C. lanatus, and C. spinosum extracts increased the antioxidant molecule glutathione (GSH) by 33.4, 21.5, and 10.5% at 50 µg/ml, respectively, in human endothelial EA.hy926 cells. C. intybus extract was also shown to induce in endothelial cells the transcriptional expression of Nrf2 (the major transcription factor of antioxidant genes), as well as of antioxidant genes GCLC, GSR, NQO1, and HMOX1. In conclusion, the results suggested that extracts from edible plants may prevent diseases associated especially with endothelium damage.

Beneficial Effects of Sideritis scardica and Cichorium spinosum against Amyloidogenic Pathway and Tau Misprocessing in Alzheimer's Disease Neuronal Cell Culture Models.

BACKGROUND: Natural products are a significantly underutilized source of potential treatments against human disease. Alzheimer's disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by recent findings. OBJECTIVE: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet. METHODS/RESULTS: After the detailed characterization of the extracts' composition using LC-HRMS methods, they were evaluated on two AD neuronal cell culture models, namely the AßPP overexpressing SH-SY5Y-AßPP and the hyperphosphorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and subsequently their downstream effects on AßPP and tau processing pathways were investigated in the SH-SY5Y-AßPP and PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly decrease total tau, the activation of the GSK3ß, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Furthermore, both extracts appear to promote AßPP processing through the alpha, non-amyloidogenic pathway, albeit through partly different mechanisms. CONCLUSIONS: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or treatment of AD, and merit further investigations at the in vivo level.